Chimara Erica, Ferrazoli Lucilaine, Ueky Suely Yoko Misuka, Martins Maria Conceição, Durham Alan Mitchel, Arbeit Robert D, Leão Sylvia Cardoso
Instituto Adolfo Lutz, São Paulo, Brazil.
BMC Microbiol. 2008 Mar 20;8:48. doi: 10.1186/1471-2180-8-48.
Identification of nontuberculous mycobacteria (NTM) based on phenotypic tests is time-consuming, labor-intensive, expensive and often provides erroneous or inconclusive results. In the molecular method referred to as PRA-hsp65, a fragment of the hsp65 gene is amplified by PCR and then analyzed by restriction digest; this rapid approach offers the promise of accurate, cost-effective species identification. The aim of this study was to determine whether species identification of NTM using PRA-hsp65 is sufficiently reliable to serve as the routine methodology in a reference laboratory.
A total of 434 NTM isolates were obtained from 5019 cultures submitted to the Institute Adolpho Lutz, Sao Paulo Brazil, between January 2000 and January 2001. Species identification was performed for all isolates using conventional phenotypic methods and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing a 441 bp fragment of hsp65. Phenotypic evaluation and PRA-hsp65 were concordant for 321 (74%) isolates. These assignments were presumed to be correct. For the remaining 113 discordant isolates, definitive identification was based on sequencing a 441 bp fragment of hsp65. PRA-hsp65 identified 30 isolates with hsp65 alleles representing 13 previously unreported PRA-hsp65 patterns. Overall, species identification by PRA-hsp65 was significantly more accurate than by phenotype methods (392 (90.3%) vs. 338 (77.9%), respectively; p < .0001, Fisher's test). Among the 333 isolates representing the most common pathogenic species, PRA-hsp65 provided an incorrect result for only 1.2%.
PRA-hsp65 is a rapid and highly reliable method and deserves consideration by any clinical microbiology laboratory charged with performing species identification of NTM.
基于表型试验鉴定非结核分枝杆菌（NTM）耗时、费力、成本高，且常常得出错误或不确定的结果。在被称为PRA-hsp65的分子方法中，hsp65基因的一个片段通过聚合酶链反应（PCR）进行扩增，然后通过限制性酶切分析；这种快速方法有望实现准确、经济高效的菌种鉴定。本研究的目的是确定使用PRA-hsp65进行NTM菌种鉴定是否足够可靠，可作为参考实验室的常规方法。
2000年1月至2001年1月期间，从提交给巴西圣保罗阿道夫·卢茨研究所的5019份培养物中总共获得了434株NTM分离株。使用传统表型方法和PRA-hsp65对所有分离株进行菌种鉴定。对于这些方法给出不一致结果的分离株，通过对hsp65的441 bp片段进行测序获得明确的菌种鉴定。表型评估和PRA-hsp65对321株（74%）分离株的结果一致。这些鉴定结果被认为是正确的。对于其余113株不一致的分离株，明确的鉴定基于对hsp65的441 bp片段进行测序。PRA-hsp65鉴定出30株具有hsp65等位基因的分离株，代表13种先前未报告的PRA-hsp65模式。总体而言，PRA-hsp65进行的菌种鉴定比表型方法显著更准确（分别为392株（90.3%）对338株（77.9%）；p <.0001，Fisher检验）。在代表最常见致病菌种的333株分离株中，PRA-hsp65仅给出了1.2%的错误结果。
PRA-hsp65是一种快速且高度可靠的方法，任何负责进行NTM菌种鉴定的临床微生物实验室都应予以考虑。
