Pneumocystis pneumonia in HIV-positive adults, Malawi (1).

In a prospective study of 660 HIV-positive Malawian adults, we diagnosed Pneumocystis jirovecii pneumonia (PcP) using clinical features, induced sputum for immunofluorescent staining, real-time PCR, and posttreatment followup. PcP incidence was highest in patients with the lowest CD4 counts, but PcP is uncommon compared with incidences of pulmonary tuberculosis and bacterial pneumonia.

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The incidence of Pneumocystis jirovecii pneumonia (PEP) in HIV-infected adults in the sub-Saharan 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 selection 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. Realtime PCR performed on sputum samples has high sensitivity 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 comprehensive diagnostic approach that included induced sputum with IF staining, real-time PCR, and follow-up after diagnosis and treatment.

The Study

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 prospective, community-based study to determine the incidence of infections that were preventable by trimethoprim-sulfamethoxazole 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 prophylaxis 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 failure to improve with antimicrobial treatment without activity 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. jtrovecii 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 performing 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 evidence alone.

Incidence rates of respiratory diagnoses per 100 person-years of follow-up were calculated with 95% confidence intervals (CIs) based on Poisson distribution. First and subsequent episodes in the same person were counted separately, except for PcP, because patients with PcP received secondary prophylaxis and exited the study. The CD4 count at the time of the episode or within the previous 6 months was used for analysis.

We used [chi square], 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 consent, and 119 (17%) were lost to follow up. A smaller proportion 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 with confirmed PcP cases (42.5 cells/[mm.sup.3], range 1-103) was not significantly lower than in those with Pneumocystis colonization (89.1 cells/[mm.sup.3], range 7-194; p = 0.28), but was significantly lower than in those with other diagnoses (97.0 cells/[mm.sup.3], 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%.

Conclusions

This is the first community-based prospective study of PcP in a developing country. We found an incidence of PcP in Malawian HIV-infected adults of 1.0/1 00 person-years, similar to the rates observed in studies that used less comprehensive diagnostic approaches in South African miners (0.5/100 person-years) (11) and the placebo arms of trials of trimethoprim-sulfamethoxazole prophylaxis in Crte d'Ivoire (12,13). The incidence in persons with CD4 counts <200/[mm.sup.3] (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/I 00 person-years [14]). In the lowest CD4 count range (<100/[mm.sup.3]), PcP was 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 diagnostic uncertainty as PcP cases would still leave the PcP incidence low in the HIV-infected population in general.

We found that Pneumocystis colonization and confirmed PcP were equally common among patients with suspected PcP. More sensitive molecular detection methods would possibly have detected higher rates of colonization. It remains uncertain why certain HIV-infected persons clear Pneumocystis colonization while others develop PcP. The level of immune suppression as indicated 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 incidence 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 bacterial pneumonia and pulmonary tuberculosis at all levels of immunosuppression. PcP rarely occurred with CD4 cell counts > 100 [mm.sup.3]. Among the most immunocompromised patients, PcP is an important diagnostic consideration.

Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.

References

(1.) Graham SM. HIV and respiratory infections in children. Curr Opin Pulm Med. 2003;9:215-20.

(2.) Fisk DT, Meshnick S, Kazanjian PH. Pneumocystis carinii pneumonia in patients in the developing world who have acquired immunodeficiency syndrome. Clin Infect Dis. 2003;36:70-8.0

(3.) Hargreaves N J, Kadzakumanja O, Phiri S, Lee CH, Tang X, Salaniponi FM, et al. Pneumocystis carinii pneumonia in patients being registered for smear-negative pulmonary tuberculosis in Malawi. Trans R Soc Trop Med Hyg. 2001;95:402-8.

(4.) Aderaye G, Bruchfeld J, Olsson M, Lindquist L. Occurrence of Pneumocystis carinii in HIV-positive patients with suspected pulmonary tuberculosis in Ethiopia. AIDS. 2003; 17:435-40.

(5.) Chakaya JM, Bii C, Ng'ang'a L, Amukoye E, Ouko T, Muita L, et al. Pneumocystis carinii pneumonia in HIV/AIDS patients at an urban district hospital in Kenya. East Afr Med J. 2003;80:30-5.

(6.) Maskell NA, Waine DJ, Lindley A, Pepperell JC, Wakefield AE, Miller RF, et al. Asymptomatic carriage of Pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study. Thorax. 2003;58:594-7.

(7.) Cruciani M, Marcati P, Malena M, Bosco O, Serpelloni G, Mengoli C. Meta-analysis of diagnostic procedures for Pneumocystis carinii pneumonia in HIV-1-infected patients. Eur Respir J. 2002;20:982-9.

(8.) van Oosterhout JJG, Laufer MK, Graham SM, Thumba F, Perez MA, Chimbiya N, et al. A community-based study of the incidence of trimethoprim-sulfamethoxazole-preventable infections in Malawian adults living with HIV. J Acquir Immune Defic Syndr. 2005;39:626-31.

(9.) Smith DE, McLuckie A, Wyatt J, Gazzard B. Severe exercise hypoxaemia with normal or near normal X-rays: a feature of Pneumocystis carinii infection. Lancet. 1988;2:104-51.

(10.) Alvarez-Martinez MJ, Miro JM, Valls ME, Moreno A, Rivas PV, Sole M, et al.; Spanish PCP Working Group. Sensitivity and specificity of nested and real-time PCR for the detection of Pneumocystis jiroveci in clinical specimens. Diagn Microbiol Infect Dis. 2006;56:153-60. Epub 2006 May 4.

(11.) Corbett EL, Churchyard G J, Charalambos S, Samb B, Moloi V, Clayton TC, et a|. Morbidity and mortality in South African gold miners: impact of untreated disease due to human immunodeficiency virus. Clin Infect Dis. 2002;34:1251-8.

(12.) Wiktor SZ, Sassan-Morokro M, Grant AD, Abouya L, Karon JM, Maurice C, et al. Efficacy of trimethoprim-sulphamethoxazole prophylaxis to decrease morbidity and mortality in HIV-1-infected patients with tuberculosis in Abidjan, Cote d'Ivoire: a randomised controlled trial. Lancet. 1999;353:146-75.

(13.) Anglaret X, Chene G, Attia A, Toure S, Lafont S, Combe P, et al. Early chemoprophylaxis with trimethoprim-sulphamethoxazole for HIV-1 infected adults in Abidjan, Cote d'Ivoire: a randomised trial. Cotrimo--CI Study Group. Lancet. 1999;353:1463-8.

(14.) Kaplan JE, Hanson D, Dworkin MS, Frederick T, Bertolli J, Lindegren ML, et al. Epidemiology of human immunodeficiency virus-associated opportunistic infections in the United States in the era of highly active antiretroviral therapy. Clin Infect Dis. 2000;30:S5-14.

(15.) Beard CB, Fox MR, Lawrence GG, Guarner J, Hanzlick RL, Huang L, et al. Genetic differences in Pneumocystis isolates recovered from immunocompetent infants and from adults with AIDS: epidemiological implications. J Infect Dis. 2005;192:1815-8. Epub 2005 Oct 13.

Address for correspondence: Christopher V. Plowe, University of Maryland School of Medicine, 685 W Baltimore St, HSF1-480, Baltimore, MD 21201, USA; email: [e-mail]

(1) Data 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 MoPeB3198).

Joep J.G. van Oosterhout,* Miriam K. Laufer, ([dagger]) M. Arantza Perez,* Stephen M. Graham,* Nelson Chimbiya,* Phillip C. Thesing,* Miriam J. Alvarez-Martinez, ([double dagger]) Paul E. Wilson, ([double dagger]) Maganizo Chagomerana,* Eduard E. Zijlstra,* Terrie E. Taylor,*([section]) Christopher V. Plowe, ([dagger]) and Steven R. Meshnick ([double dagger])

*University of Malawi College of Medicine, Blantyre, Malawi; ([dagger])University of Maryland School of Medicine, Baltimore, Maryland, USA; ([double dagger])University of North Carolina, Chapel Hill, North Carolina, USA; and ([section]) Michigan State University, East Lansing, Michigan, USA

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.

Table 1. Baseline characteristics of study participants at
enrollment, Malawi, 2002-2004

                                      No.
Characteristic                      episodes    PYO

CD4 (cells/[mm.sup.3])
    0-99                              125        83
    100-199                           159       145
    200-499                           271       268
    [greater than or                  77         75
      equal to] 500
    Missing at enrollment             28         19
  HIV clinical stage ([dagger])
    I                                 267       255
    II                                191       190
    III                               160       120
    IV                                42         25

Total                                 660       591

* PYO, person-years of observation (based on enrollment
characteristics).

([dagger]) As defined by World Health Organization criteria.

Table 2. Outcomes in 95 episodes of suspected Pneumocystis
pneumonia (PcP), Malawi, 2002-2004 *

Final diagnosist                     Clinical diagnosis$

Confirmed PcP                             PcP
Confirmed PcP                             PcP
Confirmed PcP                             PcP

Confirmed PcP                        Bronchiectasis
Confirmed PcP                          Tuberculosis
Pneumocystis colonization/           Pulmonary KS
pulmonary KS
Pneumocystis colonization/             Tuberculosis
tuberculosis
Pneumocystis colonization/           Bacterial pneumonia
bacterial pneumonia
Pneumocystis colonization/             Unspecified
unspecified respiratory illness      respiratory illness
Pneumocystis colonization/           Other diagnosis ([paragraph])
other diagnosis
Unspecified respiratory illness           PcP
Tuberculosis                            Tuberculosis
Bacterial pneumonia                  Bacterial pneumonia
Unspecified respiratory illness         Unspecified
                                     respiratory illness
Unspecified respiratory illness         Unspecified
                                     respiratory illness
Other diagnoses ([paragraph])        Other diagnoses ([paragraph])
Other diagnosis ([paragraph])        Other diagnosis ([paragraph])

Final diagnosist                         No.          IF        PCR
                                      episodes

Confirmed PcP                             2          Pos        Pos
Confirmed PcP                             1          Neg        Pos
Confirmed PcP                             1           -          -

Confirmed PcP                             1          Neg        Pos
Confirmed PcP                             1          Neg        Pos
Pneumocystis colonization/                3          Neg        Pos
pulmonary KS                         ([section])
Pneumocystis colonization/                3          Neg        Pos
tuberculosis
Pneumocystis colonization/                2          Neg        Pos
bacterial pneumonia
Pneumocystis colonization/                2          Neg        Pos
unspecified respiratory illness
Pneumocystis colonization/                1          Neg        Pos
other diagnosis
Unspecified respiratory illness           3          Neg        Neg
Tuberculosis                             19          Neg        Neg
Bacterial pneumonia                      10          Neg        Neg
Unspecified respiratory illness          20          Neg        Neg

Unspecified respiratory illness           1          Neg         NA

Other diagnoses ([paragraph])            24          Neg        Neg
Other diagnosis ([paragraph])             1          Neg         NA

Final diagnosist                     Follow-up data

Confirmed PcP                                         -
Confirmed PcP                                         -
Confirmed PcP                        Death after 2 wk of PcP treatment
                                     (IS not done due to respiratory
                                              distress)
Confirmed PcP                                 Death 1 wk after IS
Confirmed PcP                        Improvement but short follow-up
                                              (2 wk)
Pneumocystis colonization/               Death 23 wk after first IS
pulmonary KS
Pneumocystis colonization/                      Recovery
tuberculosis
Pneumocystis colonization/                      Recovery
bacterial pneumonia
Pneumocystis colonization/                      Recovery
unspecified respiratory illness
Pneumocystis colonization/                      Recovery
other diagnosis
Unspecified respiratory illness                    -
Tuberculosis                                       -
Bacterial pneumonia                                -
Unspecified respiratory illness                    -

Unspecified respiratory illness      Recovery (18 wk follow-up)

Other diagnoses ([paragraph])                      -
Other diagnosis ([paragraph])        Recovery (>1 y follow-up)

* IF, immunofluorescence stain, Pos, positive, Neg, negative, IS,
induced sputum procedure; KS, Kaposi sarcoma; NA, not available.

([dagger]) Diagnosis based on clinical evidence, IF, and PCR from
an IS sample, and follow-up after episode.

([double dagger]) Diagnosis based on clinical evidence and IF from
an IS sample.

([section]) Three episodes occurring in 1 person.

([paragraph]) Among other diagnoses were sepsis, bronchitis,
emphysema, pulmonary KS, and bronchiectasis.

Table 3. Incidence of Pneumocystis pneumonia (PcP) and other
respiratory illnesses, Malawi, 2002-2004 *

                                  All CD4 counts

                                                 Incidence, %
Diagnosis                         No. events       (95% CI)

Confirmed PcP                         6         1.0 (0.3-2.2)

Bacterial pneumonia ([dagger])       102       17.3 (14.1-21.0)

Pulmonary tuberculosis                51        8.6 (6.4-11.3)

Unspecified respiratory              127       21.5 (17.9-25.6)
illness

                                  CD4 0-99/[mm.sup.3]

                                                 Incidence, %
Diagnosis                         No. events       (95% CI)

Confirmed PcP                         5         5.7 (1.9-13.4)

Bacterial pneumonia ([dagger])        35       40.2 (28.0-56.0)

Pulmonary tuberculosis                20       23.0 (14.0-35.5)

Unspecified respiratory               46       52.9 (38.7-70.5)
illness

                                  CD4 100-199/[mm.sup.3]

                                                  Incidence,
Diagnosis                         No. events       (95% CI)

Confirmed PcP                         1         0.6 (0.01-3.8)

Bacterial pneumonia ([dagger])        42       28.6 (20.6-38.6)

Pulmonary tuberculosis                25       17.0 (11.0-25.1)

Unspecified respiratory               38       25.9 (18.3-35.5)
illness

* CI, confidence interval.

([dagger]) Diagnosis based on new consolidations shown on chest x-ray
and response to antimicrobial drugs; includes patients with and
without positive blood cultures.