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NGS checklist takes in infectious disease testing

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William Check, PhD

October 2017—The CAP issued its first accreditation checklist for next-generation sequencing in 2014, as NGS was becoming a tool used in a growing number of clinical laboratories. The list of requirements, which was a new section in the molecular pathology checklist, focused on constitutive (germline) testing and oncology testing.

Now, with the growing penetration of NGS into the infectious disease field, the CAP has issued a revised edition of the NGS requirements, one that includes, among other things, greater coverage of NGS in the microbiology laboratory.

Dr. Pinsky

Dr. Pinsky

“The main point is that we’ve added information to almost all of the notes in many existing requirements that clarify the requirements for infectious disease testing,” says Benjamin Pinsky, MD, PhD, a member of the CAP’s Microbiology Resource Committee and of its NGS Project Team. “We did not yet break out the applications of next-generation sequencing to infectious disease from the rest of the checklist.” As NGS becomes more commonly used in infectious disease, those applications are likely to be set apart, adds Dr. Pinsky, associate professor of pathology and of medicine (infectious diseases), Stanford University School of Medicine, and medical director of the clinical virology laboratory, Stanford Health Care and Stanford Children’s Health.

Sheldon Campbell, MD, PhD, a member of the CAP Checklists Committee and associate professor of laboratory medicine, Yale School of Medicine, notes there are no FDA-approved applications for NGS in infectious diseases. “However, it has become an important tool in outbreak investigations and epidemiology, and it has become clear that NGS-based techniques for strain typing and following strains in outbreaks give you more information than do older methods of bacteria, parasite, fungus, and virus identification,” he says.

In the 2014 checklist, cancer and constitutive testing of human DNA were strongly represented, says Rakesh Nagarajan, MD, PhD, chief biomedical informatics officer at PierianDx and adjunct associate professor of pathology and immunology, Washington University School of Medicine. “Over the next few years there was increasing recognition that NGS could be used in infectious diseases and pharmacogenomics. There was some information pertaining to those areas at the time of the initial checklist, but they were not a major focus.”

For the revised 2017 checklist, released in August, he and others reviewed the recently published literature and the checklist as a whole and made either structural or higher-level recommendations that can be taken on in future years. The reviewers were in four subgroups, each with its own focus: molecular oncology, inherited disease, infectious disease, and pharmacogenomics. Their members came not only from the Checklists Committee and NGS Project Team but also from the CAP Molecular Oncology, Microbiology Resource, Personalized Health Care, and Histocompatibility/Identity Testing committees and the CAP/ACMG Biochemical and Molecular Genetics Resource Committee. The CAP Council on Accreditation leads the work to reexamine and revise checklists.

Amajor change in the 2017 checklist addresses the validation of NGS testing methods and how many and what variety of samples should be used for validating NGS. “We added a note about input specimen types,” Dr. Nagarajan explains. “The specimen types you use for validation should be the same types you expect to encounter during testing. You don’t need to test all possible specimen types. However, you shouldn’t evaluate those specimen types in cell lines alone.” What’s important, Dr. Nagarajan emphasizes, is the context. “Cell lines alone are not adequate to satisfy this requirement. You also have to have primary specimen types from patients and a representation of those types you expect to get in clinical work.”

MOL.36015 on “NGS Analytical Wet Bench Process Validation” says, in part, the following: “Due to extensive microbial genetic variation and diversity, it is not possible to perform an NGS test validation that would assess the ability of the test to accurately and reliably detect every possible organism or variant that may be present in a specimen. To address this limitation, a methods-based approach can be used for validation wherein the specimens used for validation contain a representative spectrum of the types of organisms, resistance variants, pathogenic factors, and host-response markers that the test is designed to detect. For tests that are designed for organism detection, common pathogens found in a particular specimen type should be included, when feasible, in the validation to ensure their accurate detection.”

Dr. Nagarajan describes as a “huge” addition the inclusion of language illustrating that the checklist no longer addresses only sequencing the human genome but detection of sequences of microorganisms as well. “Whether you are using specific probes or a metagenomic approach, you must validate specimen types inclusive of the microbiology process,” he says.

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