Evaluation of Toluidine Blue O staining for the diagnosis of Pneumocystis jiroveci in expectorated sputum sample and bronchoalveolar lavage from HIV-infected patients in a tertiary care referral center in Ethiopia.

Background: Pneumocystis pneumonia (PCP) is becoming increasingly recognized in sub-Saharan Africa. The currently recommended diagnostic methods using induced sputum (IS) and bronchoalveolar lavage (BAL) are neither technically feasible nor affordable for a wider clinical use in developing countries. Therefore, there is a need for a simple and affordable diagnostic test.

Methods: The yield of Toluidine Blue O (TBO) stain, immunofluorescence (IF), and polymerase chain reaction (PCR) for the diagnosis of Pneumocystis jiroveci were compared in 78 expectorated sputum and 118 BAL samples of 131 HIV-infected patients presenting with atypical chest X-ray and sputum smear-negative for acid-fast bacilli.

Results: A total of 56 (42.7%) patients tested positive for P. jiroveci by PCR, 39 (29.4%) by IF, and 28 (21.4%) by TBO stain. The sensitivity of TBO as compared to IF and PCR was 71.4% and 34.5% in expectorated sputum and 68% and 41.5% in BAL samples, respectively, with a specificity approaching 100% in both. The sputum PCR showed high concordance rate with BAL PCR. The sensitivity and specificity of sputum PCR as compared to BALPCR was 78.9% and 89%, respectively. In both TBO and IF positive BAL samples, majority were from patients who could not produce sputum (p<0.001). The density of P. jiroveci clusters in BAL samples did not correlate with prior co-trimoxazole use, immunologic status of the patient or overall mortality.

Conclusion: Compared to IF, TBO staining has an acceptable sensitivity and very high specificity both in expectorated sputum and BAL samples. Expectorated sputum is, therefore, the most practical specimen and TBO staining an inexpensive diagnostic method to be recommended for high-HIV, resource-constrained settings. Bronchoscopy for the diagnosis of PCP is often not required for patients who can produce sputum. For patients who cannot produce sputum, however, the cost and efficacy of TBO in IS sample needs to be investigated in resource-poor countries.