Joep J.G. van Oosterhout,* Miriam K. Laufer,†
M. Arantza Perez,* Stephen M. Graham,*
Nelson Chimbiya,* Phillip C. Thesing,*
Miriam J. Álvarez-Martinez,‡ Paul E. Wilson,‡
Maganizo Chagomerana,* Eduard E. Zijlstra,*
Terrie E. Taylor,*§ Christopher V. Plowe,†
and Steven R. Meshnick‡
In a prospective study of 660 HIV-positive Malawian
adults, we diagnosed Pneumocystis jirovecii pneumonia
(PcP) using clinical features, induced sputum for immunoflu-
orescent staining, real-time PCR, and posttreatment follow-
up. PcP incidence was highest in patients with the lowest
CD4 counts, but PcP is uncommon compared with inci-
dences of pulmonary tuberculosis and bacterial pneumonia.
African region remains uncertain. That PcP is common in
African children <1 year of age is well documented (1),
but reported prevalence and incidence rates in adult
African populations vary widely (2). Many of these reports
were cross-sectional studies in selected populations from
tertiary hospitals (3–5), and therefore might contain selec-
tion bias that favors identifying higher rates of PcP.
To our knowledge, no large prospective studies have
been done by using broncho-alveolar lavage (BAL) in
combination with immunofluorescent (IF) staining for P.
jirovecii cysts, the diagnostic procedures of choice. Real-
time PCR performed on sputum samples has high sensitiv-
ity but low specificity for PcP (6,7). The few studies in
African adults that used PCR assays for Pneumocystis did
not distinguish subclinical colonization from infection,
mainly because of limited follow-up after diagnosis (3,4).
We describe here the incidence of PcP from a large cohort
study of HIV-infected Malawian adults that used a com-
prehensive diagnostic approach that included induced spu-
tum with IF staining, real-time PCR, and follow-up after
diagnosis and treatment.
he incidence of Pneumocystis jirovecii pneumonia
(PcP) in HIV-infected adults in the sub-Saharan
HIV-infected adults (>15 years of age), who sought
treatment at a government health center in the township of
Ndirande, Blantyre, Malawi, were enrolled in a prospec-
tive, community-based study to determine the incidence of
infections that were preventable by trimethoprim-sul-
famethoxazole prophylaxis (8). Clinical evaluations were
performed monthly and at sick visits occurring between
the scheduled monthly evaluations. CD4 counts were
determined every 4 months. Standardized diagnostic and
treatment guidelines and case definitions were used. At the
time of the study, in Malawi, antiretroviral therapy (ART)
was rarely used, and trimethoprim-sulfamethoxazole pro-
phylaxis was not recommended.
Cases of suspected PcP were identified by patients’
clinical signs and symptoms, chest x-ray results, oxygen
desaturation exercise test results (9), CD4 count, and fail-
ure to improve with antimicrobial treatment without activ-
ity against P. jirovecii. Patients’ sputum production was
induced by an ultrasonic nebulizer with hypertonic saline,
followed by IF staining for P. jirovecii cysts. A case was
classified as clinical PcP when the IF staining for P.
jirovecii cysts was positive or the participant had strong
clinical evidence of PcP and negative IF. Clinical follow-
up data were collected after the episode of suspected PcP.
After the study, real-time PCR for the P. jirovecii
dihydropteroate synthase and human RNAase P (control
DNA) was performed on DNA extracted from the stored
induced sputum slides (10). Clinicians were not aware of
the PCR results during the study, and laboratory staff per-
forming the PCR was blinded to clinical information and
IF results. A final diagnosis of confirmed PcP was made
for any episode with a positive IF result, positive PCR
result, or both, unless recovery (defined as resolution of
respiratory symptoms present at the start of the episode)
without PcP treatment was observed with a minimum of 4
weeks of follow-up. If the PCR results were positive but
the patient recovered without active treatment against PcP,
the result was interpreted as Pneumocystis colonization. A
negative PCR result ruled out PcP diagnosis in patients
who had received PcP treatment on the basis of clinical
Incidence rates of respiratory diagnoses per 100 per-
son-years of follow-up were calculated with 95% confi-
dence intervals (CIs) based on Poisson distribution. First
and subsequent episodes in the same person were counted
separately, except for PcP, because patients with PcP
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007325
*University of Malawi College of Medicine, Blantyre, Malawi;
†University of Maryland School of Medicine, Baltimore, Maryland,
USA; ‡University of North Carolina, Chapel Hill, North Carolina,
USA; and §Michigan State University, East Lansing, Michigan,
1Data from this study were presented in part at the 9th College of
Medicine Research Dissemination Conference, Blantyre, Malawi,
12 Nov 2005 (abstract DCON/05/44), and the XV International
AIDS Conference, Bangkok, Thailand, 11–16 Jul 2004 (abstract
received secondary prophylaxis and exited the study. The
CD4 count at the time of the episode or within the previ-
ous 6 months was used for analysis.
We used χ2, Mann-Whitney, and Student t tests for
analysis of age, sex, and CD4 counts among diagnoses,
respectively, using SPSS version 12 software (SPSS Inc.,
Chicago, IL, USA). The study was approved by the
Institutional Review Boards of the University of Malawi
College of Medicine, the University of Maryland, and
Michigan State University.
Beginning in September 2002, 660 adults were
enrolled in the study and followed up through August
2004. Baseline CD4 and World Health Organization stage
data are shown in Table 1. Mean age was 31.7 years (range
16–66); 437 (66%) were female. Mean duration of follow
up was 10.7 months (95% CI 10.4–11.5) per person.
Eighty-six (13%) participants died, and 37 (6%) were
withdrawn from the study because they started lifelong
trimethoprim-sulfamethoxazole prophylaxis. Sixty-three
participants (9.5%) left the area, 20 (3%) withdrew con-
sent, and 119 (17%) were lost to follow up. A smaller pro-
portion of patients from the lower CD4 strata exited the
study than from the higher CD4 content group.
Ninety-five episodes of suspected PcP occurred in 75
persons. Outcomes of these episodes are given in Table 2.
A final diagnosis of confirmed PcP was made in 6
episodes, and 9 episodes of Pneumocystis colonization
were recorded, with a mean follow up of 26 weeks (range
4–48 weeks). Table 3 shows the incidence rates of PcP and
other respiratory conditions in the cohort.
With full diagnostic workup including posttreatment
follow up as the gold standard for the diagnosis of PcP, the
sensitivity of PCR alone was 100%, the specificity 88%,
and the positive predictive value 31%. Among episodes in
which PcP was suspected, the mean CD4 count in patients
326Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007
Pneumocystis Pneumonia in HIV-positive Adults, Malawi
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007327
with confirmed PcP cases (42.5 cells/mm3, range 1–103)
was not significantly lower than in those with
Pneumocystis colonization (89.1 cells/mm3, range 7–194;
p = 0.28), but was significantly lower than in those with
other diagnoses (97.0 cells/mm3, range 1–311; p = 0.03).
Mean age and sex distribution of confirmed PcP,
Pneumocystis colonization, and other diagnoses were not
significantly different. The case-fatality rate of confirmed
PcP was 50%.
This is the first community-based prospective study of
PcPin a developing country. We found an incidence of PcP
in Malawian HIV-infected adults of 1.0/100 person-years,
similar to the rates observed in studies that used less com-
prehensive diagnostic approaches in South African miners
(0.5/100 person-years) (11) and the placebo arms of trials
of trimethoprim-sulfamethoxazole prophylaxis in Côte
d’Ivoire (12,13). The incidence in persons with CD4
counts <200/mm3(2.5/100 person-years) was clearly
lower than in AIDS patients in the United States before the
introduction of routine trimethoprim-sulfamethoxazole
prophylaxis and highly active ART (10/100 person-years
). In the lowest CD4 count range (<100/mm3), PcPwas
common, although the incidence was low compared with
that of bacterial pneumonia and pulmonary tuberculosis.
We believe it is unlikely that we missed many PcP
cases among other diagnoses or losses to follow-up because
of the intensive active and passive follow-up and because
our facility provided expeditious, high-quality care free of
charge. Allowing for reduced sensitivity of induced sputum
compared to BAL (7) and considering cases with diagnos-
tic uncertainty as PcP cases would still leave the PcP inci-
dence low in the HIV-infected population in general.
We found that Pneumocystis colonization and con-
firmed PcP were equally common among patients with
suspected PcP. More sensitive molecular detection meth-
ods would possibly have detected higher rates of coloniza-
tion. It remains uncertain why certain HIV-infected
persons clear Pneumocystis colonization while others
develop PcP. The level of immune suppression as indicat-
ed by the CD4 count is a possible explanation, although
our data do not support this. Genetic differences between
P. jirovecii strains may be relevant (15). Variation in
worldwide distribution of strains, as well as differences in
host genetics and shorter survival of patients in low CD4
count ranges, are possible causes of the lower PcP inci-
dence in Africa than in developed countries.
The incidence of PcP in HIV-infected Malawian
adults, diagnosed clinically and confirmed with molecular
analysis, was low compared with the incidence of bacteri-
al pneumonia and pulmonary tuberculosis at all levels of
immunosuppression. PcP rarely occurred with CD4 cell
counts >100 mm3. Among the most immunocompromised
patients, PcP is an important diagnostic consideration.
Dr van Oosterhout is a senior lecturer in the Department of
Medicine, University of Malawi College of Medicine, Blantyre,
Malawi. His research interests are the clinical aspects of HIV and
the treatment of HIV and tuberculosis.
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Address for correspondence: Christopher V. Plowe, University of
Maryland School of Medicine, 685 W Baltimore St, HSF1-480,
Baltimore, MD 21201, USA; email: email@example.com
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Use of trade names is for identification only and does not imply
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