Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies

Abstract

Specific single-nucleotide polymorphisms (SNPs) are recognized as important DNA sequence variations influencing the pathogenesis of Pneumocystis jirovecii and the clinical outcome of Pneumocystis pneumonia, which is a major worldwide cause of illness among immunocompromised patients. Genotyping platforms for pooled DNA samples are promising methodologies for genetic characterization of infectious organisms. We have developed a new typing strategy for P. jirovecii, which consisted of DNA pools prepared according to clinical data (HIV diagnosis, microscopic and molecular detection of P. jirovecii, parasite burden, clinical diagnosis and follow-up of infection) from individual samples using quantitative real-time PCR followed by multiplex-PCR/single base extension (MPCR/SBE). The frequencies of multiple P. jirovecii SNPs (DHFR312, mt85, SOD215 and SOD110) encoded at three distinct loci, the dihydrofolate reductase (DHFR), the mitochondrial large-subunit rRNA (mtLSU rRNA) and the superoxide dismutase (SOD) loci, were estimated in seven DNA pooled samples, representing a total of 100 individual samples. The studied SNPs were confirmed to be associated with distinct clinical parameters of infection such as parasite burden and follow-up. The MPCR/SBE-DNA pooling methodology, described in the present study, was demonstrated to be a useful high-throughput procedure for large-scale P. jirovecii SNPs screening and a powerful tool for evaluation of clinically relevant SNPs potentially related to parasite burden, clinical diagnosis and follow-up of P. jirovecii infection. In further studies, the candidate SNPs mt85, SOD215 and SOD110 may be used as molecular markers in association with MPCR/SBE-DNA pooling to generate useful information for understanding the patterns and causes of Pneumocystis pneumonia.

Multilocus Microsatellite Genotyping Array for Investigation of Genetic Epidemiology of Pneumocystis jirovecii

“…approaches has contributed significantly to our understanding of Pneumocystis biology, many questions remain unresolved (27,31). A genotyping approach with neutrally evolving markers, a high discriminatory index, and a stepwise model of allele adaptation would be a useful addition for studying P. jirovecii.…”

Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene

“…However, some studies on Pneumocystis genetic diversity have reported significant association between some singlenucleotide polymorphisms (SNPs) and clinical parameters in patient with PcP. Notably, SNPs at position 110 and 215 in the sod locus were described and associated with favourable outcome of PcP (SOD215C ) (Esteves et al 2010) or a lower parasite burden (SOD215C/SOD110T) (Esteves et al 2011(Esteves et al , 2012. The mitochondrial MnSOD is probably not the unique component of the Pneumocystis antioxidant system (Fig 6).…”

Diagnosis of Pneumocystis pneumonia: evaluation of four serologic biomarkers

“…Bio-Rad, France) and nested-PCR directed to P. jirovecii large subunit mitochondrial rRNA (mtLSU rRNA) gene [12,13,33]. In the Dutch patients, P. jirovecii was detected by direct microscopy using histochemical stainings (DM/HS) (Giemsa and Grocott's methenamine silver) and real-time PCR directed to the dihydropteroate synthase (DHPS) gene of P.…”

“…In the Spanish COPD patients, detection of P. jirovecii was performed by nested-PCR directed to the mtLSU rRNA gene [33]. [2,7,13].…”

“…Since P. jirovecii cannot be propagated in culture, microscopic visualization of cysts or trophic forms in pulmonary specimens with cytochemical staining or immunofluorescent staining with monoclonal antibodies (IF/Mab) and/or DNA amplification (PCR or PCRrelated protocols) are the standard procedures to detect this microorganism [1,6,[8][9][10][11][12][13][14][15].…”