A-294 Duplicate Testing of Samples Suspected of SARS-CoV-2 Infection Could Prevent Misdiagnosis of Covid-19
Authors: Sánchez-Sánchez J , Thapa G , Mylona A , Vuono R 
Published: September 2023

Abstract

Background
There is no doubt that the whole world has been shaken by the SARS-CoV-2 pandemic, which has affected both global health and the global economy. At the same time the importance of molecular methods for the accurate diagnosis of respiratory infections has been reinforced together with the need of testing methodologies that minimize the risk of misleading results. During the last three years of the pandemic, SARS-CoV-2 exhibited a tremendous adaptation capability through numerous mutations, leading to the appearance of the well-known Variants of Concern (VOCs) responsible of increased transmission rates. Therefore, the development of molecular methods capable of detecting all variants has become a challenge. To overcome this, potential genetic targets with low mutation rates have been identified, including ORF1ab, N gene, S gene and E gene. While Real-time PCR based detection assays of viral genomes are very accurate, testing naso/oropharyngeal samples using singlet analysis does not guarantee the error free detection of true positive results for SARS-CoV-2 infections. Furthermore, it has been documented that the probability of a false-negative testing results is influenced by a number of variables . Therefore, testing protocol adaptations, such as replicate testing, has been shown to efficiently reduce the probability of missing SARS-CoV-2 positive cases. The pilot study presented here investigates the outcome of testing 10 014 nasopharyngeal samples collected from patients with a suspected SARS-CoV-2 infection using two separate detection kits. In this study the error rate of single vs duplicate testing is discussed.

Methods
10 014 nasopharyngeal swabs, collected from three different U.K. hospital trusts and Kent based care homes, were tested at the North Kent Pathology Service (NKPS). Each swab collected was placed in sterile transport media and stored at 4°C prior to viral genome isolation using the HigherPurity™ Viral RNA extraction kit. All collected specimens were processed within 24 h from collection. Total RNA isolated was immediately analyzed using two commercial CE-IVD kits: Vitassay qPCR SARS-CoV-2 and Primerdesign™ LTD, Coronavirus (COVID-19) Genesig Real-Time PCR assay. All samples were tested in duplicate.

Results
Following the testing and subsequent analysis, 491 samples (4.9%) were detected positive for SARS-CoV-2 using both assay kits. Of these, 302 samples (61.5%) were reported as duplicate positive (on both replicate 1 and 2) whereas 189 samples (38.5%) were detected positive on either replicate 1 or 2. In particular, 85 (17.3%) were detected positive on replicate 1 and 104 (21.2%) were detected positive on replicate 2.

Conclusions
Approximately 19% of tested individuals were detected as SARS-CoV-2 positive based on a positive Real-time PCR result in one of the two reactions. This percentage represents the potential SARS-CoV-2 infections that can be missed when a single reaction Real-time PCR testing is employed for infection detection in the wider population, and equates to 940 individuals out of 100 000 potentially being falsely confirmed as SARS-CoV-2 negative The authors believe that in the presence of fast transmitting variants this is a significant number of false negative tests and should be taken into consideration when testing protocols are developed and disease modelling is employed.