• Specimens should be placed at 4 °C immediately after collection and promptly transported to the laboratory.
• Storage of clinical specimens at the site of collection and transport to the laboratory should follow guidelines as for influenza specimen collection.
• Specimens should be aliquoted in 3-4 vials of approximately 0.5 ml each on arrival at the laboratory.
• If the specimens cannot be processed within 48 hours, they should be kept frozen at or below -70 °C.
• Repeated freezing and thawing must be avoided.
• For storage and transport, triple packaging should be used in keeping with international and national biosafety recommendations  ^[1],[2].

 

LABORATORY TECHNIQUES FOR THE DETECTION AND TYPING OF RSV

Several countries and laboratories presently use different types of laboratory assays for the detection of RSV.  The WHO Global RSV Surveillance Phase 2 recommends using the below assays according to the test algorithm below and as described in the WHO Strategy.^[3]

• The CDC Real-Time RT-PCR Assay for Respiratory Syncytial Virus (RSV) - singleplex assay for RSV detection. Ver.003.2016
• The VIDRL RSV rPCR Multiplex Assay for typing of RSV A and RSV B
• The CDC RSV rPCR Multiplex Assay for the detection and typing of RSV A and RSV B

Alternatively, countries can use their existing rRT-PCR assay laboratory developed assay or commercial assay for the detection and typing of RSV provided they have validated their assay under the guidance of reference laboratories through external quality assurance program

 

LABORATORY TECHNIQUES FOR RSV SEQUENCING  

Sharing of RSV positive samples/isolates with RSV Reference Laboratories

Participating laboratories are requested to share a subset of positive RSV specimens with one of the Reference Laboratories as described in the WHO Strategy for Global Respiratory Syncytial Virus Surveillance guidelines ⁹   Sharing of specimens will allow for ongoing quality assurance, standardization of typing and sequencing methodologies, the output of which will contribute towards closing knowledge gaps in the molecular epidemiology of RSV .

Shipping procedures:  For shipping procedures and logistics for sharing of RSV positive specimens with Reference Laboratories contact WHO HQ. (Awandha link to WHO contact page)

Prior to sequencing specimens collected during Phase 2, RSV positive specimens / isolates collected in the pilot phase will undergo genetic characterization by Reference Laboratories. Countries from the WHO RSV Pilot phase are invited to select RSV positive specimens collected in the 2016-17 and 2017-18 RSV seasons, and to share positive specimens/isolates with RSV Reference Laboratories for sequencing purposes.

If countries can generate their own sequence data and would like to undertake this task, then these sequences will be added to the sequence data under this project, to make a more comprehensive overall analysis of the RSV genetic variation occurring globally. Countries implementing sequencing should follow the guidelines of the RSV reference laboratories

Retrospective sequencing of RSV specimens

The objective of retrospective sequencing is to standardize RSV sequencing methodologies through;

• protocols for virus sharing with RSV Reference Laboratories
• recommending a standard approach to virus nomenclature for genomic analysis and management purposes
• Investigate the compatibility of different amplicon-based sequencing methods at the reference laboratories for G gene, F gene or whole genome sequencing
• the development of standards, methods and bioinformatic pipelines for genome assembly and annotation
• the establishment of a platform to share sequencing outputs and analysis  

Prospective sequencing of RSV specimens

The criteria for selecting specimens for sequencing prospectively is outlined in the WHO Strategy for Global Respiratory Syncytial Virus Surveillance Phase 2 document 2019^[4]