Molecular diagnosis and detection of Pneumocystis jirovecii DHPS and DHFR genotypes in respiratory specimens from Colombian patients

Abstract

A total of 98 respiratory specimens from 88 patients suspected of having Pneumocystis jirovecii pneumonia (PcP) were evaluated using a previously reported nested polymerase chain reaction (PCR) assay for mitochondrial large subunit rRNA (mtLSUrRNA). In addition, samples from patients with other pulmonary infections and a sizeable DNA collection from other fungal pathogens were studied. A panfungal PCR assay amplifying the ITS1-ITS2 regions were also used to identify all fungal DNAs. All samples positive for mtLSUrRNA-PCR were evaluated to determine mutations in dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) genes. All PCR-amplified products were sequenced. Of the 98 clinical specimens, 13 (13.2%) were positive by GMS stain and mtLSUrRNA-PCR, while 32 (32.6%) that were GMS stain-negative gave positive results with mtLSUrRNA-PCR. All the sequences corresponding to the 45 products amplified by mtLSUrRNA-PCR showed 99% or greater identity with P. jirovecii. The mtLSUrRNA-PCR exhibited 86% sensitivity and 98% and 96.6% specificity when results were compared to those corresponding to negative controls and other proven clinical entities, respectively. We found mutations in the DHPS gene in 3 (7.7%) patients, 2 located at codon 55 and 1 at codon 57. One patient showed a synonymous substitution at nucleotide position 312 in the DHFR gene. These results suggest that mtLSUrRNA-PCR is a useful test for diagnosing PcP. In contrast to other studies, this study found a low prevalence of mutations in the DHPS and DHFR genes in Colombian patients.