Tracking SARS-CoV-2 Omicron lineages using real-time reverse transcriptase PCR assays and prospective comparison with genome sequencing.

Omicron has become the dominant SARS-CoV-2 variant globally since December 2021, with distinct waves being associated with separate Omicron sublineages. Rapid detection of BA.1, BA.2, BA.4, and BA.5 was accomplished in the province of Alberta, Canada, through the design and implementation of real-time reverse transcriptase PCR assays targeting S:N501Y, S:ins214EPE, S:H69/V70, ORF7b:L11F, and M:D3N. Using the combination of results for each of these markers, samples could be designated as belonging to sublineages within BA.1, BA.2, BA.4, or BA.5. The analytical sensitivity of these markers ranged from 132 to 2229 copies/mL and in-laboratory accuracy was 98.9-100%. A 97.3% agreement using 12,592 specimens was demonstrated for the assays compared to genome sequencing. The use of these assays, combined with genome sequencing, facilitated the surveillance of SARS-CoV-2 lineages throughout a BA.5-dominated period.

Respiratory virus coinfections with severe acute respiratory coronavirus virus 2 (SARS-CoV-2) continue to be rare one year into the coronavirus disease 2019 (COVID-19) pandemic in Alberta, Canada (June 2020-May 2021).

To assess the burden of respiratory virus coinfections with severe acute respiratory coronavirus virus 2 (SARS-CoV-2), this study reviewed 4,818 specimens positive for SARS-CoV-2 and tested using respiratory virus multiplex testing. Coinfections with SARS-CoV-2 were uncommon (2.8%), with enterovirus or rhinovirus as the most prevalent target (88.1%). Respiratory virus coinfection with SARS-CoV-2 remains low 1 year into the coronavirus disease 2019 (COVID-19) pandemic.

Precision Response to the Rise of the SARS-CoV-2 B.1.1.7 Variant of Concern by Combining Novel PCR Assays and Genome Sequencing for Rapid Variant Detection and Surveillance.

SARS-CoV-2 variants of concern (VOCs) have emerged as a global threat to the COVID-19 pandemic response. We implemented a combined approach to quickly detect known VOCs while continuously monitoring for evolving mutations of the virus. To rapidly detect VOCs, two real-time reverse transcriptase PCR assays were designed and implemented, targeting the spike gene H69/V70 deletion and the N501Y mutation. The H69/V70 deletion and N501Y mutation assays demonstrated accuracies of 98.3% (95% CI 93.8 to 99.8) and 100% (95% CI 96.8 to 100), limits of detection of 1,089 and 294 copies/ml, and percent coefficients of variation of 0.08 to 1.16% and 0 to 2.72% for the two gene targets, respectively. No cross-reactivity with common respiratory pathogens was observed with either assay. Implementation of these tests allowed the swift escalation in testing for VOCs from 2.2% to ∼100% of all SARS-CoV-2-positive samples over 12 January to 9 February 2021, and resulted in the detection of a rapid rise of B.1.1.7 cases within the province of Alberta, Canada. A prospective comparison of the VOC assays to genome sequencing for the detection of B.1.1.7, combined detection of P.1 and B.1.351, and wild-type (i.e., non-VOC) lineages showed sensitivities of 98.2 to 100%, specificities of 98.9 to 100%, positive predictive values of 76.9% to 100%, and negative predictive values of 96 to 100%. Variant screening results inform sampling strategies for regular surveillance by genome sequencing, thus allowing rapid identification of known VOCs while continuously monitoring the evolution of SARS-CoV-2 in the province. IMPORTANCE Different strains, or variants, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the virus that causes COVID-19) have emerged that have higher levels of transmission, less susceptibility to our immune response, and possibly cause more severe disease than previous strains of the virus. Rapid detection of these variants of concern is important to help contain them and prevent them from spreading widely within the population. This study describes two newly developed tests that are able to identify and differentiate the variants of concern from regular strains of SARS-CoV-2. These tests are faster and simpler than the main, gold standard method of identifying variants of concern (genome sequencing). These tests also demonstrated a high correlation with genome sequencing and allowed for the rapid and accurate detection of the rise of B.1.1.7 (one of the variants of concern) in the province of Alberta, Canada.

An improved DNA array-based classification method for the identification of Salmonella serotypes shows high concordance between traditional and genotypic testing.

Previously we developed and tested the Salmonella GenoSerotyping Array (SGSA), which utilized oligonucleotide probes for O- and H- antigen biomarkers to perform accurate molecular serotyping of 57 Salmonella serotypes. Here we describe the development and validation of the ISO 17025 accredited second version of the SGSA (SGSA v. 2) with reliable and unambiguous molecular serotyping results for 112 serotypes of Salmonella which were verified both in silico and in vitro. Improvements included an expansion of the probe sets along with a new classifier tool for prediction of individual antigens and overall serotype from the array probe intensity results. The array classifier and probe sequences were validated in silico to high concordance using 36,153 draft genomes of diverse Salmonella serotypes assembled from public repositories. We obtained correct and unambiguous serotype assignments for 31,924 (88.30%) of the tested samples and a further 3,916 (10.83%) had fully concordant antigen predictions but could not be assigned to a single serotype. The SGSA v. 2 can directly use bacterial colonies with a limit of detection of 860 CFU/mL or purified DNA template at a concentration of 1.0 x 10-1 ng/µl. The SGSA v. 2 was also validated in the wet laboratory and certified using panel of 406 samples representing 185 different serotypes with correct antigen and serotype determinations for 60.89% of the panel and 18.31% correctly identified but an ambiguous overall serotype determination.

Evaluation of Molecular Methods for Identification of Salmonella Serovars.

Classification by serotyping is the essential first step in the characterization of Salmonella isolates and is important for surveillance, source tracking, and outbreak detection. To improve detection and reduce the burden of salmonellosis, several rapid and high-throughput molecular Salmonella serotyping methods have been developed.The aim of this study was to compare three commercial kits, Salm SeroGen (Salm Sero-Genotyping AS-1 kit), Check&Trace (Check-Points), and xMAP (xMAP Salmonella serotyping assay), to the Salmonella genoserotyping array (SGSA) developed by our laboratory. They were assessed using a panel of 321 isolates that represent commonly reported serovars from human and nonhuman sources globally. The four methods correctly identified 73.8% to 94.7% of the isolates tested. The methods correctly identified 85% and 98% of the clinically important Salmonella serovars Enteritidis and Typhimurium, respectively. The methods correctly identified 75% to 100% of the nontyphoidal, broad host range Salmonella serovars, including Heidelberg, Hadar, Infantis, Kentucky, Montevideo, Newport, and Virchow. The sensitivity and specificity of Salmonella serovars Typhimurium and Enteritidis ranged from 85% to 100% and 99% to 100%, respectively.It is anticipated that whole-genome sequencing will replace serotyping in public health laboratories in the future. However, at present, it is approximately three times more expensive than molecular methods. Until consistent standards and methodologies are deployed for whole-genome sequencing, data analysis and interlaboratory comparability remain a challenge. The use of molecular serotyping will provide a valuable high-throughput alternative to traditional serotyping. This comprehensive analysis provides a detailed comparison of commercial kits available for the molecular serotyping of Salmonella.

Multi-laboratory evaluation of the rapid genoserotyping array (SGSA) for the identification of Salmonella serovars.

Salmonella serotyping is an essential first step for identification of isolates associated with disease outbreaks. The Salmonella genoserotyping array (SGSA) is a microarray-based alternative to standard serotyping designed to rapidly identify 57 of the most commonly reported serovars through detection of the genes encoding surface O and H antigens and reporting the corresponding serovar in accordance with the existing White-Kaufmann-Le Minor serotyping scheme. In this study, we evaluated the SGSA at 4 laboratories in 3 countries by testing 1874 isolates from human and non-human sources. The SGSA correctly identified 96.7% of isolates from the target 57 serovars. For the prevalent and clinically important Salmonella serovars Enteritidis and Typhimurium, test specificity and sensitivity were greater than 98% and 99%, respectively. Due to its high-throughput nature, the SGSA is a rapid and cost-effective alternative to standard serotyping for identifying the most prevalent serovars of Salmonella.