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首页> 外文期刊>Journal of Clinical Microbiology >Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology
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Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology

机译：吉氏肺孢菌性肺炎中多个单核苷酸多态性的临床相关性：多重PCR-单碱基延伸方法学的发展

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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.
机译：吉氏肺孢子虫肺炎（PcP）是艾滋病患者呼吸系统疾病的主要原因。在编码二氢叶酸还原酶（ DHFR ），线粒体大亚基rRNA（ mtLSU rRNA ）的三个截然不同的P. jirovecii位点的多个单核苷酸多态性（SNP）的鉴定，以及超氧化物歧化酶（ SOD ）是使用多重PCR（MPCR），直接测序和两种单碱基延伸（SBE）技术实现的。扩增了先前与疾病参数相关的四个SNP（ DHFR312 ， mt85 ， SOD215 和 SOD110 ）。并同时进行基因分型。标准测序技术（直接测序）与SBE分析之间的结果一致性，丙烯酰胺凝胶电泳为96.9％，毛细管电泳为98.4％。交叉遗传分析在所研究的SNP之间建立了几种统计关联： mt85C-SOD110T ， SOD110T-SOD215C 和 SOD110C-SOD215T 。这些结果通过聚类分析得到证实。数据显示，在具有低至中等寄生虫负担的分离株中， DHFR312C ， mt85C ， SOD110T 和 SOD215C < / em>，而对于高寄生虫负担病例， DHFR312T ， mt85T ， SOD110C 和 SOD215T 。研究表明，所研究的多态性是与PcP临床数据潜在相关的合适遗传靶标，可以用作进一步研究结果的预测指标，以帮助管理PcP的临床决策。在本研究中首次描述的MPCR / SBE方案被证明是一种快速，高精度的基因分型方法，用于对由不同基因座编码的jirovecii SNP进行基因分型，可用于流行病学研究以及作为预后分类的附加程序和诊断。

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