J. Med. Microbiol. - Vol.

48 (1999), 973-975
0 1999 The Pathological Society of Great Britain and Ireland
ISSN 0022-26 15

EDITORIAL

Penicilliosis marneffei

Penicillium marneffei causes one of the most important

opportunist infections in South-east Asia. Clinicians in
other parts of the world are often not well acquainted
with the disease, sometimes resulting in delayed
diagnosis and treatment. Interestingly, the first documented human case was a laboratory-acquired infection
in France in 1959 [l]. In 1973 a natural infection was
reported in an American suffering from Hodgkin's
disease who had a history of travel to South-east Asia
[2]. Since then, cases have been reported in several
countries in this area. With the advent of the AIDS
pandemic there was a dramatic increase in the number
of penicilliosis marneffei patients, and the epidemiology, pathology and clinical management of the disease
have been clarified.

Mycology
I! marneffei is a dimorphic fungus with a restricted
distribution. Most infections are seen in indigenous
inhabitants of the south-western part of China (e.g., the
Guangxi Province), Vietnam, Thailand and Hong Kong,
or in those with a history of travel to these areas.
Although the fungus was initially isolated from
bamboo rats [3], the role of the rodents in the natural
history of the fungus has not been established. It is
postulated that I! marneflei exists as a saprophyte in
soil, and that human beings, as well as bamboo rats,
become infected through inhalation of the conidiophores [4]. However, attempts at isolation of I!
marneffei from soil have not been very successful.

P marneffei is unique among over 400 Penicillium

species in being the only one that demonstrates thermal
dimorphism [5]. The fungus grows well on Sabouraud
dextrose agar. When grown at 25"C, the fresh culture
appears similar to other Penicillium spp. : rapidly
growing greenish-yellow mycelial colonies. The most
characteristic feature is a soluble brick-red pigment that
diffuses into the medium. Few Penicillium spp. produce
such a pigment. For a definitive mycological identification, demonstration of thermal dimorphism is essential.
At 37C the fungus assumes a yeast form, in culture
and in vivo. Colonies are glabrous, beige-coloured and
do not produce red pigment.
Microscopically, the mycelial form resembles other
Penicillium spp. with smooth-walled conidiophores

West meets East

borne on terminal verticils of aerial hyphae. The yeast
forms are ovoid or elongated measuring 2-3 pm X
26.5 pm. Similar forms are observed within macrophages or extracellularly in tissue samples from
patients. In contrast to the yeasts of other fungi, those
of I! marneflei divide not by budding, but by fission, so
that a transverse septum is often seen in dividing cells.
This helps to differentiate I! marneffei from other
dimorphic fungi, especially Histoplasma capsulatum.

Clinical features and diagnosis

Penicilliosis marneffei is characterised by systemic
infiltration of the cells of the reticulo-endothelial
system, resulting in a progressive febrile illness that
is almost always fatal if untreated. Common presentations included fever (up to 98% of patients), anaemia
(75%), weight loss (72%), skin lesions (70%) and
lymphadenopathy (52%) [6]. The skin lesions are
particularly distinctive: molluscum contagiosum-like
papules, commonly on the forehead, trunk, upper
extremities and abdomen, that may be skin-coloured
or pigmented with central umbilication [7]. Occasionally the lesions may appear vesicular, necrotic, or
pustular, and may appear as ulcers on the skin or
mucosal surfaces. Microscopy of the lesions reveals
numerous yeast cells both intra- and extra-cellularly.
Pulmonary involvement can result in cough, infiltration, abscess, or cavitation. Outside the lungs and the
skin, disease may be found in the bones and joints,
lymph nodes, pericardium, liver, and even the central
nervous system [6].
HIV infection is the single most important underlying
disease for penicilliosis marneffei [81. In Thailand, for
example, >90% of the documented cases were HIVinfected [9]. Indeed, among HIV-infected patients,
penicilliosis marneffei is the third commonest opportunist infection in parts of South-east Asia after
tuberculosis and cryptococcosis [9]. Some patients
may have other underlying diseases or may be
receiving corticosteroids; a few have no recognisable
condition that impairs their immune status. The relative
predominance of different types of patients can vary in
different centres: in our hospital only 3 1% of patients
with penicilliosis marneffei have AIDS, while 54%
have other underlying diseases, including systemic
lupus erythematosus and haematological malignancy,

974

S. S. Y. WONG, H. SIAU AND K. Y. YUEN

or were on corticosteroids; 15% were not obviously

immunocompromised.
Clinical diagnosis in endemic areas is not usually
difficult, especially in AIDS patients with characteristic
skin lesions. Elsewhere, diagnosis may not be immediately obvious as there are few, if any, pathognomonic signs and symptoms. The only clue may be a
history of travel to endemic areas, which should arouse
clinical suspicion, especially in immunocompromised
patients. Most patients present with pyrexia of unknown origin, and an initial diagnosis of typhoid fever,
tuberculosis, melioidosis or autoimmune disease is
often entertained, reflecting the local incidences of
these diseases. In c. 80% of our patients, initial
coverage with broad-spectrum antibiotics, including plactam antibiotics and the newer quinolones, was given;
60% of patients were given empirical anti-tuberculosis
treatment, as tuberculosis remains endemic in much of
South-east Asia. Laboratory confirmation of the diagnosis is therefore essential.

Laboratory diagnosis
The only way to make a definitive diagnosis is
conventional fungal culture of a good clinical specimen. I? marneffei can be readily differentiated from
other Penicillium spp. by the production of the
diffusible red pigment and by demonstration of thermal
dimorphism. P. marneffei is isolated most frequently
from skin biopsies and peripheral blood, followed by
bone marrow, lymph nodes and other tissue biopsies
[6]. It may be found rarely on direct examination of a
peripheral blood smear in those with heavy, disseminated infections [ lo]. However, 24-45% of patients
may not have a positive blood culture, especially if the
disease is localised, and the time for a positive culture
to appear varies from 2 days to 4 weeks. In patients
without fungaemia, broncho-alveolar lavage, biopsies
and fine needle aspiration, or even pericardial aspiration, are necessary to obtain tissues from the suspected
sites of infection.
The yeast form of I? marneffei may be stained by
methenamine silver or periodic acid-Schiff stains, but
this may not differentiate it from other dimorphic fungi
such as H. capsulatum in histological sections. A
monoclonal antibody to galactomannan (EB-A 1) may
be used to detect I? marneffei (and Aspergillus spp.) in
formalin-fixed, paraffin-embedded tissues [ 111. Various
serodiagnostic tests have been developed for the
detection of antibodies or antigens in the serum and
body fluids of infected patients. In the early studies
culture filtrates or whole cell extracts were used as
antigens. I? rnarneffei was cultured in liquid media, and
the culture filtrate, or anti-I? marneffei sera raised in
rabbits, was incorporated in an immunodiffusion test to
detect antibody or antigens respectively.

We have used an indirect immunofluorescent antibody

test for serodiagnosis, with yeast hyphae (representing
the tissue multiplication phase) or germinating conidia
(representing the initial tissue invasion phase) as
antigens [12]. Much higher IgG titres were found in
all eight patients with culture-positive penicilliosis
marneffei (including three with HIV), than in 78
healthy controls. Cross-reactivity to other dimorphic
fungi was not tested, because of the lack of such cases
locally, but sera from two patients with cryptococcosis
and two with candidaemia showed IgG titres similar to
those of healthy controls. In our experience, an IgG
titre >80 is indicative of penicilliosis marneffei, and a
thorough investigation of the patient is warranted.
However, this test may be less useful for diagnosis in
patients living in endemic areas.
A latex agglutination test, with polystyrene beads
coated with rabbit anti-I? marneffei globulin, has also
been developed; antigenaemia was detected in 13 of 17
I? marneffei culture-positive HIV patients [ 131. Similarly, purified hyperimmune IgG, from rabbits immunised with yeast cells, has been used in an enzymelinked immunosorbent assay (ELISA) to quantify I?
marneffei yeast antigens in urine samples. Urine
samples from all of 33 I? marneffei culture-positive
HIV patients yielded high titres [ 141.
There have been several attempts to characterise I?
marneffei antigens. The gene encoding one of them, the
Mplp mannoprotein, has been sequenced, and the
protein has been expressed and used for diagnosis [ 151.
Among 17 I? marneffei culture-positive HIV patients
and two culture-positive HIV-negative patients, 82%
had antibodies against Mplp. However, 71% of 24 I?
marneffei culture-positive HIV-positive patients and
neither of the two culture-positive HIV-negative patients had Mpl mannoprotein in their sera [16]. It is
likely that antibody and antigen detection tests are
required for diagnosis in non-immunosuppressed and
immunocompromised patients, respectively.
Although encouraging results have been obtained with
tests to detect antigen or antibody in blood and body
fluids, the sensitivity of these tests, and their crossreactivity in other fungal, mycobacterial and bacterial
infections must be evaluated further. Test kits must be
available commercially, before wide acceptance and
common use is possible.
The detection of the I? marneflei genomic DNA in
clinical specimens has also been reported. Primers to
amplify a 347-bp fragment of the internal transcribed
spacer region between 18s rDNA and 5.8s rDNA have
been described [ 171. A PCR-Southern hybridisation
format, with amplification of a 63 1-bp fragment of the
18s rDNA, followed by hybridisation with a F!
marneff-specific 15-oligonucleotide probe has also
been used [ 181. We are presently testing the possibility
of using a one-tube nested PCR to detect the Mpl gene.

EDITORIAL

Treatment
Penicilliosis is very amenable to antifungal treatment.
P murneffei is susceptible to itraconazole and amphotericin B in vitro, although the susceptibility to
fluconazole and 5-fluorocytosine is less uniform [ 191.
There have been few well-controlled clinical trials
comparing the efficacy of different antifungal regimens.
Theoretically, patients with disseminated disease and
fungaemia should benefit from an initial course of
amphotericin B, as it is fungicidal. If there is a high
fungal load, with or without impairment of host
immune response, this is a definite advantage. In one
non-randomised study, intravenous amphotericin B
(0.6 mg/kg daily) for 2 weeks, followed by oral
itraconazole (400 mg/day) for 10 weeks, resulted in
clinical and microbiological cure in 97.3% of the
patients [20]. This seems a reasonable regimen in most
cases, based also on previous clinical experience and
in-vitro susceptibility results. In the only double-blind,
placebo-controlled randomised trial published [2 13, the
efficacy of secondary prophylaxis with itraconazole
was demonstrated in HIV-positive patients with penicilliosis marneffei: after an initial 2-week regimen of
intravenous amphotericin B, 0.6 mg/kg daily, oral
itraconazole was given at a dose of 200 mg twice
daily for 10 weeks. The relapse rate in the placebo
group within 1 year was 57%, with no relapse in the
treatment group.
S. S. Y. WONG, H. SIAU, K. Y. YUEN
Department of Microbiology,
University of Hong Kong,
Queen Mary Hospital,
Hong Kong
Corresponding author: Professor K. Y. Yuen
(e-mail: kyyuen@hkucc .hku.hk)

References
Segretain G. Description dune nouvelle espece de penicillium:
Penicillium marneffei n. sp. Bull Soc Mvcol Fr 1959; 75:
412-41 6.
DiSalvo AF, Fickling AM, Ajello L. Infection caused by
Penicillium marneffei: description of first natural infection in
man. Am J Clin Pathol 1973; 60: 259-263.
Capponi M, Sureau P, Segretain G. [Penicillos from Rhizomys
Sinensis]. Penicillos de Rhizomys sinensis. Bull Soc Path Exot
1956; 49: 418-421.
Deng ZL, Yun M, Ajello L. Human penicilliosis marneffei and

975

its relation to the bamboo rat (Rhizomyspminosus). J Med kt

M-jcol 1986; 24: 383-389.
5. Kwon-Chung KJ, Bennett JE. Medical mycology. Philadelphia,
PA, Lea and Febiger. 1992.
6. Duong TA. Infection due to Penicillium marneflei, an emerging
pathogen: review of 155 reported cases. Clin Infect Dis 1996;
23: 125-130.
7. Borradori L, Schmit JC, Stetzkowski M, Dussoix P, Saurat JH,
Filthuth I. Penicilliosis marneffei infection in AIDS. J Am
Acad Dermatol 1994; 31: 843-846.
8. Tsang DNC, Li PCK, Tsui MS, Lau YT, Ma KF, Yeoh EK.
Penicillium marneffei: another pathogen to consider in patients
infected with human immunodeficiency virus. Rev Inject Dis
1991; 13: 766-767.
9. Supparatpinyo K, Khamwan C, Baosoung V, Nelson KE,
Sirisanthana T. Disseminated Penicillium marnejfei infection in
southeast Asia. Lancet 1994; 344: 1 10- 1 13.
10. Supparatpinyo K, Sirisanthana T. Disseminated Penicillium
marneffei infection diagnosed on examination of a peripheral
blood smear of a patient with human immunodeficiency virus
infection. Clin Znfect Dis 1994; 18: 246-247.
11. Pierard GE, Estrada J, Pierard-Franchimont C, Thiry A, Stynen
D. Immunohistochemical expression of galactomannan in the
cytoplasm of phagocytic cells during invasive aspergillosis. Am
J Clin Pathol 1991; 96: 373-376.
12. Yuen K-Y, Wong SS, Tsang DN, Chau P-Y. Serodiagnosis of
Penicillium marneffei infection. Lancet 1994, 344: 444-445.
13. Kaufman L, Standard PG, Jalbert M, Kantipong P, Limpakarnjanarat K, Mastro TD. Diagnostic antigenemia tests for
penicilliosis marneffei. J Clin Microbiol 1996; 34: 2503-2505.
14. Desakorn V, Smith MD, Walsh AL et al. Diagnosis of
Penicillium murneffei infection by quantitation of urinary
antigen by using an enzyme immunoassay. J Clin Microbiol
1999; 37: 117-121.
15. Cao L, Chen D-L, Lee C et al. Detection of specific antibodies
to an antigenic mannoprotein for diagnosis of Penicillium
marnefei penicilliosis. J Clin Microbiol 1998; 36: 3028-303 1.
16. Cao L, Chan K-M, Chen D et al. Detection of cell wall
mannoprotein Mp lp in culture supernatants of Penicillium
nzarnefjei and in sera of penicilliosis patients. J Clin Microbiol
1999; 37: 981-986.
17. LoBuglio KF, Taylor JW. Phylogeny and PCR identification of
the human pathogenic fungus Penicillium murneffei, J Clin
Microbiol 1995; 33: 85-89.
18. Vanittanakom N, Merz WG, Sittisombut N, Khamwan C,
Nelson KE, Sirisanthana T. Specific identification of PenicilEium marneffei by a polymerase chain reaction/hybridization
technique. Med Mycol 1998; 36: 169-175.
19. Supparatpinyo K, Nelson KE, Merz WG et al. Response to
antifungal therapy by human immunodeficiency virus-infected
patients with disseminated Penicillium marneffei infections and
in vitro susceptibilities of isolates from clinical specimens.
Antimicrob Agents Chemother 1993: 37: 2407-241 1.
20. Sirisanthana T, Supparatpinyo K, Perriens J, Nelson KE.
Amphotericin B and itraconazole for treatment of disseminated
Penicillium marneffei infection in human immunodeficiency
virus-infected patients. Clin Znfect Dis 1998; 26: 1107- 11 10.
21. Supparatpinyo K, Perriens J, Nelson KE, Sirisanthana T. A
controlled trial of itraconazole to prevent relapse of Penicillium marneffei infection in patients infected with the human
immunodeficiency virus. N Engl J Med 1999; 339:
1739- 1743.

CALL FOR ABSTRACTS

ANAEROBE 2000
A Congress of the Confederation of Anaerobe Societies
July 10-12, 2000
Manchester, UK
ANAEROBE 2000 is a congress of the Confederation
of Anaerobe Societies to be held July 10-12, 2000 in
Manchester, UK. This meeting will include invited
speakers, oral presentations, and poster presentations.

To receive a program prospectus, contact the Anaerobe

Society of the Americas: by mail: ASA, PO. Box
452058, Los Angeles, CA 90045, USA; by e-mail:
asa@anaerobe.org; or by Fax: 1 (310) 216-9274. For
more information, visit the web site: www.anaerobe.org.
This meeting is sponsored by the Anaerobe Society of
the Americas, the Society for Anaerobic Microbiology,
and the Japanese Association for Anaerobic Infection
Research.

Submissions are requested on topics including: New

Antimicrobials, In-Vitro Susceptibility, Molecular
Biology, Anaerobic Diarrhoea, Veterinary, Clinical
Infections, and other topics related to Anaerobes.
The abstract submission deadline is February 1, 2000.

LIPPINCOTT WILLIAMS 0WlLKlNS

and

r4Tcombine to help you save on your advertising costs

Adobe

Use the combined experience and expertise of Lippincott Williams & Wilkins and our printers to save money
We now accept PDF (Portable Document Format) files produced using Adobe Acrobat (version 3.0 or later), and
you no longer have to pay film preparation costs
PDF files are easy to prepare and easy to transport!

Most films are outputted from digital files after on-screen design. We suggest that you supply us with digital files rather
than going LO ihe lirne and expense of producing f i l ~ and
~ i transporting it Lo us

PDF files can be sent electronically and the quality of printing from digital tiles is higher!

LWW have produced a detailed specification to produce your PDF file

For a copy please contact us on 4 4 (0) 171 940 7551

To book an advertising space, please contact
Dick Bower on tel: +44(0)181 880 8122; fax: +44 (0)181 543 3810; e-mail: dick.bower@btinternet.com
L l p p l ~ ~ o ~ or
~ Kelly Adamitis on tel. +1 215 413 4051;fax: +1 215 238 4461; e-mail: kadamitis@phl.lrpub.com

KILLLZMS tn WlLKlhS
4 Wdten Klm*arC~npa
Y

Please note that we can still accommodate film or bromide if necessary

LIPP[NC~TT

WIl.LIAL.(5 f5ivll KIN5

A &&em

X l v r w r CUI p n )