Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline.

During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans. We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays. For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral clade and was present in 18.8 % of the genomes we analyzed. Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.

Kuchinski KS ，Jassem AN ，Prystajecky NA 《-》

Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR.

The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources. Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity. Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction. Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput.

Perchetti GA ，Nalla AK ，Huang ML ，Jerome KR ，Greninger AL ... -  《-》

Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia.

The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem unprecedented in the recent history of humanity. Different in-house real-time RT-PCR (rRT-PCR) methods for SARS-CoV-2 diagnosis and the appearance of genomes with mutations in primer regions have been reported. Hence, whole-genome data from locally-circulating SARS-CoV-2 strains contribute to the knowledge of its global variability and the development and fine tuning of diagnostic protocols. To describe the genetic variability of Colombian SARS-CoV-2 genomes in hybridization regions of oligonucleotides of the main in-house methods for SARS-CoV-2 detection, RNA samples with confirmed SARS-CoV-2 molecular diagnosis were processed through next-generation sequencing. Primers/probes sequences from 13 target regions for SARS-CoV-2 detection suggested by 7 institutions and consolidated by WHO during the early stage of the pandemic were aligned with Muscle tool to assess the genetic variability potentially affecting their performance. Finally, the corresponding codon positions at the 3' end of each primer, the open reading frame inspection was identified for each gene/protein product. Complete SARS-CoV-2 genomes were obtained from 30 COVID-19 cases, representative of the current epidemiology in the country. Mismatches between at least one Colombian sequence and five oligonucleotides targeting the RdRP and N genes were observed. The 3' end of 4 primers aligned to the third codon position, showed high risk of nucleotide substitution and potential mismatches at this critical position. Genetic variability was detected in Colombian SARS-CoV-2 sequences in some of the primer/probe regions for in-house rRT-PCR diagnostic tests available at WHO COVID-19 technical guidelines; its impact on the performance and rates of false-negative results should be experimentally evaluated. The genomic surveillance of SARS-CoV-2 is highly recommended for the early identification of mutations in critical regions and to issue recommendations on specific diagnostic tests to ensure the coverage of locally-circulating genetic variants.

Álvarez-Díaz DA ，Franco-Muñoz C ，Laiton-Donato K ，Usme-Ciro JA ，Franco-Sierra ND ，Flórez-Sánchez AC ，Gómez-Rangel S ，Rodríguez-Calderon LD ，Barbosa-Ramirez J ，Ospitia-Baez E ，Walteros DM ，Ospina-Martinez ML ，Mercado-Reyes M ... -  《-》

Rapid Serological Assays and SARS-CoV-2 Real-Time Polymerase Chain Reaction Assays for the Detection of SARS-CoV-2: Comparative Study.

Real-time polymerase chain reaction (RT-PCR) testing for the identification of viral nucleic acid is the current standard for the diagnosis of SARS-CoV-2 infection, but technical issues limit its utilization for large-scale screening. Serological immunoglobulin M (IgM)/IgG testing has been proposed as a useful tool for detecting SARS-CoV-2 exposure. The objective of our study was to compare the results of the rapid serological VivaDiag test for SARS-CoV-2-related IgM/IgG detection with those of the standard RT-PCR laboratory test for identifying SARS-CoV-2 nucleic acid. We simultaneously performed both serological and molecular tests with a consecutive series of 191 symptomatic patients. The results provided by a new rapid serological colorimetric test for analyzing IgM/IgG expression were compared with those of RT-PCR testing for SARS-CoV-2 detection. Of the 191 subjects, 70 (36.6%) tested positive for SARS-CoV-2 based on RT-PCR results, while 34 (17.3%) tested positive based on serological IgM/IgG expression. Additionally, 13 (6.8%) subjects tested positive based on serological test results, but also tested negative based on RT-PCR results. The rapid serological test had a sensitivity of 30% and a specificity of 89% compared to the standard RT-PCR assay. Interestingly, the performance of both assays improved 8 days after symptom appearance. After 10 days had passed since symptom appearance, the predictive value of the rapid serological test was higher than that of the standard molecular assay (proportion of positive results: 40% vs 20%). Multivariate analysis showed that age >58 years (P<.01) and period of >15 days after symptom onset (P<.02) were significant and independent factors associated with serological test positivity. The rapid serological test analyzed in this study seems limited in terms of usefulness when diagnosing SARS-CoV-2 infection. However, it may be useful for providing relevant information on people's immunoreaction to COVID-19 exposure.

Paradiso AV ，De Summa S ，Loconsole D ，Procacci V ，Sallustio A ，Centrone F ，Silvestris N ，Cafagna V ，De Palma G ，Tufaro A ，Garrisi VM ，Chironna M ... -  《JOURNAL OF MEDICAL INTERNET RESEARCH》

Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit.

Nearly 400,000 people worldwide are known to have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) beginning in December 2019. The virus has now spread to over 168 countries including the United States, where the first cluster of cases was observed in the Seattle metropolitan area in Washington. Given the rapid increase in the number of cases in many localities, the availability of accurate, high-throughput SARS-CoV-2 testing is vital to efforts to manage the current public health crisis. In the course of optimizing SARS-CoV-2 testing performed by the University of Washington Clinical Virology Lab (UW Virology Lab), we evaluated assays using seven different primer-probe sets and one assay kit. We found that the most sensitive assays were those that used the E-gene primer-probe set described by Corman et al. (V. M. Corman, O. Landt, M. Kaiser, R. Molenkamp, et al., Euro Surveill 25:2000045, 2020, https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045) and the N2 set developed by the CDC (Division of Viral Diseases, Centers for Disease Control and Prevention, 2020, https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf). All assays tested were found to be highly specific for SARS-CoV-2, with no cross-reactivity with other respiratory viruses observed in our analyses regardless of the primer-probe set or kit used. These results will provide valuable information to other clinical laboratories who are actively developing SARS-CoV-2 testing protocols at a time when increased testing capacity is urgently needed worldwide.

Nalla AK ，Casto AM ，Huang MW ，Perchetti GA ，Sampoleo R ，Shrestha L ，Wei Y ，Zhu H ，Jerome KR ，Greninger AL ... -  《-》