Successful Detection of Delta and Omicron Variants of SARS-CoV-2 by Veterinary Diagnostic Laboratory Participants in an Interlaboratory Comparison Exercise

Background Since the beginning of the COVID-19 pandemic veterinary diagnostic laboratories have tested diagnostic samples for SARS-CoV-2 not only in animals, but in over five million human samples. An evaluation of the performance of those laboratories is needed using blinded test samples to ensure that laboratories report reliable data to the public. This interlaboratory comparison exercise (ILC3) builds on two prior exercises to assess whether veterinary diagnostic laboratories can detect Delta and Omicron variants spiked in canine nasal matrix or viral transport medium. Methods Inactivated Delta variant at levels of 25 to 1,000 copies per 50 μL of nasal matrix were prepared for participants by the ILC organizer, an independent laboratory, for blinded analysis. Omicron variant at 1,000 copies per 50 μL of transport medium was also included. Feline infectious peritonitis virus (FIPV) RNA was used as a confounder for specificity assessment. A total of 14 test samples were prepared for each participant. Participants used their routine diagnostic procedures for RNA extraction and real-time RT-PCR. Results were analyzed according to International Organization for Standardization (ISO) 16140 - 2:2016. Results The overall results showed 93% detection for Delta and 97% for Omicron at 1,000 copies per 50 μL (22-200 copies per reaction). The overall specificity was 97% for blank samples and 100% for blank samples with FIPV. No differences in Ct values were significant for samples with the same virus levels between N1 and N2 markers, nor between the two variants. Conclusions The results indicated that all ILC3 participants were able to detect both Delta and Omicron variants. The canine nasal matrix did not significantly affect SARS-CoV-2 detection. Impact Statement Ensuring accurate detection methods for SARS-CoV-2 is critical as veterinary diagnostic labs are testing both human and animal samples. This exercise used blinded test samples and provided high confidence in the sensitivity of methods in twenty-nine laboratories for detection of SARS-CoV-2 variants while addressing the impact of sample matrix. Importantly, the results indicated that variants and matrix do not impact detection results. Additionally, this article examined decision-making criteria for Ct cut-off values from different laboratories and encouraged them to review and potentially reassess their criteria to improve future performance. This knowledge will lead to higher confidence in laboratory detection of current and new SARS-CoV-2 variants and aid in establishing reasonable cut-off parameters for these diagnostics tests. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The ILC3 was funded by FDA's Vet-LIRN program, and laboratories were not charged to participate in this exercise. Vet-LIRN laboratories may have also used infrastructure grant funds provided via Vet-LIRN funding opportunity PAR-17-141 to cover the cost of supplies. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript * ILC : Interlaboratory comparison RT–qPCR : quantitative reverse transcriptase–PCR or real-time reverse transcriptase–PCR MTM : molecular transport medium Ct : threshold cycle ROD : rate of detection LOD : level of detection s.d. : standard deviation FIPV : Feline infectious peritonitis virus.