Results from the second WHO external quality assessment for the molecular detection of respiratory syncytial virus, 2019-2020

Abstract

Background: External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019-2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019-2020 WHO RSV EQA.

Methods: Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories.

Results: An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping.

Conclusions: The WHO RSV EQA 2019-2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets.

Keywords: laboratory diagnostics and systems; respiratory diseases; strengthening laboratory capacity for infectious disease control.

FIGURE 1

(A) Respiratory syncytial virus (RSV) genome schematic organisation, and location of the CDC detection (M gene) and Victorian Infectious Disease Reference Laboratory (VIDRL) (L gene) subtyping primers/probes. (B) Mismatches between the CDC detection pan RT‐PCR primers/probe and the M gene for the 2019 EQA sequences. The forward primer matches all 8 RSV‐A and RSV‐B M gene sequences. The probe matches all the RSV‐B samples but has two mismatches shared by all the RSV‐A sequences. The reverse primer matches with all the RSV‐A sequences, but there are 2 mismatches with the RSV‐B sequences. (C) Mismatches between the VIDRL duplex subtyping primers/probe and the L gene for the 2019 EQA sequences. The forward primer shows two mismatches with all the available RSV‐B samples, at nucleotide positions 8 and 9. The RSV‐B detection probe and reverse primer matches all four sequences exactly. The forward primer matches with all three RSV‐A available sequences, as does the reverse primer. The RSV‐A detection probe shows one mismatch (shared by all the available RSV‐A sequences). Mismatched nucleotides shown in bold red with overlying asterisk (*); primer sequences aligned with gene sequence, using reverse complement if required.