Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology

Abstract

Pneumocystis jirovecii pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct P. jirovecii loci encoding dihydrofolate reductase (DHFR), mitochondrial large-subunit rRNA (mtLSU rRNA), and superoxide dismutase (SOD) was achieved using multiplex-PCR (MPCR) followed by direct sequencing and two single-base extension (SBE) techniques. Four SNPs (DHFR312, mt85, SOD215, and SOD110), correlated previously with parameters of disease, were amplified and genotyped simultaneously. The concordance of results between the standard sequencing technique (direct sequencing) and SBE analysis was 96.9% for the acrylamide gel electrophoresis and 98.4% for the capillary electrophoresis. The cross-genetic analysis established several statistical associations among the SNPs studied: mt85C-SOD110T, SOD110T-SOD215C, and SOD110C-SOD215T. These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages of DHFR312C, mt85C, SOD110T, and SOD215C were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates with DHFR312T, mt85T, SOD110C, and SOD215T. The polymorphisms studied were shown to be suitable genetic targets potentially correlated with PcP clinical data that can be used as predictors of outcome in further studies to help clinical decision-making in the management of PcP. The MPCR/SBE protocol described for the first time in the present study was shown to be a rapid, highly accurate method for genotyping P. jirovecii SNPs encoded by different loci that could be used for epidemiological studies and as an additional procedure for the prognostic classification and diagnosis of PcP.

Prevalence and Genotype Distribution of Pneumocystis jirovecii in Cuban Infants and Toddlers with Whooping Cough

“…It has been suggested that the epidemiological factors inherent in geographical locations may influence the transmission and circulation of different P. jirovecii genotypes (8,28,37).…”

Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

“…Studies from other sites have failed to find nonsynonomous mutations in DHFR from P. jirovecii, suggesting failure of therapy is not uniquely linked to mutations in DHFR (7)(8)(9)(10). Although DHPS mutations apparently contribute to clinical failure by conferring resistance to sulfamethoxazole (1,2), the appearance of mutations in both DHPS and DHFR in clinical isolates and the apparently more frequent development of DHFR mutations among patients who did receive prophylaxis that included an inhibitor of DHFR suggest a selection pressure is exerted on DHFR by exposure to trimethoprim or pyrimethamine (2).…”

“…Mutations leading to amino acid substitutions in DHPS have been linked to resistance to sulfamethoxazole (1). Mutations leading to amino acid substitutions in dihydrofolate reductase of Pneumocystis jirovecii have also been observed worldwide for over a decade, but attempts to link clinical outcomes to these mutations have been inconclusive (2)(3)(4)(5)(6)(7)(8)(9)(10).…”

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).…”