Multicenter Clinical Validation of a Cartridge-Based Real-Time PCR System for Detection of Coccidioides spp. in Lower Respiratory Specimens.

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.