Laboratory Sciences of Arizona/Sonora Quest Laboratories, Banner University Medical Center-Phoenix, University of Arizona, Phoenix, Arizona, USA
Laboratory Sciences of Arizona/Sonora Quest Laboratories, Banner University Medical Center-Phoenix, University of Arizona, Phoenix, Arizona, USA.
J Clin Microbiol. 2018 Jan 24;56(2). doi: 10.1128/JCM.01277-17. Print 2018 Feb.
Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for spp. directly in lower respiratory secretions and compared them to today's "gold standard," fungal culture. The GeneSTAT assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at -70°C. These samples were tested by the GeneSTAT assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of , except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.
现有的球孢子菌病诊断方法在准确性和及时性方面存在明显缺陷。我们回顾性和前瞻性地评估了一种新的基于试剂盒的实时 PCR 检测方法对 spp. 的诊断性能和可重复性，该方法直接检测下呼吸道分泌物，与当今的“金标准”真菌培养进行了比较。GeneSTAT 检测使用一个 106bp 的目标序列，每个基因组重复多次（约 60 次），从而将提取 DNA 的检测限（LOD）降低到 10 个基因组当量/ml。共检测了 332 例前瞻性和回顾性患者个体标本。回顾性样本包括 100 例支气管肺泡灌洗或支气管冲洗（BAL/BW）（培养阳性 51 例，阴性 49 例）标本，这些标本以前采集并储存在-70°C。这些标本在三个临床检测地点通过 GeneSTAT 检测进行检测。敏感性为 100%，特异性在 93.8%至 100%之间。三个地点的百分比一致性差异极小，为 95.6%至 100%。此外，总共 232 份新鲜（前瞻性）未经鉴定的 BAL/BW 标本在三个临床地点进行了检测，其中包括来自南加州的一些标本，以提供各种分离株。标本通过真菌培养进行检测，除一个标本外，所有的分离株均通过分子手段（AccuProbe）确认。该检测方法在三个地点的敏感性为 100%（4/4），对阳性新鲜标本的总体特异性为 99.6%（227/228），范围为 98.1%至 100%。在进行交叉反应性检测时，该检测方法对 47 种不同的细菌、病毒和真菌种属的筛查特异性为 100%。
