Development of a Sensitive and Specific Novel qPCR Assay for Simultaneous Detection and Differentiation of Mucormycosis and Aspergillosis by Melting Curve Analysis.

Rachna Seth,Janya Sachdev,Manoranjan Mahapatra,Viveka P Jyotsna,Ashit Bhushan Xess,Immaculata Xess,Bhaskar Rana,Prashant Mani,Arun Kumar Jain,Mragnayani Pandey,Dibyabhaba Pradhan,Gagandeep Singh,Sameer Bakhshi,Reshu Agarwal,Lalit Dar,Rakesh Kumar,Usha Yadav

doi:10.3389/ffunb.2021.800898

Abstract

Molecular diagnostic assays can expedite the diagnosis of fungal infections, and subsequently help in early interventions and appropriate management of patients. The aim of this study was to develop a single set of primers for a real-time quantitative polymerase chain reaction (qPCR) assay to detect and identify commonly reported, clinically relevant molds i.e., Aspergillus spp, Mucorales and Fusarium spp., up to genus level by melting curve analysis. This assay was evaluated in whole blood from patients with suspected invasive aspergillosis (IA), and in tissue biopsy, bronchoalveolar lavage (BAL) fluid and other site-specific samples from patients with suspected invasive mucormycosis (IM). The limit of detection (LoD) was determined as 10 copies/μl for all three molds. The mean coefficient of variation (CV) across all sets of intra- and inter-assay data was 0.63% (ranging from 0.42 to 1.56%), showing high reproducibility of the assay. Sensitivity and specificity of the assay were 93.3 and 97.1% respectively for diagnosis of IA, and 99.29 and 83.84% respectively for diagnosis of IM. Fusarium was not detected in any of the clinical samples included and the few laboratory confirmed cases of fusariosis did not meet the inclusion criteria of the study. Hence no ROC curve or cutoff value could be generated for the same. This newly developed qPCR assay therefore appears to be a promising tool in detection of IA and IM.

Highlights

Limitations in conventional diagnostic methods often lead to delays in diagnosis of invasive fungal infections (IFIs), which in turn delays treatment and is a key risk factor for poor patient outcomes (Barnes, 2008)
The real-time PCR assay using newly designed primers evaluated in this study is superior to standard conventional methods of diagnosis and is a fast, sensitive, and specific method for the diagnosis of invasive mold infections
It can detect and differentiate all three clinically relevant molds prevalent in this geographical area, without the need for amplicon sequencing, this would be especially useful in cases of hematological malignancy, who can be affected by invasive fungal infections due to all three molds, and in detection of cases of mixed infection

Summary

Introduction

Limitations in conventional diagnostic methods often lead to delays in diagnosis of invasive fungal infections (IFIs), which in turn delays treatment and is a key risk factor for poor patient outcomes (Barnes, 2008). Real-time and nested PCR assays have been designed for the specific detection of single or multiple fungal pathogens (Kami et al, 2001; Cornet et al, 2002; Costa et al, 2002; Sanguinetti et al, 2003; Simoneau et al, 2005; Meersseman et al, 2007; Botterel et al, 2008; Imbert et al, 2016, 2018). Unlike conventional PCR, realtime PCR techniques can quantify the load of fungal DNA in a sample and can distinguish between fungal colonizers and invasive pathogens (Li et al, 2016; Valero et al, 2016). There are a variety of commercially available PCR assays currently in use, but only a few have been investigated in large cohort studies (e.g., AsperGenius R , MycAssay Aspergillus R , MycoGENIE R ) (Guinea et al, 2013; White et al, 2015; Dannaoui et al, 2017; Salzer et al, 2019)

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Conclusion