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This technical specification details the requirements, and provides guidance for the application of advanced sequencing procedures for direct identification and characterisation without culturing of microbial causes of human infectious disease; sometimes termed clinical metagenomics. This document covers considerations associated with specimen collection, processing, nucleic acid isolation, targeting/enrichment, modification/manipulation, library preparation, sequencing, sequence analysis and reporting for the examination of sequences from microbes (including viruses, bacteria, protists and fungi) directly from human specimens (including blood, urine, cerebral spinal fluid and swabs in appropriate buffer) for the purposes of diagnosis, management and/or epidemiological monitoring of infectious disease.
This document is applicable to institutions, e.g. medical diagnostic and public health laboratories, that use sequencing methods to detect clinical microbial pathogens as well as the biotechnology and IVD industry that serves them and those tasked with regulating their application.
Sequencing intended to interrogate the genomes of cultured isolates, host derived nucleic acids or to comprehensively profile the microbiome (such as for estimating diversity indices) is not covered by this document. While there are shared considerations that will be accounted for, this document is not intended to directly cover microbial detection from the environment or foods furthermore, while not directly intended for veterinary applications many considerations detailed here will apply to broader one health considerations associated with animal disease.
Molecular in vitro diagnostics (IVD) tests for infectious diseases increasingly use targeted methods like the polymerase chain reaction (PCR) which can identify pathogen genomic materials in patient specimens or isolates. As a result a number of standards and guidelines have been written to assist in deploying these approaches both generally and specifically for infectious diseases. A complementary strategy used to provide additional pathogen genetic information, to aid patient management or epidemiological decisions, or, increasingly, as a diagnostic method, uses advanced sequencing technology, either alone or in combination with other methods like PCR. When considering microbial analysis sequencing methods can be applied to:
- A) identify the pathogen genetic material present within the patient specimens as a route to determine the causative aetiology and provide a diagnosis,
- B) cultured isolates of the pathogen,
- C) the microbial community to provide information associated with the human microbiome.
This technical specification focuses on A above and will highlight challenges associated with the analytical steps of the process involved in sampling, preparing, nucleic acid targeting and/or enrichment, sequencing and analysis of pathogen derived nucleic acids direct from patient specimens. As well as highlighting associated pitfalls, this technical specification will also provide solutions to improve the accuracy associated with using sequencing technology to directly detect and diagnose pathogens. Aspects associated with B and C may be addressed in future technical specifications. Sequencing differs from many other diagnostic methods in that the readout (the genomic sequence of the pathogen) can provide additional information (such as genetic determinants of strain type or resistance) and does not necessarily require suspicion of the specific aetiological cause to guide diagnostic test selection. Consequently, ensuring accuracy and best practice when using sequencing for the diagnosis, management and epidemiological evaluation of infectious disease-causing microbes requires some unique considerations. This is especially relevant when considering validation of analytical specificity and how to address incidental findings.
This technical specification outlines the routes for ensuring quality, safety and high performance when using sequencing approaches for clinical analysis of infectious disease and how to report these findings. This will benefit medical and epidemiological laboratories, developers of the methods and related stakeholders: such as regulatory authorities and/or accreditation bodies monitoring the quality and performance of these laboratories.
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