Results from the WHO external quality assessment for the respiratory syncytial virus pilot, 2016‐17

Abstract Background External quality assessments (EQAs) for the molecular detection of respiratory syncytial virus (RSV) are necessary to ensure the provision of reliable and accurate results. One of the objectives of the pilot of the World Health Organization (WHO) Global RSV Surveillance, 2016‐2017, was to evaluate and standardize RSV molecular tests used by participating countries. This paper describes the first WHO RSV EQA for the molecular detection of RSV. Methods The WHO implemented the pilot of Global RSV Surveillance based on the WHO Global Influenza Surveillance and Response System (GISRS) from 2016 to 2018 in 14 countries. To ensure standardization of tests, 13 participating laboratories were required to complete a 12 panel RSV EQA prepared and distributed by the Centers for Disease Control and Prevention (CDC), USA. The 14th laboratory joined the pilot late and participated in a separate EQA. Laboratories evaluated a RSV rRT‐PCR assay developed by CDC and compared where applicable, other Laboratory Developed Tests (LDTs) or commercial assays already in use at their laboratories. Results Laboratories performed well using the CDC RSV rRT‐PCR in comparison with LDTs and commercial assays. Using the CDC assay, 11 of 13 laboratories reported correct results. Two laboratories each reported one false‐positive finding. Of the laboratories using LDTs or commercial assays, results as assessed by Ct values were 100% correct for 1/5 (20%). With corrective actions, all laboratories achieved satisfactory outputs. Conclusions These findings indicate that reliable results can be expected from this pilot. Continued participation in EQAs for the molecular detection of RSV is recommended.


| INTRODUC TI ON
The World Health Organization (WHO) Global Influenza Program (GIP) launched a pilot in 2016 to build global respiratory syncytial virus (RSV) surveillance on the well-established WHO Global Influenza Surveillance and Response System (GISRS). 1 RSV is a significant public health problem, causing an estimated annual 33 million acute lower respiratory infections (ARI) in children under 5 years. 2 Very young children and the elderly are at risk of experiencing severe RSV infections. 3,4 Particularly in low-income countries, RSV in infants causes considerable mortality, the magnitude of which is not be fully understood. 5 Although monoclonal antibodies as palivizumab have existed for some time, there are several RSV vaccines and monoclonal antibodies in phase I, II, and III clinical trials. Therefore, new RSV interventions may be on the market within the next 5-10 years. 6 The establishment of a WHO Global RSV Surveillance is essential to support countries' understanding of RSV epidemiology in different regions and associated disease burden and is instrumental to understand the global circulation of different strains of RSV. A key objective of the WHO Global RSV Surveillance is to standardize RSV molecular detection and surveillance using the already established GISRS platform.
Although several commercial and RSV LDTs are available for RSV detection, there are no agreed reference standards for RSV molecular testing and ongoing evaluations of performance through external quality assessments (EQAs) [7][8][9][10] Fourteen countries from all 6 WHO regions where National Influenza Centers (NIC) have demonstrated reliable quality of molecular detection of influenza viruses through the annual influenza WHO EQA, were selected to participate in the pilot.
In addition, three laboratories with long-standing experience in RSV detection and research agreed to serve as RSV Reference Laboratories, primarily to provide technical support to the 14 pilot laboratories.11 An established real-time (r) RT-PCR was provided to the pilot participant laboratories as the standard assay for the RSV detection by the CDC, USA. 12 Pilot laboratories were given the option to use LDTs or commercial real-time RT-PCR assays following validation against the CDC RSV rRT-PCR assay. 12

| CDC RSV rRT-PCR assay
The CDC RSV rRT-PCR assay was selected as the reference assay

| Preparation and composition of the CDC RSV EQA panel
Each CDC panel consisted of 20 freeze-dried samples prepared from RSV-infected or uninfected cell cultures (Table 1)

| Distribution of RSV EQA panel to participating laboratories
RSV EQA panels were dispatched at ambient temperature by courier service between December 2016 and March 2017 by CDC. The RSV rRT-PCR kits were shipped on dry ice. Laboratories were requested to return their results within 4 weeks after receiving the materials. Upon shipping, each laboratory received a message with a unique number, a copy of the protocols and package inserts and a fillable result form.
The unique assigned number was used for further communications with each laboratory. Laboratories reported results electronically to CDC for analysis using unique assigned numbers, safeguarding participant laboratories confidentiality. Following analysis, CDC reported results to participating laboratories and to WHO maintaining confidentiality.  (Table 1). Laboratory 13 obtained 3 false-negative results for specimens containing lower viral loads of RSV B_Strain 209_GB3 with a commercial assay but correctly identified RSV B_Strain 209_GB3 with the CDC assay (Table 1)  It is possible that the EQA specimens could have been handled with special care although such specimens should be processed according to standard laboratory practices. It is challenging to coordinate an EQA panel testing such that the EQA specimens should be blinded for the laboratory staff. Although not required, the CDC EQA samples, upon reconstitution, provided enough material for a second nucleic acid extraction and rRT-PCR testing in anticipation of an unsuccessful first test.

| Other molecular methods for RSV detection
The overall results presented during this exercise indicated that the pilot laboratories, particularly when using the recommended RSV assay and reagents provided by CDC, performed very well. Pilot laboratories used information regarding their performance, which was generated from the CDC RSV EQA panel to make appropriate changes and improvements in the laboratory detection of RSV.
Corrective action for one laboratory included that a change was made to use one of the real-time PCR platforms and software versions which had been validated and recommended by CDC. This resulted in that laboratory achieving the expected output. Taking corrective actions and investigation of test non-conformities, inconsistencies, and deficiencies provides an opportunity for standardized laboratory detection improvements. Although several types of laboratory tests are available for the diagnosis of RSV, it is necessary to have a standard reference protocol and assessments of performance by EQAs. Rapid diagnostic antigen detection tests although useful are generally reliable in young children but less useful in older children and adults. The relevance of EQAs continues to be important with the growing trend to use molecular point of care tests at emergency care instead of antigen tests. 22,23 Highly sensitive RT-PCR assays should be considered when testing adults, due to their ability to detect low viral loads in clinical specimens.
Although several RT-PCR RSV LDTs and commercial assays are available, the sensitivity and specificity of these assays should be periodically evaluated against a reference standard to avoid the underor over-reporting of RSV infections. This pilot focused on the quality of detection of RSV and therefore used primers and probes to highly conserved regions in the RSV M gene.
Following satisfactory performance of laboratories in the EQA using either the CDC or non-CDC assay, participating laboratories were given the option to conduct RSV surveillance using the assay of their choice. As CDC receives clinical specimens and RSV isolates from many different countries, they can closely monitor the evolution or RSV genes targeted by their assay and can alert participating laboratories should mutations in the targeted sequences affect the performance of the assay. It is unlikely however that only one reference assay will be used in ongoing RSV surveillance hence the necessity for EQAs. The participation of laboratories in RSV EQAs is critical to compare and evaluate the performance of various molecular assays. Furthermore, there is a danger in using one primer design, as when this fails all laboratories will fail.

| CON CLUS IONS
The comparison of EQA results with a variety of assays is a good surrogate measure of performance as there are no international molecular standards available to date for the molecular detection of RSV. 24 The molecular detection of RSV was standardized through the CDC RSV Proficiency Panel in 13 pilot countries of the 6 WHO regions. Laboratories performed well using the CDC RSV molecular assay in comparison with LDTs and commercial assays. These countries were the first participants of the pilot of the WHO Global RSV Surveillance. With the expected introduction of RSV vaccines in the future and the need to provide accurate data to close knowledge gaps in the epidemiology of RSV, the standardization of molecular