Open AccessClinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology
Author(s) -
Francisco Esteves,
Jorge Gaspar,
Bruno de Sousa,
Francisco Antúnes,
Mehnaz Kamal,
Olga Matos
Publication year - 2011
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.02303-10
Subject(s) - pneumocystis jirovecii , multiplex polymerase chain reaction , single nucleotide polymorphism , biology , multiplex , polymerase chain reaction , pneumonia , virology , genotype , microbiology and biotechnology , computational biology , genetics , medicine , gene , human immunodeficiency virus (hiv)
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 distinctP. 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 , andSOD110 ), 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 , andSOD110C-SOD215T . These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages ofDHFR312C ,mt85C ,SOD110T , andSOD215C were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates withDHFR312T ,mt85T ,SOD110C , andSOD215T . 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 genotypingP. 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.