Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas, Medellín, Colombia.
Diagn Microbiol Infect Dis. 2012 Mar;72(3):204-13. doi: 10.1016/j.diagmicrobio.2011.11.015.
A total of 98 respiratory specimens from 88 patients suspected of having Pneumocystis jirovecii pneumonia (PcP) were evaluated using a previously reported nested polymerase chain reaction (PCR) assay for mitochondrial large subunit rRNA (mtLSUrRNA). In addition, samples from patients with other pulmonary infections and a sizeable DNA collection from other fungal pathogens were studied. A panfungal PCR assay amplifying the ITS1-ITS2 regions were also used to identify all fungal DNAs. All samples positive for mtLSUrRNA-PCR were evaluated to determine mutations in dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) genes. All PCR-amplified products were sequenced. Of the 98 clinical specimens, 13 (13.2%) were positive by GMS stain and mtLSUrRNA-PCR, while 32 (32.6%) that were GMS stain-negative gave positive results with mtLSUrRNA-PCR. All the sequences corresponding to the 45 products amplified by mtLSUrRNA-PCR showed 99% or greater identity with P. jirovecii. The mtLSUrRNA-PCR exhibited 86% sensitivity and 98% and 96.6% specificity when results were compared to those corresponding to negative controls and other proven clinical entities, respectively. We found mutations in the DHPS gene in 3 (7.7%) patients, 2 located at codon 55 and 1 at codon 57. One patient showed a synonymous substitution at nucleotide position 312 in the DHFR gene. These results suggest that mtLSUrRNA-PCR is a useful test for diagnosing PcP. In contrast to other studies, this study found a low prevalence of mutations in the DHPS and DHFR genes in Colombian patients.
共评估了 88 例疑似卡氏肺孢子菌肺炎（PcP）患者的 98 份呼吸道标本，采用先前报道的线粒体大亚基 rRNA（mtLSUrRNA）巢式聚合酶链反应（PCR）检测方法。此外，还研究了来自其他肺部感染患者的样本和来自其他真菌病原体的大量 DNA 样本。还使用扩增 ITS1-ITS2 区的泛真菌 PCR 检测方法来鉴定所有真菌 DNA。所有 mtLSUrRNA-PCR 阳性的样本都进行了二氢喋呤合成酶（DHPS）和二氢叶酸还原酶（DHFR）基因突变分析。所有扩增的 PCR 产物均进行测序。在 98 份临床标本中，13 份（13.2%）GMS 染色和 mtLSUrRNA-PCR 阳性，32 份（32.6%）GMS 染色阴性的标本 mtLSUrRNA-PCR 阳性。mtLSUrRNA-PCR 扩增的 45 个产物的所有序列与卡氏肺孢子菌的同源性均为 99%或更高。与阴性对照和其他已证实的临床实体相比，mtLSUrRNA-PCR 的敏感性分别为 86%、98%和 96.6%。我们在 3 名（7.7%）患者的 DHPS 基因中发现了突变，其中 2 个位于密码子 55，1 个位于密码子 57。1 名患者在 DHFR 基因的核苷酸位置 312 处出现同义替换。这些结果表明，mtLSUrRNA-PCR 是诊断 PcP 的一种有用的检测方法。与其他研究不同，本研究发现哥伦比亚患者的 DHPS 和 DHFR 基因突变率较低。
