1.What is the step-by-step process in confirming suspected MERS-CoV Cases?

Laboratory confirmation of suspected MERS CoV cases at the RITM is based on the detection of unique sequence targets of the MERS CoV viral RNA, based on the WHO-recommended CDC protocol for Real-Time PCR detection of MERS Coronavirus. This involves initial real-time PCR assays to determine if the samples are positive for MERS CoV. If negative in the initial screening assays, the result is released as negative. However, if positive in the initial test, additional tests will be performed using real-time PCR and additionally, DNA sequencing for some cases, to confirm that the sample is positive for MERS CoV. Details of the procedure is described below:

Specimen preparation
All clinical respiratory specimens for MERS CoV testing are inactivated prior to molecular diagnostic testing.

Molecular diagnostics

• Viral RNA is extracted from the inactivated clinical specimen.
• We follow the WHO testing algorithm for MERS CoV detection, involving detection of multiple gene regions in the MERS CoV genome, such as: (a) upE (upstream of the E gene), (b) ORF1a (open reading frame 1a gene), (c) N2 (nucleocapsid 2 gene), (d) N3 (nucleocapsid 3 gene), (e) RdRp (RNA dependent RNA polymerase gene) and (f) NSeq (N gene)
  • According to WHO and CDC, a MERS CoV positive sample should test positive for 2 separate gene targets (eg. upE and N2 or N2 and N3 or upE and N3, etc). A MERS CoV negative sample on the other hand should test negative for the screening assays (the Tier 1 assays below)
  • We follow the following testing algorithm:

• After testing, results are analyzed, encoded in the test registry, verified and then released.
• If a sample is negative in the Tier 1 assays, no additional tests will be done and the result is released as negative. If the sample is positive in Tier 1, Tier 2 assays (and Tier 3 assays, for some cases) are performed as additional confirmatory tests before the result is released as positive.

2. How long does it usually take for the result to be confirmed?

• For regular samples, the turnaround time for MERS CoV testing at RITM is 48 hrs (from specimen inactivation to results reporting), with running times confined to regular office hours (Monday to Friday, 8:00 am to 5:00pm).
• This turnaround time is based on the estimated time it takes to complete all procedures according to the standard process flow for molecular detection of MERS CoV, with allowance for extra number of samples (in instances where multiple samples are received, more samples translates to longer time before procedures are completed) as well as for repeat testing.
• If there are no repeats and the sample is negative for MERS CoV, the result may be released within 7 hours from the start of sample processing.
• However, if the sample is positive in the initial screening assays described above, results may be released within 24 hours or up to 7 days, if sequencing is needed as an additional confirmatory test.
• Although samples are usually processed within office hours (8am to 5pm), we have on-call staff who go on duty once we receive samples for urgent processing.

Additional information regarding the MERS-CoV test at RITM:

• The RITM MBL employs a comprehensive Quality Assurance program, to ensure accuracy, efficiency and timeliness in results reporting. The Quality Assurance program includes:
  • Annual participation of RITM MBL in an internationally-recognized (QCMD) External Quality Assurance Program for MERS CoV detection (perfect scores each year since we started participating)
  • Annual Proficiency Testing of MBL laboratory staff
  • Use of WHO-recommended PCR assays
  • Validation of assays prior to routine use at MBL
  • Lot testing for reagents
  • Inclusion of internal controls in each run
  • Control performance monitoring
  • Use of calibrated equipment in testing
  • Verification of data and results prior to results reporting