RT-PCR/MALDI-TOF Diagnostic Target Performance Reflects Circulating SARS-CoV-2 Variant Diversity in New York City.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern have emerged. New variants pose challenges for diagnostic platforms because sequence diversity can alter primer/probe-binding sites (PBSs), causing false-negative results. The MassARRAY SARS-CoV-2 Panel (Agena Bioscience) uses RT-PCR and mass spectrometry to detect five multiplex targets across N and ORF1ab genes. Herein, we use a data set of 256 SARS-CoV-2-positive specimens collected between April 11, 2021, and August 28, 2021, to evaluate target performance with paired sequencing data. During this time frame, two targets in the N gene (N2 and N3) were subject to the greatest sequence diversity. In specimens with N3 dropout, 69% harbored the Alpha-specific A28095U polymorphism that introduces a 3'-mismatch to the N3 forward PBS and increases risk of target dropout relative to specimens with 28095A (relative risk, 20.02; 95% CI, 11.36 to 35.72; P < 0.0001). Furthermore, among specimens with N2 dropout, 90% harbored the Delta-specific G28916U polymorphism that creates a 3'-mismatch to the N2 probe PBS and increases target dropout risk (relative risk, 11.92; 95% CI, 8.17 to 14.06; P < 0.0001). These findings highlight the robust capability of MassARRAY SARS-CoV-2 Panel target results to reveal circulating virus diversity, and they underscore the power of multitarget design to capture variants of concern.

Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multitarget approach.

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

Food for thought: Eating before saliva collection and interference with SARS-CoV-2 detection.

Saliva is a promising specimen for the detection of viruses that cause upper respiratory infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to its cost-effectiveness and noninvasive collection. However, together with intrinsic enzymes and oral microbiota, children's unique dietary habits may introduce substances that interfere with diagnostic testing. To determine whether children's dietary choices impact SARS-CoV-2 molecular detection in saliva, we performed a diagnostic study that simulates testing of real-life specimens provided from healthy children (n = 5) who self-collected saliva at home before and at 0, 20, and 60 min after eating 20 foods they selected. Each of 72 specimens was split into two volumes and spiked with SARS-CoV-2-negative or SARS-CoV-2-positive clinical standards before side-by-side testing by reverse-transcription polymerase chain reaction matrix-assisted laser desorption ionization time-of-flight (RT-PCR/MALDI-TOF) assay. Detection of internal extraction control and SARS-CoV-2 nucleic acids was reduced in replicates of saliva collected at 0 min after eating 11 of 20 foods. Interference resolved at 20 and 60 min after eating all foods except hot dogs in one participant. This represented a significant improvement in the detection of nucleic acids compared to saliva collected at 0 min after eating (p = 0.0005). We demonstrate successful detection of viral nucleic acids in saliva self-collected by children before and after eating a variety of foods. Fasting is not required before saliva collection for SARS-CoV-2 testing by RT-PCR/MALDI-TOF, but waiting for 20 min after eating is sufficient for accurate testing. These findings should be considered for SARS-CoV-2 testing and broader viral diagnostics in saliva specimens.