Optimization and clinical evaluation of a 5.8S rRNA quantitative PCR assay for the diagnosis of talaromycosis

Abstract Talaromyces marneffei is a dimorphic fungus endemic in Southeast Asia which causes the invasive fungal disease talaromycosis in immunocompromised individuals. Detection of T. marneffei nucleic acid in blood by PCR has demonstrated potential as a diagnostic tool for talaromycosis, but previously developed assays have limited sensitivity. This study described the optimization of a quantitative PCR (qPCR) assay for the diagnosis of talaromycosis. Our assay performance was maximized by testing different primers, methods of cell lysis and DNA extraction, whole blood versus plasma, and methods of specimen treatment, using mean quantification cycle (Cq) values to compare performance. Our qPCR assay achieved the highest analytical sensitivity of 1 yeast cell per mL of whole blood, using primers targeting the 5.8S ribosomal DNA, cell lysis by bead beating, and DNA extraction by the MasterPure Yeast Purification kit. There was no cross-reactivity observed with six Penicillium species and nine clinically related fungal isolates. In a case-control, diagnostic validation study of 138 cases of talaromycosis and 30 controls with other invasive fungal diseases and opportunistic infections, our 5.8S qPCR assay detected T. marneffei in 99.0% (101/102, 95% CI: 94.6–99.9%) of blood culture-positive and 55.6% (20/37, 95% CI: 38.1–72%) of blood culture-negative patients. Overall, our 5.8S qPCR assay had significantly higher sensitivity compared to conventional BACTEC blood culture, 87.7% (95% CI: 80.7–92.5%) versus 73.9% (95% CI: 65.6% - 80.8%, P < 0.001), and the specificity was 96.7% (95% CI: 80.9–99.8%). Our 5.8S qPCR assay has potential as a non-invasive and rapid rule-in test for talaromycosis.