Evaluation of Extraction Methods for Clinical Metagenomic Assay.

Suha A Farraj,Shreif A El-Kafrawy,Esam I Azhar,Taha A Kumosani,Jehad M Yousef

doi:10.3390/microorganisms8081128

Suha A Farraj, Shreif A El-Kafrawy + Show 3 more

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https://doi.org/10.3390/microorganisms8081128

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Abstract

(1) Background: Clinical metagenomics is a promising approach that helps to identify etiological agents in cases of unknown infections. For the efficient detection of an unknown pathogen, the extraction method must be carefully selected for the maximum recovery of nucleic acid from different microorganisms. The aim of this study was to evaluate different extraction methods that have the ability to isolate nucleic acids from different types of pathogens with good quality and quantity for efficient use in clinical metagenomic identification. (2) Methods: A mock sample spiked with five different pathogens was used for the comparative evaluation of different commercial extraction kits. Extracted samples were subjected to library preparation and run on MiSeq. The selected extraction method based on the outcome of the comparative evaluation was used subsequently for the nucleic acid isolation of all infectious agents in clinical respiratory samples with multiple infections. (3) Results: The protocol using the PowerViral® Environmental RNA-DNA Isolation Kit with a 5-min bead beating step achieved the best results with a low starting volume. The analysis of the tested clinical specimens showed the ability to successfully identify different types of pathogens. (4) Conclusions: The optimized extraction protocol in this study is recommended for clinical metagenomics application in specimens with multiple infections from different taxa.

Highlights

Infectious diseases are still the leading cause of human morbidity and mortality [1]
next-generation sequencing (NGS) can be utilized for cases in the field known as clinical metagenomics (CMg), which expected to become the leading diagnostic method in the near future for the identification of pathogens associated with infection, especially during outbreak investigations [5,10]
The mock sample used in this study includes the following microorganisms; two viruses isolated at Special Infectious Agents Unit (SIAU) with a full genome characterization [13], adenovirus (AdV), a nonenveloped double-stranded DNA virus, isolated in HeLa cells with a virus titer of 4.2 × 105 fifty-percent tissue culture infective dose/mL (TCID50/mL); and Alkhumrah virus (ALKV), an enveloped single-stranded RNA virus, isolated in Vero cells with a virus titer of 5.6 × 105 TCID50

Summary

Introduction

Infectious diseases are still the leading cause of human morbidity and mortality [1]. The limited identification of a wide range of infectious agents that are associated with human infections using currently available methods makes it really difficult to diagnose diseases, which highly affects the clinical management of such cases and might lead to potentially adverse reactions due to improper treatment [7]. Next-generation sequencing (NGS) may provide the solution for such problem as it has the ability to identify almost all microorganisms in a clinical sample without prior knowledge of the target [8], allowing the identification of known and novel infectious agents in different human specimens regardless of the organism type associated with the infection. NGS can be utilized for cases in the field known as clinical metagenomics (CMg), which expected to become the leading diagnostic method in the near future for the identification of pathogens associated with infection, especially during outbreak investigations [5,10]

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