Molecular Methods for Identifying Mycobacteria, Nocardiae, and Fungi

诺卡氏菌分离株的研究进展。已经清楚地表明，分子方法对于准确鉴定临床分离株的种级是必要的。我们以前的研究表明，通过扩增16S rRNA和热休克蛋白基因的部分片段，然后对扩增出的扩增产物进行限制性片段长度多态性分析，可以快速、准确地进行鉴定。我们最近对鉴定诺卡氏菌的另一个基因区域secA1的研究结果。已经发表，并表明这个相对较短的区域通常比16S区域更有助于准确识别这些生物。我们现在使用secA1基因区域的测序作为鉴定诺卡氏菌分离株的常规程序；如果结果有任何不明确的地方，我们还会对完整的16S rRNA基因区域进行测序，以获得额外的遗传信息，试图解决这种不确定问题。为了最大限度地提高我们使用secA1基因区域进行鉴定的可靠性，我们正在获取所有新描述的具有临床意义的诺卡氏菌物种的模式菌株。 在疾病控制和预防中心和德克萨斯大学卫生中心泰勒分校的研究人员的合作下，我们发表了对两个具有临床意义的诺卡氏菌物种的描述：黑诺卡氏菌和沃勒诺卡氏菌。 焦磷酸测序是临床实验室快速鉴定微生物病原体的一种很有前途的新分子技术。利用我们收集的大量具有临床意义的不同物种，我们调查了这一程序在鉴定诺卡氏菌物种方面的实用性，并发现该技术不能准确地识别目前被认为具有临床意义的所有物种。 诺卡氏菌属之间存在种间差异。他们对抗菌剂的敏感性，可能还有物种内的变异性。然而，由于它们的生长相对较慢，而且在液体介质中倾向于形成块状，而不是更均匀的悬浮液，在解释药敏试验结果时，很难获得实验室内和实验室间的一致性。我们正在参与诺卡氏菌药敏检测的机构间研究，目的是更好地标准化此类检测的性能和解释。对五个不同中心的所有分离株的测试已经完成，研究数据目前正在分析中。 目前，我们没有计划对快速生长的分枝杆菌进行任何额外的研究。然而，我们正在应用类似于我们对诺卡氏菌和快速生长菌所使用的技术来研究具有临床意义的真菌。大多数霉菌分离物的鉴定仍然几乎完全基于形态。然而，霉菌隔离可能需要一段时间才能形成能够进行识别的结构，在某些情况下，这种结构永远不会形成。为了提高霉菌鉴定的速度和准确性，我们将探索对不同基因区域进行焦磷酸测序的可行性，特别是将不同基因和基因区域用于不同组霉菌的可行性。 我们也一直在研究焦磷酸测序技术在酵母分离鉴定中的应用。在形态上，许多酵母种类彼此非常相似，而且只有少量的表型试验可用于酵母鉴定，这使得表型试验的判别力相对较差。我们已经发现，焦磷酸测序，利用一个高度可变的内部转录间隔区，可以可靠地识别最常见的酵母菌临床分离株，虽然略低于传统的循环测序，但成本更低。一份总结我们研究结果的手稿最近出版了。

Ongoing studies of isolates of Nocardia spp. have clearly demonstrated that molecular methods are necessary for the accurate species-level identification of clinical isolates. Our previous studies showed that rapid and accurate identifications could be obtained by amplification of portions of the 16S rRNA and heat-shock protein genes, followed by restriction fragment length polymorphism analysis of the resulting amplicons. Results of our recent studies of another gene region, secA1, for the identification of Nocardia spp. have been published, and have demonstrated that this relatively short region is generally even more useful than the 16S region for accurate identification of these organisms. We now use sequencing of the secA1 gene region as our routine procedure for identification of Nocardia isolates; if the results are in any way ambiguous, we also sequence the full 16S rRNA gene region to obtain additional genetic information to try to resolve the ambiguity. To maximize the reliability of our identifications using the secA1 gene region, we are acquiring the type strains for all newly-described Nocardia species of clinical significance. In collaboration with investigators from the Centers for Disease Control and Prevention and the University of Texas Health Center at Tyler, we have published descriptions of two clinically significant species of Nocardia, N. blacklockiae and N. wallacei. Pyrosequencing is a promising new molecular technique for the rapid identification of microbial pathogens in clinical laboratories. Using our large collection of different species of clinical significance, we have investigated the utility of this procedure for identification of Nocardia species, and have found that the technique cannot accurately identify all of the species that are currently considered to be clinically significant. There are inter-species differences among Nocardia spp. in their susceptibility to antimicrobial agents, and probably intra-species variability as well. However, because of their relatively slow growth and their tendency to form clumps, rather than more homogeneous suspensions, in liquid media, there have been difficulties in obtaining intra- and inter-laboratory concordance in interpretation of susceptibility testing results. We are participating in an inter-institutional study of susceptibility testing of Nocardia spp., with the goal of better standardizing the performance and interpretation of such testing. Testing of all isolates at five different centers has been completed, and the data from the study are currently being analyzed. We are not planning any additional studies of rapidly growing mycobacteria at present. We are, however, applying techniques similar to those we have used with both Nocardia and rapid growers to the study of clinically significant fungi. The identification of most mold isolates is still based almost entirely on morphology. However, it may take some time for a mold isolate to develop the structures that will allow an identification to be made, and in some cases such structures never develop. In an attempt to enhance both the speed and accuracy of mold identification, we will be exploring the feasibility of using pyrosequencing of various gene regions, and will in particular investigate the feasibility of using different genes and gene regions for different groups of molds. We have also been investigating the utility of pyrosequencing for the identification of yeast isolates. Morphologically, many yeast species are quite similar to one another, and the fact that only a small number of phenotypic tests are available for yeast identification makes discrimination by phenotypic testing relatively unreliable. We have found that pyrosequencing, utilizing a hyper-variable internal transcribed spacer region, can reliably identify most common clinical isolates of yeast species, and, while somewhat less precise than conventional cycle sequencing, is less costly. A manuscript summarizing the results of our studies has recently been published.