key: cord-0757644-otew0gcz
authors: Daum, Luke T; Fischer, Gerald W
title: Rapid and Safe Detection of SARS-CoV-2 and Influenza Virus RNA using Onsite qPCR Diagnostic Testing from Clinical Specimens Collected in Molecular Transport Medium
date: 2021-06-22
journal: J Appl Lab Med
DOI: 10.1093/jalm/jfab073
sha: 824cdef21fc543a53d4fb9d8811950778c317942
doc_id: 757644
cord_uid: otew0gcz

BACKGROUND: The ability to rapidly detect SARS-CoV-2 and influenza virus infection is vital for patient care due to overlap in clinical symptoms. Roche’s cobas® Liat(®) SARS-CoV-2 & Influenza A/B Nucleic Acid Test used on the cobas(®) Liat(®) was granted approval under FDA’s Emergency Use Authorization (EUA) for nasopharyngeal (NP) and nasal swabs collected in viral/universal transport medium (VTM/UTM). However, there is a critical need for media that inactivates the virus, especially when specimens are collected in decentralized settings. This study aimed to investigate the use of PrimeStore Molecular Transport Medium(®) (PS-MTM(®)), designed to inactivate/kill and stabilize RNA/DNA for ambient transport and pre-processing of collected samples. METHODS: A limit of detection (LOD) using serially diluted SARS-CoV-2 RNA in PS-MTM(®) and routine UTM was established using standard qPCR. Additionally, a clinical panel of NP and oral swabs collected in PS-MTM(®) collected during the 2020 coronavirus disease 2019 (COVID-19) pandemic were evaluated on the cobas(®) Liat(®) and compared to ‘gold standard’ qPCR on an ABI-7500 instrument. RESULTS: SARS-CoV-2 RNA LOD using standard qPCR was equivalent on the cobas(®) Liat(®) instrument. cobas(®) Liat(®) detection from oral/NP swabs in PS-MTM(®) media exhibited equivalent positive percent agreement (100%) and negative percent agreement (96.4%). CONCLUSION: PS-MTM(®) and the Roche cobas(®) Liat(®) are compatible and complimentary devices for respiratory specimen collection and rapid disease detection, respectively. PS-MTM(®) is equivalent to standard VTM/UTM with the added benefit of safe, non-infectious sample processing for near-patient testing.

As of: 5/10/2021 amplification (~20-minute run time) from nasopharyngeal (NP) or nasal swabs collected in 103 commercial viral or universal transport mediums (VTM and UTM) [6] [7] [8] . 104

For routine respiratory collection and testing, most laboratories collect swabs (NP, nasal, 105 or throat swabs) in VTM and UTM manufactured by Copan, Becton Dickinson and Thermo 106

Fisher. The various media recipes were designed and patented in the 1990's for the purpose 107 of maintaining microbial integrity until collected specimens can be cultured at reference 108 labs 9 . However, in the late 1990s, many laboratories began to transition from detection 109 solely by culture to molecular-based approaches including quantitative polymerase chain 110 reaction (qPCR). Many diagnostic labs, including those testing SARS-CoV-2, exclusively 111 employ qPCR due to increased sensitivity over culture 10 . However, some reagents in 112 VTM/UTM intended to maintain microbial viability, i.e., gelatin and BSA (Daum et al., 113 unpublished) may inhibit or reduce qPCR cycle threshold (CT) values when co-extracted 114 during nucleic acid extraction amplification. Thus, most diagnostic testing manufacturers 115 list specific VTM/UTM products on the package insert to ensure that test performance is not 116 altered based on the media used for specimen collection. 117

PrimeStore Molecular Transport Medium ® (PS-MTM ® ) is a microbial nucleic acid storage 118 and transport device cleared in 2018 by the U.S. Food and Drug Administration (US FDA) 11 . 119 PS-MTM ® is indicated for rapid killing/inactivation of viruses (including Influenza), and 120 bacteria (including Mycobacterium tuberculosis) or other respiratory pathogens, i.e., SARS-121 CoV-2 virus within a collected respiratory sample 11-13 . Importantly, RNA and DNA from 122 collected samples are subsequently stabilized and preserved to provide safer and more 123 efficient workflow for automated extraction, qPCR and sequencing 14-16 . 124

As of: 5/10/2021 PS-MTM ® functions by disrupting and shearing lipid membranes, inactivating cellular 125 nucleases, and preserving released genetic material--RNA and DNA--at ambient temperature 126 or higher for extended periods. The nucleic acids from patient samples collected in PS-127 MTM ® do not require cold-chain and microbes are lysed (viruses in >5 minutes, bacteria in 128 >30 minutes) for safe shipping and transport using standard delivery 11-14 . Importantly, the 129 majority of collected samples do not require processing in BLS-II or III facilities. PS-MTM ® 130 is compatible with most commercial extraction kits, e.g., Roche's MagNA Pure, Qiagen kits 14-131 17 . The collection medium is suited for respiratory specimens collected/tested at POC 132 diagnostic centers and has added utility for field collection in remote areas, triage centers, 133

and border crossings where cold-chain, transport, and dissemination of potentially 134 infectious pathogens are a concern 18-19 . During the 2020-21 COVID-19 pandemic, more than 135 For this experiment, triplicate reactions of each 10-fold reduction (e.g., 1,000 copies/µL to 1 189 copy/µL) were evaluated. Results

Limit of Detection (LOD) 208 209

Using the CDC's N2 assay, a qPCR LOD was performed to establish equivalency using 210 UTM-RT medium at 1 copy/mL. For these dilutions, the mean CT value according to qPCR 223 using the CDC's N2 assay was 39.2 and 39.6, respectively. The average CT value at each 10-224

As of: 5/10/2021 fold SARS-CoV-2 RNA dilution was lower (i.e., more targets detected) for PS-MTM ® samples; 225 however, no significant difference was noted in triplicate samples analyzed. 226 there is limited data correlating CT value with transmission events versus residual nucleic 274 acid from a previous infection. This is an area that will need to be explored further but is 275 out of the scope of this study. In this retrospective clinical study, the Liat ® test-failure rate 276 was 2% (two tests). Both were from clinical samples testing negative by CDC's qPCR test 277 and both were determined as negative upon re-testing. 

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