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Background: We identified two cases of Omicron BA.5.2.1 that exhibited characteristics of open-reading frame 1a (ORF1a) gene target failure (OGTF) in RNA detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The purpose of this study was to determine the cause of the OGTF in the two samples., Method: Samples were specimens obtained from a 37-year-old male and a 43-year-old female who were suspected to have Coronavirus disease 2019. SARS-CoV-2 RNA detection using μTAS Wako g1 system (FUJIFILM Wako Pure Chemical, Osaka, Japan) was performed and it detected S gene but did not detect ORF1a gene., Results: The results of whole genome sequencing of these samples showed that SARS-CoV-2 genome in these two samples matched to Omicron BA.5.2.1. SARS-CoV-2 genomes in the two samples had a deletion of 15 bases and a deletion of 9 bases, so that they overlapped with the 3'-terminal side of the primer binding region in ORF1a gene, and these deletions were considered to be the cause of OGTF., Conclusions: Since such a gene target failure due to base deletion is not considered rare, it was considered desirable to implement a multi-target detection in SARS-CoV-2 RNA molecular diagnostic system to address the mutation or deletion of the viral gene.

Background: Nucleic acid amplification testing is the gold standard for SARS-CoV-2 diagnostics, although it may produce a certain number of false positive results. There has not been much published about the characteristics of false positive results. In this study, based on retesting, specimens that initially tested positive for SARS-CoV-2 were classified as true or false positive groups to characterize the distribution of cycle threshold (CT) values for N1 and N2 targets and number of targets detected for each group., Methods: Specimens that were positive for N-gene on retesting and accompanied with S-gene were identified as true positives (true positive based on retesting, rTP), while specimens that retested negative were classified as false positives (false positive based on retesting, rFP)., Results: Of the specimens retested, 85/127 (66.9%) were rFP, 16/47 (34.0%) specimens with both N1 and N2 targets initially detected were rFP, and the CT values for each target was higher in rFP than in rTP. ROC curve analysis showed that optimal cutoff values of CT to differentiate between rTP and rFP were 34.8 for N1 and 33.0 for N2. With the optimal cutoff values of CT for each target, out of the 24 specimens that were positive for both N1 and N2 targets and classified as rTP, 23 (95.8%) were correctly identified as true positives. rFP specimens had a single N1 target in 52/61 (85.2%) and a single N2 target in 17/19 (89.5%). Notably, no true positive results were obtained from any specimens with only N2 target detected., Conclusions: These results suggest that retesting should be performed for positive results with a CT value greater than optimal cutoff value for each target or with a single N1 target amplified, considering the possibility of a false positive. This may provide guidance on indications to perform retesting to minimize the number of false positives.