key: cord-0973941-ksj377th authors: Falasca, Francesca; Sciandra, Ilaria; Di Carlo, Daniele; Gentile, Massimo; Deales, Alberto; Antonelli, Guido; Turriziani, Ombretta title: Detection of SARS-COV N2 Gene: Very low amounts of viral RNA or false positive? date: 2020-10-14 journal: J Clin Virol DOI: 10.1016/j.jcv.2020.104660 sha: c4fac10a2fc92b3bece543fb65fee69d969f6b32 doc_id: 973941 cord_uid: ksj377th BACKGROUND: The detection of a low amount of viral RNA is crucial to identify a SARS-CoV-2 positive individual harboring a low level of virus, especially during the convalescent period. However, the detection of one gene at high Cycle threshold (Ct) has to be interpreted with caution. In this study we address this specific issue and report our real-life experience. STUDY DESIGN: A total of 1639 nasopharyngeal swabs (NPS) were analyzed with Xpert® Xpress SARS-CoV-2. Positive samples showing high Ct values (Ct>35) were concentrated by centrifugation and re-tested with Cepheid or other methods (RealStar SARS-CoV2 RT-PCR, Altona Diagnostics; GeneFinder COVID-19 Plus RealAmp Kit, Elitech). RESULTS: 1599 (97.5%) negative samples, 36 (2.3%) positive samples and 4 (0.2%) presumptive positive samples were detected. In 17 out of 36 positive patients, very low viral RNA copies were suspected since positivity was detected at high Ct. We confirmed positivity for patients who showed both E and N genes detected and for patients with only N detected but with Ct <39. On the contrary, samples with only gene N detected with Ct values >39 were found negative. NPS taken 24 hours after the first collection confirmed the negativity of the 12 samples. Clinical data sustained these results since only 2 of these 12 patients showed COVID-19-like symptoms. CONCLUSIONS: These data support our consideration that detection of the N2 gene at high Ct needs to be interpreted with caution, suggesting that collaboration between virologists and clinicians is important for better understanding of results. The timely diagnosis of COVID-19 cases and subsequent infection control are essential to prevent transmission in healthcare facilities and the community. Rapid SARS-CoV-2 testing, and particularly molecular assays, can have a considerable impact on the ability to make immediate decisions regarding management of the infected patient, including his isolation or the assessment of risk of transmission to healthcare workers performing invasive procedures on critically ill patients. So far, nucleic acid amplification testing is still the gold standard for the diagnosis of SARS-CoV-2 in respiratory samples [1, 2] . Despite the good performance of validated nucleic acid amplification assays, a risk of false-negative results still exists. The negativity of the assay may be due to inappropriate sample collection as well as to extraction/Real Time-PCR workflow and to sensitivity of the assays used. Concerning the latter, it is worth noting that the ability of molecular assays to detect SARS-CoV-2 infection can be limited by low amounts of viral RNA (e.g., early or late in COVID-19 disease). Xpert® Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA) is a rapid molecular diagnostic test utilizing real time RT-PCR technology to detect the nucleocapsid gene (N2 region of the N-gene) and envelope gene (E) in respiratory specimens. The limit of detection (LoD) was reported at 100 copies/ml. Studies have assessed the N2 target's high specificity and sensitivity for SARS-CoV-2 detection, and cases testing positive with only the N2 region have often been observed in samples containing very low viral RNA copies [3] . The detection of a low amount of viral RNA is crucial to identify a positive individual harboring a low level of virus, especially during the convalescent period. However, the detection of one gene at high Cycle threshold (Ct) introduces problems of interpretation, and the results should be handled with caution. In this study we address this specific issue and report our real-life experience on the above assay A total of 1639 nasopharyngeal swabs (NPS) were analyzed with Xpert® Xpress SARS-CoV-2 (Cepheid). The Xpert Xpress SARS-CoV-2 test provides positive results when a signal for the N2 region or signals for both nucleic acid targets (N2 and E) have a Ct within the valid range (<45 Ct) and endpoint above the minimum setting. A presumptive positive result is given when the SARS-CoV-2 signal for only the E nucleic acid target has been detected. Positive samples showing high Ct values (Ct>35) were concentrated by centrifugation (2 hours at 14000 rpm at 4°C) and re-tested with Cepheid or other methods (RealStar SARS-CoV2 RT-PCR, Altona Diagnostics; GeneFinder COVID-19 Plus RealAmp Kit, Elitech). A total of 1639 NPS were analyzed with Xpert® Xpress SARS-CoV-2 from 01 April 2020 to 31 July 2020. Surprisingly, we confirmed positivity for Pt 2, Pt 9 and Pt 16, which showed both E and N genes detected and for patients with only N detected but with Ct <39 (Pt 7 and Pt 8). On the contrary, samples with only gene N detected with Ct values >39 were found negative. NPS taken 24 hours after the first collection confirmed the negativity of the 12 samples. Notably, only 2 of these 12 patients were admitted to hospital with COVID-19-like symptoms; the rest were screened preoperatively or prior to admission to hospital wings, as recommended in order to identify possible asymptomatic infections. The qualitative real time PCR (qRT-PCR) assay is being widely used for detection of SARS-CoV-2 infection. However, the problem of qRT-PCR with inaccurate results is increasingly reported. Studies have shown that a significant number of SARS-CoV-2 false negative samples is inevitably due to low viral load in patients' throats and to the limited sensitivity of PCR technology. PCR is easily affected by sample inhibitors, poor amplification efficiency, and less precision in low-concentration samples [4] . False negative results may compromise the timely diagnosis, early treatment, prevention of transmission, and assessment of discharge criteria [5] . For all these reasons, several publications have focused their attention on false negative results [6, 7] . However, false positive results have been observed, even if less frequently, due to contaminations of commercial primers/probe sets or poor test specificity [8, 9] . These data support our consideration that detection of the N2 gene at high Ct needs to be interpreted with caution and highlight the importance of virologist-clinician collaboration for better understanding of results. Laboratory diagnosis of emerging human coronavirus infectionsthe state of the art An assessment of realtime RT-PCR kits for SARS-CoV-2 detection ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens Real-time RT-PCR in COVID-19 detection: issues affecting the results Sources of pre-analytical, analytical and post-analytical errors in the microbiology laboratory Variation in false negative rate of reverse transcriptase polymerase chain reaction-based SARS-CoV-2 tests by time since exposure Interpret with caution: An evaluation of the commercial AusDiagnostics versus in-house developed assays for the detection of SARS-CoV-2 virus Pitfalls in SARS-CoV-2 PCR diagnostics False-positive reverse transcriptase polymerase chain reaction screening for SARS-CoV-2 in the setting of urgent head and neck surgery and otolaryngologic emergencies during the pandemic: Clinical implications Authorship contribution statement FF and IS design the study, interpreted data and drafted the article.