Researchers collected demographic and clinical information with a structured, pre-tested questionnaire and examined respiratory specimens obtained as nasopharyngeal aspirates or expectorated sputum. The participants ranged from newborn infants to 17 years old; 129 were boys and 103 were girls. All 116 children with HIV were receiving antiretroviral therapy and cotrimoxazole prophylaxis, compared with 16 of 116, or 14%, of the HIV-negative children receiving prophylaxis.

The clinical groups also differed in their respiratory presentations: fever was recorded in 77% of the HIV-negative children and 27% of the children with HIV, while breathlessness at rest was more prominent in the HIV-negative group. Both differences had p-values below 0.001. These symptom differences describe the enrolled groups and do not establish pneumocystis pneumonia as the cause of fever or breathlessness.

Real-time PCR targeted the mitochondrial large-subunit ribosomal RNA gene of P. jirovecii. All 150 samples tested by PCR were negative, corresponding to a positivity rate of 0% with an upper 95% confidence interval of 2.0%. Giemsa staining was also negative in all 232 samples examined, yielding a positivity rate of 0% with an upper 95% confidence interval of 1.3%.

In contrast, calcofluor white fluorescence microscopy classified 24 of 232 samples as positive, a rate of 10.3%. The apparent positives included 15 of 116 samples from children with HIV, or 13%, and nine of 116 samples from HIV-negative children, or 8%; the difference between the groups was not statistically significant, with p = 0.19. No sample was positive by more than one diagnostic method.

All 24 calcofluor-positive samples were PCR-negative and Giemsa-negative, and the fluorescing material displayed atypical branching morphology that the investigators said was inconsistent with Pneumocystis cysts. They identified non-specific binding as the most likely explanation for these unconfirmed results, indicating that the microscopy findings should not be treated as evidence of 24 additional cases or used as stand-alone evidence of pneumocystis pneumonia. The authors described the apparent burden of pneumocystis pneumonia as very low in the cohort despite the large gap in prophylaxis coverage.

They said this finding challenges the automatic extrapolation of international prophylaxis guidance to the local setting and supports locally grounded epidemiological surveillance. The study does not show that cotrimoxazole prophylaxis caused the absence of confirmed cases, because all children with HIV were receiving prophylaxis while most HIV-negative children were not, and the groups differed in their underlying illnesses and clinical profiles. Nor does a cross-sectional survey establish that withholding prophylaxis prevents infection.

The result instead documents what was observed in a defined clinical population: no molecularly confirmed cases, substantial underuse of prophylaxis among HIV-negative participants, and a series of fluorescence-microscopy results unsupported by the other two methods. Several design features limit how far the findings can be generalized. The investigation covered only two tertiary hospitals in Enugu and one calendar year, and its selected hospital-based population may not represent all immunocompromised children in Nigeria.

The cross-sectional design cannot measure changes in incidence over time or directly compare outcomes between protected and unprotected children. Only 150 of the 232 samples were tested by PCR, and the respiratory specimens were nasopharyngeal aspirates or expectorated sputum rather than bronchoveolar lavage samples. The reported confidence limits quantify uncertainty around the observed positivity rates, while the absence of confirmed cases in this cohort does not establish that the disease is absent elsewhere in South-East Nigeria.

Within these constraints, the diagnostic comparison remains the study’s clearest result: PCR detected no Pneumocystis DNA in the samples tested, Giemsa staining detected none in all 232 samples, and calcofluor white microscopy generated 10.3% unconfirmed apparent positives that require molecular or immunofluorescence confirmation.

Subject of Research: Evaluation of pneumocystis pneumonia diagnostics and cotrimoxazole prophylaxis among immunocompromised children in Enugu, Nigeria

Article Title: Absence of pcr-confirmed pneumocystis pneumonia among immunocompromised nigerian children despite suboptimal cotrimoxazole prophylaxis: a prospective epidemiological and diagnostic evaluation in South-East Nigeria

Article References: Okeke, P. C. U., Nwafia, I. N., Nwachukwu, P. T., Ebede, S. O., Ndubueze, C. P., Okwor, C. J., Izundu, D. O., Ozumba, U. C., Ohanu, M. E., & Oladele, R. O. (2026). Absence of pcr-confirmed pneumocystis pneumonia among immunocompromised nigerian children despite suboptimal cotrimoxazole prophylaxis: a prospective epidemiological and diagnostic evaluation in South-East Nigeria. BMC Pediatrics. https://doi.org/10.1186/s12887-026-07598-5

Image Credits: AI Generated

DOI: 10.1186/s12887-026-07598-5

Keywords: Pneumocystis jirovecii, pneumocystis pneumonia, real-time PCR, calcofluor white microscopy, Giemsa staining, cotrimoxazole prophylaxis, HIV-positive children, immunosuppressive treatment, Enugu, Nigeria, Absence, pcr-confirmed