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. 2021 Sep:225:153579.
doi: 10.1016/j.prp.2021.153579. Epub 2021 Aug 4.

SARS-CoV-2 detection by reverse transcriptase polymerase chain reaction testing: Analysis of false positive results and recommendations for quality control measures

Lester J Layfield  1 , Simone Camp  2 , Kelly Bowers  2 , Douglas C Miller  2
Affiliations

SARS-CoV-2 detection by reverse transcriptase polymerase chain reaction testing: Analysis of false positive results and recommendations for quality control measures

Lester J Layfield et al. Pathol Res Pract. 2021 Sep.

Abstract

Testing for SARS-CoV-2 has become a critical component for the management of the COVID-19 pandemic. Reverse transcriptase polymerase chain reaction (RT-PCR) assays are currently the predominate method for testing. Quality control (QC) measures utilize known positive and known negative controls to ensure the adequacy of extraction and RT-PCR steps but do not evaluate all components of testing. We have conducted a quality assurance review of our RT-PCR testing for COVID-19 to determine the rate of false positive results in asymptomatic patients and causes for these errors.

Design: We have developed a quality control procedure in which all specimens from asymptomatic unexposed persons with SARS-CoV-2 positive tests were retested. When a second test was "non-detected" a third test was performed and a root cause analysis of the erroneous result undertaken.

Results: In the study period, 24,717 samples were tested and 6251 were from asymptomatic patients. Of the 288 initial positive tests, 20 (6.9%) were negative on retesting. Review of cycle threshold curves, technologists' records, location of specimen on testing plates and relationships with high viral load specimens was undertaken. Analysis revealed technologists' errors (misplacement of specimen in testing plate or contamination) and cross contamination from high viral load specimens in adjacent wells of testing plates were common causes for false positive results.

Discussion: SARS-CoV-2 RT-PCR testing is associated with a small number of false positive results, most easily recognized in asymptomatic non-exposed patients. Implementation of a limited retesting protocol identifies clinically significant testing errors and allows review and improvement of laboratory procedures.

Keywords: COVID-19; False positive results; Polymerase chain reaction; Quality control; SARS-CoV-2.

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Figures

Fig. 1
Fig. 1
Protocol for Reporting positive PCR Results for SARS-CoV-2 Testing in asymptomatic and non-exposed patients.
Fig. 2
Fig. 2
Root cause analysis for false positive results.
See this image and copyright information in PMC

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