False positive results in real-time reverse transcription-polymerase chain reaction (rRT-PCR) for SARS-CoV-2? [25/01/22]

The following post was written to update a previous Short from 2020. Please see the past Short on this topic here.

There are multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergency use authorization (EUA) tests among clinical laboratories and a large amount of cross-assay variation between different assays. Moreover, these assays were rapidly developed, minimally standardized and there is no well recognized external quality assessment program (EQA). As a result, good estimates of the diagnostic sensitivity and specificity are not available. There has been more focus on the diagnostic sensitivity and it is known that sensitivity is poor if the test is performed too early and that the viral RNA may be detectable for a period of time after active infection although the virus is no longer viable. Therefore, the CDC does not recommend PCR retesting after recovery, but CDC suggests symptomless persons who are immunologically normal are no longer infectious about 10 days after symptom onset, although very recently, in more complicated guidelines, CDC has shortened the recommended time for isolation and quarantine to 5 days, with an additional 5 days of mask wearing in most cases and less sensitive protein antigen testing in some cases (1).

There is less information about diagnostic specificity (false positives). Among others, false positives will depend on the length of the DNA probes, how many and which genes are measured and technical errors. The DNA probes used in the CDC rRT-PCR test kits for SARS-CoV-2 assay are only about 25 bases long which does not meet the FDA recommendation for nucleic acid-based molecular diagnostics for viral disease infections where 100 contiguous bases is desirable (2). Various methods use different genes and different probes that may not be equivalent. There is a 100-fold difference in limit of detection (LoD) between some assays (3). Technical error, especially due to DNA contamination, may cause false positives. Seventy-seven professional baseball major league players initially tested positive in one lab but negative elsewhere (4) in what was deemed Lab error. Except that they had multiple sources for testing, they might have been classified as asymptomatic. Many other persons may have been classified in error from this incident. Moreover, false positive PCR results have been reported in the literature. For example a patient who presented with history of fever with dry cough, and body pain who had been in contact with a COVID-19 patient and tested positive for SARS-CoV-2 by PCR was found to be negative for SARS-CoV-2 IgG antibody with repeated testing (5). It is possible that this patient was infected with influenza. Thus, CDC now encourages laboratories to consider adoption of a multiplexed method that can facilitate detection and differentiation of SARS-CoV-2 and influenza viruses (6).

Originally, PCR was followed by a second step where a separation technique was used to confirm that the amplified substance was correct. rRT-PCR is usually not followed by a second step. RRT-PCR is usually applied for diagnostic purposes, not for screening. For acute viral infections, after symptoms appear, a rRT-PCR test battery may be performed. In diagnostic testing, symptoms or high-risk behavior cause an increase in prevalence because those with certain symptoms are classified into characterized groups and false positives are few.

Diagnostic applications are usually applied for chronic viral infections such as HCV, HIV and chronic HBV where symptoms or high-risk behavior initiates testing. In all these chronic diseases antibody concentrations are high and serology usually precedes rRT-PCR, so that false positives are rare. COVID-19 testing is primarily without confirmation.

For SARS-CoV-2 rRT-PCR, cycle threshold (Ct) of 24 or less has been shown to be highly predictable for identifying active COVID-19 cases (7), but since LoD of various methods drastically differ it is unclear which methods this applies to. Generally, methods do not amplify more than 40 cycles, but some systems go beyond 40 Ct. It seems likely that short probes in such systems could lead to amplification errors. There is one report (8), of EQA in clinical laboratories for other RNA virus. The authors compiled 43 EQAs of rRT-PCR assays, conducted between 2004-2019. Three hundred and thirty-six of 10,538 negative samples (3.2%) were reported as positive. The authors defined the lowest percentage of the interquartile range which was 0.8% as a conservative estimate of the false positive rate. In another report, Sin Hang Lee found that 3 of 10 positive proficiency samples in the State of Connecticut were negative containing no SARS-CoV-2 RNA by a confirmatory assay (2). The Foundation for Innovative New Diagnostics (FIND) examined 22 rRT-SARS-CoV-2 diagnostic tests (6) and found diagnostic specificities ranging between 100% and 96% for 100 specimens assayed by each test. Although the great majority showed 100% specificity, given the small number assayed, the lower 95% confidence limit which was 95% for almost all assays would seem to be a better estimate (possible 5% error). Moreover, these were tested under controlled conditions, not similar to high output clinical laboratories running thousands of tests.

Read More – False positive results in real-time reverse transcription-polymerase chain reaction (rRT-PCR) for SARS-CoV-2? [25/01/22]