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

IF 2.7 3区 医学 Q3 INFECTIOUS DISEASES
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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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.

5.8S rRNA定量PCR诊断塔氏菌病的优化及临床评价。
马尔尼菲塔芳香菌是东南亚特有的一种二态真菌,可引起免疫功能低下个体的侵袭性真菌疾病塔芳香菌病。通过PCR检测血液中的马尼菲T.核酸已被证明有潜力作为一种诊断塔氏菌病的工具,但以前开发的检测方法灵敏度有限。本研究描述了一种用于诊断talaromyosis的定量PCR (qPCR)方法的优化。通过测试不同的引物、细胞裂解和DNA提取方法、全血与血浆以及标本处理方法,使用平均定量周期(Cq)值来比较性能,我们的检测性能得到了最大化。我们的qPCR检测获得了最高的分析灵敏度,每mL全血中有1个酵母细胞,使用5.8S核糖体DNA为引物,通过敲打头裂解细胞,并使用MasterPure酵母纯化试剂盒提取DNA。与6种青霉菌和9种临床相关真菌分离株无交叉反应。在一项病例对照、诊断验证研究中,138例塔兰菌病和30例其他侵袭性真菌疾病和机会性感染的对照中,我们的58s qPCR检测在99.0% (101/102,95% CI: 94.6%-99.9%)的血培养阳性患者和55.6% (20/37,95% CI: 38.1%-72%)的血培养阴性患者中检测到马氏弓形虫。总体而言,与传统BACTEC血培养相比,我们的5.8S qPCR检测具有显著更高的敏感性,分别为87.7% (95% CI: 80.7%-92.5%)和73.9% (95% CI: 65.6%-80.8%)
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来源期刊
Medical mycology
Medical mycology 医学-兽医学
CiteScore
5.70
自引率
3.40%
发文量
632
审稿时长
12 months
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.
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