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Paradigm Shift Intervention Monitoring
Asymptomatic Cases Raise More Testing Concerns
Detection end points were two copies per reaction for the upE assay, and 10 copies per reaction for the confirmatory, ORF1b gene, assay.
The above translation (in red) provide additional detail on the testing of the asymptomatic cases in Italy which generated weak positives in the PCR testing done by the Laboratory of Virology by the University of Florence. The samples were subsequently tested by Italy’s Superior Health Institute (ISS) which used a different set of primers which produced a negative result. Since the samples came from asymptomatic cases, the viral RNA levels would be expected to be near the limits of detection and if two labs used assays with different sensitivities, the more sensitive test would generate weak positives, while the less sensitive test would generate false negatives.
As seen in the comments (in blue) titration of various sets of MERS-CoV primers produce sensitivities which differ by five fold. Thus, the ISS should retest the samples using the primers used by the University of Florence, which would produce confirmatory positives if there wasn’t extensive cross contamination in the positive tests.
Full sequences have been generated for four previously confirmed MERS-CoV cases and partial sequences have been generated for at least four additional cases by sequencing the inserts generated in PCR tests. The sequences have been identical or closely related to the consensus sequence and sequencing of the inserts from the weak positives in Italy would confirm that the weak positives were due to MERS-CoV RNA.
The testing protocol requires positive results in two sets of primers and the likelihood that another coronavirus would generate positives for both sets is near zero. However, testing samples with RNA levels at the limits of detection would generate weak positives for the more sensitive assay and false negatives for the less sensitive assay.
MERS-CoV RNA in samples collected from asymptomatic cases may be detected during a small window of opportunity, and use of the most sensitive sets of primers may be required.
Retesting using the primers which generated the weak positives should be done immediately, and the inserts should be sequenced.