Tropical Medicine and International Health

doi:10.1111/tmi.12087

volume 18 no 5 pp 564577 may 2013

Bacillus thuringiensis israelensis (Bti) for the control of dengue

vectors: systematic literature review
R. Boyce1, A. Lenhart2, A. Kroeger2,3, R. Velayudhan4, B. Roberts5 and O. Horstick6
1
2
3
4
5
6

Department of Internal Medicine, Massachusetts General Hospital, Boston, MA, USA

Liverpool School of Tropical Medicine, Liverpool, UK
Special Programme for Research and Training in Tropical Diseases, World Health Organization, Geneva, Switzerland
Department for the Control of Neglected Tropical Diseases, World Health Organization, Geneva, Switzerland
London School of Hygiene, Tropical Medicine, London, UK
Institute of Public Health & University of Heidelberg, Heidelberg, Germany

Abstract

objective To systematically review the literature on the effectiveness of Bacillus thuringiensis

israelensis (Bti), when used as a single agent in the field, for the control of dengue vectors.
method Systematic literature search of the published and grey literature was carried out using the
following databases: MEDLINE, EMBASE, Global Health, Web of Science, the Cochrane Library,
WHOLIS, ELDIS, the New York Academy of Medicine Gray Literature Report, Africa-Wide and
Google. All results were screened for duplicates and assessed for eligibility. Relevant data were
extracted, and a quality assessment was conducted using the CONSORT 2010 checklist.
results Fourteen studies satisfied the eligibility criteria, incorporating a wide range of interventions
and outcome measures. Six studies were classified as effectiveness studies, and the remaining eight
examined the efficacy of Bti in more controlled settings. Twelve (all eight efficacy studies and 4 of 6
effectiveness studies) reported reductions in entomological indices with an average duration of control
of 24 weeks. The two effectiveness studies that did not report significant entomological reductions
were both cluster-randomised study designs that utilised basic interventions such as environmental
management or general education on environment control practices in their respective control groups.
Only one study described a reduction in entomological indices together with epidemiological data,
reporting one dengue case in the treated area compared to 15 dengue cases in the untreated area
during the observed study period.
conclusion While Bti can be effective in reducing the number of immature Aedes in treated
containers in the short term, there is very limited evidence that dengue morbidity can be reduced
through the use of Bti alone. There is currently insufficient evidence to recommend the use of Bti as a
single agent for the long-term control of dengue vectors and prevention of dengue fever. Further
studies examining the role of Bti in combination with other strategies to control dengue vectors are
warranted.
keywords dengue, vector control, Bacillus thuringiensis israelensis, Aedes

Introduction
Dengue is the most rapidly advancing vector-borne disease in the world (Special Programme for Research and
Training in Tropical Diseases, WHO/TDR 2007). Overall, it is estimated that 2.5 billion people, or roughly onethird of the worlds population, live in dengue-endemic
areas. Annually, an estimated 50100 million cases of
dengue fever and several hundred thousand cases of
severe dengue occur.
In the absence of any vaccine and preventive drugs,
controlling the mosquito vectors of dengue (principally
564

Aedes aegypti) is the only way to prevent and control

dengue transmission. Chemical and biological agents
remain important components of dengue vector control
programmes. Unfortunately, the widespread use of chemical insecticides has contributed to increasing resistance to
these agents among A. aegypti, especially in the Americas
and the Caribbean (Rodriguez et al. 2005; Harris et al.
2010).
Biological control, based on the introduction of organisms that prey upon, parasitise, compete with or otherwise reduce populations of the target species, is
considered a practical alternative to the application of

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chemical insecticides in controlling mosquito vectors of

disease (Special Programme for Research and Training in
Tropical Diseases, WHO/TDR 2009). In addition to
reducing both toxicity to non-target species and environmental contamination, biological control also offers
reduced potential for resistance development. However,
biological control agents can be comparatively expensive
and logistically more challenging to deploy and maintain
compared to traditional chemical agents.
Bacillus thuringiensis var. israelensis (Bti) is a grampositive, spore-forming entomopathogenic bacterium first
isolated in 1976 (Goldberg & Margalit 1977). As a biological control agent, Bti has demonstrated high efficacy
against target organisms, primarily mosquito and black
fly larvae (Mittal 2003; Lacey 2007). Bacillus thuringiensis israelensis exerts its lethal effects through producing a
variety of toxic proteins that are ingested by the larvae of
susceptible organisms. These toxins are then activated in
the gut of the larvae where they cause disruption of the
cell membranes and death of the organism. The specificity of this mechanism has been demonstrated in multiple
studies with no adverse effects on non-target invertebrates and vertebrates (Lee & Scott 1989; Merritt et al.
1989; Lacey & Mulla 1990; Saik et al. 1990). Because of
the complex mechanism of action involving many proteins, the potential for resistance development is greatly
reduced. Bacillus thuringiensis israelensis is available in a
number of formulations that can be applied by hand or
with conventional spray equipment (Lacey 2007), allowing Bti to be utilised in a variety of breeding habitats.
To date, no systematic review of the scientific literature
has been undertaken to examine the evidence for the
effectiveness of Bti against dengue vectors. The objective
of this study was to provide a systematic review of the
effectiveness of Bti, when used as a single agent in the
field, for the control of dengue vectors and prevention of
dengue fever.
Methods
This review follows the reporting guidelines set forth in
the PRISMA statement for systematic reviews and metaanalyses (Liberati et al. 2009).
Eligibility criteria
The eligibility criteria for the reviewed literature included
the following: (i) research with an experimental design
producing primary quantitative data, (ii) research conducted in the field, defined as any community or environment where dengue vectors naturally occur, (iii) the use
of Bti as a single agent to control dengue vectors, (iv)

2013 Blackwell Publishing Ltd

clear information on Bti formulation and dosing, (v)

outcome measures reported as immature indices (i.e.
Stegomyia indices, oviposition indices and/or presence/
absence of immature stages of Aedes) and (vi) a minimum follow-up period of at least 20 days. Only literature reported in English was included. Conference
abstracts and proceedings from conferences were
excluded.
Search strategy
The literature search and analysis was developed and carried out through March 2012, by two data extractors.
The search terms derived from three major categories: (i)
dengue disease, which included dengue and dengue
hemorrhagic fever, (ii) the Bti intervention and (iii) the
outcome, which centred on reductions in dengue vector
density as measured by various entomological indices.
The search included both free text and subject heading
terms.
The search strategy was applied to the following databases to locate peer-reviewed studies: MEDLINE, EMBASE, Global Health, Web of Science, the Cochrane
Library and WHOLIS. The following databases were
searched for grey literature: ELDIS, the New York Academy of Medicine Gray Literature Report, Africa-Wide
and Google. Because of the limited search options available in many of the grey literature databases, a broad
search strategy was employed, typically including only
the terms dengue and/or Bacillus thuringiensis.
Study selection, data extraction
All results were screened for duplicates by author, title,
journal and publication date. In the first stage, results
were screened based on the title and abstract only. The
full text of those studies that were not excluded was
then reviewed for final assessment. The reference section
of each of the selected publications was reviewed to
identify additional relevant studies. Relevant information, including study design, setting and Bti formulation,
from each of the selected studies was extracted, and
when information was unclear from the reported results,
an attempt was made to contact the corresponding
author. Studies were first classified as either efficacy
studies, where Bti was placed in targeted containers that
were observed for reductions in immature stages, or
effectiveness studies, where Bti was used to treat containers or peridomestic areas, and surveyed for reductions in classical entomological indices. Study designs
were subsequently divided into three categories:
randomised or quasi-randomised controlled trials (RCT),
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cluster-randomised controlled trials (CRCT) and nonrandomised controlled trials (NRCT).

Results
Study selection

Analysis

After screening out duplicates, the literature search identified 355 articles for assessment. Fourteen articles met the
eligibility criteria. Of these, nine were identified from the
published literature databases and three from the grey
literature search (Phan-Urai et al. 1995; Haq et al. 2004;
Lam et al. 2010). The most common reasons for exclusion were as follows: interventions did not use Bti (190
studies), the manuscripts were review articles and/or of
non-experimental design (93 articles) or the studies took
place in laboratory or semi-field settings (33 articles). A
review of the references cited in the articles that met the
eligibility criteria yielded one additional study (Dua et al.
1993). One more article (Tan et al. 2012) that was

Because the majority of the included studies were nonrandomised, it was not possible to utilise an existing, validated instrument for assessing risk of bias. Instead, the
CONSORT 2010 checklist served as a framework to
describe limitations in the conduct and reporting of the
included studies. No studies were excluded for quality
reasons if the eligibility criteria were met, but limitations
and possible biases are reported in the results section.
Different Bti formulations and methods of application,
together with varying statistical analyses and outcome
measures across the studies, precluded any attempt at
meta-analysis.

Academic databases

Gray literature

Medline, EMBASE, Global Health,

WHOLIS, Cochrane Library

ELDIS, NY Academy of Medicine Gray

Literature Report, Africa-Wide, Google

Screening for duplicates

Combined results (n = 354)

Excluded (n = 309)
Screening by title & abstract

-Bti not studied (n = 190)

-Review or non-experimental design (n = 87)

Included (n = 45)

-Abstract only (n = 18)

-Laboratory or semi-field design (n = 14)
Excluded (n = 33)

Screening by full text

-Laboratory or semi-field design (n = 19)

-Combination intervention (n = 7)

Met eligibility criteria (n = 12)

-Review or non-experimental design (n = 6)

-Non-dengue vector (n = 1)

New study list of references (n = 13)

Newly published study (n = 14)

Included (n = 14)

Figure 1 Flow chart describing paper selection and inclusion/exclusion process.

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R. Boyce et al. BTI and dengue: a systematic review

published during manuscript preparation was included in

the review. No further studies were included after reviewing the references of this additional article. Figure 1 summarises the selection process.
The 14 included studies are summarised in Tables 1
and 2, in which they are assigned a unique identifier for
reference purposes in the remainder of this manuscript.
General study characteristics
All studies were published between 1993 and 2012. Ten
of the studies were conducted in South-East Asia, with
India being the most common location (1, 2, 3, 5 in
Table 1). Three studies were conducted in South America
(4, 6, 11 in Tables 1 and 2) and one in the Caribbean
(10 in Table 1). In most studies, the setting was selected
because of a high incidence of dengue or elevated preintervention entomological indices. No study incorporated economic analysis or provided cost data for consideration.
Study designs
Six studies were classified as effectiveness studies; the
remaining eight examined the efficacy of Bti in more
controlled settings. The most common study design was
the NRCT, which was used in 10 studies. Three were
CRCTs (4, 10, 11 in Tables 1 and 2), often considered
the gold standard for such research, and 1 was a RCT
(13 in Table 1). Eight studies used various breeding containers (water tanks, water jars, discarded tires, plants)
as the unit of allocation, 4 used households (4, 8, 11,
12 in Table 2), and 2 used land areas (7 and 14 in
Table 2). All studies incorporated a control group, the
majority of which received only entomological surveillance. Two of the control groups received education
and/or environmental management interventions (4, 11
in Table 2), and one control group received standard
control strategies consisting of weekly temephos treatment (7 in Table 2). Two control areas were unexpectedly treated with extensive space spraying mid-way
through the study due to a dengue outbreak (8, 14 in
Table 2).
The sample size varied significantly across studies. The
smallest study (5 in Table 1) examined only five cement
tanks in each of the two intervention groups compared to
eight control tanks, while the largest study (4 in Table 2)
incorporated more than 1000 households. The duration
of follow-up ranged from 20 days (5 in Table 1) to
15 months (4 in Table 2) with a median follow-up of
6 weeks. Three studies (9, 10, 14 in Tables 1 and 2) prespecified the period of follow-up.

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Nearly all studies provided a rudimentary description

of the study setting, often limited to geographical location and previous estimates of dengue incidence. Less
commonly reported was information on potential confounding factors such as the socio-economic status of residents, housing construction and infrastructure. Weather
conditions, either historical or during the intervention
period, were reported in only five studies (4, 7, 10, 11,
12 in Tables 1 and 2).
Outcome measures
The most commonly reported outcome measures in the
efficacy studies were larval and pupal densities, defined as
the mean number of larvae or pupae per container, and
per cent reduction in infestation using Mullas formula
(Mulla et al. 1971). Four studies (6, 9, 12, 14 in Tables 1
and 2) also monitored the average larval free period or
the amount of time after application of the intervention
that potential habitats remained free from Aedes larvae.
In the community effectiveness trials, oviposition indices
and the Stegomyia indices (the house index (number of
positive houses per 100 houses), container index (number
of positive containers per 100 containers) and Breteau
index (number of positive containers per 100 houses))
were more commonly reported (WHO/SEARO 1999).
Only one study reported clinical outcomes from routinely
collected epidemiological data (14 in Table 2).
Entomological surveillance protocols were clearly
reported in 10 of the studies, while four studies (2, 4, 6,
13 in Tables 1 and 2) provided no or limited information
regarding entomological sampling methods. Five studies
(1, 2, 3, 5, 9 in Table 1) did not incorporate statistical
precision estimates or significance tests in the reporting of
outcomes.
Interventions
A wide range of formulations and application methods
were utilised in the studies. Seven studies (2, 4, 6, 9, 10,
11, 12 in Tables 1 and 2) incorporated slow-release Bti
tablets or briquettes, seven studies (1, 3, 5, 7, 8 14 in
Tables 1 and 2) used manual or motorised spray equipment to apply Bti, and three studies (2, 8, 13 in Tables 1
and 2) applied Bti granules or powder directly into positive breeding sites. The majority of the efficacy trials utilised only a single application of Bti in the study
containers. In contrast, all of the effectiveness trials
repeated application of Bti in differing frequencies as outlined in Table 2. Seven studies incorporated multiple
intervention groups, comparing the effect of various formulations or dosages of Bti (1, 2, 5, 6 in Table 1) or
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Table 1 Summary of efficacy studies evaluating the use of Bti for the control of dengue vectors
No.

Reference

Setting

Objectives

1.

Ansari and
Razdan
(1999)

Delhi, India

Evaluate the Bt H-14

granule formulation
under laboratory and
field conditions
against Aedes aegypti

2.

Batra et al.
(2000)

Delhi, India

3.

Dua et al.
(1993)

5.

Haq et al.
(2004)

568

Study design
and duration

Bti formulation

Intervention group(s)

NRCT
4 weeks

Bti H-14 granules

Potency not reported
Manufacturer not reported

Single spray
application
of Bti H-14 applied to
20 evaporation
coolers and 36
discarded tires at
three different doses
(0.25, 0.50 and
1.0 g/m2)

Evaluate the
effectiveness of 3
formulations of Bti
against immature
A. aegypti in coolers
and tires

NRCT
6 weeks

Bacticide (1000 ITU/mg)

Biotech International, Delhi
Vectobac DT (1900 ITU/mg)
Vectobac G (3500 ITU/mg)
Abbott Laboratories

Single application
of Bti in 5 different
formulations/doses:
(1) 15 desert coolers
with Vectobac
G at 2 g/cooler
(2) 65 desert
coolers with
Vectobac DT at
0.75 g/cooler
(3) 10 desert
coolers with
Bacticide powder
at 1.0 g/cooler
(4) 35 discarded tires
with Vectobac DT
at one tablet
(0.375 g) per tire
(5) 20 discarded tires
with Vectobac DT at
two tablets
(0.75 g) per tire

Hardwar,
India

Evaluate Bactoculicide
for its efficacy to
control mosquitos
(Aedes, Anopheles,
Culex) breeding in
factory scraps in an
industrial area

NRCT
6 weeks

Bactoculicide
Potency not reported
Bordsk Chemical,
Moscow

Single application of
Bactoculicide
sprayed over the
standing water in
73 industrial
scrap containers
positive for mosquito
larvae
at a dosage of 0.5 g/m2

Surat City,
India

Evaluate spraying of
two bacterial larvicide
formulations for
efficacy against
Anopheles, Culex and
Aedes mosquitoes
under the operational
conditions of an urban
malarial control
programme

NRCT
20 days

Bacticide
WP (1000 ITU/mg)
Biotech International,
Delhi

(1) Bacticide sprayed

at 5 kg/ha in 5
cemented tanks and
chambers at
15 construction sites;
retreatment at 10 days
(2) Vectobac
sprayed at 11 kg/ha
in 5 cemented
tanks and chambers
at 16 construction sites;
retreatment at 10 days

Vectobac 12AS
(1200 ITU/mg)
Aventis Crop
Sciences

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Control group(s)

Principal outcomes and

statistical analysis

Results

Study conclusions

No treatment of
4 evaporation
coolers and
6 discarded tires

Larval density and

per cent reduction
(Mulla)
No statistical testing
of results

Reduction at 4 weeks:
Evaporation coolers:
0.25 g/m2 = 46.3%
0.5 g/m2 = 100%
1.0 g/m2 = 100%
Discarded tires:
0.25 g/m2 = 47.7%
0.5 g/m2 = 100%
1.0 g/m2 = 100%

The formulation was most

effective at a dose of 0.5 g/m2
with larvicidal activity persisting
for up to 4 weeks. Bt H-14 may be
used for control of dengue,
but studies examining relative
efficacy and cost-effectiveness
are required

No treatment
of 10 desert
coolers

Per cent of breeding

sites positive for
3rd/4th stage instars
and pupae; per cent
reduction (Mulla)
No statistical testing
of results

Per cent reduction

Intervention 1: Vectobac
G 2 weeks = 100%
4 weeks = 66.7%
6 weeks = 16.7%
Intervention 2: Vectobac DT
2 weeks = 100%
4 weeks = 86.5%
6 weeks = 28.8%
Intervention 3: Bacticide
2 weeks = 100%
4 weeks = 25.0%
6 weeks = 12.5%
Intervention 4: Vectobac DT
2 weeks = 100%
4 weeks = 60.9%
6 weeks = 6.3%
Intervention 5: Vectobac DT
2 weeks = 100%
4 weeks = 100%
6 weeks = 0%

Results showed that Bti

formulations provide complete
control of A. aegypti
larvae for 24 weeks. Study
highlights the practical
community-based
application of Bti.
Use of these formulations is
effective and more
user-friendly
than conventional methods

No treatment of
10 industrial
scrap containers
positive for
mosquito larvae

Larval density of
3rd/4th stage instars,
per cent reduction
(Mulla)
No statistical testing
of results

Per cent Reduction:

24 hrs: 100%
10 days: 100%
14 days: 97.7%
24 days: 98.4%
5th week: 100%
6th week: 90%

Bactoculicide appeared to
be the best solution for
controlling mosquito breeding
in such problematic habitats,
controlling mosquito breeding
for up to 5 weeks

No treatment of
8 cemented
tanks and
chambers at
15 construction
sites

Pupal/larval densities
and per cent
reduction
(Mulla)
No statistical
testing
of results

(1) Bacticide provided 100%

reduction for duration
of the study
(2) Vectobac provided 100%
reduction through day 3; 53.1%
reduction on day 17 after
retreatment

Study demonstrated that

biolarvicides should be used
at an interval of 710 days.
Liquid Vectobac had a relative
ease of operation compared
to Bacticide. Biolarvicides
can be incorporated
as part of integrated vector
control program

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Table 1 (Continued)
No.

Reference

Setting

Objectives

6.

Kroeger et al.
(1995)

Cucuta,
Columbia

Three objectives: (1)

describe peoples
knowledge, attitudes and
practices regarding
dengue fever, (2) analyse
infestation of the
community with
A. aegypti and (3) test
the efficacy of Bti with
respect to the level and
duration of larval
reduction in water tanks

Study design
and duration
NRCT
48 days

Bti formulation
Culinex tablets
(33 000 ITU/mg)
Valent BioSciences

Intervention group(s)

2 tablets of Culinex
(33 000 ITU/mg)
per 50 l in 143 household water
tanks used for laundry

1 tablet of Culinex
(33 000 ITU/mg)
per 50 l in 6 household
water tanks used for laundry

2 tablets of Culinex
(33 000 ITU/mg) per 50 l in
61 household water
tanks used for laundry

2 tablets of Culinex
(33 000 ITU/mg) per 50 l in
11 household water tanks
used for laundry that were
regularly emptied during
first 10 days of observation

9.

Mahilum
et al. (2005)

Cebu City,
Philippines

Evaluate the present

dengue situation and
control strategies to
include a survey of
mosquito breeding zsites
and infestation rates, an
evaluation of peoples
knowledge, attitude and
practices towards
dengue infection and the
efficacy of Bti tablets
against A. aegypti larvae

NRCT
21 days

Vectobac DT/Culinex
tablets (2700 ITU/mg)
Valent BioSciences

11 positive breeding sites,

including drums, pails,
cement basins, discarded tires
and water jars, were treated
at a single application of
1 tablet for sites <50 l capacity
and 2 tablets for sites >50
l capacity

10.

Marcombe
et al. (2011)

Vauclin,
Martinique

Characterise the
resistance status of
A. aegypti larvae from
Martinique to
conventional and
alternative insecticides
and to assess their
efficacy and residual
activity under simulated
and field conditions

CRCT
105 days

Vectobac DT
(3400 ITU/mg)
Abbott Laboratories

Three communities each had 5

positive breeding sites selected
for a single treatment with one
of 4 insecticides: Bti (5 mg/l),
diflubenzuron, pyriproxyfen
or spinosad, for a total of
15 interventions sites
per insecticide

13.

Sulaiman
et al. (1999)

Kuala
Lumpur,
Malaysia

Compare the efficacy of

Abate (temephos) and
Vectobac G against
Aedes albopictus in
bromeliads in the field.

RCT
4 weeks

Vectobac G
(200 ITU/mg)
Abbott Laboratories

Forty-five bromeliad plants chosen

at random and divided into
3 groups of 15 plants assigned
to receive a single application
of either (1) Vectobac G at 1 g
per plant, (2) Abate at 1 g per
plant or (3) no treatment

Bti, Bacillus thuringiensis israelensis; RCT, randomised controlled trials; CRCT, cluster-randomised controlled trials; NRCT,
non-randomised controlled trials.

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Control group(s)
(1) Culinex vs. no
larvicide
No treatment of 22
household water tanks
used for laundry

Principal outcomes and

statistical analysis
Length of time until tank
reinfested with 3rd or 4th
larval instar

Results

Study conclusions

Intervention 1:
>90% of treated tanks remained free of
reinfestation for 30 days, compared to <30%
of untreated tanks (no P-value reported)

Application of Bti led to a rapid and total elimination of

mosquito larvae with an effect that lasted for more than a
month. Lower doses were less efficacious and should not
be recommended. Community acceptance of Bti was
higher than that of temephos, largely because of the lack
of effect on water quality and taste. Further studies are
required to evaluate the relative reliability and costs
compared to traditional larvicides

Wilcoxon rank-sum test and

chi-square test

(2) Culinex (33 000

ITU/mg) vs. Culinex
(8000 ITU/mg)
1 tablet of Culinex
(8000 ITU/mg) per 50 l
in 20 household water
tanks used for laundry
(3) Two vs. one tablet
of Culinex
(33 000 ITU/mg)
per tank
1 tablet of Culinex
(33 000 ITU/mg) per
50 l in 60 household
water tanks used for
laundry
(4) Effect of changing
water
2 tablets of Culinex
(33 000 ITU/mg) per
50 l in 39 household
water tanks used for
laundry that were not
emptied during first ten
days of observation

Intervention 2:
100% of 33 000 ITU treated tanks free of
reinfestation for 28 days, compared to only
6 days in those tanks treated with 8,00 ITU
formulation (P = 0.025)

Intervention 3:
100% of tanks treated with 2 tablets free of
reinfestation for 28 days, compared to only
6 days in tanks treated with only 1 tablet
(P = 0.02)

Intervention 4: 100% of tanks in both

groups free for 10 days; >90% of tanks in
both groups free for 28 days with decrease to
approximately 50% at 34 days in tanks that
are not emptied; slightly higher proportion of
emptied tanks free at all time periods
(P < 0.01)

Three positive breeding

sites including a 200-l
drum, a 25-l can and a
200-l plastic container
received no treatment

Mortality rates of larvae

observed at 3-day intervals
No statistical testing of results

Control sites were infested throughout the

duration of the study, while the intervention
sites showed 100% mortality for the initial
6 days of the study. Reinfestation was noted
in some containers as early as 9 days, but the
majority of containers remained free of
larvae for up to 18 days

Duration of Bti efficacy was less in field conditions than

under semi-field conditions, perhaps due to heavy rains. Bti
should be incorporated in integrated mosquito control
programmes and is a useful supplement or replacement for
conventional insecticides

3 communities each
had 5 positive breeding
sites to serve as an
untreated control (15
total)

Larval and pupal density;

relative density (Mulla)
Log transformation and ANOVA

There was a strong and significant difference

over time between treatment groups
(P < 0.001). Bti demonstrated a fast killing
effect with RD decreasing from 100% to 1%
(95% CI 0.44%), but low residual activity
with larval densities returning to 22% (95%
CI 749%) of initial size after 28 days

A lower residual activity for all insecticides in field trials

compared to semi-field trials was observed. The poor
efficacy of Bti may render control of A. aegypti
populations in Martinique difficult. Mosquitos were,
however, fully susceptible to Bti despite more than
12 years of use

Fifteen plants assigned

to the no treatment
group

Per cent mortality of immatures

Vectobac G yielded 97.8% mortality 24 hrs

after treatment, 76.2% 1 week after
treatment and 65.4% 2 weeks after
treatment. This reduction was not
significantly different from Abate (P > 0.05),
but was significantly different from the
control (P < 0.01). After 2 weeks, there was
no significant differences between the
intervention and control groups

Both insecticides would be effective in a similar

environment with residual activity of up to 1 week

ANOVA

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Table 2 Summary of effectiveness studies evaluating the use of Bti for the control of dengue vectors
No.

Reference

Setting

Objectives

4.

Favier et al.
(2006)

Brazilia, Brazil

Determine the influence of climate

and of environmental vector
control with or without insecticide
on Aedes aegypti larval indices and
pupal density

7.

Lam et al.
(2010)

Western
Singapore

8.

Lee et al.
(2008)

11.

Study design and

duration

Bti formulation

Intervention group(s)

CRCT
18 months

Mosquito Dunks (7000

ITU/mg)
Summit Chemical

Four intervention groups, each containing

environmental management plus:
(1) Temephos (Abate) at 1 ppm in
all containers
(2) Temephos (Abate) at 1 ppm in all
containers in all premises
(3) Bti (Mosquito Dunks) in all
containers in positive premises only
(4) Methoprene-S (Altosid) in all
containers in positive premises only

Investigate the efficacy of Bti

against Aedes albopictus in a
forested military training ground
where source reduction in natural
breeding sites is difficult and nonspecific insecticides may cause
harm to the ecosystem

NRCT
3 months

Vectobac WG (3000 ITU)

Valent BioSciences

130 ha sprayed with Bti at a dosage of

500 g/ha every 2 weeks using motorised
back-pack and vehicle-mounted sprayers

Selangor State,
Malaysia

Determine the impact of larviciding

with a Bti formulation, Vectobac
WG, on the adult mosquito
population in a dengue endemic
site in Selangor State, Malaysia

NRCT
12 weeks

Vectobac WG
(3000 ITU/mg)
Valent BioSciences

Two residential areas (20 houses each)

received a single indoor treatment of
Vectobac WG in all water containers
>50 l at a dosage of 8 mg/l combined
with back-pack spraying of all outdoor
larval habitats at a dosage of 500 g/ha
every 2 weeks

Ocampo
et al. (2009)

Cali, Columbia

Evaluate two control methods for

A. aegypti that can be used by the
community: lethal ovitraps and Bti
briquettes

CRCT
4 months

Bactimos briquettes
(7000 ITU/mg)
Summit Chemicals

Four neighbourhoods, each had one

block (40 houses) randomly selected to
receive education and either (1) lethal
ovitraps, (2) Bti briquettes in main
breeding sites, (3) lethal ovitraps + Bti
briquettes or (4) no insecticide (control);
Bti dosage was 1/4 briquette in each
water storage tank (76 total), which
were replaced monthly

12.

Phan-Urai
et al. (1995)

Chanthaburi
Province,
Thailand

Evaluate the Bti H-14 formulated

tablet for Aedes larvae in a rural
village of Chanthaburi Province

NRCT
17 weeks

Larvitab 1-g tablet

(600 ITU/mg)
Manufacturer
not reported

Bti at a dosage of 1 g per 200 l was

applied to all potential breeding sites,
including water storage containers,
cement baths and ant guards, in a single
village (61 houses) in Chanthaburi
Province. Treatment was repeated when
containers were reinfested with mosquito
larvae

14.

Tan et al.
(2012)

Shah Alam,
Malaysia

Comparing the efficacy of BTI

(Vectobac) against Aedes
albopictus and A. aegypti

RCT
1 year

Vectobac WG
(3000 ITU/mg)
Valet Biosciences
Corporation

One residential area (300 houses) received

7 biweekly cycles of Vectobac WG,
followed by 7 weekly cycles, followed by
4 biweekly cycles in all mapped
potential and actual outdoor larval
habitats

Bti, Bacillus thuringiensis israelensis; RCT, randomised controlled trials; CRCT, cluster-randomised controlled trials; NRCT,
non-randomised controlled trials.

572

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R. Boyce et al. BTI and dengue: a systematic review

Control group(s)

Principal outcomes and statistical

analysis

Only environmental
management, defined as
incitation to container
removal when possible or
emptying water
in storage containers
in all premises

House index, container index,

Breteau index, pupal density

128 ha treated with

standard control
strategies comprised of
weekly oiling of ground
larval habitats and
monthly treatment of
permanent water bodies
with temephos

Ovitrap index, larval density,

per cent reduction (Mulla)

One untreated residential

area that was treated with
extensive space spraying
6 weeks into the trial due
to a dengue outbreak

Ovitrap index and larval density

One block from each of

the 4 neighbourhoods
was randomly selected to
receive no insecticide,
but only education
(environmental control,
emptying water jars)

No treatment of similar
breeding sites in a single
village (92 houses) in
Chanthaburi province.

Results

Study conclusions

No significant differences in larval indices

and pupal density between control and
intervention groups

In moderately infested areas, insecticides do not

improve upon environmental vector control.
Infestations could be further reduced by focusing on
residences and containers particularly at risk

Per cent reduction in ovitrap index at

intervention site:
Month 1 = 14.2% (P < 0.05)
Month 2 = 47.9% (P < 0.05)
Month 3 = 66.0% (P < 0.05)

Wide application of Bti into vegetation to treat all

natural breeding sites produced a significant decline
of the adult A. albopictus population compared to
the control This is an innovative approach that can
be easily adapted to all communities to successfully
suppress the A. albopictus adult population

Nonparametric tests with 95% CI

for indices and MannWhitney
U-test for proportions

t-test for OI and larval density

Per cent reduction in larval density at

intervention site:
Month 1 = 59.3% (P < 0.05)
Month 2 = 53.2% (P < 0.05)
Month 3 = 80.0% (P < 0.05)
The OI significantly decreased in both study
sites 4 weeks after initiating the
intervention, declining from 57.5% to
19.1% (P < 0.05) at one site and 66.7% to
30.3% (P < 0.05) at another. This decline
in the OI was paralleled by a similar decline
in both A. aegypti and Aedes albopictus
larval densities. An increase in OI and larval
density was observed in both
sites following cessation of the
intervention. The OI at the control site
remained high until the initiation of
space spraying following a dengue outbreak

Widespread application of Vectobac WG at targeted

larval habitats is able to provide control of dengue
vectors. Space spraying of Bti was found to be
superior to traditional methods of application in
terms of effectiveness, coverage and labour.
Additionally, Bti did not produce any undesirable
environmental consequences

House index, pupal index (mean

pupae per house) and adult index
(per cent of houses infested with
adult Aedes)
Poisson regression

Entomological indices obtained during the

intervention period were not significantly
different between treatment groups and
controls. During the entire study period
(341 visits), only one water tank treated
with Bti was positive for larvae. Positive
containers consisted mostly of plants in
water and small containers. Bti briquettes
were not used routinely in 40% of houses

Pre-intervention education and environmental

management may have contributed to the lack of
effect seen with interventions. Decrease in pupal
density did not eliminate presence of adult
mosquitoes in homes to a level sufficient to prevent
transmission suggesting larger buffer zone may be
required to address breeding sites outside of the
residential areas. Use of Bti briquettes may have
induced people to change water more often

House index, container index,

Breteau index, per cent reduction
(Mulla), average larva free period,
landing and biting rates
F-test, t-test

Significant reductions (P < 0.05) in the

intervention area were observed, with
averages of:
HI = 69.8%
CI = 84.1%
BI = 84.4%
Landing rate = 73.9%
Biting rate = 73.6%

The Bti formulation was very practical and effective

for the control of A. aegypti larvae in Thai
communities. The intervention was readily accepted
by the community

t-test for OI and larval density

No similar trend observed in control area.

Average larval free period was longest in
drinking containers (16.4 + / 2.5 weeks),
followed by ant guard, washing and bathing
containers
One residential area
without BTI application.
However, intensive
pyrethroid fogging
operation in study weeks
3754 due to a dengue
outbreak

2013 Blackwell Publishing Ltd

Ovitrap index and larval density

Routine epidemiological data on
dengue cases
Chi-square test for OI
t-test for larval density

OI was suppressed to below 10% and

maintainted up to 4 weeks post-treatment
Outdoor OI remained at 40% in the
untreated site (P < 0.05)
1 dengue case in the treated area
15 dengue cases in the untreated area

BTI application can suppress A. aegypti and Aedes

albopictus populations at a dengue endemic site with
a temephos-resistant population and potentially
interrupt dengue transmission in humans

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R. Boyce et al. BTI and dengue: a systematic review

comparing the effect of Bti to other insecticides (4, 10 in

Tables 1 and 2) or lethal ovitraps (11 in Table 2) with a
standard control. The most common insecticide of comparison was temephos, which was used in two of the
studies (4, 10 in Tables 1 and 2).
Efficacy and effectiveness of Bti interventions
Each of the eight efficacy studies reported a post-intervention reduction in the observed density of immature
stages compared to the respective control group. In general, these were non-randomised studies using containers
as the unit of allocation. In the community-based effectiveness studies, the results were less clear, with 4 of the
6 studies (7, 8, 12, 14 in Tables 1 and 2) reporting significant but time-limited reductions in entomological indices. The two effectiveness studies that did not show
significant reductions employed basic interventions such
as environmental management (4 in Table 2) or general
education on environment control practices (11 in
Table 2), in the respective control groups. The other
study that used standard dengue vector control practices
as the control comparison (7 in Table 1), did, however,
report a relative reduction in entomological indices in the
Bti intervention area.
In the efficacy studies looking only at targeted breeding
sites, Bti demonstrated a rapid killing effect, typically
eliminating all larvae from treated containers within
24 h. The vast majority of containers remained free of
larvae for the first 2 weeks of observation. However,
reinfestation of some containers did occur within 7
9 days of treatment (5, 9 in Table 1). Only one study (3
in Table 1) reported 100% reduction in larval density for
more than 4 weeks with a single application of Bti. Similar results were demonstrated in another study (1 in
Table 1), but the study period was limited to 4 weeks.
Five studies examined the effect of repeated Bti treatments (7, 8, 11, 12, 14 in Table 2). Two of these studies
sprayed Bti every 2 weeks (7, 8 in Table 2), in 1 study,
Bti briquettes were replaced every month (11 in Table 2),
in another study, Bti briquettes were replaced when the
containers were reinfested (12 in Table 2), and in 1 study
(14 in Table 2), Bti was sprayed in biweekly and weekly
cycles. In four of the studies (7, 8, 12, 14 in Table 2), the
repeated application of Bti at various frequencies resulted
in significant (P < 0.05) reductions in the density of
immature stages compared to the control over the course
of treatment. Each of these four studies also examined
the effect on adult dengue vector populations, either
through ovitrap indices or landing and biting rates. In
each study, a decline in the adult mosquito population
was observed, typically only after the second application
574

of Bti, which occurred in the fourth to sixth week of the

trial. This delayed effect is thought to be due to the survival of already existing adult mosquitoes, which would
not have been affected by larvicidal applications of Bti.
The study by Lee et al. (8 in Table 2) showed a rebound
in the adult and larval indices within 6 weeks of cessation of treatment. Although the study by Ocampo et al.
(11 in Table 2) did not observe any significant reductions
in entomological indices, only one Bti-treated container
was ever found to be positive for mosquito larvae.
The four studies that compared different formulations
of Bti (1, 2, 5, 6 in Table 1) did not suggest evidence of
superior efficacy of any one commercial product. The
results did, however, suggest that higher doses of Bti provide a longer duration of effect (1, 2, 6 in Table 1). The
per cent reduction in the density of immature stages at
4 weeks nearly doubled with a proportional increase in
Bti concentration in these three studies. The study by Ansari and Razdan (1 in Table 1) demonstrated the decreasing marginal benefit of this approach, as both of the
higher concentrations used (0.5 and 1.0 g/m2) achieved
similar results at 4 weeks, suggesting that a similar effect
could be achieved with less cost at the lower dose.
In the two studies where Bti formulations were compared with other insecticides (4, 10 in Tables 1 and 2),
the results were mixed. The study by Favier et al. (4
Table 2) found no significant reductions attributable to
any of the interventions, which included Bti, temephos
and methoprene-S. Here, the authors concluded that
insecticides may not improve on environmental control
practices, which served as the control comparison. In
contrast, the study by Marcombe et al. (2011) showed
that both Bti and pyriproxyfen were effective in initially
lowering larval densities but had a notably lower residual
activity compared to diflubenzuron and spinosad.
Only one study (14 in Tables 1 and 2) linked routinely
collected epidemiological data to the entomological data.
When a dengue outbreak occurred in the study area, only
one case was reported in the Bti intervention area and 15
cases were reported in the control area, suggesting that
the lower mosquito densities in the Bti intervention area
could have had a protective effect.
Acceptability of Bti
Very few of the included studies formally evaluated the
acceptability of Bti application. The study by Haq et al.
(5 in Table 1) surveyed field staff and noted some operational challenges and reports of skin irritation after contact with Bacticide. Lee et al. (8 in Table 2) monitored
for potential effects on non-target organisms, but did not
observe any undesirable consequences. Ocampo et al.

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volume 18 no 5 pp 564577 may 2013

R. Boyce et al. BTI and dengue: a systematic review

(11 in Table 2) reported that the Bactimos briquettes left

a visible residue on water storage containers and speculated that this may have induced residents to clean the
containers more often.
In general, most studies reported that the Bti intervention was readily accepted and even preferred over other
measures such as temephos applications because it did
not affect water quality or taste. The study by Ocampo
et al. (11 in Table 2) was the only one to quantify acceptance, reporting that 40% of the houses did not routinely
use the briquettes, because householders did not like the
residue the briquettes left in their water.
Discussion
The evidence presented from the efficacy studies suggests
that Bti can be effective in controlling the immature
stages of dengue vector mosquitoes in a variety of breeding sites. The killing effect is rapid, typically eliminating
all immature forms from treated containers within 24 h,
with a residual effect that ranges between 2 and 4 weeks.
These results, however, only suggest that Bti is effective
in specifically targeted containers that actually receive
treatment. Given the large number of potential habitats,
the widespread application of Bti to all potential breeding
sites may not be practical.
A better estimate of the overall impact of Bti as single
agent is provided by the effectiveness studies (4, 7, 8, 11,
12, 14 in Table 2). The findings from these studies are
mixed and do not provide conclusive evidence that Bti,
when used a single agent, produces significant reductions
in entomological indices. There are a number of reasons
that might explain the lack of effect. First, investigators
may have failed to identify and treat all potential breeding sites within each household. Thus, while Bti may
have achieved significant reductions in treated containers,
the existence of even a few untreated and productive
breeding sites may have masked the effect of Bti. The
study by Ocampo et al. (11 in Table 2) highlights this
phenomenon, where over the course of the study, only
one of 76 water storage tanks that were treated with Bti
was ever positive for mosquito larvae. However,
untreated smaller containers and containers holding
aquatic plants were often positive for larvae.
The comparison of experimental results with untreated
control groups is also important in determining the
impact of Bti as a sole control agent. In all studies where
individual containers were the unit of allocation, the control containers received no treatment. However, in 2 of
the 4 household and community-level studies (4, 11 in
Table 2), the control group received an intervention in
the form of education and/or environmental management.

2013 Blackwell Publishing Ltd

Both these studies found no significant differences

between the Bti intervention groups and the control
groups, suggesting that Bti alone provided an equivalent
level of control as what was achieved through education
and/or environmental management.
The study by Ocampo et al. (11 in Table 2) also raised
the issue of buffer zones. While it may be possible to
greatly reduce breeding sites within targeted residential
areas, treatment must also extend beyond these areas to
prevent the immigration of adult mosquitoes from nontreated areas. The flight range of A. aegypti is restricted:
mosquitoes rarely disperse further than 100 m from their
emergence location (Muir & Kay 1998; Harrington et al.
2005). Despite this limited flight range, Ocampo et al.
(11 in Table 2) did not observe a reduction in the presence of adult mosquitoes in the intervention households.
This result suggests that their buffer zone, which consisted of treatment of one house beyond the intervention
block in every direction, was insufficient. Lee et al. (8 in
Table 2) experienced a similar challenge because they
were unable to treat a factory that was adjacent to one
of their study sites. While they still observed a reduction
in the ovitrap index at this location, the magnitude of the
effect was less pronounced than that of another site,
which was not adjacent to the untreated factory.
The reviewed studies did not provide a generalisable
estimate of community acceptance and uptake of the Bti
interventions. In the majority of the studies, vector surveillance and Bti application were carried out by the
investigators rather than vector control authorities or
members of the community. Given the limits on national
budgets for vector control programmes, it is unlikely that
routine control efforts will achieve levels of intensity
comparable to those obtained in these studies.
The majority of the studies failed to assess and compare baseline characteristics between the control and
intervention groups. Additionally, five studies (1, 2, 3, 5,
9 in Table 1) did not incorporate any statistical methods
into their data analyses for the purpose of estimating precision or comparing results between groups. Without this
information, it is difficult to interpret the significance of
the reported results.
Finally, only one study (14 in Table 2) linked routinely
collected epidemiological data from both the Bti intervention and control sites. Although fewer dengue cases were
reported in the intervention area, no information was
available regarding the method or quality of routine epidemiological data collection or possible sites of dengue
infection outside of the study area.
Publication bias should be considered as a final limitation of this study, as likely more studies with a positive
outcome are reported in the literature. The diversified
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R. Boyce et al. BTI and dengue: a systematic review

search strategy limits this publication bias; however, it

cannot be completely eliminated.
In summary, there is evidence that Bti is effective in
reducing the density of immature dengue vectors when it
is applied to targeted containers as demonstrated by the
efficacy studies. However, the evidence to suggest that
Bti is effective as a single agent, when used in a community setting, is limited. Given the increasing prevalence of
insecticide resistance in dengue vectors in many parts of
the world, understanding the control implications of
using alternatives to chemical insecticides such as Bti is
becoming increasingly important. However, there is a
clear need for further studies that utilise cluster-randomised controlled designs to investigate the efficacy and
effectiveness of Bti and to further link entomological outcomes to dengue transmission measures.

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Corresponding Author Olaf Horstick, Institute of Public Health, INF 324, University of Heidelberg, 69120 Heidelberg, Germany.
E-mail: Olaf.Horstick@Uni-Heidelberg.de

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