The application of a novel 5-in-1 multiplex RT-PCR assay for rapid detection of SARS-CoV-2 and differentiation between variants of concern.

OBJECTIVES: To rapid detect for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immediately distinguish whether positive samples represent variants of concern (VOCs), we established a novel 5-in-1 VOC assay. METHODS: This assay could distinguish among five VOCs: Alpha, Beta, Gamma, Delta, and Omicron, in a single reaction tube. The five variants exhibit different single nucleotide polymorphisms (SNPs) in their viral genome, which can be exploited to distinguish them. We selected target SNPs in the spike gene, including N501Y, P681R, K417N, and deletion H69/V70 for the assay. RESULTS: The limit of detection of each gene locus was 80 copies per PCR reaction. We observed a high consistency among the results when comparing the performance of our 5-in-1 VOC assay, whole gene sequencing, and the Roche VirSNiP SARS-CoV-2 test in retrospectively analyzing 150 clinical SARS-CoV-2 variant positive samples. The 5-in-1 VOC assay offers an alternative and rapid high-throughput test for most diagnostic laboratories in a flexible sample-to-result platform. CONCLUSION: The assay can also be applied in a commercial platform with completion of the SARS-CoV-2 confirmation test and identification of its variant within 2.5 hours.

Monitoring algorithm of hospitalized patients in a medical center with SARS-CoV-2 (Omicron variant) infection: clinical epidemiological surveillance and immunological assessment.

Purpose: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major healthcare threat worldwide. Since it was first identified in November 2021, the Omicron (B.1.1.529) variant of SARS-CoV-2 has evolved into several lineages, including BA.1, BA.2-BA.4, and BA.5. SARS-CoV-2 variants might increase transmissibility, pathogenicity, and resistance to vaccine-induced immunity. Thus, the epidemiological surveillance of circulating lineages using variant phenotyping is essential. The aim of the current study was to characterize the clinical outcome of Omicron BA.2 infections among hospitalized COVID-19 patients and to perform an immunological assessment of such cases against SARS-CoV-2. Patients and Methods: We evaluated the analytical and clinical performance of the BioIC SARS-CoV-2 immunoglobulin (Ig)M/IgG detection kit, which was used for detecting antibodies against SARS-CoV-2 in 257 patients infected with the Omicron variant. Results: Poor prognosis was noted in 38 patients, including eight deaths in patients characterized by comorbidities predisposing them to severe COVID-19. The variant-of-concern (VOC) typing and serological analysis identified time-dependent epidemic trends of BA.2 variants emerging in the outbreak of the fourth wave in Taiwan. Of the 257 specimens analyzed, 108 (42%) and 24 (9.3%) were positive for anti-N IgM and IgG respectively. Conclusion: The VOC typing of these samples allowed for the identification of epidemic trends by time intervals, including the B.1.1.529 variant replacing the B.1.617.2 variant. Moreover, antibody testing might serve as a complementary method for COVID-19 diagnosis. The combination of serological testing results with the reverse transcription-polymerase chain reaction cycle threshold value has potential value in disease prognosis, thereby aiding in epidemic investigations conducted by clinicians or the healthcare department.

Clinical Comparison of Three Sample-to-Answer Systems for Detecting SARS-CoV-2 in B.1.1.7 Lineage Emergence.

PURPOSE: Accurate molecular diagnostic assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, are needed for epidemiology studies and to support infection-control measures. We evaluated the analytical sensitivity and clinical performance of three sample-to-answer molecular-diagnostics systems for detecting SARS-CoV-2 using 325 nasopharyngeal swab clinical samples from symptomatic patients. METHODS: The BioFire Respiratory Panel 2.1 (RP2.1), cobas Liat SARS-CoV-2 and Influenza A/B, and Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV platforms, which have been granted emergency-use authorization by the US FDA, were tested and compared. RESULTS: The positive percent agreement, negative percent agreement, and overall percent agreement among the three point of care testing systems were 98-100%, including for the wild-type SARS-CoV-2 (non-B.1.1.7) and a variant of concern (B.1.1.7). Notably, the BioFire RP2.1 may fail to detect the SARS-CoV-2 S gene in the B.1.1.7 lineage because of the spike protein mutation. CONCLUSION: All three point of care testing platforms provided highly sensitive, robust, and almost accurate results for rapidly detecting SARS-CoV-2. These automated molecular diagnostic assays can increase the effectiveness of control and prevention measures for infectious diseases.

Novel dual multiplex real-time RT-PCR assays for the rapid detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus using the BD MAX open system.

SARS-CoV-2 has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens, including SARS-CoV-2, in nasopharyngeal specimens from individuals with symptoms of upper respiratory tract infection. We developed and validated a rapid total nucleic acid extraction method based on real-time reverse transcription-polymerase chain reaction (RT-PCR) for the reliable, high-throughput simultaneous detection of common cold viral pathogens using the BD MAX Platform. The system was evaluated using 205 nasopharyngeal swab clinical samples. For assessment of the limit of detection (LoD), we used SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) RNA standards. The BD MAX dual multiplex real-time RT-PCR panel demonstrated a sensitivity comparable to that of the World Health Organization-recommended SARS-CoV-2 assay with an LoD of 50 copies/PCR. The LoD of influenza A/B and RSV was 100-200 copies/PCR. The overall percent agreement between the BD MAX panel and laboratory-developed RT-PCR test on 55 SARS-CoV-2-positive clinical samples was 100%. Among the 55 positive cases of COVID-19 analysed, no coinfection was detected. The BD MAX rapid multiplex PCR provides a highly sensitive, robust, and accurate assay for the rapid detection of SARS-CoV-2, influenza A/B, and RSV.

Novel automated sample-to-result SARS-CoV-2 laboratory-developed RT-PCR assay for high-throughput testing using LabTurbo AIO 48 system.

BACKGROUND AND AIMS: Immediate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for preventing the spread of coronavirus disease 2019 (COVID-19). The LabTurbo AIO 48 system is an automated platform that allows nucleic acid extraction and sample analysis on the same instrument, producing faster results without affecting their accuracy. We aimed to independently evaluate the LabTurbo AIO 48 (all-in-one system) for SARS-CoV-2 detection. MATERIALS AND METHODS: Comparative limit of detection (LOD) was assessed on both the LabTurbo AIO 48 and current standard detection system based on real-time reverse transcriptase polymerase chain reaction (RT-PCR), using SARS-CoV-2 RNA control. Additional 125 primary clinical samples were assessed using both the protocols in parallel. RESULTS: The turnaround time from sample to results for 48 samples analyzed on LabTurbo AIO 48 was approximately 2.5 h, whereas that analyzed using the in-house RT-PCR protocol was 4.8 h. LabTurbo AIO 48 also demonstrated higher sensitivity than our reference RT-PCR assay, with a LOD of 9.4 copies/reaction. The overall percentage agreement between both the methods for 125 samples was 100%. CONCLUSION: LabTurbo AIO 48 is a robust detection option for SARS-CoV-2, allowing faster results and, consequently, aiding in better control and prevention of COVID-19.

Novel rapid identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by real-time RT-PCR using BD Max Open System in Taiwan.

Coronavirus disease 2019 has become a worldwide pandemic. By April 7, 2020, approximately 1,279,722 confirmed cases were reported worldwide including those in Asia, European Region, African Region and Region of the Americas. Rapid and accurate detection of Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) is critical for patient care and implementing public health measures to control the spread of infection. In this study, we developed and validated a rapid total nucleic acid extraction method based on real-time RT-PCR for reliable, high-throughput identification of SARS-CoV-2 using the BD MAX platform. For clinical validation, 300 throat swab and 100 sputum clinical samples were examined by both the BD MAX platform and in-house real-time RT-PCR methods, which showed 100% concordant results. This BD MAX protocol is fully automated and the turnaround time from sample to results is approximately 2.5 h for 24 samples compared to 4.8 h by in-house real-time RT-PCR. Our developed BD MAX RT-PCR assay can accurately identify SARS-CoV-2 infection and shorten the turnaround time to increase the effectiveness of control and prevention measures for this emerging infectious disease.