Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture

K Wagner,B Springer,P M Keller,V P Pires

doi:10.1038/s41598-018-25129-w

Abstract

The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

Highlights

In recent years an enormous increase in the frequency and severity of fungal infections has been observed[1,2]
Diagnostic performance of 18S rDNA RT-PCR compared to internal transcribed spacer (ITS) PCR
We found a concordance of 91% between 18S rDNA RT-PCR and culture results for the diagnosis of fungal infections (Table 3)

Summary

Introduction

In recent years an enormous increase in the frequency and severity of fungal infections has been observed[1,2]. Diagnostic performance of microscopic examination and culture are highly dependent on the quality of the clinical specimens and the experience of the laboratory personnel These classical methods have previously shown lower sensitivity in fungal detection compared to molecular methods[7,8,9]. A more appropriate approach is a compilation of clinical and microbiological data as gold standard to evaluate the diagnostic performance of new molecular methods In this retrospective study, we used clinical specimens that have been previously analysed by culture and/or internal transcribed spacer (ITS) PCR and could be categorized as “confirmed fungal infection” or “no fungal www.nature.com/scientificreports/. 18S rDNA RT-PCR ITS PCR positive negative positive negative clinical and microbiological data positive negative infection” These specimens were retrospectively analysed by RT-PCR amplification of the fungal ribosomal small subunit (18S) and amplicon sequencing to rapidly and accurately identify fungal pathogens (Fig. 1)

Objectives

Methods

Results

Discussion

Conclusion