TY - JOUR
T1 - Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies.
AU - Esteves, Francisco Vaz de Carvalho
AU - Gaspar, J.
AU - Sousa, Bruno Cecílio de
AU - Antunes, F.
AU - Mansinho,, K.
AU - Matos, Olga Maria Guerreiro de
PY - 2012/1/1
Y1 - 2012/1/1
N2 - Clin Microbiol Infect 2012; 18: E177-E184 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.
AB - Clin Microbiol Infect 2012; 18: E177-E184 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.
KW - TIME PCR EXPERIMENTS
KW - RIBOSOMAL-RNA
KW - PROPHYLAXIS
KW - SINGLE-NUCLEOTIDE POLYMORPHISMS
KW - HIV-INFECTED PATIENTS
KW - molecular diagnosis
KW - CARINII-PNEUMONIA
KW - multilocus genotyping
KW - epidemiology
KW - BASE EXTENSION
KW - TRANSMISSION
KW - MUTATIONS
KW - DIHYDROPTEROATE SYNTHASE GENE
KW - DNA pools
KW - immunocompromised patients
KW - Pneumocystis pneumonia
U2 - 10.1111/j.1469-0691.2012.03828.x
DO - 10.1111/j.1469-0691.2012.03828.x
M3 - Article
C2 - 22487139
VL - 18
SP - 177
EP - 184
JO - Clinical Microbiology and Infection
JF - Clinical Microbiology and Infection
SN - 1198-743X
IS - 6
ER -