• Clinical Validation of a Sensitive Test for Saliva Collected in Healthcare and Community Settings with Pooling Utility for Severe Acute Respiratory Syndrome Coronavirus 2 Mass Surveillance

      Sahajpal, Nikhil S.; Mondal, Ashis K.; Ananth, Sudha; Njau, Allan; Ahluwalia, Pankaj; Kota, Vamsi; Caspary, Kevin; Ross, Ted M.; Farrell, Michael; Shannon, Michael P.; et al. (Elsevier, 2021-07)
      The clinical performance of saliva compared with nasopharyngeal swabs (NPSs) has shown conflicting results in healthcare and community settings. In the present study, a total of 429 matched NPS and saliva sample pairs, collected in either healthcare or community setting, were evaluated. Phase-1 (protocol U) tested 240 matched NPS and saliva sample pairs; phase 2 (SalivaAll protocol) tested 189 matched NPS and saliva sample pairs, with an additional sample homogenization step before RNA extraction. A total of 85 saliva samples were evaluated with both protocols. In phase-1, 28.3% (68/ 240) samples tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from saliva, NPS, or both. The detection rate from saliva was lower compared with that from NPS samples (50.0% versus 89.7%). In phase-2, 50.2% (95/189) samples tested positive for SARS-CoV-2 from saliva, NPS, or both. The detection rate from saliva was higher compared with that from NPS samples (97.8% versus 78.9%). Of the 85 saliva samples evaluated with both protocols, the detection rate was 100% for samples tested with SalivaAll, and 36.7% with protocol U. The limit of detection with SalivaAll protocol was 20 to 60 copies/mL. The pooled testing approach demonstrated a 95% positive and 100% negative percentage agreement. This protocol for saliva samples results in higher sensitivity compared with NPS samples and breaks the barrier to using pooled saliva for SARS-CoV-2 testing.
    • Clinical validation of innovative, low cost, kit-free, RNA processing protocol for RT-PCR based COVID-19 testing

      Sahajpal, Nikhil Shri; Mondal, Ashis K; Njau, Allan; Ananth, Sudha; Kothandaraman, Arvind; Hegde, Madhuri; Chaubey, Alka; Padala, Sandeep; Kota, Vamsi; Caspary, Kevin; et al. (medRxiv, 2020-07-30)
      The current gold-standard molecular diagnosis for COVID-19 is based on a multi-step assay involving RNA-extraction and RT-PCR analysis for the detection of SARS-CoV-2. RNA34 extraction step has been a major rate-limiting step in implementing high-throughput screening for COVID-19 during this pandemic. Moreover, clinical laboratories are facing several challenges that include cost, reagents, instrumentation, turn-around time, trained personnel, and supply-chain constraints to efficiently implement and sustain testing. Cognizant of these limitations, we evaluated the extraction-free methods described in the literature and have developed an innovative, simplified and easy protocol employing limited reagents to extract RNA for subsequent RT-PCR analysis. Nasopharyngeal-swab samples were subjected to the following individual conditions: 65°C for 15 minutes; 80°C for 5 minutes; 90°C for 5 minutes or 80°C for 1 minute, and processed for direct RT-PCR. These groups were also compared with a supplemental protocol adding isopropanol-ethanol-water elution steps followed by RT-PCR assay. The direct RT-PCR assay did not detect SARS-CoV-2 within the various temperature incubation only groups, whereas, the 90°C for 5 minutes-isopropanol-ethanol-water method was found to be comparable to the FDA-EUA method. Evaluation of the performance metrics for 100 clinical samples demonstrated a sensitivity of 94.2% and a specificity of 100%. The limit of detection was ascertained to be ~40 copies/ml by absolute-quantification. The protocol presented for this assay employs limited reagents and yields results with high sensitivity. Additionally, it presents a simplified methodology that would be easier to implement in laboratories in limited resource countries in order to meet the high current COVID-19 testing needs.