ORIGINAL ARTICLE

Cytological Diagnosis of Penicillium

marneffei Infection
I-Shiow Jan,1 Ping-Fung Chung,1 Jann-Yuan Wang,2 Ming-Hsiang Weng,1
Chien-Ching Hung,2 Li-Na Lee1,2*

Background/Purpose: Penicillium marneffei is an emerging opportunistic pathogen. The goal of this study
was to study its clinical and radiographic presentation, and the diagnostic value of a cytological study of
penicilliosis.
Methods: A total of 24 patients with penicilliosis were found by culture in an 8-year period. Thirteen patients
had cytological examination, which were retrospectively reviewed to analyze the morphological characteristics
of P. marneffei and diagnostic yields of different cytological specimens.
Results: Twenty (83%) of the 24 patients (20 males) had human immunodeficiency virus (HIV) infection.
In eight (40%) patients, penicilliosis was the initial manifestation of HIV infection. Penicilliosis was diagnosed
in all patients antemortem from culture of blood or other body sites. One (4%) died. Six of 13 patients
who had cytological study had intra- or extracellular yeast-like organisms with transverse septum found in
fine needle aspirate, imprint or sputum cytology studies. The cytological diagnostic yields for P. marneffei
from lung biopsy imprint smears, lung aspirates, neck lymph node aspirates and sputum were 2/2, 2/4,
2/2 and 2/4, respectively. The cytological diagnosis could be made immediately after aspiration, much
quicker than diagnosis from cultures.
Conclusion: P. marneffei causes disseminated infection, and can be the initial manifestation in HIV-infected
patients in Taiwan. Penicilliosis can be diagnosed rapidly with cytological study of lung biopsy imprint
smears, lung aspirates, neck lymph node aspirates or sputum. [J Formos Med Assoc 2008;107(6):443447]

Key Words: cytology, HIV infection, Penicillium marneffei

Penicillium marneffei is an emerging opportunistic Fortunately, the yeast form of P. marneffei has a
pathogen that causes focal or disseminated infec- characteristic morphology including a transverse
tion in immunocompromised hosts. Penicilliosis septum, which can be demonstrated by cytology,
is an important infection in patients with acquired and the diagnosis can be made rapidly.4,711 Here,
immunodeficiency syndrome in Southeast Asia we report our experiences in 24 patients with
including Taiwan, with a high mortality if not di- penicilliosis in an 8-year period, and in six of
agnosed and treated early.16 The diagnosis, how- them, P. marneffei was demonstrated in the fine
ever, is difficult and often delayed due to its clinical needle aspirate, lung biopsy imprint or sputum
and radiographic similarities to tuberculosis and cytology. We describe the clinical presentations
non-tuberculous mycobacterium (NTM) infection, of penicilliosis, the morphological characteristics
and the slow growth rate of Penicillium species.1,6 of the organism in cytological specimens, and

2008 Elsevier & Formosan Medical Association

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Departments of 1Laboratory Medicine and 2Internal Medicine, National Taiwan University Hospital, National Taiwan
University College of Medicine, Taipei, Taiwan.

Received: January 16, 2008 *Correspondence to: Dr Li-Na Lee, Department of Laboratory Medicine, National
Revised: January 30, 2008 Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 100, Taiwan.
Accepted: February 12, 2008 E-mail: linalee@ntu.edu.tw

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I.S. Jan, et al

evaluate the yield rates of cytological studies of Results

different specimens.
Case identification
From 1999 through 2006, 24 patients (male: 20,
Methods 83%) were diagnosed to have penicilliosis by
culture of blood (n = 15), skin pus (n = 8), sputum
Patient population (n = 5), bronchial washing (n = 4), lung aspirate
Twenty-four patients with culture-proven P. marn- (n = 4), throat swab (n = 2), neck lymph node
effei infection were identified retrospectively from biopsy tissue (n = 2) or bone marrow biopsy tis-
the database of the microbiology laboratory in sue (n = 2). The mean age was 39.5 years (range,
our hospital between 1999 and 2006. We then 2181 years). All patients were tested for HIV in-
searched the database of our cytology laboratory fection, and 20 (83%) were positive. The other
and found that 13 had a cytological examination. four patients had underlying diseases including
A total of 27 cytological specimens, including bronchiectasis, renal transplantation, disseminated
17 exfoliation cytology specimens (8 sputum, 3 infection due to Mycobacterium fortuitum and M.
bronchial washing, 4 pleural effusion, 2 cerebro- avium-intracellulare (1 of each).
spinal fluid), and 10 fine needle aspiration cytology
specimens (4 aspiration from neck lymph nodes, Clinical and radiographic presentations
4 aspiration from lung, 2 imprint from needle lung The most common symptoms and signs were fever,
biopsy) were collected from these 13 patients. malaise, cough (each 58%), enlargement of neck
The clinical presentation, laboratory data, radiolog- lymph nodes (38%), skin eruptions (33%), body
ical findings, and histopathology were reviewed. weight loss (33%), dyspnea (17%), diarrhea (12%)
and hemoptysis (8%). Chest radiography revealed
Cytological specimen preparation multiple nodules and/or consolidation, often in-
Neck lymph node aspiration was done using the volving upper lobes (67%). In nine (38%) patients,
capillary method by a 21- or 22-gauge needle.12 the nodules were cavitated. Thoracic computed
Percutaneous lung aspiration or biopsy using an tomography was performed in 15 patients, and in
18-gauge needle or a Trucut type biopsy needle was eight (53%) of them, the nodules or consolida-
performed by a chest physician under the guidance tions were shown to be located subpleurally, and
of ultrasonography, or by a radiologist under the thus were accessible by ultrasound-guided biopsy.
guidance of computed tomography.711 Sputum Because of the clinical and radiographic presen-
and body fluid specimens were prepared according tations, the initial diagnosis was often disseminated
to routine laboratory methods. Rius stain (a mod- NTM infection, pulmonary tuberculosis, lung ab-
ified Romanowsky stain) and Papanicolaou stain scess or lung cancer. The diagnosis of penicilliosis
was performed on all cytology smears.1315 was usually made within 2 weeks after admission,
after P. marneffei was isolated from one or more
Microbiological identification of P. marneffei clinical specimens. In eight (40%) patients, peni-
P. marneffei was identified according to the stan- cilliosis was the initial presentation of HIV infec-
dard mycological criteria.1,5,16 Clinical specimens tion. All 24 patients had antemortem diagnosis of
were inoculated onto Sabouraud dextrose agar penicilliosis and received antifungal therapy during
and incubated at 25C in ambient air. Mature admission. One patient died of penicilliosis (mor-
colonies with the characteristic reddish-brown tality, 4%). The cause of death in a 28-year-old
mycelium and diffusible reddish-brown pigment male with penicilliosis as the initial manifestation
were subcultured onto brain-heart infusion agar of HIV infection was septic shock. He did not un-
slants and were incubated at 37C to yield the dergo cytological study despite the presence of neck
yeast phase.16 lymph nodes and skin eruptions. P. marneffei was

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 Cytology of Penicillium marneffei infection

A B

50 m 50 m

Figure. (A) Lung imprint cytological smear shows aggregates of macrophages engorged with numerous yeast-like
organisms 38 m in diameter. These yeast-like organisms are spherical, oval or sausage-shaped and have an eccentric
or central dot and occasional septum (arrows) (Rius stain, original magnification 1000). (B) Lung imprint wet-fixation
smear shows small cellular size (arrow) and air-dried artifact (arrowhead) (Papanicolaous stain, original magnification
1000).

isolated from blood and bone marrow 8 days Table. Diagnostic yields of cytology and mycology studies from
after admission. various clinical specimens in patients with Penicillium
marneffei infection

Cytological presentation Specimens Positive cytology Positive culture

Thirteen patients had a cytological examination, Percutaneous lung biopsy 100%* (2/2) 50% (1/2)
and six had a cytological diagnosis of P. marneffei imprint
infection. Figure A shows an imprint smear of Percutaneous lung aspirate 50% (2/4) 100% (4/4)
percutaneous needle lung biopsy. It contains ag- Neck lymph node aspirate 100% (2/2) 100% (1/1)
gregates of macrophages that were engorged with Sputum 50% (2/4) 50% (4/8)
numerous yeast-like organisms 38 m in diam- Bronchial washing 0% (0/3) 75% (3/4)
Pleural effusion 0% (0/4) 0% (0/5)
eter. These organisms could also be observed ex-
Cerebrospinal fluid 0% (0/2) 0% (0/2)
tracellularly within a necrotic background. These
*Imprint cytology.
yeast-like organisms were spherical, oval or sausage-
shaped, and had an eccentric or central dot and
occasional septum. The transverse septum, which The Table shows that cytological findings from
represents the fission form of reproduction, is percutaneous lung biopsy imprint smears, lung
characteristic of P. marneffei.8 The morphology of aspirates, neck lymph node aspirates and sputum
yeast forms was demonstrated more clearly in had a good diagnostic yield comparable to that of
air-dried cytology smears stained with Rius mycology culture. The diagnostic yield of bronchial
method than in wet-fixed smears stained with washing cytology was poor compared with culture.
Papanicolaous method (Figure B). Rius stain, a None of the pleural fluid or cerebrospinal fluid
modified Romanowsky stain, is simple and rapid, samples demonstrated the presence of P. marneffei
requiring only 2 minutes, and can be done im- by cytology or culture.
mediately at the bedside after lymph node aspi-
ration or percutaneous lung aspiration/biopsy. In
one patient, the diagnosis was made immediately Discussion
after ultrasound-guided percutaneous lung aspira-
tion, several days before P. marneffei was isolated P. marneffei is dimorphic and can be isolated from
from her aspirates. lungs, spleen, liver and lymph nodes of infected

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I.S. Jan, et al

hosts. It reproduces by fission rather than budding. 25 m in size, is the microorganism that is most
A central transverse septum, which represents often confused with P. marneffei.9,21 The presence
fission of the yeast, can be demonstrated by of the budding form indicates Histoplasma, while
Papanicolaou or Riu stains.6,10 P. marneffei ranks a transverse septum (the marker of binary fis-
as the third most common opportunistic infection sion), oval and sausage forms indicate Penicillium.
next to extrapulmonary tuberculosis and crypto- P. jiroveci can be identified by its extracellular loca-
coccosis in HIV-infected patients in Thailand.4 In tion, the presence of crescent forms, and the lack
our series, penicilliosis was the initial presentation of septation.20
of HIV infection in 40% of HIV-infected patients. Although the diagnosis of penicilliosis by
The diagnosis of penicilliosis, however, is difficult needle aspiration cytology has been reported pre-
due to the similarity of its clinical presentation viously, none of those studies had evaluated the
(fever, neck lymph node enlargement, skin erup- diagnostic yields of different cytological speci-
tions) to that of disseminated NTM infection, mens.711,19 Our study, though small in sample
and the resemblance of radiographic presentation size, showed that lung aspirate, lung biopsy imprint
(upper lobe nodules and/or consolidations with smear, lymph node aspirate and sputum cytology
cavitation) to that of tuberculosis. Thus, most of have good diagnostic yields (50100%), similar to
our patients were initially diagnosed as having that of culture. The results suggest that P. marneffei
disseminated NTM infection, pulmonary tuber- causes a suppurative infection in the lungs and
culosis, lung abscess or lung cancer, until proven lymph nodes, with the presence of plenty of organ-
otherwise. Conventional diagnostic methods for isms which lead to feasible cytological or cultural
penicilliosis, including fungal culture and histo- diagnosis. The success of lymph node aspiration
logic examination, are time-consuming and can be cytology can spare patients from a more invasive
too slow for the critically ill patient with invasive excision biopsy. The findings also suggested that
fungal infection.1,17,18 Cytological study, none- sputum of good quality provided a better sample
theless, can offer the correct diagnosis rapidly. of pulmonary penicilliosis for cytological study
Although screening for malignant neoplastic cells than bronchial washing. The poor cytological di-
is the primary issue of clinical cytology, more and agnostic yield of bronchial washing, compared
more evidence indicates that morphologic diag- with culture, may have been caused by the vigorous
nosis of fungal infection is another important suction during the washing procedure which could
function of clinical cytology.711,19 Accurate diag- disrupt fungal particles and distort the morphology.
nosis of fungal infections by clinical cytology has Failure of both cytological and cultural methods in
been reported.711,19 Our study showed that air- demonstrating P. marneffei in pleural fluid and
dried cytological smears stained by a modified cerebrospinal fluid suggests that the microorgan-
Romanowsky method, with sharper contrast be- ism is scarce in these specimens.
tween white septum and deep-purple yeast form In conclusion, P. marneffei causes disseminated
and larger particle size than the Papanicolaou stain infection and can be the initial manifestation in
(Figure B), can clearly demonstrate the character- HIV-infected patients in Taiwan. Penicilliosis can
istic morphology of P. marneffei immediately after be diagnosed rapidly with a cytological study of
aspiration. The need for a rapid diagnosis is shown lung aspirate, lung biopsy imprint smear, neck
by our lone fatal case, in whom the diagnosis of lymph node aspirate, or sputum.
penicilliosis was made when the blood culture
grew P. marneffei. The initiation of amphotericin-B
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