Reliability of RT-PCR tests to detect SARS-CoV-2: risk analysis

The rapid escalation of the number of COVID-19 (Coronavirus Disease 2019) cases has forced countries around the world to implement systems for the widest possible testing of their populations. The World Health Organization (WHO) has in fact urged all countries to carry out as many tests as they can. Clinical laboratories have had to respond urgently to numerous and rising demands for diagnostic tests for SARS-CoV-2. The majority of laboratories have had to implement the RT-PCR (Reverse Transcriptase − Polymerase Chain Reaction) test method without the benefit of adequate experimental feedback. It is hoped that this article will make a useful contribution in the form of a methodology for the risk analysis of SARS-CoV-2 testing by RT-PCR and at the same time result reliability analysis of diagnostic tests, via an approach based on a combination of Fishbone Diagram and FMECA (Failure Mode, Effects, and Criticality Analysis) methods. The risk analysis is based on lessons learned from the actual experience of a real laboratory, which enabled the authors to pinpoint the principal risks that impact the reliability of RT-PCR test results. The probability of obtaining erroneous results (false positives or negatives) is implicit in the criticality assessment obtained via FMECA. In other words, the higher the criticality, the higher the risk of obtaining an erroneous result. These risks must therefore be controlled as a priority. The principal risks are studied for the following process stages: nucleic acid extraction, preparation of the mix and validation of results. For the extraction of nucleic acids, highly critical risks (exceeding the threshold set from experimentation) are the risk of error when depositing samples on the extraction plate and sample non-conformity. For the preparation of the mix the highest risks are a non-homogenous mix and, predominantly, errors when depositing samples on the amplification plate. For the validation of results, criticality can reach the maximum severity rating: here, the risks that require particular attention concern the interpretation of raw test data, poor IQC (Internal Quality Control) management and the manual entry of results and/or file numbers. Recommendations are therefore made with regard to human factor influences, internal contamination within the laboratory, management of reagents, other consumables and critical equipment, and the effect of sample quality. This article demonstrates the necessity to monitor, both internally and externally, the performance of the test process within a clinical laboratory in terms of quality and reliability.