Aftryp SWG PDF

Scientific Working Group
Report on
African trypanosomiasis
(sleeping sickness)
4-8 June 2001

Geneva, Switzerland
www.who.int/tdr
TDR/SWG/01
Original: English
Report of the Scientific Working Group
meeting on African trypanosomiasis
Geneva, 4-8 June, 2001
TDR/SWG/01
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Programme for Research and Training in Tropical Diseases (2003)
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Contents
Message from the Executive Director, CDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
Message from the Director, TDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface ............................................................................. viii
1 Executive summary ................................................................ 1
2 Overview and objectives .......................................................... 3
3 Epidemiology, disease surveillance and control

EMERGENCE AND RE-EMERGENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
EPIDEMIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Reservoir Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Incidence and Prevalence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
SURVEILLANCE AND INTERVENTION FOR CONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Changing Institutional Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
SOCIOECONOMIC AND BEHAVIOURAL ASPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4 Drug development, preclinical and clinical studies,

and drug resistance
PRECLINICAL STUDIES
Resistance to Arsenicals and Diamidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Blood–Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Role of CNS Trypanosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Drug Discovery and Drug Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
CLINICAL STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Clinical Aspects of Treatment Failure and Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Application of Existing Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Early-stage drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Late-stage drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Other potential drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
New Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
TDR/SWG/01 • Report of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2 0 0 1 iii

Combination Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Follow-up of Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Prevention and Management of Encephalopathy Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Coordination of Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5 Pathogenesis, genomics and applied genomics

PATHOGENESIS
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Trypanosome Biological Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
APPLIED GENOMICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Trypanosoma brucei Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
TSETSE GENETICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
6 Cross-cutting issues
RESOURCE FLOW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
ADVOCACY AND MARKETING FOR SLEEPING SICKNESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
INSTITUTIONAL DEVELOPMENT AND CAPACITY BUILDING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
SOUTH-SOUTH COLLABORATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Annex 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
RESOURCE FLOW FOR AFRICAN TRYPANOSOMIASIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Annex 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
A POSITION PAPER ON AFRICAN TRYPANOSOMIASIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Annex 3
EMERGENCE AND RE-EMERGENCE OF HUMAN AFRICAN TRYPANOSOMIASIS . . . . . . . . . . . . . . . . 42
I The situation in Angola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
II The situation in Tanzania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
III The situation in Uganda and Sudan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
iv R e p o r t of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2001 • TDR/SWG/01
Annex 4
EPIDEMIOLOGY, DISEASE SURVEILLANCE AND CONTROL, AND VECTOR CONTROL . . . . . . . . . . . 54
I Epidemiology and control of human African trypanosomiasis . . . . . . . . . . . . . . . . . . . . 55
II Vector control in relation to human African trypanosomiasis . . . . . . . . . . . . . . . . . . . . 73
III A basis for financial decision-making on strategies for the control of
human African trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Annex 5
DRUG DEVELOPMENT, PRECLINICAL AND CLINICAL STUDIES, AND DRUG RESISTANCE . . . . . 90
I Drug development for African trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
II Drug resistance in sleeping sickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
III Human African trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
IV Treatment and clinical studies: Working paper for the Scientific Working
Group on Human African Trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Annex 6
PATHOGENESIS, GENOMICS AND APPLIED GENOMICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
I Discussion document on pathogenesis/applied genomics . . . . . . . . . . . . . . . . . . . . . . 121
II Applied genomics: Prospects for control of African trypanosomiasis via
the tsetse vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
III Applied genomics and bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Annex 7
INSTITUTIONAL CAPABILITY STRENGTHENING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Institutional development and capacity building in countries endemic for
sleeping sickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Annex 8
LIST OF PARTICIPANTS .................................................................. 167
TDR/SWG/01 • Report of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2 0 0 1 v
Message from the Executive Director,

Communicable Diseases
Following the appointment of Dr Brundtland as Director-General of the World Health
Organization (WHO) in 1998, functional restructuring placed both the control of infectious
diseases and tropical diseases research under one cluster. This has permitted better
identification of priorities and the linking of research with prevention and control activities,
resulting in joint fundraising. The recent agreement with the drug industry on chemotherapy of
African trypanosomiasis is a product of such joint activities. It is hoped that other agreements
emphasizing sleeping sickness will also be signed.
In addition, there has been a concerted effort to move infectious diseases higher in the
economic development agenda in various world economic fora, resulting in political commit-
ment by governments of both developed and disease endemic countries, and in financial com-
mitment by members of the G8, in particular France and the United States of America, to the
Global Fund for AIDS and Health. Currently, the Global Fund is directed at the three major
(based on morbidity and mortality data) infectious diseases – namely malaria, tuberculosis
and AIDS. This will lead to improved health delivery systems that will be better placed to deal
with other diseases such as sleeping sickness. It is envisaged that the health delivery systems
will undergo diversification, allowing governments, NGOs and the private sector to work
together to deliver health services more effectively. This will be of particular importance for
diseases such as sleeping sickness, where NGOs have had to be depended on for continued
support during periods of decreased resources.
This meeting aims to draw up a well thought out research agenda, provide data that
can be used to convince policy-makers to place infectious diseases at the forefront of
development activities, and show the world that the job on infectious diseases is not
yet done.
David L Heymann
World Health Organization
Geneva, June 2001
vi R e p o r t of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2001 • TDR/SWG/01
Message from the Director, TDR
In 1998, the UNDP/World Bank/WHO Special Programme for Research and Training
in Tropical Diseases (TDR) underwent an external review. It was noted that, with the reorgani-
zation of the Programme into functional units, certain aspects of disease focus had been
neglected. It was decided, therefore, that TDR should hold scientific working group (SWG)
meetings to address each of the ten diseases handled by the Programme. Two of these SWG
meetings, this SWG on African trypanosomiasis being the first, will be held each year in order
that all ten diseases are covered in five years. It is expected that this first meeting will set a
research agenda for African trypanosomiasis, closely linked to control needs and open to the
opportunities that science and technology can provide, which will act as a guide not only to
TDR but also to other parties interested in research on African trypanosomiasis.
Funding through TDR is on the increase, and a full-time disease research coordinator for
African trypanosomiasis is to be recruited. This is a reflection of growing donor
interest in the disease, for which a significant amount of funding has already been assured.
However, the funding available is largely restricted to particular projects and limited in geo-
graphical coverage to a small proportion of the 250 known foci of sleeping sickness in Africa.
Furthermore, the number and mix of donors is limited, as is the period covered by the current
funding agreement, which is only for five years. Securing resources for the period following
these five years is therefore a priority. Strategies to attract more funding will include creating
a supportive environment, joining forces with others of like mind, and using the voices of
affected countries. The current matrix approach to programme management in TDR lays great
emphasis on accountability and gives a certain amount of funding security to diseases that are
unlikely to receive additional funding from alternative sources.
Carlos M Morel
Director, TDR
Geneva, June 2001
TDR/SWG/01 • Report of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2 0 0 1 vii

Preface
During the last ten years, sleeping sickness has only been marginally recognized as a health
problem and a research priority, but the recent re-emerging outbreaks and increasing drug
resistance have painfully demonstrated the potential danger in any endemic area. Although
sleeping sickness was brought to low endemic levels practically everywhere in Africa during
the sixties, since then, most national sleeping sickness control programmes have gradually
been sacrificed in favour of more prominent health needs. As a result, the core of national
know-how simply disappeared while dependency on external support was rendered even
greater. Recent outbreaks have opened eyes worldwide as to how serious the situation is, and
increasing awareness is manifest in scientific publications, the layman’s press, and television
programmes. The donor community, as well as industry, has been alerted, and is now provid-
ing unprecedented levels of support to both control and research. As this report states, it was
an opportune time to convene a Scientific Working Group.
This TDR Scientific Working Group was asked to provide technical guidance to donors,
responsible officers at national level, and research institutions as to the concrete directions
research should take, in the Group’s opinion, and where resources should be made available.
The relatively large group of approximately 30 participants permitted wide geographical rep-
resentation and ample coverage in terms of scientific discipline. In order to guarantee that the
recommendations issuing from the meeting were critical, the SWG decided to restrict these to
the two of highest priority for each subsection of the report.
This report briefly reviews the recent emergence and re-emergence of trypanosomiasis, and
elaborates on as yet unanswered questions and issues such as the role of animal reservoirs in
T. b. gambiense transmission, the need for epidemiological indicators for monitoring control
interventions, and the lack of a “gold standard” for new and existing diagnostic tests. One
research area pertinent to the identification of the reservoir of infection and transmission pat-
terns would be the improvement and validation of the current bloodmeal analysis technology.
New tools for vector control per se are not considered a priority at this moment – “the tool
box is full” – but research into the question of why relatively little use is being made of the
currently available tools is indicated.
In the past, practically everywhere, trypanosomiasis control was in the hands of vertical pro-
grammes, which meant a great deal of adaptation was needed, both mentally and logistically,
to integrate personnel and equipment into the general health services. Although in a few coun-
tries this took place gradually and more or less satisfactorily, in countries with a large num-
ber of endemic foci and where large numbers of personnel and facilities were involved, inte-
gration was, and remains, a complicated process. In such cases, local studies on new delivery
pathways are urgently needed.
viii R e p o r t of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2001 • TDR/SWG/01

The disability adjusted life year (DALY) has finally found grace in socioeconomic reasoning
and is now being applied in several economic analyses for trypanosomiasis control. However,
the data collected so far indicate that further refinement is needed to be able to compare the
cost-effectiveness of the different control approaches. Also, little is yet known of the personal
costs a patient spends on seeking care, his/her participation in cost sharing, or the costs of
death and incapacity for the family.
The arsenal of trypanocidal drugs, being scarce already, is becoming more and more limited
due to increasing drug resistance. As an immediate relief measure, applied research to help
develop standard protocols for optimal use of combinations of currently available compounds
is needed. As a long-range objective, the highest priority should be given to laboratory
research to develop new molecules.
When Mott published, in 1899, his classic report demonstrating perivascular infiltration to be
the main histopathological characteristic of sleeping sickness, he probably would not have
imagined how, a good hundred years later, specialists would still be busy following up the
pathogenesis of these brain lesions. Although only a few research groups have been involved
up till now, this field of interesting research is relevant to the possible development of non-
invasive tests for determining the stage of disease. Pathogenesis research could also lead to
prevention and treatment of the fatal reactive encephalopathies which occur during treatment.
Much attention has been paid in this report to genomics as a means of refining parasite identi-
fication, and to genetics for studies on the vectorial capacity of tsetse flies. The T. brucei
genome network of collaborating centres, initiated by TDR jointly with other institutions, is
expected to strengthen functional links at an international level. Emphasis is placed on
strengthening laboratories in Africa for participation in the work on bioinformatics and
genomics.
As a result of the lack of career openings, a high proportion of ex-trainees has left the try-
panosomiasis field. One of the recommendations of the Group is to improve the position of
local scientists in African research institutes by allowing for salary supplements. Making
research careers more attractive and stable should eventually result in establishing an accept-
able core of scientists in the endemic countries. Although the drain of specific knowledge and
experience to elsewhere has been disappointing, the Group has no doubt that continued train-
ing is the only solution to filling the gap in availability of scientists and technicians in endemic
countries.
Considering the improved funding perspectives for both control and research, it is now oppor-
tune to make efforts to enhance collaboration between the two. In order to be able to generate
TDR/SWG/01 • Report of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2 0 0 1 ix
valid disease data from each endemic country, it is recommended that a research nucleus
group be identified and that inter-country collaboration and comparison of results between
endemic countries be strongly advocated.
With this up-to-date review of the present epidemiological situation and the actual control
needs, the Scientific Working Group calls for a response from the scientific community all over
the world. The improved funding possibilities that exist at the present time provide the neces-
sary momentum for introducing new research and control projects and for strengthening rele-
vant ongoing programmes.
Peter de Raadt,
SWG Chairperson
x R e p o r t of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2001 • TDR/SWG/01
1 Executive summary
The Scientific Working Group (SWG) meeting It was noted that recent scientific advances in
on African trypanosomiasis brought together a applied genomics and bioinformatics provide
multidisciplinary group of scientists, partners opportunities that can be exploited to provide
and collaborators from academia, public and new tools for control of disease, and recent sup-
private sectors, sleeping sickness control pro- port from the pharmaceutical industry and a pri-
grammes, and both disease endemic and disease vate foundation has given impetus to tackling the
non-endemic countries. The objectives of the problem of African trypanosomiasis. However,
meeting were to chart out a global research the challenges of obtaining adequate donor sup-
agenda on African trypanosomiasis, closely port and commitment of governments of endemic
linked to control needs and open to opportunities countries, and of personnel recruitment and
arising from basic science, to guide TDR and retention, are daunting. Research on African try-
other parties interested in research on African panosomiasis is an international effort and the
trypanosomiasis and provide data that can be need for partnerships cannot be overemphasized.
used in advocacy to convince policy-makers and
donor agencies to place control of this disease The meeting provided an opportunity to identify
higher on their agenda. knowledge that could be exploited for developing
new, and improving existing, tools for manage-
The Group reviewed the current status of know- ment of disease and vectors, and to determine the
ledge and made recommendations on what tools needs for research capability strengthening in
are needed for appropriate and effective manage- basic sciences in disease endemic countries.
ment and control of sleeping sickness. Three TDR’s comparative advantage in enhancing exist-
broad areas were considered: ing, and developing new partnerships for maxi-
• Epidemiology, disease surveillance and mal application of knowledge was highlighted.
control.
The following are the highest priority recommen-
• Drug development, preclinical and clinical
dations made by the SWG. The SWG:
studies, drug resistance.
• Pathogenesis and applied genomics. • noted with concern that sleeping sickness is a
re-emerging disease which is not given due
attention by governments of endemic countries
Considerable progress has been achieved in
or the international donor community until it
research on African trypanosomiasis in the fol-
attains epidemic proportions, and recommend-
lowing areas: diagnosis and development of
ed that the disease burden and cost effective-
diagnostic tests; epidemiology, host-parasite-
ness of control strategies be calculated to show
vector relationships, animal reservoirs; develop-
that the social and economic consequences of
ment of tsetse traps and screens; understanding
epidemics outweigh the cost of maintaining
of the pathology of the disease; and, drug target-
surveillance;
ed biochemistry of trypanosomes. However, this
progress has not been matched in control of the • noted that lack of appropriate field applicable
disease, due to lack of capacity to sustain diagnostic tools for disease detection, and
improved interventions and to civil disorder in stage of disease, critically affect the control of
some endemic countries. sleeping sickness, and recommended that sim-
ple non-invasive, single-format, field-applica-
ble tests for diagnosis and determination of
stage of disease be developed and validated;
TDR/SWG/01 • Report of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2 0 0 1 1
• considered the small number of anti-try- • noted the possible co-existence of T. b. rhode-
panosomal drugs available, and recommended siense and T .b. gambiense sleeping sickness
that synthetic and natural product libraries be patients in foci where refugees are settled,
integrated for use in drug development; the difficulties in differentiating the two
• acknowledged that the development of drugs trypanosome species, and the different treat-
for late-stage disease is hindered by the blood- ment schedules for both forms of the disease,
brain barrier, which prevents the delivery of and recommended that genomics be applied
drugs to the central nervous system (CNS), to comparing T. brucei sub species, strains and
and recommended that CNS-penetration mod- life cycle stages for their differentiation and
els be used in drug development strategies for disease management;
human African trypanosomiasis, and that • appreciated the important role that vector
strategies which facilitate the delivery of drugs control plays in reducing the transmission of
across the blood brain barrier be developed; vector borne diseases, and recommended that
• took into account the limited evidence suggest- tsetse-trypanosome interactions be investigated
ing that combination therapy with late-stage to determine the molecular basis of refractori-
drugs has an additive effect, and, in view of ness for trypanosome transmission and mecha-
the urgent need to have alternative treatments nisms for driving desirable genes into vector
for melarsoprol refractory patients, recom- populations;
mended that combination chemotherapy using • noted with concern the absence, within TDR,
late-stage drugs be optimized; of a full-time staff member responsible for
• acknowledged the difficulties associated with research activities on African trypanosomiasis,
the treatment of sleeping sickness patients, and and recommended recruitment of such a
the lengthy post-treatment follow-up, and rec- person;
ommended investigating and applying new • recognized the inadequacy of infrastructure for
information on immune parameters to (i) the research in different endemic countries, and
determination of stage of disease, (ii) the pre- recommended networking and cross country
vention and/or amelioration of the encephalitis comparison of research progress to assist in
and encephalopathy associated with the dis- capacity building and stimulate cross border
ease and its treatment respectively, and (iii) the interest and advocacy;
development and validation of a non-invasive • noted with concern the low priority given to
protocol for determining cure and shortening African trypanosomiasis by governments of
the duration of after-treatment follow-up; endemic countries, and recommended strong
• expressed concern that the limited number of advocacy to persuade disease endemic country
suitable centres in Africa created a situation governments to accord priority attention to
where research was increasingly compartmen- research and control of African trypanosomia-
talized, and recommended that the capacity of sis amidst their other health priorities.
laboratories/centres within Africa be strength-
ened in the basic sciences, including in bioin-
Other high priority recommendations, with sug-
formatics, genomics and applied genomics,
gestions for studies and/or actions that should be
drug discovery and development;
undertaken to meet the objectives of the meeting,
are listed in the text.
2 R e p o r t of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2001 • TDR/SWG/01
2 Overview and objectives
African trypanosomiasis is caused by protozoan trypanosomes, and signif icantly, in T. b.

parasites, trypanosomes, which are transmitted rhodesiense sleeping sickness, domestic and
by tsetse flies (of the genus Glossina). The dis- wild animals. In T. b. gambiense disease, the
ease occurs in two forms: a chronic form caused classical human-fly-human transmission cycle
by Trypanosoma brucei gambiense, which occurs occurs in both endemic and epidemic situations.
in West and Central Africa; and an acute form,
caused by T. b. rhodesiense, which occurs in Sleeping sickness is a re-emergent disease,
Eastern and Southern Africa. The chronic infec- but does not get due attention, probably because
tion lasts for years, whilst the acute disease may its impact is regional. The disease occurs in
last for only weeks before death occurs, if treat- 36 sub-Saharan countries, within the area of
ment is not administered. distribution of the tsetse fly. Over 60 million
people living in some 250 foci within this region
The epidemiology of sleeping sickness is com- are at risk of contracting the disease (see figure 1).
plex and transmission cycles are subject to
interactions between humans, tsetse flies and
Figure 1 - The focal distribution of human African trypanosomiasis
Left of dotted line:

Trypanosoma brucei gambiense foci in
West and Central Africa
Right of dotted line: T. b. rhodesiense foci

in Eastern and South Africa
Source: WHO picture library
The number of cases reported annually is over The social and economic impact of sleeping
40 000, but this is highly underestimated, due sickness is often underestimated. During epi-
to difficulties in diagnosis and remoteness of demics, large proportions of communities are
affected areas. It has been estimated that the affected, with loss of life and untold suffering.
actual number of cases is at least 300 000, the These have serious social and economic conse-
vast majority of whom are not diagnosed or quences, which far outweigh the cost of main-
treated (WHO 1998). These figures are relatively taining surveillance. The disease has been a
small compared to other tropical diseases, but major cause of depopulation of large tracts of
African trypanosomiasis, without intervention, Africa. The fear it causes has led to abandon-
has the propensity to develop into epidemics, ment of fertile lands, and is an impediment to
making it a major public health problem. development. Some affected countries have agri-
Furthermore, the case fatality rate in untreated culture-based economies, and workers in cocoa
patients is 100%. This fact, combined with the and coffee plantations are always at risk of con-
focal nature of the disease, means that the dis- tracting the disease, consequently decreasing the
ability adjusted life years (DALYs) averted per labour force. This is reinforced by the fact that
infection cured or prevented are very high. As a the disease mainly strikes the active adult popu-
result, control of this disease in areas at risk is lation.
highly cost-effective, falling well below the
accepted threshold value for money of US$25 Regular medical surveillance, involving accurate
per DALY averted (Dr A. Shaw, personal case detection and appropriate treatment, and
communication, see attached working document, tsetse control where applicable, is the backbone
Shaw and Cattand, Annex 4 section III). of the strategy for the control of sleeping sick-
ness (WHO, 1998). With the available tools, con-
At the beginning of the last century, huge sleep- trol is a continuing effort rather than eradication.
ing sickness epidemics devastated large areas of Experience has shown that where control is inter-
the continent. In the 1960s, the prevalence of the rupted, for a variety of reasons, resurgence of the
disease had been successfully reduced to less disease occurs sooner or later.
than 0.1% in all endemic countries, through his-
toric campaigns by the former colonial powers. Over recent years, human trypanosomiasis has
Soon after independence, however, national gov- been the subject of renewed interest among the
ernments failed to sustain such programmes due donor community and scientists. Substantial vol-
to lack or diversion of resources to other more untary contributions have been made by Belgium
pressing health problems. Breakdown of special- and France for research and control in the
ized mobile teams and health facilities in several endemic countries, as well as by the pharmaceu-
countries, as a consequence of war and civil tical industry, but these contributions only partly
strife or change in health policy, resulted in dra- cover the current needs, and the number of
matic resurgence of the disease, the distribution donors is still very limited. Nongovernmental
of which corresponds closely with that of major organizations (NGOs) have clearly committed
conflicts in sub-Saharan Africa. efforts to participate in control. Special articles
on the trypanosomiasis problem have appeared
in both the scientific press and public media
(Figaro, June 2001; New York Times, 9 February
2001).
The objectives of the scientific working group

(SWG) were to:
• Identify areas where there are gaps in knowl-
edge, and studies that are necessary to fill
these gaps.
• Identify research that is directly relevant to
control programmes and treatment centres as a
priority.
• Promote development of new tools for control
and new methods for use of old tools, and
effective approaches to disease control.
• Set objectives for research capability strength-
ening for basic science, genomics and applied
genomics, drug discovery and development in
disease endemic countries.
References
WHO Expert Committee on Control and Surveillance
of African Trypanosomiasis. Geneva, World Health
Organization, 1998 (WHO Technical Report Series,
No. 881).
3 Epidemiology, disease surveillance and control
EMERGENCE AND RE-EMERGENCE
The persistence and re-emergence of sleeping may increase human-fly contact and hinder
sickness in Africa is attributable to various regular medical surveillance of the population
factors including lack of surveillance, shortage at risk. In rhodesiense sleeping sickness, cattle
of drugs, and several other determinants which movements also increase the risk of infection.
operate at different levels. Means for regular Agro-ecological changes may alter tsetse habitat
surveillance are often inadequate. At the individ- and increase human-fly contact. A significant
ual and family levels, there may be inadequate resurgence of the disease has occurred, notably
knowledge about disease symptoms, transmis- in Angola, the Democratic Republic of the
sion dynamics and treatment. Population move- Congo, Sudan and Uganda. New foci have also
ments, such as seasonal migration and refugees, emerged in recent years.
Table 1 - Number of sleeping sickness patients treated by year per country
Sleeping sickness Country 1995 1996 1997 1998 1999 2000

Angola 6 786 8 275 7 373 5 351 4 546
D.R. Congo 18 158 19 342 25 200 26 318
T. b. gambiense
Sudan 157 737 1 800 18 684 16 975
N.W. Uganda 1 062 980 1 069 967 1 020
S.E. Uganda 497 271 287 299
T. b. rhodesiense
Tanzania 400 531 421 588 627
Source: Compiled from the working

documents of the various countries.
Ministries of health, research organizations Central governments often accord sleeping

and services often lack, or do not have, adequate sickness a low priority, until it assumes epidemic
economic resources for sleeping sickness proportions. In addition, political upheavals, civil
control programmes due to competing health pri- strife and wars lead to the breakdown of health
orities. Recruitment of medium-level personnel services and of control programmes.
is inhibited by lack of incentives and career
prospects. Ministries may lack funds for the The international community is prepared to
purchase of diagnostic tests and drugs, except mobilize resources when epidemics occur but
as part of externally-funded programmes. is often unable to provide long-term support
for surveillance and preventive measures in
Other factors that increase the risk of infection endemic situations. Until recently, the continued
and human-fly contact include agricultural devel- production of anti-trypanosomal drugs was in
opment such as coffee and cocoa plantations, and question. This problem has been resolved for
the tourism industry. the next five years through generous donations
by pharmaceutical companies. However, arrange- networks, such as the Programme Against

ments need to be made to ensure a sustained African Trypanosomiasis (PAAT) managed by
supply beyond this period. the Food and Agriculture Organization (FAO),
Organization of African Unity (OAU) and
In order to ensure that research results are com- WHO, and new networks, such as the WHO
parable over a wide area, advantage should be surveillance network, should be supported.
taken of the potential of networking (for more
general data collection) and using consortia of
investigators, as appropriate, including public/
private partnerships. Avenues should be explored
as to how to make better use of existing
EPIDEMIOLOGY
Reservoir Studies
Reservoir hosts play an important part in sus-

taining endemicity and in the re-emergence of • Assessment of the epidemiological sig-
epidemics. In the case of T. b. rhodesiense nificance of an animal reservoir for
sleeping sickness, several wild and domestic gambiense sleeping sickness using new
animal reservoir hosts have been identified and approaches such as the molecular
certain outbreaks brought under control by treat- approach.
ing nearby cattle. Although natural infections
with T. b. gambiense have been reported in
• Assessment of the epidemiological and
clinical significance of “unconfirmed
domestic animals such as pigs, dogs, sheep and
cases”, defined as individuals with clin-
cattle, and may occur in wild animals as well,
ical signs, or as cases where indirect
the role of an animal reservoir in the epidemiol-
evidence of infection such as sero-posi-
ogy of T. b. gambiense remains as yet undeter-
tivity or polymerase chain reaction
mined. Another important group of reservoir
(PCR)-positivity exists but where the
hosts, especially in T. b. gambiense epidemiolo-
parasite cannot be demonstrated by
gy, are the human carriers: infected individuals
microscopy.
who remain undiagnosed due to inadequate sur-
veillance and/or the limitations of current diag-
nostic tools in demonstrating low levels of try-
panosome infections. The following areas for
research were identified:
Incidence and Prevalence
To design optimal control strategies and The validation of diagnostic tests is needed
estimate the burden of disease, knowledge of to obtain more accurate estimates of the real
current prevalence and the impact of control disease prevalence.
measures is required. This could be obtained by:
• Standardizing and extending reporting

systems using geographic information
systems (GIS).
• Collating and analysing existing
epidemiological data on the impact of
past control schemes on prevalence.
• Introducing a standardized protocol
for monitoring the impact of control
measures on epidemiological indicators.
• Epidemiological modelling to better
understand and predict the disease’s
behaviour, e.g. to obtain better estimates
of the ratio of unreported to reported
cases, or of the basic reproductive
number, Ro.
SURVEILLANCE AND INTERVENTION FOR CONTROL
Diagnostics
Currently, the screening tests used are only avail- tics based on molecular techniques (e.g. PCR)
able in bulk presentation, which limits their use for epidemiological and clinical studies is strong-
where small numbers of people or individuals ly recommended. The need for field applicable,
need to be tested, e.g. in rural health centres, for non-invasive diagnostic methods is recognized,
surveillance on a small scale. There is a need for especially to avoid lumbar punctures.
further development of simple, field applicable,
single test formats (such as dipsticks), with high The main constraint to the development and vali-
specificity. A multicentre validation of diagnos- dation of diagnostic tests is the lack of a gold
standard. Statistical methods exist for the evalua- Changing Institutional Environment
tion of diagnostic tests in the absence of a gold
standard, but so far these have not been applied Far-reaching institutional changes have occurred
to the diagnosis of human African trypanosomia- during the last decade which have had a major
sis. It is recommended that the use of these impact on the organization and implementation
methods should be explored. of sleeping sickness control. These include
decentralization, integration of human African
For stage determination and follow-up, the trypanosomiasis (HAT) control into the primary
following investigations should be undertaken: health care (PHC) system, cost recovery/sharing,
and varying degrees of community involvement
in surveillance and control. Capacity building in
• Multicentre validation of latex/IgM policy and health systems, to help identify and
and PCR tests on cerebrospinal fluid. remedy the changes that have adversely affected
• Identification of new markers of disease control, is recommended. The following
neuropathogenesis, e.g. cytokines. areas of research were identified:
• Evaluation of the use of antigen
detection tests for follow-up.
• Evaluation of the impact of changes in
delivery pathways for sleeping sickness
control on the effectiveness of control
measures.
Vectors
• Investigation of the effects of changes
in cost-sharing arrangements on disease
A range of tools for the control of tsetse flies
detection rates, compliance, and effec-
have been developed over the last twenty years,
tiveness of control.
some of which can be applied by communities.
However, their adoption and application by com-
munities in endemic areas has not been sustain-
able. The exact role of the vector in relation to The monitoring protocols recommended above
transmission of the disease from the various ani- should be applied here.
mal reservoir hosts is unclear. The following
research areas were identified:
• The constraints affecting sustained use

of vector control tools by affected com-
munities.
• Comparison, validation and improve-
ment of available tools for blood-meal
analysis.
SOCIOECONOMIC AND BEHAVIOURAL ASPECTS
Controlling sleeping sickness is a highly cost- It is recommended that social science research
effective intervention, with the cost per DALY address the following issues:
comparing very favourably with other health
interventions, and falling well below the
accepted value of US$25 per DALY averted • Costing of the different control strategies
(Dr A. Shaw, personal communication). This to cover both endemic and epidemic sit-
reflects the focal nature of the disease, and the uations, NGO and national programmes,
fact that the case fatality rate in untreated and a range of countries.
patients is 100%. Although the funding situation
• The direct and indirect costs to patients
has improved somewhat, and greater awareness
and their families of obtaining diagnosis,
of sleeping sickness as a public health priority
treatment, hospitalization, and follow-up
exists, it is vital to reinforce and extend this by
examinations, as well as the costs of
generating appropriate socioeconomic informa-
permanent disability.
tion in order to:
• Refinement of the work done so far on
• Determine the financial resources that are
calculation of DALYs, and its extension
required for control.
to other settings.
• Choose the most appropriate and cost-effec-
• Clarification of issues influencing
tive control strategies.
community and individual support of,
• Promote advocacy through better understand- and involvement in, control measures
ing of the economic burden of the disease. including:
• Provide guidelines for allocating resources - The development of approaches for
amongst competing health needs. enhancing and sustaining community
participation in the control and surveil-
lance of sleeping sickness in endemic
areas.
- The possible existence of gender issues
in the diagnosis and treatment of sleep-
ing sickness, focusing on knowledge,
practice and health care seeking patterns.
When combined with the epidemiological infor-

mation outlined in the section above (on epi-
demiology), such studies would enable the bur-
den of disease to be estimated and the cost-
effectiveness of different interventions to be cal-
culated and compared.
RECOMMENDATIONS
The following are the highest and high priority recommendations from this section:
Highest priority
• Development and validation of non-invasive, field-applicable, single-test format diagnostics
tests, including tests for disease-stage determination.
• Calculation of burden of disease and cost-effectiveness of control strategies.
High priority
• Assessment of the epidemiological and clinical significance of ‘unconfirmed suspects’.
• Systematic monitoring of disease incidence and prevalence, especially in relation to control
measures.
• Identification of issues influencing individual and community participation in control
measures.
4 Drug development, preclinical and clinical
studies and drug resistance
PRECLINICAL STUDIES
The frequency and extent of use of the standard with little success. But with the advent of com-
drugs against African trypanosomiasis, melarso- binatorial chemistry and the sequencing of the
prol and pentamidine, is likely to lead to the trypanosome genome, new techniques can now
development of resistance. Indeed, there has be applied to the development of novel, specific
been an increase of late-stage cases refractory and non-toxic agents for HAT. In addition, to
to melarsoprol treatment in the past decade foster continuation of research and development
(Legros et al, 1999). The availability of these of new drugs after the end of the currently
agents, and of eflornithine (DFMO), was not assured five-year period, capacity strengthening
assured until recently. Even now, it is only of laboratories and/or centres within Africa for
assured for the next five years. With the excep- drug discovery and development is recommend-
tion of a new pentamidine-related drug (DB ed.
289), which is due to enter phase II clinical
trials in 2001, no new candidate agents are
currently in the advanced stage of development.
Resistance to Arsenicals
All the drugs are expensive and require hospital-
and Diamidines
ization for administration by the parenteral
route. In addition, melarsoprol is associated
with a 10% incidence of reactive encephalopa- Laboratory studies have identified the P2
thy that is fatal in up to 5% of the victims. amino-purine transporter as one route of entry
Therefore, there is an urgent need for novel, into trypanosomes for both melarsoprol and
safe, rapidly-acting and inexpensive agents for pentamidine. Loss or alterations to this trans-
the treatment of human African trypanosomiasis porter, caused by genetic modifications to the
(HAT) in the 21st Century. TbAT1 gene, can contribute to the development
of resistance. However, other transporters have
During the late stages of African trypanososmia- been implicated in the uptake of pentamidine,
sis, parasites lodge in privileged sites within the and possibly other analogues, since parasites
central nervous system, causing encephalitis. lacking the P2 transporter have been shown to
The biological nature of such parasites, the be sensitive to pentamidine. Moreover, geneti-
mechanisms by which they, and the drugs that cally modified parasites, lacking the TbAT1
cure late-stage infections, cross the blood-brain gene, are only marginally less sensitive to
barrier, are unknown. There is evidence that melarsoprol than wild type trypanosomes.
eflornithine acts additively with non-permeating
drugs in late-stage infections and suppresses the Treatment failure with melarsoprol may also be
encephalitis. The nature of these drug interac- attributed to the level of melarsoprol permeating
tions is not known but understanding them is into the cerebrospinal fluid (CSF), which varies
critical to the development of new approaches to considerably between individuals. Concentra-
chemotherapy. tions of this drug within the central nervous
system (CNS) are close to the minimum
In the past decade, drug discovery has proceed- inhibitory concentration (MIC) of the drug
ed along biochemical target-based approaches against T. b. gambiense. Hence, modest increas-
es in MIC values in parasites can increase the of drugs for the treatment of Alzheimer`s dis-
proportion of late-stage patients showing refrac- ease, brain tumours and neuro-psychiatric con-
toriness to treatment with melarsoprol. ditions, where novel in vitro methods to screen
Therefore, the tripartite relationship between for compounds that cross the BBB or deliver
drug, host and parasite, and cross-resistance drugs across the BBB have been utilized. It is
between veterinary trypanocides, e.g. dimi- recommended that the relevance of these
nazene aceturate, and HAT drugs, needs consid- approaches in the treatment of HAT be investi-
eration in determining treatment failure in the gated and applied as the first approach in the
field. drug screening pathway, followed by more
focused animal model studies.
Three areas of research are recommended:
The following investigations are recommended:
• Identification of modes of uptake, and

of action and mechanisms of resistance • The use of models of CNS penetration
to arsenicals and diamidines in veteri- (BBB models) for inclusion in the HAT
nary and human use. The influence of drug development pathway, in particu-
both parasite and host factors on treat- lar:
ment failure requires consideration. - In silico models that use the physico-
• Comparison of the mechanisms under- chemical properties of compounds to
lying treatment failure in field isolates determine which are most likely to
of the parasite with the mechanisms distribute from the blood to the brain
underlying resistance in laboratory (primary screen).
strains of the parasite in which resist- - In vitro cell culture models (e.g.
ance has been induced. MDCK and endothelial cells) that
• Reliable diagnosis of drug resistance in enable the study of permeation of
the field. drugs through a cell layer that has
"tight" junctions (secondary screen).
• Strategies that facilitate the delivery
of drugs across the blood-brain barrier
in animal models using known
trypanocides, including:
Blood-Brain Barrier
- A bradykinin agonist (RMP-7,
Cereport) that is on clinical trial for
The development of therapeutic drugs for the
delivery of anticancer and antiviral
treatment of late stage HAT is limited by the
drugs into the brain.
blood-brain barrier (BBB) that prevents the free
distribution of drugs into the CNS. An essential - The role of drugs that modulate the
component in the development of drugs for the CNS inflammatory response (for exam-
treatment of late stage HAT is to design or ple, DFMO and azathioprine) on drug
select compounds that can cross the BBB. This access and activity in animal models.
approach has been a priority in the development
Role of CNS Trypanosomes drugs without support for the synthesis and test-
ing of new molecules. The use of contemporary
Drug action and efficacy depend on the physio- drug discovery methods should be encouraged to
logical/metabolic state of the trypanosome, which find new molecules for the treatment of HAT.
may be different for stages in the CNS and CSF These methods should follow current drug dis-
from those in the bloodstream. Metabolically covery practices utilized by the pharmaceutical
inactive and non-dividing forms tend to be less industry. Building facilities and training scientists
sensitive to drugs, and, depending on the mode of to carry out drug discovery and development
action of the drug, can even be completely insen- research in endemic countries should be strongly
sitive. Apart from parasites in the brain and CSF, encouraged.
trypanosomes in other tissue niches that are less
accessible to the drugs, must be taken into The following areas of research are recommended:
account. Research therefore should be directed at:
• Drug discovery efforts which integrate
synthetic and natural product libraries
• Development of suitable models for stud- with structure-based modelling, computa-
ies on CNS and CSF trypanosomes, tional chemistry and high-throughput
including in vitro models and animal screening (HTS) both for efficacy and
models. toxicity. Target based libraries will initial-
• Studies on the biology of CNS and CSF ly require specific biochemical assays
parasites, their localization and means of while diverse synthetic and natural prod-
entry from the vascular sites, and their uct libraries will require whole cell
inter-exchange. assays.
• Studies on the metabolic state, and • High-throughput screening of synthetic
susceptibility to drugs, of CSF and CNS combinatorial libraries designed based on
trypanosomes. existing leads or obtained from existing
combinatorial libraries in pharmaceutical
companies.
Drug Discovery and Drug Targets • Investigation of target enzymes or path-

ways which, when inhibited, render para-
sites non-viable. Examples of such tar-
While agencies funding scientific research devote
gets are S-adenosylmethionine decar-
considerable resources to carrying out research
boxylase, myristate metabolism and vari-
on the basic biology of trypanosomes, limited
able surface glycoprotein (VSG) synthe-
funding goes to drug discovery and development
sis, and farnesyltransferase.
projects. Likewise, drug discovery efforts by the
pharmaceutical industry, directed specifically • Identification, validation by genetic
towards new agents for the treatment of HAT, are manipulation, and production of new tar-
nearly non-existent. A wealth of knowledge about gets based on exploitation of information
the biology of the organism, and the promise of from the genome project. Validated tar-
new drug targets resulting from genome research, gets should enter HTS screens.
will have little impact on the discovery of new
RECOMMENDATIONS
Highest priority
• Drug discovery efforts which integrate synthetic and natural product libraries with structure-
based modelling, computational chemistry and high-throughput screening, and whole cell
assays, both for efficacy and toxicity.
• Investigation of the use of models of CNS penetration for inclusion in the HAT drug devel-
opment pathway, and development of strategies that facilitate the delivery of drugs across the
blood-brain barrier.
High priority
• Characterization of modes of uptake and action of melarsoprol and diamidines, and identifi-
cation of mechanisms of treatment failure with these drugs in the field.
• Studies on the biology of CNS and CSF parasites, their localization and means of entry from
the vascular site, and their inter-exchange.
CLINICAL STUDIES
Clinical Aspects of Treatment Failure 2. Eflornithine
and Monitoring The ongoing pharmacokinetic study of oral
eflornithine monotherapy should be completed,
and the results used in planning further develop-
Treatment failures with melarsoprol have been ment. In addition, development of a new route
observed in up to 30% of patients in some foci of synthesis should be considered in view of the
in north-western Uganda, southern Sudan and technical difficulties with the current route.
northern Angola (Legros, 1999). The WHO
coordinated Sleeping Sickness Treatment and 3. Nifurtimox
Drug Resistance Network is conducting sentinel Nifurtimox is being used on a compassionate
surveillance for treatment failures. Reasons basis for the treatment of melarsoprol refractory
underlying treatment failures should be investi- cases. However, it is not registered for treatment
gated. of sleeping sickness. Complete development of
the drug up to registration is recommended.
Application of Existing Drugs Currently available data suggest that nifurtimox
may be more suitable for use in combination
Early-stage drugs than as monotherapy.
1. Pentamidine
Recent data from pharmacokinetic studies Other potential drugs
suggest that the half-life of pentamidine is suff- 1. Diminazene aceturate
iciently long to allow shorter treatment regimens. Diminazene aceturate has been used by sleeping
2. Suramin sickness control programmes in several coun-
The efficacy of a shorter course for the treatment tries, but has not been registered for human use.
of early stage T. b. rhodesiense should be Development for human use as well as an oral
explored. preparation should be considered.
2. Benznidazole
Late-stage drugs Limited experimental data suggest that
1. Melarsoprol benznidazole has some activity against strains
Currently, the 10-day (concise) melarsoprol of T. brucei. It has an established safety record
regimen is being reviewed in 17 centres in in humans in the treatment of American
7 countries. A full report is expected at the end trypanosomiasis (Chagas disease). It should be
of 2002. considered as a possible alternative compound
for use in combination therapy of sleeping
Preclinical and clinical evaluation of the concise sickness.
melarsoprol regimen for treatment of T. b.
rhodesiense infections is recommended. New Compounds
The possibilities of a new formulation for melar- 1. DB 289
soprol should be explored in order to avoid the An international consortium is conducting clini-
adverse extravascular effects at the site of admin- cal research to develop DB 289 for oral use
istration caused by the currently used solvent against first-stage sleeping sickness. Phase I tri-
propylene glycol. CNS penetration of new for- als have been concluded and a Phase IIa (proof-
mulations should be equivalent or superior to the of-principle) trial is planned. Results of the
current formulation. investigations will be made available to TDR.
2. Megazol • Combination of melarsoprol and nifur-

Limited studies in animal models suggest that timox vs. monotherapy with each of the
megazol is effective as monotherapy in first- drugs. Preliminary results show that the
stage sleeping sickness and in combination with combination therapy with low-dose con-
suramin in second-stage T. gambiense infections. secutive melarsoprol combined with
Toxicity studies should be completed. Should the short-duration nifurtimox was superior to
data from these studies be favourable, further monotherapy with either melarsoprol or
investigations on the efficacy of monotherapy in nifurtimox.
first- and second-stage disease, using appropriate • Trials comparing melarsoprol and nifur-
animal models, should be carried out. timox, melarsoprol and intravenous (iv)
eflornithine, iv eflornithine and nifur-
timox, have recently started.
Combination Therapy
In the longer term, combination treatment regi-
There is experimental and limited clinical evi- mens should be optimized (i.e. maximum effica-
dence suggesting that combinations of presently cy, minimum toxicity, shortest possible duration,
available late-stage drugs, melarsoprol, eflor- simplicity, with preferably oral administration,
nithine and/or nifurtimox, act additively and minimal cost). Further pharmacological and
(Jennings, 1988, 1993). The current simultaneous preclinical investigations are necessary, including
availability of these drugs gives an unprecedent- experimental studies on the optimal proportions
ed opportunity to establish optimal combination of drugs in combination treatment regimens.
treatment regimens. Although there are indica- Those investigations should be followed by
tions that the early-stage drug suramin, in combi- appropriate clinical trials, preferably including
nation with several other compounds, can cure pharmacokinetic data collection. Oral eflor-
sleeping sickness, priority should be given to nithine would be preferred for combination ther-
studies of combinations of late-stage drugs. apy as the current complicated iv regimen of
eflornithine is not suitable for large-scale treat-
In view of the urgent need to have available alter- ment in rural areas. Drug combinations with
native treatments for melarsoprol refractory melarsoprol should be based on the concise
patients, short-term as well as longer-term solu- melarsoprol treatment regimen.
tions are needed. To identify alternative treatment
for melarsoprol-refractory patients, the following
clinical trials are currently being conducted:
Follow-up of Treatment
One of the major problems of clinical trials and

case management is the long two-year follow-up
period, requiring multiple lumbar punctures.
Appropriately planned studies to determine the
interval between treatment and relapses, which
may allow a reduction of the follow-up period,
are recommended. Research on less invasive
markers for stage determination and cure should
be given high priority, with emphasis on their
applicability in the field.
Prevention and Management of

Encephalopathy Syndromes
Encephalopathy syndromes, which occur during

administration of late-stage drugs, are a major
problem in the treatment of sleeping sickness.
Research into the underlying mechanisms is criti-
cal for the development of improved strategies
for prevention and management of the complica-
tions of treatment.
Coordination of Clinical Trials
The current number of suitable centres (with ade-

quate equipment, trained staff, accessibility and
security) to conduct clinical trials in the field of
sleeping sickness is very limited. In view of the
anticipated number of clinical trials, coordination
will be necessary. It is recommended that a clini-
cal trial group be created within the WHO sleep-
ing sickness treatment and drug resistance net-
work. This group should coordinate clinical trials
and provide appropriate training (good clinical
practice and ethics).
RECOMMENDATIONS
Highest priority
• Development and optimization of a protocol for combination therapy using late-stage drugs.
• Development of tools for shortening the duration of after-treatment follow-up and for dis-
ease-stage determination.
High priority
• Development of nifurtimox up to registration for use against African trypanosomiasis.
• Preclinical evaluation of the 10-day concise melarsoprol regimen for treatment of
T. b. rhodesiense (evaluation in an appropriate monkey model).
• If the toxicological data are favourable, appropriate preclinical studies on the efficacy of
megazol in first- and second-stage infections.
• Elucidation of the underlying mechanisms of encephalopathy syndromes in view of their
prevention and management.
• Exploration of the possibility of better formulations of melarsoprol.
References
Legros D et al. [Therapeutic failure of melarsoprol
among patients treated for late stage
T. b. gambiense human African trypanosomiasis
in Uganda]. Bulletin de la Société de Pathologie
Exotique, 1999, 92(3):171-2 [in French].
Jennings FW. Chemotherapy of trypanosomiasis: the

potentiation of melarsoprol by concurrent difluoro-
methylornithine (DFMO) treatment. Transactions of
the Royal Society of Tropical Medicine and Hygiene,
1988, 82(4):572-3.
Jennings FW. The potentiation of arsenicals with

difluoromethylornithine (DFMO): experimental
studies in murine trypanosomiasis. Bulletin de la
Société de Pathologie Exotique et de ses Filiales,
1988, 81(3 Pt 2):595-607.
Jennings FW. Combination chemotherapy of

CNS trypanosomiasis. Acta Tropica, 1993,
54(3-4):205-13.
5 Pathogenesis and applied genomics
PATHOGENESIS
Background is typified by a loss of Th1 cells secreting IFNg

and the production of very high levels of the
Proper and safe chemotherapy of trypanosomia- anti-inflammatory cytokine IL-10. The high IL-
sis is acutely dependent upon accurately identify- 10 levels are associated with loss of tissue resist-
ing the clinical stage of infection (staging). ance and spread of trypanosomes to the CNS.
Staging has been difficult in the past because of
a lack of accurate, sensitive and relatively non- Early- and late-stage trypanosomiasis can there-
invasive clinical or parasitological tools for diag- fore be characterized by the levels of specific
nosis. However, recent advances in the immunol- cytokines in patients. High IFNg but low IL-10
ogy of trypanosomiasis and in parasite cell biolo- levels are associated with the early non-invasive
gy suggest new avenues for accurate staging. disease, and high IL-10 but low IFNg levels are
Immunological results from both experimental associated with late-stage invasive disease. Since
and clinical trypanosomiasis studies have shown preliminary clinical measurements show that
that infection may be broken down into three rel- there is concordance with respect to serum and
atively well-defined stages: early innate CSF levels of these cytokines, the prospects for
response; early acquired immune response; late determining early- or late-stage disease by test-
acquired immune response. Each of these ing serum for IFNg or IL-10 levels, respectively,
responses exhibits distinguishing prognostic is clear. Furthermore, examination for cytokine
characteristics. levels, which are very short-lived in serum and
CSF, may help clarify whether a seropositive
The early innate response is triggered by the individual has had a recurrence of disease.
presence of trypanosomes within tissues, in suf- Moreover, information concerning immune status
ficiently high numbers, leading to the release of and trypanosome-derived inflammatory factors
pro-inflammatory mediators including the (e.g. glycosyl phosphatidyl inositol) may impact
cytokines interleukin-1 (IL-1), IL-6, IL-12 and on how patients are treated for late-stage disease.
tumour necrosis factor alpha (TNFa) as well as However, the picture is clouded by new informa-
nitric oxide (NO). The early release of these pro- tion about the trypanosome itself. The following
inflammatory factors helps set the stage for early areas of study were identified:
acquired immune responses to the parasite vari-
ant surface glycoprotein (VSG). The early
release of IL-12 promotes T helper (Th) to • Validation of experimental and prelimi-
release cytokines, particularly interferon-gamma nary clinical results suggesting that stage
(IFNg), which is responsible for host resistance of disease can be determined by the
against parasites spreading throughout host tis- presence of specific cytokines including
sues. This may be more important than anti-VSG IFNg, IL-10 and TNF.
antibodies which are only found within blood • Definition of the pathogenesis of the
vessels. This early resistance is, however, super- encephalitis associated with late-stage
seded by later immune responses that are associ- sleeping sickness and the encephalopa-
ated with the spread of trypanosomes to the thy associated with treatment, and
CNS. The late-stage acquired immune response
Trypanosome Biological Phenotype

investigation of ways to prevent or ame-
liorate these phenomena using in vitro Trypanosomes have evolved mechanisms to reg-
and in vivo disease models. ulate their biological virulence phenotype at the
• Utilization of the data and technical clonal level. There is evidence that clonally
approaches of the human genome project derived “virulent variants” are capable of enter-
to unravel the composite immune ing the CNS at an accelerated rate in non-human
response to trypanosomes at each stage primates, suggesting the possibility for develop-
of the disease process. ment of discrete potential for CNS invasion
within the host. The molecular mechanisms
behind such clonal changes are unknown. This
information might be useful in choosing the
appropriate clinical treatment at any stage of dis-
ease. For example, the presence of trypanosomes
expressing “CNS invasion markers” at any stage
of disease might be used to initiate late-stage
chemotherapy. In addition, such information has
the potential to detect drug resistant variants in
patients and aid the choice of drugs used to treat
such patients.
RECOMMENDATIONS
Highest priority
• Identification of parameters within the host immune system for disease-stage determination
and improved management of post treatment encephalopathy.
High priority
• Determination of the biological phenotypes of trypanosomes lodging in different tissue com-
partments to identify tissue tropism and drug-resistance characteristics.
APPLIED GENOMICS
Trypanosoma brucei Genomics and functional analysis of genes. Two resource

centres have been established: DNA-based
The T. brucei genome network was formed by resources in Cambridge, and derivative and
TDR with a brief to coordinate the analysis and mutant lines of trypanosomes in Glasgow. These
sequencing of the nuclear genome. This huge centres are funded to the end of the sequencing
task can progress most efficiently if the commu- phase, at which time a re-appraisal of resource
nity works together with open sharing of data and provision will be required.
resources. The network has sufficient funds
(from the Wellcome Trust UK and the US The TDR planning meeting for the Parasite
National Institute of Allergy and Infectious Genome Initiative assigned some activities to
Diseases/National Institutes of Health be undertaken in Africa, but these activities were
[NIAID/NIH] USA) to complete the sequencing not sustained for various reasons which included
of the megabase chromosomes, where the majori- reduced funding for African trypanosomiasis
ty of genes are located, which will take until by TDR and changes in available technologies
2004. Approximately 85-90% of the genome is and priorities within the genome networks. The
currently available as fragmentary sequences in involvement of African scientists and TDR is
databases in the sequencing centre websites, and crucial in ensuring the application of information
will ultimately be available in public web-based from genomics into disease control. African insti-
databases. tutions must develop the capacity to fully exploit
the resources from the genome projects. It is
The Wellcome Trust has provided funds to estab- anticipated that capacity strengthening in African
lish a genome database, at the Sanger Centre, institutions will impact on the applications from
that will contain all data related to sequencing both the parasite and the vector genome projects.
RECOMMENDATIONS
Highest priority
• Strengthening of capacity of research laboratories/centres in Africa in bioinformatics,
genomics and applied genomics.
• Application of genomics to comparison of Trypanosoma brucei subspecies, strains, and life
cycle stages.
High priority
• Identification, application and database collation of DNA-based polymorphic markers for
species differentiation, reservoir identification and determination of drug resistance.
• Application of bioinformatics and experimental methods of determination of gene function
to identify novel drug targets.
• Use of resources available from the human genome project to investigate host response to
infection.
TSETSE GENETICS
Recent advances in molecular technologies, and

their application to insects, are being widely Tsetse-trypanosome interactions
explored because they have the potential to • Determination of the molecular
result in the development of novel strategies for basis of refractoriness for try-
control of vector borne diseases. The technolo- panosome transmission.
gies needed to undertake such studies have been • Engineering of trypanosome refrac-
developed for other insect vectors and thus can tory traits into strains of tsetse fly.
facilitate rapid application to tsetse biology.
• Investigation of gene spreading
mechanisms that can be used for
Limited field data indicate extensive genetic
population replacement studies, e.g.
sub-structuring in populations, which display
cytoplasm incompatibility mediated
differences in vectoral capacity. Information on
by Wolbachia symbionts of tsetse.
population genetics would facilitate more effec-
tive and targeted disease management strategies
by identifying sub-populations of flies responsi- Population genetics of tsetse
ble for disease transmission. There exists a mid- • Development of molecular polymor-
gut symbiont-based genetic transformation sys- phic DNA markers and their appli-
tem which allows expression of gene products cation to field flies to understand
that confer trypanosome refractory phenotypes. the extent of genetic sub-structuring
Candidate genes with either anti-trypanosomal existing in tsetse populations.
traits or which, when expressed, can block para-
• Investigation of mating incompati-
site transformation and /or differentiation,
bilities existing among field popula-
should be identified for expression in the tsetse
tions.
mid-gut. The eventual genetic engineering of
anti-trypanosomal traits into tsetse populations • Investigation of differences in vector
would result in new vector control strategies. competence abilities of genetically
The laboratory and field-based research recom- isolated sub-populations of flies.
mended below is necessary to develop the
applied approaches that can lead to novel vector-
based disease-control strategies.
RECOMMENDATIONS
Highest priority
• Determination of the molecular basis of refractoriness for trypanosome transmission and
development of mechanisms for driving the desired genes/traits into tsetse populations.
High priority
• Development and application of molecular markers to determine genetic sub-structuring and
mating incompatibilities in tsetse populations, and vector competence of genetically isolated
sub-populations.
• Development of a tsetse-parasite genome network to obtain and coordinate information on
expressed sequence tags (EST), genomic sequences, physical map locations of selected
genes, and eventual proteomics approaches.
• Coordination of the network by TDR, whose comparative advantage is evidenced by the suc-
cessful management of the mosquito-parasite genome networks.
6 Cross-cutting issues
RESOURCE FLOW
Funding support for control, and for research The SWG was held at an opportune time when
and development, requires long-term commit- the funding level through TDR is increasing and
ment. Over the past 20 years, many countries when African trypanosomiasis is re-surfacing in
have provided support for control of, and the global health agenda. The funding and assur-
research on, African trypanosomiasis; in particu- ance of a five-year drug supply by the private
lar the governments of Belgium, France and UK sector provides a unique opportunity to deal with
are major supporters. The European Union, the current epidemic in Africa as well as to
USA, Canada, the Organization of Petroleum develop protocols for combination chemotherapy
Exporting Countries (OPEC) Fund and China are in the face of increased melarsoprol refractori-
also partners. A list of countries and institutions ness. In addition, the five years provides a period
(by no means complete) that provide financial of grace during which TDR and the scientific
support is attached (Annex 1). community can lay the foundations for ensuring
continued availability of medicaments beyond
Considerable sums of money are invested in the five years by investing in research and capa-
basic research on trypanosomes by institutions in bility strengthening within Africa for drug dis-
USA, and EU countries, because the try- covery and development as well as for genomics,
panosome is a good model for basic research on applied genomics and proteomics. The SWG
cell biology. Consequently, there is probably acknowledged the comparative advantage that
more information on the cellular structure, bio- TDR has in coordinating and networking the
chemistry and molecular biology of try- activities of various scientific groups, laborato-
panosomes than any other non-mammalian cell ries and centres in different parts of the world.
type, and a great deal is known about the differ- Consequently, the group recommended recruit-
ences between trypanosomes and mammalian ment of a full-time professional staff member
cells. However, only a small amount of this within TDR to coordinate the various activities
knowledge is being applied directly in the man- on African trypanosomiasis.
agement and control of the disease. Some of the
knowledge is exploitable for development of new
tools for disease and vector control as well as for
improved patient management. The SWG meet-
ing provided an opportunity to identify the
knowledge that could be exploited for develop-
ment of new tools and improvement of existing
ones for disease and vector management, as well
as to determine the needs for research capability
strengthening in disease endemic countries for
basic sciences. TDR’s comparative advantage in
enhancing existing, and developing new, partner-
ships for maximal application of the available
knowledge was highlighted.
ADVOCACY AND MARKETING FOR SLEEPING SICKNESS
The SWG noted with appreciation the increase in NGOs and national systems). A need to enhance
funding coming to TDR for African trypanosomi- collaboration between the groups working in
asis. However, there was concern that, in the pre- research and those working in control, and also
vious six years or so, there had been little interest between groups working in disease and non-dis-
from the donor community to fund activities on ease endemic countries, was identified. This
African trypanosomiasis. A need to present the would increase the much needed credibility when
disease in a way that changes donor perception of requesting donor support. The SWG noted that
the problem was identified. The Group was scientists working in disease endemic countries
informed that one way of changing is to present often work in isolation and have difficulties
projects that are convincing, focused and concise, developing the required credibility for donor sup-
emphasizing donor/researcher partnerships in port, and identified a need to support good ideas
project management and follow-up and the bene- from young scientists, enabling them to seek
fits to the target communities. Networking independent funding, i.e. to support them to a
appears to be more attractive to donors than sin- point where donors have confidence in them. In
gle, isolated projects. addition, a mechanism to assist young scientists
to develop their ideas into fundable proposals,
The Group indicated a number of networks where their ideas are likely to provide vital infor-
already existed, both formal and informal (with mation, should be put in place.
INSTITUTIONAL DEVELOPMENT AND CAPACITY BUILDING

Given the re-emergent nature of African try- try, was recommended. A need to train technical
panosomiasis, it is necessary to identify and staff for specific needs in research, disease sur-
strengthen a nucleus of research groups and veillance and management was also identified.
institutions in different endemic countries to The Group further noted the difficulties of sus-
generate data, on disease burden and surveil- taining trained staff within the field of African
lance, for advocacy. Where whole institutions trypanosomiasis in disease endemic countries,
are difficult to maintain as disease specific and suggested lack of career development
research institutes, their disease mandates could opportunities as one of the factors contributing
be expanded to enable them to add on other dis- to this shortage.
ease research activities while retaining core
activities in the area of sleeping sickness. Staff retention is a global problem, not peculiar
to the field of African trypanosomiasis. Those
The SWG commended TDR for the great num- working in this field are in the public sector,
ber of scientists it has supported for post-gradu- where salaries are not attractive and career
ate training. However, continued training of advancement is slow. Lack of resources to carry
research personnel in African trypanosomiasis, out research, and to attend short-term training
and linking of this training to specific techno- courses, is a disincentive to remain in the sector.
logical needs, which vary from country to coun- Funding for research, on a more sustainable
basis and for longer periods, is a necessity; while disease endemic areas have identified scientists
linking of national institutions with overseas or technicians in scientific institutions as a
ones, with donor support, is an area for possible national resource with unique operational needs.
expansion. It was agreed that advocacy would be crucial,
and international forums, such as the
The SWG welcomed the recent TDR initiative of Organization of African Unity (OAU)
institutional strengthening grants; a call for let- International Scientific Council for
ters of intent was already issued. TDR is also Trypanosomiasis Research and Control (ISC-
working on strategies for strengthening whole TRC), would be used to send the message. The
national health research systems in countries that presence of TDR at such forums, and in other
do not yet have a strong research culture, and a African national systems, would also be useful.
mechanism is being worked out to give priority At the same time, there is a need to ensure
to funding of research-strengthening proposals. appropriate networking in order to avail the nec-
essary assistance in writing good proposals. The
The commitment of African governments to sup- possibility of providing salary support with
port trypanosomiasis research and control, with research grants should also be given considera-
improved terms of service for those working in tion by TDR.
the sector, is important. Very few countries in the
SOUTH-SOUTH COLLABORATION
In March 2001, WHO held a meeting in Harare, America, was emphasized. This networking will
Zimbabwe, to discuss an initiative aimed at enhance the application of molecular biology
increasing discussion and scientific interaction techniques and genomics (functional and
between investigators in disease endemic coun- applied) to developing solutions to public health
tries. The meeting was attended by scientists problems. Initiatives for multicentre projects,
from several countries in Africa, Asia and Latin which incorporate capacity building and training,
America, who discussed ways of increasing the will be developed. Additionally, a number of
collaboration that already exists between investi- short-term training courses in basic and applied
gators in the South and collaborators in more biology, that incorporate the application of the
advanced laboratories of the North. The need to latest technologies, are planned. It is anticipated
develop more sustainable pathways for training, that the results of this network will include
that will make more efficient use of available process indicators (academic qualifications,
resources by exploiting opportunities for the highest quality publications), products (e.g.
exchange of complementary expertise present tools, chemotherapeutic agents), and an impact
within laboratories in Africa, Asia and Latin on alleviating disease.
RECOMMENDATIONS
• Recruitment by TDR of a full-time professional staff member to coordinate African try-

panosomiasis activities.
• Identification and strengthening of a nucleus of research groups and institutions in different
endemic countries to generate data on disease surveillance and advocacy.
• Networking and cross-country comparison of research progress to assist in capacity building
and stimulate cross border interest and advocacy.
• Strong advocacy to persuade DEC governments to give priority to research and control of
African trypanosomiasis amidst other health priorities.
• Payment of a salary component within TDR funded grants to enhance retention of disease
endemic country scientists in the field.
Annex 1
RESOURCE FLOW FOR AFRICAN
TRYPANOSOMIASIS
BCT Bureau Central de la LIRI Livestock Research Institute
Trypanosomiose
MEMISA Medische Missie Samenwerking
BMS Bristol-Myers Squibb
MSF Médecins sans Frontières
CAR Central African Republic
NIAID National Institute of Allergy and
DFID Department for International Infectious Diseases
Development
OAU Organization of African Unity
DRC Democratic Republic of Congo
OCCGE Organisation de Coordination
EU European Union et de Coopération pour la lutte
contre les Grandes Endemies
FAO Food and Agriculture
Organization OCEAC Organisation de Coordination
pour la lutte contre les
FITCA Farming in Tsetse Controlled
Endemies en Afrique centrale
Areas
PATT Programme against African
FOMETRO Fonds Médicale Tropicale
Trypanosomiasis
IBAR InterAfrican Bureau for Animal
STI Swiss Tropical Institute
Resources
UN United Nations
ICCT Instituto de Combate e Controlo
das Trypanosomiases UNC University of North Carolina
IPMP Instituto Português de Medecina UNDP United Nations Development
Preventiva Programme
IRD Institut de Recherche et WHO World Health Organization
Développement
ITM Institut Tropical de Médecin
KETRI Kenya Trypanosomiasis
Research Institute
Annex 2
POSITION PAPER
A POSITION PAPER ON AFRICAN should be explored for drug discovery and develop-
TRYPANOSOMIASIS ment of tools for trypanosomiasis control and patient
management.
Felix A.S. Kuzoe 5. Institutions in the endemic countries should be
strengthened to enable implementation research.
WHO/TDR, Geneva
Clinical studies should be conducted according to the
concept of good clinical practice (GCP). A few clinical
MEETING THE CHALLENGE research centres or treatment centre networks should
1. New tools are needed for the control of African try- be identified and strengthened and clinical investiga-
panosomiasis (sleeping sickness); however, these on tors given appropriate training.
their own will not improve the trypanosomiasis situa-
tion. Factors that militate against the effective use of
existing tools, such as continued worsening economies
and structural adjustment programmes in the affected SUMMARY
countries, will remain. This position paper provides an overview of the
• Operational research should be conducted on the main features of African trypanosomiasis and the
effective use of available tools with existing capaci- problems relating to its control. It gives a historical
ties, such as the health services, strong control pro-
account of the disease, its epidemiology and socioe-
grammes in other diseases, NGOs, etc.
• Development of simple surveillance systems with conomic impact. It describes the strategy for control,
available tools that can be integrated into available namely case detection, chemotherapy and vector
capacities to improve case detection and active control, and the limitations. It discusses global fund-
screening of populations at risk will permit early ing of basic research on African trypanosomes, and
diagnosis of cases and increase the number of peo-
the pivotal role of WHO/TDR in facilitating the
ple under medical surveillance.
• Long-term commitment by the international com- evaluation of any spin-offs from the research
munity to national sleeping sickness control pro- towards development of public health tools.
grammes, rather than support for crisis manage- Considerable progress has been achieved in
ment, will ensure sustainability. research on African trypanosomiasis, but this
2. Social, economic and cultural factors enhance or progress has not been reflected in the field due to
hinder efforts to control sleeping sickness. There is
lack of interest in development of new tools and
need for studies on social, socio-cultural and anthro-
pological aspects of endemicity of sleeping sickness. lack of sustainability of control methods, resulting
The effects of decentralization of health services on largely from inadequate resources for public health
African trypanosomiasis need to be studied for and wars and civil strife in some endemic countries.
improved management of control programmes. With the decrease in resources for African try-
3. The private sector has the expertise for drug devel-
panosomiasis in TDR, which started in 1994, there
opment. However, for diseases such as African try-
panosomiasis that have no market for drugs, develop- have been grave consequences in endemic coun-
ment must be based on public sector financing. tries, both in terms of reduction in human resources
• The recent award of US$ 15 million by the Bill & and degrading of facilities for research and evalua-
Melinda Gates Foundation to a consortium of scien- tion of new tools for African trypanosomiasis. To
tists towards the development of drugs for African meet the challenge of African trypanosomiasis in the
trypanosomiasis and Leishmaniasis is a welcome
development that has no precedence in the history
21st century, this position paper makes a number of
of African trypanosomiasis. Furthermore, the World proposals.
Health Organization (WHO) and Aventis Pharma
have announced a major initiative to control African AFRICAN TRYPANOSOMIASIS AS
trypanosomiasis, whereby Aventis Pharma has com- A PUBLIC HEALTH PROBLEM
mitted US$25 million to support WHO’s activities in
At the beginning of the last century, sleeping sick-
this field for a period of five years.
• TDR has links with the outside world with special ness was perceived by the colonial powers as by far
reference to product development. Over its lifetime, the most important public health problem in Africa.
it has generated multiple partnerships for product Huge epidemics devastated large areas of the conti-
development and has gained considerable experi- nent. In the 1960s, the prevalence of sleeping sick-
ence. Thus, it has been successful and taken some ness was successfully reduced in all endemic coun-
products up to registration in collaboration with the
private sector, for example, mefloquine, eflornithine,
tries to less than 0.1%, through historic campaigns
AmBisome and artemether. More partners are need- by the former colonial powers. Soon after inde-
ed in drug discovery research and drug develop- pendence, however, national governments were
ment for African trypanosomiasis. either lacking in resources or had diverted
4. Basic research on trypanosomes continues to be resources to other pressing health problems.
funded with considerable sums of money in the North.
Breakdown of specialized mobile teams and health
No drugs have yet come out of basic research by
rational drug design. Functional genomics, bioinfor- facilities in several countries, as a consequence of
matics and proteomics provide new opportunities that war and civil strife or change in health policy,
resulted in dramatic resurgence of African try-
panosomiasis, the distribution of which corre- T. b. rhodesiense, which occurs in Central and
sponds closely with that of major conflicts in sub- Southern Africa. The chronic infection lasts for
Saharan Africa. In the Democratic Republic of the years, whilst the acute infection may last only for
Congo (DRC), the number of cases being reported weeks.
yearly has now reached levels comparable to the
1930s, and may result in as many deaths in adults Other forms of trypanosomiasis, called nagana,
as HIV/AIDS (Ekwanzala et al 1996). And yet affect livestock, and are considered the most impor-
African trypanosomiasis is curable. Other countries tant infectious disease holding back the develop-
affected by the resurgence/epidemics are Angola, ment of livestock production in much of Africa. The
the DRC, Central African Republic, Sudan and annual losses in meat production attributed to try-
Uganda. Sleeping sickness due to T. b. rhodesiense panosomiasis are estimated to be US$5 billion
seems to be quiescent at present and less wide- (ILRAD 1993). No new drug has entered the market
spread, with active foci occurring in Tanzania and for over 30 years because the disease does not affect
Uganda. African trypanosomiasis is a re-emergent livestock in Western developed countries.
disease, but does not get due attention, probably
because its impact is regional. Epidemiology
The epidemiology of sleeping sickness is complex
The disease occurs in some 36 sub-Saharan coun- and the transmission cycles are subject to interac-
tries, within the area of distribution of the tsetse fly. tions between humans, tsetse flies, trypanosomes
Over 60 million people living in some 250 foci with- and, significantly in T. b. rhodesiense sleeping sick-
in this region are at risk of contracting the disease. ness, domestic and wild animals. In T. b. gambiense
The number of cases reported annually is over 40 disease, the classical human-fly-human transmis-
000, but this is highly underestimated due to the dif- sion cycle occurs in both endemic and epidemic sit-
ficulty of diagnosis and remoteness of affected uations. Humans are the important reservoir and
areas. It has been estimated that the actual number hence it is possible to reduce transmission through
of cases is about 300 000 (WHO 1998). Though these diagnosis and treatment of the infected population.
figures are relatively small compared to other tropi- Although it has been demonstrated by biochemical
cal diseases, African trypanosomiasis, without inter- and DNA techniques that trypanosomes identical to
vention, has the propensity to develop into epi- those which cause T. b. gambiense disease in humans
demics. This characteristic makes it a major public occur in domestic animals and some game, the sig-
health problem. During epidemics, large propor- nificance of these potential reservoir hosts on trans-
tions of communities are affected with great loss of mission is not clear. In T. b. rhodesiense disease, on
life and untold human suffering. Epidemics have the other hand, it has been recognized that infec-
serious social and economic consequences, which tions in humans in endemic situations are acquired
far outweigh the cost of maintaining surveillance. from savannah species of tsetse flies, all of which
Sleeping sickness has perhaps been a major cause of feed preferentially on a wide variety of game. The
depopulation of large tracts of Africa, and fear of the game-fly-human cycle is typical. Endemic T. b.
disease has led to abandonment of fertile lands and rhodesiense situations are sporadic in nature and
is an impediment to development. The World Bank patchy in distribution, and adult men tend to be
Report (1993) estimated that, in 1990, there were 55 predominantly infected. In epidemic T. b. rhode-
000 deaths due to African trypanosomiasis and 1.8 siense, however, human-fly-human or domestic ani-
million disability adjusted life years (DALYs) lost mal-fly-human cycles predominate. There is equal
due to the disease. More recent estimates are rather distribution of infection among men, women, and
similar, with 2.1 million DALYs lost and 66 000 children. For T. b. rhodesiense disease, chemoprophy-
deaths in 1999. As a comparison, the number of mil- laxis of the domestic animal reservoir has been rec-
lions of DALYs lost is estimated at 45.0 for malaria, ommended as a new approach to control. Available
4.9 for lymphatic filariasis, 2.0 for leishmaniasis, 1.9 evidence suggests that HIV infection has had little
for schistosomiasis, 1.1 for onchocerciasis and 0.7 for impact on the epidemiology of T. b. gambiense
Chagas disease (WHO 2000). African trypanosomiasis (Louis et al 1991; Pepin
et al 1992; Meda et al 1995).
THE DISEASE
African trypanosomiasis is caused by protozoan Medical Surveillance
haemoflagellates, trypanosomes that are transmit- Regular medical surveillance, involving case detec-
ted by tsetse flies (Glossina spp). The disease occurs tion and treatment, and tsetse fly control, where
in two forms: the chronic form caused by applicable, is the backbone of the strategy for con-
Trpanosoma brucei gambiense, which occurs in West trol of sleeping sickness (WHO, 1998). With avail-
and Central Africa; and the acute form, caused by able tools, control is a continuing effort rather than
eradication. Experience over the last 50 years has T. b. rhodesiense sleeping sickness. All these drugs
shown that, where control efforts are interrupted, have adverse effects, melarsoprol causing reactive
e.g. due to civil strife, political upheavals, economic encephalopathy in 5-10% of patients with a fatal
constraints, or out of complacency, sooner or later, outcome in 1-5%. Increasing numbers of patients -
there will be resurgence of the disease. between 20-25% in certain foci - do not respond to
melarsoprol treatment, probably due to resistance of
Diagnosis the parasite to the drug (Legros et al 1999). The term
For unequivocal diagnosis in humans, it is essential ‘drug resistance’ covers host and parasite-related
to demonstrate trypanosomes in lymph node aspi- factors. Host related factors include poor distribu-
rate, blood or cerebrospinal fluid (CSF). A number tion of drug to infected tissues and intracellular
of tools exist for the diagnosis of patients. In T. b. sites, variation in drug metabolism between indi-
gambiense areas, the Card Agglutination Test for viduals, and diminished activity of drug. Parasite
Trypanosomiasis (CATT), a serological test detect- related factors that contribute to resistance include
ing antibodies, is used for mass screening. reduced drug accumulation in the parasite, a change
Serologically positive cases are then confirmed in enzyme target through increase in enzyme levels,
using parasitological tests. Since parasitemia varies increase in metabolite production or retention, or
between foci and disease stage, it is necessary to use of alternative pathways to bypass the site of
adopt blood concentration techniques, such as the inhibition. A number of these factors could be
Haematocrit Centrifugation technique (HCT), the involved and therefore the mechanism of resistance
Miniature Anion Centrifugation Technique needs to be elucidated and strategies to combat it,
(MAECT), or the Quantitative Buffy Coat (QBC) such as the use of drug combinations, developed.
technique. A study on the treatment of serologically The development of a simple test to quickly diag-
positive patients who are parasitologically negative nose resistance in parasite isolates is also needed for
with one injection of pentamidine is in progress in successful chemotherapy. Clinical trials with a com-
DRC. The use of the CATT for screening popula- bination of melarsoprol and eflornithine, whose
tions in T. b. gambiense areas has greatly improved synergism has been demonstrated in the mouse
the potential for community diagnosis. Despite the model, should be given priority (Jennings 1988).
advances, techniques, especially the CATT, have Combinations of other drugs are also envisaged.
rarely been put into practice in endemic areas except The varying treatment schedules of available drugs
as part of externally funded programmes. This were developed empirically and, therefore, opti-
relates in part to the costs (Smith et al 1998). The mization of current treatment regimens is needed.
Card Indirect Agglutination Test for Burri et al (2000) have shown that, with a new regi-
Trypanosomiasis (CIATT) is another serological test men of melarsoprol, it is possible to reduce the dura-
which detects antigens and therefore active infection of treatment from 40 days to 10 days. A study
tion. However, the very high frequency of positive sponsored by TDR on treatment with pentamidine,
results in low endemic areas, indicates that it may 7 days versus 3 days, in DRC has been interrupted
not be suitable for screening in control programmes. due to rebel forces taking over that part of the coun-
The role of the polymerase chain reaction (PCR) in try. This occurrence underscores the difficulties of
diagnosis remains to be determined. A test is need- carrying out field research on African trypanosomi-
ed to diagnose late-stage disease, which presently asis.
relies on lumber puncture that is painful and not
well accepted by people. A test is also needed to Eflornithine, developed in 1990, is the only available
determine cure after chemotherapy. The current alternative drug to treat T. b. gambiense patients who
requirement for a 2-year period of follow-up of do not respond to melarsoprol. It is not effective in
treated patients is cumbersome, costly, and leads to T. b. rhodesiense sleeping sickness. The drug has
loss of many patients. many drawbacks: a complicated mode of adminis-
tration (intravenously every 6 hours at a dose of
Chemotherapy 100mg/kg/day for 14 days), which limits its use to
The chemotherapy of African trypanosomiasis is a hospital setting; it cannot be used for mass treat-
unsatisfactory, relying on a few drugs which have ment; and the cost of treatment is US$300-500 per
adverse side effects. Pentamidine, a diamidine patient, which makes it unaffordable by the affected
developed in 1937, is used for early-stage T. b. gam- countries. To reduce costs, a shorter course of eflor-
biese sleeping sickness. Suramin, a sulphanated nithine (7-day treatment) was compared to the stan-
naphthylamine developed in 1922, is used to treat dard 14-day treatment. However, the results
early stage T. b. rhodesiense. Melarsoprol, a trivalent showed that the 7-day treatment is effective only in
arsenical derivative developed in 1948, is used for patients who have relapsed on melarsoprol and
the treatment of late-stage of both T. b. gambiense and that, for new patients, the 14-day course is superior
(Pepin et al 2000). In recent years, there have been brain barrier in humans. The functional integrity of
doubts about the future availability of eflornithine. the blood-brain barrier and its role in pharmacoki-
However, through the collaboration between netics and pathogenesis is pivotal to understanding
Aventis, the manufacturer, WHO and Médecins the disease. Attention should be given to new
sans Frontières (MSF), potential manufacturers have approaches to drug delivery, such as across the
been identified, and it is likely that a suitable pro- blood-brain barrier. In 1999, two leading com-
ducer will be selected to produce the drug and that pounds, both of them diamidines that gave satisfac-
funds will be provided through donor contributions tory results against trypanosomes in animal models
to guarantee production for the next five years. in acute infection, failed to cure the chronic infec-
Nifurtimox, nitrofurasan, was used for acute tion, due to inability to cross the blood-brain barrier.
Chagas disease. Although it was not registered for A number of lead compounds have suffered the
human African trypanosomiasis, it has been used same fate in the past. The ideal trypanocide must be
experimentally and on a compassionate basis to safe and effective, and have a simple mode of
treat T. b. gambiense sleeping sickness patients. administration to allow its use under rural condi-
Bayer, the manufacturer, has agreed to continue pro- tions, where health facilities are usually poor, and
duction for treatment of African trypanosomiasis; above all should be affordable. The occurrence of T.
however, registration of the drug for this purpose is b. rhodesiense sleeping sickness outside the tradition-
an urgent issue that needs to be addressed. al focus in south-eastern Uganda (Enyaru et al
1999), justifies fears of mixing T. b. gambiense and
Vector control T. b. rhodesiense due to human population move-
A variety of traps and screens impregnated with ments between foci in Uganda, the DRC and
insecticide have been shown to be effective in reduc- Tanzania (Kigoma), and underlines the need for a
ing tsetse populations by 99% in control pro- new drug that can treat both T. b. gambiense and T. b.
grammes and are suitable for rural community par- rhodesiense. A multicentre clinical trial with eflor-
ticipation. In any outbreak of sleeping sickness, nithine showed that eflornithine was relatively less
tsetse control in combination with diagnosis and effective in Uganda than in three other countries
treatment should arrest transmission. Besides, these (Pepin et al 2000). It should be noted that Uganda is
devices can also be used as preventive measures to the only country where both T. b. gambiense and T. b.
reduce human-fly contact. In vector-borne diseases, rhodesiense sleeping sickness foci exist and, there-
vector control plays an important role in reducing fore, this observation needs further investigation.
transmission. For example, Chagas disease caused
by T. cruzi has been successfully controlled by vec- SOCIAL AND ECONOMIC IMPACT
tor control as a result of national government com- OF SLEEPING SICKNESS
mitment to a long-term programme. In onchocercia- The social and economic impact of sleeping sickness
sis endemic areas of West Africa, blindness is no is often underestimated. Some affected countries
longer a public health problem as a result of vector have agriculture-based economies, and workers on
control and donated drug (Mectizan) distribution. cocoa and coffee plantations are at risk of contract-
The effect of deforestation and climatic changes on ing the disease; consequently the labour force is
tsetse populations in West Africa may be responsi- reduced. At community and family levels, mental
ble for the lull in sleeping sickness in countries like confusion, personality and behaviour changes,
Ghana and Nigeria. Operational issues, such as which often characterize central nervous system
motivation of communities and recurrent costs, involvement in late-stage disease, may lead to
which militate against sustainability in the use of divorce and break up in homes and present an
impregnated traps and screens on regular basis, unfavourable climate for bringing up children. In
need to be studied and solutions found. some cases, such people become mentally dis-
turbed, suicidal and violent, and constitute a danger
DRUG DEVELOPMENT to themselves and to the community. Aroke et al
An oral formulation of eflornithine has many (1998) reported that, in the past, T. b. gambiense
advantages over the injectable form and will allow sleeping sickness in children had an influence on
use on a wider scale. A pharmacokinetic study of their physical growth and attainment of sexual
oral eflornithine is in progress in Côte d’Ivoire and maturity. In Central Africa, there are important
it is anticipated that Phase 3 clinical trials will take problems regarding treatment, particularly the
place in 2001, to evaluate its safety and efficacy, severe social consequences of long-term hospitaliza-
toward eventual submission of data for registration tion. Important behavioural factors contributing to
to a regulatory authority. The need for new drugs is the risk of death from sleeping sickness, such as
fundamental. One of the obstacles to finding new negative attitudes towards hospital treatment, have
drugs is the difficulty of transport across the blood- often led to the patient absconding and not com-
pleting treatment. In studies on the impact of try- activities. TDR’s initiatives in bringing together sci-
panosomiasis on land occupancy systems, on popu- entists in Scientific Working Groups, meetings and
lation movements and social conditions in Burkina workshops to deliberate on specific issues, paid off
Faso and Côte d’Ivoire, the extreme mobility of the on several occasions. A few examples follow.
people due to migrant labour was identified as one
of the major problems in case detection, case report- A Glossina trapping meeting held in Brazzaville,
ing and control of the disease. There is need to con- Congo, in March 1985, brought together scientists,
tinue monitoring and mapping population move- from West, Central and Southern Africa, interested
ments, as well as incidence of trypanosomiasis, par- in developing Glossina trapping technology. This
ticularly as the impacts of the infection are rather meeting opened the way for better interaction
latent but extremely serious in the long run. Studies between the scientists and gave impetus to
in Uganda demonstrated that African trypanosomi- improvement in trapping technology for tsetse con-
asis had an adverse impact on the functioning of trol. Five years later, the pyramidal trap impregnat-
households at Iganga, south-east Uganda. Adverse ed with insecticide used at 10 traps per km2 was
impacts included increased poverty, decline in agri- effectively employed in reducing tsetse populations
cultural activities often leading to famine or lack of by over 95 per cent within 3-4 months during the
basic food security, disruption of children ‘s educa- epidemics of sleeping sickness in Busoga, south-east
tion, and general reversal of role obligations, which Uganda, at an estimated cost of US$0.9 per head of
more often than not enhanced women’s and chil- population protected (Lancien, 1991).
dren’s burdens. The debilitating nature of the dis-
ease also poses more problems for women, who The rational development of control and treatment
may be stigmatized and/or rejected by their spous- requires thorough knowledge of the pathology of
es even after recuperation. The extent of disruption the disease. At the inception of TDR in 1975, there
of household social and economic activities is great- were less than 25 autopsies of African trypanosomi-
ly influenced by such factors as the household’s eco- asis reported in the literature. The need for system-
nomic status, composition and level of organization atic autopsy backed by expert histopathology led
(Kyomunhendo 1995, 1998). TDR, in collaboration with the University of
Glasgow, to establish a network of clinical centres
GLOBAL RESEARCH ON AFRICAN that were provided with kits and protocols for
TRYPANOSOMES autopsy. From this effort, evidence was provided
The trypanosome has many unique biological char- from both laboratory and clinical data showing that
acteristics that make it one of the most studied par- reactive encephalopathy, which occurs in 3-5% of
asites. It offers many opportunities for basic patients treated with melarsoprol, points to a drug-
research: it is easy to cultivate and purify to yield related immune response rather than to toxicity
large amounts of protein and nucleic acid, it is a (Haller et al 1986). A set of slides showing the fea-
eukaryotic experimental model for research on the tures of neuropathology in African trypanosomiasis
control of gene expression, etc. Though it is difficult was prepared and made available to universities for
to get funds for the control of sleeping sickness, teaching purposes.
large sums of money are invested annually, particu-
larly in the North, on basic research on African try- New strategies for managing patients with central
panosomes. There is probably more information on nervous system involvement in African trypanoso-
the biochemistry and molecular biology of try- miasis are needed. In 1986, TDR organized a work-
panosomes than on any other non-mammalian cell shop in collaboration with the Institut de Neuro-
type, and a great deal is known about the differ- logie Tropicale, Limoges, France, which brought
ences between trypanosomes and mammalian cells. together clinicians, neurologists, neuropathologists
Yet no drugs have come out of basic research. TDR and scientists. Following the recommendations of
provides an essential link between research institu- this group, a number of studies funded by TDR in
tions in the North and endemic countries, through collaboration with other institutions, resulted in sig-
access to a network of national field projects and nificant progress in understanding the molecular
control programmes, where spin-offs from basic mechanisms underlying pathogenesis, including
research can be evaluated as tools for the control brain dysfunction and neuropsychiatric symptoms
and prevention of sleeping sickness. It is necessary associated with the disease. The trypanosome lym-
for TDR to preserve this unique role (Kuzoe, 1993). phocyte triggering factor, TLTF, a molecule which
binds to CD8+ cells and triggers the production of
For institutions in the South, TDR was conspicuous- gamma interferon, which is growth factor for T. b.
ly a major source of funding for research on African brucei, was reported by the Karolinska Institute,
trypanosomiasis and institutional strengthening Sweden, in 1991(Olsson et al 1991). The gene for
TLTF has since been identified, cloned and ical studies. One should, however, not overlook the
sequenced, and a recombinant TLTF produced support, no matter how small, that has been provid-
(Bakhiet et al 1993; Vadya et al 1997; Hamadien et al ed by other institutions in the North to these centres.
1999). The exploitation of TLTF for immunotherapy
needs to be followed up. Other studies elsewhere The Bill & Melinda Gates Foundation, in December
have shown that proinflammatory cytokines, 2000, awarded US$15.1 million to treat African try-
including tumor necrosis factor (TNF), interleukins panosomiasis and leishmaniasis to an international
1 and 6 (IL-1, IL-6), and prostaglandins, play an consortium of researchers led by Dr Richard R.
important role in the pathogenesis of central nerv- Tidwell, a scientist at University of North Carolina
ous system disease (Hunter et al , 1992; Alafiatayo at Chapel Hill, to develop new drugs to fight
et al, 1994; Jennings et al 1997). Furthermore, this African sleeping sickness and leishmaniasis. This
pathology has been shown to be prevented and welcome news is without precedence in the history
ameliorated by drugs such as eflornithine (Jennings of African trypanosomiasis. Some of the scientists
et al, 1997) and megazole (Enanga et al, 1998). involved in this consortium already collaborate with
Consideration should be given to the use of such TDR in drug discovery research and drug develop-
drugs in the management of African trypanosomia- ment. TDR should establish links and maintain col-
sis. Further, preclinical research with such drugs is laboration with this consortium. The primate facili-
needed. ty at the Kenya Trypanosomiasis Research Institute
(KETRI), that was established with TDR’s support
The availability of clinical centres in endemic coun- many years ago, is an asset that will be available for
tries supported by TDR facilitated the evaluation of the evaluation of potential lead compounds in pri-
the diagnostic tests MAECT, CATT and CIATT, as mates. There are few clinical research centres in
well as clinical trials of eflornithine which produced endemic areas where clinical trials can be conducted
data that were presented to the US FDA for registra- and, therefore, these will be in demand for drug
tion of the drug in 1990. combination trials, oral eflornithine phase 3 clinical
trials, and trials with any potential candidate com-
Under the auspices of TDR and its collaborators, pounds. Two years ago, TDR started an initiative to
considerable progress was made in research on train clinical investigators and monitors worldwide
African trypanosomiasis in: diagnosis and develop- to conduct clinical trials according to the good clini-
ment of diagnostic tests; epidemiology, host-para- cal practice (GCP) concept. Training will be required
site-vector relationships, animal reservoirs; devel- for clinical investigators in the clinical centres that
opment of tsetse traps and screens; better under- will be involved in clinical trials. TDR has a com-
standing of the pathology of the disease, and drug parative advantage in institutional strengthening
targeted biochemistry of trypanosomes. However, and can make a significant contribution to the work
this progress has not been matched in the control of of this consortium towards the evaluation of candi-
the disease, due to lack of capacity to sustain date compounds. TDR has links with the outside
improved interventions as well as civil disorder in world, with special reference to product develop-
some endemic countries. These factors were largely ment, which should be maintained.
responsible for the current problems of African try-
panosomiasis. BIOINFORMATICS
During 1993/94, TDR initiated a number of parasite
From 1994, when reorganization took place in TDR genome networks - for T. b. brucei, T. cruzi, Leishma-
along disciplines instead of diseases, the resources nia major, Schistosoma mansoni, Brugia malayi. The
allocated to African trypanosomiasis decreased, and networks are now oriented towards post genomics,
continued to decrease tremendously in subsequent and bioinformatics networks are being expanded
years, up to 64% in 2000. In view of TDR’s unique for data mining annotation and in-depth analysis.
role in research on African trypanosomiasis, the con- The T. b. brucei network should be assessed and the
sequences of this lack of funds were grave. Several necessary inputs provided to move it forward in the
trained researchers left trypanosomiasis research for genome analysis of T. b. brucei.
HIV/AIDS and malaria, where they could get
research funds. It is not surprising that the current COORDINATION NETWORK
chairman of the Task Force on African A Human African Trypanosomiasis Treatment and
Trypanosomiasis now works on a project on Drug Resistance Network was formed in 1999 in
HIV/AIDS. One tsetse trap expert has turned his WHO (Communicable Disease Surveillance and
ingenuity to making and supplying bednets for a Response unit). It has as objectives: to assess the
malaria control project. Clinical research centres are effectiveness of current treatment regimens; col-
rundown and have reduced capacity to conduct clin- lect/disseminate information on refractoriness to
treatment; ensure availability and affordability of miasis in mice. Tropical Medicine and International
existing drugs; provide guidelines for treatment; Health 1998, 3(9):736-41.
promote research on causes of treatment failures,
drugs and treatments (WHO 1999). The Go- Enyaru JCK, et al. Evidence for the occurrence of
vernment of France provides funds for running the Trypanosoma brucei rhodesiense sleeping sickness out-
secretariat of the network. Participants in the net- side the traditional focus in south-eastern Uganda.
work come from WHO (Communicable Diseases Annals of Tropical Medicine and Parasitology, 1999,
cluster, the Regional Office for Africa); MSF; the 93(8):817-22.
United States Centers for Disease Control and
Prevention (CDC), Atlanta; the Swiss Tropical Insti- Hamadien M, Lycke N, Bakhiet M. Induction of the
tute, Basel; the Institute of Tropical Medicine, Ant- trypanosome lymphocyte triggering factor (TLTF)
werp. TDR should play an active role in the network and neutralizing antibodies to the TLTF in experi-
and participate in its meetings. mental trypanosomiasis. Immunology, 1999,
96(4):606-11.
Acknowledgements
I wish to thank Professor David H. Molyneux, Haller L, et al. Clinical and Pathological Aspects of
Professor of Tropical Health Sciences, Liverpool Human African trypanosomiasis (T. b. gambiense)
School of Tropical Medicine, Pembroke Place, with particular reference to reactive arsenical
Liverpool, and Dr Jacques Pepin, Associate encephalopathy. American Journal of Tropical Medicine
Professor, Infectious Diseases Division, Centre for and Hygiene, 1986, 35(1):94-99.
International Health, University of Sherbrooke,
Quebec, Canada, for their criticisms and comments Hunter CA, et al. Astrocyte activation correlates
on this article. I also wish to thank Dr Paul Nunn, with cytokine production in central nervous system
Dr Johannes Sommerfeld and Dr Charity Gichuki, of Trypanosoma brucei brucei-infected mice.
WHO, for reviewing the paper. Laboratory Investigations, 1992, 67(5):635-42.
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Annex 3
THE EMERGENCE AND RE-EMERGENCE
OF HUMAN TRYPANOSOMIASIS
(SLEEPING SICKNESS) IN AFRICA
I THE SITUATION IN ANGOLA∗ already stated, and the continual movement of the
Theophile Josenando population, which includes carriers of the parasite
Instituto de comabate e controlo de Tripanosomiase, who act as a reservoir for its dissemination. In addi-
Luanda, Angola tion, socioeconomic factors including poverty are
without doubt implicated in the spread and persist-
Angola is a country situated at the interface between ence of sleeping sickness.
Central Africa and Southern Africa and has an esti-
mated population of 12 million. Sleeping sickness is At the symposium organized by Fundanga, the
a major public health problem in the country today, Angolan Foundation for Solidarity and Deve-
with more than 32 000 new cases having been lopment, and held in September 1998 on the prem-
detected during the last five years. In contrast, in ises of the Angolan National Assembly in Luanda,
1974, when surveillance was more active and better sleeping sickness was clearly identified as a major
organized, there were only three new cases. public health problem. As a result, the Angolan
Government committed itself financially to taking
Tsetse flies, or Glossina, the vectors of the disease, are action against the disease, with annual financing of
present in 14 of the country’s 18 provinces. Two mil- about $1 million in local currency (Kwanza) and $1
lion out of the 4.5 million people living in the seven million in convertible currency (dollars) since 1998
provinces affected - Zaire, Uige, Kwanza Norte, for activities of the National Programme for control
Malanje, Bengo, Kwanza Sul and the peripheral of human trypanosomiasis.
areas of Luanda - are exposed to the risk of direct
infection. The number of patients has been rising The National Programme has been gradually
continuously for more than ten years. The number of strengthened in its leadership role, and its efforts
new cases identified in 1997 was 8275, the highest have borne fruit with the transformation of the
figure ever reported in the history of the disease in National Programme into the Institute for the
Angola. The figures reported reflect the case detec- Control of Human and Animal Trypanosomiasis
tion and treatment activities introduced over the last (Instituto de Combate e Controlo das Trypano-
few years. Paradoxically, the rapid increase in the somiases, or ICCT) by Government decree 2/00 of
number of patients identified is witness to the efforts 14 January 2000. The newly created Institute enjoys
made to manage the disease by the national health autonomy in management and its position has
services with the help of non-governmental organi- changed in the structural chart of the Ministry of
zations (NGOs). In the period 1996-2000, 32 445 Health. This autonomy first found expression in the
patients were treated (Table 1). delegation of management of salaries of personnel
working at ICCT headquarters and at the Viana
Table 1. The number of patients treated Reference Centre.
in the years 1996-2000
Control of trypanosomiasis addresses one of five
1996 1997 1998 1999 2000 priority endemic diseases (AIDS, trypanosomiasis,
tuberculosis, leprosy, malaria) for the Ministry of
6786 8275 7373 5351 4546 Health in 2001. In Angola, the inclusion of ICCT
national staff in the activities of NGOs is effective,
enabling it to be present at most places where
Since African trypanosomiasis is invariably fatal patients are managed.
when left untreated, the increase in number of treat-
ed patients means a decrease in mortality. So the 32 This year, the Ministry of Health assigned five
331 patients treated durng the period 1996-2000 physicians to the ICCT and is presently trying to
escaped from certain death. The treatment centres obtain greater participation by the State in the
are in areas of military confrontation (war front) and financing of the Programme. There is also effective
this might explain the fluctuations in number of financial participation by the autonomous provin-
patients treated each year. In addition, this has led to ces, especially the province of Zaire, which ensures
low surveillance coverage and inadequate screening supplies of specific drugs for the disease.
of the population at risk, suggesting that the true
number of patients is very high. Since the refurbishment of the screening and treat-
ment centre and the Viana Reference Centre, some
The main reasons for the persistence of sleeping staff have been transferred from the headquarters of
sickness in Angola are inaccessibility of most of the ICCT to the Viana Reference Centre, including one
territory affected by trypanosomiasis, the scant sur- physician in charge, two clinicians, one head of lab-
veillance coverage of endemic areas, for reasons oratory, and one clinical assistant. This doubling of
* The orginal manuscript is in French
staff has enabled us to open a reference treatment RECOMMENDATIONS FOR ACTION
centre, supported by the research projects of the • Combat poverty at all levels. This is one of the
Swiss Tropical Institute. We can therefore say that, in major objective, poverty being one of the factors
addition to the refurbishment and the equipment of that aggravate the socioeconomic status of the
the laboratories, functional practice and expertise population, including those suffering from try-
have been developed. panosomiasis.
A large number of NGOs operate in Angola. Most of • Adopt a strategy of permanent, regular, epidemi-
them were originally working independently of ological surveillance, consisting of active diagno-
each other, applying different methodologies to the sis of new cases of the disease, with a view to
control of African trypanosomiasis, but numerous early detection, treatment and effective follow-up
territorial disputes arose and it became necessary of sufferers.
for the Ministry of Health to take over direction of
trypanosomiasis control. As a result, coordination of • Mobilize resources to combat the disease, such as
the national and international NGOs through ICCT drugs, reagents, equipment and traps.
was initiated. Many meetings were held for consul-
tation and coordination (on average, one every three • Strengthen vector control actions, using the most
months), while project visits by ICCT staff also appropriate techniques to combat tsetse flies.
helped to enhance the legitimacy and credibility of
the Institute, facilitating the coordination of inter- • Draw up an information, education and commu-
ventions. Currently, there is no more rivalry and the nication (IEC) plan for the population involving
role of the ICCT is understood by all, such that the political leaders, leaders of civil society and health
number of visits to partner treatment centres has officials in order to take concrete steps to control
substantially increased. The NGOs have accepted this disease.
standardization of the methods for case detection,
treatment and data collection, while a growing • Draw up a plan of work for animal trypanosomi-
number of technical and medical staff belonging to asis and update Glossina mapping of the country.
ICCT now work with NGOs, where they are replac-
ing expatriate staff. • Strengthen cooperation between the ICCT and its
national and international partners.
The NGOs that have worked, and are still working, in
Angola are shown in Table 2. • Prepare coherent and feasible projects that are
guaranteed to receive national and international
Table 2. NGO presence in endemic areas between funding.
1996 and 2000
• Look for ways and means to motivate ICCT work-
Organization Location 1996 1997 1998 1999 2000 ers by improving working and social conditions.
MSF-B N’dalatanado + + + +
MSF-F Maquela do
Zombo +
Quiculungo +
Caritas M’banza-Congo + + +
Uige
Quitexe + + + + +
Lucala + + +
Negage + + +
APN Dondo + + + + +
Casuala + + + + +
MMC Quiçama + + + +
FUNDANGA Caxito + +
ADRA Cacuso
II THE SITUATION IN TANZANIA The worst affected area is along the Malagarasi
Valley, where the vegetation is characterized by big
Stafford Kibona, trees overhanging open grassland. The population
National Institute of Medical Research, Tabora, Tanzania in this large area is, for the most part, concentrated
in villages on the Kigoma–Kibondo trunk road.
GENERAL OVERVIEW Sleeping sickness infections are contracted mainly
In the United Republic of Tanzania, sleeping sick- by those going into the bush to hunt, fish, collect
ness, also known as human African trypanosomiasis honey, beeswax, etc. There are few cases of perido-
(HAT), is one of the major public health problems. mestic infection.
Sleeping sickness was first recorded in 1922 in
Maswa district, south of Lake Victoria (Kilama et al Each village has a population of over 2000. Some of
1981). It then spread throughout mainland Tanzania the villages are remote from the main road, for
and is currently endemic in eight regions, namely example Kagera and Mvinza in the north-east and
Arusha, Lindi, Ruvuma, Kagera, Kigoma, Tabora, Kitanga and Heru-Ushingu in the extreme north-
Mbeya, Rukwa. The annual average number of west.
cases for the last decade was 264. There are isolated
foci, most of which have been producing cases for Villages Affected by Sleeping Sickness in
many years. Heavy foci exist in Kigoma Region Kasulu District
(Table 1). Thirty-two villages are exposed to the risk of infec-
tion in the endemic district of Kasulu. Of these, the
Table 1. Number of sleeping sickness incidences diag- following are regarded as highest risk areas: Heru,
nosed from district hospitals in six regions of Tanzania Kitagata, Kitanga, Makere, Mugombe, Mvugwe,
for the past five years. Mwali, Nyachenda, Nyakitonto, Nyamidaho,
Nyarugusu, Shingu. Others include Kagera,
Region District 1996 1997 1998 1999 2000 Kaguruka, Kitema, Mvinza, Rungwe, Mpya, Titye.
KIGOMA Kibondo 212 286 206 410 376 Overall, the estimated population at risk of infection
ARUSHA Kasulu 155 198 172 156 191 is 230 000. Each village has a health clinic. There is
Babati 12 19 15 12 34 one health centre for every six villages.
Monduli 19 6 2 9
Hanang 5 3 8 2
TABORA Urambo 1 7 2 9
Villages in Kibondo District
RUKWA Nkansi 4 1 7 8 6
Sleeping sickness occurs in the following villages in
Mpanda 5 1 - - - this district: Bitare, Biturana, Busunzu, Kanembwa,
MBEYA Chunya 6 4 - - - Kazira mihunda, Kifura, Kilemba, Kingoro,
TOTAL 400 531 421 588 627 Kitahana, Kumbanga, Kumhasha, Kumshindwi,
Malagarasi, Mkabuye, Mvugwe, Nduta, Nyankwi,
Although sleeping sickness was present in eight Nyaruyoba, Rusohoko. Each village has a dispensa-
regions during the past ten years, the disease was ry. Each health centre serves six or seven villages.
more concentrated in Kasulu and Kibondo districts
of Kigoma Region, which accounted for about 90% Refugee Camps in Kigoma
of the cases. These figures are underestimates due to There are about 280 000 refugees, mainly from the
lack of accurate diagnosis and under-reporting. Eastern part of the Democratic Republic of Congo
(DRC), settled in camps in Kigoma region. Sleeping
KIGOMA SITUATION sickness cases have already been detected in these
An outbreak of sleeping sickness is currently being camps and there is concern that an overlap of gam-
experienced in Kigoma Region of Western Tanzania biense and rhodesiense sleeping sickness exists in
along Lake Tanganyika. The disease represents a the region.
continuing threat to the health and morale of many
communities in the area. All the districts of Kigoma CAUSES OF EMERGENCE AND
Region are affected by sleeping sickness, with RE-EMERGENCE
Kibondo and Kasulu districts producing a great pro- The current situation of sleeping sickness in
portion of the total cases over the past few years. Tanzania, especially the outbreak in Kigoma region,
The whole of Kigoma Region is on the Western Fly is caused by the following factors:
Belt, a portion of a large forest which extends, with • Poor surveillance due to inadequate funding and
few breaks, over an area of 10 368 sq. km. and lies staff.
between 31°E and 24.7°W, 3.5°N and 5.2°S. Here, • Inadequate and erratic supply of specific try-
game is fairly abundant, and the people regard panocidal drugs.
sleeping sickness as an old disease. • Poorly equipped field laboratories for diagnosis.
• Opening up of new unauthorized settlements and IDENTITY OF THE HUMAN INFECTIVE
farms in the endemic areas of sleeping sickness. In TRYPANOSOME IN TANZANIA
the past, the people in Nyakitonto area, for exam- The occurrence of the chronic syndrome of sleeping
ple, had wished to live in the valley (Kitome area), sickness, together with the marked variation in effi-
which is wooded and well watered but also heavi- cacy of chemotherapeutic treatments, in Tanzania,
ly infested with tsetse fly. Permissions to do so had may be cited as indications that the trypanosomes
been consistently denied. However, a few families constitute a heterogeneous complex of organisms
had occupied the area by 1983 and, as was perhaps including perhaps a mixture of T. b. rhodesiense and
inevitable, many infections occurred thereafter. T. b. gambiense.
• Lack of monitoring and health education cam-
paigns. Old records show that the Malagarasi focus
• Increased forest activities including cultivation (Kigoma region), for example, in north-western
outside the villages or on the buffer zone. Some of Tanzania, is an old gambiense sleeping sickness
the planned villages were, unfortunately, located focus with the last case of gambiense infection hav-
in a rather dry and infertile area causing the vil- ing been reported in 1958 (Kihamia et al. 1991). The
lagers to open new farmlands outside the villages. area is now considered endemic for rhodesiense
• Apparent bush regeneration with resultant tsetse sleeping sickness. In Tanzania there are considerable
encroachment. differences in the clinical types of human try-
• The highly probable introduction of a virulent panosomiasis, with the severity of disease varying
strain of T. b. rhodesiense. according to geographical location and becoming
less virulent the further one goes south. While this
The serious outbreaks of sleeping sickness in the may be attributed to the heterogeneity of T. b. rho-
district are a disappointing setback in the disease desiense strains (Komba et al. 1997), there is concern
control efforts of the country, and indicate a neces- that it may be due to the occurrence of the two
sity for review of sleeping sickness control meas- species of trypanosome. Furthermore, the presence
ures in the area. A study in Kigoma region sug- of large numbers of refugees from the DRC, a coun-
gests the following factors are important in the try known to be endemic to gambiense sleeping
current outbreak: sickness, increases this possibility.
• Farming activities carried on outside the protect-
ed area. Further studies are required to:
• Inter-village visits through tsetse infested bushes • Investigate why the disease is localized in specif-
in search of the basic necessities of life. ic foci despite the tsetse fly and reservoir animals
• Increased forest activities – honey and beeswax being present in large areas of the country.
collection, fishing, hunting, etc.
• Peridomestic activities e.g. firewood gathering, • Investigate the distribution of T. b. rhodesiense
fetching water, cutting poles for building. strains in Tanzania.
• Visits into the wildlife areas where no human set-
tlement is allowed. • Investigate the possibility of an overlap of rhode-
siense and gambiense sleeping sickness, especial-
The problem that now confronts Tanzania is that ly in Kigoma focus, which has an influx of
sleeping sickness is still endemic and there is lax- refugees from the DRC.
ity of control measures. Compared to other
endemic diseases, the number of human deaths References
from sleeping sickness appears insignificant, but Kilama WL, Mtera KNM, Paul RK. Epidemiology of
even temporary exacerbation of the disease fright- human trypanosomiasis in Tanzania. In: Proceedings
ens the local people. of the 17th Meeting of the International Scientific
Council of Trypanosomiasis Research and Control, 1981,
While it is impossible to predict the future, the Publication No. 112, pg. 187.
possibility of a larger outbreak must be consid-
ered. With the evidence currently available, it Kihamia CM et al. Trypanosomiasis. In: Health and
would be a reasonable precaution to step up con- diseases in Tanzania. 1991, Harper Collins Academic,
trol measures. UK.
Komba EK, et al. Genetic diversity among

Trypanosoma brucei rhodesiense isolates from
Tanzania. Parasitology, 1997, 115:571-579.
III THE SITUATION IN UGANDA animals in Zululand by David Bruce two centuries
AND SUDAN ago (WHO, 1995). Later, morphologically identical
trypanosomes were identified in the blood of a
D.B. Mbulamberi, European from The Gambia, West Africa (Dutton,
Ministry of Health, P.O. Box 7272, Kampala, Uganda. 1902), and transmission by riverine tsetse (Glossina
palpalis) was confirmed. Subsequent studies
SUMMARY revealed the extensive, often focal, distribution of
Sleeping sickness is a disease of the rural poor the disease, the substantial endemicity and the
which tends to emerge and re-emerge in epidemics chronic progressive nature of human infection in
in some 36 sub-Saharan African countries, where West and Central Africa.
close to 50 million people are at risk of contracting
the disease. Together, these 36 countries report only The disease was called Gambian trypanosomiasis
about 25 000 new cases of the disease to WHO annu- and the parasite, T. gambiense (later T. brucei T. brucei
ally. This is an obvious underestimate attributed to gambiense). In 1908, a severe rapidly fatal trypanoso-
poor reporting, difficulty in diagnosing the disease, mal infection was identified in the Luangwa valley,
and poor accessibility of the affected areas. The true Zambia. Further investigation confirmed its clinical
figure is currently estimated to exceed 300 000 new severity, the distinct epidemiology with transmis-
cases annually. sion via savannah tsetse, and the zoonotic nature of
infection from game animals harbouring T. brucei.
In the recent past, Uganda and Sudan have not been This led to the description of Rhodesian trypanoso-
spared epidemics of this disease. A devasting epi- miasis due to T. rhodesiense (T. b. rhodesiense). Other
demic of T. b. rhodesiense sleeping sickness has members of the T. brucei T. brucei group, that are
occurred in south-eastern Uganda and one of T. b. non-infective to humans and occur in game and
gambiense in the West Nile region of the country domestic animals, were designated T. brucei (subse-
(Uganda). In Southern Sudan, along the border with quently T. b. brucei).
Uganda, there is another epidemic of T. b. gambiense
sleeping sickness. The General Epidemiology of the Disease
Trypanosomes, the parasites which cause the dis-
The causes of the emergence and re-emergence of ease, occur in the blood of man and animals as the
epidemics of this disease are varied, but can be con- trypomastigote.
veniently grouped into political, economic, behav-
ioural factors, and the effects of climate and vegeta- All members of the T. brucei group are morphologi-
tion on tsetse fly distribution. cally identical. The parasites have evolved mecha-
nisms for evading host immune responses through
To limit the impact on human lives in these two variation of their surface antigen glycoproteins. The
countries, external support will be required to zoonotic nature of T. b. rhodesiense was initially
implement strategies for disease and vector control. established by inoculation of ‘volunteers’ with para-
On the one hand, donor agencies, NGOs and mis- sites from a bushbuck (Heish et al, 1958), and later
sion organizations could play an important role in from domestic cattle.
supporting these control efforts. On the other hand,
national authorities will need to control and coordi- Subsequently, the blood incubation infectivity test
nate these efforts with assistance from WHO and the (BIIT) was developed to assess the human infective
international community. potential of parasites from a range of wild and
domestic animals. More recently, a number of
This paper presents a general introduction to the molecular techniques, especially isoenzyme analy-
disease (sleeping sickness) and a brief account of sis (Stevens and Godfrey, 1992) and restriction frag-
factual epidemic outbreaks in Uganda and the ment length polymorphism (RFLP), have been used
Sudan. The paper then proceeds to discuss, in gen- as markers for parasite strains to explore the molec-
eral terms, possible factors for the emergence and ular epidemiology of this complex group of para-
re-emergence of epidemics of the disease. sites. These techniques allow T. b. gambiense to be
distinguished from T. b. rhodesiense.
INTRODUCTION In West and Central Africa, sleeping sickness is

transmitted by riverine species of tsetse fly (palpalis
Historical Aspects of the Disease group), which require sustained levels of humidity
Trypanosoma brucei, and the role of the tsetse fly and prefer dense riverine habitats. These tsetse flies
(Glossina spp.) as its vector, was identified in game feed preferentially on man, especially where man-
fly contact is high, such as at water collection and tination test. Lancien in Uganda (1991) confirmed
bathing points, river crossings and sacred groves. that insecticide impregnated traps can be used, with
For the riverine species of tsetse fly, man provides community participation, to control sleeping sick-
the reservoir of infection, although both wild and ness epidemics.
domestic animals may play a minor role in particu-
lar foci. Throughout most of the range, T. b. rhode- Until recently, the treatment of sleeping sickness
siense transmission is effected by savannah species relied essentially on three drugs, namely pentami-
of tsetse (morsitans group). This group of tsetse flies dine, suramin and melarsoprol. Pentamidine, a
survives in drier, more open areas of woodland, diamidine introduced in 1937, is currently available
savannah and acacia thickets, and prefers to feed on as pentamidine isethionate and is effective against
game animals and domestic stock. Human infection early infections of T. b. gambiense. Suramin, which
occurs sporadically in individuals coming into con- was introduced in 1922, is effective against early
tact with the zoonotic cycle, for example poachers, infections of both T. b. gambiense and T. b. rhode-
hunters, honey gatherers, firewood collectors and siense. Melarsoprol, a trivalent arsenical introduced in
tourists. A wide spectrum of animals, notably game 1949, was the only drug available, until 1990, for the
animals and domestic cattle, provide a reservoir of treatment of late infections of both T. b. gambiense
infection. However, in East Africa, the epidemiology and T. b. rhodesiense. All these drugs have adverse
is different in that T. b. rhodesiense is transmitted by side effects, melarsoprol causing reactive encepha-
a riverine species of tsetse, namely G. fuscipes lopathy in 5—10% of patients treated, with a fatal
fuscipes, and domestic cattle are the main reservoir. outcome in 1 -5%. Resistance of T. b. gambiense to pen-
This situation creates a lot of potential for epidemic tamidine, and of both T. b. gambiense and T. b. rhode-
outbreaks of the disease. siense to melarsoprol, occurs. Nifurtimox, a 5—nitro-
furan, is currently being used, either singly or in com-
Clinical Manifestation of the Disease bination with other drugs, to treat late-stage gambi-
In most endemic areas, T. b. gambiense causes a pro- ense infections on a compassionate basis.
tracted, often initially unrecognized, illness with
episodes of fever, headache and malaise, accompa- Eflornithine (DFMO), a potent inhibitor of
nied by progressive lymphadenopathy and followed polyamine synthesis, was developed for the treat-
later by the development of a progressive, fatal, ment of sleeping sickness through collaboration
meningoencephalitis. This contrasts with the acute, between Marion Merrel Dow Inc., USA, and the
severe, febrile disease observed with T. b. rhodesiense, UNDP/World Bank/WHO Special programme for
with rapid progression to meningoencephalitis. Research and Training in Tropical Diseases (TDR).
There is relentless deterioration to a stuporous state, However, while this drug provides the best alterna-
with cachexia, wasting and progressive malnutrition, tive treatment to melarsoprol for gambiense sleeping
deepening coma and death, within a few months in sickness, alone it is ineffective against T. b. rhodesiense
the case of T. b. rhodesiense and extending for months infection. Therefore, no alternative treatment for
or even years in the case of T. b. gambiense. late-stage rhodesiense infection is yet available.
AVAILABLE OPPORTUNITIES FOR THE The availability of all these drugs is currently high-
CONTROL OF SLEEPING SICKNESS ly uncertain, with the various manufacturing firms
The principle of control and prevention of sleeping either threatening to stop, or having already
sickness relies on an integrated strategy of continu- stopped, their production. It is evident that the
ous surveillance, involving diagnosis and treatment treatment of sleeping sickness is still unsatisfactory.
of the population at risk, and vector control where The ideal trypanocide should be safe and effective.
applicable (de Raadt, 1986). A number of tools for It must have a simple mode of administration to
diagnosis and vector control have been developed allow its use under rural conditions where health
through research during the past decade and are, facilities are usually of a poor standard, and, above
indeed, field applicable by national health services. all, it should be affordable.
These include the card agglutination test for try-
panosomiasis (CATT) (Magnus et al, 1978) for sero-
diagnosis, and the miniature anion-exchange cen- THE PAST AND PRESENT SLEEPING
trifugation technique (MAECT) (Lumsden et al, SICKNESS SITUATION IN UGANDA
1979) for parasitological diagnosis. The antigen AND SUDAN
ELISA, developed by Nantulya (1989) and evaluat-
ed for detection of gambiense (Nantulya et al, 1992) Uganda
and rhodesiense (Komba et al, 1992) sleeping sick- The sleeping sickness epidemic which devastated
ness, was subsequently modified into a latex agglu- the shores of Lake Victoria at the beginning of the
last century is a famous event in the annals of tropi- and EATRO, the department probably in the best
cal medicine. It is famous because an estimated one position to help contain this epidemic during its
quarter to one third of a million people lost their outset, lost valuable logistics to a partner state and
lives (Langlands, 1967). The same epidemic brought became helpless. The Ministry of Health posted
controversy as to whether Dr Castellani or Colonel microscopists to the area and later opened up treat-
Bruce first identified the trypanosome as the cause ment centres in the area. However, the epidemic
of the epidemic. continued to increase in magnitude from 52 cases in
1976 to over 8000 cases in 1980 (Fig. 1).
The cause of the epidemic was attributed to T. gam-
biense introduced to this part of the country by a Figure 1.
Annual incidence of sleeping sickness due to
party accompanying the explorer Lugard from the REPORTED
CASES
T. b. rhodesiense in Eastern Uganda, 1976-March 2000
Congo basin on relief of the Emin Pasha expedition 9000
8000
in 1894 (Christy, 1903). However, it is more accurate 7000
to say that the cause lay in the general increase in 6000

5000
social, commercial and military mobility which 4000
developed throughout tropical Africa in the late 3000

Cases up to March
nineteenth and early twentieth centuries. 2000
1000
0
1976 77 78 79 1980 81 82 83 84 85 86 87 88 89 1990 91 92 93 94 95 96 97 98 1999 (March)
Another outbreak involving about 2500 persons YEAR
occurred in the same area from Jinja eastwards to

the border with Kenya between 1939 and 1945 In the West Nile region, a new outbreak of T. b. gam-
(Mackichan, 1944-45). The most striking feature of biense sleeping sickness occurred along the Dacha
this epidemic was the virulence and rapidity of the river. During the first year of the outbreak, a total of
fatal course of the disease observed on animal inoc- 12 cases were recorded. In the following year (1958),
ulation. Thus, it is believed, the epidemic was this new focus produced 7 cases and it seemed as if
caused by T. rhodesiense. The first cases detected the outbreak had ceased. However, the 1959 inci-
were among migrant workers employed on Kakira dence of 30 cases, the highest annual figure since the
sugar estates. Since these immigrants came from big epidemics had been finally checked 12 years
areas of reasonable proximity to the infected areas of before, made it evident that the outbreak was by no
Tanganyika (now Tanzania), this epidemic was means under control.
thought to have been introduced by them.
However, this outbreak was finally brought under
Since that outbreak, cases continued to be reported control and the situation remained largely stable
from within the infected area, though not in epi- thereafter until the early 1980s, when the current
demic numbers. In 1971, infection spilled north of outbreak in the region started. This current outbreak
the usual focus and involved up to 169 persons. is associated with the war of liberation against Idi
Amin in 1979, when most local residents in the
Following the control of the small epidemic of 1971, region fled into exile in Southern Sudan where there
surveillance programmes were not instituted was a ravaging epidemic of T. b. gambiense sleeping
because of the prevailing political and economic sickness, then as now. When these local residents
atmosphere in the country at that time. There was returned to the West Nile region in the early 1980s,
indiscriminate and haphazard movement of people some of them had the infection, which they intro-
and livestock across the traditional trypanosomiasis duced into the area. The epidemiological trend of
barrier zone. Besides, smuggling of commodities, the disease in the region (West Nile), over the years,
including cattle, between Kenya and Uganda, across is shown in Fig. 2.
the zone, became a means of livelihood. It was there-
fore difficult for the Ministry of Health teams to
Figure 2.
enforce surveillance measures. The Tsetse Control Annual incidence of sleeping sickness due to
REPORTED T. b. gambiense in North Western Uganda, 1981-1999
Department could not carry out control programmes CASES
2500
due to lack of insecticide, transport and human
2000
resources. Thus, there was total breakdown of control
measures and hence, by 1976, the stage was set for 1500
another epidemic outbreak of the disease in the area. 1000
500
This new outbreak started in Luuka County of

0
Iganga district in August 1976. Unfortunately, in 1981 82 83 84 85 86 87 88 89 1990 91 92 93 94 95 96 97 98 99
YEAR
June 1977, the East African Community collapsed
Sudan uncontrolled movement of people into areas that
Sleeping sickness due to T. b. gambiense has been may have been previously abandoned because of
known to occur in the Southern Sudan since the epidemics, thereby promoting circulation of the par-
early 20th century. Epidemics of the disease have asite in the population and the risk of contact
occurred mainly in the Southern and South-western between people and the tsetse flies. Civil distur-
parts of the country bordering Uganda, the bance and war will, in the final analysis, lead to a
Democratic Republic of the Congo (DRC), and the breakdown in vital social services including med-
Central African Republic. In addition, cases of the ical and vector surveillance programmes. This is
disease have been reported along the Ethiopian bor- obviously the most important factor in the case of
der, in Raga since 1909, in Yei since 1910, in Kajokaji the current epidemics in both Uganda and
since 1914, in Nimule since 1915, in Tambura since Southern Sudan, as is the case in most other sub-
1918, and in Yambio since 1924. Saharan African countries, e.g. Angola, Mozambi-
que, the DRC.
Almost all the aforementioned foci are still active.
Duku (1979) attributed these epidemic outbreaks to: Declining Economies
the presence of active foci in neighbouring coun- Declining economies, as is the case in most sub-
tries, particularly those where tribal settlements Saharan African countries, will dictate reduced
straddled the borders; widespread distribution of financing of disease control programmes. This, in
the vectors; and political upheavals, instability and turn, will affect field control activities as well as the
civil disturbance. implementation of advances so far made in diagno-
sis, treatment and vector control. This is mainly
The current flare-up of the disease started after the because these new advances are not available on the
signing of the Addis Ababa peace agreement in local market and therefore require importing into
1972. The relative peace which followed the signing the country, for which foreign currency is required.
of this agreement made it possible for some control In addition, reduced financing is likely to lead to the
measures to be instituted with external assistance temptation of progressively dismantling vertical
from WHO and the Government of the Kingdom of disease control programmes in preference for inte-
Belgium between 1974 and 1978, hence the avail- grated, community-based programmes, thus lead-
ability of the information shown in Fig. 3. Otherwise ing to loss of focus.
information on the current epidemic in the Southern
Sudan is not easily available. Behavioural Factors
One of the factors to be considered here is the low
priority rating accorded to sleeping sickness on the
Figure 3.
Self-reporting cases of sleeping sickness in Yambio district, part of both donors and national governments. This
SELF-
S. Sudan, 1974-1978
is despite the negative impact of the disease on
REPORTING
CASES
development. One possible explanation is that
1600
1400
sleeping sickness control programmes do not have
1200 much appeal for international aid donors due to
1000
various factors including: the regional distribution
800
600
of the disease and mainly rural nature of the prob-
400 lem; the relatively small number of new cases
200 reported annually compared to other diseases; the
0
1974 1975 1976 1977 1978 requirement for long-term input to control pro-
YEAR grammes for sustainability in the absence of the
prospect of eradication. Paradoxically, when epi-
FACTORS RESPONSIBLE FOR THE demics of the disease occur, financial support is
EMERGENCE AND RE-EMERGENCE OF made easily available in amounts which are usually
SLEEPING SICKNESS disproportionately higher than those required for
the regular preventive measures (Kuzoe, 1993).
Factors responsible for the emergence and re-emer-
gence of sleeping sickness are varied and diverse. Another pertinent behavioural factor, in this respect,
Mbulamberi (1989) gave an outline of these factors. is the population density of the tsetse flies and their
A brief account, a modified version of these factors, feeding behaviour. A tsetse fly feeding on a number
is given below. of animals, and possibly also on man, may become
infected with many different strains of try-
Civil Disturbance and War panosome. Most of these strains will be non-patho-
Civil disturbance and war cause extensive and often genic for man and, even if a man-infective strain is
acquired by the fly, the tendency will be for it to be recruited for large-scale construction work (tropical
so diluted by the non-pathogenic strains that it will aggregation of labour) and pilgrims attending major
not be passed on in sufficient number to cause infec- religious festivals.
tion in man.
A new population in an area may spark off an epi-
Another important behavioural factor is increased demic outbreak of sleeping sickness as a result of
man-fly contact. This phenomenon occurs most imported cases among them, which may be suffi-
commonly during the hot, dry season with the ciently large to increase the reservoir of infection
result that transmission is enhanced. Indeed, a peri- available to the insect vector and so, in a quantita-
od of drought almost invariably means an increase tive manner, promote transmission. An imported
in the number of infections because the few sources strain may also show quantitative differences such
of water are shared by man, tsetse flies and game as enhanced virulence or ability to spread, or may
animals, in close association; there is also more be one to which the indigenous population has not
hunting and more searching for wild forest products been previously exposed and to which no resistance
at times when crops are bad. This phenomenon is has been acquired. This phenomenon can also oper-
particularly applicable to T. rhodesiense infections ate vice versa. Further, as with some other diseases,
(Willet, 1965, and many other workers). the periodicity of epidemics of sleeping sickness
may be associated with the growing up of a new
The presence of domestic and wild animal reservoir generation of people with no previous experience of
hosts is another important factor. The pig in the case the disease.
of T. gambiense, and cattle in the case of T. rhodesiense,
have been incriminated as domestic animal reser- The occurrence of sub-acute cases of the disease is
voir hosts, while the kob and hartebeest in the case yet another important factor. The presence of an
of T. gambiense and the bushbuck in the case of T. undetected and perhaps unsuspected reservoir of
rhodesiense have been incriminated as wild game infection in the form of human “healthy carriers” of
reservoir hosts. The bushbuck is particularly impor- the disease, which has been reported by several
tant because it tends to live in thickets near human workers (Buyst, 1977; Rickman, 1974; Woodruff
habitation, which puts it in close contact with man. et al, 1982), has important epidemiological implica-
tions.
The appearance of different forms of the parasite is
another important factor. The appearance of such Under conditions in which man-biting tsetse are
parasites may be due either to the parasites being common and where people congregate, the ambu-
introduced from outside the area or to genetic lant human carrier assumes a powerful potential for
changes in the parasite. There is at least a suspicion, the onward transmission and spread of sleeping
based on field observations, that zymodemes of try- sickness. The occurrence of asymptomatic carriers
panosome introduced into fresh localities may of rhodesiense sleeping sickness is certainly low.
exhibit an enhanced ability to spread through the However, sleeping sickness cases with non-specific
community. Scott (1961) reported two instances in symptoms (fever, headache) who remain ambulant
which the introduction of infected persons from an for several weeks are common, and they too may be
established epidemic area resulted in outbreaks of important reservoirs of infection where man-fly
the disease in endemic localities far removed from contact is intense (Wurapa et al, 1984). This threat is
the original focus of infection. There are other simi- also present among many early cases of the gambi-
lar observations suggesting that severe local out- ense disease, in which the initial stages are general-
breaks which quickly follow the introduction of ly relatively mild and the victim may continue to
infected persons to fresh localities are, in some way, work for many months or even years before he is
connected with enhanced ability of the zymodeme eventually driven, by increasing illness, to seek
to spread. Indeed, the possible existence of epidem- treatment or to retire to his home. During this time,
ic trypanosome zymodemes has been advanced by he is a constant source of infection to tsetse so that
some workers. the very nature of the illness provides great oppor-
tunities for its spread. In both the Ugandan and
Another factor of importance are the changes in Southern Sudan situation, the question of delayed
population movements and population growth. It is diagnosis and treatment is a big factor.
generally supposed that population movements are
liable to precipitate epidemics. Refugees displaced Another critical factor in this category is human
as a result of war, famine, earthquakes and other behaviour and activities in the fly’s habitat. Often,
similar occurrences are notoriously prone to disease man becomes infected during travel, hunting, fish-
in epidemic form, as are immigrant labour forces ing, collection of honey or when working in the bush
which the fly inhabits. Fishing and “honey-hunting” vive for protracted periods. This enhances the
are particularly hazardous occupations. While fish- potential for these flies to transmit the disease, of
ing in riverine pools surrounded by thickets, people course depending on their infection rates.
may be in close contact with tsetse flies for many
days at a time, a situation in which the association References
between humans, bushbuck and tsetse fly is likely to Christy C. The Epidemiology and Etiology of the
be significant and conducive to transmission and sleeping sickness epidemic in the Equatorial East
spread of the disease. Wyatt et al. (1985), working in Africa with clinical observations. Report of the
north-east Zambia, found fishing to be more com- Sleeping Sickness Commission of the Royal Society,
mon among cases of sleeping sickness than controls. 1903, 3:3-32.
Fishing represented a hazard both while walking to
the stream and while engaged in the activity itself. Buyst H. The epidemiology of sleeping sickness in
the historical Luangwa Valley. Annales de la Société
Climate, Vegetation and Tsetse Fly Belge de Médecine Tropicale, 1977, 57(4-5):349-359.
Distribution Factors
Climate appears to be of more than ordinary impor- De Raadt P. Integrated sleeping sickness control. In:
tance. At higher temperatures, there is an increased Proceedings of the CEC International Symposium, Ispra,
salivary gland infection rate in the tsetse fly, and, in Italy, 1986, 4:147-151.
addition to this direct effect, there are many ways in
which climate influences a closer association Duku MO. Human trypanosomiasis in the Southern
between man and fly. Sudan: Present situation and control measures in:
International Scientific Council for Trypanosomiasis
In the current epidemic of sleeping sickness in south- Research and Control 16th meeting, Yaounde Cameroon,
eastern Uganda, heavy rains coupled with the abun- 1979, 139-145.
dant growth of Lantana camara thickets provided G.
f. fuscipes with suitable conditions well outside its Dutton JE. Preliminary note upon a trypanosome
usual riverine habitat, so it was able to live and breed occurring in the blood of man. Thomas Yates
in the vegetation surrounding homesteads. Laboratory Report, 1902, 4:455.
Climate also has an influence on where people Ford J. The role of the trypanosomes in African ecology -
choose to live, and on the population density of both a study of the tsetse fly problem. Oxford, Clarendon
flies and human beings, as discussed by Ford (1971). press, 1971, 568 pp.
This is relevant to the proper use and full develop-
ment of land, which is the ultimate aim of eradicat- Heisch RB, McMahon JP, Manson Bahr PEC. The
ing the tsetse fly and trypanosomiasis. isolation of Trypanosoma rhodesiense from a bush-
buck. British Medical Journal, 1958, ii:1203.
Infection rates in tsetse flies, and their infectivity, are
affected by climate. Wijers (1960) observed that Komba EK, et al. Multicentre evaluation of an anti-
infection rates were highest in flies taking an infec- gen-detection ELISA for the diagnosis of Trypanosoma
tive blood-meal on the day on which they emerged, brucei rhodesiense sleeping sickness. Bulletin of the
somewhat lower on the second day after emergence, World Health Organization, 1992, 70:57-61.
and did not occur thereafter. Thus, the fact that flies
emerging during the hot season are likely to feed Kuzoe FAS. Current situation of African trypanoso-
early in their adult life means that infection rates in miasis. Acta Tropica, 1993, 54:153-162.
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However, the number of trypanosomes inoculated Lancien J. Lutte contre la maladie du sommeil dans
by an infected tsetse fly varies greatly, even among le sud-est Ouganda par le piégeage des glossines.
flies infected from the same host and in the same fly Annales de la Société Belge de Médecine Tropicale, 1991,
at different times. 71 (Suppl.1): 35-47.
Climate also affects tsetse longevity. Flies emerging Langlands BW. The sleeping sickness epidemic in
at the end of the hot, dry season are particularly Uganda 1900-1920: a study in historical geography.
receptive to trypanosome infection since they will Department of Geography, Makerere University
feed early in adult life. With the onset of the rainy College, Kampala, 1967 (unpublished document).
season, the expectation of life of a tsetse fly is maxi-
mal, so that a combination of these factors produces Lumsden WGR et al. Trypanosoma brucei: miniature
a situation in which infected flies are liable to sur- anion exchange centrifugation for detection of low
parasitemias: adaptation for field use. Transactions of Woodruff AW, Evans DA, Owino NO. A healthy car-
the Royal Society of Tropical Medicine and Hygiene, rier of African trypanosomiasis. Journal of Infection,
1979, 73:312-317. 1982, 5:89-92.
Mackichan IW. Rhodesian sleeping sickness in east- World Health Organization. Planning overview of
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Tropical Medicine and Hygiene, 1944-45, 38:49-60. Diseases, Geneva, 1995.
Magnus E, Vervoort T, Van Meirvenne N. A card Wurapa FK et al. A healthy carrier of Trypanosoma
agglutination test with stained trypanosomes rhodesiense: a case report. Transactions of the Royal
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Médecine Tropicale, 1978, 58:169-176.
Wyatt GB, Boatin BA, Wurapa FK. Risk factors asso-
Mbulamberi DB. Possible causes leading to an epi- ciated with the acquisition of sleeping sickness in
demic outbreak of sleeping sickness: facts and north-east Zambia: A case-control study. Annals of
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Tropicale, 1989, 69 (Suppl. 1): 173-179. 392.
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lower Luangwa valley, Eastern Province, Zambia.
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Annex 4
EPIDEMIOLOGY, DISEASE SURVEILLANCE
AND CONTROL, AND VECTOR CONTROL
I EPIDEMIOLOGY AND According to the World Health Organization (1998),
CONTROL OF HUMAN AFRICAN there remain within the “tsetse belt” more than 200
TRYPANOSOMIASIS* active foci, located between latitudes 15° North and
15° South. Within this area, 60 million individuals liv-
Honoré A. Méda1 and Jacques Pépin 2 ing in 36 countries are exposed to the infection. Due
1 Project SIDA 2, Benin (ACDI-Canada) B.P. 900 Tri to shortages of financial and human resources, less
Postal, Cotonou, Republique du Benin. than 4 million benefit from an adequate surveillance
2 University of Sherbrook, Infectious Diseases Division, and control programme; all endemic countries are
Centre for International Health, 3001, 12th Avenue, characterized by shortages of the financial and
Sherbrook, Quebec, Canada. human resources necessary to implement or sustain a
comprehensive control programme. Reports from
INTRODUCTION national control programmes can only give a rough
Human African trypanosomiasis (HAT) is caused by idea of the epidemiological situation, because of the
hemoflagellates of the Trypanosoma genus, difficult security situation and the decay of commu-
Trypanozoon subgenus and brucei species, which nication systems in many parts of high-incidence
classically includes three subspecies: Trypanosoma countries. Year-to-year incidence for the 1977-1994
brucei brucei, T. b. gambiense and T. b. rhodesiense. period in all endemic countries can be found in a
These subspecies are morphologically identical but recent WHO report (WHO, 1998), where Figure 1
differ in their ability to infect various hosts. T. b. bru- shows the geographical distribution of the cumula-
cei is essentially a parasite of domestic and game tive number of new cases reported between 1977 and
animals and is not pathogenic to humans because it 1997 for the endemic countries. T. b. gambiense try-
is lysed by a haptoglobin-like molecule (Smith et al, panosomiasis is now a major public health problem
1995). Only T. b. rhodesiense and T. b. gambiense are in Central Africa, specially in the Democratic
considered to be human pathogens. There are two Republic of Congo (DRC), Angola and Southern
clinical variants: an acute syndrome attributed to T. Sudan, where the ongoing civil war hampers control
b. rhodesiense and a chronic one caused by T. b. gam- efforts to such an extent that national statistics give
biense. Both diseases result from complex interac- only a very incomplete view of the problem. In DRC,
tions between the parasite and its tsetse fly where relatively better information is available, the
(Glossina) vector and vertebrate hosts. total number of people at risk is estimated, by the
national control programme, to be 12 500 000. The
During the last couple of decades, considerable number of new cases reported each year has now
progress has been made toward the improvement of reached levels comparable to those seen in the early
epidemiological knowledge. This has led to the 1930s, despite substantial underdiagnosis due to
development of new tools suitable for control. A inadequate coverage of endemic regions; this situa-
recent paper by Pépin et and Méda (2001) provides details tion may result in the death of as many adults as
of the advances in the epidemiology and control of AIDS (Ekwanzala et al, 1996). Underdiagnosis is also
HAT. However, these advances have not been suffi- exacerbated by the poor sensitivity of diagnostic
ciently used in the field for what they were intended. methods. The number of cases reported annually
The aim of this paper is to try to summarize what we increased dramatically from about 10 000 in 1980 to
know of the epidemiology and control and to identi- more than 27 000 in 1998. In the most endemic
fy some of the most important gaps that need to be regions (e.g. Equateur and Bandundu), many com-
urgently tackled by the scientists and programme munities have been found to have a prevalence of
managers involved in research and control activities. over 10% during recent case-finding surveys. In 1994,
an extra-ordinary prevalence of 72% in a small village
CURRENT EPIDEMIOLOGICAL of the Bandundu region was reported.
SITUATION AND DISEASE BURDEN
HAT is the only vector-borne parasitic disease whose Angola is the country with the second highest inci-
geographical distribution is limited to the African dence of HAT, respectively 8275 and 6610 new cases
continent. T. b. gambiense is seen in West and Central were reported in 1997 and 1998 by the national con-
Africa, and T. b. rhodesiense in East and Southern trol programme. Variations in the annual number of
Africa. Uganda is the only country where both sub- reported cases must be interpreted with caution due
species are found: T. b. gambiense in the north-west to the impact of the civil war on case-finding. The
and T. b. rhodesiense in the south-east. This distribu- disease is endemic in the north-west provinces. The
tion has probably remained constant over time. prevalence rates reported vary between 1.3% and
* The contents of this paper are drawn largely from Pépin J. Méda AH. The Parasitology, 2001, 49:71-132, by permission of the publisher Academic
epidemiology and control of human African trypanosomiasis. Advances in Press.
9.7%. Uganda is the only country where both T. b. 30 days on average; it varies, according to species
gambiense and T. b. rhodesiense are found, the former and the ambient temperature, between one and
in the north-west of the country, near the Sudanese eight weeks. When Glossina takes a blood-meal from
border, and the latter in the south-east, without over- an infected host, it ingests bloodstream trypo-
lap. Between 1000 and 2000 T. b. gambiense cases were mastigote forms in its salivary glands. Trypanoso-
reported annually in 1990-1994, but this has at least mes then move to the fly’s midgut where, over a few
stabilized with 978 cases reported in 1998, more than days, they transform into the procyclic stage. The
half of them from Arua district. A major epidemic of variant surface glycoprotein (VSG), the dominant
Rhodesian HAT devastated SE Uganda from the constituent of the surface of bloodstream try-
mid-1970s, with a cumulative total of about 40 000 panosomes, is replaced by an invariant surface pro-
new cases over 15 years (Smith et al, 1998; Hide, tein. After two to three weeks of maturation and
1999). There has been some improvement recently, multiplication, trypanosomes migrate to the sali-
after many years of efforts by national authorities vary gland, where other transformations lead to
with substantial external support. Only 271 cases their development into metacyclic trypanosomes,
were reported in 1998 from the SE of the country which require VSG to be infective for a susceptible
(Busoga). In Sudan, where reliable statistics are not human or animal host during the next blood-meal.
available, the foci of T. b. gambiense HAT are located These forms are the metacyclic trypomastigotes, the
in the southern part of the Equatoria region, west of only stage which is infective to vertebrates. Once
the Nile, within 100 kilometres of the borders with infected, a tsetse fly remains so for the rest of its life
Central African Republic (CAR), the DRC and span of between three and four months. While tak-
Uganda. Extrapolations suggest that there must be at ing subsequent blood-meals, the fly is capable of
least a few thousand cases per year. In other coun- inoculating another vertebrate host with the meta-
tries of Central Africa, HAT is more or less an emerg- cyclic trypanosomes. Within the human host, the
ing public health problem. Elsewhere in East and parasite multiplies at the site of inoculation, where a
Southern Africa, the incidence of T. b. rhodesiense try- chancre might develop. Inoculation chancres are
panosomiasis remains low. In West Africa, the dis- rarely recognized on African skin. From this site, the
ease has regressed or disappeared from several parasite gets into the bloodstream and lymph nodes
countries as ecological changes reduced the intensity and multiplies through binary scissiparity with
of man-fly contact. Only Côte d’Ivoire and Guinea three morphological forms: the short and stumpy,
still report a significant number of cases. Only a few the intermediate, and the long and slender forms.
dozen cases are seen each year in Burkina Faso and
Mali, corresponding both to importation of cases and After infection of a human host, there is a switch
local transmission. The situation is less well known from the expression of metacyclic VSG to blood-
in other West African countries. stream VSG. To evade the host’s immune response,
trypanosomes can successively express different
It is difficult to estimate the overall burden of HAT. VSG, but only one at a time. This antigenic variation
There are about 100 000 new cases per year, with is a unique feature of trypanosomes (Barry, 1997); it
between one third and one half of cases remaining has direct consequences for the epidemiology of the
undetected and untreated. Rhodesian trypanosomi- disease. Up to a thousand different VSGs are genet-
asis represents less than 5% of the overall burden of ically encoded, and it is thought that the VSG cur-
the disease. Recent estimates (WHO, 2000) are rently expressed protects invariant constituents
rather similar, with 2.05 million disability adjusted from the host’s immune response. The mechanisms
life years (DALYs) lost, and 66 000 deaths, in 1999 through which trypanosomes switch to expressing a
due to HAT. As a comparison, the number of mil- different VSG are complex, but it allows the parasite
lions of DALYs lost is estimated at 45.0 for malaria, to escape from antibodies directed against the pre-
4.9 for lymphatic filariasis, 2.0 for leishmaniasis, 1.9 vious VSG. This complex process explains the inter-
for schistosomiasis, 1.1 for onchocerciasis, 0.7 for mittent parasitaemia and the very long, largely
Chagas disease. asymptomatic, incubation period. Thus, there is an
alternation between periods of higher parasitaemia
following expression of a new VSG, during which
EPIDEMIOLOGY OF T. B. GAMBIENSE the human host might be more infectious, and peri-
HUMAN AFRICAN TRYPANOSOMIASIS ods of lower or undetectable parasitaemia, during
which infectivity must be lower. Variations in the
The Life Cycle of T. brucei virulence of T. b. gambiense strains were noted early
The complex life cycle undergone by T. brucei in its by Van Hoof (1947). More recently, biochemical,
tsetse fly vector and human host is described in var- molecular and immunological methods have been
ious standard textbooks. The whole cycle lasts about developed for the identification of trypanosomes by
examining their genetic material (Gibson, 1994). savannah of Central Africa. The savannah is the
These methods include two-dimensional gel elec- exclusive habitat of G. morsitans in various regions
trophoresis and isoenzyme, chromosome and DNA of Africa. Its geographical distribution is limited to
analysis using polymerase chain reaction (PCR) gallery forests bordering streams, where optimal
techniques, which have substantially contributed to survival conditions are found. In a given geograph-
a better understanding of the parasite’s taxonomy ical area, the distribution of tsetse flies varies
and the epidemiology of the disease, especially in depending on the species and is mainly determined
studies on animal reservoirs of human trypano- by the climate, presence of water, vegetation and
somes. Cellular and molecular characteristics of availability of sources of blood-meals (humans and
human trypanosomes have been reviewed else- animals). Studies conducted in the forest zone of
where (El-Sayed and Donelson, 1997; Barry 1997). West Africa have shown that G. p. palpalis is a very
mobile vector found in different biotopes and that
its distribution is closely linked to human occupa-
The Vectors tion patterns (Baldry, 1980; Challier and Gouteux,
1980; Gouteux and Laveissière, 1982). Highest
Vector species and sub-species apparent densities per trap (ADT) per day were
The tsetse fly is a vector of trypanosomes that infect observed on the edges of villages where the swine
man as well as wild (antelopes, giraffes, etc.) and population provides an abundant, easily accessible
domestic (pigs, cattle, sheep, goats, dogs, horses, food source. High densities have also been found
etc.) animals. It belongs to the genus Glossina, which near sources of potable water, in coffee and cocoa
includes about 30 known species divided into three plantations, especially those located on the edge of
groups: the Glossina sub-genus which includes forests or gallery forests, and along paths separating
species of the morsitans group; the Nemorhina sub- plantations from the remaining forest used by
genus (palpalis group) and the Austenina sub-genus humans and some wild animals, especially the
(fusca group). A software has been developed for the bushbuck, which are food sources for Glossina.
identification of glossinids by species and sub-
species and the determination of their epidemiolog- The tsetse fly is only active for a short time when
ical importance (Brunhes, 1994). Several factors looking for blood-meal (35 minutes on average per
related to the vectors determine the transmission of day). It spends most of the time resting to digest or
trypanosomes to humans: vector biology and ecolo- gestate. The amount of activity of each species
gy, vectorial capacity, man-fly contacts, longevity, determines its chances of encountering a host on
dispersal, feeding behaviours, etc. which a blood-meal may be obtained and varies
depending on climatic factors (temperature, humid-
Distribution and ecology of Glossina ity, amount of light, wind, rain), olfactory and visu-
A large variety of traps have been tested for tsetse al stimuli (smelling and seeing a potential feeding
sampling and control, a dozen of which were host), and on intrinsic factors (physiological age,
reviewed by Leak (1999). The favourite technique nutritional status, gravidity).
currently used to study Glossina habitats, densities
and ecodistribution is the biconical trap developed Vectorial capacity, competence and
by Challier and Laveissière (1973). Turner (1980) susceptibility of Glossina
proposed the “marking-release-recapture” tech- The vectorial capacity of Glossina is determined by
nique using a radioactive compound (e.g. Fe59) and its ability to infect itself while feeding on a verte-
scintillometer to study its bioecological characteris- brate host, and to subsequently develop an infection
tics such as Glossina habitats, behaviour and dynam- and transmit the trypanosome to another verte-
ics, population size, densities, dispersal, survival brate host (Challier, 1982). According to these crite-
rates, resting sites. ria, only the palpalis and morsitans groups contain
species and sub-species that are vectors of T. b. gam-
The palpalis group contains two excellent vector biense. It has been demonstrated that not all flies in a
species of T. b. gambiense and of animal trypanoso- given area have the same capacity to transmit the
miases in West and Central Africa: G. palpalis palpalis parasite. It is therefore important to determine whe-
in forest areas and G. p. gambiensis in savannah ther or not there are local conditions that may
areas. The former inhabit the forest areas and the reduce or increase this capacity. The tsetse fly’s abil-
moist savannah, whereas the latter is found in the ity to infect itself while feeding on a parasitized host
semi-arid savannah. G. tachinoides and G. fuscipes depends on several poorly understood factors. The
fuscipes are related to the palpalis group and are vec- number of trypanosomes ingested by the fly during
tors of sleeping sickness in the savannahs of West its blood-meal could be one such factor. However, it
and Central Africa while G. pallidipes is found in the has been demonstrated that a single trypanosome
can infect G. m. morsitans (Maudlin and Welburn, distinguish between meals of human and animal ori-
1989; Baker, 1991). The concept that a blood-meal gin. Glossinids of the palpalis group are notable for
taken from an individual with low-level para- their eclecticism and opportunism: they feed indis-
sitaemia is less infectious for the vector than one criminately on many species and are therefore very
taken from a host with high-level parasitaemia dangerous to man. In the forest zone of Côte d’Ivoire,
needs to be investigated. Under natural conditions, Gouteux et al (1982) found that at least 75% of the
the infection rate among tsetse flies is quite low. On blood-meals were from suidae around the villages. In
average, less than 1% of tsetse flies are infected with plantations in the same zone, however, Laveissière
T. brucei ssp (Molyneux, 1980b). Teneral flies are et al (1985) found that 46% of the blood-meals were
more susceptible to infection by trypanosomes. from man. This observation was confirmed by recent
results of Sané et al (2000).
As reviewed by Leak (1999), it has been suggested
that lectins present in the haemolymph and midgut A comparison of the results of analyses of blood-
of the fly are responsible for increasing the resist- meals collected in the five main foci in Côte d’Ivoire
ance of the tsetse fly, with age, to infection with showed that the proportion of human blood-meals
T. brucei. Lectins are absent or blocked in unfed ten- varies significantly from one focus to the other,
eral flies, thus permitting infection, while in older although the socio-geographic conditions in these
flies lectin production seems to be stimulated by foci appear to be identical: a human origin corre-
blood-meals, preventing subsequent infections. The sponded to 42% of blood-meals of G. p. palpalis at
trypanosome also has effects on Glossina. Jenni et al Vavoua, 73% at Daniafla, 55% at Zoukougbeu, 91% at
(1980) have shown that the infected insect has a ten- Gagnoa and 27% at Sinfra. Humans, therefore, seem
dency to bite more often and feed more voraciously, to be the favourite host, in differing degrees, of
which can have a substantial impact on its transmis- G. p. palpalis in plantation zones of Côte d’Ivoire. This
sion potential. According to Maudlin et al (1998), the preference for man was greater in the foci with low
risk of death increases with age, but much more transmission rates (Daniafla, Gagnoa) than in the
quickly in infected than non-infected tsetse flies. more active foci (Sinfra, Vavoua and Zoukougbeu).
Infected flies are much more susceptible to insecti- Domestic animals appear to play an important role in
cides than non-infected flies (Nitcheman, 1990). feeding tsetse flies at Vavoua and Zoukougbeu, the
most active foci, whereas, in the low-incidence foci,
Feeding behaviour and trophic preferences the percentage of animal blood-meals is insignificant.
The first concern of the teneral Glossina is to find a At Zoukougbeu, apart from on humans, G. p. palpalis
host on which a blood-meal may be taken. The feeds freely on domestic swine (30% of meals),
young adult needs a blood-meal to complete its whereas at Vavoua it prefers the bushbuck which
maturation. This first meal takes place between 24 provides an equal percentage of its food as man
and 72 hours or even later, and depends on intrinsic (42%). The diversity of the feeding regimen of G.
(diurnal rythm, sex, gravidity, species, etc.) and p. palpalis might explain to some extent the varia-
environmental (climatic conditions, visual, mechan- tions in levels of incidence of human disease. In foci
ical and olfactory stimuli) factors (Colvin and where transmission is more intense, G. p. palpalis
Gibson, 1992). The tsetse’s feeding ground may be feeds on both man and animals and proper case-find-
restricted by the relatively limited dispersion of ing and treatment of infected humans is not sufficient
potential sources of blood-meals. In forest areas, to control the disease. With a diversified regimen
G. p. palpalis confines itself to the outskirts of vil- alternating between man and animals as sources of
lages where swine hide, along paths through plan- blood-meals, tsetse flies are able not only to transmit
tations, near sources of potable water, and around trypanosomes to humans, but also to maintain the
farm camps and other places used by man putative animal reservoir. In contrast, in low trans-
(Laveissière and Hervouët, 1981). mission foci, the tsetse fly depends on man, trans-
mission to and from animals is rare, and case-finding
Most studies on the trophic preferences of Glossina prevents the accumulation of human cases. This West
were conducted in West Africa and based on analyses African paradigm should probably not be systemati-
of blood-meals collected by dissecting the insect. cally extrapolated to high-incidence foci of Central
Methods with different degrees of sensitivity have Africa, where the role of animals is less clear. If one
been reviewed by Leak (1999), including the precip- of its usual mammal hosts is not available, G.
itin test, the agglutination inhibition test, the comple- tachinoides replaces it with reptiles as observed in the
ment fixation test, direct and indirect ELISA assays, gallery forest of the savannah: snakes or lizards
tests based on the latex agglutination technique, and, account for between 54% and 67% of the blood-meals
more recently, one based on electrophoresis of super- of G. tachinoides, while only 8% of its meals are from
oxide dismutase (Diallo et al, 1997) which can only man (Laveissière and Boreham, 1976). The feeding
preferences of this vector also vary seasonally The number of trypanosomes inoculated into the
because of changes in the availability of hosts. In the mammal host during a blood-meal is probably one
hot season, 30%-55% of its meals come from mammal of the factors that determine the probability of trans-
hosts (mainly humans and bushbucks) whereas in mission. It has been estimated that, to infect man,
the cold season, the only animals available are rep- the inoculum must contain between 300 and 500 try-
tiles, and these provide over 50% of its meals panosomes (Challier, 1982). Transmission is also
(Laveissière and Boreham, 1976). influenced by factors intrinsic to the vector (physio-
logical age, nutritional status, gravidity, etc.) and by
Transmission cycles of T. b. gambiense climatic and other environmental conditions. The
In the forest zone of Côte d’Ivoire, man-fly contacts low infection rate naturally observed among tsetse
occur in almost all biotopes but especially in planta- flies limits the devastating epidemic potential of
tions and peridomestic areas where it is easier for African trypanosomiasis, and explains the apparent
the fly to find hosts (Challier and Gouteux, 1980; paradox between the abundance of the G. p. palpalis
Laveissière et al, 1985). Some authors attribute the and G. morsitans vectors, and the relative rarity of
persistence of residual foci of HAT to the existence human disease. There is no direct correlation
of other cycles adjacent or parallel to the common between the densities of G. p. palpalis, the major vec-
man-fly-man cycle (Molyneux, 1980a; WHO, 1998), tor living in close contact with man, and the inci-
i.e. cycles where the parasite travels between dence of sleeping sickness.
domestic and/or wild animals (swine, sheep, goats,
bushbuck, etc.) and man. They identify two such In endemic foci, the nature, frequency and intensity
transmission cycles: a) a domestic cycle where the of man-fly contacts are the major determinants of
Glossina transmits the parasite between human the risk of African trypanosomiasis. In the forest
hosts and domestic animals; b) a sylvatic cycle zone of Côte d’Ivoire, there is virtually no ecological
where the vector transmits the parasite between zone where humans are safe from being bitten by
wild animals, sometimes with humans or domestic G. p. palpalis. Man-fly contacts can occur in all botan-
animals entering this cycle, resulting in sporadic ical zones and are affected not only by the vegetal
cases or even epidemic outbreaks. The domestic environment but also, and especially, by human
cycle hypothesis is supported by similarities hosts. Multidisciplinary studies have shown that
observed between parasites isolated in humans, ani- human activities and behaviours have an important
mals and the vectors (Gibson et al, 1978; Mehlitz impact on the epidemiology of sleeping sickness,
et al, 1982), whereas there is little evidence that wild through an increase in the frequency and intensity
animals are infected with T. b. gambiense. of man fly-contact (Laveissière et al, 1985, 1986a,
1986b; Hervouët and Laveissière, 1987; Méda et al,
Tsetse flies and the epidemiology of African 1993). For example, coffee-growing, which requires
trypanosomiasis the planter to spend more time in the plantation
The transmission of infectious trypanosomes to than if growing cocoa or food crops, is associated
humans depends on many factors: the density of with a high risk of infection due to the increased
Glossina populations, Glossina longevity, the vector’s contact time with the vector. This risk is heightened
susceptibility to infection, Glossina infestation rates by residing in farm camps and by procuring water
and the factors that influence these, and human from natural water sources located near edges of
behaviours and activities in the biotopes of the flies plantations and gallery forests. Many other activi-
that determine the frequency of man-fly contacts. As ties, such as collecting firewood, washing and fish-
described in the previous sections, the vector’s biol- ing, bring human hosts into contact with tsetse flies.
ogy, ecology and feeding behaviour have direct con- The availability of other sources of blood-meals (e.g.
sequences on transmission of the parasite. Thus, pigs) reduces the chances of transmission.
through its biological cycle, the Glossina allows the
maturation, development, multiplication, transmis- The Human Reservoir
sion and dissemination of the parasite. The feeding Humans are the main reservoir of T. b. gambiense.
behaviour of various species of Glossina determines Four factors potentially influence man’s potential in
their epidemiological contribution to the transmis- transmitting T. b. gambiense: the duration of infec-
sion of T. b. gambiense to the humans, and to some tion, the degree of parasitaemia, the number and
extent the animals, that constitute the parasite’s distribution of individuals who are infected, and the
reservoir. The female G. p. palpalis, because of its intensity of contact with the vectors.
more aggressive feeding behaviour and longer life
span, plays an essential role in the transmission of The duration of infection in humans
the parasite. Given that the tsetse fly is a relatively ineffective
vector, the very long duration of infection (from a
few months to several years) during which the take most blood-meals on non-human sources and
human host can maintain normal activities, and transmission to humans is unlikely. On the other
provide blood-meals for the vectors, must be the hand, high human population density results in
key determinant behind the endemic features and modifications of the habitat that reduce the tsetse
epidemic potential of Gambian HAT (Baker, 1974). population. Thus, an intermediate population den-
Fortunately, this long duration of infection offers a sity is optimal for Gambian trypanosomiasis to
golden opportunity for intervention. Control pro- prosper. A recent study in Côte d’Ivoire showed a
grammes focusing on the identification and treat- strong correlation between the epidemiological risk
ment of asymptomatically infected humans, and and settlement density (Laveissière and Méda,
thus on a shortening of the duration of infection, are 1999). Whether or not there are secular or seasonal
extremely effective if an overwhelming majority of variations in the frequency with which humans
the population shows up during case-finding sur- become infected with T. b. gambiense is unknown but
veys and if sensitive diagnostic methods are used. plausible through changes in tsetse densities, man-
fly contact, presence of alternative sources of blood-
The distribution of infection in human popula- meals, etc.
tions
Prevalence of trypanosome-infected individuals in Familial aggregation
human populations varies tremendously according Familial clustering of trypanosomiasis has been rec-
to socio-demographic factors. Trypanosomiasis is ognized since the beginning of the century. Compa-
not found equally in males and females. The appar- red to children of mothers without a past history of
ent preponderance of cases among females would HAT, the risk of a child having had trypanosomiasis
not correspond to substantial differences in inci- was four times higher if the mother had had the dis-
dence or prevalence if reliable denominators were ease, while it was two times higher in brothers and
available (which is rarely so). Females often partici- sisters of a case than in their half-brothers and half-
pate more regularly in case-finding sessions, which sisters. Such clustering could be due to either genet-
can also lead to a higher number of cases (Ason- ic susceptibility or to shared exposure to the vector.
ganyi and Ade, 1994). Variations in incidence and Several arguments reviewed elsewhere (Khonde
prevalence between age and ethnic group have also et al, 1997) suggest that the latter is the most plausi-
been noted. This probably relates to differences in ble explanation. Shared exposure could result from
occupational exposure and other determinants of simultaneous contact with an infective tsetse whose
man-fly contact rather than to genetically deter- blood-meal on a first individual is interrupted and
mined susceptibility (Laveissière et al, 1986a; Her- resumed on a nearby relative, or from members of a
vouët and Laveissière, 1987; Méda et al, 1993). Other same family sharing an ecological microcosm and
risk factors have been described, which are probably being similarly but not simultaneously exposed to
all markers of exposure to infective tsetse flies: lack the vector bites (Gouteux et al, 1989).
of formal education, absence of pigs (an alternative
source of blood-meals for tsetse) in the habitat, etc. The influence of human behaviour
(Méda et al, 1993; Méda et al, 1995). Human behaviour plays an important role in the
epidemiology of Gambian trypanosomiasis. Health
Occupation is also related to exposure and to inci- seeking behaviour might delay the recognition of an
dence. For instance, in Côte d’Ivoire, HAT is more epidemic and enhance transmission of the parasite
frequent in coffee and cocoa plantation workers or if symptomatic individuals wait for months before
people who fetch water than in other inhabitants reaching a health facility where trypanosomes can
(Laveissière et al, 1986a and b; Méda et al, 1993). be detected, maybe because they first attributed the
More than 80% of cases occur in people who not disease to other, supra-natural, causes and sought
only work but also live in small plantation settle- traditional treatment. Participation in case-finding
ments; a case-control study showed that such peo- surveys varies from place to place and over time,
ple were five times more likely to develop try- and is a key determinant in the success or failure of
panosomiasis than their counterparts who resided such programmes.
in villages (Méda et al, 1993). In some foci of the
DRC, a higher prevalence was found in fishermen, Migrations have contributed to trypanosomiasis
while elsewhere, farmers were more likely to get epidemics, as they favour the circulation of try-
infected (Henry et al, 1982; Mentens et al, 1988). panosomes from high-incidence to low-incidence
areas where the population is more susceptible
Human population density influences the risk of (Prothero, 1963). Although this remains controver-
epidemics in gambiense sleeping sickness (Scott, sial, the explosive epidemics seen in Uganda and
1970). If population density is very light, tsetse flies the Congo a century ago have been attributed by
many authors to the large-scale circulation of (Van Hoof, 1947; Schutt and Mehlitz, 1981). These
workers organized by the colonizers to suit their infections generally resulted in low parasitaemia
needs (Leak, 1999). A recent example of the impact that lasted less than a year. The study of naturally
of migrations is the epidemic in NW Uganda, which occurring infections in animals was facilitated by
resulted from the exodus of Ugandan refugees to the development of appropriate laboratory meth-
Sudan and Zaire where they got infected, followed ods: the blood incubation infectivity test (BIIT),
a few years later by the migration to Uganda of isoenzyme electrophoresis, and DNA analysis.
infected Sudanese refugees and the return of Domestic animals were found to be infected with
Ugandans back home (Paquet et al, 1995). parasites enzymatically identical to T. b. gambiense.
Pigs have generated more interest because they are
Immunity a frequent source of blood-meal for tsetse flies, and
It was generally thought that no immunity followed have been found infected with T. b. gambiense in
a first diagnosis of HAT, because of the parasite’s Liberia (a country with little human disease), Côte
antigenic variation and its repertoire of VSG which d’Ivoire, Congo and the DRC (Gibson et al, 1978;
includes hundreds of different antigens. However, Schutt and Mehlitz, 1981; Mehlitz et al, 1982;
observational and experimental studies in animal Noireau et al, 1989; Truc et al, 1991). In Côte d’Ivoire,
models have shown these animals to be more resist- 52 sympatric T. brucei strains were characterised by
ant to homologous trypanosomes when re-chal- isoenzyme electrophoresis: among 12 zymodemes
lenged after adequate treatment of a previous infec- revealed, the most frequent was found both in
tion. This protection resulted from exposure to humans and pigs (Penchenier et al, 1997). In Congo,
metacyclic trypanosomes rather than to blood- sheep were also found to be infected with T. b. gam-
stream forms (Nantulya et al, 1984; Akol and biense, and the prevalence of Trypanozoon infection in
Murray, 1985; Vos et al, 1988). The existence of pro- domestic animals was estimated to be 0.5%
tective immunity in humans was investigated in a (Noireau et al, 1989; Truc et al, 1991). A dog was
very-high incidence community of the DRC where found infected with T. b. gambiense in Liberia
38% of adults had a past history of trypanosomiasis (Zillmann et al, 1984). A more recent study
(Khonde et al, 1995). The results suggest that a first using PCR showed the simultaneous presence
episode of trypanosomiasis confers to adults about of T. b. gambiense in humans and animals (a dog and
85% protection against subsequent reinfection. a pig) from the Lower Congo province of the DRC
(Schares and Mehlitz, 1996). However, the epidemi-
The impact of HIV ological significance of the animal reservoir is
Little is known about the interactions between the unknown. Whether or not animals may become a
human immunodeficiency virus (HIV) and Gam- threat for reintroduction or persistence of the para-
bian trypanosomiasis. Three studies performed in site in foci where near elimination of HAT has been
Central Africa (Louis et al, 1991; Pépin et al, 1992) achieved remains an important research question
and Côte d’Ivoire (Méda et al, 1995) suggest that (Molyneux, 1980a).
HIV infection has so far had little impact on the epi-
demiology of Gambian trypanosomiasis, to some EPIDEMIOLOGY OF T. B. RHODESIENSE
extent because the prevalence of HIV remains rela- TRYPANOSOMIASIS
tively low in rural communities where HAT is HAT caused by T. b. gambiense and T. b. rhodesiense
endemic. No data are available for T. b. rhodesiense differs in its epidemiological features. We will only
areas. Given that HIV infection is getting more stress these diffences. T. b. rhodesiense is a zoonosis
prevalent in rural areas, it is worthwhile undertak- sporadically transmitted to humans when they ven-
ing a well designed nested case control study to fur- ture into bush infested by tsetse fly vectors whose
ther investigate these interactions. It has been blood-meals are normally taken on game animals.
observed that HIV co-infected HAT patients Humans enter this sylvatic cycle as an incidental
respond less well to eflornithine treatment than host. Except during epidemics, human-fly-human
seronegatives (Milord et al, 1992), but this is unlike- or domestic animal-fly-human transmission is
ly to have any impact on transmission. thought to be rare. The epidemics usually involve
G. f. fuscipes, a peridomestic fly, and transmission
The Animal Reservoir occurs around the villages. The disease, character-
The existence of an animal reservoir of T. b. gambi- ized by an acute or subacute malaria-like syndrome
ense has been investigated for a long time with high parasitaemias, progresses over weeks or
(Makumyaviri et al, 1989; Leak, 1999). Earlier stud- months rather than months or years. The vectors
ies showed that many species of domestic animal differ from those of T. b. gambiense. The parasite is
could be experimentally infected with T. b. gambi- transmitted mostly by three species of the morsitans
ense: pigs, dogs, goats, sheep and even chickens group: Glossina morsitans morsitans and G. m. cen-
tralis in East Africa, G. pallidipes in East and The limiting factor is the relatively low sensitivity of
Southern Africa and G. swynnertoni in Kenya and the standard parasitological techniques. A more fre-
Tanzania. G. fucipes fucipes, which belongs to the pal- quent use of finer serological and parasitological
palis group, is a vector of human and animal try- techniques could lead to a rapid and sustainable
panosomiasis in Central and East Africa. reduction of the human reservoir. In order to reduce
man’s exposure to infectious bites, the tsetse flies
The existence of animal reservoirs was established must be destroyed. This must be carried out with
in the 1950s. Cattle are the major reservoir (Hide the active involvement of the community, who can
et al, 1996), and played a major role in an epidemic take on trap laying, spraying, bush clearing, etc.
in south-east Uganda, with 23% of cattle carrying There are a variety of vector control techniques, the
human infective strains. Other domestic (dog, choice of which depends on financial and human
sheep, maybe pig) and various game (warthog, resources, the epidemiological situation, and the
bushbuck, hartebeest, impala, lion, zebra, hyena) programme duration. Within the African context,
animals can harbour the parasite in their blood; vector control must be carried out with cheap, cost-
some of these animals are well adapted to the para- effective and easy-to-use methods. Control integrat-
site and can remain infected for more than two years ed into primary health care and targeted on groups
without overt disease. Conversely, some other at risk has been found feasible, cost-effective and
species (e.g. dog) rapidly succumb from the infec- cheaper, provided village health workers (VHWs)
tion. In contrast to T. b. gambiense trypanosomiasis are motivated and the beneficiary population partic-
which results from peridomestic infections in ipates fully. Community involvement, both in case
Central Africa, rhodesiense trypanosomiasis is, in finding and vector control, through the implication
endemic situations, acquired far from the village, of VHWs, has been successfully tested on a large
where the animal reservoir lives. It is mainly an scale in various epidemiological contexts.
occupational disease of adult men: hunters, fisher-
men, firewood collectors and honey gatherers have Case Detection
been found to be at higher risk. Tourists visiting Case detection has been the cornerstone of HAT con-
game parks are also exposed to the infection. trol. For many years, specialized case-finding mobile
However, in epidemic situations, when transmis- teams have relied essentially on the presence of
sion becomes peridomestic, all groups, including swollen cervical lymphs nodes. Lymph node fluid,
males and females are at similar risk. Indeed, it has when present, is examined for evidence of try-
been observed that an increase in the number of panosomes. In some very active foci, the mobile
cases in children and women is an indication that an teams carry out wet film and giemsa-stained thick
outbreak is developing (Apted, 1970). As it is in gam- smear examinations. Because of the low sensitivity of
biense sleeping sickness, familial aggregation has these methods, better serological and parasitological
been noted (Okia et al, 1994). In contrast to Gambian tools have been developed over the last two decades.
trypanosomiasis, there is a marked seasonality of
disease occurrence, with a higher incidence during Serological methods
the warmer season (Smith et al, 1998). Population Various serological assays were developed to help
movements and political upheavals played an case-finding teams identify a small number of anti-
important role in development of recent epidemic body carriers on whom to concentrate efforts for try-
in south-east Uganda (Mbulamberi, 1989a; Smith panosome detection using parasitological methods.
et al, 1998). Changes in agricultural practices also In the 1970s, the indirect fluorescent antibody test
led to more favourable conditions for the develop- (IFAT) was deemed the most reliable technique for
ment of G. fucipes, and resulted in the recent perido- epidemiological surveillance of T. b. gambiense try-
mestic epidemic, which was brought into control by panosomiasis. However, it required relatively expen-
the surveillance and early diagnosis through sleep- sive equipment and qualified staff, and its imple-
ing sickness orderlies and vector control (Smith mentation was possible only in laboratories. Delays
et al, 1998; Okoth, 1999) in obtaining results were such that some seropositive
suspects could not be located again to undergo the
CONTROL OF HUMAN AFRICAN parasitological confirmation test. From the 1980s
TRYPANOSOMIASIS onwards, the IFAT was supplanted by the card
Sleeping sickness control relies on two principles: agglutination test for gambiense trypanosomiasis
reduction of the parasite reservoir through case (CATT) (Magnus et al, 1978), the advent of which
detection and treatment, and reduction of man- considerably enhanced detection of cases of Gam-
fly contact through vector control. In the case of bian trypanosomiasis. The CATT is a latex aggluti-
T. b. gambiense, reduction of the human reservoir can nation test relying on the detection of antibodies
be achieved through case-finding and treatment. using the variable antigen type (VAT) LiTat 1.3 anti-
gen, which is expressed by several T. b. gambiense some in biological fluids. This confirmation can be
stocks. This is the only serological assay currently obtained through the examination of wet blood or
used by control programmes. It can be performed in giemsa-stained blood smears, or by direct examina-
the field, without electricity and without specialized tion of the lymph node aspirate when a typical lym-
staff, and the results are available within 10 minutes. phadenopathy is palpable (Cattand and de Raadt,
It is cheap, at approximately 0.40 USD per test, and 1991; Miézan et al, 1994). These classical methods
is generally performed on whole blood. Its sensitivi- have a rather low sensitivity in gambiense try-
ty varies between 92% and 100% when assessed in panosomiasis. Demonstrating the presence of try-
patients parasitologically confirmed in hospital or panosomes in blood and cerebrospinal fluid (CSF)
other settings, and its specificity is thought to be 94- has been considerably facilitated by the develop-
97% (Noireau et al, 1988; Miézan et al, 1991). The ment of concentration methods. The micro-haemat-
CATT proved highly specific during testing in non- ocrit centrifugation technique (Woo’s test) is much
endemic areas (Bafort et al, 1986). Some strains of more effective in the rhodesiense disease (and in vet-
T. b. gambiense do not express the LiTat 1.3 antigen, erinary medicine) than in T. b. gambiense HAT. The
but their distribution seems fairly limited (Dukes miniature anion-exchange centrifugation technique
et al, 1992). The positive predictive value of the (mAECT) is deemed at present to be the most sensi-
CATT depends on the prevalence of disease in the tive parasitological method for the detection of
population. This prevalence ranges between 1 and blood parasites (Miézan et al, 1994). Because of the
5% in most endemic foci and the positive predictive cost (about US$2) and its relative complexity, it is
value generally varies between 14% and 40% used selectively to test CATT-positive suspects
(Miézan et al, 1991). The CATT can be performed on among whom the diagnosis could not be confirmed
filter paper, using smaller volumes of reagents, thus by classical methods. Two new parasitological tech-
reducing costs (Miézan et al, 1991). However, the niques have recently been added to the diagnostic
reliability of this micro-CATT is less satisfactory arsenal: the quantitative buffy coat technique (QBC)
under field conditions than in a research setting. The (Bailey and Smith, 1992) and the kit for in vitro iso-
CATT can also be used on diluted serum rather than lation of trypanosomes (KIVI) (Aerts et al, 1992).
whole blood, resulting in higher specificity at the Neither the QBC, which requires relatively expen-
expense of lower sensitivity (WHO, 1998). sive equipment, nor the KIVI, which is a parasite
isolation method rather than a screening test, has
The card indirect agglutination trypanosomiasis test proved to be superior to the mAECT (Truc et al,
(CIATT) is an indirect assay that was newly pro- 1994).
posed for the detection of circulating trypanosome
antigens in patients’ blood (Nantulya, 1997). The In the CSF, the most sensitive technique is that of
parasite antigen detected is an internal, invariant double centrifugation (Cattand et al, 1988; Miézan
molecule that is common to both T. b. gambiense and et al, 1994). A variation of the latter, the single cen-
T. b. rhodesiense. This test can be used to detect cur- trifugation of cerebrospinal fluid in a sealed Pasteur
rent infection in both forms of the disease. It has pipette, has been recently developed (Miézan et al,
been found to be highly sensitive in endemic areas 2000). It makes detection of trypanosomes in the
(Asonganyi et al, 1998). The test was shown to be CSF simpler, quicker and more sensitive, and is spe-
easy to use in field conditions and does not need cially suitable for passive diagnosis in suspects who
chain maintenance for the storage of reagents. A consult in health facilities with symptoms sugges-
recent evaluation in non-endemic areas revealed a tive of sleeping sickness. The combination of all
specificity ranging from 61% to 98% depending on these seroparasitological methods ensures increased
the proximity of the endemic zone (Meda et al, sub- sensitivity, but falls short of perfectly reliable para-
mitted for publication). Specificity is improved by sitological diagnosis in all seropositive persons,
titration of seropositive specimens. Apart from their hence the need to pursue efforts to develop more
diagnostic potential, the CATT and CIATT have also sensitive parasitological assays and more specific
been found to have potential for use in patient fol- serodiagnostic techniques.
low-up to determine chemotherapeutic cure.
Further operational studies aimed at testing the use- Treatment and Drug Resistance
fulness of the CIATT in clinical settings and for field Pentamidine remains the standard treatment for
use by national control programmes should be car- early-stage patients and melarsoprol for late-stage
ried out. cases. The treatment for HAT is selected by first
establishing the stage of infection. The diagnosis of
Parasitological methods late-stage trypanosomiasis is based on at least one of
Confirmation of diagnosis among seropositive indi- the following criteria (WHO, 1998): CSF white cell
viduals depends on demonstrating the trypano- count (WCC) > 5/mm3 or CSF proteins >37 mg/100
ml (as measured by dye-binding protein assay), or bioecological and epidemiological studies. Before
on both criteria, with or without the presence of try- the advent of insecticides, vector control depended
panosomes in CSF. Miézan et al (1998) reported that primarily on elimination of the wooded vegetation
the CSF WCC is, by itself, as sensitive for diagnosis which constitutes the habitat of Glossina. Nowadays,
of central nervous system involvement as is the insecticides are applied to various types of traps and
combination of the above criteria. Therefore, the screens to destroy the vector. A number of improved
WCC was recommended in patients with confirmed biconical, monoconical and pyramidal traps,
infection, especially in poorly equipped facilities. inspired by the Challier-Laveissière biconical trap
Advances have been made in the treatment of HAT. (Challier and Laveissière, 1973), have been tested for
Between 5% and 10% of late-stage patients treated the control of G. p. palpalis, G. morsitans and
with melarsoprol, an arsenical derivative, succumb G. f. quanzensis in West and Central Africa. Lancien
to its undesirable effects. Until recently, melarsoprol designed the monoconical trap (Lancien, 1981),
was the only available treatment for late-stage which was followed by the pyramidal trap
patients. A new drug, eflornithine, has been devel- (Gouteux and Lancien, 1986), and later by the
oped (Pépin and Milord, 1994). Results obtained Vavoua trap (Laveissière and Grébaut, 1990). To
with eflornithine are excellent, but its future avail- enhance trap efficacy, especially against G. morsi-
ability remains doubtful. tans, olfactory attractive baits are attached to them
(carbonic gas, acetone, urine phenols and host skin
Drug resistance has been a relatively uncommon secretions) (Leak, 1999). These act at a greater dis-
phenomenon in Gambian trypanosomiasis, despite tance than purely visual baits.
suramin, pentamidine and melarsoprol having been
used for five decades (Pépin and Milord, 1994), and, Pilot studies conducted in various epidemiological
as a consequence, has not had any impact on the settings have shown that trapping is effective.
epidemiology of the disease. Suramin is little used Efficacy is measured in terms of the apparent densi-
in the treatment of Gambian trypanosomiasis. ty of flies captured per trap per day (ADT), and
Suramin and Pentamidine are given throughout varies according to the type of trap, the species or
Africa to patients with early-stage of T. b. rhodesiense sub-species of Glossina, the environmental and cli-
and T. b. gambiense disease respectively. So far, the matic conditions, etc. (Laveissière, 1988). In the for-
treatment failure rate remains fairly low. The situa- est areas of Côte d’Ivoire, it was demonstrated that
tion is quite different for melarsoprol. There are at the black/blue/black screen is about twice as effi-
least two foci where melarsoprol resistance is more cient as the simple blue one (Laveissière et al, 1987).
frequent than elsewhere; clearly this is an issue that In the West African savannah, the ADT of G. tachi-
will need better investigation and monitoring over noides and G. p. gambiensis populations was reduced
the next few years. One is the Mbanza Kongo focus by 88-92% using the blue screens (Mérot et al, 1984).
of northern Angola, close to the border with Lower- The same screens reduced the ADT of G. tachinoides
Congo: a 40% failure rate was reported 25 years ago by 98% in only 15 days (Laveissière and Couret,
(Ruppol and Burke, 1977) and similar failure rates 1981). In contrast, in the forest areas of Congo, the
have been observed in recent years. Limited epi- screens did not yield satisfactory results. The ADT
demiological data suggest that there has been little was more drastically reduced (99%) after five
spread of resistant strains over time. In the Arua dis- months in the forest areas of Côte d’Ivoire using
trict of northern Uganda, a 27% failure rate has biconical traps (Laveissière and Hervouët, 1981).
recently been reported among new cases treated Later on, in the same areas, about 16 000 blue
with melarsoprol (Legros et al, 1999); a 10-fold screens sited in coffee and cocoa plantations
lower failure rate has been documented in the adja- reduced the ADT of G. p. palpalis by 90% in one
cent focus of Adjumani. In the DRC, where melarso- week, and by 98% at the end of five months
prol failures are not specially frequent, statistically (Laveissière et al, 1986c).
significant differences in failure rates were found. It
has been also observed that HIV co-infected HAT Traps and screens have thus replaced insecticide
patients respond less well to eflornithine treatment spraying. These vector control methods have gener-
than seronegatives (Milord et al, 1992), but this is ated much interest: they are effective, simple, envi-
unlikely to have any impact on transmission as ronmentally friendly and suitable for use by the
eflornithine is little used and relapsing cases of HAT communities themselves. Pilot projects in Burkina
are often not parasitaemic. Faso (Mérot et al, 1984), Côte d’Ivoire (Laveissière
et al, 1994b), Uganda (Lancien, 1991) and Congo
Vector Control (Gouteux and Sinda, 1990) have demonstrated the
The tsetse fly is one of the rare insects for which sev- feasibility and efficiency of traps and screens used
eral control methods have been developed, based on with the participation of rural communities under
the supervision of specialized teams. The number of equipment maintenance, especially during the
traps and/or screens required for a particular loca- farming season, and to lower efficacy due to the
tion depends on the type of vegetation in the tsetse insecticide being washed away by rains and the
habitats, and on the presence or number of edges, screens being concealed by weeds which grow
water sources, paths and encampments which quickly during the rainy season. Two years later, the
determine the frequency and intensity of man-fly impact of the control programme on the incidence of
contacts. The communities are in a position to pro- human disease was nevertheless obvious: no new
vide information on locations where the villagers patient had been detected during the case-finding
are most often bitten. The life span of a trap depends survey carried out in the study area where the over-
on the quality of materials and of maintenance, and all prevalence was initially higher than 1%. Globally,
on the environmental conditions. In experiments this experiment had shown that about a year’s vec-
conducted in West Africa, it was estimated that tor control was needed to substantially reduce trans-
about 10% and 20% of the traps or screens needed to mission. Similar results were recorded in Congo
be renewed each year in the forest and savannah (Gouteux and Sinda, 1990) and Uganda (Lancien,
zones respectively (Laveissière, 1988). It is not pos- 1991). Clearly, efficient case-finding must be con-
sible to give the precise number of traps or screens ducted simultaneously with vector control, other-
to be installed per hectare, since each microcosm wise the persistence of the human reservoir will
will have its own features. The number of traps or lead to a rapid resurgence of disease when vector
screens to be installed does not depend on the den- control is pursued less vigorously. The cost of a
sity of the human population to be covered. In the screen and a trap (Vavoua type) was estimated to be
Vavoua pilot study, traps and screens were installed US$3 and US$6 respectively (Laveissière and Méda,
as follows: one screen every 100 metres, one or two 1992). The cost of one hectare protected was esti-
screens per water-point or encampment, one screen mated at US$1 for the first year, and much less for
at each working place in the plantation, one trap the second year. Costs vary depending on the type
every 300 metres in forest areas and one every 100 of trap or screen used and on whether or not the
metres around villages (Laveissière et al, 1994). equipment is insecticide-impregnated.
Traps are preferable to screens if re-impregnation Genetic control by the release of sterile males is
and surveillance cannot be carried out by the popu- unsuitable for control in epidemic situations; it
lation. Traps need to be re-impregnated once a year, poses a series of technical, financial and logistic
at the end of the rainy season. Screens have to be re- problems which do not facilitate its implementation
impregnated once during the first year of the cam- in endemic countries.
paign, and twice a year later on. Traps should be
installed as far beyond the endemic area as possible, GAPS IN KNOWLEDGE AND
so as to provide an effective barrier. In the gallery RESEARCH NEEDS
forest of savannah areas, open and sunny places fre- Over the last two decades, new and much more
quently visited by people, such as washing and effective tools for HAT control have been developed
bathing places, water collection points, bridges and compared to those that existed some forty years ago
banks of rivers, are the preferable sites. In forest when the disease was almost eliminated. This
areas, the tsetse flies must be intercepted at the progress contrasts with the present epidemiological
interfaces between different ecological patterns, i.e. situation. The disease remains a serious emerging
the edges that are considered as epidemiologically public health problem in Central, East and West
dangerous areas (Laveissière et al, 1986b), such as Africa. WHO, industrial firms, bilateral and multi-
areas around villages, paths separating wooded lateral organizations have decided to mobilize ade-
areas and other types of ecological patterns, espe- quate resources to combat it. Many gaps in knowl-
cially cocoa or coffee plantations, etc. edge relevant to control of the disease need to be
urgently filled. Some of these gaps have been iden-
Vector control with community involvement, as part tified as priority areas by the Working Group on
of a comprehensive sleeping sickness control pro- Operational Research on African Trypanosomiasis
gramme, was organized in the forest areas of (Geneva, 1997) and the International Colloquium on
Vavoua (Côte d’Ivoire) where Laveissière et al Operational research priorities on African try-
(1985) recorded a 90% reduction of ADT in the vil- panosomiasis (Antwerp 1998). Scientists and pro-
lages and farms a week after setting up the traps gramme managers are invited to pay much more
and screens, and over 99% after three months. These attention to the multiple questions related to the
results remained stable for the first six months. development of new tools for control or better use of
Subsequently, ADTs have increased. This phenome- existing ones. Most perspectives in HAT research
non was linked to the gradual abandonment of and control proposed by Kuzoe (1991) still need to
be considered. In the short and intermediate terms, was evaluated. This test has been found to be high-
some of the research priorities include the following ly sensitive and sufficiently specific for both types of
fields. HAT. Its suitability and predictive value in clinical
management need to be assessed in different epi-
Improvement of Epidemiological demiological settings. The test is easy to use in field
Knowledge and Disease Surveillance and conditions and does not need cold chain mainte-
Control nance for storage; therefore, it is worthwhile evalu-
Little is known of the basic epidemiology of HAT ating its suitability for screening in primary health
disease in many endemic countries, especially care programmes. The last meeting of the Task Force
Tanzania, Southern Sudan, Angola, Chad, Central on Operational Research on African Trypano-
African Republic and Guinea. It has been speculat- somiasis elaborated guidelines for designing the fol-
ed that T. rhodesiense sleeping sickness occurs among lowing studies on the CATT and CIATT:
tourists in East Africa; if this is true, it does mean • Assessment of chemotherapeutic cure of sleeping
that maybe the disease is rising to an epidemic level sickness using the CIATT in T. b. gambiense and
among local populations. Improvement in knowl- T. b. rhodesiense HAT.
edge of the host-parasite and vector-parasite rela- • Comparison of the CIATT and CATT in the diag-
tionships is of great interest for the development of nosis of Trypanosoma brucei gambiense sleeping
new tools and products for control, including new sickness in a clinical setting.
drugs for treatment. The zoonotic nature of T. rhode- • Applicability of the CIATT in the diagnosis of
siense was confirmed about forty years ago. The sit- Trypanosoma brucei rhodesiense sleeping sickness in
uation is completely different for T. gambiense; it has a clinical setting.
been shown that domestic animals such as pigs, • Operationality of the CIATT versus CATT in a
dogs, sheep and cattle can, or do, carry parasites field survey of T. b. gambiense sleeping sickness.
whose isoenzyme features match those of parasites
infecting humans. An important question arises as The question of the treatment of CATT-positive but
to the epidemiological importance of these animal aparasitaemic individuals is under study in the DRC
reservoirs in T. b. gambiense trypanosomiasis. At and NE Uganda. The significance of the results of
present, the exact role of these reservoirs is these studies need to be assessed for impact on the
unknown; it is difficult to establish the relative epidemiology of T. b. gambiense infections in humans.
importance of transmission between animals and
humans. Some studies suggest that such transmis- The usefulness of remote sensing as a tool for sup-
sion could play a significant role by maintaining a porting vector control and surveillance needs to be
minimum level of transmission when the level of explored further (Kuzoe, 1991). Is it a reliable tool
endemicity is low. If this transmission is epidemio- for the identification of high risk areas for human
logically significant, what are the factors that influ- trypanosomiasis? Is it an adequate tool for surveil-
ence the infective contacts between animals and lance of the disease? Some questions, specifically
humans? Is there a sylvatic cycle which contributes related to surveillance and treatment and requiring
to the persistence of Gambian trypanosomiasis? investigation, are given in the handbook by Leak
How can this cycle be interrupted? Several methods (1999), including: Is there a level of vector density
have been proposed for blood-meal analysis. What that ensures a low endemic level of trypanosomia-
is the most reliable, easy to use in field conditions, sis? Is trypanosome prevalence, as determined by
and inexpensive, technique for the determination of passive surveillance, a reliable indication that more
blood-meal origin of the vectors? resources are needed for active surveillance? What
should be the interval between visits of mobile
Surveillance and Management of teams in the context of active surveillance?
Sleeping Sickness
The card agglutination test for trypanosomiasis PCR has recently been introduced as a new tool for
(CATT) is a simple, easy to use, sensitive and rela- the identification of trypanosomes; however its use-
tively effective tool for the diagnosis of T. b. gambi- fulness in the diagnosis and management of the dis-
ense trypanosomiasis, which has been available for ease in humans, and its application in studies on
two decades. However, there is no equivalent test animal reservoirs, remain unsatisfactory. The tech-
for T. b. rhodesiense sleeping sickness; it has been nology needs to be refined, adapted for field use,
found that T. b. gambiense in Central Africa does not and transferred to field workers who need to be
express the antigens used in the CATT. Therefore trained in its application.
there is need to improve the reagents presently
used. More recently, another simple test, the card Geographical information system (GIS) and map-
indirect agglutination trypanosomiasis test (CIATT), ping technology offers a cost-effective and rational
tool for monitoring and control of disease, and is mining the epidemiology of resistance to the drug,
now available for spatial analysis of data collected in are lacking. Nifurtimox is a synthetic nitrofuran
endemic foci. This tool has been successfully applied which was primarily used for treatment of Chagas
in the control of animal trypanosomiasis in savannah disease due to T. cruzi. In HAT, it has only been
areas. In the context of limited resources, GIS is more used in the former Zaire and in Sudan, mostly in
and more perceived as an efficient tool for the iden- patients with infection refractory to melarsoprol, in
tification and mapping of high risk areas where con- combination with other trypanocidal drugs. Fur-
trol efforts should be directed. However, its applica- ther studies using an improved protocol should be
tion in HAT has been very limited so far. Therefore, carried out in T. b. gambiense areas where malarso-
there is a need to provide opportunities for multidis- prol resistance is expanding (the DRC, Angola,
ciplinary research teams, including geographers, to Sudan) in order to identify effective combinations
further use GIS in pilot control programmes. of drugs. Controlled clinical trials on prednisone
versus placebo, to assess reduction of incidence of
Another gap mentioned by some young researchers melarsoprol-associated encephalopathy, might also
is that there is no functional network for scientists be interesting. No data are available on the efficacy
working in the field of trypanosomiasis at regional of new drugs (nifurtimox, steroids) in T. b. rhode-
or sub-regional levels. Such a network would link siense sleeping sickness.
senior and young scientists together, allow efficient
use of human resources and strengthening of the Research on Tsetse Flies and Vector Control
scientific capabilities of young researchers. The biotopes most suitable for tsetse flies are known
for almost every biogeographical area, except man-
Research on Existing and New Drugs, grove swamps. There is a need to collect basic epi-
and Drug Resistance demiological information on types of sites in order to
The ideal drug is one that is safe, effective, afford- determine which areas present the greatest risk to
able, and can be taken orally. Scientists and indus- humans in West and Central Africa. A better under-
trialists are invited to collaborate in the develop- standing of the variability of vectorial capacity of the
ment of new effective compounds for the treatment tsetse fly, including age, population structure, and
of HAT. Pharmacokinetic studies have shown that feeding patterns, might lead to improvement of con-
it is worthwhile evaluating pentamidine adminis- trol; populations that are genetically different might
tered for tree days versus seven days in the first have different vectorial capacities in different epi-
stage of the disease. Studies in laboratory animals, demiological contexts. What are the sociological fac-
using several combinations of existing drugs such tors that affect man-fly contact and are responsible
as pentamidine and eflornithine, suramin sodium for the outbreak of epidemics? A recent meeting held
and melarsoprol, or melarsoprol and nifurtimox, in Harare recommended setting up a quality control
have shown them to be synergistic. However, few system for new insecticides to be used for tsetse con-
data on the effects of drug combinations on either trol. What is the role of population mobility in the
type of African trypanosomiasis are available. In emergence and maintenance of epidemics of sleep-
view of the increasing development of resistance to ing sickness in various epidemiological settings?
melarsoprol monotherapy in some areas, studies on
the available drugs, given separately or in combi- Data collected from catching tsetse flies with traps
nation (eflornithine/melarsoprol, nifurtimox/me- are classically analysed using latin squares. Howe-
larsoprol, etc.), should be undertaken in humans, ver, this method is questionable; there is a need to
based on the latest pharmacokinetic information. identify more adequate statistical techniques for the
Relapses following treatment with melarsoprol, analysis of these particular types of data.
with or without pentamidine, occur on a large scale
in the DRC and Angola. Studies on factors associat- Social and Economic Impact of
ed with treatment failure with melarsoprol should Sleeping Sickness
be designed to identify preventive measures. Trials Little information is available in the literature on the
on alternative regimens of melarsoprol (e.g. 14 days social and economic impact of HAT. Therefore, there
versus 10 days) might improve its efficacy and tol- is a need for social and economic studies to evaluate
erance. Many hypothetical reasons have been given the sustainability and long-term cost-effectiveness of
for treatment failure, such as possible reinfection, integrated control programmes (Kuzoe, 1991). Some
misclassification of the disease, lack of compliance evidence suggests that sleeping sickness can reduce
with treatment, and individual variation in the physical attributes and socio-economic potential at
pharmacokinetics of the drug. Studies on the mag- individual, family and community levels; but physi-
nitude of the phenomenon, and on the risk factors cians, demographers, social scientists and econo-
and biological and environmental conditions deter- mists have so far been unable to quantify and quali-
fy the effects of the disease. Operational research is Bafort JM, Schutte CH, Gathiram V. Specificity of the
also needed to assess the impact of the current struc- Testryp CATT card agglutination test in a non-sleep-
tural health sector reforms on sleeping sickness con- ing-sickness area of Africa. South African Medical
trol programmes. Community involvement in tsetse Journal, 1986, 69(9):541-2.
control and disease surveillance is of crucial impor-
tance. However, human factors which motivate com- Barry JD. The biology of antigenic variation in
munities to participate may vary considerably African trypanosomes. In: Hide G et al, eds.
(according to socio-cultural factors, areas) and need Trypanosomiasis and leishmaniasis. Biology and control.
further investigations. What kind of message and Oxon, CAB International, 1997:89-107.
what is the best way to communicate information to
rural communities? Multidisciplinary research Brunhes J. Logiciel d’identification : Glossine expert.
teams including social scientists, economists and Manuel illustré d’utilisation [Identification software:
basic scientists should be involved in such studies. Glossina expert. Illustrated user’s manual]. Paris:
Centre for International Cooperation in Agrono-
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II VECTOR CONTROL IN ense epidemics were brought under control in the
RELATION TO HUMAN AFRICAN francophone region of West Africa. This francopho-
TRYPANOSOMIASIS ne approach was also adopted during the 1930s to
control epidemics in Nigeria and Ghana with the
I. Maudlin, E. M. Fèvre, P. G. Coleman, newer arsenical tryparsamide. The different colo-
Centre for Tropical Veterinary Medicine, The University nial administrations appear to have became locked
of Edinburgh, Easter Bush Veterinary College, into ‘their’ strategies of either treating the cases or
Midlothian, EH25 9RG, UK dealing with the vector (de Raadt and Jannin, 1999).
It is, nonetheless, clear that both approaches were
CONTROLLING VECTOR-BORNE effective.
DISEASES
Controlling vector-borne diseases represents a spe- In the case of sleeping sickness, as with other vector-
cial challenge to societies in developing countries. borne diseases, the first line of defence in the face of
Apart from the sheer size of the public health prob- an epidemic would appear to be tsetse control –
lems posed by diseases such as malaria and sleeping why then, was this approach not universally adopt-
sickness, vector-borne diseases are especially diffi- ed across colonial Africa? And why were the alter-
cult to control. One of the main reasons for this is native strategies adopted in francophone Africa suc-
that vector-borne diseases are quantitatively differ- cessful? It is instructive to examine the reasoning
ent from directly transmitted diseases in that the behind the decisions governing interventions for
basic reproductive number (R0 - the number of new sleeping sickness control, not only to explain the
infections which arise from a single current infec- events of history but also to inform policy-makers
tion, introduced into a population of susceptibles, faced with the resurgence of sleeping sickness
during the period of its infectiousness) for vector- (World Health Organization 2001).
borne diseases is often an order of magnitude
greater than for directly transmitted diseases. This TWO DISEASES – TWO APPROACHES
means that, generally, vector-borne diseases are TO CONTROL
more difficult to control and may bounce back from In retrospect, we tend to view this division between
control faster than directly transmitted diseases. francophone and anglophone, in approaching what
However, it follows from our theoretical under- was apparently the same problem, as merely anoth-
standing that targeting control activities at the vec- er example of the failure of communications
tor has potentially the greatest impact on disease between two competitive colonial powers. This may,
transmission (see below). however, be simplistic. Assuming that there was
some logic to this dichotomy of approach, it is prof-
CONTROLLING SLEEPING SICKNESS itable to first consider how this position arose on the
It is over a century since it was first recognized that two sides of the continent, as this may influence
tsetse flies were responsible for transmitting try- how we might best proceed to control sleeping sick-
panosomes from wild animals to domestic livestock ness in the future.
(Bruce, 1895). When it subsequently became clear
that tsetse were also responsible for the transmis- The reasons for the differing approaches to disease
sion of human sleeping sickness (Bruce and control have their origins in the biology of two quite
Nabarro, 1903), control of these insects assumed distinct diseases. These differences may be expres-
massive importance for the colonial powers because sed within a theoretical framework of vector-borne
of the threat the disease posed to the economic disease transmission, as discussed below.
development of Africa south of the Sahara.
Subsequently the largely anglophone countries of AN EPIDEMIOLOGICAL FRAMEWORK
East, Central and Southern Africa aimed a large part TO EVALUATE SLEEPING SICKNESS
of their research and control efforts at elimination of CONTROL STRATEGIES
the vector combined with passive case detection. By The basic reproductive number, R0, is central to
contrast in West Africa, and particularly among the understanding how the parasite spreads through,
francophone countries, the more direct approach of and is maintained, in a population. A threshold con-
controlling the disease in humans became estab- dition of R0>1 must be achieved if a parasitic
lished practice. This followed from the success of species “is capable of invading, and establishing
Jamot, who pioneered large-scale case detection and itself within, a host population” (Anderson and
treatment with the arsenical drug Atoxyl (de Raadt May 1992). Ideally, disease control will force R0<1,
and Jannin 1999). By combining large-scale arsenical and so eventually lead to the eradication of the par-
chemotherapy with institutional interventions such asite from the population. Determination of this
as regulating the movement of people, T. b. gambi- threshold condition for transmission is, therefore,
important for the design and implementation of Two well recognized differences between sleeping
control strategies. (If the threshold condition cannot sickness caused by T. b. gambiense and T. b. rhodesiense
be met, then control activities should aim to main- are the duration of infection and the role of the animal
tain R0 at as low a value as possible.) reservoir. T. b. rhodesiense infections are characteristi-
cally acute, with death often occurring within a few
The study of vector-borne disease epidemiology months of infection (Odiit et al, 1997; Apted, 1970;
and the design of control programmes has benefited Wellde et al, 1989). The role of a non-human animal
from the development of a basic mathematical theo- reservoir in T. b. rhodesiense transmission has long
ry by Macdonald (based on Ronald Ross’ earlier been recognized (Heisch et al, 1958; Onyango et al,
work), which captures the essentials of vector trans- 1966). Indeed, movement of cattle from the sleeping
mission (Macdonald, 1957). From this theory, the sickness endemic areas of south-east Uganda has been
reproductive number of a vector borne infection, R0, implicated in the origins of a T. rhodesiense outbreak in
is defined as: an area previously free from the disease (Fèvre et al,
2001). By contrast, T. b. gambiense infections tend to be
chronic, with the time between onset of symptoms,
development of late-stage disease and subsequent
where m is the ratio of vectors to humans; a is the death often occurring over several years (Apted,
feeding rate of vectors, such that the average time 1970). Also, humans are generally considered to be the
between feeds is 1/a; c is the probability that a sus- main host for T. b. gambiense infections with the animal
ceptible vector acquires infection after feeding on an reservoir less important than in T. b. rhodesiense.
infectious human; b is the probability that a suscep- However, the role of the animal reservoir in gambiense
tible human acquires infection after being bitten by infections is still unresolved. Rogers (1988) argued
an infected vector; r is the rate at which humans that domestic animals may be essential in the mainte-
recover from infection, with the average duration of nance of T. b. gambiense as R0 in humans alone may
infection being 1/r; T is the time taken for the para- fall below unity, leading to suggestions that the exis-
site to mature in the vector; and u is the vector mor- tence of a non-human animal reservoir in T. b. gambi-
tality, such that 1/u is the average vector life ense infections may have contributed to the failure of
expectancy. A fuller explanation of how this expres- human population surveillance and treatment cam-
sion is derived, both mathematically and intuitively, paigns to eradicate sleeping sickness in certain set-
is given by Anderson and May (1992). tings (Morris, 1946; Rogers, 1988). However, it has
also been argued that the existence of an animal pop-
The epidemiological impact of a particular control ulation on which tsetse flies preferentially feed may
intervention may be investigated by assigning its result in humans receiving infectious bites at a lower
effects to one or more of the parameters in the rate than if flies only feed on humans (Goutex,
expression for R0. For example, vector control will Laveissièrre and Breham, 1982).
kill vectors, and so increase u and decrease m; while
active case detection and treatment would increase r. These differences in the natural history of the dis-
Thus the expression for R0 provides insights into eases may be incorporated into the expression for
vector-borne disease epidemiology and a frame- R0, and their impact on the relative effectiveness
work in which to evaluate the relative impact of dif- (expressed in terms of reducing R0) of strategies to
ferent interventions. An important qualitative con- control the two types of sleeping sickness demon-
clusion from the simple expression for R0 is that strated (Welburn et al, 2001). It was shown that
killing adult vectors is more effective than the early detection and treatment of cases profoundly reduces
treatment of cases. The time a case is infected enters the transmission of T. b. gambiense but has relatively
into the expression for R0 linearly via the parameter little impact on the transmission of T. b. rhodesiense.
r. In contrast, adult vector mortality enters in a non- This is because the duration of T. b. rhodesiense is rel-
linear fashion via the term exp(-uT). atively short, and the animal reservoir is a relatively
important component of R0.
This general model has subsequently been devel-
oped to specifically model the African trypanosomi- In the light of these theoretical and biological con-
ases, in both animals and humans, by Rogers (1988). siderations, the different approaches adopted by the
The model provides a theoretical basis to investigate colonial authorities of the time are seen to be quite
how differences in the natural history of T. b. gambi- logical.
ense and T. b. rhodesiense will effect the epidemiology
of sleeping sickness caused by the two pathogens, CONTROL IN THE 21ST CENTURY
and how these differences will impact on the relative While, theoretically, vector control will proportion-
effectiveness of the same control interventions. ally have the greatest impact on disease transmis-
sion, it may not always be the approach of choice in ered, as in reality these resources are always limited.
the field when other variables are taken into Indeed, in countries affected by sleeping sickness,
account: the many resources necessary for implementation of
• Treatment of infected people has to be carried out certain control strategies are often non-existent or
for humanitarian reasons, and funding may usu- severely constrained. Control policies should be
ally being found for such emergency interven- based on the optimal utilization of the available
tions. resources. To help determine this optimal utiliza-
• In the war zones of Africa, sustaining a tsetse con- tion, cost-effectiveness analysis is an important tool
trol operation is very difficult and logistically (Murray et al, 2000; Shaw et al, 2001). Such analysis
more demanding than case finding. Tsetse control must be based on a sound understanding of the nat-
operations in these circumstances may be merely ural history of the disease, to best predict the likely
token, having little impact when carried out by epidemiological effects of the same resources invest-
poorly equipped and under-trained personnel. ed into alternative interventions.
Given limited resources and societal instability
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III A BASIS FOR FINANCIAL DE- have only been studied sporadically. Thus, whilst
CISION-MAKING ON STRATEGIES most people working on the disease have a very
FOR THE CONTROL OF HUMAN clear idea of what the most cost-effective strategies
AFRICAN TRYPANOSOMIASIS are in their locality, given their resources and price
structure, little has been done to bring together the
APM Shaw,1 P Cattand,2 PG Coleman,3 M John4 information gained in order to help decision-makers
1
AP Consultants, Upper Cottage, Abbotts Ann, design strategies and prioritize in new areas or
Hampshire, SP11 7BA, UK. under new circumstances.
2
Association against Trypanosomiasis in Africa, 1 rue de
l’Hôtel Dieu, F-74200 Thonon, France. This paper is the first step in an ongoing exercise to:
3
CTVM, University of Edinburgh, Easter Bush, Roslin, • identify the main factors influencing the cost-
Midlothian, EH25 9RG, UK. effectiveness of the different approaches to con-
4
Le Kerrio, 56230, Berric, France. trolling the disease.
• review the current literature on this subject.
INTRODUCTION • identify potential research areas and information
This discussion paper seeks to identify the main needs.
issues involved in designing cost-effective strategies
for the control of human African trypanosomiasis For 1999, the burden of HAT was estimated at 66 000
(HAT) and to highlight areas where more informa- deaths and 2 million DALYs lost (World Health
tion needs to be compiled or new research initiated. Organization, 2000). Although only 45 000 new
cases were reported (World Health Organization,
Some form of control of the disease has been ongo- 2001), the likely number of people affected is proba-
ing in most parts of Africa since the beginning of the bly ten times greater, thus approaching half a mil-
last century. Thus many schemes have been funded lion. Of the 60 million people thought to be ‘at risk’,
and budgeted for. However, the economics of the only 3–4 million are covered by some form of dis-
different approaches and their cost-effectiveness ease surveillance.
Figure 1. Control options
Control of Disease Reservoirs Point of Control of the vector

contact
Human Point of Animal

contact
TREATMENT TSETSE
CONTROL
of patients found
- 1st/2nd stage Traps, targets, screens,
rhodesiense / gambiense aerial spraying,
insecticide treatment
of cattle
TREATMENT
OF LIVESTOCK
DETECTION cattle and, in the past,

some wildlife control
finding patients:
passive/fixed-post
surveillance,
active surveillance
The control of HAT is based on combinations of the years in the case of gambiense sleeping sickness, and
four different approaches illustrated in Figure 1. It have made several attempts to have their symptoms
involves: treated and obtain a correct diagnosis. Usually this
• treating those human patients diagnosed with the will have involved several trips to their rural health
disease. centre, possibly also to a nearby hospital or treat-
• trying to improve diagnosis, by some level of sur- ment centre, a visit to a local healer, and being treat-
veillance, to find patients and ensure that they are ed for malaria and other diseases before being diag-
treated earlier, hopefully while still in the first nosed as having sleeping sickness. Both during trips
stage of the disease. to the health centre and while the patient is hospi-
• proceeding to more active forms of case-detec- talized, it will usually be necessary for a relative to
tion, aimed not only at finding and diagnosing accompany and look after the patient. As the disease
patients but also at reducing the size of the human progresses, the patient in search of a diagnosis will
reservoir of the disease. This applies to the gambi- have become more and more of a burden on his fam-
ense form of the disease. ily, requiring care and being unable to undertake
• trying to reduce the chance of people picking up normal activities.
the infection from domestic animals or wildlife, as
in Uganda, where cattle are routinely treated A very rough estimate of the possible cost to
around new outbreaks to control the rhodesiense patients trying to obtain a diagnosis was made by
form of the disease (personal communication, I Landell Mills (2000) for the rhodesiense situation in
Maudlin). Uganda. This came to US$25 per patient, including
• controlling tsetse fly populations so as to reduce estimates for the cost of transport to rural health
transmission of both forms of the disease. centres and hospitals, treatments for malaria,
painkillers, and the time taken by relatives to
Finally, as indicated in Fig. 1 by the two boxes accompany and care for the person. For those rhode-
marked ‘point of contact’, control of HAT involves: siense patients who are never correctly diagnosed
• avoiding areas likely to lead to infection - a strat- but who do receive some treatments, this cost would
egy that people have used since time immemori- rise to a minimum of US$50, including consultations
al. This strategy applies, above all, to avoiding with a local healer, further trips to treatment centres,
contact with the tsetse fly and is an approach that possibly a short stay in hospital, and care by rela-
has mainly been employed by livestock keepers to tives.
prevent their cattle becoming infected and either
dying or becoming less productive. As people For those correctly diagnosed, the costs of treat-
become aware of the dangers of working in cer- ment, as estimated by WHO in 1998, are given in
tain tsetse-infested thickets, they avoid them. In Table 1. These figures are based on the then current
the past it was thought that people could avoid drug costs, treatment regimes in use, and estimates
contracting the rhodesiense form of the disease of the cost of hospitalization.
simply by avoiding areas containing wildlife. In
the early stages of an epidemic of sleeping sick- Table 1 Costs of treatment
ness, the disease tends to be found among people Item First-stage disease Second-stage disease
whose occupations put them at risk by bringing Pentamidine Suramin Melarsoprol Eflornithine
them into contact with infected flies, particularly Estimate of total cost 107 114 253 675
at certain times of the day (e.g. when collecting Cost excluding
drug cost 87 79 190 367
water, washing clothes, entering game reserves -
Drug cost as %
as in the case of beekeepers, hunters, park of total cost 19 31 25 46
rangers). Thus, avoidance, whilst not specifically Source: WHO, 1998
discussed here, is a control strategy of sorts, However, these costings will need to be re-evaluat-
although in practice it has mainly been used by ed in the light of current plans to make drugs avail-
cattle herders to protect their stock. able free of cost from WHO for a specified number
of years. The costs of transport and administration
FINDING AND TREATING PATIENTS will remain, but, for the relevant drugs, the cost paid
As cited above, WHO estimates are that only about by recipient countries or programmes will consist
10% of sleeping sickness patients are correctly diag- only of transport and drug administration. As the
nosed and receive treatment. This proportion may table indicates, treatment costs could be reduced by
be larger in endemic foci where active surveillance significant percentages, but nevertheless, in eco-
is undertaken, but it can also be a great deal smaller. nomic as against purely financial terms, the use of
Typically, patients who are detected passively have these drugs will still represent a resource cost which
suffered from symptoms for some time, possibly should be taken into account. Furthermore, drug
availability on these terms will alter with time. used innovative techniques of filter paper sampling
Taken together therefore, this implies that the aver- by trained community health workers who either
age cost for patients (a high proportion of whom visit the community or are based at rural health cen-
will already be in the second stage of the disease) tres:
passively diagnosed and correctly treated would be • Fixed-post or passive surveillance. Using this
in the order of US$50–300 each. strategy, patients who present with symptoms
that are difficult to diagnose, or who don’t
In order to analyse this aspect further, there is a need respond to treatments e.g. for malaria, are eventu-
to: ally referred to a treatment centre and tested for a
• analyse case histories of individuals being treated variety of diseases, including trypanosomiasis, so
for the disease in order to determine by what that those with the disease are eventually diag-
process they obtained a diagnosis, how long it nosed. The initial screening test is performed on
took, and how much it cost them and the health wet blood.
services. • Filter paper sampling at rural health centres.
• collate information on currently used treatment Under this strategy, community health workers
regimes and the costs of hospitalization at try- (CHWs) based at rural health centres receive
panosomiasis treatment centres. some training in collecting samples on filter paper
and routinely test all new patients presenting
ECONOMICS OF CONTROLLING themselves at the health centre for whatever rea-
THE HUMAN RESERVOIR: son.
GAMBIENSE FORM OF THE DISEASE • Filter paper sampling by community health
Turning next to active surveillance, which has been workers. CHWs trained in collecting filter paper
the mainstay of programmes to combat the gambi- samples spend 20% of their time collecting sam-
ense form of the disease, calculated costs are given ples and following up seropositive individuals -
in the World Health Organization (WHO) Expert as based on experience in Uganda (John, 1995)
Committee report of 1998 (see series of tables in and Côte d’Ivoire (Laveissière et al, 1995).
Annex 9 of that report). The discussion below is • Monovalent mobile teams. These are the classic
based on these calculations, as originally present- surveillance teams. The card agglutination test for
ed by Shaw et al (1995) and following the same trypanosomiasis (CATT) is performed on whole
approach as those prepared for the previous WHO blood, and all the parasitological tests, except
Expert Committee on HAT (Shaw and Cattand, lumbar punctures, are conducted in the field.
1985). In order to produce a coherent set of cost- Monovalent teams work only on trypanosomia-
ings, it was necessary to use a real situation as a sis.
basis while making some adjustments to produce a • Polyvalent mobile teams. These operate in the
scenario in line with generally accepted norms, same way as monovalent teams except that only a
and the figures and prices used were based main- third of their work consists of screening for try-
ly on work conducted in the Moyo District of panosomiasis.
Uganda (John, 1995). The figures used in 1985 were
based on WHO’s work in the Daloa area of Côte In order to standardize the results, calculations were
d’Ivoire. based on areas with a population of 100 000 people,
containing 10 rural health centres, and where 20
WHO’s calculations of 1998 were used to create a community health workers were operating. The
spreadsheet (Microsoft Excel®) model, so allowing numbers screened by each strategy were assumed to
results to be calculated for any starting prevalence. be as given in Table 2.
The population covered, number of units involved
in surveillance, sampling intensity, sensitivity and Table 2 Numbers screened for trypanosomiasis using
specificity of screening and diagnostic tests, as well different surveillance strategies in an area with a pop-
as all prices and other costs, can also be varied. The ulation of 100 000
results of repeated runs of this model enable the rel-
10 rural 20 community One One
ative cost-effectiveness of different sampling strate- Surveillance Fixed-post health health monovalent polyvalent
strategy surveillance
centres workers mobile team mobile team
gies at different prevalences to be analysed. These Number
are presented and discussed in the series of graphs screened per
annum
300 3000 24 000 36 000 20 000
below.
The cost calculations covered:
The original analyses were based on five alternative • initial screening using the CATT test.
surveillance strategies, including the classic mobile • parasitological confirmation using gland punc-
teams, fixed-post surveillance, and the less widely tures, the capillary tube centrifugation technique
(CTC) test, the miniature anion centrifugation Firstly, the relative performance of the different sur-
technique (m-AECT), and lumbar punctures. veillance strategies was analysed in terms of the cost
• training of staff in specialized techniques needed of finding gambiense patients. The results, in terms of
for diagnosing trypanosomiasis. US$ per trypanosomiasis patient found, are given in
• administrative overheads. Figure 2. Obviously the cost of finding a patient
• depreciation (annual cost) of capital items (vehi- declines very rapidly as the prevalence increases,
cles, laboratory equipment, etc.). since a higher proportion of those screened are
• travel allowances and a share of salaries of all staff infected. This clearly does not reflect any increase in
in proportion to the amount of time they spend on efficiency, simply that more and more of those
trypanosomiasis control. screened are infected, so the costs of the operation
• running costs for vehicles, specialized equipment average out over a larger number of individuals.
and other recurrent costs for each surveillance Fig. 2 is given in three sections so that the differen-
strategy. tials between the costs of each sampling strategy can
be seen more clearly.
Figure 2a. Figure 2b.

Cost of detection per Cost of detection per patient
patient at low prevalences: 0.01% to 1% at medium prevalences: 1% to 5%
US$ PER US$ PER

PATIENT PATIENT
FOUND FOUND
3000 160
140
2500
120
Rural
2000 Health centre
100
Community
health worker
Fixed post
1500 surveillance 80
Monovalent
mobile team
60
Polyvalent
1000 mobile team
40
500
20
0 0
0 0.5 1 0 1 2 3 4 5
PREVALENCE (%) PREVALENCE (%)
At very low prevalences (of 1% or less, see Fig. 2a), person, while surveillance using CHWs costs just
such as those encountered in past years in areas under US$100 per person, and using mobile teams
where surveillance was reasonably regular and the or rural health centre costs between US$120 and
disease was considered to be under control, the US$140.
costs (at a 0.05% prevalence) are well over US$2000
per patient identified using rural health centres or At medium level prevalences (from 1% to 5%, see
mobile teams, and drop to just under US$2000 using Fig. 2b), the costs per patient found continue to fall,
CHWs. Passive or fixed-post detection costs only US and the differentials between strategies narrow fur-
$50 per patient identified since there are virtually no ther, with the cost of passive detection being US$14,
overheads. When the prevalence reaches 1%, the of detection by CHWs being US$22, and of the other
cost of passive detection falls to just over US$20 per three options being about US$30.
At high level prevalences (over 10%, see Fig. 2c), the Given the way in which the figures were calculated,
cost of patients found passively falls to around by independently building up the cost of each strat-
US$10, as it does for all surveillance strategies at a egy using local norms and prices, it is surprising
prevalence of 20%. When the prevalence reaches how similar the costs for finding patients using dif-
50%, the cost of detection becomes very low, around ferent strategies are. Setting aside passive detection,
US$5 for all surveillance strategies, except passive of the four active detection strategies, CHWs using
detection where it remains at about US$10. filter paper is consistently the most cost-effective.
Figure 2c.
Cost of detection
Figure 2c. per patient
Cost of detection per5%
at high prevalences: patient
and over
at high prevalences: 1% to 5%
US$ PER
PATIENT
FOUND 35
30
25
Rural
Health centre
Community
20 health worker
Fixed post
surveillance
15 Monovalent
mobile team
Polyvalent
mobile team
10
0
0 10 20 30 40 50 60 70
PREVALENCE (%)
The purpose-built mobile team concerned only with what proportion of the population could be sam-
trypanosomiasis surveillance is consistently the pled in a year using the different approaches and
most costly. reflecting the assumptions made about the possible
workload that each form of active surveillance can
The almost total convergence of costs at high preva- tackle. The figure shows the situation in the hypo-
lences is due to the fact that, at higher prevalences, thetical area with a human population of 100 000, a
more than half of all costs are diagnostic costs for fixed number (10) of rural health centres, and a fixed
initial screening and parasitological examinations number (20) of CHWs who can assign a significant
(ranging between US$3.50 and US$2.50 per person). proportion of their time to active case detection for
At these prevalences, most individuals are sero-pos- sleeping sickness. In such a situation, only the
itive and have to be re-examined, so the running inputs by the mobile teams can be varied, for exam-
costs and overheads associated with each strategy ple increased as illustrated, from spending half their
are spread over a large number of patients. time in the area to having two teams working there
full time. Based on experience, it was also assumed
In order to further examine the relative effectiveness that mobile teams were able to undertake further
of the different surveillance strategies, Fig. 3 shows examinations on a higher proportion of CATT-posi-
tive patients than was possible under the surveil- Under these conditions, as illustrated in Fig. 3, find-
lance strategies based on CHWs. The filter paper ing and treating a majority of the patients in the
based sampling strategies involve a delay before the population can only be done using one or more
results are known, after which individuals testing monovalent mobile teams. The other active surveil-
positive are recalled and taken to a treatment centre lance strategies could be effective in detecting the
for further testing. In contrast, mobile teams can presence of the disease, or in gradually eroding the
undertake many of the parasitological tests them- size of the human reservoir, provided that the inci-
selves, and transport the suspected patients to a dence is not high.
treatment centre for final confirmation of disease
status.
Fig. 3
Percentage of population sampled and
percentage of all trypanosomiasis patients found according
to surveillance strategy used
% people
80,00
70,00
60,00
50,00
% 40,00
30,00
20,00
10,00
0,00
10 Health Centres
20 CHW's
% people
Fixed Post
0.5 Monovalent Teams
0.5 Polyvalent Teams
% patients
1 Monovalent Team
1 Polyvalent Team
2 Monovalent Teams
2 Polyvalent Teams
Fig. 4
Turning from the costs per individual found with the
Total trypanosomiasis specific costs for
disease to the total investment required for finding
case-finding by surveillance strategy
trypanosomiasis patients, Fig. 4 illustrates these for
the five different surveillance strategies for preva- 60 000
lences of 1% and 10%. As would be expected, the
50 000
costs largely reflect the proportion of population
screened by each strategy (Fig. 3 and Table 2). 40 000
Although the costs would vary from country to
country, and have probably increased somewhat US $ 30 000
since these figures were published in 1998, they give
an idea of the orders of magnitude involved, ranging 20 000
10% Prevalence
from US$50 000 to US $60 000 for a monovalent 1% Prevalence
10 000
mobile team, to a minimal investment for fixed
post/passive detection. 0
s rs e
nt
re ke nc am am
or lla Te Te
Ce W ve
i
i le i le
lth lth ur ob ob
ea a S
tM tM
lH He st
en en
ur
a ty Po al al
ni v
R u
ixe
d
no lyv
10 m
m F M
o Po
Co 1 1
20
In order to better understand how the situation nate the total costs at very low prevalences. But once
evolves at high prevalences, details of the calcula- a prevalence of 1% is reached, all other costs are
tion for intervention by a mobile team are shown in dwarfed by the cost of treating patients. Given the
Fig. 5. Here, the cost of treating the identified uncertainty about how to value drug costs, these
patients has been added to produce the total costs, have been included at their recent commercial cost
which have been broken down into four categories. but separated from other treatment costs (adminis-
The costs of surveillance (logistics, share of salaries, tration of drugs and hospital care). Thus the graphs
etc.), which are given as ‘sampling strategy’, and the can be read so as to exclude the cost of drugs. A
costs of diagnostic tests, which cover both initial notional figure for transport, or for their economic
screening and parasitological confirmation, domi- cost, could be included in subsequent analyses.
Fig. 5a
Breakdown of costs of detection and treatment of patients
at low to medium prevalences
(one mobile team sampling 36 000 people)
400,000
300,000 CostofofDrugs
Cost Drugs
US$ 200,000 Other
Other treatmentcost
treatment cost
Diagnostic Tests
100,000 Diagnostic Tests
Sampling Strategy
0 Sampling Strategy
0.1 0.5 1 2 3 4 5
% Prevalence
Fig. 5b
Breakdown of costs of detection and treatment of patients
at high prevalences
(one mobile team sampling 36 000 people)
4 500 000
4 000 000
3 500 000
3 000 000
2 500 000 CostofofDrugs
Cost Drugs
US$ Othertreatment
treatmentcost
cost
2 000 000 Other
DiagnosticTests
Diagnostic Tests
1 500 000
SamplingStrategy
Sampling Strategy
1 000 000
500 000
0
10 20 30 40 50 60 70
% Prevalence
The costs of controlling the disease in the area pos- A preliminary and very approximate economic
tulated, with one monovalent mobile team screen- analysis of the control of rhodesiense disease in
ing 36% of the population in a year, thus range from south-eastern Uganda was undertaken as part of a
just over US$50 000 where the prevalence is 0.1%, to DFID-commissioned review of its research work
over US$4 million where the prevalence is 70%. (Landell Mills, 2000). Although based on extremely
approximate assumptions, this analysis highlighted
This analysis, based on conditions in Uganda and the potentially very favourable situation where it is
extrapolation of the situation encountered there, possible to control the disease by treating cattle.
thus examines the ways in which the costs of con- Drug treatments cost US$1.75 to US$2 per dose, and
trolling the human reservoir vary with the sampling it is thought that between 5% and 20% of cattle carry
strategy, sampling intensity, and prevalence. The cattle pathogenic trypanosomes (personal commu-
spreadsheet model produced provides a basis for nication, Paul Coleman). Based on work done on the
extending this analysis to cover other surveillance impact of trypanosomiasis on livestock production,
protocols, price sets, test sensitivities, etc. It is likely and current milk and cattle prices in Uganda, it was
that the costings for the extremely high prevalences estimated that treating 1000 cattle around a disease
need revising upwards, since in the model, labour focus could yield a benefit of between US$500 to
and time requirements were not fully adjusted to a US$3000, as compared to a cost of US$750 to
situation where virtually all individuals test positive US$2000. Furthermore, if this expenditure on cattle
to the screening test and need parasitological confir- treatment was successful in reducing the incidence
mation. in humans, the financial benefit in terms of sleeping
sickness treatment cost avoided would probably
To update and validate this analysis in different cir- more than justify the expenditure.
cumstances, what is required is:
• collation and examination of data from budgets In this preliminary analysis of the economics of dis-
and actual expenditures from a range of surveil- ease control over the past decade in south-eastern
lance activities in different countries. Uganda, four categories of costs were considered:
• to find out what protocols are used in different research, vector control, medical surveillance and
field situations and what results are obtained, cattle treatment (Landell Mills, 2000). The benefits
particularly in terms of what sequence of tests are were calculated by considering three likely alterna-
used to obtain parasitological confirmation and tive scenarios for what the disease incidence might
how many patients are detected by each test at have been if there had been no control activities. The
different prevalences. monetary benefits consisted of costs saved for treat-
ing patients, benefits to cattle production, and
ECONOMICS OF CONTROLLING THE patients’ costs incurred while seeking treatment (see
ANIMAL RESERVOIR: RHODESIENSE section Finding and treating patients above). The non-
FORM OF THE DISEASE monetary benefits were calculated in terms of
Turning to the rhodesiense form of the disease, its DALYs averted. The monetary benefits produced
more acute course means that patients usually pres- benefit-cost ratios ranging from 1.08 to 1.98. Because
ent with symptoms shortly after infection. the project produced a net financial gain, the cost
Nevertheless, diagnosis is often slow and inaccurate. per DALY was actually negative, ranging from -
Control of this form of the disease has relied less on US$1.50 to -US$7.50. These results should be treated
active surveillance and more on vector control. with caution, as they depend on very rough
assumptions. However, they do point to the likeli-
Recent research results have added a powerful and hood that this control approach, where it is feasible,
cost-effective tool to the armoury of control methods could be extremely cost-effective.
for this form of the disease. The proof that cattle
have now become the main reservoir of the disease Research into the involvement of cattle as a reser-
(Hide et al, 1996) in south-eastern Uganda has, as its voir of T. b. rhodesiense is ongoing. From the eco-
corollary, shown that treating cattle would control nomic point of view, one key issue is, what propor-
the reservoir, and, if at suitable level, would stop tion of the cattle population will need to be treated,
transmission to humans. Although T. b. rhodesiense is and with what frequency, in order to generate finan-
not pathogenic to cattle, the drugs used to control it cial benefits which cover the costs of controlling the
also kill T. vivax and T. congolense, which are patho- disease in humans? Another important variable is
genic to cattle and are prevalent among the cattle in the ratio of reported to unreported cases of the dis-
the area. Thus, treating cattle generates an econom- ease in humans. Obtaining this information
ic benefit which is independent of the control of the depends on the results of epidemiological studies.
disease in humans. The economic gains to cattle production need to be
more accurately estimated, using data on the preva- The cost of pour-on formulations currently ranges
lence of cattle pathogenic trypanosomes and their around US$1.50 to US$2 for an adult bovine.
impact on livestock productivity. Insecticide treatment of cattle reduces fly density,
but whether or not this would be of use in pre-
VECTOR CONTROL venting HAT depends entirely on the local epi-
At this stage of the work, it has not been possible to demiology of the disease.
review existing information about the costs of vec-
tor control in detail. Recent years have seen few A useful series of comparative costings for tsetse
initiatives that use vector control to deal with control methods for one country, along with details
human trypanosomiasis. Costs are available from of how they were calculated, can be found in Barrett
recent work in Uganda, where pyramidal traps (1997) for the case of Zimbabwe. Current research
were very effective in reducing tsetse density and and information needs include updating costings
disease incidence; these costs (personal communi- for the various strategies, and collating information
cation R. Floto) amounted to ECU 781 000, exclud- on the impact that vector control operations under-
ing technical assistance - at today’s prices, about taken in the past have had on the incidence of sleep-
US $1.1 million. ing sickness. The extent to which community
involvement and inputs, especially of labour, can be
The costs of vector control will tend to be very spe- sustained over long periods of time also needs to be
cific to the situation, varying with terrain (especial- revisited.
ly for target and spraying operations), type of
organization (especially for targets, traps or It is difficult, especially for gambiense disease, to
screens), and type of settlements and rural economy separate the effects of controlling the human reser-
(being affected by the availability of labour for voir from those of vector control, since the two are
maintaining traps and targets, and the presence of usually undertaken at the same time. The cost-
cattle where these are to be treated with insecti- effectiveness of vector control versus case-finding
cides). Estimates of the cost of vector control opera- and treatment was modelled by Shaw (1989). At this
tions, such as those cited below, almost invariably time, it appeared that case-finding and treatment
include only the marginal costs, i.e. the extra costs was the more cost-effective at lower incidences and
involved in field work, but neglect the considerable when dealing exclusively with a human reservoir.
overheads, which can double or triple the costs per The conclusion, then as now, was that epidemiolog-
sq. km. Vector control operations include: ical models and economic models need to be inte-
• aerial spraying, using fixed wing aircraft and the grated in order to be of use in decision-making.
sequential aerosol technique, which typically
involves spraying the area in a cycle of five at DALYS AND THE COST-EFFECTIVENESS
carefully timed intervals. Apart from the sterile OF SLEEPING SICKNESS CONTROL
insect technique, aerial spraying tends to be the Finally, in order to examine the wider economics of
most expensive control method. It has the great controlling sleeping sickness in relation to the con-
advantage of achieving a very rapid reduction in trol of other diseases, an estimate of the cost per
the fly population. Costs are likely to be well DALY averted is needed. So far there have been very
upwards of US$500 per sq. km. few comprehensive attempts to calculate the actual
• traps, screens and/or targets. The cost of these and potential DALYs lost due to either form of
operations is far more variable than for aerial sleeping sickness.
spraying, depending on the number deployed per
sq. km. or per linear km. and on the way in which Recent work in Uganda has produced calculations
they are deployed and serviced. The costs for tar- of DALYs for rhodesiense (Odiit et al, 2000a and
get operations probably range between US$300 2000b). This work will be integrated into more
and US$400 per sq. km., and low-cost trap- or comprehensive economic analyses. The authors
screen-based operations could cost as little as point out that the age distribution of trypanosomi-
US$100 to US$150 per sq. km. asis patients very closely follows that of the active
• treating cattle with insecticides (not to be con- adult population, so that the disease tends to hit
fused with treating cattle with trypanocides, as the most economically productive group of society
discussed above). Here, various ‘pour-on’ formu- hardest, affecting family livelihoods and communi-
lations are used, but again the costs are very diffi- ty prosperity very much. These data confirm obser-
cult to estimate since the pour-on formulations vations made throughout Africa for both forms of
also protect against tick-borne diseases, and the the disease. Odiit et al estimate that, at the time of
number of cattle to be treated per sq. km. is also a diagnosis, patients will have been suffering from
variable as is the number of treatments per year. symptoms of the disease for an average of 61 days
and will then require hospitalization for an average to look at the economics of alternative treatments for
of 34 days. For patients correctly diagnosed and second-stage gambiense patients was based on the
treated, the case fatality rate is 5.3%, but for unre- age-at-death distribution calculated for rhodesiense
ported cases, the outcome is assumed to be patients in Uganda (Politi et al, 1995). These authors
inevitably fatal. Based on the age distribution of also concluded that the standard treatment for sec-
patients, Odiit et al estimate that the number of ond-stage patients represented a very attractive cost
DALYs lost for unreported, and thus untreated, per DALY averted, ranking with the most cost-effec-
patients is just over 20 years (personal communica- tive interventions such as childhood immunization
tion Dr Paul Coleman). This figure was used to and blood-screening for HIV.
underpin the economic analysis discussed in the
section on Economics of controlling the animal reser- Figure 6 takes this discussion a bit further by mod-
voir above. In this case, because controlling the ani- elling how the situation could be analysed if more
mal reservoir reduced the rate of transmission to data were available. The cost of treatment and
humans, and it was assumed that for every report- detection of patients using a mobile team at differ-
ed case there was one unreported case, the full ent prevalences, as shown in Figure 2, is divided by
twenty years applied to half of the cases averted. a conservative estimate of the number of DALYs
This DALY figure thus also means that the cost- averted. The ‘zero’ baseline figure, which gives the
effectiveness of controlling rhodesiense sleeping highest cost, is based on each patient treated, i.e.
sickness compares very favourably with that of each premature death prevented, representing 15
other high priority health control activities, such as DALYs averted. This figure was selected as a con-
malaria (Goodman et al, 2000), the expanded pro- servative estimate, taking into account the long
gramme on immunization (EPI), and human asymptomatic period for gambiense disease, and cal-
immunodeficiency virus (HIV). ibrated to be rather lower than the figure for rhode-
siense. On this estimate, once the prevalence has
For gambiense disease, no published DALY figures reached 1%, the cost per person found and treated
based on detailed field records are available. falls to US$330, and thus the cost per DALY averted
However, ongoing work in Southern Sudan indi- falls below the ‘good value for money’ threshold of
cates that a similar situation probably exists (per- US$25. At higher prevalences, the cost per DALY
sonal communication Dr Anne Moore). An attempt averted stabilizes at between US$10 and US$12.
Figure 6.
US$ per DALY averted at different prevalences
(Surveillance for gambiense Figure 6. using a monovalent
US$ per DALY mobile
averted at team)
different prevalences
(Surveillance for gambiense using a monovalent mobile team)
200
175
150
0
125 0.5
US$
1
100
1.5
75 Multiplier applied
to DALYs averted
50
25
0
0.05 0.5 2 4 10 20 30 40 50 60 70
PREVALENCE
In order to add another dimension to the analysis,
three further lines have been drawn showing what
the effect would be if the number of DALYs averted
per patient found and treated was increased by 0.5,
1 or 1.5. This analysis needs to be developed when
more data have been analysed or collected to show:
• what the actual figure for DALYs averted per
patient treated is likely to be.
• how the prevalence evolves from year to year in
gambiense foci.
The latter brings the discussion back to epidemiolo-
gy, since, at low prevalences, this multiplier can be
seen to be a measure of Ro, the basic reproductive
rate of the disease.
There is thus a clear agenda for analysing existing

data on the progress of epidemics and for collecting
new data in order to add to the knowledge of the
epidemiology of the disease. The low proportion of
all cases actually recorded has meant that knowl-
edge of the year-to-year changes in prevalence is
often patchy or anecdotal. Nevertheless, for foci
which have been the subject of more intensive con-
trol work over a number of years, data do exist. In
this context, epidemiological models (e.g. Rogers,
1988) have an important role to play.
CONCLUSIONS
This paper has tried to cover the main issues
involved in the economics of controlling both gambi-
ense and rhodesiense sleeping sickness. Some aspects,
notably vector control, have only been superficially
treated. Nevertheless, it is hoped that the paper pro-
vides a sufficient basis for discussion on what
research and information gathering is needed to
help plan funding and resource allocation and
gauge what returns to expect from these invest-
ments.
Returning to the information requirements that

were noted at the end of each section, these fall into
one of two categories: economic and epidemiologi-
cal. Some of the issues and needs identified have
been included in the table below, which provides a
simple framework for categorizing the information
required.
Table 3
Framework for identifying information requirements and sources
Epidemiological Economic
• Analyse changes in preva- • Look at finances of past con-

lence over time, relating to trol programmes, budgets,
control strategies (including expenditure, overheads.
vector control) being used.
• Compare prices and costs of
• Collate diagnostic protocols surveillance between coun-
used in different tries and in different epi-
countries/situations. demiological situations.
• Collate information on sensi- • Update costings on vector
Analyse existing data tivity and specificity of dif- control.
ferent tests in the field.
• Cost out different surveil-
lance strategies, and refine
existing spreadsheet model
or develop new ones.
• Cost out different treatment
protocols, determine appro-
priate cost for drugs, esti-
mate hospitalization costs.
• Determine the ratio of • Determine the costs of the

reported to unreported disease to the local economy.
cases.
Initiate new data
• Determine changes in preva-
collection
lence over time and with
respect to control work
undertaken.
From the point of view of those allocating funds of controlling rhodesiense. Any errors remain the
within the health sector, the recent emergence of responsibility of the first author,who would also like
DALY calculations for sleeping sickness has made it to thank Ian Maudlin, Anne Moore, Jean Jannin and
very clear that control of this disease represents an Martin Odiit for their helpful comments and
extremely cost-effective investment. This is linked to encouragement.
two factors. The first is the inevitably fatal outcome of
the disease, which has been discussed above. The sec- References
ond is the focal nature of the disease, which means Barrett JC. Economic Issues in Trypanosomiasis
that although the population at risk is large, the dis- Control. Bulletin 75, Natural Resources Institute,
ease is nevertheless location specific, so that control Chatham Maritime, UK, 1997.
operations can target circumscribed geographical
areas where the disease is known to be present. Goodman C, Coleman P, Mills A. Economic
Analysis of Malaria Control in Sub-Saharan Africa.
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over many years by Pierre Cattand, the joint work
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John, and Paul Coleman’s many suggestions and of Trypanosoma brucei rhodesiense epidemics in East
generous inputs into the analysis of the economics Africa. Parasitology Today, 1996, 12: 50-55.
John M. Utilisation of testryp catt applied to samples of World Health Organization. The world health report.
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Sibanda, 2000.
Odiit M et al. Incorporating the burden of human sleep-

ing sickness in an economic impact assessment of try-
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Odiit M et al. The burden of sleeping sickness in

Southeastern Uganda – geographical variations. Paper
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Politi C et al. Cost-effectiveness analysis of alterna-

tive treatments of African gambiense trypanosomia-
sis in Uganda. Health Economics, 1995, 4:272-287.
Rogers DJ. A general model for the African try-

panosomiases. Parasitology, 1988, 97:193-212.
Shaw APM, Cattand P. The costs of different approach-

es towards the control of human African trypanosomia-
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benefits of alternative disease control strategies: vec-
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ment. Annales de la Société Belge de Médecine Tropicale,
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Shaw APM, John M, Cattand P. The financial implica-

tions of different approaches to the detection and treat-
ment of human African trypanosomiasis. Paper
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Annex 5
DRUG DEVELOPMENT, PRECLINICAL AND
CLINICAL STUDIES AND DRUG RESISTANCE
I DRUG DEVELOPMENT FOR developed in the late 1970s (at the Merrell Dow
AFRICAN TRYPANOSOMIASIS research laboratory) as an antitumour agent, it
proved to be minimally active in that context but was
Cyrus Bacchi effective against laboratory infections of T. b. brucei,
Pace University, Haskins Laboratories, 41 Park Row, and later against T. b. gambiense in clinical trials. It
New York, NY 10038, USA cured late-stage and melarsoprol-resistant CNS infec-
tions, but was not active against T. b. rhodesiense infec-
tion. Its side effects are generally mild and reversible
INTRODUCTION upon withdrawal of the drug (Van Nieuwenhove
There is an urgent and obvious need for novel, effec- 1992; Pepin and Milord 1994; Burri et al, in press). It
tive, non-toxic drugs for treatment of human was approved by the US Food and Drug Admini-
African trypanosomiasis (HAT). This is evident stration (FDA) in November 1990. Three problems
from: arise with its use: expense (about US$500 per
• Dramatic recent increases in incidence of the dis- patient), availability, and dosing (four times a day,
ease due to civil unrest, warfare, and general intravenously, for 14 days). Aventis (current holder of
breakdown of health monitoring and delivery the patent) now has a production agreement with
infrastructure in large areas of Africa. Bristol-Myers Squibb for the synthesis of DFMO to be
• An increase in resistance to standard trypanocidal used in a depilatory cream (Vaniqa).
agents.
• Toxicity of standard agents. In a recent development, WHO and Aventis have
entered into an agreement in which Aventis will
Of the standard agents available, pentamidine is supply 60 000 vials of eflornithine each year for five
used for early-stage Trypanosoma brucei gambiense years. WHO will continue to explore avenues to
infections but is not generally recommended for maintain production after the five-year period.
early-stage T. b. rhodesiense. Pentamidine has been in WHO is conducting clinical studies to determine the
use since 1940, is available from Aventis, and is also efficacy and safety of oral dose eflornithine, which
used for treatment of Pneumocytis carinii in AIDS. would allow its use on a wide scale (FAS Kuzoe,
Resistance is due in part to inability to transport the personal communication).
agent. Suramin, a polysulphonated naphthylurea,
has been in use since 1920 and is presently used for New agent in Phase I clinical trials:
early-stage T. b. rhodesiense. It does not penetrate the DB 075/289
blood-brain barrier and is not used for the central A number of 2,5-bis(4 amidinophenyl)furan caba-
nervous system (CNS) disease. It has the longest mates are reported to have activity agains
half-life of any routinely used trypanocide due to Pneumocysitis carinii (Rahmathhulla, et al 1999). One
binding to serum proteins and there is no consistent of these compounds (DB 075/289) has nearly com-
appearance of resistance. Melarsoprol (Mel B, pleted Phase I testing (toxicity, pharmacokinetics),
Arsobal) is the first choice for treatment of late-stage aimed at development against Pneumocystis carinii
CNS infection. Developed in 1949, it has saved mil- pneumonia (PCP) and African trypanosomiasis. In
lions of lives but suffers from toxicity problems case of a favourable outcome, first field trials (Phase
(encephalopathic syndrome) in about 10% of IIA: proof of principle in patients) are to be con-
patients, which are life-threatening in about 5% of ducted starting in May 2001. Studies in try-
patients. Recently, there has been an increase in the panosome infected monkeys are ongoing and are
number of patients who are refractory to treatment, thus far promising (C. Burri, Pesonal communica-
which can largely be attributed to lack of uptake by tion). DB075/289 does not pass the blood-brain bar-
the parasite (Burri et al, in press). rier and is only active against the first phase of the
disease. However, as an orally dosed agent, it would
In a recent development, Aventis and WHO have be ideal for treatment of actue disease as an alterna-
signed an agreement in which Aventis will supply tive to pentamidine and potential additional means
pentamidine and melarsoprol free of charge for five for disease control in high transmission areas. The
years. There are indications that Bayer will enter development of DB 075/289 is conducted by an
into a similar agreement for suramin (FAS Kuzoe, international consortium lead by Dr Richard
personal communication). Tidwell of the University of North Carolina, and
funded by a 15.1 million grant from the Bill and
APPROVED NEW AGENT Melissa Gates Foundation.
α-difluoromethylornithine (DFMO, Orni-
DL-α
dyl®‚) is an inhibitor of ornithine decarboxylase, the
lead enzyme of polyamine biosynthesis. Initially
LEADS TO OTHER AGENTS of 150 mg/kg for seven days (infusion pumps).
Antagonists of Polyamine Metabolism. In HETA costs about US$2/g to synthesize under labo-
addition to DFMO, a number of other agents target- ratory conditions. It deserves further study and
ing polyamine metabolism have shown promise. should be examined in preclinical trials.
These include MDL 73811 (5’-{[(Z)-4-amino-2-
butenyl]methylamino}-5’-deoxyadenosine), an enzy- Trypanothione Reductase Inhibitors.
me-activated inhibitor of S-adenosylmethionine Trypanosomes produce a unique glutathione ana-
(AdoMet) decarboxylase which supplies decarboxy- logue, N1,N8-bis(glutathionyl)spermidine, for use
lated AdoMet, the source of aminopropyl groups for as a redox defence mechanism in combination with
spermidine and spermine synthesis. This agent was a specific trypanothione reductase, which restores
developed by Marion Merrell Dow in the late 1980s. oxidized trypanothione to the reduced state. The lat-
It cures acute laboratory infections of T. b. brucei and ter is thus a logical drug target. Many inhibitors of
T. b. rhodesiense clinical isolates (Bitonti et al, 1990). It this enzyme have been developed, and some have
is active against late-stage model infections when proven highly effective in vitro against bloodstream
used in combination with low dose DFMO (Bacchi form trypanosomes, yet none have been shown to
et al, 1994). MDL-73811 is not toxic to mice at >10 have significant activity in model infections. Classes
times the curative dose levels used. It is rapidly of compounds include phenothiazines, tricyclic
transported by the parasite through the P2 adeno- compounds, diphenylsulphides, phenylpropyl and
sine site (Bitonti et al, 1990; Goldberg et al, 1998). It is naphthylmethyl β-substituted polyamines. Dif-
given intraperitoneally once a day at 10-25 mg/kg. ficulties with bioavailability, pharmacokinetics, and
Supplies are now limited. However, the absence of metabolism of these compounds need to be
toxicity and the strong activity against T. b. rhode- addressed (Werbovetz, 2000; Keiser et al, 2001).
siense isolates indicate steps should be taken to
obtain further supplies and initiate preclinical trials. Cysteine Protease Inhibitors. Cysteine pro-
teases have been detected in most parasitic proto-
CGP 40215 is a bicyclic analogue of methylglyoxal zoans and are considered important potential tar-
bis(guanylhydrazone) (MGBG), an inhibitor of gets for drug development. Initial studies examined
AdoMet dc. This agent also resembles the diamidines cruzain, the major cysteine protease activity in T.
berenil and pentamidine. It was the most active, both cruzi, while more recently trypanopain-Tb (the
in vitro and in vivo, of a series of derivatives produced major lysosomal cysteine protease of T. b. brucei) has
by Ciba-Geigy in the 1990s, curing laboratory infec- been the subject of extensive study. In this context,
tions of T. b. brucei, T. b. rhodesiense, T. congolense, and treatment with the cysteine protease inhibitor Cbz-
T. b. gambiense - a total of 19 isolates (Brun et al, 1996; Phe-Ala-CHN2 at 250 mg/kg/day for three days
Bacchi et al, 1996). Used singly, CGP 40215 was not reduced parasitemia to undetectable levels in
curative to a CNS model, but was curative when used T. b. brucei infected mice (Scory et al, 1999).
in combination with DFMO. Unfortunately, when However, the parasitemia returned and animals
tested in vervet monkeys with a CNS infection, the relapsed after treatment had ceased. This study, cou-
compound was not curative and pharmacokinetic pled with similar results obtained with T. cruzi, indi-
studies indicated that it did not cross the blood-brain cate these inhibitors have potential, but will need
barrier (BBB) (Keiser et al, 2001). extensive refinement to adjust the availability and
pharmacokinetics (Werbovetz, 2000).
Methionine Recycling. Methylthioadenosine
phosphorylase (MTA-Pase) is the lead enzyme of a Nitro-imidazoles. Megazole is a 5-nitroimida-
salvage pathway which regenerates adenine and zole (2-amino-5-[1-methyl-5-nitro-2-imidazolyl]-
methionine from MTA, the by-product of amino- 1,3,4-thiadiazole) which was first synthesized in
propyl group transfer from decarboxylated AdoMet. 1968, but not studied because of risk of mutagenic-
African trypanosomes have an MTA-Pase with a ity (Keiser et al, 2001). Recent studies, however,
broad substrate specificity. The MTA substrate ana- have demonstrated significant activity of megazole
logue hydroxyethylthioadenosine (HETA) is cleaved against acute murine infections of T. b. brucei and T.
by MTA-Pase and the ribose moiety is metabolized, b. gambiense. Megazole was also effective in elimi-
possibly to a keto acid, which appears to be the nating a murine model CNS infection when used in
active agent (Bacchi et al, 1999). HETA is able to cure combination with suramin. Suramin prolonged
acute T. b. brucei infections and infections caused by megazole’s elimination half-life and altered other
6 of 11 T. b. rhodesiense isolates in mice. HETA was 500 pharmacokinetic parameters including the reten-
times less toxic to mammalian cells in culture than to tion time (doubled) and serum profile (extended
trypanosomes (Sufrin et al, 1996; Bacchi et al, 1997). peak). In most studies, single-dose oral administra-
In mice, it has given no evidence of toxicity at doses tion of megazole was used (Enanga et al, 1998). In
a primate model, cerebrospinal fluid (CSF) levels studies are planned with this series (JRL Pink and
in an animal dosed with a combination of mega- FAS Kuzoe, personal communication).
zole and suramin were found to be 5-10% of plas-
ma levels after a single 100 mg/kg dose - a level VSG Synthesis. African trypanosomes incorpo-
about 10 times that of the MIC100 for T. b. gambi- rate myristic acid into the glycosyl phosphatidyli-
ense. This animal was cured of a CNS infection nositol (GPI), which serves as an anchor for the vari-
(Enanga et al, 2000). Excretion of megazole is pri- ant surface glycoproteins (VSGs) which cover the
marily (80%) in the urine in the primate model, and trypanosome. Myristate is obtained by try-
four metabolites were consistently found in signif- panosomes either by scavenging from the host by
icant amounts (Enanga et al, 1999). Although myristate exchange, or by fatty acid remodelling.
promising, further preclinical studies will be need- The critical nature of VSG in the trypanosome’s exis-
ed to determine the structure of the metabolites tence in the host makes the myristoylation step an
and their overall mutagenic potential before clini- attractive chemotherapeutic target (Werbovetz,
cal studies can begin. 2000). Fatty acid analogues of myristate gave IC50
values <100 nM in vitro against T. b. brucei and
Inhibition of Glycolysis. The predominant T. b. rhodesiense bloodform trypanosomes. These
form of African trypanosomes in the blood is the analogues were inactive in vivo however, because
long slender trypomastigote, which depends on gly- they were rapidly metabolized by the host
colysis for energy production. Recent molecular (Werbovetz et al, 1996). The second pathway is one
modelling studies on glyceraldehyde-3-phosphate in which other fatty acids are remodelled to myris-
dehydrogenase (GAPDH) have detected differences tate and then preferentially incorporated into GPI
in the nicotinamide adenine dinucleotide (NAD)+ and not into other lipids (Morita et al, 2000). This
binding site between the trypanosome and human pathway can be inhibited by the antibiotic thiolacto-
enzyme, and an overall sequence identity of only mycin, which kills trypanosomes in vitro with an
28.5% with the trypanosome enzyme (Suresh et al, IC50 concentration of 150 µM. However, in pharma-
2000). From these observations, adenosine ana- cokinetic studies in mice, thiolactiomycin was found
logues modified in the C2’ ribose and N6 adenine to be rapidly excreted. Peak serum levels were
positions have been synthesized. The most active of obtained 15 minutes after intramuscular injection
these analogues was an N6 derivative, N6-(1- and then rapidly declined until, at 60 minutes, they
napthalenemethyl)-2’-(3-methoxygenzamido) were 1/10th that of peak levels. This compound
adenosine, with an IC50 value of 12 µM against could be given orally with similar rapid elevation
T. b. brucei and inhibition of pyruvate excretion in and decline of serum levels and tissue distribution
in vitro incubations. This compound did not inhibit patterns (Miyakawa et al, 1982). It would be impor-
human GAPDH at 50 µM, but had an IC50 of tant to pursue these lines of study and develop
0.28 µM against L. mexicana GAPDH (Aronov et al, effective myristate analogues which are not metabo-
1999). Although promising, no in vivo studies have lizable by the host and/or thiolactomycin analogues
been reported with these compounds. which have an extended serum half-life.
SIPI 1029. This agent is a triazine derivative Amidine Prodrugs and Cis-platinum
(Trybizine.HCl) which is used against T. evansi in Derivatives. A number of 2,5-bis (4 amidino-
buffaloes in China. SIPI 1029 was effective both phenyl) furan carbamates are reported to have activ-
in vitro and in acute mouse model infections against ity against Pneumocystis carinii, and one is undergo-
T. b. brucei, T. b. gambiense and T. b. rhodesiense ing Phase I clinical trials for this target. As an orally
(Bacchi et al, 1998; Kaminsky and Brun, 1998). In dosed diamidine, it would be of significant interest
combination with low-dose DFMO, it cured a model to examine these agents for treatment of acute T. b.
CNS infection (Bacchi et al, 1998), although used gambiense infections as an alternative to pentami-
singly it was not active in this model, or in a vervet dine (Rahmathhulla et al, 1999).
monkey CNS model (Keiser et al, 2001). Pharma-
cokinetic studies in the latter model revealed only A group of organometallic complexes derived from
low concentrations of the agent in CSF. pentamidine have been evaluated for activity in vivo
against acute T. b. brucei in mouse and sheep model
Over 200 analogues of SIPI 1029 were tested at the infections. Cis-platinum pentamidine-bromide,
Swiss Tropical Institute against African try- –thiocyanate and -seleniocyanate were curative in a
panosomes in vitro. Over 40 were tested in an acute single dose of 1-3 mg/kg. The bromide derivative
mouse model and the most active compounds (5-10) was curative at 1 mg/kg and had a chemotherapeu-
were examined for activity in a CNS model. None tic index of 200 compared to pentamidine, which
showed activity in this model and no additional was curative at 3.5 mg/kg and had a chemothera-
peutic index of 13. Further studies are planned with diamidines and melarsoprol has been the focus of
a CNS model infection and with pentamidine-resist- much study (Hasne and Barrett, 2000; Barrett and
ant isolates (Loiseau et al, 2001). Fairlamb, 1999). Thus P2 carries adenosine and ade-
nine, while melarsopol and pentamidine interfere
Combination Chemotherapy. A number of with their uptake. Trypanosomes resistant to me-
compounds have been identified in the past seven- larsoprol and to berenil have lost P2. Megazole, a
eight years which are excellent trypanocides both 5-nitromidazole active against African trypano-
in vitro and in vivo, with the exception that they do somes (see above) is also transported through P2
not penetrate the BBB and hence do not cure model (Hasne and Barrett, 2000). It now appears that there
CNS infections. In light of the lack of lead com- are other transporters, in addition to P2, for dia-
pounds, and the fact that even current promising midines and melarsoprol (DeKoning, 2001).
compounds have not undergone extensive preclini-
cal toxicology studies, which may very well elimi- Another aspect of trypanosome nucleoside uptake
nate them from consideration, it would be prudent is the demonstration of an AdoMet transporter sep-
to examine drug combinations for CNS activity. arable from P1 and P2 (Goldberg et al, 1997).
Which compounds however, should be the starting AdoMet transporter is not a usual component of
points? In light of the many studies and clinical mammalian cell nucleoside uptake. Its appearance
activity, eflornithine (DFMO) at low dose levels has in trypanosomes should be exploited in terms of
proven active in curing model CNS infections in transport of currently active agents and in develop-
combination with: suramin, melarsoprol, SIPI 1029, ment of novel agents with the ability to transverse a
MDL 73811, HETA, CGP 40215, 9-deazainosine. wide range of transporters. For example, the MTA-
Clearly, the ability of DFMO to act synergistically Pase analogue HETA is taken up by both P2 and the
with so many chemically distinct agents does not AdoMet transporter (Goldberg et al, 1998). Overall,
imply a common biochemical basis of action. Rather, the function and substrate specificity of try-
the literature on artificially induced BBB injury indi- panosome nucleoside/drug transporters should be
cates that polyamine metabolism is significantly out made a priority for study and drug development.
of balance after injury and that DFMO has a role in
correcting this (Croft, 1999). A recent review indi- Acknowledgements
cates that a number of different types of injuries to I thank Michael Barrett, Reto Brun, Christian Burri,
the brain result, after a complex cascade of events, in Felix Kuzoe, and Karl Werbovetz for supplying
the enhanced synthesis, degradation and release of recent references, helpful discussions, and/or other
polyamines in brain tissue. Ultimately, damage may information essential for this document.
be attributable to formation of toxic reaction prod-
ucts as a result of the oxidative degradation of References
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this injury, and in so doing a situation may develop micromolar, biologically active inhibitors of try-
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II DRUG RESISTANCE IN However, reports have been mounting that many
SLEEPING SICKNESS cases in the current epidemics of sleeping sickness
are not responding to melarsoprol, the principal drug
Michael P Barrett, used against stage II disease. This report aims to dis-
Institute of Biomedical and Life Sciences, University of cuss the current status of drug resistance in human
Glasgow, Glasgow G12 8QQ, UK infective trypanosomes, to consider mechanisms by
which parasites become resistant to drugs, and to dis-
INTRODUCTION cuss how resistance is detected and how detection
No vaccines exist against sleeping sickness, and the may be improved. Suggestions as to how to deal with
prospects of prophylactic immunization are poor drug resistant parasites and how to delay the onset
since the parasites change their surface coat period- and spread of resistance are also made.
ically in a process known as antigenic variation.
Drugs remain the principal means of intervention. EPIDEMIOLOGY OF SLEEPING
Five drugs are currently used against Human SICKNESS: IMPLICATIONS FOR THE
African Trypanosomiasis (HAT) (Pépin and Milord, SPREAD OF RESISTANCE
1994), the drug of choice depending on which sub- A full understanding of the epidemiology and pop-
species of T. brucei is involved and on whether the ulation structures of human infectious try-
disease is diagnosed before or after the parasites panosomes, and their relationship with non-human
have become established within the cerebrospinal infectious parasites, is crucial in understanding pos-
fluid (CSF). Problems associated with the current sible routes to the spread of drug resistance.
therapies for sleeping sickness include toxicity, Currently the epidemiology of the disease is not suf-
resistance, and a lack of a guaranteed supply ficiently well understood to make solid conclusions.
(Barrett, 2000) (although it seems likely that licensed However, recent data have started to clarify the sit-
drugs will be available for at least the next five uation in the field, allowing room for some specula-
years). It is unlikely that new formulations will be tion on this topic (MacLeod et al, 2001).
available for at least ten years. This report deals
with the problem of drug resistance. T. brucei parasites are able to undergo genetic
exchange inside the tsetse fly, although this process
Antimicrobial drug resistance is widespread. Many is not obligatory. It has recently been shown that
antibiotics are obsolete and the antimalarial drug many infected tsetse flies in the field carry mixed
chloroquine has been rendered useless in many populations of trypanosome (MacLeod et al, 1999),
parts of the world due to the emergence of resist- thus genetic exchange is possible and studies on the
ance. Drug resistance has also become a serious population genetics of trypanosomes have revealed
problem in the treatment of animal trypanosomias- that genetic exchange does occur sufficiently fre-
es (Geerts and Holmes, 1998). The epidemiology of quently to be an important determinant of genetic
sleeping sickness and recommended regimens for diversity.
the administration of drugs against this disease
appear to be unfavourable for the development of However, it is not yet clear whether the human
resistance. HAT is relatively uncommon when infectious Trypanosoma brucei gambiense does engage
compared to malaria (estimates of 300 thousand in genetic exchange. Moreover, in East Africa it
cases of sleeping sickness compared with 300 mil- appears that T. b. rhodesiense may have a predomi-
lion of malaria). No drugs are currently used pro- nantly clonal structure (MacLeod et al, 2000), sug-
phylactically against sleeping sickness and admin- gesting that it does not frequently engage in genetic
istration of all drugs is parenteral and should occur exchange, although this does not mean that it can-
in a clinical setting and not through self-adminis- not do so. While T. b. gambiense (type I) appears to be
tration. Therefore the conditions which have nor- genetically distinct from other T. brucei sub-species,
mally been implicated in the selection of drug- T. b. brucei and T. b. rhodesiense may simply be con-
resistance, i.e. widespread use, significant under- sidered as host-range variants of local T. b. brucei
dosing associated with self-administration and populations that become genetically isolated. Type
improper prophylactic use, do not appear to be rel- II T. b. gambiense found in West and Central Africa
evant to sleeping sickness. In addition, the zoonot- also appear to closely resemble T. b. brucei (MacLeod
ic nature of the trypanosomes which cause human et al, 2001).
disease could mean that drug selection would be
relieved as parasites are transmitted to untreated Studies on trypanosome population genetics are still
animals, thus diminishing the pressure to maintain in their infancy and it cannot be ruled out that
resistance genes in the parasite population. human infectious parasites can engage in genetic
exchange with non-human infectious organisms.
Moreover, human infectious parasites are not limited • Treatment failure is increasingly being reported
in host-range to humans, and many trypanosomes from epidemic loci (30% failure in northern
(around 20%) isolated from cattle in Uganda and Uganda compared with 5-10% normally).
Western Kenya are human infectious (Hide, 1999). In • T. b. gambiense isolates from treatment failures in
West Africa it appears that T. b. gambiense may also north-west Uganda have recently been shown to
have a host range beyond humans, with pigs repre- have reduced sensitivity to the drug.
senting a particularly important reservoir • While plasma levels of melarsoprol appear to
(Penchenier et al, 1999). A critical corollary of the remain at levels high enough to kill the parasites,
essential zoonotic nature of trypanosomes is that the same may not be true in the CSF and other
resistant strains selected by drug use in animals extravascular compartments.
could be transferred to humans (this will be dis- • The amino-purine transporter P2, encoded by the
cussed later). In addition, human infectious parasites TbAT1 gene, is at least partially responsible for
could also acquire resistance from non-human infec- uptake of melamine-based arsenicals. Loss of the
tious parasites as a result of genetic exchange during transporter can contribute to resistance. One
mixed transmission in tsetse flies. It is important to study has shown that removal of the TbAT1 gene
stress, however, that this has yet to be demonstrated renders parasites only fourfold less sensitive to
in experimental or population genetic studies. melarsen oxide than wild-type trypanosomes.
However, this degree of resistance might be
However, it is clear that even in instances where try- enough to allow survival in the CSF (and relapses
panosomes in humans are not exposed to the classi- appear to depend on CSF involvement).
cal risk factors for the development of drug resist- • Many of the isolates from treatment failures have
ance, the possibility exists that human infectious an altered TbAT1 gene.
parasites are exposed to these classic factors in ani- • It is possible that a combination of reduced drug
mals. Domestic animals do receive large doses of sensitivity (in part due to reduced uptake because
various trypanocides, often administered at the dis- of changes to the P2 transporter) and variability in
cretion of farmers, and frequently given over pro- accumulation of drug in the CSF can explain cur-
longed periods as prophylaxis. While trypanocides rent treatment failures in Africa.
used to treat humans and animals do differ, the
potential to develop cross-resistance between The drug and its use
human and animal trypanocides does exist. For Melarsoprol (Mel B) is a melaminophenyl-based
example, diminazene is used extensively for the organic arsenical which was introduced as an anti-
treatment of cattle. Cross–resistance between dimi- trypanosomiasis reagent in 1949 (Friedheim, 1949).
nazene and the related diamidine pentamidine can It was Paul Ehrlich who promoted the idea that
be induced in trypanosomes (Barrett and Fairlamb, arsenicals could be useful drugs for use against
1999). Cross-resistance between diminazene and sleeping sickness at the turn of the century. His com-
melarsoprol can also be selected with relative ease pound, salvarsan or ‘606’, developed for use against
(reviewed in Barret and Fairlamb, 1999). This is syphilis, is often considered as the prototypic
because all of the drugs can enter trypanosomes via chemotherapeutic reagent. Ernst Friedheim devel-
the P2 amino-purine transporter. Loss of this trans- oped the melamine-based arsenicals in the late
porter can diminish uptake of these drugs into par- 1940s after the dangers of serious side effects associ-
asites, which decreases sensitivity. Although the sit- ated with tryparsamide and high rates of treatment
uation with respect to cross-resistance is not entire- failure decreased confidence in this product.
ly straightforward, as outlined in the sections about Interestingly, melarsoprol has recently been used in
resistance with respect to each of the human drugs, clinical trials against leukaemia (Soignet et al, 1999).
it should be considered when looking at treatment The trials were abandoned when it became clear
of animal trypanosomiases in regions where the that patients were suffering from seizures at more or
human disease is endemic. less the same rate as in sleeping sickness treatment.
However, different regimes might be tried.
THE STATUS OF RESISTANCE TO
CURRENTLY USED DRUGS IN Melarsoprol itself is amphipathic and will diffuse
SLEEPING SICKNESS across cellular membranes. However, the drug is
very rapidly converted to the highly hydrophilic
Melarsoprol melarsen oxide in plasma (96% clearance of melar-
soprol within 1 hr) (Burri et al, 1993). Melarsen
Summary points on melarsoprol resistance oxide levels peak within 15 minutes and have a half-
• Melarsoprol has been widely used in the field life of 3.9 hours. Relatively little melarsoprol, or its
since the 1950s. metabolites, accumulate across the blood-brain bar-
rier, with maximum levels being equal only to Trypanosomes exposed to arsenicals lyse very rap-
around 1-2% of maximum plasma levels. The new idly. A mode of action has yet to be established. Loss
pharmacokinetic information (Burri et al, 1993; of ATP due to inhibition of glycolysis could under-
Keiser et al, 2000) has been very useful in establish- lie lysis caused by the drug, as bloodstream form
ing a new regimen for administration of the drug trypanosomes depend solely upon glycolysis for
which is likely to engender better patient compli- ATP production. However, it seems that the cells
ance (Burri et al, 2000). Detailed information on lyse before ATP supplies are seriously depleted,
pharmacokinetics may also be crucial in permitting leading several workers to question whether glycol-
an understanding of the factors that underlie clinical ysis is a target for arsenical action (Van Schaftingen
drug failure. It seems that levels of drug that reach et al, 1987).
the CSF might be insufficient to kill parasites that
are only a few fold less sensitive to drug than wild- Another suggested target of melarsoprol was try-
type trypanosomes. panothione, a key low molecular weight thiol found
in trypanosomatids but not mammalian cells. Since
Of all the trypanocides, toxic effects are worst with arsenic is known to form stable interactions with
melarsoprol; up to 10% of patients suffer a frequent- thiols, trypanothione was also proposed as the
ly fatal reactive encephalopathy (Pépin and Milord, definitive target for these compounds (Fairlamb
1994). A high proportion of leukaemia patients suf- et al, 1989). Arsenicals, however, interact more tight-
fered neurological seizures when treated with ly with other thiols including lipoic acid and, at the
melarsoprol (Soignet et al, 1999), demonstrating that point of arsenical-induced lysis, only a small frac-
melarsoprol is itself responsible for the reactive tion of trypanothione is conjugated with the drug
encephalopathy associated with drug treatment. (Fairlamb et al, 1992). Thus it seems unlikely that
trypanothione is the in situ target of these drugs.
Exfoliative dermatitis has also been observed.
Hypersensitivity, renal and hepatic dysfunction are Resistance
also known. Myocardial damage, albuminuria and Treatment failure with melarsoprol has been report-
hypertension can also occur. Headache, fever, gen- ed in the field. There has always been a cohort of
eral malaise, urticaria, abdominal pains, vomiting 5–10% of treated patients who relapsed after treat-
and acute diarrhoea are all less severe but common ment, although it was never clear what factors were
side effects (Pépin and Milord, 1994). responsible for this. However, in northern Uganda
relapse rates after melarsoprol treatment of around
Mode of uptake and action 30% have been reported (Legros et al, 1999).
It has been proposed that both the melaminophenyl Anecdotal reports of similar failure rates in northern
arsenicals and diamidine classes of drug enter T. Angola are also in circulation. The incidence of
brucei by the P2 amino-purine transporter (Carter relapse after treatment does not, at present, seem to
and Fairlamb, 1993). Trypanosomes selected for be as serious in southern Sudan, although around
resistance to sodium melarsen had lost the P2 trans- 16% failure rate has also been reported here. The
porter (Carter and Fairlamb, 1993), and a T. equiper- recent build up of data on the pharmacokinetics of
dum line selected for resistance to diminazene, the drug (Burri et al, 1993; Keiser et al, 2000), cou-
which displayed some cross-resistance to arseni- pled with advances in understanding of the molec-
cals, had a P2 transporter with markedly reduced ular and biochemical basis of resistance and an
affinity for substrate (Barrett et al, 1995). These data analysis of field isolates from relapse cases in
suggested that the P2 transporter is involved in Uganda (Matovu et al, in press), has allowed the
uptake of arsenicals (Carter and Fairlamb, 1993) development of a model for the likely causes of
and diamidines including diminazene (Barrett et al, treatment failure in the field.
1995) (the situation for pentamidine is more com-
plicated, as described later). A simplistic model pro- Precise quantification of drug levels in a patient’s
posing that loss of the P2 transporter was necessary plasma and CSF is difficult, although best results
and sufficient to induce resistance to have been obtained using a bioassay for trypanoci-
melaminophenyl arsenicals and diamidines was dal activity in fluids containing melarsoprol or its
developed (Barrett and Fairlamb, 1999; Carter and active metabolites (Burri and Brun, 1992). The logis-
Fairlamb, 1993; Barrett et al, 1995). In vitro, unme- tics of drug delivery in primary health care centres
tabolized melarsoprol is likely to cross the mem- is difficult, so administration of the drug can differ
brane by passive diffusion (Scott et al, 1997) from patient to patient in terms of both total deliv-
although melarsen oxide does not. The possibility ered dose and timing between doses. Moreover, the
of additional modes of uptake should not be metabolism of the drug and also its distribution
excluded. within the body will vary from patient to patient
(i.e. accumulation of the drug in the CSF and other Experiments that led to the identification of a role
extravascular compartments can be highly vari- for the P2 transporter in resistance (Carter and
able). Fairlamb, 1993) involved laboratory derived isolates
selected for high level resistance to sodium
These variables can be crucial to the success of the melarsen (which, like other melaminophenyl arseni-
drug. Absolute quantities vary between patients; cals, is probably rapidly metabolized to melarsen
however, maximum serum levels following four oxide in vivo). However, recent data involving
injections of the drug according to the “classic” pro- genetic manipulation of trypanosomes, removing
tocol were 5 – 6 µg ml-1 (Burri et al, 1993). This falls the TbAT1 gene, revealed that loss of the P2 trans-
to 0.22 µg ml-1 120 hours after the final injection in porter yields only around fourfold reduced sensitiv-
the series. Drug detected in the CSF 24 hours after ity to melarsen oxide (Matovu et al, 2001).
the final injection varied between individuals. The Additional mechanisms must therefore be at play in
maximum concentration was 260 ng ml-1 but this determining high level resistance in laboratory
descended to undetectable quantities in some derived lines. The modest change in sensitivity
patients and average levels in the CSF are estimated relating to impairment of P2 function, however,
to be in the order of 50-fold lower than those in plas- could render parasites resistant to levels of melarsen
ma (Burri et al, 1993). Melarsoprol is rapidly con- oxide accumulating in the CSF and other extravas-
verted to melarsen oxide (and possibly other cular compartments. It appears that the majority of
metabolites) in plasma (Keiser et al, 2000). relapses reported in Uganda do involve small num-
bers of CSF parasites re-invading the bloodstream
There is also some variability in intrinsic sensitivity post-treatment (Matovu et al, 2001). It is also note-
to melarsoprol (or melarsen oxide) among different worthy that genetic removal of TbAT1 had no
isolates of trypanosome. Sensitive parasites appear impact on parasite viability.
to have minimal inhibitory concentration (MIC) val-
ues of around 1-30 ng ml-1 (De Koning, 2001). Many of the drug resistant T. b gambiense isolates
from northern Uganda have mutations in the TbAT1
The quantity of active melamine-based arsenical gene that encodes the P2 transporter (Matovu et al,
reaching extravascular parasites, combined with the
2001). Many of these mutations are common with
MIC of the trypanosomes, will determine whether
mutations found in laboratory derived drug resist-
they are killed by the drug. The MIC values of most
ant isolates (Maser et al, 1999). Although some of the
parasites are only marginally lower than estimated
resistant isolates apparently do not have changes to
concentrations of drug that are achieved in the CSF
the sequence of TbAT1, it cannot be ruled out that
of most patients. Data regarding melarsoprol in
other genetic alterations, beyond the open reading
other extravascular compartments that also harbour
frame, would downregulate expression of that gene.
trypanosomes are absent. The 5-10% of melarsoprol
One example of a T. b gambiense line lacking the
refractory cases could conceivably represent a
TbAT1 gene has been reported (Matovu et al, 2001).
cohort of individuals in whom extravascular accu-
A multi-drug resistant T. b rhodesiense isolate from
mulation of the drug is at the lower end of a nor-
south-east Uganda (Matovu et al, 1997) had an iden-
mally distributed curve and does not reach steriliz-
tical set of mutations as the T. b gambiense series and
ing levels, thus allowing relapse.
an Angolan isolate (Matovu et al, 2001).
Since the achievable CSF dose is close to the MIC of
normal sensitive trypanosomes, a mere halving in Many instances of cross-resistance between mela-
sensitivity of a line of parasite could mean that a mine-based arsenicals and diamidines have been
larger number of individuals fail to accumulate ster- reported (reviewed in Barrett and Fairlamb, 1999).
ilizing levels of arsenical in the CSF. As the degree of The fact that the P2 transporter appears to be
sensitivity declines, the number of refractory cases responsible for the uptake of both of these classes of
will rise in proportion. drug has suggested that transporter alterations
could be the basis of cross-resistance. The fact that
According to this model, a combination of parasite some lines of pentamidine resistant parasite have no
drug-sensitivity and host factors, including the per- reduction in P2 transporter activity was explained
meability of the blood-brain barrier to the drug, will by the discovery of additional pentamidine trans-
determine whether a case is sensitive or refractory porters (De Koning, 2001) and by the fact that other,
to arsenicals. This model is supported by recent data non-transport, related events could underlie pen-
from northern Uganda which indicate that alter- tamidine resistance (Berger et al, 1995). The recent
ations to the TbAT1 gene that encodes the P2 trans- discovery that loss of the P2 transporter through
porter correlate with resistance to melarsoprol gene knockout rather than drug selection leads to a
(Matovu et al, 2001). rather modest decrease in sensitivity to melamine-
based arsenicals suggests that other biochemical Elimination is slow, with estimates of 70-80% of the
changes must be occurring in resistant cells. These drug reported binding to plasma proteins (Sands
other factors contributing to resistance remain to be et al, 1985). The plasma half life is 12 days (but
identified. varies), and the drug is probably metabolized by
humans since only around 11% is eliminated in the
urine. The drug is thought not to cross the blood-
Pentamidine brain barrier, although there are reports suggesting
that small amounts may enter the CSF.
Summary points on pentamidine resistance
• Use of the drug in the field against early-stage Intramuscular injection can cause reactions at the
sleeping sickness has been extensive, although its site of administration. Other reactions include
withdrawal from use as a prophylactic in the mid- hypotension, abdominal pain, hypersalivation, ver-
twentieth century might have slowed the appear- tigo, nausea and chest pain. Nephrotoxicity is com-
ance of resistance. mon; hypoglycaemia is also seen in significant num-
• Anecdotal reports about resistance in the field are bers of patients. Diabetes mellitus may ensue sever-
on the increase. al months after therapy. Rapid intravenous injection
• Laboratory isolates resistant to the drug have should be avoided as it induces a number of effects
been selected. including hypotension, tachycardia, nausea and
• There can be cross-resistance to other drugs, vomiting (Anon, 1998).
including melamine-based arsenicals and other
diamidines, which may to be related to loss of Mode of uptake and action
drug uptake via the P2 transporter. Pentamidine is concentrated to high levels by the
• Resistance need not necessarily involve cross- parasites. It seems that pentamidine can enter T. bru-
resistance. For example, one laboratory derived cei via the same P2 amino-purine transporter which
line selected for melarsen resistance which had accumulates melaminophenyl arsenicals (Carter
lost the P2 transporter was not cross resistant to et al, 1995). Loss of this transporter can render para-
pentamidine. Moreover, another line selected for sites cross-resistant to both diamidines and arseni-
pentamidine resistance was not cross resistant to cals. However, some parasites without P2 remain
arsenicals or other diamidines. sensitive to pentamidine (Carter and Fairlamb,
1993). In T. brucei, three transporters that can carry
• Pentamidine can enter T. brucei via several trans-
pentamidine into the cell have been identified
porters (P2, HATP1, LAPT1) and it accumulates to
(De Koning, 2001). In addition to P2, a high affinity
high intracellular levels. Resistance in some cases
transporter HAPT1 (Km for pentamidine = 36nM)
correlates to reduced uptake, but in others
and a low affinity transporter LAPT1 (Km for pen-
reduced uptake does not appear to be involved.
tamidine = 56 µM) are responsible for the uptake of
• The mode of action is not known.
pentamidine. This could explain why the P2 defi-
cient line, RU15, which is resistant to melamine-ba-
The drug and its use sed arsenicals and some diamidines, was not resist-
Pentamidine is an aromatic diamidine that has been
ant to pentamidine (Carter and Fairlamb, 1993).
in use for treatment of trypanosomiasis for over fifty Moreover, another line selected for pentamidine
years (Sands et al, 1985). It is supplied as a white resistance was not resistant to other diamidines
powder in 200 mg ampoules. It was developed after (Berger et al, 1995); a better understanding of the
the observation that a related compound, synthalin, different routes of uptake for different diamidines is
which induces hypoglycaemia in mammals, had desirable.
prolific anti-trypanosomal activity. Diamidines
actually work directly against the parasites, inde- The mode of action of the drug has not been estab-
pendently of their physiological action on the host. lished. As a polycation, the molecule interacts elec-
Pentamidine is active against early stages of the trostatically with cellular polyanions, including the
gambiense form of sleeping sickness. It is also used unique intercatenated network of circular DNA
against antimony refractory leishmaniasis and molecules which make up the mitochondrial
Pneumocycstis carinii pneumonia (Sands et al, 1985). genome of all kinetoplastid flagellates termed the
kinetoplast. In the case of African trypanosomes, an
Maximum plasma concentrations are reached with- interaction with the kinetoplast may appear to be of
in an hour of intramuscular injection. Extensive limited interest as these organisms do not have a
variation in plasma concentration is found between classical mitochondrial metabolism. However, it is
individuals (0.2-4.4 mg l-1 following a 4 mg kg-1 wrong to consider the mitochondrion as inert since
injection have been reported). Each daily dose leads some kinetoplast genes are known to be expressed
to an increase in residual drug concentration. and the membrane is maintained in an energized

form indicating that several mitochondrial enzyme • Suramin resistance in T. evansi lines appears to be
systems must be active. stable in the field.
• Numerous laboratory isolates (T. brucei and T.
T. brucei can retain viability when the kinetoplast evansi) have been selected for suramin resistance.
has been removed (dyskinetoplastidy). On the other • Most laboratory resistant isolates are not cross-
hand, dyskinetoplastid parasites have been shown resistant to other drugs.
to be somewhat less sensitive than wild-type cells to • Neither a mode of action nor mechanisms of
diminazene (Agbe and Yielding, 1995). Fluorescent resistance are known.
analogues of the diamidines, e.g. DB75 and stil-
bamidine, accumulate rapidly in the kinetoplast and The drug and its use
have also been shown to accumulate in small vesic- Suramin, a colourless polysulphonated symmetrical
ular structures in the cytosol. naphthalene derivative, was first used against
sleeping sickness in 1922 (Voogd et al, 1993). It is
Numerous other potential targets have been pro- useful for the treatment of early-stage infection due
posed but none have been verified. Given that the to either T. b. gambiense or T. b. rhodesiense, when
drug reaches millimolar concentrations within try- there is no central nervous system involvement.
panosomes, it could be that its toxic effect arises Other naphthalene dyes, including trypan red and
from inhibition of multiple cellular targets, although trypan blue, were initially developed for their
the fact that one resistant line (Berger et al, 1995) marked trypanocidal activity. The drug has recently
does accumulate drug to millimolar concentrations been used in clinical trials against hormone-refrac-
without adverse effects might indicate that there is a tory prostate cancer and has also been used in the
specific target that has yet to be identified. chemotherapy of some helminth infections. During
the 1950s, there were reports that treatment failure
Resistance in patients infected with T. b gambiense was relative-
Pentamidine resistance did not emerge during ly high (around 30% [Neujean and Evens, 1958]).
large-scale chemoprophylaxis campaigns in the Consequently the drug lost favour as a treatment for
middle part of the twentieth century in west Africa, gambiense sleeping sickness although the factors
underlying these treatment failures were never
possibly because the drug was withdrawn from
identified with any certainty.
widescale use in the 1950s thus removing selection
pressure. In this regard, it is perhaps of note that one
Poor intestinal absorption, and a local irritation if
laboratory derived line resistant to pentamidine had
given intramuscularly, mean that the drug should be
substantially diminished viability in mammals
administered by slow intravenous injection (Anon,
(Berger et al, 1995).
1998). Dosing at 1 g per week over six weeks main-
tains levels at 150-200 mg l-1. Most of the drug binds
It is not clear, nor easy to ascertain from available lit-
to serum proteins. It does not cross the blood-brain
erature, whether melarsoprol resistant field isolates
barrier to levels capable of killing trypanosomes in
are pentamidine cross-resistant. However, it seems the CSF at doses given during treatment of early-
that both pentamidine and melarsoprol enter try- stage disease. Plasma concentrations decline expo-
panosomes via the P2 transporter and anecdotal evi- nentially with a half-life of up to 60 days. About 80%
dence has indicated that treatment failures with of the dose is eliminated in the urine.
pentamidine are growing more common as they are
with melarsoprol. Possibly the presence of trans- Nausea, vomiting, urticaria and loss of consciousness
porters, in addition to P2, which can accumulate can be immediate side-effects (Anon, 1998). Fever (up
pentamidine means that changes to P2 will not to 40oC within a few hours) is common as are photo-
underlie pentamidine resistance in the field. phobia and lacrimation. Renal damage can occur sev-
Mechanisms for resistance to pentamidine are cur- eral days after treatment. Other adverse reactions
rently not known. include exfoliative dermatitis, stomal ulceration,
agranulocytosis and, rarely, haemolytic anaemia.
Suramin
Mode of uptake and action
Summary points on suramin resistance The drug is a highly charged molecule containing
• Use of the drug in the field against sleeping sick- six negative charges at physiological pH and it
ness is not extensive. binds with high avidity to many serum proteins
• Few reports about resistance in the field have including low density lipoprotein (LDL), for which
been published. trypanosomes have a receptor (Vansterkenburg
• Veterinary use was more widespread than human et al, 1993). Suramin accumulates in trypanosomes
use in the mid to late twentieth century. relatively slowly and may be taken up bound to

LDL by receptor-mediated endocytosis, although been reported. Information from these species (par-
definitive evidence proving this is absent. The same ticularly T. evansi) might be useful in helping to
experiments that showed that uptake might depend ascertain resistance mechanisms in man.
on LDL also showed that suramin inhibits LDL
uptake and that this inhibitory effect could be the The relative scarcity of reports of suramin resistance
cause of suramin’s toxic effect on trypanosomes in sleeping sickness has made it difficult to compile
(these organisms get most of their lipids from the field data on this subject. The large incidence of
breakdown of exogenous LDL). The highly charged reported treatment failures of T. b gambiense in west
nature of the drug enables it to bind to many pro- Africa in the 1950s could have been due to multiple
teins through electrostatic interaction. factors of which parasite resistance is just one
Consequently, when the drug is tested for inhibition (Neujean and Evens, 1958). Many laboratory
of a variety of purified enzymes, it shows activity. derived lines have been selected for resistance to
This has led to many hypotheses regarding its mode suramin over the years (since the 1930s). Several
of action. studies have focused on T. evansi (Mutugi et al,
1995). One laboratory study pointed to the fact that
Treatment of trypanosomes with the drug does lead resistant lines were more difficult to clone and grow
to a reduction in the glycolytic rate. This inspired a than wild-type T. evansi. This prompted suggestions
study into its inhibitory effect against the enzyme that resistance might not be a stable phenotype
glycerol phophate oxidase, which is present in try- (Mutugi et al, 1995). However, T. evansi isolated
panosomes but absent from the mammalian host from the Sudan some twenty years after suramin
(Fairlamb and Bowman, 1977). Suramin inhibited had been withdrawn from use due to the advent of
the enzyme and some textbooks mistakenly report resistance (El Rayah, 1999) was still highly resistant
that this enzyme is the target. The discovery of clus- to this drug, indicating that resistance can be very
ters of positively charged amino-acids within sever- stable.
al of the T. brucei glycolytic enzymes led to the
hypothesis that the drug’s action was dependent No evidence for a reduction in drug uptake associ-
upon this interaction. However, none of these spec- ated with resistance has been reported and mecha-
ulative ideas have been proven and, at this stage, it nisms of resistance are not known. Most laboratory
should be emphasized that the drug’s mode of studies, stretching back 70 years or more, have
action is not known. failed to identify cross-resistance between suramin
and other trypanocides. One study did show that a
The drug is highly active against bloodstream forms line selected for resistance to melarsen oxide (33-
of the parasite in vitro, but around a hundredfold fold resistance) (Fairlamb et al, 1992) had a nearly
less active against procyclic forms of the organisms sixfold decrease in susceptibility to suramin.
(Scott et al, 1996). This indicates that either its However, most other studies have concluded that
uptake, or its direct or indirect targets, are differen- there is no cross-resistance between suramin and the
tially regulated in the different forms of the parasite. melamine based arsenicals or other drugs.
Early suggestions that receptor mediated endocyto-
sis did not occur at all in procyclic forms have
proven to be incorrect as this form of the parasite Eflornithine
does endocytose a number of macromolecules (Liu Summary points on eflornithine resistance
et al, 1999). However, specific receptors may be dif- • Use of the drug in the field against sleeping sick-
ferent. The fact that procyclic form organisms, ness has not been extensive.
unlike bloodstream forms, are not totally dependent • No reports about resistance in the field have been
upon glycolysis has also been used to fuel ideas on published.
modes of action. • T. b. rhodesiense is innately refractory to eflor-
nithine, possibly due to a shorter half life of the
Fang et al (1994) recently re-introduced the old target enzyme ornithine decarboxylase compared
notion that the immune system may play a role in to the susceptible T. b. gambiense.
the action of suramin, since immunosuppressed • Laboratory isolates resistant to the drug have
mice needed higher doses (around threefold) to been derived.
clear T. evansi infections. • Model organisms (e.g. Leishmania, Neurospora)
resistant to the drug have also been derived.
Resistance • In model organisms, resistance has been shown to
Field reports on sleeping sickness resistant to relate to different phenomena e.g.:
suramin are rare. In animal diseases, however, para- - Increase in ornithine decarboxylase levels (gene
sites of the brucei group resistant to this drug have amplification).

- Decreased drug uptake (loss of basic amino acid the membrane (Erwin and Pegg, 1982). It has been
transporter in Neurospora; unknown mechanism reported that uptake of DFMO in T. brucei also
behind reduced uptake in procyclic T. brucei). occurs via passive diffusion across the plasma mem-
• T. brucei resistant to DFMO have not been report- brane (Bitonti et al, 1986; Bellofatto et al, 1987).
ed to be cross-resistant to other drugs. These observations were based on the fact that
• The mode of action of the drug relates to its inhi- uptake appeared to be unsaturable over a wide
bition of ornithine decarboxylase. A functional range of DFMO concentrations and that internal
immune system is required to kill cells in vivo. concentration of drug equilibrated with external
Whether it is simply loss of polyamines or other concentration. Other features reported in these stud-
indirect effects, e.g. increase in decarboxylated ies, e.g. temperature sensitivity of uptake, might be
S-adenosyl methionine and inappropriate methy- interpreted as evidence for transport although the
lation, that is behind cytotoxicity is not clear. authors did not consider this. A separate report did
Uptake has been reported to be via passive diffu- note a saturable process typical of transport-associ-
sion in bloodstream forms and via carrier mediat- ated uptake in procyclic organisms with a Km of
ed uptake in procyclic forms, although further 244 µM (Phillips and Wang, 1987). In the yeast
studies on this question are needed. Neurospora crassa, a basic amino-acid transporter has
been implicated in uptake of DFMO (since this
The drug and its use transporter is lost in strains selected for resistance to
Eflornithine, or D,L-α-difluoromethyl ornithine the drug) (Davis et al, 1994). None of the studies in
(DFMO), is an analogue of ornithine which acts as a trypanosomes have indicated that DFMO shares an
specific suicide inhibitor of the enzyme ornithine uptake mechanism with ornithine, arginine or
decarboxylase (ODC). It was developed as an anti- lysine. The mode of uptake into trypanosomes
cancer reagent, however, it remains at the trial stage remains uncertain.
against neoplastic disease. The drug also has activi-
ty against sleeping sickness caused by T. b gambiense, DFMO has similar affinity for both the mammalian
even in the late CNS-involved stage. It was regis- and trypanosomal ornithine decarboxylases. Its
tered in the USA in 1990 and the UK in 1991 (Pépin specificity against the parasite apparently arises
and Milord, 1994). By 1995 it was registered in seven because T. b. gambiense ODC is degraded within the
African countries. Fourteen daily intravenous injec- cell and replenished at a rate much slower than its
tions of 400 mg per kg of body weight are recom- mammalian counterpart (Phillips et al, 1987). Thus,
mended (Anon, 1998). It has also been licensed for a pulse of DFMO can deprive trypanosomes of ODC
use as a topical application to prevent facial hair and polyamine synthesis for a prolonged period
growth and is now marketed for this purpose compared with mammalian cells, leading to a cessa-
(Hickman et al, 2001). Fifty four per cent of the dose tion of growth.
becomes bioavailable after oral administration
(Anon, 1998). The mean half-life in plasma follow- Inhibition of ornithine decarboxylase has other
ing intravenous injection is 3 hours, with 80% of the results besides a reduction in putrescine and further
drug excreted unchanged in urine after 24 hours. polyamine biosynthesis. For example, it leads to an
Little of the drug binds to serum proteins. increase in cellular levels of S-adenosyl methionine,
Immediately after a 14-day course, the CSF to plas- which might have toxic effects (Byres et al, 1991).
ma ratio is 0.91 in adults and 0.58 in children. Inappropriate methylation of proteins, nucleic
Children retain less of the drug than adults. acids, lipids and other cell components is thus being
implicated.
Few side effects are apparent although anaemia and
other blood cell reductions (leukopenia, thrombocy- Trypanothione is a glutathione-spermidine conju-
topeania) are known. Diarrhoea is a common prob- gate unique to trypanosomatids and it plays a criti-
lem to those on oral eflornithine. Convulsions, fever cal role in maintenance of cellular redox potential
and vomiting have also been reported in low num- (Fairlamb and Cerami, 1992). Trypanothione levels
bers of cases (Anon, 1998). are diminished after DFMO treatment, which might
render parasites more vulnerable to oxidative stress.
Mode of uptake and action A functional immune system is required to kill the
Eflornithine is a specific suicide inhibitor of the growth-arrested trypanosomes (De Gree et al, 1983).
enzyme ornithine decarboxylase (ODC), which is a It has also been reported that T. brucei lacks
key enzyme in the biosynthesis of polyamines polyamine transporters, rendering the parasite aux-
(McCann and Pegg, 1992). Some early studies in otrophic for polyamines (Fairlamb and Le Quesne,
mammalian cells indicated that DFMO uptake was 1997). Conversely, many mammalian cells can scav-
a passive process involving simple diffusion across enge polyamines from plasma using transporters,
allowing them to bypass the lack of endogenous
biosynthesis while T. brucei cannot tolerate this situ- boxylase activity associated with an amplification in
ation. copy number of the gene (probably associated with
amplification of an episome) (Sanchez et al, 1997).
In order to be effective against sleeping sickness, the Elevated putrescine uptake in L. infantum exposed
drug needs to be given in large doses. An addition- to DFMO has also been reported (Balana-Fouce et al,
al drawback is the drug’s lack of activity against 1991).
rhodesiense sleeping sickness (Iten et al, 1995), which
appears to contain an ornithine decarboxylase that Increased ornithine decarboxylase activity (associat-
is relatively quickly turned over (Iten et al, 1997). A ed with a rise in transcript levels but not, this time,
seven-day course appears to be somewhat less effi- gene copy number) has also been reported in arsen-
cacious than the 14-day course; however, the cost- ite resistance, associated with increased trypanoth-
benefit ratio appears to favour the shorter course ione biosynthesis in Leishmania tarentolae (Haimeur
(Pépin et al, 2000). et al, 1999). The significance of this observation lies
in the fact that, should a similar route to arsenical
Resistance resistance be possible in T. brucei, the possibility of
No reports of resistance in the field were found in cross-resistance to DFMO would materialize.
the literature, which is not surprising since the drug However, it should be stressed that so far a similar
has not been widely used in very large-scale treat- mechanism has not been noted in trypanosomes,
ment regimes. T. b. rhodesiense appears to be innate- and to date, lines selected for resistance to DFMO
ly less susceptible to the drug than T. b. gambiense were not cross-resistant to other trypanocides.
(Iten et al, 1995) since it has a higher overall ODC
activity and the enzyme has a shorter half life than
the gambiense counterpart (Iten et al, 1997). Another
Nifurtimox
explanation involving relative levels of S–adenosyl
methionine, which accumulate to lower levels in
The drug and its use
refractory but not sensitive cells treated with
Nifurtimox was originally licensed for use against
DFMO, has been proposed. Alternative explana-
South American trypanosomiasis. The drug con-
tions related to drug uptake, or polyamine uptake
tains a nitro group which is central to its activity. It
allowing bypass of the inhibitory effect, have not
has also been used in trials, with only limited suc-
been subject to extensive analysis.
cess (50-80% cure), against T. brucei gambiense in
West Africa, although since it is apparently active
In procyclic cells selected for DFMO resistance,
putrescine uptake was noted to be three-four times against melarsoprol refractory parasites it may still
higher than in wild-type lines (Phillips and Wang, be used and as treatment failures with arsenical
1987). Putrescine at >1 mM allowed parasites to sur- increase (Pépin et al, 1992).
vive DFMO treatment in vitro while 0.1 mM did not
(Phillips and Wang, 1987). Moreover, T. brucei para- Serum levels are reportedly low when given orally,
sites from which the ornithine decarboxylase gene peaking one-three hours after administration. The
had been removed were also viable and capable of drug can accumulate across the blood-brain barrier
growth provided external putrescine was abundant (Anon, 1998).
(Li et al, 1988) (far more abundant than in mam-
malian serum, where it is around 220 nM [Cooper Toxic effects to the central nervous system and
et al, 1978]). A similar result was obtained with peripheral nervous system have been reported.
Leishmania parasites from which ODC had been
knocked-out, i.e. putrescine enabled bypass of Mode of uptake and action
DFMO inhibition of polyamine synthesis, and also No reports about the mechanism of action against
enabled the cells to dispose of accumulated T. brucei could be found in the literature, although
S–adenosyl methionine (Jiang et al, 1999). reports relating to activity against T. cruzi exist.
Uptake of nifurtimox into Trypanosoma cruzi has
Lines selected in the laboratory for resistance to the been reported to occur via passive diffusion across
drug have been studied. Reduced drug accumula- the plasma membrane (Tsuhako, et al.,1991). Studies
tion was noted in procyclic parasites resistant to have not yet been extended to T. brucei but it is also
eflornithine (Bellofatto et al, 1987). However, likely to enter these cells via passive diffusion.
whether this was due to decreased uptake or
increased efflux was not determined. Nifurtimox is a nitrofuran compound. One electron
reduction of the nitro-group generates a potent free
A Leishmania line selected for resistance to DFMO radical which may interact with cellular con-
was shown to have an increase in ornithine decar- stituents or generate reduced oxygen metabolites

believed to cause death of the parasite (Docampo The drug can be given orally. Peak plasma levels fol-
and Moreno, 1984). The reduction potential of the lowing a 100 mg kg-1 dosing (Enanga et al, 2000) in
compound (-260 mV) is such that it is relatively eas- primates yielded plasma levels of between 0.2 µg ml
ily reduced in many cell types. The specificity and 46 µg ml-1 24 hr after dosing. The drug or its
towards the parasite (which is not great, nifurtimox metabolites can be found in the CSF at levels 5.5%-
being quite toxic to mammals) is thought to be asso- 10.6% of those in plasma. The elimination half time
ciated with its being more readily reduced by the was around 2.5 hours. Suramin substantially
parasite than the host cells. Moreover, mammalian increases the half-life of the drug and also increases
cells may have better protection against oxidative the amounts which can accumulate across the
damage. Specific targets or enzymes capable of blood-brain barrier.
reducing the drug cannot be ruled out. Some path-
ways which might lead to the preferential reduction No published reports of the effect of megazol on
of these compounds in trypanosomes have been humans exist. When the drug was administered to
studied. An intriguing hypothesis was that trypan- two patients with Chagas’ disease, anecdotal evi-
othione reductase might be responsible for the dence indicated there was little toxicity. However,
reduction (Henderson et al, 1988). Certainly, a num- the compound is positive in Ames’ tests (Ferreira
ber of nitro-containing compounds can act as “sub- and Ferreira, 1986) and this fact alone appears to
versive-substrates” for the enzyme. However, nifur-
have served as a deterrent to further development,
timox was one of the less successful substrates
in spite of the excellent safety record of another
(Henderson et al, 1988). and it is unlikely that try-
Ames’ test positive anti-protozoal 5-nitroimidazole,
panothione reductase-mediated nitro-reduction
metronidazole. Toxicological studies in mammals
underlies the activity of the clinically used nitro-
should be performed under recognized good labo-
heterocyclics.
ratory practice (GLP) conditions as a matter of
urgency. If toxicity proves to be no worse than for
Resistance
nifurtimox, then the case for pursuing the drug as a
African trypanosome lines selected for resistance to
potential reagent for clinical use would be strong.
nifurtimox have not been reported in the literature.
It is not clear why treatment failure is high. Further
pharmacokinetic studies should be made to deter-
Mode of uptake and action
mine the degree to which the drug reaches the CSF. Megazol possesses part of the motif recognized by
the P2 amino-purine transporter which is responsi-
Trypanosoma cruzi isolates show various levels of ble for the uptake of several anti-trypanosomal
sensitivity to the drug (Filardi and Brener, 1987). drugs (Barrett et al, 2000). Should this drug share the
There appears to be a correlation between drug P2 transporter as a portal of entry, it would be of
uptake and sensitivity (with lines accumulating limited use against arsenical resistant parasites.
least drug being least sensitive to it). No systematic However, strains of parasite lacking the P2 trans-
study on susceptibility of different sub-species or porter and resistant to other drugs which use this
strains of T. brucei have yet been conducted. portal of entry into trypanosomes, were not cross-
Interestingly, another nitroheterocycle called mega- resistant to megazol drugs (Barrett et al, 2000).
zol (discussed below) was equally active against Uptake of radiolabelled megazol revealed that this
lines of T. cruzi showing different sensitivities to drug, although capable of interacting with the P2
nifurtimox (Filardi and Brener, 1987). transporter, enters cells predominantly via passive
diffusion (Barrett et al, 2000).
Megazol The mode of action of the drug is not clear. The fact
that a nitro group is central to its function does not
The drug and its use necessarily imply that the mode of action will be the
Megazol is a 5-nitroimidazole which has good effi- same as for nifurtimox. Indeed, its reduction poten-
cacy against both T. cruzi (Filardi and Brener, 1987) tial of -438 mV (Viodé et al, 1999) is far lower than
and T. brucei (Enanga et al, 2000). Its synthesis was that of nifurtimox (-260 mV), and 5-nitroimidazoles
first reported in 1968 (Berkelhammer and Asato, are not normally reduced by aerobic cells. However,
1968). The activity of the drug against African try- megazol is susceptible to nitro-reduction in the
panosomes is striking. A single dose clears parasites presence of several enzymatic systems including
from the blood of rodents and a primate model. some found in T. cruzi extracts. How it exerts a lethal
Administration of the drug following a single dose action against parasites, however, is not certain
of suramin cleared parasites from the CSF of an although it seems likely that trypanosomes possess
infected primate (B. Enanga, personal communica- a specific enzyme capable of reducing this com-
tion). pound.

Resistance T. b. gambiense is much more difficult to grow in
Both procyclic and bloodstream forms of the para- rodents, although Mastomys rats do allow prolifera-
sites have been selected for resistance to megazol in tion of some isolates (only 20% of T. b. gambiense iso-
the laboratory (Enanga & Barrett, unpublished lates from a recent study in northern Uganda could
results) (procyclic forms with >100-fold resistance, be grown in Mastomys - Matovu, personal commu-
bloodstream forms with about 20-fold resistance to nication). Standardization of rodent protocols is
the drug). Modest levels of cross-resistance to the important, i.e. similar parasite numbers should be
other nitroheterocycle, nifurtimox, were apparent inoculated, similar drug dosing post-inoculation
(about 7-fold in both cases). Even more modest lev- should be given, and there should be similar follow
els of cross-resistance to diamidines (2 to 4-fold) and up with regard to checking mice for parasitemia.
melamine based arsenicals (3 to 5-fold) were also
observed. Preliminary evidence suggests that there The protocols established at the Swiss Tropical
is not a role for an efflux pump or for increased lev- Institute for each of these procedures may be rec-
els of trypanothione in the procyclic lines. The ommended as the standards which should be fol-
mechanism of resistance is currently unknown but lowed, although consensus agreement is required
under investigation. for this and other procedures may be considered
(for example those recommended for testing of vet-
DETECTION OF DRUG RESISTANCE erinary trypanocides in a recent PAAT document)
Treatment failure and drug resistance are by no (Geerts and Holmes, 1998).
means synonymous. Resistance is best defined as
“the heritable, temporary or permanent loss of the Drug Sensitivity Testing in Vitro
initial sensitivity of the population of microorgan- It is also possible to cultivate some lines in vitro
isms against the active substance” (Schnitzer and using established culture media. Laboratory adapt-
Grunberg 1957). It is essential that drugs are admin- ed lines are relatively easy to establish in culture.
istered according to the recommendations put for- However, many field isolates, particularly of
ward based on regimens optimal for activity, T. b gambiense, do not adapt readily to in vitro culture
although further studies on optimizing the dose are and few reports of axenically cultured T. b. gambiense
required for most drugs. This is particularly impor- could be found. Lines which have successfully been
tant in late-stage sleeping sickness where the quan- passed through Mastomys can proliferate in rich
tity of drug that accumulates in the extravascular medium (containing human serum) over a mono-
compartment may not extend far beyond the MIC layer of Mastomys embryonic fibroblast cells (Brun
required to kill the parasites. Treatment failure can et al, 1989). Until better conditions for the in vitro
come about for a number of reasons including cultivation of T. b. gambiense are established, it is
administration of sub-curative doses of drug or host important that all drug tests against all isolates
factors including metabolism and distribution with- should be performed under the same conditions. If
in the body. T. b. gambiense is to be compared with T. b. rhode-
siense, it is of limited use to use different cultivation
It is important to determine whether patients who
systems to determine drug sensitivity. This is partic-
have not cleared all parasites after treatment actual-
ularly the case when using mammalian cell mono-
ly carry drug resistant trypanosomes. Therefore
layers as part of the culture system since these may
tests for resistance should be performed.
affect the drug and its activity against parasites.
Isolation of Parasites, Propagation and
Drug Sensitivity Testing in Rodents Molecular Approaches to the
Blood (or CSF) can be taken from infected patients
Identification of Drug Resistance
Alterations to the gene TbAT1 that encodes the P2
and injected directly into a suitable rodent model. In
the case of T. b. rhodesiense, standard laboratory transporter have been identified, (Maser et al, 1999)
white mice or rats are adequate for this process. and many parasite lines which are less sensitive than
High parasitemias are readily achieved in these normal to melarsen-based arsenicals possess similar
hosts and highly parasitemic blood isolated from mutations (Matovu, personal communication).
these hosts can be isolated, mixed with a suitable Particular mutations appear to have been selected on
cryopreservant, and frozen in liquid nitrogen. multiple independent occasions. This opens the pos-
Preserved stocks can be re-injected into rodents and sibility of using a polymerase chain reaction (PCR)
then treated with trypanocidal drugs over a range of based approach to detect the presence of particular
concentrations to determine the MIC and effective mutant alleles which correlate to drug resistance.
dose 50 (ED50) of drug useful against these para- This approach will be limited if different types of
sites in vivo. Protocols must be standardized for this mutation affect the status of expression of the TbAT1
purpose. gene. More studies to investigate the frequency of

particular mutations that correlate to resistance are pentamidine appears to have alternative routes of
needed if such a test is to be useful. The possibility entry into the cells (De Koning, 2001). Nevertheless,
that different types of mutation could affect the P2 pentamidine should also be administered prudent-
transporter in such a way as to reduce sensitivity to ly, and curative doses ensured, to restrict the oppor-
drug, and the possibility that other biochemical tunities for selection of resistance to this drug which
changes not related to the P2 transporter could also might cause cross-resistance to melarsoprol.
induce resistance to arsenicals, means that a simple
PCR based test might produce an unacceptably high A Possible Role for Veterinary
number of false negative results. Trypanocide Use in the Selection of
Melarsoprol Resistance in Sleeping
GUIDELINES ON THE DELAY OF THE Sickness
DEVELOPMENT OF DRUG RESISTANCE Microbes selected for resistance to drugs during
Recent evidence indicates that the rise in the num- unsupervised treatment of livestock have become a
ber of sleeping sickness cases proving refractory to major route of introduction of resistance and resist-
melarsoprol treatment (Legros et al, 1999) is at least ance genes into human pathogens.
partially caused by the emergence and spread of
parasites resistant to the drug in the field (Matovu It seems that cross-resistance between diminazene
et al, in press). However, the global quantities of and melamine based arsenicals occurs more consis-
melarsoprol administered are far lower than, for tently than between the latter and pentamidine
example, those for chloroquine in malaria prophy- (Barrett and Fairlamb, 1999; Barrett et al, 1995). This
laxis, or for many of the antibiotics to which resist- could be because diminazene appears to enter pre-
ance is now widespread. It is also recommended dominantly via the P2 transporter and not via the
that melarsoprol is administered in a clinical setting, alternative transporters that carry pentamidine
thus improving the likelihood that a full curative (De Koning, 2001). The emergence of diminazene-
dose is given. As well, the dynamics of transmission melarsoprol cross-resistance can have profound
via tsetse flies make it far less likely that human try- implications for the development of drug resistant
panosomes will be transmitted between human sleeping sickness in the field.
hosts at the same frequency as are Plasmodium para-
sites by anopheline mosquitoes. Therefore, it is per- To date, no studies have been conducted to assess
haps surprising that resistance to the drug has whether a drug resistant parasite selected in an ani-
emerged, albeit apparently with a substantially mal can be transferred to humans – this is a specu-
slower time of onset than with chloroquine (chloro- lative scenario. However, it is clear that diminazene
quine was introduced in 1945 and melarsoprol in is administered to trypanosome infected cattle
1949). These factors also need to be set against the (Geerts and Holmes, 1998) and that several generic
fact that the quantities of melarsoprol reaching the versions of this product have appeared on the mar-
parasites within the CSF are only marginally higher ket which are of highly variable quality. Ad-hoc,
than the MIC of the drug, so that parasites which are unsupervised administration of sub-curative doses
only slightly less sensitive than wild-type to drug of diminazene to cattle is reportedly widespread,
may be selected with relative ease. While the param- and resistance to diminazene (in T. congolense and
eters that can lead to selection of resistance are com- possibly other trypanosome species) is rife in parts
plex, there is no doubt that it is critical to ensure of Kenya and elsewhere.
that the recommended dose of the drug is given to
every patient. Up to 20% of trypanosomes isolated from cattle in
western Kenya and parts of Uganda are infectious to
The potential of cross-resistance between pentami- humans (MacLeod et al, 2001). Therefore, human
dine and melarsoprol must also be considered. This infectious trypanosomes are present in areas where
is because both of these trypanocides can enter conditions for selection of resistance to diminazene
T. brucei via the P2 nucleoside transporter (Barrett are prevalent. Indeed, one isolate from south-eastern
and Fairlamb, 1999). Selection of resistance to one Uganda was clearly resistant to diminazene and
drug could therefore, in principle, lead to cross- isometamidium (Matovu et al, 1997). It has yet to be
resistance to the other. Interestingly, the relatively demonstrated whether resistance is associated with
widescale use of pentamidine as a prophylactic in loss of the P2 transporter, or whether cross-resistance
west Africa up until the 1950s appears not to have to melarsoprol occurs. In spite of this lack of data,
selected for resistance. Some laboratory derived there are reasons for concern about the selection of
lines have been shown to be pentamidine-melarso- drug resistant parasites in animals which can then be
prol cross-resistant but this has not been shown to transferred to human infections. It should be stressed
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RDC). Proceedings of the trypanosomiasis and leishma-
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niasis seminar and 3rd COST-B9 meeting in anti-proto-
due to the lack of the P2 transporter is a possibility.
zoal chemotherapy. Bruges, Belgium, 28-31 May, 2000.
Acknowledgments Bitonti AJ et al. Uptake of alpha-difluoromethylor-

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III HUMAN AFRICAN that more than 15% of all patients in the nervous
TRYPANOSOMIASIS system phase of the disease, treated in Côte
(Original manuscript in French) d’Ivoire, can be excluded from the fatal side-effects
of arsobal therapy.
Felix Doua
Clinical Research Project into Trypanosomiasis, BP 1425 Treatment and Clinical Trials using
Daloa, Côte d’Ivoire Melarsoprol
Treatment regimens using melarsoprol in countries
INTRODUCTION where sleeping sickness is endemic date back to
In the last 20 years there has been a resurgence of colonial times and consist of the administration of
interest in human African trypanosomiasis (HAT) three to four series of two to four injections of melar-
following a recrudescence of the disease in most soprol spaced out at seven to ten day intervals.
countries of sub-Saharan Africa. Although there are These regimens, developed on an empirical basis,
thousands of new cases every year in countries such have helped control HAT epidemics but could be
as the Democratic Republic of the Congo (DRC), the cause of the growing number of recorded treat-
Angola and Uganda, no noteworthy progress has ment failures. Recent pharmacokinetic data on
been recorded in the development of new drugs to arsobal, obtained by bioassay and computer simula-
fight the condition. tions (Burri et al, 1993), have shown that an alterna-
tive, more rational, therapy regimen, consisting of
The drugs currently used in the treatment of sleep- ten injections at low doses, can be used without
ing sickness were first marketed in the 1950s. increasing the risk of fatal accidents. The new regi-
Pentamidine and suramin are the drugs of choice men (Burri et al, 1995) is currently under clinical
for the early-stage disease, while melarsoprol evaluation in seven African countries.
(arsobal), despite its toxicity, is the drug of choice for
the late-stage disease, when the central nervous sys- Treatment and Clinical Trials using DFMO
tem is involved. However, a rise in treatment fail- DFMO (ornidyl, eflornithine) is an irreversible spe-
ures with melarsoprol has been noted in the last few cific inhibitor of ornithine-decarboxylase, a key
years (Ginoux et al, 1984; De Gros et al, 1999), there- enzyme in the synthesis of polyamines, which are
by hampering efforts to combat sleeping sickness physiological substances implicated in cell multipli-
despite the development of sensitive diagnostic cation (Sjoerdsma et al, 1984).
tools and versatile low-cost anti-vectoral control
methods such as insecticide impregnated screens. Clinical trials carried out using DFMO show that
a seven-day regimen is effective in treatment of
Difluoromethylornithine (DFMO, ornidyl, eflor- T. b. gambiense HAT. The side effects, recorded in
nithine) has been undergoing clinical trials in the parenteral DFMO administration, are generally re-
last two decades but is not yet on the market versible (Doua et al, 1987; Taelman et al, 1987) and
because its cost is considered prohibitive for user the relapse rates after treatment are normally about
countries. There is therefore an urgent need to use 1% (Doua et al, 1993). However, the cost of DFMO
better the available trypanocides and to develop remains a limiting factor to its wider use. Clinical
new ones that are cheap, well tolerated and effective trials are ongoing in Côte d’Ivoire, to determine the
at every phase of the disease. efficacy after oral administration, and the pharma-
cokinetics. The intended result is to develop an oral
formulation of the product at an affordable cost to
TREATMENT AND CLINICAL TRIALS patients.
CONDUCTED ON THE T. B. GAMBIENSE
FORM OF SLEEPING SICKNESS Treatment and Clinical Trials using
Nifurtimox
Treatment and Clinical Trials using Pentami- This use of nifurtimox for the treatment of T. b. gam-
dine biense HAT has not been studied in large-scale clini-
Pentamidine, an aromatic diamidine, has a success cal trials, although results obtained with four late-
rate of 95% when used for early stages of T. b. gam- stage patients in the DRC show that the product is
biense HAT (Doua et al, 1993), and of 94% when effective but very toxic (Jansens et al, 1977).
used for patients suffering from the early nervous
system phase (Doua et al, 1996). These results indi- CONCLUSIONS AND
cate that pentamidine passes the blood-brain barri- RECOMMENDATIONS
er and is capable of halting the multiplication of In the last 20 years there has been rapid develop-
trypanosomes in cerebrospinal fluid. This suggests ment of both serological and parasitological HAT

diagnostic techniques, but in 2001, treatment of the Doua et al. Human tryponosmiasis in the Ivory
disease is still based only on pentamidine and Coast: therapy and problems. Acta tropica, 1993,
suramin for the early stages of the infection, and on 54:163-168.
melarsoprol for the central nervous system stages.
Treatment schedules with these trypanocides were Doua et al. Efficacy of pentamidine in the treatment
derived empirically, without pharmacokinetic stud- of early late stage trypanosoma brucei gambiense try-
ies. Thus, the treatments applied to patients vary panosomiasis. American journal of Tropical Medicine
from one country to the next and sometimes from and Hygiene, 1996, 55:586-588.
one treatment centre to another in the same country,
which only aggravates the high number of failures Ginoux et al. Les échecs du traitement de la try-
observed, in particular with melarsoprol, the drug panosomiase à T. b. gambiense au Congo. [Failures of
of choice for HAT. T.b. gambiense trypanosomiasis treatment in the
Congo]. Médecine tropicale, 1984, 22:149-154.
DFMO, which has given rise to great hopes for the
treatment of T. b. gambiense HAT, still has not been Jansens et al. Clinical trials with Nifurtimox in
commercialized because of its cost. To make efforts human trypanosomiasis. Annales de la Société Belge
to control HAT more effective, and to make for de Médecine Tropicale, 1977, 57:475-479.
rational treatment of patients, we recommend the
following: Sjoerdsna A, Schecter PJ. Chemotherapeutic impli-
• Encouragement of research aimed at developing cations of polyamine biosynthesis inhibition. Cli-
new, effective, well-tolerated, cheap trypanocides. nical Pharmacology and Therapeutics, 1984, 35:287-300.
• Harmonization of treatment schedules for HAT,
particularly those for melarsoprol. Taelman H et al. Difluoromethylornithine, a new ef-
• Conduct of multicentre clinical trials using pen- fective treatment of Gambian trypanosomiasis. Re-
tamidine for patients in the early nervous system sults in five patients. The American Journal of
stage of HAT, with a view to evaluating its effica-
Medicine, 1987, 82:607-614.
cy in different HAT foci.
• Conduct of multicentre clinical trials using orally
administered DFMO, after the current study on
pharmacokinetics is completed.
• Conduct of multicentre clinical trials using melar-
soprol-DFMO combination in patients suffering
relapse after treatment with melarsoprol.
• Introduction of nifurtimox for the treatment of
HAT cases that are resistant to melarsoprol.
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IV TREATMENT AND ed to be more than US$100 per adult patient (WHO
CLINICAL STUDIES 1998). In addition, sleeping sickness patients are
almost always from poor rural backgrounds within
Martin Odiit their countries (Okia et al 1994). Drugs for the treat-
Livestock Health Research Institute, P.O. Box 96, Tororo, ment of sleeping sickness are not readily available in
Uganda drug stores and are most often procured by the gov-
ernments and non-governmental organizations
SUMMARY through the assistance of the World Health Organi-
Diagnosis and treatment of sleeping sickness zation. Stocks of drugs are carefully monitored at
remains the major control strategy of the disease. national levels. However, it is conceivable that, in
The current diagnostic kits are insensitive, the diag- the event of an epidemic, it may not be possible to
nostic and treatment centres inadequately equipped have enough drugs immediately available, a con-
and staffed, and the patients poor and often in inac- straint already experienced by some African coun-
cessible rural locations. The drugs used for the treat- tries during sleeping sickness epidemics. Most drug
ment of human African trypanosomiasis are toxic, companies appear to be reluctant to produce drugs
necessitating a positive demonstration of parasites, for sleeping sickness because they will not obtain
including the invasive, painful lumbar punctures returns that are adequate to finance further drug
for disease stage determination. The treatment of development (Barrett, 2000; Stephenson and
the disease is lengthy, parenteral and costly, and the Wiselka, 2000).
drugs used toxic and not readily available. These
traditional problems of sleeping sickness are com- SLEEPING SICKNESS CONTROL
pounded by the emergence of drug resistance to Sleeping sickness control has traditionally been car-
melarsoprol in T. b. gambiense patients, the increas- ried out through case detection and treatment, and
ing uncertainty of sustained production of drugs for through vector control when epidemics occur
sleeping sickness due to economic justification, and (WHO, 1998). Early diagnosis and treatment is the
the increasing prices of some drugs due to their goal of control programmes, but is often hampered
newly found use for HIV treatment. Clinical studies by the paucity of resources required to implement
that address these problems which face the major these programmes. Most programmes of sleeping
control strategy for sleeping sickness are urgently sickness control are donor dependent because the
needed. For melarsoprol treatment of T. b. gambiense costs of sleeping sickness interventions are beyond
late-stage patients, studies on reduction in length of the budgets of the affected countries. The need for
treatment schedules, and therefore of costs, and cheaper control strategies is therefore a priority.
improved compliance, are in the advanced stages. Decentralization of services, including health servic-
Research into cost-effective and sustainable strate- es, is being advocated. In some countries, planning
gies of improving diagnosis and treatment are nec- and implementation of the sleeping sickness control
essary to reduce the level of under-reporting that is programmes has been moved to the district level.
associated with certain death of sleeping sickness However, current sleeping sickness control strategies
patients if untreated. This scientific working paper are expensive and often complicated, requiring qual-
reviews some of the current problems of treatment ified personnel. Research into the roles of the nation-
of sleeping sickness and suggests clinical studies al and sub-national levels in the decentralization of
that may address these problems in the near future. sleeping sickness control should be reviewed.
INTRODUCTION DIAGNOSIS
Human African trypanosomiasis (sleeping sickness) Detection of the parasite in gland or lymph node
is found in the tsetse belt of tropical Africa, where it aspirate or blood is always followed by a lumbar
is estimated that over 50 million people live (WHO, puncture to determine whether there is involvement
1998). Approximately 45 000 cases are reported of the central nervous system (CNS) (also known as
annually, though it is believed that 300 000 persons the late stage), because drugs used when the CNS is
are infected each year (WHO, 1998). There are two involved are different from those used when the
types of sleeping sickness, caused by: Trypanosoma parasite is still restricted to the haematolymphatic
brucei gambiense, found in west and central Africa, system (also known as the early stage). However,
and by T. b. rhodesiense, reported in east and south- most of the currently used confirmatory diagnostic
ern Africa. The gross national product per capita for techniques such as the thick blood smear and the
the sleeping sickness affected countries (< US$500) haematocrit centrifugation test have poor sensitivity
ranks them as amongst the least developed coun- (WHO 1998). The polymerase chain reaction (PCR)
tries of the world (World Bank, 2000). However, the has been found to be very sensitive and specific
costs of treatment for sleeping sickness are estimat- (Kyambadde et al, 2000; Penchenier et al, 2000), but

it is rather complicated and expensive for applica- series of injections each separated by an interval of
tion as a routine diagnostic test, which restricts its one week, are used (WHO, 1998). One regime starts
use to research laboratories and referral hospitals. with 2.5ml and the other with 0.5ml, but both total
35 mls of a 36g/litre solution of melarsoprol.
The criteria used for determination of CNS involve-
ment are as follows: cell counts above 5 cells/mm3 The most serious side effect of melarsoprol is reac-
or the demonstration of trypanosomes or protein tive encephalopathy occurring between the third
levels above normal (25mg per cent) in the cere- injection and the beginning of the second course.
brospinal fluid (CSF). The first two criteria are gen- The onset may be sudden or the condition may
erally used because they are simpler and do not develop slowly with fever, headache, tremor, slur-
require reagents. ring of speech, convulsions, and finally coma. It
occurs in 5% of patients, and the incidence of fatali-
CHEMOTHERAPY OF SLEEPING ty due to these reactions is approximately 1% (Odiit
SICKNESS et al, 1997). However, the fatality of untreated dis-
Most of the affected countries use broad guidelines ease is believed to be 100%, making the decision to
for the treatment of sleeping sickness as recommend- treat an ethical necessity. The treatment of these
ed by the World Health Organization (WHO, 1998). reactions includes the use of corticosteroids, hyper-
However, there is an urgent need for novel approach- tonic solutions to combat cerebral oedema, rapidly
es in sleeping sickness treatment and control. acting anticonvulsants, and subcutaneous adrena-
line. There are a few cases of diarrhoea, jaundice and
Pentamidine is used for treating the early stage of dermatitis, but these are generally not life threaten-
sleeping sickness caused by T. b. gambiense. It is ing.
effective for treatment so long as there is no
detectable involvement of the CNS. The dosage PATIENT FOLLOW-UP
used is 4mg of pentamidine base per kg of body It is mandatory to systematically follow up all treat-
weight. A total of seven injections are given daily, ed patients to ascertain that they have been cured. It
intramuscularly. Drug resistance appears not to be is recommended that patients be seen every six
an important problem in the use of pentamidine for months over a two-year period. In addition to a full
the treatment of T. b. gambiense. The side effects clinical and parasitological examination, a lumbar
include pain and induration or sterile abscess at the puncture should be carried out on the patients, to
site of injection, vomiting, abdominal pain, hypo- allow the CSF to be examined for possible increase
tension, syncope, hypoglycaemia, and peripheral in leukocyte counts or presence of trypanosomes
neuritis. Treatment failure attributable to resistance that would indicate a relapse. However, due to the
to pentamidine appears not to be an important pub- painful lumbar puncture procedure, most patients
lic health problem. Studies of the pharmokinetics of resent returning for follow-up once they begin to
pentamidine are required to determine if the length feel better, while active follow-up to ensure compli-
of the prescribed treatment regime can be reduced. ance is not affordable. Therefore, less invasive and
painful methods of ascertaining cure need to be
Suramin is used for the treatment of the early stage developed. Changes in CATT titres or CIATT titres
of T. b. rhodesiense because, though the duration of and haematological parameters such Hb, ESR etc.
treatment is longer than that for pentamidine and it are suggested for investigation in assessing cure. In
is given by intravenous treatment, no primary addition, the duration of post-therapeutic follow-up
resistance to suramin has been reported for should be reviewed, especially for T. b. rhodesiense,
T. b. rhodesiense. The dosage used is 20mg/kg body in the expectation that it may be shorter than that
weight. Intravenous injections - a maximum of se- required for T. b. gambiense.
ven - are given every seven days. Side effects obser-
ved include pyrexia, pains in the joints and soles of DRUG RESISTANCE
the feet, skin rash and desquamation, hypersensitiv- There is no recent evidence for resistance of T. b. rho-
ity reactions. Pharmacokinetic studies of suramin desiense to melarsoprol. In the early 1970s, melarso-
should be undertaken to determine if the treatment prol treatment failure of 12 (3.4%) out of 358 treated
regime can be shortened. cases was reported (Ogada, 1974). It is possible that,
with low treatment failure rates and lack of system-
Melarsoprol is the drug used to treat late-stage atic follow-up, patients with treatment failure may
sleeping sickness due to both T. b. rhodesiense and be missed. There should be periodic active follow-
T. b. gambiense in Uganda. The maximum dosage for up of cohorts of sleeping sickness cases to monitor
each injection is 3.6 mg/kg body weight. Currently, drug efficacy.
two regimes for the treatment, comprising several

Treatment failure of melarsoprol in T. b. gambiense east, the teams are used to increase the awareness
disease is emerging. Legros et al (1999a and 1999b) of the population and detect cases who may not yet
reported a melarsoprol treatment failure rate of have sought health care. The costs of maintaining
26.9% among 428 patients treated in north western mobile teams are not affordable by the countries
Uganda. Melarsoprol treatment failure rates needs affected by sleeping sickness, and therefore the
to be monitored and documented regularly to estab- teams are often used in epidemic situations and
lish the magnitude of the problem. The cause of this with donor support. Operational research is
treatment failure is under investigation to deter- required to optimize the distribution of fixed post
mine whether it is due to variation of melarsoprol diagnosis and treatment. This strategy may be a
pharmacokinetics between individuals or if it is more sustainable strategy, especially in between
associated with reduced susceptibility of try- epidemics of sleeping sickness.
panosomes to melarsoprol. Legros et al (1999b)
emphasize the need for second-line drugs to treat NEWLY DESIGNED TREATMENT
patients that have already received one or several REGIMES
full course(s) of melarsoprol. In Uganda, melar- Pharmacokinetic data from studies carried out on
soprol treatment failure is a more significant prob- T. b. gambiense patients indicate that the course of
lem to the west of the river Nile than to the east, in melarsoprol treatment in an adult can be reduced
the T. b. gambiense affected area in the north-west of from a period of 25-36 days (WHO, 1998) to a peri-
the country. Possible risk factors explaining this dif- od of just ten days, greatly cutting the costs of hos-
ference in geographical distribution here and else- pitalization (Burri et al, 1995). A randomized trial
where on the continent should be examined. Sub- comparing the standard treatment schedule with
clinical, inadequate treatments in the history of this new concise regimen showed the same levels of
these melarsoprol treatment failure foci could be an parasitological cure (100%) and adverse effects (16-
explanation. 17%) as well as better compliance with the new
regime (Burri et al, 2000). Adaptation of the new
HEALTH SYSTEMS AND SLEEPING concise regime is to be monitored. It would not be
SICKNESS CASE MANAGEMENT advisable to adapt the model to treatment of late-
Demonstration of the trypanosome is mandatory stage T. b. rhodesiense infection because the pharma-
before treatment can be administered to a sleeping cokinetics may be dissimilar given that this disease
sickness case, due to the toxicity of the drugs used. is much more severe than the gambiense form. The
blood-brain barrier may be more affected, allowing
Due to the costs of staffing and equipping sleeping
for higher levels of melarsoprol in the central nerv-
sickness treatment facilities, few health units are
ous system and possible neurological side effects. A
equipped for treating the disease. The endemic
study of the pharmacokinetics of melarsoprol in
area for sleeping sickness in most countries is vast,
late-stage T. b. rhodesiense infections is therefore nec-
with poor coverage by fixed post health units for
essary. However, a seven-day course of eflornithine
diagnosis and treatment of the disease. Therefore,
for the treatment of late-stage T. b. gambiense infec-
most sleeping sickness patients travel more than
tion was found to be inferior to the standard 14-day
5km to receive treatment. The problem of poor
regimen (Pépin et al, 2000).
accessibility to diagnosis and subsequent treat-
ment may be alleviated by the use of mobile teams. There have been attempts to adjust the criteria for
However, this is more rewarding where a valid treatment and so reduce the number of patients
screening test is commercially available. The card receiving melarsoprol treatment that is very toxic.
agglutination test for trypanosomiasis (CATT) is To determine CNS involvement, it has been pro-
such a test. It has good sensitivity and specificity posed that the number of cells be raised to 20
(Magnus et al, 1978) but it is only useful for the cells/mm3 (Doua et al, 1996). There is not yet
gambiense form of sleeping sickness. A similar test enough evidence to change the cut-off point for the
for the rhodesiense form of the disease is required number of cells/mm3 in the CSF (Doua et al, 1996);
before mobile teams can provide a rewarding strat- therefore the WHO recommended criteria for stag-
egy for routine diagnosis and treatment. A new ing are still maintained.
test, the card indirect agglutination trypanosomia-
sis test (TrypTectCIATT®), is reported to have good Chemoprophylaxis is not currently used as a sleep-
sensitivity for both forms of sleeping sickness ing sickness control strategy. A multicentre evalua-
(Nantulya, 1997) and is to undergo further field tion of the positive predictive value of increasing
evaluation (TDR, 1999). In Uganda, mobile teams dilutions of the CATT (Magnus et al, 1978) is being
are used in the north-west with support from a carried out; results of this study will be used to see
non-governmental organization (Médecins sans if mass chemoprophylaxis is indicated in instances
Frontières, France); during outbreaks in the south- of high sero-prevalence.

Eflornithine (difluoromethylornithine, or α-DFMO, lating in humans and livestock is similar. The cost-
or Ornidyl®) is not effective against T. b. rhodesiense effectiveness and acceptability of the strategy to
(Iten et al, 1995; Matovu et al, 1997) but is useful for control T. b. rhodesiense sleeping sickness by mass
the treatment of late-stage T. b. gambiense infection chemotherapy of livestock should be investigated,
(Doua et al, 1987). It is presently available for the taking into account other potential benefits to the
treatment of relapse following melarsoprol treat- farmer.
ment, but the production of this drug was recently
suspended. It has been suggested that nifurtimox be In conclusion, the current problems in the treatment
used in desperate situations where DFMO is not of sleeping sickness in Africa include: melarsoprol
available for treating melarsoprol refractory cases. treatment failure of T. b. gambiense sleeping sick-
Nifurtimox is very toxic but is reported to be effec- ness; unavailability of alternative drugs; unafford-
tive for curing some T. b. gambiense patients, includ- able treatments; poor coverage by diagnostic and
ing those with late-stage disease refractory to melar- treatment facilities. Studies to identify practical
soprol treatment (WHO, 1998). solutions to these problems are urgently required to
reduce the current high fatality due to sleeping
Nifurtimox (Lampit®, Bayer) has been tested for the sickness, a disease that is regarded as always fatal if
treatment of sleeping sickness with conflicting not treated.
results (Pépin et al, 1992; Van Niewenhove, 1992;
Doua and Yapo, 1993). It is a cheap drug and easy to SUGGESTED STUDIES
administer but is not yet registered for use in the • Research into new, safe, effective and cheap drug
treatment of this disease. With the current emer- combinations that are active for all stages of sleep-
gence of melarsoprol resistance, the efficacy and ing sickness. Suggested drug combinations to
safety of nifurtimox in the treatment of sleeping compare are melarsoprol and eflornithine, melar-
sickness caused by T. b. gambiense should be re-eval- soprol and nifurtimox, nifurtimox and eflor-
uated. nithine.
REDUCTION OF DRUG PRESSURE • Continued screening of new compounds that are

In areas where drug resistance to melarsoprol exists, less toxic, easy to administer, cheaper and effec-
the need to use the drug can be reduced by putting tive for all stage of sleeping sickness.
emphasis on preventive control strategies such as
vector control and mass chemotherapy of the • Research on cost-effective means to prevent out-
domestic animal reservoir of the disease. breaks of sleeping sickness through methods such
as targeted vector control, and to avoid the high
TSETSE CONTROL costs of treatment, problems of drug manufacture,
Although tsetse control is reported to reduce the and complications associated with treatment fail-
transmission of sleeping sickness, it is rarely main- ure. Identification of markers for determining the
tained because of the costs of the methods applied. probability of occurrence of sleeping sickness out-
Currently, tsetse traps and targets are considered the breaks in space and time for use as a basis for dis-
cheapest, and an environmentally friendly option, ease prevention.
applicable through community participation, but
they are still not widely used to control sleeping • Active follow-up of cohorts of sleeping sickness
sickness. Pour-on insecticide for cattle is another cases to accurately monitor drug efficacy, and
vector control option that may involve community document the results. Mapping of foci of melarso-
participation. However, use of pour-on insecticides prol treatment failure to study the possible risk
is dependent on the distribution and movement of factors of its evolution.
livestock.
• Research into other methods of control to reduce
MASS CHEMOTHERAPY OF LIVE- the pressure on human trypanocidal drugs, given
STOCK IN T. B. RHODESIENSE AREAS the development of drug resistance. Evaluation of
Research on the role of the livestock reservoir in the the cost-effectiveness of mass chemotherapy of live-
epidemiology of T. b. rhodesiense sleeping sickness stock in the control of T. b. rhodesiense epidemics.
indicates that chemotherapy of livestock in endemic
areas is an important policy for the control of sleep- • Operational research to optimize the geographical
ing sickness. There is molecular evidence for the distribution of sleeping sickness diagnostic and
similarity of the parasites of man and livestock treatment facilities, taking into account factors
(Enyaru et al, 1992; Hide and Tait, 1991; Hide et al, such as current disease distribution, population
1994), suggesting that the parasite population circu- distribution, availability of health infrastructure,

and the effects of distance on case detection and Enyaru JCK et al. Characterisation by isoenzyme
early diagnosis. electrophoresis of trypanozoon stocks from slee-
ping sickness endemic areas of south east Uganda.
• Diagnosis of suspect referrals, not positive by tra- Bulletin of the World Health Organization, 1992, 70:631-
ditional methods and requiring immediate atten- 636.
tion, by equipping research institutes and referral
centres with PCR technology. PCR can serve as a Hide G, Tait A. The molecular epidemiology of par-
gold standard for evaluating diagnostic tests. asites. Experentia, 1991, 47:128-142.
• Pharmacokinetics studies of melarsoprol and Hide G et al. Epidemiological relationship of Try-

suramin in T. b. rhodesiense patients and of pen- panosoma brucei stocks from South East Uganda; evi-
tamidine in T. b. gambiense patients. dence for different population structures in human
and non-human trypanosomes. Parasitology, 1994,
• Continued monitoring of the ten-day melarsoprol 109:95-111.
protocol for treatment of sleeping sickness due to
T. b. gambiense, making the results readily avail- Iten M et al. Innate lack of susceptibility of Ugandan
able to all health policy-makers in affected coun- Trypanosoma brucei rhodesiense to DL-alpha-difluo-
tries. romethyl ornithine (DFMO). Tropical Medicine and
Parasitology, 1995, 46:190-194.
• Market research aimed at creating interest in the
private sector for the manufacture of eflornithine. Kyambadde JW et al. Detection of trypansomes in
suspected sleeping sickness patients in Uganda using
• Review of the roles of national and sub-national the polymerase chain reaction. Bulletin of the World
control programmes. Health Organization, 2000, 78:119-124.
• Review of the parameters for, and duration of, Legros D et al. Therapeutic failure of melarsoprol
post-treatment follow-up of sleeping sickness among patients treated for late stage of T. b. gambi-
patients. ense human African trypanosomiasis in Uganda.
Bulletin de la Société de Pathologie Exotique , 1999 (a),
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trial. The Lancet, 2000, 355:1419-1425. agglutination test with stained trypanosomes
(CATT) for the serological diagnosis of T. gambiense
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Coast. Experimental Parasitology, 1993, 77:306-14. infections. Transactions of the Royal Society of Tropical
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Annex 6
PATHOGENESIS, GENOMICS AND
APPLIED GENOMICS

I DISCUSSION DOCUMENT ON • Late-stage anti-inflammatory and immunosup-
PATHOGENESIS / APPLIED pressive events that modulate the protective
GENOMICS effects of host T and B cell responses:
- Elucidation of mechanisms that promote type 2
John M. Mansfield cytokine responses, and that suppress host tis-
Department of Bacteriology, University of Wisconsin- sue specific resistance.
Madison, 1925 Willow Drive, FRI Building Madison, WI - Role of these modulatory events in controlling
53706, USA or exacerbating tissue pathology.
For the purposes of group discussion, I have arbi- 2. Changes in parasite cell biology during infection
trarily selected several research areas relevant to that impact on host resistance, with an emphasis on:
human African trypanosomiasis (HAT)-associated • Molecular basis of changes in trypanosome viru-
pathogenesis; these areas are outlined below. Some lence for the host
background information on trypanosome immunol- - Relatedness to other clonal differentiation events.
ogy and cell biology is provided in the text that fol- - Association with tissue specific residence/tro-
lows (excerpted from recent reviews by Mansfield pism.
and Olivier (2001), Mansfield et al. (2001), Paulnock - Identification and targeting of parasite genes
and Coller (2001), and related resources (Imboden and molecules that are differentially expressed
et al, submitted for publication, 2001; Mansfield et al, in highly virulent trypanosomes.
in press; Paulnock et al, 2000). It is anticipated that
Melville and colleagues will provide more detailed • Molecular basis for the resistance and susceptibil-
and appropriate information on the trypanosome ity of certain trypanosomes to the cytolytic effects
genomics project and its practical applications. of human serum high density lipoproteins (HDL)
and of tumour necrosis factor-alpha (TNFα).
DISCUSSION OUTLINE 3. Identification of stage-specific parasite molecules

through genomics and proteomics approaches that
1) Stage-specific immunobiological changes that might be exploited, with an emphasis on:
occur during infection, with an emphasis on the fol- • Potential trypanosome molecules/cell biological
lowing subtopics: systems that could be targeted by highly specific
• Early pro-inflammatory events that trigger innate chemotherapeutic agents:
immune system responses: - BSF trypomastigote specific.
- Glycosylphosphatidylinositol (GPI) substituents - Tsetse fly stage specific.
of the variant surface glycoprotein (VSG) mole-
cule shed into host tissues. • Highly conserved invariant antigens that could be
- Trypanosome lymphocyte triggering factor (TLTF) used for either more accurate immunodiagnosis
release from trypanosomes and induction of in- or for targeted immunotherapy.
terferon-gamma (IFN-γ) production.
- Macrophage activation events associated with
exposure to parasite activation molecules and BACKGROUND INFORMATION
host activation factors.
The background information that follows is an opin-
• Early-stage acquired immune mechanisms associ- ionated view of trypanosomiasis, excerpted from
ated with parasite control in the vascular and this author’s writings and other resources, that is
extravascular tissue sites: meant to serve only as background information for
- Distinct roles of Th1 cells and B cells in provid- group discussion on immunology and selected
ing tissue specific resistance to trypanosomes. aspects of parasite biology. As pointed out in the
- Macrophage release of trypanocidal factors/ passages below, there may be some disagreement as
cytokines. to the biological significance of certain observations.
Additional background information on the geno-
• Interrelationship of host innate and acquired mics projects will be provided by others.
immune responses to host pathology throughout
infection: Stage-Specific Immunobiological Changes
- Elucidation of immunological events that pro- that Occur During Infection
mote tissue specific pathology at all stages of Macrophages and innate immunity
infection. Cells of the macrophage lineage provide the first line
of host defense against infectious diseases, and also

modulate downstream events that impact on the tion, 2001; Paulnock et al, 1989); and, upregulation
development of acquired immunity. Macrophages of mRNA or proteins for other markers that include
are present in all tissues and possess the ability to TNFα, IL-1, IL-6, inducible nitric oxide synthase
recognize and eliminate many microbes. It is well (iNOS), prostaglandins and IL-10 (Beschin et al,
established that recognition of microbes by 1998; Darji et al, 1992; Imboden et al, submitted for
macrophages results in cellular activation following publication, 2001; Magez et al, 1999; Mathias et al,
the uptake or binding of microbial components to 1990; Schleifer and Mansfield, 1993; Sileghem et al,
specific membrane receptors (Hoffman et al, Mosser, 1989). More importantly, the expression or release of
1992; Mosser and Karp, 1999). Receptor-mediated several of these activation markers is associated
activation of macrophages represents one of the first with modulation of host immunity and resistance.
events in the innate immune response to many For example nitric oxide (NO), prostaglandins and
microbial infections, leading to the production of TNFα have been implicated in the suppressor cell
pro-inflammatory cytokines that initiate an inflam- activity exhibited by macrophages at different time
matory response and affect the downstream devel- points of infection (Beschin et al, 1998; Borowy et
opment of activated T cells as well as other parame- al, 1990; Hertz and Manfield, 1999; Schleifer and
ters of host immunity. Cytokines produced by acti- Mansfield, 1993; Sileghem et al, 1991; Sileghem et
vated T cells, primarily IFN-γ, provide additional al, 1989; Sternberg and McGuigan, 1992; Sternberg
activation signals for macrophages, unleashing effec- and Mabbot, 1996); although NO and TNFα have
tor functions that can destroy a wide range of intra- been shown to kill trypanosomes in vitro and are
and extracellular microorganisms (Bendelac and thought to be important for trypanosome control in
Fearon, 1997; Fearon and Locksley, 1996; Medzhitov extravascular tissue sites (more below on this topic)
et al, 1997). Thus, the processes of innate resistance (Lucas et al, 1994; Magez, et al 1997; Magez et al,
and acquired immunity are intimately interdepend- 1999; Mnaimneh et al, 1997; Vincendeau and
ent, with macrophages playing a dual role as the ini- Daulouede, 1991; Vincen-deau et al, 1992), neither
tiators of acquired responses and as a major effector factor alone has been linked definitively to protec-
component of cell-mediated immunity. tion in vivo (Hertz and Mansfield; 1999; Magez et al,
1999) and the expression of these factors may be
Macrophage activation in linked to pathological changes during infection
trypanosomiasis (Mabbott and Sternberg, 1995; Mabbott et al, 1994;
Macrophage activation is one of the hallmarks of Sternberg and Mabbott, 1996). However, the pro-
infection with the African trypanosomes (Askonas, inflammatory pattern of macrophage activation
1984; Askonas, 1985; Bancroft et al, 1983; Beschin appears to change over the course of infection to
et al, 1998; Borowy et al, 1990; Clayton et al, 1979; become a counter-inflammatory pattern of activa-
Darji et al, 1992; De Gee et al, 1985; Fierer and tion in which IL-10 predominates and Type 2
Askonas, 1982; Fierer et al, 1984; Grosskinsky and cytokine responses appear to emerge (Namangala
Askonas, 1981; Grosskinsky et al, 1983; Mayor-Wi- et al, 2000; Namangala et al, 2000; Namangala et al,
they et al, 1978; Murray and Morrison, 1979; 2001); these events have been associated with late
Paulnock and Coller, 2001; Paulnock et al, 1989; stage disease (Imboden et al, 2001; Paulnock and
Sacks et al, 1982; Schleifer and Mansfield, 1993, Coller, 2001; Sternberg, 2001).
Sileghem et al, 1989; Wellhausen and Mansfield,
1979). There is extensive evidence that the numbers
and activity of macrophages increase dramatically
in the tissues of trypanosome infected animals, and
are associated with tissue pathology. Within the first
two weeks of experimental Trypanosoma brucei rhode-
siense infection, for example, a large percentage of
cells in the enlarged spleen exhibit membrane and
functional characteristics associated with activated
macrophages. These include: increases in the release
of interleukin-12 (IL-12) and IL-18, known to be
important in the development of the early polarized
Th1 cell responses to trypanosome antigens Figure 1. Glycosylphosphatidylinositol (GPI) mem-
(Mansfield, 1994; Mansfield et al, submitted for pub- brane anchor substituents of the trypanosome variant
lication; Schleifer et al, 1993; Schopf et al, 1998), an surface glycoprotein (VSG) molecule. GPIs have been
enhanced ability to serve as antigen processing cells termed “…one of the most potent microbial pro-inflam-
coupled to increases in expression of membrane I- matory agents known”(Almeida et al, 2000). The GPI
Aα, B7-1 and B7-2 (Imboden, submitted for publica- anchor of the Trypanosoma brucei rhodesiense LouTat 1 VSG

molecule is depicted in this figure, showing glycosylinos- mechanisms, the amounts of GPI substituents (GPI-
itolphosphate (GIP) substituents associated with sVSG mfVSG, GIP-sVSG and DMG) saturating host tis-
after cleavage from membrane-anchored GPI-mfVSG by a sues during infection are quite substantial.
trypanosome membrane-associated phospholipase C Conservative calculations estimate that experimen-
(GPI-PLC), as well as dimyristoylglycerol (DMG) sub- tally infected animals may be exposed to 15-20 µM
stituents that remain associated with the trypanosome VSG with each wave of parasitemia, which is an
membrane. Figure adapted from Menon (1999 and 1994), inordinate amount of parasite material with intrin-
Magez (1998) and others (Varela-Nieto et al, 1996). sic macrophage activation potential to be released
into host tissues.
Role of the variant surface glycoprotein
GPI anchor in macrophage activation Limited in vitro studies by several labs have begun
The source(s) and mode of action of activating fac- to characterize the activating effects of GPI sub-
tors delivered to macrophages during trypanosome stituents on macrophages. It appears that GIP-sVSG
infection are only partially understood. One major and GPI-mfVSG (containing the DMG lipid sub-
activation factor is of parasite origin; this is the gly- stituent) have similar macrophage activating capa-
cosylphosphatidylinositol (GPI) membrane anchor bilities in terms of TNFα, IL-6 and NO production,
of the trypanosome VSG molecule (see Figure 1). but that there may be subtle differences in the abili-
The GPI anchor precursor is synthesized in the ty of GPI-mfVSG to more effectively induce IL-1 and
endoplasmic reticulum and subsequently is cova- IL-12 production (Magez et al, 1998; Schofield et al,
lently attached to newly synthesized VSGs after 1996; Schofield and Tachado, 1996; Tachado et al,
proteolytic cleavage of a VSG C-terminal GPI 1996; Tachado and Schofield, 1994). An extension of
attachment signal sequence (Bangs et al, 1988; these initial studies regarding the ability of GIP-
Cross, 1990; Doering et al, 1989; Doering et al, 1990; sVSG to interact with macrophages demonstrates
Englund, 1993; Ferguson et al, 1988; Masterson that the GIP-sVSG component binds directly to
et al, 1989; Menon, 1999; Menon, 1994, Menon et al, macrophages and induces expression of a specific
1997; Menon et al, 1990; Menon et al, 1990; Patnaik subset of activation genes in an IFN-γ independent
et al, 1993; Raper et al, 1993; Sharma et al, 1999; manner (Imboden et al, submitted for publication
Vidugiriene and Menon, 1995; Werbovetz and 2001; Paulnock and Coller, 2001). Studies have also
Englund, 1997). After further modifications in both shown that GPI substituents exhibit signaling activ-
the glycoprotein and GPI anchor residue, the ities (Tachado et al, 1997). That specific signal(s) are
mature VSG is transported to and anchored in the delivered to the cell is apparent from the resultant
trypanosome plasma membrane as membrane-form activation phenotype of macrophages exposed to
VSG (GPI-mfVSG). During the course of infection, a GPIs, and by recent in vitro studies showing that
trypanosome membrane-associated phospholipase GIP substituents (specifically the core glycan
C (GPI-PLC) becomes activated and cleaves the GPI sequence) activate a specific protein tyrosine kinase,
anchor as shown in Figure 1; this results in the while the DMG substituent may independently acti-
release of substantial soluble VSG (GIP-sVSG) that vate a protein kinase C isoform in macrophages
retains only the glycosylinositolphosphate (GIP) (Tachado et al, 1997). However, it is not yet known
substituent of the original GPI anchor and leaves the how GPIs interact with the macrophage membrane
dimyristoylglycerol (DMG) lipid component nor what receptor(s) may be important in delivering
remaining behind in the membrane (Armah and GPI-mediated activation signals to the cell nucleus.
Mensa-Wilmot, 2000; Bulow et al, 1989; Butikofer
et al, 1996; Hereld et al, 1988; Hereld et al, 1986; During trypanosome infection, however, it is clear
Mensa-Wilmot and Englund, 1992; Mensa-Wilmot, that host cells are exposed to biologically active lev-
1995; Paturiaux-Hanocq et al, 2000). Parasite num- els of GIP-sVSG, DMG and GPI-mfVSG, although
bers routinely fluctuate during infection, both in the the timing of exposure to these molecules and the
blood and extravascular tissues, primarily as the nature of their delivery to the macrophage mem-
result of host B and T cell responses to variant deter- brane could be very different (see Figure 2). While
minants of the VSG molecule. Since a) trypanosome the effects of such GPI substituents on uninfected
numbers may approach 108 organisms per ml blood macrophages have been partially characterized
and may also be quite high in the extravascular tis- in vitro, the impact of tissue saturation with these
sues during peak parasitemias, b) there are approx- activating agents in vivo during infection is just
imately 107 VSG molecules per cell, c) GIP-sVSG is being fully appreciated (Imboden et al, submitted
clipped and released from both viable and for publication 2001; Paulnock and Coller 2001).
stressed/damaged trypanosomes, and d) try- Also, macrophages are not the only cell type that
panosomes are episodically destroyed by antibody- can be targeted by GPIs; recent evidence demon-
(Ab-) and Th1 cell/macrophage-dependent effector strates that natural killer (NK) 1.1 T cells may

express T cell receptors (TCR) specific for GPI deter- panosome growth (Bakhiet et al, 1996; Bakhiet et
minants and that B cells may be stimulated to under- al, 1990; Hamadien et al, 1999; Olsson et al, 1991;
go cell differentiation by GPIs (Bento et al, 1996; Scho- Olsson et al, 1993; Vaidya et al, 1997). Thus a factor
field, et al, 1999). Thus, GPI substituents released by secreted from the parasite, TLTF, is visualized as
pathogens such as the African trypanosomes may inducing an essential trypanosome growth factor,
have broad effects on the host immune system that IFN-γ, from host cells.
surpass central activating effects on macrophages.
Experiments partially in conflict with these pro-
Role of IFN-γ in macrophage activation posed hypotheses exist, however. First there has
and early host protection and/or pathology been no independent confirmation that IFN-γ serves
The other major macrophage activation factor pro- as a growth factor for African trypanosomes;
duced during trypanosomiasis is IFN-γ, which is of unpublished studies from several labs have failed to
host origin. Small amounts of IFN-γ may be pro- substantiate the claim that IFN-γ serves as a growth
duced very early during infection as the result of factor and no critical biochemical studies of IFN-γ
TLTF activation of CD8 T cells (Bakhiet et al, 1996; binding to or utilization by trypanosomes have yet
Bakhiet et al, 1993; Schleifer and Mansfield, submit- been published. Furthermore, parasitemias are
ted for publication 2001; Vaidya et al, 1997). First higher in IFN-γ knockout mice, which are highly
described by Bakhiet, Olsson and colleagues susceptible rather than more resistant to infection
(Bakhiet et al, 1993; Bakhiet et al, 1996; Bakhiet et al, with T. b. rhodesiense (see discussion below on the
1990; Olsson et al, 1991; Olsson et al, 1993; Olsson role of IFN-γ in early host resistance [Hertz et al,
et al, 1992) and subsequently cloned by the Do- 1998]). One might reasonably expect that parasi-
nelson laboratory (Vaidya et al, 1997), TLTF is a 453 temias would be lower in IFN-γ deprived animals if
amino acid protein with potentially important bio- this cytokine served in any substantial manner as a
logical effects. TLTF was discovered when resear- growth factor. Additionally, TLTF expression is
chers noted that rodents injected with T. b. brucei, or identical in trypanosomes expressing high and low
lymphoid cells cultured with trypanosomes in vitro, virulence attributes (Mansfield, 2001), suggesting
exhibited an increase in the number of antigen non- that there is no modulation of the gene or protein in
specific IFN-γ secreting cells; depletion of CD8+ T organisms known to exhibit rapid growth character-
cells in animals or cultures abrogated the effect and, istics and virulence attributes for mammalian hosts.
interestingly, also resulted in less trypanosome
growth (Bakhiet et al, 1990). Use of a chamber sys- Initial studies asserted that TLTF was a secreted pro-
tem separating lymphoid cells and trypanosomes tein (Vaidya et al, 1997), but subsequent characteri-
showed that a soluble factor was responsible for zations showed that the amino acid sequence does
induction of IFN-γ synthesis (Olsson et al, 1991). not contain a hydrophobic region typical of mem-
brane-transported trypanosome proteins, though it
Several Trypanosoma species appear to express TLTF did appear to be targeted to the flagellar pocket
but may possess different IFN-γ stimulating abilities region through an apparent conformational signal
as measured by the relative increase of IFN-γ pro- within a specific 144 amino acid domain (Hill et al,
ducing cell numbers in the presence of extracts or 1999). To date there is no clear evidence that TLTF is
culture filtrates of species including T. evansi, T. b. rho- a secreted protein, though the predicted protein
desiense, and T. b. gambiense (Bakhiet et al, 1996). Subse- does possess unique internal targeting sequences.
quent characterization of CD8+ T cell IFN-γ activa- More recent studies on the cell biology of TLTF have
tion by TLTF showed that tyrosine protein kinases suggested an alternate (or coincident) role for the
are necessary for activation but protein kinase C and protein. The gene sequence identified as TLTF is
protein kinase A specifically are not (Bakhiet et al, expressed in both insect and bloodstream forms of
1993). Interestingly, TLTF may stimulate other cells T. b. brucei and the protein appears to be tightly asso-
to release IFN-γ, such as rat dorsal root ganglia, and ciated with the flagellar cytoskeleton (present in
this secretion apparently also is dependent on tyro- detergent-resistant and Ca2++-resistant cytoskeletal
sine kinase(s) (Eltayeb et al, 2000). These types of fractions of trypanosome extracts) (Hill et al, 2000);
studies and their experimental extension over the modification of TLTF gene expression in the pro-
past decade have led investigators to posit the fol- cyclic form resulted in an unusual motility defect,
lowing hypotheses with respect to the role of TLTF: suggesting that TLTF may be an integral part of the
trypanosomes secrete TLTF which binds to CD8 trypanosome cytoskeletal architecture. Surprisingly,
molecules expressed on CD8+ T cells, thereby in- TLTF-like genes are present in a number of diver-
ducing antigen non-specific activation and produc- gent eukaryotes including Drosophila and zebra fish.
tion of IFN-γ; TLTF-induced release of IFN-γ subse- Notably, the human growth arrest specific gene
quently serves as a growth factor that promotes try- (GAS)11, closely related to TLTF (Vaidya et al, 1997),

is a possible tumor suppressor molecule with a sub- linked to early host resistance during T. b. rhodesiense
molecular region that may localize to cellular micro- infection, and is thought to be associated with
tubules. macrophage activation characteristics responsible
for control of the parasites in extravascular tissue
Thus, it is difficult to see how TLTF, a tightly bound sites (De Gee et al, 1985; Hertz et al, 1998; Imboden
cytoskeleton-associated molecule, would be secret- et al, submitted for publication, 2001; Paulnock and
ed or released in biologically active levels during Coller, 2001).
infection or in cell cultures containing viable try-
panosomes to affect the release of IFN-γ from host
cells. Yet, it is clear that trypanosome infections and Figure 2. Macrophage activation in African try-
trypanosome extracts are capable of inducing IFN-γ panosomiasis is mediated by exposure to host and
release from naive host lymphoid cells in an antigen parasite factors.
nonspecific manner; the levels are low and occur
independently of antigen specific induction of IFN-
γ from host Th1 cells (Mansfield, 2001; Schopf et al,
1998). Thus, IFN-γ secretion induced by parasite Trypanosomes
material(s) has been a repeatable phenomenon and
is clearly of some interest; there is the distinct possi-
RELATIVE LEVELS
bility that release of biologically active TLTF (or a DMG
similar molecule with closely related effects) occurs
during periods of cataclysmic elimination of try- GIP-sVSG
panosome variant antigen types (VATs) by host Ab
and Th1/macrophage cell responses throughout
infection (see below), rather than by an active secre- IFN-γ
tory pathway, to induce IFN-γ. Given that try-

panosomes / trypanosome extracts are capable of 0 1 2 3 4 5 6 7 8 9 10
inducing nonspecific IFN-γ release from host cells, DAY OF INFECTION
and that IFN-γ may be an early and critical factor in
host protection (probably important in regulating
parasite numbers in the extravascular tissues—see (Figure adapted from Paulnock and Coller, 2001)
Figures 2 and 3), it may be that TLTF is important in
a different context than originally suggested: by The theoretical timing of exposure to host- and parasite-
inducing low levels of IFN-γ that control an early derived macrophage activating factors during an early
potentially explosive spread of trypanosomes in peak of parasitemia in trypanosome infection is shown
infected host tissues, regardless of the genetically- here. GIP-sVSG homodimers, released from the try-
based resistance status of the host. Such an early panosome plasma membrane by the action of GPI-PLC,
control over parasite expansion and, in susceptible are detectable within several days of infection throughout
animals or individuals, a delay in host death, would the body (Diffley and Jayawardena, 1982; Diffley and
permit the host to survive for a period of time so Straus, 1986; Diffley et al, 1980; Norton et al, 1986).
that the possibility of trypanosomes being taken up Although DMG is liberated by the same GPI-PLC cleav-
in a blood-meal and transmitted to new hosts could age event as GIP-sVSG, host tissues may be exposed ear-
occur. Regardless of one’s view of TLTF, the hard lier to GIP-sVSG since effective membrane transfer of
DMG would largely be dependent on high concentrations
work of providing functional and genetic linkages
of parasites in contact with macrophage membranes. GIP-
between TLTF expression and biological effects on
sVSG can activate macrophages independently of residual
the host remain.
DMG (Imboden et al, submitted for publication 2001;
Magez et al, 1998; Paulnock and Coller, 2001; Schofield
Clearly, however, the major source of IFN-γ during
and Tachado, 1996; Tachado et al, 1997). During immune
infection appears to be parasite antigen stimulated
clearance of trypanosomes, macrophages take up para-
Th1 cells, which appear in significant numbers
sites and presumably are also exposed to intact GPI-
somewhat later than early TLTF induced IFN-γ mfVSG as well as DMG remaining from GPI-PLC cleav-
responses. Th1 cell responses to VSG (and to invari- age of the anchor. Low levels of IFN-γ may also serve as
ant antigens of the parasite) have now been charac- an early activating agent during infection, presumably
terized and result in strong IFN-γ responses in derived from TLTF-stimulated CD8 T cells. Cytokine lev-
genetically more resistant animals but not in sus- els subsequently rise substantially over time due to CD4
ceptible animals (Hertz et al, 1998; Hertz and Th1 cell stimulation by VSG and other parasite antigens.
Mansfield, 1999; Schleifer et al, 1993; Schopf et al, The patterns shown here may differ markedly later in
1998). The IFN-γ response has been definitively infection.

The IFN-γ/IFN-γ receptor interaction and down- that susceptible mice, which normally do not make
stream subcellular signaling pathways have already a sufficient Ab response to VSG and do not clear
been well characterized in other model systems, and VATs from the blood, were afforded by donor cells
macrophage activation events triggered by IFN-γ from resistant mice a functional B cell response that
have been extensively studied (Adams and enabled them to clear parasitemia during infection;
Hamilton, 1984; Adams and Hamilton, 1987; Adams however, despite the ability to eliminate try-
and Hamilton, 1986; Hamilton, 1989; Paulnock, panosomes from the blood, these animals were just
1992; Paulnock, 1994; Paulnock-King et al, 1985). In as susceptible as mice receiving susceptible donor
African trypanosomiasis, it is clear that some bone marrow cells that failed to make protective
macrophage activation events are dependent on VSG-specific B cell responses (De Gee and
IFN-γ exposure and others on exposure to try- Mansfield, 1984). Subsequent genetic studies with
panosome-derived molecules, including GIP-sVSG crosses between Ab+ resistant and Ab- susceptible
(Imboden et al, submitted for publication 2001; mouse strains showed that the F1 hybrids all were
Paulnock and Coller, 2001). The activation patterns able to make VAT-specific Ab responses and control
observed in the presence of both factors are different parasitemias, but all such hybrids were as suscepti-
from either one alone, are dependent on the genetic ble as the susceptible parental strain (De Gee et al,
background of the infected host, and may be impor- 1988). Taken together, these types of results showed
tant in the control of infection. Why trypanosomes that the VSG-specific B cell response, although
induce such broad macrophage activation effects is linked to trypanosome clearance from the blood,
unknown. It may be important for trypanosomes to was not by itself functionally or genetically linked to
induce early temporal protection against the infec- overall host resistance.
tion regardless of the genetically-based resistance
status of the host (e.g. so the host isn’t killed by Th cell responses to trypanosome antigens
infection before natural transmission of the disease This information led the way to studies that first
can effectively occur). Alternatively, it might be elucidated Th cell responses to VSG and other try-
linked to the early generation of suppressor panosome antigens during infection (Hertz et al,
macrophage activity so as to depress host T cell 1998; Hertz and Mansfield, 1999; Mansfield, 1994;
responses to parasite antigens. Or it may result in Schleifer et al, 1993; Schopf et al, 1998). T cell
deregulation of IFN-γ-induced activation events in responses to trypanosome antigens were not discov-
macrophages in order to avoid parasite elimination. ered previously because of several interesting char-
Clearly, a goal of unraveling the cellular and molec- acteristics of trypanosome infections. First, a non-
ular basis of macrophage activation by try- specific immunosuppression of T cell responses in
panosome-derived antigens or other factors (e.g. trypanosomiasis had been recognized for many
DNA released from dead trypanosomes [Shoda years (Mansfield and Wallace, 1974), and, although
et al, 2001]) in the context of overall host resistance earlier studies revealed that T cell responses to try-
to infection and tissue pathology is important. panosome antigens could be induced in immunized
animals (Campbell et al, 1982; Finerty et al, 1978),
Tissue specific immune control such responses were not readily detectable in infect-
mechanisms in early infection ed animals (Mansfield and Kreier, 1972; Paulnock
There are clear differences in the ability of various et al, 1989). For example, not only were spleen or
host species, and strains within species, to display lymph node T cells from infected mice unable to
relative resistance to African trypanosomiasis proliferate in response to mitogens or antigen, they
(Levine and Mansfield, 1981; Mulligan, 1970). also failed to produce significant amounts of IL-2 or
Studies over the past twenty years have revealed IL-4, and these events could be shown to impact on
that the host Ab response plays only a partial role in T-dependent B cell responses to a variety of antigens
such relative resistance against trypanosomes. (Mansfield and Bagasra, 1978). This generalized
While VSG-specific Ab clearly is responsible for the immunodeficiency was shown to result in part from
cataclysmic elimination of VATs from the blood- the presence of macrophage “suppressor cells” in
stream of infected hosts, it is now known that this lymphoid tissues (Sacks et al, 1982; Wellhausen and
event is not linked, functionally or genetically, to Mansfield, 1980; Wellhausen and Mansfield, 1979;
overall host resistance (De Gee et al, 1988; De Gee Wellhausen and Mansfield, 1980); in fact,
and Mansfield, 1984; Mansfield, 1995; Mansfield, macrophages from infected mice had the capacity to
1990; Mansfield and Olivier, 2001). The seminal actively suppress the proliferative responses of nor-
studies were those in which H-2 compatible radia- mal T cells to mitogens and antigens in vitro and in
tion chimera mice, reconstituted with reciprocal vivo. A breakthrough in recognizing that Th cell
bone marrow cell transplants from relatively resist- responses to trypanosome antigens occurred during
ant or susceptible donors, revealed the following: infection came with the finding that functional com-

partmentalization of such responses occurred sons for the relative tissue compartmentalization of
(Schleifer et al, 1993). It was revealed that Th cells Th cell cytokine responses (e.g., IL-2 and IFN-γ pro-
reactive with VSG were predominant in the peri- duction by peritoneal Th cells, but mostly IFN-γ pro-
toneal T cell population; when stimulated with duction by Th cells in the peripheral lymphoid tis-
VSG, these cells made a substantial IL-2 and IFN-γ sues) have not been elucidated, the reason for inhi-
cytokine response but failed to proliferate. Subse- bition of T cell clonal expansion has now been
quently, it was discovered that Th cells in the resolved. Suppressor macrophages were shown to
peripheral lymphoid tissues also made an IFN-γ elaborate several factors that inhibited the prolifera-
response (but little IL-2) when stimulated with VSG. tive (but not the cytokine) responses of VSG activat-
Thus, it was apparent that VSG-reactive T cells were ed Th1 cells: NO, prostaglandins and TNFα (Darji et
present in infected animal tissues but that they al, 1996; Hertz and Mansfield, 1999; Schleifer and
exhibited a restricted cytokine response and mini- Mansfield, 1993; Sternberg and McGuigan, 1992).
mal evidence for clonal expansion (Schleifer et al, Macrophages were activated to produce these sup-
1993). Since these VSG-reactive T cells displayed a pressive factors primarily as the result of exposure
CD4+ αβ TCR+ membrane phenotype, expressed to GIP-sVSG and to IFN-γ released by parasite anti-
Type 1 cytokines, were major histocompatibility gen stimulated Th cells (Hertz and Mansfield, 1999;
class II antigen (MHC II) restricted and antigen pre- Mansfield et al, submitted for publication; Schleifer
senting cell (APC) dependent (Hertz et al, 1998; and Mansfield, 1993). The full impact of NO and
Schleifer et al, 1993; Schopf et al, 1998), it was clear prostaglandins on host immunity to trypanosomes
that they represented a classical Th1 subset of T cells has not been completely resolved, but studies with
that recognized VSG during infection. More recent iNOS KO mice have shown that, although NO is the
work has begun to elucidate the submolecular tar- main “suppressor” factor that limits clonal expan-
gets of VSG-reactive Th cells. In preliminary studies sion of T cells (and maybe also modulates cytokine
it has been shown that Th cell specificities are direct- responses to a degree) the absence of NO did not
ed against a defined hypervariable subregion of affect overall host resistance (Hertz and Mansfield,
VSGs that is not exposed when VSG homodimers 1999).
are assembled into the surface coat structure
(Mansfield, 2001; Mansfield and Olivier, 2001), ful- Early and strong trypanosome-specific Th1 cell
filling earlier predictions that VSG sequence vari- responses may provide an essential component of
ability in nonexposed regions of the molecule might host resistance; this realization emerged from stud-
be driven by T cell selection (Blum et al, 1993; Field ies with cytokine gene knockout mice. The central
and Boothroyd, 1996; Reinitz et al, 1992). finding in one study was that mice with a resistant
genetic background but which lacked a functional
The extreme polarization of the Th1 cell cytokine IFN-γ gene were as susceptible as scid mice to try-
responses seen in some experimental systems is due panosome infection, even though those mice pro-
in part to the early production of IL-12 by duced Abs sufficient to control parasitemia (Hertz et
macrophages exposed to trypanosome GPI sub- al, 1998). In contrast, the same genetic strain of
stituents (Mansfield et al, submitted for publica- mouse with the IL-4 instead of the IFN-γ gene
tion). That IL-12 is not the only polarizing factor is knocked out were as resistant as wt mice to infection
seen from preliminary studies with IL-12 knockout (Hertz and Mansfield, 1999). These results under-
(KO) mice and mice exposed to Abs against IL-12; in scored earlier studies demonstrating that the VAT-
each case, early temporal depression of the Type 1 specific Ab response and control of parasitemia
cytokine response did not result in a compensatory were not capable of providing resistance alone, and
Type 2 cytokine response and, after a period of 10 that the production of a single cytokine, IFN-γ, in
days or so, the Th1 cell response emerged in both response to infection was found to be a critical ele-
groups (Mansfield et al, submitted for publication). ment in host resistance. The mechanism(s) associat-
That IL-12 is not the only polarizing factor is seen ed with IFN-γ-mediated resistance are not yet clear,
from preliminary studies with IL-12 knockout (KO) but seem to involve macrophage factors induced by
mice and mice exposed to Abs against IL-12; in each IFN-γ activation. Several candidate factors have
case, early temporal depression of the Type 1 been proposed, such as NO and TNFα, both of
cytokine response did not result in a compensatory which have been shown to kill trypanosomes in vitro
Type 2 cytokine response and, after a period of 10 (Lucas et al, 1994; Lucas et al, 1993; Magez et al,
days or so, the Th1 cell response emerged in both 1997; Magez et al, 1999; Mnaimneh et al, 1997;
groups (Mansfield et al, submitted for publication). Vincendeau et al, 1992). Recent studies suggest,
Thus, there are complex features of infection that however, that neither factor alone is capable of
promote the production of Type 1 cytokines and the mediating resistance in vivo; results with try-
outgrowth of antigen-reactive Th1 cells. While rea- panosome infected iNOS KO mice and TNFα KO

mice showed that such mutations on a resistant phenotype of pro-inflammatory responses and parasitici-
mouse genetic background do not significantly dal macrophage activation to a phenotype of counter-
affect the course of infection (Hertz and Mansfield, inflammatory responses and Type 2 cytokine responses
1999; Magez et al, 1999; Millar et al, 1999), although that do not promote parasite elimination from tissues and
it is possible that the combination of NO and TNFα perhaps also from the blood. Adapted from Mansfield and
is required for functional resistance. Clearly, IFN-γ Olivier (2001).
inducible events in macrophages must carefully be
evaluated for their impact on trypanosomes during Thus, relative resistance to African trypanosomes
early stages of infection. Since these events occur may be mediated by two major components of host
independently of B cell mediated resistance mecha- immunity, neither one of which by itself is adequate
nisms that are known to control trypanosomes in to provide resistance (Figure 3A). First, VSG specif-
the vasculature, and since IFN-γ activated ic Ab responses control trypanosomes present in the
macrophage control mechanisms are presumed to blood. Second, T cell production of IFN-γ and sub-
be important in regulating trypanosome numbers in sequent macrophage activation events are necessary
the extravascular tissue spaces (but this by itself is to control trypanosomes in the extravascular tissues.
inadequate to provide protection [Mansfield, 2001]), Animals that make weak B cell and/or T cell
it appears that multiple arms of the host immune responses to trypanosome variant antigens invari-
system are required to control trypanosomes and to ably will demonstrate relative susceptibility; in con-
provide relative resistance during infection. trast, animals making pronounced B and T cell
responses (including appropriate macrophage acti-
Figures 3A and 3B. Innate and acquired immune vation events) will display relative high resistance.
elements in early- versus late-stage trypanosomiasis. However, this picture is evolving considerably due
to evidence that the later stages of experimental
infection display a different pattern, a pattern that is
associated with loss of resistance to trypanosomes
(see Figure 3B).
Overview
Events that impact on B, T or macrophage cell
responses during infection can be expected to cause
modulations in host resistance and tissue pathology.
These events need to be clarified in both animal
model systems and in clinical HAT.
Changes in Parasite Cell Biology during

Infection that Impact on Host Resistance
The African trypanosomes display considerable bio-
logical variation during their life cycle. This biolog-
ical variation is directed by specific patterns of gene
and protein expression. For example, bloodstream
trypomastigote forms display a number of different
surface antigenic phenotypes during infection of
their mammalian hosts; this phenotypic variation
has as its basis the differential expression of VSG
genes and molecules (Borst et al, 1998; Borst and
Rudenko, 1994; Cross, 1990; Donelson, 1987;
Van der Ploeg et al, 1992; Vickerman and Luckins,
1969). Additionally, the differentiation of long slen-
der (LS) trypomastigote forms to short stumpy (SS)
These figures portray the cell and molecular elements trypomastigotes during infection results in pro-
important in controlling trypanosome infection of host tis- found morphological and functional changes in
sues. In early infection stages, IFN-g cytokine responses these cells. Such changes include mitochondrial bio-
activate macrophages to produce factors cytotoxic for try- genesis and the acquisition of new metabolic path-
panosomes, limiting the spread or survival of parasites ways; it is the differential expression of specific
outside the vasculature. B cell responses appear to selec- genes and proteins that presage these biological
tively control trypanosomes within the vasculature. In changes (Bienen et al, 1983; Bohringer and Hecker,
late-stage infections, there appears to be a shift from the 1975; Hua et al, 1997; Mulligan, 1970; Mutomba and

Wang, 1998; Tschudi, 1995; Vanhamme and Pays, genetically distinct populations of trypanosomes, or
1995; Vickerman, 1971; Vickerman et al, 1993). whether there is intraclonal biological variation
Furthermore, the differentiation of SS forms to pro- within trypanosome populations that impacts on
cyclic forms in the insect vector also results from the the course of disease in a genetically defined host.
differential expression of specific genes and proteins
(Butikofer et al, 1997; Roditi, 1996; Ruepp et al, 1997; This question was addressed in an earlier study in
Vanhamme and Pays, 1995; Vickerman et al, 1988). which mice of a relatively resistant phenotype were
Finally, the transformation of insect forms to meta- infected with a single trypanosome of T. b. rhode-
cyclic trypomastigotes results in different morpho- siense clone LouTat 1 (Inverso and Mansfield, 1983).
logical and functional changes in the parasites that Several different VATs were isolated from para-
permit infection of a new mammalian host; these sitemic peaks at intermediate and late time points
changes also occur as the result of differential gene during infection of a single mouse; these VATs were
and protein expression (Turner et al, 1986; subcloned and characterized as to VSG phenotype.
Vanhamme and Pays, 1995; Vickerman, 1985; Three different VATs, which represented antigeni-
Vickerman et al, 1993). Thus, all trypanosomes cally distinct daughter cell populations clonally
exhibit considerable clonal plasticity in terms of derived from a single LouTat 1 parental cell, were
their antigenicity, morphology and biological func- used to infect the same mouse strain; the courses of
tion during the life cycle; this plasticity occurs as the infection were monitored in comparison with mice
direct result of differential expression of specific infected with LouTat 1. The interesting result was
subsets of genes and proteins. It follows that the fail- that each of the daughter cell populations exhibited
ure of trypanosomes to undergo biological variation a different virulence profile compared to the
at critical points in the life cycle may result in elimi- parental clone (Inverso and Mansfield, 1983). For
nation by the mammalian host, failure to differenti- example, LouTat 1 caused death in approximately
ate within the intermediate host and vector, or 62 days post-infection, while LouTat 1.3, LouTat 1.4
inability to establish new animal infections. and LouTat 1.5 caused death in approximately 44,
30, and 28 days, respectively. These results demon-
The molecular mechanisms that regulate changes in strated that VATs arising during infection expressed
VSG phenotype and stage of cellular differentiation virulence phenotypes different from the infecting
are shared in common among African trypano- VAT. In essence, daughter cells arising within a try-
somes; however these are not the only mechanisms panosome population expressed the capacity to
that regulate biological differences in these para- transcend host genetic resistance characteristics and
sites. New evidence is emerging that differential render a relatively resistant animal into a more sus-
susceptibility of brucei group trypanosomes to host ceptible one.
factors such as trypanosome lytic factor (TLF)
(De Greef and Hamers, 1994; Hager et al, 1994; These seminal observations have been repeated with
Hajduk et al, 1992; Rickman and Robson, 1970; consistent results, and related observations on clonal
Rifkin, 1978; Smith et al, 1995); may occur as the heterogeneity among trypanosomes have been made
result of clonal variation among trypanosomes in other studies (Diffley and Mama, 1989; Inverso
(Hager and Hajduk, 1997; Hajduk et al, 1995); the et al, 1988; Joshua, 1990; Mamman et al, 1995; Ortiz
basis for such changes has not yet been defined, but et al, 1994; Reinitz and Mansfield, 1988; Sacks et al,
is believed to encompass specific clonal modifica- 1980; Seed, 1978; Seed and Sechelski, 1996; Turner,
tions in gene or protein expression. Host specificity 1990; Turner et al, 1995). Additionally, the general
may also be defined by the clonal expression of dif- observation has been made that many other daugh-
ferent transferrin receptor genes (Bitter et al, 1998). ter cells/VATs arising naturally from LouTat 1 also
While these examples reflect on trypanosome infec- expressed differences in virulence (see Table 1,
tivity for a host species, related observations and below), and that the most virulent VATs seemed to
mathematical modeling predict that considerable arise at later time points in infection, just prior to
biological variation occurs among trypanosomes host death. Thus, the apparent result of infection
during infection that is not related directly to infec- with relatively low virulence trypanosomes is a pro-
tivity or cyclical differentiation in the life cycle gressive increase in population virulence with time,
(Turner et al, 1995; Vassella et al, 1997). For example, rather like the turning up of a “virulence rheostat”.
it is well known that different isolates, species and Based on these observations, it was speculated that
subspecies of trypanosomes exhibit remarkable the longer a trypanosome population existed in a
variation in pathogenicity and virulence for geneti- mammalian host, the more virulent that population
cally defined host species (Mulligan, 1970). A key might become. Such a virulence rheostat may have
question has been whether such differences in viru- evolved as a programmed mechanism to overcome
lence are immutable characteristics associated with different levels of host resistance that might be

encountered in nature, where there is a pool of coat structure (Inverso et al, 1988), transcribed iden-
genetically disparate mammalian hosts available for tical VSG genes (Reinitz et al, 1992; Uphoff et al, sub-
infection. Implicit in this speculation, however, is the mitted 2001) and expressed their VSG genes by a
idea that a virulence rheostat must somehow be duplicative transposition event from the same chro-
reset, perhaps upon cyclical passage through the mosomal telomeric expression site (Uphoff et al, sub-
intermediate host and vector, the tsetse fly. mitted 2001).
Since virulent trypanosomes seem to arise after a Table 1. Virulence phenotypes of

prolonged period of replication in an infected host, LouTat 1-derived VATs.
it may be possible to generate highly virulent try-
panosomes by rapid subpassage of low virulence Variant Antigenic Types Virulence
trypanosomes through mice for a substantial time (Mean Survival Time)
period; in essence, the effect would be one mimick- (A) VATs isolated from natural infections*
ing a single, prolonged course of infection. This was LouTat 1 (parental clone) 62
achieved by infecting irradiated mice with LouTat 1 LouTat 1.9 46
LouTat 1.3 44
and subpassaging the trypanosomes into different LouTat 1 4 30
mice every three days for approximately six months. LouTat 1.5 28
At the end of this time, trypanosome stabilates were LouTat 1.7 25
LouTat 1.2 13
made from sublines and subclones, and were LouTat 1.6 11
assessed for their virulence characteristics. One rep- LouTat 1.10 6
resentative subclone, designated LouTat 1A, was

(B) VATs derived by subpassage**
examined in some detail. These trypanosomes dis-
played a single uncontrolled peak of parasitemia LouTat 1A 4
LouTat 1D 4
and were able to kill a resistant mouse strain (as well LouTat 1X 4
as all other resistant or susceptible strains of mice) in LouTat 1n 6-60
approximately four days post-infection; in contrast
the parental clone LouTat 1 gave rise to multiple Therefore, it is unlikely that either VSG molecules or
peaks of parasitemia and a prolonged survival time the active VSG gene expression site are important
of over 60 days in the same mouse strain (Inverso for virulence regulation in trypanosomes (see
et al, 1988). Thus a model system of comparative Figures 4-6). Confirmation that VSG genes and VSG
trypanosome virulence was developed from this gene expression sites are not involved in virulence
approach, in which the relatively low virulence came from additional experimental approaches.
clone LouTat 1 and the relatively high virulence sub- New sublines and subclones were derived by rapid
clone LouTat 1A represent different ends of a viru- subpassage of LouTat 1 through irradiated mice, as
lence spectrum, with the virulence of other natural- above, and examined for VSG phenotype and gene
ly arising VATs existing somewhere between these expression. Many of the subclones expressed the
two extremes (Table 1). same VSG gene as LouTat 1 and all were highly vir-
ulent like LouTat 1A (Table 1). In another approach,
A natural question that arose from these types of LouTat 1A was used to infect rabbits and goats;
studies was whether the VSG molecules expressed although these animals exhibited pathology sooner
by virulent VATs acted as virulence factors, with spe- than LouTat 1 infected controls, they did not die as
cific VSG isotypes exerting defined biological effects early as mice and trypanosomes were able to under-
on the host. This idea was not unfounded since sev- go antigenic variation. The VATs generated from
eral biological traits associated with VSG molecules LouTat 1A in these animals were isolated and sub-
have been described in the literature (Mathias et al, sequently used to infect mice; the results showed
1990; Musoke and Barbet; 1977; Schofield et al, 1999; that the LouTat 1A-derived VATs were as virulent
Tachado et al, 1997; Tizard et al, 1978). Alternatively, for mice as LouTat 1A (Inverso et al, 1988). Thus
one could also speculate that expression site-associ- high levels of virulence, once expressed in mam-
ated genes (ESAGs) co-transcribed with certain VSG malian hosts, appear to be a constitutive trait that is
genes at specific chromosomal expression sites may unaffected by further VSG gene switching.
be responsible for virulence expression or regulation.
This idea was based on observations of others con-
cerning potential growth or differentiation regulato-
ry roles associated with ESAGs (Cross, 1990;
Vickerman et al, 1993). However, an analysis of the
LouTat 1/LouTat 1A model system revealed that
both organisms displayed the same antigenic surface

Figure 4. clones. A few chromosomes were altered in size, as
Amino acid sequence of the T. b. rhodesiense LouTat 1/ LouTat 1A determined by pulsed field gel electrophoresis;
VSG molecule. The protein sequence was deduced from the iden-
tical full-length nucleotide sequences (Accession no. X56643)
however, there was no net loss of cellular DNA nor
(Reinitz et al, 1992) as well as from partial amino acid sequencing were any differences apparent in restriction frag-
data (Inverso et al, 1988). The N–terminal signal sequence and the ment length polymorphism (RFLP) patterns utiliz-
C-terminal hydrophobic extension are underlined; threonine-27
ing a number of random and known non-VSG
is the start site of the mature protein.
cDNA probes. Thus, the chromosomal size varia-
tions observed may largely be subtelomeric or sim-
ply do not involve chromosome regions to which
the probes hybridized. Two-dimensional gel elec-
trophoresis of 35S-labeled proteins showed not
only that different proteins were expressed in
LouTat 1A compared to LouTat 1, but also that
there were different proteins expressed in LouTat 1
compared to LouTat 1A. Competitive Northern
analyses in which labeled total cDNA from LouTat
1A, in the presence of excess unlabeled LouTat 1
competitive total cDNA, was hybridized to mRNA
from LouTat 1A showed that that there were
numerous mRNA species unique to LouTat 1A.
Taken together, these observations suggested that a
Figure 5. Southern blot analysis of LouTat 1
subset of genes and proteins was being expressed
and 1A VSG genes.
Genomic DNA from LouTat 1, 1.5 and 1A was digested with in LouTat 1A that was not being expressed at the
XmnI and hybridized in a Southern blot with a labeled LouTat 1 same level in LouTat 1.
VSG cDNA probe (BC, basic copy of VSG gene; ELC, expression
linked copy).
Overall, preliminary observations on the biological
behaviour of LouTat 1 and LouTat 1A, and on the
subcellular differences detectable between LouTat 1
and LouTat 1A, as well as biological variation
observed with other subspecies of T. b. brucei in
terms of TLF susceptibility or TNFα sensitivity, have
led to the hypothesis that African trypanosomes
have the capacity to regulate clonal expression of
virulence. Specifically, it has been proposed (a) that
trypanosomes have evolved the programmed
capacity for clonal upregulation of virulence as a
means to successfully subvert host resistance mech-
anisms, regardless of host genetic background; (b)
that this capacity to modify virulence phenotype
occurs independently of changes in VSG gene
expression; and (c) that the level of virulence
expressed is determined by differential gene and/or
protein expression during trypanosome growth in
an infected host.
Overview
Figure 6. Restriction map of the LouTat 1 It seems therefore an important goal to characterize
and LouTat 1A VSG gene expression sites. the molecular mosaic associated with the virulence
The expressed copies are in a telomeric site while the basic copy phenotype and to determine whether or not specific
of the VSG gene is found at an internal chromosomal site that is
subsets of genes or proteins linked to virulence can
the same for both trypanosomes. The solid bar denotes the VSG
gene. be identified; the prediction is that such approaches
may open new doors in the understanding of try-
panosome-mediated pathogenesis, and candidate
Subsequently LouTat 1 and 1A were examined for virulence factors could be targeted for specific
non-VSG related cellular differences (Mansfield, immuno- or chemotherapies.
2001). Comparative analysis of several traits re-
vealed significant differences between the two

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II APPLIED GENOMICS: PROSPECTS and for research on the development of new drugs.
FOR CONTROL OF AFRICAN TRYPANO- While this is encouraging news, treatment alone for
SOMIASIS VIA THE TSETSE VECTOR a zoonosis for which there is no evidence of acqui-
red immunity is likely to be extremely costly in the
Serap Aksoy1, Wendy Gibson2, Ron H. Gooding3, long run even if effective drugs were to be available.
Elliot Krafsur4, Anna Malacrida5, Michael J. The fact that the parasite relies on a single insect for
Lehane6, Scott L. O’Neill1, Terry Pearson7, Alan S. its transmission opens up many avenues for control
Robinson8, Philip Solano9, Antigone Zacharo- via the control of its vector. It is important to note
poulou10 and Liangbiao Zheng.1 that trypanosomiasis is a disease based on interac-
tions among at least three organisms, the human,
1Department of Epidemiology and Public Health, Section the parasite and the tsetse fly. Interference with any
of Vector Biology, Yale University School of Medicine, 60 of these interactions can prevent disease. In fact,
College St., 606 LEPH, New Haven, CT 06510, USA. tsetse control strategies have been widely used for
2School of Biological Sciences, University of Bristol, management of animal diseases. Most of these
Bristol BS8 1UG, U.K. efforts however have been on a small scale, involv-
3Department of Biological Sciences, University of ing trapping and the use of insecticide sprays, and
Alberta, Edmonton, Alberta, Canada. have been costly and difficult to sustain. A recent
4Department of Entomology, Iowa State University, 407 study looking into the cost/benefit analysis of vari-
Science 2, Ames, Iowa 50011, USA. ous strategies clearly identifies large-scale, area-
5Dipartimento di Biologia Animale, Zoological wide methods as being far more efficient and affor-
Laboratory, Universita di Pavia, Pavia, Italy. dable in the long run for tsetse control (Budd, 1999).
6School of Biological Sciences, University of Wales, In Lome, Togo, July 2000, the Organization of
Bangor LL57 2UW, U.K. African Unity (OAU) endorsed a strategy of tsetse
7Department of Biochemistry and Microbiology, fly and trypanosomosis eradication on the African
University of Victoria, Victoria, BC V8W3P6, Canada. continent by using an integrated approach includ-
8Entomology Unit, FAO/IAEA Agricultural and ing area-wide control strategies. The recent advan-
Biotechnology Laboratory, A-2444 Seibersdorf, Austria. ces in molecular technologies and their application
9Institut Pierre Richet, 01 BP 1500 Bouke 01, Côte to insects have revolutionized the field of vector
d’Ivoire. biology although progress in the tsetse field has
10University of Patras, Department of Biology, been slow. In this paper, we review the current sta-
Laboratory of Genetics, Patras, Greece. tus of knowledge on the molecular aspects of tsetse
with particular attention given to interaction with
INTRODUCTION trypanosomes. We highlight areas where research
Despite many decades of research on vector-borne efforts are needed and are likely to contribute either
parasites and their development in mammalian to the development of new vector control strategies
hosts, effective strategies have yet to materialize for or to the improvement of the efficacy and afford-
control of any of the diseases with which they are ability of existing approaches.
associated. At the same time, a heavy reliance on
insecticides and therapeutic drugs has resulted in TSETSE GENOMIC STUDIES
the spread of insecticide resistance in many vectors The trypanosome has been most studied in efforts to
and the emergence of drug resistance in parasites, develop a mammalian-based vaccine. While these
threatening the availability of effective tools to com- research efforts have turned trypanosomes into a
bat these diseases. This scenario is also apparent for model eukaryote to study novel mechanisms of
sleeping sickness in Africa where there are current- gene expression and cell biology, there are no effec-
ly at least half a million people estimated to have tive products forthcoming for disease control in the
contracted the fatal disease. Antigenic variation in foreseeable future. There are now genome sequenc-
the mammalian host has hampered efforts for vac- ing programmes for the parasite, and the genome of
cine development. The current strategies for man- the human host is being deciphered. In contrast, to
agement of trypanosomiasis depend on active sur- date, only a handful of tsetse genes and proteins
veillance and treatment of infected hosts, and on have been characterized. This is also in sharp con-
limited vector control measures. These efforts are, trast to the vast level of knowledge available for
however, restricted by the lack of effective drugs, mosquitoes such as Anopheles gambiae, for which an
their high cost, adverse side effects, and the emer- international genome sequencing effort is now in
gence of drug resistance in patients (McNeil, 2000). place, and Aedes aegypti, where a similar genome
Thanks to recent international effort, there are now project is currently being planned. The many molec-
several programmes in place that will allow contin- ular markers developed in A. gambiae have formed
ued provision of the existing drugs for treatment the basis of a rapidly increasing number of popula-

tion genetics and ecological studies. The availability na, maternally inherited factors appear to be in-
of contemporary genomics information will provide volved in hybrid male sterility (Gooding, 1990). Suc-
us with essential tools for investigating parasite- cess in hybridizing members of the subgenus Glos-
host interactions as well as vector population struc- sina is often highly asymmetric, with one cross being
tures. This information is central for vector-based significantly more productive than the reciprocal
control strategies to be effectively developed. cross. Gooding (1987) attributed this asymmetry to
maternally inherited factors, which are now
TSETSE POPULATION GENETICS believed to be Wolbachia (O’Neil, et al, 1993). These
Population Structure factors can lead to unusual situations. For example,
The recent availability of several microsatellite loci using G. m. morsitans and G. m. centralis it has been
and their application to population analysis has possible to produce backcross males with maternal-
revealed extensive genetic structuring in tsetse pop- ly inherited factors from G. m. morsitans and chro-
ulations of the morsitans and palpalis groups at vari- mosomes from G. m. centralis, and yet such males
ous geographical scales (Krafsur, et al, 2000; Krafsur can fertilize G. m. morsitans but not G. m. centralis
and Griffiths, 1997; Krafsur, et al, 2000; Luna, et al, (Gooding, 1987). It was suggested that such flies
2001; Solano, et al, 2000; Wohlford, 1999). In the case could be used as genetic control agents against
of G. palpalis gambiensis, these populations were G. m. centralis, but the phenotypic expression of
shown to transmit different species of trypanosomes these maternally inherited factors is unstable
with different efficiencies (Solano, et al, 2000; Solano, (Gooding, 2000; Gooding, 1990). However, if the sta-
et al, 1997). Additional microsatellite loci and the bility problem can be overcome, this approach may
development of efficient oligonucleotide primers are be useful for control of certain members of the sub-
required to achieve the level of genetic resolution genus Glossina.
necessary to measure accurately the differentiation
of taxa and populations and gene flow within and Most hybridization studies on tsetse have used clo-
among them. In addition, randomly amplified poly- sely related species and subspecies. There have been
morphic DNAs (RAPDs) have been used successful- few “intra-taxon crosses”, using flies from colonies
ly to examine paternal lines of gene flow, an impor- that were established from widely separated geo-
tant tool in studying historical lines of dispersion graphic areas. Without exception, the latter studies
(Malacrida, et al, 1999). These studies are important found that F1 males were fertile and this has been
for vector control approaches such as the sterile interpreted as indicating that, within a nominal
insect technique (SIT), which is being considered for taxon, there are no genetic barriers between geo-
use in upcoming tsetse eradication campaigns. For graphically separated populations. However, hy-
the successful application of SIT, information is re- brid breakdown has recently been found in tsetse
quired on the tsetse species represented in the area, (Gooding, unpublished). When G. p. palpalis from
the degree of reproductive isolation among field colonies that originated from widely separated geo-
populations, the existence of natural barriers to dis- graphic areas were crossed, the first generation off-
persion, and the locations of dense, stable popula- spring had normal fertility, but a high proportion of
tions from which immigration is likely. males in the second and subsequent generations,
and in backcrosses to the parental lines, were sterile.
Hybrid Sterility The molecular basis for the hybrid breakdown is
Hybrid sterility has been considered as a potential unknown, but its elucidation will be most helpful if
control method for tsetse ever since Vanderplank hybrid breakdown is to be exploited for tsetse con-
eradicated an isolated population of G. swynnertoni trol. Hybrid breakdown may be a useful genetic
by release of G. m. centralis into its habitat (Vander- control approach for tsetse, especially if its manifes-
plank, 1948; Vanderplank, 1944). In the two subgen- tations include reduced fecundity of females and/or
era of Glossina that contain the most important try- reduced longevity of adult flies. In any case, the
panosome vectors, Glossina sensu stricto and Nemo- existence of hybrid breakdown in tsetse suggests
rhina, there are several groups of closely related taxa that there may be cryptic species of tsetse, a possi-
that hybridize readily both in the field and in the bility that could have implications for evaluating
laboratory (Gooding, 1990). Hybridization of tsetse what now appears to be simply geographic varia-
almost always results in production of hybrid fema- tion in vector competence. Further work with other
les with lower than normal fecundity and males that widely distributed species is clearly needed.
are completely sterile (Gooding, 1990). The sterility
in males is attributable mainly to incompatibility Polytene Chromosomes
between sex chromosomes from two taxa, but auto- Polytene chromosomes have proven especially va-
somal genes are also involved in some cases. In the luable in studies of chromosome structure and func-
subgenus Glossina, but not in the subgenus Nemorhi- tion. They provide a means for the accurate map-

ping of chromosome rearrangements and for the (Acosta-Serrano, et al, 2001). It has also been shown
precise localization of genes by using both chromo- that the binding of a lectin (concavanalin A) to the
some rearrangement analysis and the technique of procyclin coat molecules of the procyclic forms
in situ hybridization. This has been technically chal- induces multinucleation, a disequilibrium between
lenging in tsetse, but photographic polytene maps nuclear and kinetoplast replication and a unique
have now been constructed for three species, form of cell death (Pearson, et al, 2000). Those sur-
G. austeni, G. m. submorsitans, and G. pallidipes. Com- viving procyclic cells eventually proliferate in the
parison of the homologous chromosomes between gut (establishment phase) and flies can be scored
the three species indicates that, in addition to simi- with infections seven-ten days after acquiring an
larities in their banding patterns, there are also var- infectious meal. The subsequent maturation phase
ious major differences, especially between G. austeni occurs in the salivary glands for T. brucei (Vicker-
and the other two species (Dr A. G. Papalexiou, man, et al, 1988). Here, they first differentiate into
University of Patras, unpublished). Studies can now attached proliferating epimastigote forms which
be undertaken to identify and characterize chromo- then yield the infective, free-living metacyclic forms
somal rearrangements in field populations to which are transmitted to the next host during blood-
understand genetic variation. The development of feeding by the fly (Vickerman, et al, 1988). It is at
in situ hybridization using FISH analysis with this stage that parasites are thought to undergo
cloned tsetse genes and selected molecular probes genetic exchange (Gibson and Whittington, 1993).
such as microsatellites will facilitate the eventual The factors triggering this differentiation step are
mapping of genes of interest (e.g. genes affecting unknown. There is believed to be a critical period
vector competence, refractoriness, etc.). for maturation between days 8 and 11 after infection
(Ruepp, et al, 1997; Sinkins, et al, 1995). One sugges-
VECTORAL CAPACITY OF NATURAL TSETSE tion is that lectins have a role, since feeding lectin
POPULATIONS inhibitory sugars can block maturation (Sinkins, et
Trypanosome infections with T. brucei ssp. complex al, 1995). However, it is clearly crucial to investigate
parasites are typically detected in less than 1% of the the role of other components of the immune system.
field population of tsetse flies (Lehane, et al, 2000;
Msangi, et al, 1998; Woolhouse, et al, 1994). Even The first physical barrier to ingested parasites in the
under ideal conditions in the laboratory, transmis- gut is the peritrophic matrix (PM), which is a promi-
sion rates are between 1-10% depending on the fly nent feature of the digestive tract of insects. While in
species/strain and parasite strain (Moloo, et al, many adult insects, PM components are secreted by
1994; Moloo and Kutuza, 1988; Moloo, et al, 1992). midgut cells in response to a blood-meal, tsetse
The basis for this refractoriness is not known, but adults have a PM constitutively synthesized from
depends on tsetse species/strains and their symbi- cells in the proventriculus (cardia) in the foregut
otic bacteria as well as the genotype of the strain of prior to feeding (Lehane, et al, 1996; Lehane and
the parasite acquired. In laboratory infections, the Msangi, 1991). How trypanosomes cross the PM to
age and sex of the fly and the source of the blood- move from the endo- to the ectoperitrophic space of
meal also contribute to the outcome of the infection. the gut has been controversial, with penetration of
Field data are limited, however infections with mul- the thick chitinous PM and migration around its
tiple parasites are common, indicating that they open posterior end in the hindgut both having been
were acquired in different blood-meals, hence fly proposed (Ellis and Evans, 1977; Welburn and
age may not be as significant a factor as once Maudlin, 1999). It is generally thought that during
thought. normal development in the fly there are no intra-
cellular stages, although reports of intracellular
The life cycle of T. brucei in the tsetse fly begins T. b. rhodesiense (Ellis and Evans, 1976; Evans and
when it feeds from an infected mammalian host. Ellis, 1975) and T. congolense (Ladikpo and Seureau,
The non-proliferating short stumpy parasites that 1988) in anterior midgut cells have been published.
are pre-adapted for life in tsetse fly rapidly differen- It is also thought that during normal infection, try-
tiate into procyclic forms in the gut lumen, lose their panosomes do not cross an epithelial barrier in the
variant surface glycoprotein, and express a new coat fly, although once again there are several reports of
composed of procyclin proteins. The procyclin coat trypanosomes in the hemolymph of flies (Mshel-
contributes to the establishment of infections in the bwala, 1972; Otieno, 1973). Also unknown is how
fly (Ruepp, et al, 1997). It has recently been shown procyclic parasites move from the gut to the mouth
that procyclic cells express different procyclin coats parts and/or the salivary glands of the fly. The clas-
during establishment in the gut and that the N-ter- sical route involves crossing back into the gut lumen
minal domain of all procyclins are quantitatively across the proventriculus in the foregut and thence
removed by proteolysis in the fly, but not in culture to the mouthparts and salivary glands (Ruepp, et al,

1997; Turelli and Hoffmann, 1991). This is indeed an panosomes can be significantly reduced (Zhen-
incredible journey, which few trypanosomes appar- grong, et al, 2001). Further characterization of these
ently embark on or complete (Ruepp, et al, 1997). immune mechanisms or products and their interac-
Presumably the increased length and motility of the tion with trypanosomes are needed. In addition, a
trypanosomes observed en route are adaptations study of other tsetse molecules, such as receptors
enabling them to succeed (Ruepp, et al, 1997). involved in interactions with parasites, or molecules
that are simply required for survival of tsetse or that
TSETSE VECTOR COMPETENCE influence their fecundity, will be important for
At the center of vector competence in insects are developing strategies to interfere with transmission
immune reactions, which include a diverse set of of trypanosomes. These studies are of applied inter-
mechanisms ranging from phagocytosis, activation est as they support the development of strategies to
of proteolytic cascades, such as coagulation and block parasite transmission in vivo via transgenic
melanization and production of various antimicro- approaches.
bial peptides (Barillas-Mury, et al, 2000; Dimo-
poulos, et al, 2001). While much of this immune TRANSGENESIS IN TSETSE
response is initiated in the fat body, effector mole- Much effort has gone into developing genetic trans-
cules expressed in the midgut are increasingly being formation systems for medically and agriculturally
recognized as playing a role in immune reactions important insect vectors. There is no doubt that the
(Dimopoulos, et al, 1997; Dimopoulos, et al, 1998; availability of this technology will revolutionize in-
Lehane, et al, 1997). Discrimination between patho- sect genetics by allowing basic studies relating to
gen groups is well known in Drosophila and mosqui- the functional characterization of various genes and
toes, where it is explained by the use of different their products. It might also allow for the develop-
receptor/signalling pathways. Because dipterans ment of alternative control strategies such as the use
including mosquitoes and tsetse are largely refracto- of transgenic refractory insects. It is thought that
ry to parasite transmission, much effort has gone such genetically engineered refractory insects can be
into understanding the immune mechanisms of driven into natural populations to replace their sus-
mosquitoes. If the genetic basis of this refractoriness ceptible counterparts and hence reduce disease tran-
could be understood, then flies might be engineered smission. While the efficacy and feasibility of this
to completely block parasite transmission using var- strategy are being widely debated among scientists
ious recombinant approaches currently being devel- at large, in the case of tsetse, the development of try-
oped. Immunity genes are also being studied as they panosome-refractory strains will have an immediate
are strong candidates to confer such transmission application for at least one effective control strategy,
blocking phenotypes for inducing refractoriness. As the sterile insect technique (SIT), as described below.
a result of the application of molecular approaches,
a good deal of information is now available about At the core of transgenesis is the process of genetic
the response of mosquitoes to Plasmodium species. transformation, which in many insects relies on the
microinjection of transposable elements that insert
There is little known about the genetic basis for themselves into insect DNA, resulting in germ-line
refractoriness in tsetse or the molecular basis for the transformation. Marker genes carried by the trans-
wide ranging differences observed in parasite transposable element help identify transgenic individu-
mission rates of different tsetse species. Lectins, als. It has now been possible to introduce foreign
which are known immune molecules, might play a genes into several important insect vectors includ-
role in the attrition of trypanosomes during estab- ing one important malaria vector in Asia, Anopheles
lishment (Welburn and Maudlin, 1999). There is also stephensi (Cateruccia, et al, 2000), and others are like-
some information demonstrating antimicrobial ly to follow using similar technologies. Tsetse, how-
activity (Kaaya and Darji, 1988; Kaaya, et al, 1987; ever, have an unusual reproductive biology. There is
Kaaya, et al, 1986), the presence of the phenoloxi- no free egg stage, females retaining each egg within
dase cascade (Nigam, et al, 1997) in hemolymph. the uterus. Following hatching and in utero develop-
Preliminary results indicate that the immune re- ment of the larva, one young larva matures and is
sponse to different pathogens in tsetse is specific. finally expelled as a fully developed third instar
These results also suggest that trypanosomes may larva. Each female can deposit four-six offspring
use unprecedented novel mechanisms to achieve during its five-eight week average life span in the
their transmission through the fly by blocking the field. This viviparous reproductive biology would
expression of some of its immune responsive genes undoubtedly complicate any attempts to transform
early in the infection process (Zhengrong, et al, tsetse through egg microinjection. However, tsetse
2001). When the immune system is upregulated flies naturally harbor a number of symbiotic micro-
prior to an infectious meal, the transmission of try- organisms that have been exploited to express for-

eign gene products (Aksoy, 2000). Through such an symbionts to prevent parasite survival in the mid-
approach, the insect cells are not transformed as in gut. The identification of monoclonal antibodies
germ-line transformation, but instead, foreign genes (mAbs) with parasite transmission blocking charac-
are expressed in the symbiotic bacteria. Since the teristics and their expression as single-chain anti-
symbionts naturally live in close proximity to the body gene fragments in the symbionts provides an
developing trypanosomes, anti-pathogenic gene alternative approach. Towards this end, several
products introduced and expressed in these cells transmission-blocking antibodies targeting the
could adversely affect trypanosome transmission. major surface protein of the insect stage procyclic
trypanosomes have already been reported (Nantu-
TSETSE SYMBIONTS AND EXPRESSION lya and Moloo, 1988). Recently, midgut-specific mo-
OF FOREIGN GENES IN VIVO noclonals with transmission blocking activity have
Many insects with limited diets such as blood, plant been characterized from A. gambiae (Lal, et al, 2001).
sap or wood, rely on symbiotic microorganisms to It seems likely that similar molecules can be identi-
fulfill their nutritional requirements. It has been fied in tsetse and ultimately expressed in the gut
shown that tsetse harbour three distinct microor- symbionts. The relative ease of transformation and
ganisms (Aksoy, 2000). Two of these are present in gene expression in bacteria, and the multitude of
the gut tissue: genus Wigglesworthia (Aksoy, 1995; potential antiparasitic targets which can be explo-
Aksoy, et al, 1995) and genus Sodalis, (Aksoy, et al, red, make this a desirable system for transgenic
1995; Cheng and Aksoy, 1999; Dale and Maudlin, approaches. Should resistance develop in parasites
1999) and both are closely related to Escherichia coli. against any of the expressed foreign gene products,
The third symbiont harboured in certain tsetse it could be relatively easy to switch to express a dif-
species is Wolbachia, an obligate intracellular bacteri- ferent gene product. Alternatively, several target
um which is closely related to the genus Ehrlichia genes can potentially be expressed simultaneously
(O’Neill, et al, 1993). During its intrauterine life, the in the symbionts to prevent the development of
tsetse larva receives nutrients along with the two resistance against any one individual target.
gut symbionts from its mother, via milk-gland secre-
tions (Aksoy, et al, 1997; Ma and Denlinger, 1974), FIELD APPLICATIONS OF TRANSGENESIS
while Wolbachia is vertically transmitted by transo- In order to interfere with disease transmission, the
varial transmission. eventual goal of any transgenic approach is to
replace the naturally susceptible population with
It has been possible to culture the Sodalis symbiont their engineered refractory counterparts in the field.
in vitro (Beard, et al, 1993; Welburn, et al, 1987) and At present, there are no proven mechanisms to
a genomic transformation system has been devel- achieve this spread. One powerful potential driving
oped (Beard, et al, 1993). Recently, a homologous system involves the use of Wolbachia symbionts,
recombination approach has also been established which confer a reproductive advantage to their
so that foreign genes can now be directly inserted hosts, including the engineered females.
into the symbionts chromosome (Dale, et al, 2001). It
has also been possible to reconstitute tsetse with the The functions of Wolbachia in their various hosts are
recombinant Sodalis, which has been found to be variable. One common reproductive abnormality
successfully acquired by the intrauterine progeny they induce has been termed cytoplasmic incompat-
when microinjected into the haemolymph of the ibility (CI). This, when expressed, results in embry-
female parent (Cheng and Aksoy, 1999). It is now onic death due to disruptions in early fertilization
necessary to identify effective gene products which events (Bandi, et al, 2001). In an incompatible cross,
have anti-trypanosomal effects when expressed in the sperm enters the egg but does not successfully
Sodalis in tsetse midgut. Using a similar symbiont- contribute its genetic material to the potential
based insect transformation approach, it has been zygote. In most species, this results in very few eggs
possible to block the transmission of Trypanosoma hatching. Wolbachia infected females have a repro-
cruzi in Rhodnius prolixus in vivo by expressing the ductive advantage over their uninfected counter-
antimicrobial peptide cecropinA (cecA) in its sym- parts because they produce progeny after mating
biont, Rhodococcus rhodnii, in the hindgut of the bugs with both infected and uninfected males. This repro-
(Durvasala, et al, 1997). It has also been possible to ductive advantage allows Wolbachia to spread into
express a single-chain antibody gene fragment in populations. In Drosophila simulans in the central
the bacterial symbionts of R. prolixus (Durvasala, California valley, a natural Wolbachia infection
et al, 1999). If the tsetse immune-responsive mole- invading naive uninfected populations has spread
cules, which the trypanosomes apparently down- at a rate of over 100 km per year simply through the
regulate to achieve their transmission, can be identi- expression of CI (Turelli and Hoffmann, 1991). To
fied, they could be constitutively expressed in the understand the functional role of Wolbachia in

insects, most insects can be cured of their Wolbachia Cuisance, 1984; Vreysen, et al, 2000). Improvements
infections by administering antibiotics in the diet. in two aspects of current tsetse SIT technology have
This approach, however, is not feasible in tsetse the potential to enhance the efficacy of future pro-
since antibiotic treatment results in the clearing of grammes (Aksoy, et al, 2001).
all bacterial symbionts, including the obligate sym-
biont Wigglesworthia, in the absence of which the The first is the development of parasite refractory
flies become sterile. In order to study CI expression strains. Since the large numbers of male flies re-
in tsetse, uninfected flies need to be collected from leased can potentially contribute to a temporary
the field and colonized so that appropriate mating increase in disease transmission, the incorporation
experiments can be performed in the laboratory. As of refractory traits into the SIT release strains will
Wolbachia infected insects replace naive populations greatly enhance the efficacy of this approach, espe-
by virtue of the CI phenomenon, they can drive cially in human disease endemic foci. In the current
other maternally inherited elements of tsetse, such field SIT programmes, male tsetse are provided,
as the maternally inherited gut symbionts Sodalis, before release, with a blood-meal containing a try-
into that same population (Beard, et al, 1993). It has panocide; no infections have so far been found in
been proposed that multiple Wolbachia infections, in released sterile males caught in traps. The second is
which an insect contains two or more different Wol- the use of Wolbachia mediated CI, or hybrid sterility,
bachia strains that are incompatible with each other, as a method of inducing sterility - as an alternative
could be used to generate repeated population re- to irradiation. With CI, the released strain of tsetse
placements or to spread Wolbachia into target species would carry a Wolbachia infection that would induce
that already contain an existing infection (Sinkins, CI when males mate with wild females. The com-
et al, 1995; Sinkins, et al, 1997). The analysis of petitiveness of these males would be expected to be
Wolbachia strain types infecting different species of much higher compared with irradiated males, and,
tsetse has shown that they are different, and as such as a result, fewer insects would need to be released
represent independent acquisitions (Cheng, et al, in order to achieve the same level of sterility in the
2000). wild population. This strategy is dependent on the
use of a very efficient sexing system. If Wolbachia-
In addition to CI, certain Wolbachia strains, such as infected females were released in sufficient quanti-
wMelPop characterized from Drosophila melanogaster, ties, then Wolbachia would have the opportunity to
induce an age-shortening effect in their host (Min invade the target population, which would render
and Benzer, 1997). This age-shortening effect was subsequent releases ineffective. If it were impossible
reversed when infected flies were cured of their to guarantee extremely low quantities of released
Wolbachia infections with antibiotics (Min and females, then it would be possible to incorporate
Benzer, 1997). It remains to be seen whether such low levels of irradiation with Wolbachia induced
Wolbachia infections can be documented in tsetse. It sterility to prevent released females from successful-
has also been possible to introduce Wolbachia into ly reproducing. This approach has been successfully
insect species which do not harbour natural infec- tested in Culex mosquitoes (Shahid and Curtis,
tions, and these approaches can be pursued in 1987).
tsetse. Since the T. brucei group parasites require 14-
30 days to complete their developmental cycle in the CONCLUSIONS
fly, tsetse flies need to be at least this age to transmit • Understanding the molecular/cellular basis of
disease. Reductions in the life span of individual trypanosome transmission in tsetse is of funda-
flies might have a large effect on disease transmis- mental significance, and will allow development
sion in the field (Sinkins and O’Neill, 2000). of new applications for vector control. Since the
symbiont-based transformation system can be
STERILE INSECT TECHNIQUE AND used to express gene products in tsetse midgut,
TRANSGENIC INSECTS such will aid in the identification of candidate
SIT is a genetic population suppression approach genes that can be expressed to confer refractori-
and involves sustained, systematic releases of irra- ness in tsetse.
diated sterile male insects among the wild popula-
tion. The sterile males fertilize wild females, which • A research plan should be developed to coordi-
are then unable to produce progeny. By continually nate information on expressed sequence tag (EST)
releasing sterile males in high numbers over a peri- sequences, genomic sequences and the physical
od of three-four generations, after having previous- map locations of selected genes. For EST analysis,
ly reduced the population density by other tech- the cDNAs to be sequenced can be obtained from
niques (trapping, insecticide spraying, etc.), the tar- tsetse exposed to infected blood-meals containing
get population can be eradicated (Politzar and eukaryotic pathogens, including trypanosomes,

and from tissue specific (midgut, fat body and lineage related to Enterobacteriaceae. Insect Molecular
salivary glands) normalized libraries as well as Biology, 1995, 4:15-22.
from different developmental stages (larvae). The
ESTs deemed to be of interest should be physical- Bandi C et al. Inherited microorganisms, sex-specif-
ly mapped to metaphase chromosomes using in ic virulence and reproductive parasitism. Trends in
situ hybridization (FISH) technology. A bacterial Parasitology, 2001, 17(2):88-94.
artificial chromosome (BAC) library could be con-
structed in order to obtain single pass sequences. Barillas-Mury C, Wizel B, Han YS. Mosquito immu-
Some of these resources are already available and ne responses and malaria transmission: lessons from
these efforts would promote and foster collabora- insect model systems and implications for verte-
tion and intellectual input from the international brate innate immunity and vaccine development.
tsetse community. The availability of the complete Insect Biochemistry and Molecular Biology, 2000,
and annotated D. melanogaster genome, and the 30:429-42.
anticipated genome projects for A. gambiae and A.
aegypti, will constitute an important resource for Beard C et al. Modification of arthropod vector com-
gene discovery efforts in Glossina. It follows that a petence via symbiotic bacteria. Parasitology Today,
proteomics approach to protein identification will 1993, 9:179-183.
allow further exploitation of the genomic infor-
mation. This aspect of molecular entomology will Beard CB et al. Genetic transformation and phy-
be a powerful approach in the post-genomic era. logeny of bacterial symbionts from tsetse. Insect
Molecular Biology, 1993, 1:123-31.
• Molecular approaches for population genetics
stand to improve our understanding of vector Budd LT. Tsetse fly and trypanosomosis research and
populations and are of significance for the suc- development since 1980 - an economic analysis. Kent,
cess of area-wide control strategies. These field- United Kingdom, DFID Livestock Production Pro-
based studies could easily be coupled with gramme, 1999.
efforts to better understand the prevalence and
phenotypic effects of natural Wolbachia infections Catteruccia F et al. Stable germline transformation
in tsetse. of the malaria mosquito Anopheles stephensi. Nature,
2000, 405:959-62.
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III APPLIED GENOMICS AND contain the majority of genes, but the DNA content
BIOINFORMATICS of the mini- and intermediate chromosomes should
be investigated in the future.
Sara E. Melville1 and Daniel Masiga2.
1Cambridge University, Department of Pathology, Most megabase chromosomes differ in size from
Microbiology and Parasitology Division, University of their homologues by up to 15%, but homologous
Cambridge,CB2, IQP, U.K. chromosomes in different stocks vary more - con-
2International Centre of Insect Physiology and Ecology siderably more than reported in other organisms.
(ICIPE), Molecular Biology and Biotechnology Unit, PO Nevertheless, mapping studies show that syntenic
Box 30772, Nyayo Stadium (00506), Nairobi, Kenya. groups are maintained in all stocks studied (Mel-
ville, et al, 1998). Despite remarkable genomic plas-
ticity (Melville, et al, 1999), studies reveal a signifi-
OVERVIEW OF THE T. BRUCEI GENOME cant level of conservation of genome structure.
AND THE HISTORY OF TRYPANOSOME Initial apprehension that the genome was so poly-
GENOMICS morphic as to make sequencing of one strain of little
use for cross-comparison to other strains has been
The African trypanosome genome network was assuaged – although further studies could investi-
formed by the UNDP/World Bank/WHO Special gate the role this plasticity is playing in the survival
Programme for Research and Training in Tropical strategy of the trypanosome.
Diseases (TDR) with a brief to coordinate the ana-
lysis and sequencing of the nuclear genome. It con- There is no perfect isolate that should form the basis
sists of research laboratories worldwide, contribut- of all molecular experiments on T. brucei. It is indeed
ing to different aspects of genome analysis. At the preferable that some genomic analyses are carried
time of its formation, some aspects of genome out on multiple stocks, for example karyotyping
structure and organization were known, especially and random gene sequencing. However, we can
from studies on antigenic variation and transcrip- only aim to sequence one complete genome in the
tion, but only one group had actively initiated immediate future. T. brucei stock TREU927/4
global genomic analysis. In the last five years, (GPAL/KE/70/EATRO1534) was cloned from a
much has been learnt about the organization of the population of trypanosomes isolated from a tsetse
nuclear genome of T. brucei, which consists of at fly in Kenya and was chosen as the reference
least eleven diploid megabase chromosomes (1-6 genome for a variety of reasons, some optimal and
Mb), a variable number of intermediate-sized chro- some pragmatic. The original stock exists and the
mosomes of indeterminate ploidy (200-900 Kb), history of its isolation is documented. It was not iso-
and 50-100 minichromosomes (25-100 Kb) lated from an infected human, but laboratory tests
(Melville, et al, 1998). indicate that it has intermediate resistance to human
serum (CMR Turner, personal communication). It is
With the exception of some variant surface glyco- pleomorphic and may be replicated as the blood-
protein (VSG) genes, most expressed genes are stream or procyclic form in laboratory animals,
located in the megabase chromosomes. These are tsetse flies or culture. It has been used as a parent in
inherited in a Mendelian fashion (Turner, et al, 1990) laboratory-controlled genetic crosses with other
and have been assigned Roman numerals in order stocks, and cloned hybrids have been isolated
of increasing size in TREU927/4 (Turner, et al, 1997). (Turner, et al, 1990; Tait, et al, in press), allowing the
Many, perhaps most, genes are expressed in poly- creation of a genetic map. A high quality, arrayed
cistronic transcripts (Vanhamme and Pays, 1995). genomic library of the megabase chromosomal
Some chromosome ends carry VSG expression sites: DNA of TREU927/4 already existed (Melville, et al,
only one is active at one time, resulting in a uniform 1996), and was used to determine the structure of a
surface protein coat. Gene- or promoter-switching megabase chromosome (Melville, et al, 1999).
results in antigenic variation (Barry and McCulloch,
2001), which is an effective way for the parasite to Participation in the network is dynamic because, as
evade destruction by the host immune system and priorities change, researchers with different skills
results in the characteristic fluctuating parasitemia are required. Many contribute without direct fund-
and accompanying fever. The intermediate chromo- ing, and the network is dependent on the support
somes also carry expression sites (Rudenko, et al, and active participation of the research community.
1998), and most mini-chromosomes contain non- It has been vital to promote universal commitment
transcribed VSG and simple repeat sequences to the idea that this huge task can progress most effi-
(Weiden, et al, 1991). The network has prioritized ciently if the community works together with open
sequencing of the megabase chromosomes, as they sharing of data and resources.

The availability of the DNA sequence will benefit gle-pass sequence reads to aid gene discovery and
many research programmes. Some benefits are al- facilitate the completion of contiguous chromosome
ready obvious, some will only become clear as more sequences (http://ftp.sanger.ac.uk/pub/databas-
researchers begin to use the available data. This is es/T.brucei-sequences).
our task: to be forward-looking and imaginative in
our plans for the use of this tremendous resource. The EST and GSS sequences have been clustered
by collaborators at the Sanger Institute to provide
CURRENT STATUS OF THE contigs. In total, 96 474 sequences (~45.87Mb)
TRYPANOSOMA BRUCEI GENOME PROJECT were used for the clustering, achieved using the
Expressed Sequence Tags (ESTs) sequence assembly programme Phrap - 12 251
Rapid gene discovery was achieved in the early contigs were generated and 8242 sequences could
phase of the genome project by sequencing of ran- not be placed in a contig (singletons). One of the
domly selected cDNA clones (El-Sayed, et al, 1995; contigs has 1926 constituent members (contig
Djikeng, et al, 1998). At the time of writing, there length 9.342Kb), but this is probably due to
are 5133 T. brucei ESTs in the public databases from repeated DNA and is an example of the care that
four cloned stocks of T. brucei (dbEST: must be taken in interpreting these automatically
http://www.ncbi.nlm.nih.gov/dbEST/index.html). generated data. The cluster data are available as a
Most EST sequences were generated from cDNA searchable subcomponent of the T. brucei BLAST
clones of bloodstream-form mRNA. There has been server (http://www.sanger.ac.uk/Projects/T_bru-
no concerted effort to produce large EST datasets cei Toolkit/blast_server.shtml) and from the ftp site
from each of the life-cycle stages as it was decided to as a gzip file (hftp://ftp.sanger.ac.uk/pub/databas-
divert effort and resources to sequencing of genomes/T.brucei_sequences/GSS/clusters).
ic DNA. The determination of life-cycle stage-spe-
cific expression will be undertaken by other meth- Physical and Genetic Mapping
ods (see section 3.5). In addition to discovery of One chromosome was mapped thoroughly and to
many novel T. brucei genes, the ESTs have provided completion (Melville, et al, 1999) prior to submitting
a rich source of markers (Melville, et al, 1998) and sequencing grants. However, rapid progress in
aid sequence annotation. methods for completion of genome sequences has
reduced the requirement to produce prior contigu-
Genome Survey Sequences (GSSs) for Gene ous physical maps of each chromosome (El-Sayed,
Discovery et al, 2000). Nevertheless, mapping information is
In a pilot project, approximately 500 random required for seeding BAC sequencing and to aid
genomic clones were sequenced (El-Sayed and final reconstruction of the chromosome. Many
Donelson, 1997) to show that this led to equally effi- hybridization data have been amassed by the
cient gene discovery, due to the lack of introns and Cambridge group and also by the community
the close spacing of genes in the T. brucei genome. through using centralized resources. So far, these
Therefore, it was decided that a portion of the funds data have been made freely available but only by
obtained for high-throughput sequencing at The email request. A database is being established to
Institute for Genomic Research (TIGR) should be make the data web-accessible (see 4.4).
allocated to sequencing of random, short pieces of
DNA (GSSs). Sequencing of both ends of almost TDR and The Wellcome Trust have also supported
25000 clones (almost 50 000 short sequences) pro- the creation of a genetic map using classical genetic
vided a total of 29 Mb of the TREU927 genome analysis of F1 hybrids (Tait and Turner, Glasgow).
(http://www.tigr.org/tdb/mdb/tbdb/status.html). F1 hybrids have been isolated following simultane-
A proportion of these sequences derive from the ous passage of genotypically distinct stocks through
ends of large genomic clones (in bacteriophage P1 tsetse flies. This group has developed amplified
and bacterial artificial chromosome (BAC) vectors), restriction fragment polymorphism (AFLP), mini-
and these contribute to the mapping and sequenc- and micro-satellite markers for use in genetic analy-
ing of whole chromosomes (see section 2.4) by pro- sis and, together with the genomics group
viding paired markers of about 500 base pairs every (Melville), has been able to combine some of the
2.5Kb across the chromosomes (excepting the data with chromosome maps. The aims are to deter-
minichromosomes). Many researchers have report- mine crossover frequency, estimate the physical size
ed finding T. brucei homologues of known genes in of the recombination unit (Centimorgan), and inves-
the GSSs, and this generated great enthusiasm for tigate variation in crossing-over in different genom-
the rapid provision of more such sequences. In ic regions. Initial data of this type indicate that it is
response to requests from the community, the indeed feasible to obtain sufficient hybrid progeny
Sanger Institute has provided a further 47 000 sin- and genetic markers to aim towards using a genetic

map for positional cloning of genes underlying com- the data accessible and informative, and to stimulate
plex phenotypes (Tait, et al, in press). new ideas in the search for novel approaches to com-
bat African trypanosomiasis.
Genomic Sequencing
Sequencing of chromosome I of T. brucei strain 927/4
GUTat 10.1 commenced at the Sanger Institute in Access to Data
1998. Sequence was obtained from shotgun clones of The fields of genomics, databases and bioinformatics
chromosomal DNA eluted from a pulsed field gel are dynamic and researchers may find it difficult to
(PFG) (> 8 X coverage) and from mapped P1 clones learn how best to access the most recent data, or
(1 X coverage), a combination of methodologies pio- where to find the most innovative new programmes
neered by the malaria sequencing consortium. While for analysis. The field is developing rapidly and here
approximately 75% of the chromosome sequence we aim to provide an outline of where data may be
was contiguous by year two, 25% of the chromo- found at different stages of the sequencing projects.
some has proved difficult due to repetitive DNA: Complementary DNA (cDNA) sequencing has been
genes in VSG expression sites, gene families, retro- carried out in individual research laboratories and
transposons, and some tandem repeats (Melville, the sequences are made available via the database
et al, 1999). Following the random sequencing phase for expressed sequence tags (dbEST) at the National
(see 2.3), TIGR commenced the sequencing of chro- Center for Biotechnology Information (NCBI). All
mosome II in 1998. This sequence is derived from genomic sequence data are made available immedi-
BAC clones, mapped to chromosome II using ately via the websites at the respective sequencing
cDNAs from the EST project (Melville, et al, 1998; centres (TIGR and Sanger Institute). These are sin-
El-Sayed, et al, 1995) and sequenced to ca. 7 X cover- gle-pass sequences and, although no error correction
age. The end-sequences determined in the first or annotation are offered at this stage, this is an
phase of the project (section 2.2) allow the selection important resource for researchers who are looking
of BACs with minimum overlap for maximum effi- for genes in T. brucei that have homologues in other
ciency. The progress on chromosome II has been sim- organisms. The clustering data provided by the
ilar to that on chromosome I, with similar problems Sanger Institute are also generated automatically
in regions of clustered multicopy sequences. and any errors will be incorporated. Therefore, use
Chromosomes IV and VI are currently approximate- of such data always requires verification by the
ly 75% completed at TIGR. Preliminary (and there- researcher. Search engines are provided, allowing
fore in parts inaccurate and incomplete) annotation researchers to look for sequences with high similari-
is provided by the sequencing centres prior to com- ty to the gene they seek (http://www.ebi.ac.uk/
pletion. It is best to follow progress by regularly b l a s t 2 / p a r a s i t e s . h t m l ;
monitoring both sequencing centre websites http://www.tigr.org/tdb/mdb/tbdb/seq_search.ht
( h t t p : / / w w w. s a n g e r. a c . u k / P r o j e c t s / T- b r u c e i / ; ml). Data on significant similarities to genes in the
http://www.tigr.org/tdb/mdb/tbdb/index.html). databases are provided but researchers should note
that new information becomes available in the cen-
The funds to complete chromosomes IX, X and XI tral databases (European Molecular Biology Labora-
were awarded to Barrell (Sanger Institute), Melville tory, EMBL/GENBANK/Database of Japan,
and the network in 2000. Chromosome X shotgun DDBJ) all the time, and should check when the
sequence is now available (http://ftp.sanger.ac.uk/ most recent BLAST searches were performed. At
pub/databases/T.brucei-sequences) and chromo- intervals, sequences are submitted in batches to the
somes IX and XI are in library preparation. The public databases at the European Bioinformatics
funds to complete chromosomes III, V, VII and VIII Institute (EBI) and NCBI. Random genomic shotgun
were awarded to El-Sayed at TIGR (collaborators sequences and end-sequences of genomic clones are
Donelson, Ullu, Melville) in 2001. Each sequencing submitted to the database for genome survey
centre will therefore provide approximately 50% of sequences (dbGSS) (http://www.ncbi.nlm.nih.
the megabase chromosome DNA sequence. The proj- gov/dbGSS/index.html). Sequences of shotgun
ects run until 2004 and all the sequence is likely to be clones derived from whole BACs are submitted to
in the databases by 2003 at the latest; however it can- the database for high-throughput genomic se
not be foreseen how long it will take to complete quences (dbHTGS) (http://www.ncbi.nlm.nih.
contiguation. gov/dbHTGS/index.html) in three stages (at 3 X
and 7 X coverage, and at completion). All these data
To make full use of the substantial investment in will eventually be mirrored in the GENBANK,
genome sequencing it is necessary to complete the EMBL and DDBJ databases, ensuring that all avail-
task, to ensure there is complete information on all able T. brucei sequences may be found in a single
enzyme pathways. It is also absolutely imperative to database. However, there are some advantages in
invest substantial effort into bioinformatics to make looking at the data in the specialized genome data-

bases, as annotation is more extensive and sequence Predicted timeline to 2002-2004:
similarity assignments are provided. • 1 x sequencing of IX, X and XI-specific BACs.
• Chromosomes III, V, VII and VIII to be sequenced
On completion of a chromosome sequence, the most BAC by BAC and appearing in the databases
careful annotation is carried out. Great emphasis is piecemeal over the next two years.
laid on ensuring the sequence is accurate and con- • Contiguation and annotation of chromosomes III
tiguous (some areas of ambiguity may be tolerated in – XI over at least three years.
the final sequence, if too many resources are required
to provide final clarification), and on trying to identi- The timeline to complete sequencing is the most dif-
fy all open reading frames (ORFs). Researchers ficult to predict, and depends to some extent on the
should be aware that, however careful the annota- determination to complete subtelomeric regions,
tion, some protein coding genes may not be predict- VSG arrays, etc.
ed from sequence analysis alone and a few of those
predicted may not be transcribed. The analysis of CURRENT STATUS OF FUNCTIONAL
the chromosome is published in a peer-reviewed AND APPLIED GENOMICS PROJECTS
journal and the sequence, with full annotation, can The African trypanosome genome network has ini-
then be viewed with the Entrez browser at NCBI tiated discussions on the “post-genomic” agenda.
(http://www.ncbi.nlm.nih.gov/Entrez/Genome/ This requires careful coordination and the involve-
org.html). The sequencing centre responsible for the ment of appropriately skilled researchers. At the
sequencing of the chromosome also places the same 1999 and 2000 network meetings, lists of priorities
data on its website. drawn up (http://parsun1.path.cam.ac.uk/net-
work.htm) included microarrays for transcription
In an ideal world it would be possible to carry out analysis, techniques for gene knockouts, generation
searches of all data at a single site but the curation of of mutants, RNAi and phenotype characterization,
such a database would necessitate delay in access to and use of the genetic map for positional cloning of
the data. Immediate access to raw, unfinished se- genes (some to proceed in collaboration with other
quence is highly prized by a research community kinetoplastid genome networks). Recommendations
waiting impatiently for gene sequences, and the of interest to this discussion include identification of
sequencing centres have responded to this need. trypanosome-specific genes essential for infection
This is only really possible via deposition on home and development, characterization of molecular
websites prior to batch submission to NCBI. It is structures and parasite-specific metabolic path-
therefore important for researchers to consider ways, elucidation of unique trypanosome-specific
which databases contain the most valuable data (e.g. mechanisms of gene regulation and RNA process-
the public genome databases because of their exten- ing, and analysis of the protein profile of the para-
sive annotation, or the sequencing centre databases site to identify functionally important genes (pro-
because they may contain sequences not yet proteomics). Perhaps one or more of these approaches
cessed for transfer to NCBI) and, in some cases, to could lead to the identification of novel drug tar-
perform searches at several sites. Long-term curation gets, and/or vaccine candidates, but only if those
of interrelated genomic and functional data is a dif- researchers skilled in, and committed to, their deve-
ferent consideration (section 3.1). lopment join the global collaboration and contribute
to the post-genomic projects of the future.
Summary and Predicted Timeline. Sequence Analysis and Relational Databases

The following sequence data are available in 2001: The Wellcome Trust Functional Genomics Initiative
• 5133 EST sequences, providing approximately has provided funds to create a trypanosome data-
2000+ unique genes. base (genome databases for Schizosaccharomyces
• 96 474 GSS sequences (~45.87Mb), including 10 pombe, Leishmania major and T. brucei ) to Drs B
990 BAC and P1 end-sequences. Barrell, Sanger Institute; M Rajandream (S. pombe),
• Clustering data of all GSS and EST sequences, A Ivens (L. major); M Carrington and S Melville
with data on significant similarities. (T. brucei). These databases will contain all genomic
• The complete sequence of chromosomes I and II information, e.g. primary sequence annotation from
(to be fully annotated in 2001). the sequencing centres, secondary (ongoing) se-
• 75% of chromosomes IV and VI as complete BAC quence annotation, references, expression data, pro-
sequences. tein characterization, knockout and RNAi pheno-
• Chromosome X shotgun sequence (04/01); chro- type analyses. They will be relational databases
mosomes IX and XI shotgun sequence to follow. using SQL (structured query language) and hosted
by the Sanger Institute. The T. brucei database will

be developed in close collaboration with TIGR, and ture (M Turner, personal communication). The kary-
is overseen by a management committee. Genome otypes are identical. The P1 library is prepared from
databases of this type are long-term projects, pro- 927/4 and the BAC library from 10.1. The primary
viding a single site to review ongoing genomic and sequencing substrate is 10.1 procyclic genomic
functional genomic analyses long after sequencing DNA.
of the reference strain is complete, and as such serve
a different function to the rapid access sequence Stock 927 has the smallest nuclear genome of all
deposition on the sequencing centre websites or the T. b. brucei or rhodesiense stocks examined so far with
parasite genome BLAST server (see 2.5). News on approximately 10 Mb less DNA (Melville, et al,
progress will be posted on relevant websites. 1998), reducing the amount of funding and sequenc-
ing required quite considerably. It is capable of com-
The importance of ensuring that this database is pleting the life cycle, indicating that all vital genes
readily available to African scientists hardly needs are present and it is likely that much of the extra
explaining - they should not need to rely on collab- DNA consists of expansion of multicopy DNA –
orations with laboratories in Europe or the Americas gene familes and repeats. Nevertheless we must
to gain direct access to such comprehensive data. In always remember that trypanosomes are phenotyp-
the long-term, the enabling of molecular biological ically variable, and that the basis of this variation
research on trypanosomes will require that African must lie in the genome. It will require thought and
laboratories have secure Internet connections of innovation to find methods to compare the geno-
wide band-width. Until such time as this is possible, types of phenotypically variable strains.
we should consider providing CD versions of the
database at regular intervals. This may be proposed Both 927/4 and the derivative 10.1 may be trans-
to the database committee via Dr Melville, and will formed with foreign DNA at the procyclic and
require some coordination. bloodstream-form life cycle stages. Transformation
of the bloodstream-form is less efficient than in
Bioinformatics Projects strain 427, but this is common to most strains that
The field of bioinformatics is changing rapidly and have not been replicated in vitro for long periods. A
the databases described here will not remain static. line of GUTat10.1 expressing the tetracycline (TET)
NCBI, EBI, the sequencing centres (and many oth- repressor has also been produced and tested. This is
ers, including academic institutions) are actively useful for any experiment involving inducible
developing better analysis tools, and it is necessary expression or inducible ablation of transcription/
to watch their websites for innovations and devel- translation, and this line is currently available from
opments. Professor Christine Clayton, Heidelberg (van Deur-
sen, et al, 2001).
In addition, there is certainly scope for specific
bioinformatic analysis projects, either based on the As stated in the introduction to this section, stock
examples from more advanced genome projects 927/4 is found to be ‘intermediate’ in its resistance
(yeast, C. elegans) or on novel approaches based on to lysis by human serum (Turner, personal commu-
parasite-specific interests. For example, investiga- nication). The SRA (serum resistance-associated)
tion of the components of metabolic enzyme path- gene has been found in the genome databases (Pays,
ways (requiring skilled biochemists, but also novel personal communication), although it is not yet
bioinformatics approaches to reduce the time known if it is expressed in the correct form. The-
required for the largely manual approach currently refore, bearing in mind that T. b. brucei and T. b. rho-
used); comparison of metabolic pathways to the desiense may only be genotypic variants of a single
homologous pathways in humans and livestock species and that the basis of serum resistance may
(again requiring biochemists and informaticians); not lie exclusively in SRA expression, it should be
searching for commonalities in the biochemistry of assumed by all researchers using 927/4 and its
the kinetoplastid parasites, that differ from the derivatives as bloodstream forms in experiments
mammalian host; analysis of targeted or global/sin- that there may be human-infective cells in the po-
gle-pass comparative sequencing of other trypano- pulation.
some strains and species.
Transformation Technology
Biological Characteristics of the Reference Strain Transformation technology for genetic analysis of
A minimally culture-adapted line of the original T. brucei is in routine use (Clayton, 1999). There are
stock TREU927/4 has been generated with greater multiple vectors, several types of transformation
stability of variant antigen types (TREU927/4 markers (drug resistance, fluorescence) and a sys-
GUTat 10.1). This grows to a higher density in cul- tem of inducible expression. Foreign DNA inserts

into the genome by homologous recombination and post-translational controls on gene expression, and
this can be precisely targeted if the exact sequence of will provide a firmer basis for the design of many
the target site is known or determined, as recombi- experiments. For example, if transcription levels are
nation occurs preferentially at sites of exact homolo- found to be relevant in at least some cases, microar-
gy. Expression from episomal vectors is less success- ray technology could be very useful to compare
ful. At Tri-Tryp 2000, there was some discussion of transcription in strains of different clinical pheno-
the need for technology development. There was a type, or to examine mutant strains, to observe not
proposal that enhanced transformation efficiency of only the ablation of the targeted transcript but also
bloodstream forms is one requirement for high- any “knock-on” effect on other genes. For now, the
throughput analysis (e.g. of gene knockouts or discriminative power of microarray technology in
RNAi mutants) of the mammal-infective form, as some respects remains unproven. It also remains an
transformation of the procyclic forms (which is open question whether an array based on a single
more efficient) followed by transformation of the strain (927) is sufficient to detect all relevant differ-
cells to bloodstream forms requires passage through ences; it is possible that strains carry environment-
the tsetse fly. Of course, the ability to culture other specific genes and that strain 927 lacks the genes of
life cycle stages, e.g. metacyclic forms, would also interest. Nevertheless, if arrays are made available
represent a considerable improvement in the tools for use by the community, such preliminary (”look-
available for genetic analysis. and-see”) investigations could be carried out at little
cost. No doubt as production costs become less pro-
Microarrays hibitive, the number of strains from which arrays
Some doubts have been expressed regarding the are available will increase.
usefulness of expression analysis in the Kinetopla-
stidae. The observed polycistronic transcription The materials currently available for the creation of
suggests that control of gene expression is entirely standard DNA-spot microarrays are the ESTs and
post-transcriptional, a possibility supported by GSS clones. The ESTs almost all derive from blood-
reports of protein products of genes within the same stream forms. Two GSS libraries have been used:
polycistrons that were found to be up/down-regu- one with average inserts of 2 Kb and the other with
lated at different life cycle stages. Nevertheless, inserts of of 4 Kb. The GSS libraries may prove as
there clearly is some life cycle stage-specific tran- useful as EST libraries for microarray analysis due
scription and this is useful data for investigation of to the high gene density in the genome, and they are
processes such as infectivity, etc. It is easy to obtain certainly more representative of all the genes in the
large amounts of cultured procyclic RNAs from genome than are EST libraries. However, the clones
many strains, but perhaps prudent to perform some may contain fragments of more than one gene, thus
experiments to compare cultured procyclics with complicating full analysis of the dataset. The useful-
those extracted from tsetse flies. It is possible to ness of GSS arrays is being investigated (Clayton,
obtain sufficient RNA from bloodstream forms Heidelberg). Meanwhile, El-Sayed (TIGR) has pro-
replicating in laboratory rodents, although slow- duced the first ORF-specific array by amplifying
growing (e.g. less virulent) strains can prove more fragments of ORFs identified from the sequence of
problematic (but the rate of improvement in chromosome II (El-Sayed, et al, 2000). A UK group
microarray techniques is still in the exponential (Melville, Matthews, Turner, Sanger Institute) is
phase). Unfortunately, it is very difficult to obtain developing a pilot project to test the relationship
sufficient DNA from epimastigotes or the infective between post-transcriptional and post-translational
metacyclic forms. The metacyclic life cycle stage is processing. The long-term aim, if the usefulness of
arguably the most relevant for studies of infectivity, microarrays is proven, is to establish whole genome
and for vaccine development. With the involvement ORF-specific microarrays within four years as a col-
of researchers with tsetse fly colonies, it may be pos- laboration between TIGR and the UK group. These
sible to perform some careful experiments with would have to be made available as a resource -
metacyclic DNA after full optimization of the array either the arrays themselves, the DNA, the oligonu-
technology. cleotides, or simply the oligonucleotide sequence. It
is hard to predict at the present time what the
Still, for the examination of wild-type/reference demand will be or how the technology will develop.
strains, it remains necessary to carry out specific
experiments that detect changes in levels of individ- An additional aim of the network is to develop stan-
ual mRNAs after processing of polycistronic tran- dard operating procedures for microarray analysis
scripts, and to compare these to the concomitant lev- and presentation of data in collaboration with other
els of the protein products. This will provide data on organizations, including the EBI, where they are
the relative importance of post-transcriptional and establishing a public repository for microarray-based

gene expression data (http://www.ebi.ac.uk/array- database could include:
express/). These organizations are, in turn, mem- • unique marker name/identifier.
bers of the Microarray Gene Expression Database • type of marker.
Group (MGED), whose aims are to facilitate the • oligonucleotide primer sequences.
adoption of standards for DNA-array experiment • polymerase chain reaction (PCR) parameters.
annotation and data representation, as well as to • size(s) of PCR product in genome project refer-
introduce standard experimental controls and data ence strain 927.
normalization methods (http://www.mged.org/). • brief description of population studied and
results.
Proteomics • list of allele variants in named isolates with
Proteomics is a rapidly developing field that details of isolate history.
encompasses small-scale 2-D gel analysis to high- • references.
throughput techniques such as mass spectrometry • coordination with physical mapping/sequencing
that are not available to all. Analysis of the T. brucei data to provide information on genomic location,
proteome is ongoing in several laboratories (e.g. etc.
Wellcome News issue 26, Q1, 2001, p. 19). However,
for a coordinated network approach to global pro- Over time, sufficient information should accumu-
teomic analysis, it is necessary to develop standard late to facilitate the choice of suitable markers for
operating procedures and common marker proteins given areas and defined experiments, without
before any proteomic data are presented on the web. extensive literature searching or unnecessary collab-
This was discussed at some length at Tri-Tryp 2000, orations.
and the first step towards this goal in the T. brucei
network is to combine proteomic and array analysis RNA Interference as a Method to Investigate
as discussed in section 3.5. Gene Function
A common approach to the investigation of gene
Novel DNA-Based Markers for Epidemiology, function is to remove the gene from the genome
Diagnosis and/or Positional Cloning (gene knockout) or to prevent its transcription and
DNA-based markers have become increasingly translation. Gene knockout requires two rounds of
important in epidemiology and diagnosis in the last transformation and gene knockout, as each gene is
decade. The availability of genome sequence offers present as diploid alleles. RNAi functions by trans-
the capacity to identify a whole range of new mark- formation of a plasmid encoding a double-stranded
ers (microsatellites, minisatellites, single nucleotide RNA that is homologous to part of the transcript
polymorphisms or SNPs, indels) at much greater under study. By an unknown mechanism, the pres-
efficiency than before. Previously, satellite markers ence of the exogenous dsRNA prevents the transla-
were identified by cloning and hybridization of tion of the endogenous mRNA (Ngo, et al, 1998).
individual oligonucleotides, or by serendipity, This requires only one round of transformation and
whereas now they are identified using informatics. is useful for the analysis of function of single-locus
The identification of SNPs and indels requires se- and multi-locus genes. To date, RNAi is most suc-
quence from different strains, but may provide the cessful in procyclic forms, although many groups
advantages of isoenzyme analysis – stability over are now working to improve efficiency in blood-
millennia - at less cost in terms of money and resear- stream forms.
cher time. There is no doubt that numerous research
groups are considering using the DNA sequence in A group of UK researchers (M Field, Imperial
this way, and we may see more papers appearing College, et al) has recently been awarded a grant
using recently identified polymorphic markers. from the Wellcome Trust Functional Genomics
Committee to begin analysis of gene function in
It seems, then, timely to suggest that these data T. brucei by RNAi . This will involve analysis of phe-
could usefully be collated at a central site rather notype following systematic disruption of individ-
than leaving individual researchers to draw up their ual genes identified on the sequenced chromosomes
separate lists from the literature. This may also pro- I and II. It is the requirement of these types of grants
ve especially useful to scientists in institutions with that both the data and the biological resources are
less access to journals. This is not to subvert the use made available to the community. The data will be
of publication, which is vital to all researchers, but web-accessible and prepared in collaboration with
rather to suggest that, on publication, all markers the functional genomics database (see section 3.1).
should be submitted to a central database and that Again, it may be necessary to consider how this
this database should be available to all – on the web information may be accessed from areas with inse-
and, if necessary, on CD (see section 3.1). Such a cure access to the Internet. The DNA resources (con-

structs of vector plus gene sequence for transforma- leads for biochemists to investigate further at a
tion) will be available from Melville/Cambridge molecular level and/or for design of high-
(see section 4.5). It is hoped that the transformed cell throughput combinatorial screening program-
lines (i.e. 927 mutants) will be available from mes. However, the latter will require funding for
Turner/Glasgow (see section 4.6). the considerable work involved in such analyses,
even in the preliminary stages.
Applied Genomics: Discovery of • At this stage, the retention of such work in the
Novel Drug Targets academic sector due to lack of interest from phar-
The authors are not experts in drug target validation maceutical companies could be exploited to en-
and, to avoid charges of naivety, first wish to ack- sure that such studies are coordinated, data sha-
nowledge the considerable work by leading groups red, basic functional data all web-accessible, and
of biochemists on parasite-specific pathways that effort is not wasted. (Although, future problems
may represent valid targets for design of drugs of with intellectual property rights have to be con-
greater specificity. No doubt such projects will be sidered.)
brought to the discussion table by other contribu- • The establishment of the genome network has
tors. However, under the maxim that no single provided one paradigm of how the research com-
method guarantees the discovery of a better drug, munity can pull together to collaborate rather
and that all methods (rational design and global than compete. There are already well-established
approaches) have potential for both success and fal- groups with an interest in increasing funding for
se leads, we wish to pose the questions: is it neces- the discovery of new drugs for diseases of the
sary to expand our efforts now to increase the num- poor, e.g. TDR working groups, the Access to
ber of leads on the table? and how can we best use Medicine Campaign. Is it now possible to bring
the limited funds available? This discussion needs (sections of) these disparate groups together to
to take place across a wide range of researchers with discuss a new “network”?
different skills. As observed by members of the • Currently in the UK there are possibilities to
pharmaceutical industry when faced with the mass apply for funding for “functional genomics”, a
of new genomic information, we may yet need fur- fundamentally different activity to basic, hypoth-
ther breaking down of the barriers between scientif- esis-driven research. Are funding agencies in
ic disciplines (Browne and Thurlby, 1996). other countries initiating such programmes? Is
this a good moment to use these openings to con-
”Until recently genes have been cloned and sider an “applied genomics” application, aimed
expressed usually as the result of targeted pro- directly at drug discovery? If Wellcome Trust-
grammes of research, their biochemistry and phar- based, this would involve UK researchers, but the
macology evaluated, high-throughput mechanism- Trust recognizes the need to involve international
based screens devised and leads identified for opti- groups.
mization by chemists ... In this traditional model of • It is possible that an early-stage drug would be
pharmaceutical research, gene identification had more attractive to manufacturers if it were useful
often been considered rate limiting. The paradigm against several parasites, for example all kineto-
has shifted.... We need to be able to predict which plastids. Some effort should be put into bioinfor-
genes may be useful and why... there are potentially matics comparison of kinetoplastid genomes as
many, many more proteins to work with (and) the they become available. This could provide infor-
abundance of opportunities (must) impact on con- mation on common parasite pathways that differ
ventional research strategies”... (extracted from substantially from those found in humans and
Browne and Thurlby, 1996). mammals.
The following observations need critical assess- Applied Genomics: High-throughput Testing
ment: of Vaccine Candidates
• The mass of genomic sequence provides the pri- Discussion of the identification and testing of vaccine
mary data for discovery of new proteins and new targets following analysis of genomic information is
metabolic pathways. Initially, we will be faced even more subject to charges of naivety than feared in
with thousands of novel genes for which we have section 3.9. To the limited knowledge of the authors,
no function. At this stage, bioinformatic analysis attempts have been made by numerous skilled work-
(of metabolic pathways, for example) and system- ers to identify trypanosome proteins that may serve
atic (preferably high-throughput) analysis of gene as vaccine candidates, only to be thwarted by the
function (as attempted in the RNAi analysis de- dense coat of variable surface protein that prevents
scribed in 3.8) will be important. We would expect access by antibodies to invariant proteins, and the
that these kinds of analyses will provide new efficiency of antigenic variation. It is not immediately

obvious how the availability of genome sequence how we can ensure that the entire research commu-
and the discovery of novel genes and pathways may nity has access to information. In the near future this
provide new approaches, given these results. The may necessitate distribution of CDs, in which case
only high-throughput parasite vaccine testing project how should this be coordinated? In the more distant
known to the authors is that of Professor J Blackwell, future, WHO and those institutions who see a role
who is testing pooled DNA vaccines against leishma- for such web-based information must seek a path to
niasis. All the resources for such a project applied to improved institutional infrastructure and electronic
infection with T. brucei are available, but any assess- resources.
ment of applicability should involve a wider range of
researchers with appropriate skills. Access to Unannotated and Annotated
Sequence Data
All raw unannotated sequence data are made avail-
SHARING OF INFORMATION AND ACCESS able immediately on the websites of the sequencing
TO DATA AND RESOURCES: SUMMARIES centres, and then in the public databases. However,
The T. brucei Genome ‘Network’ access to annotated data is more problematic. The
It can seem difficult to obtain information from, or Sanger Institute is unable to annotate fully until
to know how to “join”, the genome network. This is close to completion of a chromosome, due to their
not in any way due to exclusivity as such, but be- shotgun approach. Their provision of preliminary
cause it works as any other network – people who annotation of chromosome I caused considerable
know each other and who have common interests confusion as researchers did not fully appreciate
come together in subgroups to seek funding. Inevi- that the chromosome was discontiguous. TIGR is
tably this is also driven by the availability of suitable able to provide some annotation of completed BACs
funding for functional/applied genomics in differ- as they are finished, but this approach does not pro-
ent countries – for example, the Wellcome Trust vide as many raw sequences as early on in the proj-
(UK) is currently the most forward planning in pro- ect. This problem is unresolved and subject to much
viding funds for global analyses, reducing its insis- discussion at network meetings. One interim solu-
tence on hypothesis-driven research etc., so, pre- tion is provided by the clustering and BLAST analy-
dictably, recent groupings have been UK-driven. For sis of discontinuous sequence data (GSS, shotgun,
this reason it is very important that WHO continues ESTs)(http://www.sanger.ac.uk/Projects/T_bru-
to direct some funds towards helping those African cei/Toolkit/blast_server.shtml; hftp://ftp.sanger.
scientists with an interest in this work to travel to ac.uk/pub/databases/T.brucei_sequences/GSS/cl
network meetings, to make contacts, to set up col- usters/). Many researchers annotate the small re-
laborations and to talk about priorities. This latter gions of immediate interest to them, but for some sci-
point is very important: there are many very good entists this requires some bioinformatics training. We
biologists working on the fascinating basic biology hope that some of these problems will be addressed
of T. brucei, but their priorities may differ somewhat by the creation of the relational genome database.
from those of the endemic countries and only the
presence of outspoken scientists whose interests lie Access to Comprehensive Genome
in controlling the disease will ensure that we do not Information in Relational Databases
lose sight of the necessity to direct funds towards As stated many times above, access to all databases
disease-applied genomics. currently under development will ultimately require
secure access to the Internet, although provision of
Obtaining Information CDs may be an interim solution. In addition to the
Until now, information dissemination has been genome database to be hosted at the Sanger Centre
underfunded and very dependent on the commit- (section 3.1), a separate Wellcome Trust-funded proj-
ment of certain individuals to spreading the word, ect (Melville, see 4.5) will also provide genome map-
maintaining websites, and being approachable and ping data in a relational database that will be com-
amenable to email requests for information and ex- munity-interactive and will facilitate some analyses
planations. But it is increasingly recognized that col- before it is superceded by the complete genome
lected information and properly run databases are sequence. Once again, this will be web-accessible
becoming vital in our brave new world of collabora- and, while it is possible to provide copies on CDs,
tive “big science”, and as more resources are made personnel resources are limited to providing full
available towards these aims, it should become eas- explanations and guidance on a website.
ier to obtain up-to-date information on the progress
of genome network-based projects. However, it is Obtaining DNA Resources for Use
also inevitable that these information resources will in Research Projects
be web-based. It is therefore vital to consider now All biological resources used by the genome net-

work are available to researchers on request, includ- treatment of trypanosome infection. All scientists
ing the TREU927/4 stock and 10.1 derivative, high- should have access to such data as will simplify
density filters of genomic libraries, genomic DNA hypothesis generation and experimentation, whe-
clones, cDNAs and karyotype blots (Melville, et al, ther their interests lie in basic biology or in applied
1998; http://parsun1.path.cam.ac.uk). The genome science. In addition to the promotion of good scien-
website provides addresses and email links. The tific experimentation, it seems likely at the current
genome network insists that hybridization and time that African scientists must help persuade inter-
sequence data derived using its resources are re- national funding bodies and the scientific communi-
turned to the database curator for inclusion in the ty to apply the data for the development of novel
genome database (http://parsun1.path.cam.ac.uk). epidemiological tools, drugs and vaccines.
These resources were provided with funding from
WHO/TDR for five years. Since the beginning of 2001, The area of genomics and applied genomics has
the Wellcome Trust Functional Genomics Initiative has expanded considerably over the past decade or so;
provided a four-year programme grant to expand this the driving force in this being the development of
facility to assist the growth of functional genomics bioinformatics tools that make access to the avail-
projects within the network (Melville, Cambridge). able data possible. Quite important also is the wide
range of publicly accessible tools developed to make
Obtaining Reference and Mutant Strains use of these data. Yet many African scientists who
for Use in Research Projects could use such information cannot, or do not, for
As part of the above project, a collection of reference many reasons. While this Scientific Working Group
strains, transformed lines and mutants will be main- (SWG) will address the problem of African try-
tained and provided to researchers on request (Tur- panosomiasis, there is no doubt that the opportuni-
ner, Glasgow). Current funds are not sufficient to ties and impediments are generic, and hence appli-
consider curating large field collections. The sharing cable to virtually all disease problems in endemic
of well-curated field collections may be of value in countries in Africa, and perhaps in most developing
certain comparative genomic projects and could be countries. Increasing the ability of African scientists
offered up for discussion. Many institutions have to participate in the application of data derived from
good collections of primary or very limited passage genome projects will stimulate research activities
isolates that could be harnessed as a valuable not only in this area, but also in a multidisciplinary
resource. WHO is supporting efforts to establish manner, since such data have to be evaluated in
good quality databases in a number of places, inclu- processes that require broad-based competencies.
ding Kenya Trypanosomiasis Research Institute
(KETRI). We propose here ways in which participation in A-
frica, outside of international institutions, can be
INVOLVEMENT OF SCIENTISTS FROM developed and strengthened.
TRYPANOSOMIASIS-ENDEMIC COUNTRIES
It is with some disappointment that we report the Bioinformatics (Present Competence
low level of participation of African institutions in and Training Needs)
the genome network to date. There may be a range Although there is some competence in bioinformat-
of reasons for this: inappropriateness of the tasks to ics within the science community in Africa, it is
institutional aims or facilities, competition from largely fragmented and inadequate. To quantify it
other laboratories with access to materials and the would be virtually impossible. However, it is known
facilities to perform high-throughput tasks with that this capacity is mainly concentrated in interna-
greater efficiency, disinterest in genomics given the tional institutes, a situation that is inappropriate,
more pressing problems of control of livestock infec- and efforts should be made to extend this to univer-
tion and human epidemics or research on pathogen- sities and national research centres. One way of
esis etc., or lack of sufficient contacts and encourage- beginning to do this is to develop a series of training
ment. Maybe we need not be too disappointed that courses in bioinformatics, either continent-wide or
African laboratories did not play a central role in the regionally, to develop Africa’s capacity in bioinfor-
process of sequence generation itself (other than the matics. The value of this is two-fold:
ESTs, the majority of which were generated in • It will equip participants with knowledge on what
Kenya; and we should also note the employment of information is available, where it is, how it can be
African scientists in European and US laboratories). accessed and utilized to develop tools and meth-
But we should consider it a scandal if this tremen- ods to address public health problems.
dous resource, initiated by TDR, is not made avail- • It would begin to expand the human resource
able to the entire global research community and is base in bioinformatics among African scientists,
not applied to the full to problems of control and hence contributing to the expansion of the

research activities in African trypanosomiasis and in the continent for within-continent exchange of
other major disease situations. This would create expertise. In some cases, such limitations exist even
an environment for continuous in-house training. within countries. One reason for this is that the
value of bioinformatics is not well recognized by
It is anticipated that such courses would bring to- managers of national institutions. Sharing of infor-
gether expertise from within Africa and elsewhere mation across areas of disease interest is also not
(in or outside the African trypanosomiasis commu- good, especially in African trypanosomiasis, where
nity). Doing this would help build the necessary many institutes have a single disease mandate.
partnerships (within and across diseases) to sustain Thus:
the knowledge base in bioinformatics. It may be pos- • there could be closer interaction between national
sible to bring in experts in computing who have lit- and the international institutions where there is
tle biological sciences training but who are willing to greater capacity to apply data derived from geno-
use their expertise in biology. me projects, to use the available resources more
efficiently.
Capacity building in bioinformatics can also be • there should be greater effort to promote broad
enhanced through providing funding support (by multidisciplinary projects constituted in part by
TDR and other donors) for Internet service provider the use of applied genomics. Such linkages will
(ISP) and access charges within projects. Addressed enhance the use of available information from
as a budget item in relevant proposals, this could various genome projects to address different bio-
include visits to laboratories with expertise in bioin- logical questions.
formatics (north or south). We refer you also to the
proposal from the Pathogenesis and Applied Geno- Retention of Trained Personnel
mics Committee for regional bioinformatics courses. It may be prudent to say that no single agency can
If the funding is secure and consistent, these may go guarantee that good quality personnel are retained in
some way to meeting these aims. research, and particularly in national systems within
disease endemic countries. However, important con-
Infrastructural Capacity tributions towards stemming the outflow of person-
Worldwide, the capacity of personal computers nel can be made, even by a single stakeholder.
(processor speed and storage space) continues to
improve phenomenally. Africa is no exception. Elec- Perhaps the flight of personnel largely accounts for
tronic access to information is often limited due to the paucity of good quality funding applications
poor communication infrastructure; good, fast and from Africa to address the problem of African try-
dedicated connections are few and far between, and panosomiasis. In the terms of reference for this
often only reliable in international institutions. With SWG, TDR recognizes that “many well trained per-
proper training, however, good quality information sonnel in African trypanosomiasis have left the field
can still be obtained by email via a telephone line, for others, such as HIV/AIDS”. Personnel have
even where institutions have a very limited number indeed left in many directions, including to other
of computer terminals with on-line access (even if countries, primarily in the north, to other research
only one terminal with limited time slots). Im- areas within Africa, or even out of science.
provement of institutional capacity should continue
to be an important objective; admittedly, maximal While it has been suggested that TDR could follow
access to databases is only achievable in an environ- a strategy of choosing a few studies and the centres
ment that recognizes the value of bioinformatics. to carry them out, the success of this will depend on
the retention of good quality personnel in the field.
TDR can help by giving small grants, perhaps about Within Africa, the SWG should consider:
US$2000 annually, to national institutes to fund elec- • allowing African investigators working in their
tronic access. Such funds would be used to pay ISP own countries to draw salaries or salary support
and connection charges to individual scientists from projects funded by TDR, since poor remu-
within national institutes and universities. Such neration is a major impediment to creating a criti-
funding would not necessarily be linked to specific cal mass for research.
project funding, although applicants could be • promoting collaborative initiatives between coun-
encouraged to budget for this within projects. tries in the south, where this is scientifically
sound, hence expanding the collaborations that
Institutional Linkages exist currently.
African scientists who have undertaken some train- • encouraging collaborative projects (north-south;
ing in bioinformatics have experience that can be south-south; inter-institutional within countries)
shared. However, there is not enough linkage with- that have a bioinformatics (and bioinformatics

training) component in order to strengthen the RECOMMENDATIONS
capacity to apply genomics data to address public • Improve institutional capacity in Africa to access
health problems. data from genome projects by acquiring comput-
ing hardware suitable for bioinformatics (through
FUNDING AND HUMAN RESOURCES: projects or independently). In the long term, this
REQUIREMENTS AND OPPORTUNITIES will be vital for full involvement in molecular bio-
The genome sequence will be a valuable resource for logical research on trypanosomes.
the research community, but there is much to be • Identify possibilities for adding value in the colla-
done to turn its potential for new directions and new tion of data, for example in a central database for
discoveries into reality. No researcher need feel there DNA-based markers for use in epidemiological
is no opportunity to become involved. However, in- studies.
volvement will depend heavily on having access to • Identify areas of bioinformatic analysis that lack
the available information and contact with other support, e.g. identification of metabolic path-
groups. One way to become involved in the genome ways, comparison of pathways across the Kineto-
network is to develop an interest group, investigate plastidae. Identify the researchers required for
the resources available, then look for funding to use such projects.
the genomic data for a purpose – such a group may • Identify now the requirement for further geno-
then be “hooked in”. The RNAi group is one exam- mic analysis and sequencing of other strains
ple, a drug discovery group may be another. (e.g. gambiense and other clinical variants) and
species (T. congolense, T. vivax). This may be
The next phase of the sequencing revolution will be complete genomic sequencing or shotgun se-
“comparative genomics”. Technologies that aim to quencing to a given level (e.g. 3x, 7x, 10x) and
simplify the resequencing of different genotypes could be based, once again, in high-throughput
(strains) are in development. Although it hardly or local centres.
seems possible that we might be able to sequence • Consider the techniques available for comparison
more than one genome, only five years ago we of strains, including the possibility of compara-
didn’t believe we could sequence even one. Now is tive sequencing (e.g. of gambiense).
the time to identify those species and strains that • Consider inclusion of genomics experts in ongo-
would provide vital information to researchers if ing drug development working groups.
comparative sequence information were available. • Ensure that the voices of African scientists and
applied scientists are heard within the genome
Funding agencies are increasingly realizing the va- network. Bring a drug development working par-
lue of large collaborative groupings and high-throu- ty into the network.
ghput techniques. It is timely to consider the poten- • Encourage the dissemination of information that
tial of novel applications of genomic methods to may lead to the formation of new interest groups
current problems, as the openings are there. The (where possible within existing groupings, e.g.
Wellcome Trust is in the forefront of such develop- TDR, the Programme Against African Trypano-
ments. The Trust also has an active international somiasis (PAAT), Global Forum on Agricultural
programme in (inter alia) tropical disease research Research (GFAR), to discourage proliferation) to
(http://www.wellcome.ac.uk/en/1/biosfgintintfu- access the genomic resources for new projects.
nunkcrg.html) and has recently indicated its desire • Consider current possibilities for funding of geno-
to achieve a higher profile for this programme. mics-based projects leading to drug target identi-
Therefore, at this point in time, north-south col- fication and validation; in this context, reevaluate
laborations may be seriously considered: both full the potential for inclusion of scientists from try-
inter-institute collaborations and relatively minor panosomiasis-endemic countries.
collaborations that facilitate technology transfer • Facilitate training in bioinformatics to increase
and the kind of training and contacts that enable competency and promote the utilization of geno-
scientists to develop an independent career. mics data in Africa. We take note of the call by
TDR for proposals in bioinformatics and applied
Some projects mooted here are large and costly, genomics to develop research and training cen-
requiring coordination and commitment. While tres/networks in Africa, Asia and Latin America.
TDR funds may not be sufficient to underpin coor- • Encourage the establishment of contacts between
dinated applied genomics projects, it is important scientists/institutions (in Africa and elsewhere),
that working groups such as this take a lead role in through exchange visits, training courses and joint
defining the problems that need to be addressed, to proposals, to maximize additive competencies.
ensure that the genomics revolution does not bypass • Improve the remuneration of African scientists
those directly affected by trypanosomiasis. working on TDR-supported projects in order to

retain trained personnel, hence improving institu- Melville SE et al. The molecular karyotype of the
tional and national research capacity. megabase chromosomes of Trypanosoma brucei and
• Promote collaboration between countries (South- the assignment of chromosome markers. Molecular
South; North-South) to take advantage of shared and Biochemical Parasitology, 1998, 94:155-173.
experiences and of possibilities for shared funding.
Melville SE et al. Selection of chromosome-specific
Acknowledgements DNA clones from African trypanosome genomic
Some of the text included here is extracted from: libraries. In: Analysis of non-mammalian genomes,
Melville SE. Characterisation and sequencing of the Birren B, Lai E, eds. New York, Academic Press,
African trypanosome genome. In: Guidelines and 1996, pp 257-293.
issues for the discovery and development of drugs against
tropical parasitic diseases, Vial H, Fairlamb A, Ridley Ngo H et al. Double-stranded RNA induces mRNA
R, eds. World Health Organization (2001). We thank degradation in Trypanosoma brucei. Proceedings of the
all our colleagues for discussion of the points pre- National Academy of Sciences, USA, 1998, 95:14687-92.
sented here and especially the members of the
genome network for their open sharing of informa- Rudenko G et al. Selection for activation of a new
tion and progress. variant surface glycoprotein gene expression site in
Trypanosoma brucei can result in deletion of the old
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able from the African trypanosome genome project.

Annex 7
INSTITUTIONAL CAPABILITY STRENGTHENING

INSTITUTIONAL DEVELOPMENT training, research and control work, must remain a
AND CAPACITY BUILDING IN priority. The first step would be to carry out an in-
COUNTRIES ENDEMIC FOR ventory of existing research and training institutions,
SLEEPING SICKNESS and identify areas of immediate and long-term con-
cern. This should assist in the identification of nation-
J. Mathu Ndung’u al and regional institutions in Africa which could be
Kenya Trypanosomiasis Research Institute (KETRI), strengthened by the international community in col-
P.O. Box 362 Kikuyu, Kenya laboration with national institutions and govern-
ments. Linkage arrangements, between national and
INTRODUCTION overseas institutions with donor support, need to be
This report is intended to stimulate discussion on the further explored, strengthened and extended.
possible solutions available for improving research
and training in, and strengthening institutions for, TRAINING
effective delivery of services in the 37 countries In the past, training specifically addressed the prob-
affected by trypanosomiasis and the tsetse vector. lem of tsetse and trypanosomosis in the laboratory
and field with a view to ensuring long-term com-
The financial crisis that occurred in Africa in the 1980s mitment and contribution towards solving the prob-
and 1990s had an almost paralysing effect, leading to: lem. However, this training did not take fully into
• lack of funds to maintain physical infrastructure, account some vital concerns, e.g. that African try-
vehicles and equipment, or to replace outdated panosomiasis is just one of the many health prob-
and broken-down equipment. lems Africans are exposed to, the solution of which
• decline or lack of funding, at both national and demands a broad understanding and approach.
institutional levels, to carry out field surveillance
and control of tsetse, and field and laboratory To retain the interest and commitment of personnel
research in African animal trypanosomiaisis and who have been trained, there is a need for career
sleeping sickness. development. In recent times therefore, efforts have
• poor remuneration of staff, hence internal and been directed at broadening of training. Yet much
external brain drain of trained manpower and, remains to be done, especially in light of the devel-
therefore, paucity of leadership. opments towards privatization of services, includ-
ing in tsetse control. It is in this light, and in light of
Given this background, and considering that deter- the competing demands by different sectors of the
mined efforts made over the years to solve the prob- economy for limited funds, that institutional devel-
lems have not had the desired impact, a more ration- opment and training in disease endemic countries
al approach to research, training, and institutional should be viewed.
development is indicated. Issues arising include:
number and size of available research and training Four types of training are generally recognized:
institutions; appropriateness or otherwise of any re- • Training at sub-professional level, leading to the
search and training undertaken; incentives for staff to award of a diploma or certificate, e.g. animal
remain on the job, e.g. career development, adequate health assistant, animal health technician.
remuneration and an enabling environment. • Training at professional level, leading to the
award of professional degrees and diplomas.
INSTITUTIONAL DEVELOPMENT • Training leading to postgraduate degrees and
One of the recommendations made at the 1974 FAO diplomas.
Expert Consultation on Animal Production and • Short-term training leading to specialization.
Health Research in Copenhagen, Denmark, was to
provide facilities for training research workers ”in To these must be added the training of:
their own environment” and to strengthen existing • auxiliary personnel, e.g. clinical officers and field
national and regional institutes. Presently, most re- assistants, who need short courses and/or in-ser-
search institutes established pre-independence or vice training for field work.
immediately post-independence in Africa are in a • field agents, whose activities are coming more
deplorable state due to breakdown of physical infra- and more into the limelight because of their clo-
structure, equipment and vehicles, lack of funds for seness to the communities in community-based
laboratory and field work, and severe depletion of primary health care.
research and field staff. • contacts and farmers (general training).
Rehabilitation and strengthening of existing national For the first four types of training listed, a prerequi-
and regional institutions to carry out appropriate site for admission is formal education at secondary

school level, while on-the-job and in-service training explore whether there is a place for private initiative
are essential to develop and sharpen skills. In the in training.
case of training of auxiliaries, the requirement is that
they should have attended a secondary school, but STAFF RETENTION
may have dropped out along the way. In the case of The problem of staff retention is a global one, not
field agents, the ideal requirement is basic primary peculiar to the field of tsetse and trypanosomosis.
school education, emphasizing numeracy and What makes the case of those in the field of tsetse
enabling them to follow simple instructions and and trypanosomosis different, however, is that
training and to be selected by the community whom career advancement can be painfully slow and frus-
they will serve. trating in the public sector. Among the causes of
inability to retain staff are:
Training Needs • slow career advancement and lack of career
The priority given to training of the different cadres prospects.
will vary from country to country but will largely • poor salary and working conditions.
depend on: • delay or non-payment of field allowances.
• the prevalence of tsetse and trypanosomiasis. • lack of transport.
• the impact of tsetse and trypanosomiasis on the • lack of self-worth.
national economy. • few or no opportunities to take part in short-term
• the human, material and financial resources avail- and/or in-service training programmes, which
able for dealing with the problem. were a major incentive in the past but which are
now scarce due to lack of funds.
While there is a great need for experienced profes-
sional laboratory and field staff, there is, at the pres- The low remuneration and lack of career prospects
ent time, much greater demand for specialized mid- makes recruitment of good staff for training very
dle-level personnel to carry out the arduous task of difficult. Young, talented people tend to decline
field and laboratory operations, and for auxiliary and appointment unless they lack other job opportuni-
field agents who are crucial to the success of com- ties. Trained staff, on the other hand, either leave
munity-based health care and field programmes. their employment in favour of more lucrative eco-
nomic activities and/or political appointments
Training Institutions totally unrelated to their training, or, because of the
While there is no problem finding institutions to need to supplement their meagre pay through other
train personnel at professional and postgraduate unrelated economic activities, tend not to devote
levels, there are serious problems with training for attention to the job.
middle-level specialized personnel. In the last few
years, many national and regional training institu- SUMMARY
tions which give emphasis to tsetse and trypanoso- Training is needed at all levels, but more so at the
mosis have either been reduced in size or closed middle personnel and auxiliary levels. It is impor-
down completely because of funding difficulties. tant to address the career prospects and other bene-
Yet this is the training area of great need. The num- fits of trainees, e.g. short courses and in-service
ber of trainees that can be deployed to serve in the training that could encourage staff to remain in the
field depends largely on the number and size of the job for which they are trained.
training institutions available, and whereas concern
has been expressed, it appears that no solution to The issue of funding research and training institu-
sustaining the necessary institutions is in sight. tions on a sustainable basis must be addressed, for
which it may be necessary to review the present
Although there are certain advantages of tying trend of guaranteeing funding of research projects
training in institutions to ongoing projects, e.g. with for a period of 12 months only. The possibility of
regard to the funding and training of personnel, the involving private initiative in training, and how this
disadvantage is that the lifespan and funding of can be implemented without sacrificing quality,
projects is uncertain. What is needed is a training should be examined. South-south research and
institution with its own lifespan and funding, and training should be encouraged. There is an urgent
with participation from ongoing projects. need to find out the present status of national
research and training institutions with a view to
What then are other viable options? Would a donor advising national governments on the role of such
consider supporting two or more training institu- institutions in tsetse and trypanosomosis research
tions in Africa on a sustainable basis? Another sug- and control.
gestion, which at first may appear remote, is to

In order to provide an enabling environment for
field and laboratory research and training, insti-
tutional development needs to be given new impe-
tus. A possible starting point would be to carry out
an inventory of the existing research and training in-
stitutions to provide insight as to what type of stren-
gthening may be needed. Linking national institu-
tions and overseas institutions with donor support
is a possible area for expansion.

Annex 8
LIST OF PARTICIPANTS

Dr Serap Aksoy, Department of Epidemiology and Professor Krister Kristensson, Division of Neurodege-
Public Health, Section of Vector Biology, Yale nerative Disease Research, Department of Neuro-
University School of Medicine, 60 College St. 606 science, Retzius vag 8, B2:5, Karolinska Institutet, SE-
LEPH New Haven CT06510 USA. Tel: 1 203 737 2180, 171 77 Stockholm. Tel: 46-8-728 7825; Fax: 46-8-32 53 25,
Fax: 1 203 785 4782, E-mail: serap.aksoy@yale.edu. E-mail: krister.kristensson@neuro.ki.se.
Professor Cyrus Bacchi, Pace University, Haskins Dr Grace B Kyomuhendo, Department of Women
Laboratories, 41 Park Row, New York, NY 10038 and Gender Studies, Makerere University, P. O. Box
–1598, USA. Tel: 1 212 346 1246, Fax: 1 212 346 1586, 7062, Kampala, Uganda. Tel: 256 774 71 600 or 256 41
E-mail: cbacchi@pace.edu. 531484, Fax: 256 41 543539, E-mail: gendermu@swif-
tuganda.com, or wgs@mak.ac.ug.
Dr MP Barrett, University of Glasgow Institute of
Biomedical and Life Sciences, Division of Infection
Dr Francis J Louis Institut de médecine tropicale, BP 46
and Immunity, Glasgow G12 8QQ, Scotland. Tel &
Le Pharo, 13007 Marseille, France. Tel : 33 4 91 15 01 46,
Fax; 44 141 330 6904, E-mail: m.barrett@bio.gla.ac.uk.
Fax : 33 4 91 15 01 43, E-mail: dfc.louis@wanadoo.fr.
Dr Kwabena Bosompem, University of Ghana, No-

guchi Memorial Institute for Medical Research, P.O. Professor John Mansfield, Department of Bacte-
Box LG581, Legon. Tel: 233 21 500 374, Fax: 233 21 riology, University of Wisconsin-Madison, 1925
502182, E-mail: bosskm@hotmail.com, kbosom- Willow Drive/FRI Bldg Madison, WI 53706 USA.
pem@noguchi.mimcom.net. E-mail: jmm@bact.wisc.edu.
Dr Reto Brun, Swiss Tropical Institute, Postfach, Dr Daniel Masiga, Kenya Trypanosomiasis Re-
Socinstrasse 57, Basel, Switzerland. Tel: 41 61 284 82 search Institute, P.O. Box 362, Kikuyu, Kenya.
31, Fax: 41 61 271 86 54, E-mail: reto.brun@unibas.ch. E-mail: dkmasiga@hotmail.com.
Dr Christian Burri, Swiss Tropical Institute, Po- Professor Ian Maudlin, Centre for Tropical Veteri-
stfach, Socinstrasse 57, Basel, Switzerland. Tel: 41 61 nary Medicine, the University of Edinburgh, Easter
284 82 47, E-mail: Christian.Burri@unibas.ch Bush Veterinary Centre, Roslin, Midlothian, UK
EH25 9RG. Tel: 44 131 650 7347, Fax: 44 131 650 7348,
Dr Philippe Büscher, Instituut voor Tropische Genee- E-mail: imaudlin@vet.ed.ac.uk.
skunde, 155 Nationalestraat, 2000 Antwerpen, Bel-
gium. Tel: 32 3 2476371, Fax: 32 3 2476373, E-mail: Dr Dawson B Mbulamberi, Ministry of Health, P.O.
pbuscher@itg.be. Box 7272, Kampala, Uganda. Tel: 256 41 43087,
E-mail: ugwep@swiftuganda.com.
Dr Simon L Croft, London School of Hygiene and
Tropical Medicine, Department of Infectious and
Tropical Diseases, London WC1E 7HT. Tel: 44 171 Dr Honoré Meda, Projet SIDA 2, Benin (ACDI-Ca-
927 23 45, Fax: 44 171 636 87 39, E-mail: nada), 08 B. P. 900 Tri Postal, Cotonou, République
simon.croft@lshtm.ac.uk. du Benin. Tel: 229 31 36 02, Mobile: 229 957 169,
Fax: 229 31 36 05, E-mail: hmeda@bow.intnet.bj.
Dr Peter de Raadt, Bruglaan 11, 3743 J.B. Baarn, The
Netherlands. Tel: 31 35 541 21 21, E-mail: Dr Sara E Melville, Cambridge University, De-
raadt@compuserve.com partment of Pathology, Microbiology and Parasito-
logy Division, Cambridge, CB2 1QP, UK. Tel: 44
Dr Felix Doua, Projet des Recherches Cliniques sur 1223 333335, Fax: 44 1223 333346, E-mail:
la Trypanosomiase (P.R.C.T.), B.P. 1425, Daloa, Côte sm160@mole.biocam.ac.uk
d’Ivoire. Tel: 225 32 78 36 10, Fax: 225 32 78 3021:
E-mail: doua_felix@yahoo.com Dr C Miaka Mia Bilengé, Ministère de la Santé,
Secrétariat Général à la Santé, B.P. 3040 Kinshasa,
Dr John K Enyaru, Livestock Health Research In-
République Démocratique du Congo. E-mail:
stitute, P.O. Box 96, Tororo, Uganda. Tel: 256 45
bctrdc@ic.cd.
45050, Fax: 256 422 1070, jenyaru@africaonline.co.ug.
Dr J Josenando, Instituto de Combate e Controlo Dr V Nantulya, Harvard School of Public Health,

das Tripanosomiases, 168, Kwenha, Ingombota, CP 665 Huntington Avenue, Boston, 02115 MA, USA.
2657, Luanda, Angola. Tel: 244 2 399610, Fax: 244 2 Tel: 1 617 432 0659 , E-mail: vmnantul@hsph.har-
399611, E-mail: icct@snet.co.ao. vard.edu.

Dr Joseph Ndung’u, Kenya Trypanosomiasis Re- Dr M Schottler, Head, Health Policy, Bayer A.G.,
search Institute (KETRI), P.O. Box 396, Kikuyu, Business Group Pharma, Europe and Overseas,
Kenya. Tel: 254 154 32960-4, Fax: 254 154 32397, D-1368 Leverkusen, Germany.
E.-mail: ketri@healthnet.or.ke or Ketri@net2000ke.com
or ketri@bidii.com. Dr J Slingenbergh, Senior Officer, Animal Pro-
duction and Health, FAO, Rome, Italy. E-mail:
Dr Martin Odiit, Livestock Health Research Insti- Jan.slingenberg@fao.org.
tute, P.O. Box 96 Tororo, Uganda. Tel: 256 45 45050,
Fax: 256 45 45052, modiit@uol.co.ug. WHO secretariat
Dr Mary Bendig, PRD/TDR
Dr Alexandra PM Shaw, A. P. Consultants, Upper Mr Erik Blas, Programme Manager, PPM
Cottage, Abbotts Ann, Andover SP11 7BA, UK. Tel: Dr Charity Gichuki, Organizer
44 1264 710 238, Fax: 44 1264 710 759, E-mail: Dr Melba Gomes TDR/IDE
alexandrashaw@compuserve.com. Dr Win Gutteridge, Team Coordinator PRD
Dr Beatrice Halpaap, TDR/PRD
Dr Richard R Tidwell, Department of Pathology &
Dr David Heymann, EXD/CDS
Laboratory Medicine, University of North Carolina,
Dr Jean Jannin, CSR/EDC
Chapel Hill, NC, USA. Te1: 1 919-966-4294, E-mail:
Dr J Karbwang, TDR/PRD
tidwell@med.unc.edu.
Dr Jane Kengeya-Koyondo Team Coordinator
TDR/IDE
Dr Philipe Truc, Institut de Recherche pour le Déve-
Dr Deborah Kioy, TDR/PRD
loppement UMR 035 “Trypanosomoses Africaines”,
Mr Felix Kuzoe TDR/PRD
OCEAC, BP288, Yaoundé, Cameroun. Tel: 237 23 22
Dr Maryinez Lyons CAH/FCH
32 or 237 84 60 57 (mobile), Fax: 237 23 00 61, E-mail:
Dr Carlos Morel, Director WHO/CDS/TDR
truc@iccnet.cm
Dr MA Mouries, TDR/PRD
Collaborators and partners Dr Paul Nunn, TDR TB & LEP Disease Coordinator
Dr Alain Aumonier, Director, International Affairs, Dr Ayoade Oduola Team Coordinator TDR/STR
Aventis Pharma International, Tri E1/385 - 20 Dr Mark Perkins TDR/PRD
Avenue Raymond Aron, F-92165 Antony Cedex, Dr Richard Pink, TDR/PRD
France. Tel: 33 1 5571 6087, Fax: 33 1 5571 4185 Dr J Sommerfeld, TDR/STR
E-mail: Alain.Aumonier@aventis.com. Dr J Vanreas, TDR/PRD
Dr Simon Van Nieuwenhove, Regional Adviser,
Dr Jean-Pierre Helenport, Consultant, Medécins WHO Regional Office for Africa, B.P 1899, Kinshasa
sans Frontières/World Health Organization 1, Republique Démocratique du Congo. Tel: 243 88
Eflornithine Project, 2 Impasse des Bambous, Villa 03204, Fax: 1 321 953 9097, E-mail: omskin@joban-
Maéva, 13600 La Ciotat, France. Tel: 33 4 42 981042, tech.cd or omsrdc@raga.net
E-mail: jeanpierre.helenport@wanadoo.fr. Mr S Wayling, TDR/RCS
Dr F Zicker, Team Coordinator, TDR/RCS
Dr Anne Moore, Division of Parasitic Diseases, F22,
Centers for Disease Control and Prevention, 4770
Buford Highway, Atlanta GA 30341, USA. Tel: 1 770
488 7776, Fax: 1 770 488 7761, E-mail: aym2@cdc.gov.
Dr TC Nchinda, Senior Public health Specialist,

Global Forum for Health Research, c/o WHO,
Geneva, Switzerland. Tel: 41 22 791 3808, E-mail:
nchindat@who.ch.
Dr Bernard Pecoul, Project Director, MSF Access to

Essential drugs Project, Médecins sans Frontières, 12
rue du Lac, C.P. 6090, 1211 Genève 6, Switzerland. E-
mail bpecoul@msf.org.
Dr Michaleen Richer, Medical Coordinator, Sudan

Program, International Medical Corps, P. O. Box
67513, Nairobi, Kenya. Tel: 254 2 574386/88/89, Fax:
254 2 573973, E-mail: mickey@imcafrica.com.

Mailing address:
TDR
World Health Organization
20, Avenue Appia
1211 Geneva 27
Switzerland
Street address:
TDR
Centre Casai
53, Avenue Louis-Casai
1216 Geneva
Switzerland
Tel: (+41) 22-791-3725

Fax: (+41) 22-791-4854
E-mail: tdr@who.int
Web: www.who.int/tdr

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Scientific Working Group

4-8 June 2001

Copyright ©World Health Organization on behalf of the Special

Message from the Executive Director, CDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

Message from the Director, TDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface ............................................................................. viii

1 Executive summary ................................................................ 1

2 Overview and objectives .......................................................... 3

3 Epidemiology, disease surveillance and control

4 Drug development, preclinical and clinical studies,

TDR/SWG/01 • Report of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2 0 0 1 iii

5 Pathogenesis, genomics and applied genomics

Message from the Executive Director,

TDR/SWG/01 • Report of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2 0 0 1 vii

viii R e p o r t of t h e S c i e n t i f i c Wo r k i n g G ro u p o n A f r i c a n t r y p a n o s o m i a s i s, 2001 • TDR/SWG/01

African trypanosomiasis is caused by protozoan trypanosomes, and signif icantly, in T. b.

Figure 1 - The focal distribution of human African trypanosomiasis

Left of dotted line:

Right of dotted line: T. b. rhodesiense foci

The objectives of the scientific working group

EMERGENCE AND RE-EMERGENCE

Table 1 - Number of sleeping sickness patients treated by year per country

Sleeping sickness Country 1995 1996 1997 1998 1999 2000

Source: Compiled from the working

Ministries of health, research organizations Central governments often accord sleeping

by pharmaceutical companies. However, arrange- networks, such as the Programme Against

Reservoir hosts play an important part in sus-

• Standardizing and extending reporting

SURVEILLANCE AND INTERVENTION FOR CONTROL

• The constraints affecting sustained use

When combined with the epidemiological infor-

• Identification of modes of uptake, and

Drug Discovery and Drug Targets • Investigation of target enzymes or path-

2. Megazol • Combination of melarsoprol and nifur-

One of the major problems of clinical trials and

Prevention and Management of

Encephalopathy syndromes, which occur during

Coordination of Clinical Trials

The current number of suitable centres (with ade-

Jennings FW. Chemotherapy of trypanosomiasis: the

Jennings FW. The potentiation of arsenicals with

Jennings FW. Combination chemotherapy of

Background is typified by a loss of Th1 cells secreting IFNg

Trypanosome Biological Phenotype

Trypanosoma brucei Genomics and functional analysis of genes. Two resource

Recent advances in molecular technologies, and

ADVOCACY AND MARKETING FOR SLEEPING SICKNESS

INSTITUTIONAL DEVELOPMENT AND CAPACITY BUILDING

• Recruitment by TDR of a full-time professional staff member to coordinate African try-

References ILRAD REPORTS, April 1993. Estimating the costs of

Olsson T, et al. Bidirectional activating signals

Pepin J et al. The impact of human immunodefi-

Pepin J, et al. Short-course eflornithine in Gambian

Pepin J, Meda H. The Epidemiology and control of

Smith DH, Pepin J, Stich HR. Human African try-

Vaidya T, et al. The gene for a T lymphocyte trigger-

World Bank, World Development Report, Investing in

Control and surveillance of African trypanosomiasis.

Human African trypanosomiasis, treatment and drug

Komba EK, et al. Genetic diversity among

INTRODUCTION In West and Central Africa, sleeping sickness is

Congo basin on relief of the Emin Pasha expedition 9000

to say that the cause lay in the general increase in 6000