Comparison of different microbiological procedures for the diagnosis of Pneumocystis jirovecii pneumonia on bronchoalveolar-lavage fluid

Abstract

Background The current diagnostic gold standard for Pneumocystis jirovecii is represented by microscopic visualization of the fungus from clinical respiratory samples, as bronchoalveolar-lavage fluid, defining “proven” P. jirovecii pneumonia, whereas qPCR allows defining “probable” diagnosis, as it is unable to discriminate infection from colonization. However, molecular methods, such as end-point PCR and qPCR, are faster, easier to perform and interpret, thus allowing the laboratory to give back the clinician useful microbiological data in a shorter time. The present study aims at comparing microscopy with molecular assays and beta-D-glucan diagnostic performance on bronchoalveolar-lavage fluids from patients with suspected Pneumocystis jirovecii pneumonia. Bronchoalveolar-lavage fluid from eighteen high-risk and four negative control subjects underwent Grocott-Gomori’s methenamine silver-staining, end-point PCR, RT-PCR, and beta-D-glucan assay. Results All the microscopically positive bronchoalveolar-lavage samples (50%) also resulted positive by end-point and real time PCR and all, but two, resulted positive also by beta-D-glucan quantification. End-point PCR and RT-PCR detected 10 (55%) and 11 (61%) out of the 18 samples, respectively, thus showing an enhanced sensitivity in comparison to microscopy. All RT-PCR with a Ct < 27 were confirmed microscopically, whereas samples with a Ct ≥ 27 were not. Conclusions Our work highlights the need of reshaping and redefining the role of molecular diagnostics in a peculiar clinical setting, like P. jirovecii infection, which is a rare but also severe and rapidly progressive clinical condition affecting immunocompromised hosts that would largely benefit from a faster diagnosis. Strictly selected patients, according to the inclusion criteria, resulting negative by molecular methods could be ruled out for P. jirovecii pneumonia.

Molecular Study of Pneumocystis jirovecii in Respiratory Samples of HIV Patients in Chile

“…We observed a robust correlation between the phenotypic diagnostic technique based on microscopic observation of cystic forms of P. jirovecii and the molecular approach, relying on conventional PCR amplification of the mtLSU region with only two cases identified solely through PCR (Cases 7 and 15) (Table 2), like what was recently described by [55]. The mitochondrial genome of P. jirovecii replicates and transcribes for the most part autonomously and encodes mainly proteins involved in mitochondrial respiration, its high conservation facilitates not only agent identification but also genotype exploration.…”