Clinical validation of a multiplex PCR-based detection assay using saliva or nasopharyngeal samples for SARS-Cov-2, influenza A and B.

The COVID-19 pandemic has resulted in significant diversion of human and material resources to COVID-19 diagnostics, to the extent that influenza viruses and co-infection in COVID-19 patients remains undocumented and pose serious public-health consequences. We optimized and validated a highly sensitive RT-PCR based multiplex-assay for the detection of SARS-CoV-2, influenza A and B viruses in a single-test. This study evaluated clinical specimens (n = 1411), 1019 saliva and 392 nasopharyngeal swab (NPS), tested using two-assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp-RT-PCR based assay that targets SARS-CoV-2, influenza viruses A and B. Of the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. This study presents clinical validation of a multiplex-PCR assay for testing SARS-CoV-2, influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow.

Making a Difference: Adaptation of the Clinical Laboratory in Response to the Rapidly Evolving COVID-19 Pandemic.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, led to unprecedented demands assigned to clinical diagnostic laboratories worldwide, forcing them to make significant changes to their regular workflow as they adapted to new diagnostic tests and sample volumes. Herein, we summarize the modifications/adaptation the laboratory had to exercise to cope with rapidly evolving situations in the current pandemic. In the first phase of the pandemic, the laboratory validated 2 reverse transcription polymerase chain reaction-based assays to test ∼1000 samples/day and rapidly modified procedures and validated various preanalytical and analytical steps to overcome the supply chain constraints that would have otherwise derailed testing efforts. Further, the pooling strategy was validated for wide-scale population screening using nasopharyngeal swab samples and saliva samples. The translational research arm of the laboratory pursued several initiatives to understand the variable clinical manifestations that this virus presented in the population. The phylogenetic evolution of the virus was investigated using next-generation sequencing technology. The laboratory has initiated the formation of a consortium that includes groups investigating genomes at the level of large structural variants, using genome optical mapping via this collaborative global effort. This article summarizes our journey as the laboratory has sought to adapt and continue to positively contribute to the unprecedented demands and challenges of this rapidly evolving pandemic.

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.

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.

SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance.

OBJECTIVES: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. METHODS: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. RESULTS: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60-180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. CONCLUSION: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints.

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

Proposal of RT-PCR-Based Mass Population Screening for Severe Acute Respiratory Syndrome Coronavirus 2 (Coronavirus Disease 2019).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing has lagged in many countries because of test kit shortages and analytical process bottlenecks. This study investigated the feasibility and accuracy of a sample pooling approach for wide-scale population screening for coronavirus disease 2019. A total of 940 nasopharyngeal swab samples (934 negative and 6 positive) previously tested for SARS-CoV-2 were deidentified and assigned random numbers for analysis, and 94 pools of 10 samples each were generated. Automated RNA extraction, followed by RT-PCR, was performed in a 96-well plate. Positive pools were identified, and the individual samples were reanalyzed. Of the 94 pools/wells, four were positive [Ct values: N (22.7 to 28.3), ORF1ab (23.3 to 27.2), and internal control (34.4 to 35.4)]. The 40 samples comprising the four pools were identified and reanalyzed individually; six samples were positive, with Ct values of N gene, ORF1ab, and internal control comparable to their respective wells. Additional experiments were performed on samples with high Ct values, and overall results showed 91.6% positive and 100% negative agreement compared with individual testing approach. Thus, 940 samples were tested in 148 reactions compared with 940 reactions in routine screening. The sample pooling strategy may help catch up with testing needs and minimal turnaround times and facilitate enormous savings on laboratory supplies, extraction, and PCR kits currently in short supply.