Dang Hoang Khanh, Lottie Brown, Phan Thi Ha My, Nguyen Thi Mai Thu, Emily Evans, Vo Trieu Ly, Nguyen Thanh Hiep, Ngo Thi Hoa, Thuy Le
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引用次数: 0
Abstract
Talaromyces marneffei is a dimorphic fungus endemic in Southeast Asia that 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 vs. 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%) vs. 73.9% (95% CI: 65.6%-80.8%, P < .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.
期刊介绍:
Medical Mycology is a peer-reviewed international journal that focuses on original and innovative basic and applied studies, as well as learned reviews on all aspects of medical, veterinary and environmental mycology as related to disease. The objective is to present the highest quality scientific reports from throughout the world on divergent topics. These topics include the phylogeny of fungal pathogens, epidemiology and public health mycology themes, new approaches in the diagnosis and treatment of mycoses including clinical trials and guidelines, pharmacology and antifungal susceptibilities, changes in taxonomy, description of new or unusual fungi associated with human or animal disease, immunology of fungal infections, vaccinology for prevention of fungal infections, pathogenesis and virulence, and the molecular biology of pathogenic fungi in vitro and in vivo, including genomics, transcriptomics, metabolomics, and proteomics. Case reports are no longer accepted. In addition, studies of natural products showing inhibitory activity against pathogenic fungi are not accepted without chemical characterization and identification of the compounds responsible for the inhibitory activity.