Dataset Information

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

ABSTRACT:

Background

Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings.

Methods

We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C.

Results

SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions.

Conclusion

eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

SUBMITTER: Banik S

PROVIDER: S-EPMC8195355 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Saliva is a promising alternative for a nasopharyngeal swab (NPS) in specimen collection to detect SARS-CoV-2. We compared the diagnostic performance and tolerability of saliva collection versus NPS in a clinical setting. Paired NPS and saliva specimens were collected sequentially from participants (n = 250) at the Turku University Hospital drive-in coronavirus testing station in the spring of 2022, with Omicron BA.2 as the dominant SARS-CoV-2 variant. Discomfort and preference for the sampling method were assessed. The specimens were analyzed for SARS-CoV-2 using real-time multiplex reverse transcriptase PCR (RT-PCR) with a laboratory-developed test (LDT) and two commercial kits (PerkinElmer SARS-CoV-2 and PerkinElmer SARS-CoV-2 Plus) for several target genes. Among the 250 participants, 246 had respiratory symptoms. With LDT, SARS-CoV-2 was detected in 135 and 134 participants from NPS and saliva, respectively. Of the 250 specimens, 11 gave a discordant outcome, resulting in excellent agreement between the specimen types (Cohen's kappa coefficient of 0.911; P = 0.763). The cycle threshold (CT) values of LDT and commercial kit target genes were significantly lower from NPS than from saliva. A total of 172 (69%) participants assessed saliva sampling as more tolerable than NPS (P < 0.0001). Our findings present saliva as an applicable alternative for SARS-CoV-2 diagnostics. However, the lower CT values obtained from NPS indicate that NPS may be a slightly more sensitive specimen type. Participants preferred saliva sampling, although delivering an adequate volume of saliva was challenging for some participants. IMPORTANCE The extensive testing of SARS-CoV-2 is vital in controlling the spread of COVID-19. The reference standard for specimen collection is a nasopharyngeal swab (NPS). However, the discomfort of NPS sampling, the risk of nosocomial infections, and global material shortages have accelerated the development of alternative testing methods. Our study demonstrates that patients tolerate saliva sampling better than NPS. Of importance, although the RT-PCR qualitative test results seem to correspond between NPS and saliva, we show significantly lower CT values for NPS, compared to saliva, thus contradicting the suggested superiority of the saliva specimen over NPS in the detection of the Omicron variants of SARS-CoV-2. Future research is still required to enable individual planning for specimen collection and to determine the effects of different SARS-CoV-2 variants on the sensitivity of the saliva matrix.