• COVID-19 RT-PCR diagnostic assay sensitivity and SARS-CoV-2 transmission: A missing link?

      Sahahjpal, Nikhil Shri; Hon, Ephrem Chin Lip; Dallaire, Stephanie; Williams, Colin; Ananth, Sudha; Mondal, Ashis K; Rojiani, Amyn M; Hegde, Madhuri; Kolhe, Ravindra; Department of Pathology (medRxiv, 2021-03-26)
      Background The sensitivity of commercially available RT-PCR assays varies over 10,000 fold, ranging from 10 to 20,000 viral copies/ml. The reporting of high Ct value results has been under scrutiny, as the clinical significance of these values is not yet completely understood. The early detection of infected individuals (high Ct results) in the pre-symptomatic phase of the disease using highly sensitive RT-PCR methods has been argued as a strategy to prevent transmission, while on the contrary, the reporting of high Ct has been criticized as false-positive results causing unnecessary testing and having several negative implications. The purpose of this study was to verify the presence of SARS-CoV-2 genomes in samples with a wide range of RT-PCR Ct values including samples with high Ct (37 to 42) using next-generation sequencing (NGS). Methods The study evaluated a total of 547 previously positive samples tested with the PerkinElmer® New Coronavirus Nucleic Acid Detection RT-PCR kit. The samples included in this study ranged from Ct values of 17-42, with 44 samples having a Ct > 37. Of the 547 samples, 149 were sequenced using PerkinElmer NEXTFLEX Variant-Seq SARS-CoV2 assay on NovaSeq 6000, and 398 samples were sequenced using Illumina SARS-CoV-2 respiratory viral panel kits using the NextSeq 500/550 system. Results Between the two clinical laboratories, a total of ~1.95 million samples were tested using the FDAEUA PerkinElmer® New Coronavirus RT-PCR assay. Of the 1.95 million samples, ~1.72 million were negative, ~250,000 positive, and ~16,500 in the range of 37-42. Of the 547 samples sequenced, the percentage of sequencing reads that aligned to the SARS-CoV-2 Wuhan-hu-1 reference genome (NC_045512.2) ranged from 25.5% to 99.69%. All samples sequenced showed high sequence specificity to the SARS-CoV-2 virus. Low Ct samples showed complete uniform coverage across the entire 29kb SAR-CoV-2 genome. The average coverage in samples with high Ct (>37) was found to be 55.5% (range 16.1-99.2%). However, as sample Ct increased, a gradual decrease in coverage uniformity was observed for few samples. Conclusion This study demonstrates for the first time that the viral RNA is present in the high Ct value range of 37- 42 and the sequence is unique to SARS-CoV-2 confirmed using two separate sequencing assays. This confirms that the detected Ct values are reflective of the presence of the SARS-CoV2 virus and they are not an artifact or contamination. In light of the recent work highlighting the majority of transmission being pre-symptomatic/ asymptomatic, and high Ct results being observed at both the early and late phases of infection warrants further investigation into the clinical utility of high Ct results to curtail the spread of the virus.
    • High-Throughput Next-Generation Sequencing Respiratory Viral Panel: A Diagnostic and Epidemiologic Tool for SARS-CoV-2 and Other Viruses

      Sahajpal, Nikhil S.; Mondal, Ashis K.; Njau, Allan; Petty, Zachary; Chen, Jiani; Ananth, Sudha; Ahluwalia, Pankaj; Williams, Colin; Ross, Ted M.; Chaubey, Alka; et al. (MDPI, 2021-10-14)
      Two serious public health challenges have emerged in the current COVID-19 pandemic namely, deficits in SARS-CoV-2 variant monitoring and neglect of other co-circulating respiratory viruses. Additionally, accurate assessment of the evolution, extent, and dynamics of the outbreak is required to understand the transmission of the virus. To address these challenges, we evaluated 533 samples using a high-throughput next-generation sequencing (NGS) respiratory viral panel (RVP) that includes 40 viral pathogens. The performance metrics revealed a PPA, NPA, and accuracy of 95.98%, 85.96%, and 94.4%, respectively. The clade for pangolin lineage B that contains certain distant variants, including P4715L in ORF1ab, Q57H in ORF3a, and S84L in ORF8 covarying with the D614G spike protein mutation, were the most prevalent early in the pandemic in Georgia, USA. The isolates from the same county formed paraphyletic groups, indicating virus transmission between counties. The study demonstrates the clinical and public health utility of the NGS-RVP to identify novel variants that can provide actionable information to prevent or mitigate emerging viral threats and models that provide insights into viral transmission patterns and predict transmission/resurgence of regional outbreaks as well as providing critical information on co-circulating respiratory viruses that might be independent factors contributing to the global disease burden.
    • Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs

      Jones, Les; Bakre, Abhijeet; Naikare, Hemant; Kolhe, Ravindra; Sanchez, Susan; Mosley, Yung-Yi C.; Tripp, Ralph A.; Department of Pathology (Public Library of Science (PLoS), 2021-09-17)
      The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RTLAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.
    • Next-Generation Sequencing (NGS) in COVID-19: A Tool for SARS-CoV-2 Diagnosis, Monitoring New Strains and Phylodynamic Modeling in Molecular Epidemiology

      John, Goldin; Sahajpal, Nikhil Shri; Mondal, Ashis K.; Ananth, Sudha; Williams, Colin; Chaubey, Alka; Rojiani, Amyn M.; Kolhe, Ravindra; Department of Pathology (MDPI, 2021-07-30)
      This review discusses the current testing methodologies for COVID-19 diagnosis and explores next-generation sequencing (NGS) technology for the detection of SARS-CoV-2 and monitoring phylogenetic evolution in the current COVID-19 pandemic. The review addresses the development, fundamentals, assay quality control and bioinformatics processing of the NGS data. This article provides a comprehensive review of the obstacles and opportunities facing the application of NGS technologies for the diagnosis, surveillance, and study of SARS-CoV-2 and other infectious diseases. Further, we have contemplated the opportunities and challenges inherent in the adoption of NGS technology as a diagnostic test with real-world examples of its utility in the fight against COVID-19.