APHL 2021 Poster Abstracts
COVID-19
Detection of SARS-CoV-2 Reinfection in Congregate Living Settings
M . Hetherington-Rauth , L . Bankers , D . Ir , S . Matzinger , S . E . Totten and E . Travanty , Colorado Department of Public Health and Environment , Denver , CO
To date , several reinfection cases of SARS-CoV-2 have been reported ; but frequency of reinfection is still unknown . Understanding reinfection has important implications for vaccine efficacy and controlling the spread of SARS-CoV-2 . Whole genome sequence data was used to identify cases of reinfection occurring in a congregate living facility . Individuals in these facilities are in close proximity with unavoidable contact increasing SARS-CoV-2 exposure risk . Diagnostic RT-PCR testing for SARS-CoV-2 was conducted on routine surveillance nasopharyngeal swabs collected from residents at the facility between April and October 2020 . Positive individuals were excluded from retesting for 90 days post initial positive result . After 90 days , patients were again included in routine surveillance . Candidate reinfection cases were characterized by two positive RT-PCR results separated by greater than 90 days and were sequenced using next generation sequencing technology followed by bioinformatic analysis of sequence data . Twenty individuals were found to have a positive diagnostic RT-PCR test for SARS-CoV-2 greater than 90 days following an initial positive test result . Of these candidate cases , high quality sequence data was obtained for seven individuals . Ten distinct sequences of SARS-CoV-2 were identified within the facility , as characterized by shared variants within a cluster and distinct clusters on the phylogenetic tree . No strong association between genomic cluster and facility unit was found , suggesting that each genomic cluster was widespread throughout the facility . The genomic clusters were , however , associated with distinct time periods representing either an early outbreak ( April- May ), late outbreak ( June-August ) or continued outbreak ( April- August ) in the facility . For four of the seven reinfection cases , the sequence from the first infection and that from the second infection belonged to separate genomic clusters that were present in the facility . Additionally , for one individual , the sequence from the first infection clustered within clade 20C and the re-infection infection sequence clustered within clade 20A , providing strong evidence for reinfection . Finally , the average number of differing nucleotides between first and second infection was 8.4 , with an average of 108 days between sample collections . This gives an average mutation rate that exceeds the current SARS-CoV-2 rate estimate of two substitutions per month , suggesting that continued infection is unlikely . Seven examples of positive SARS-CoV-2 RT-PCR tests separated by greater than 90 days were identified . Sequence data , including identification of temporally separated samples clustering within two distinct clades ( 20A and 20C ), demonstrate likely SARS- CoV-2 reinfection occurring within a congregate living facility .
Presenter : Shannon Matzinger , Colorado Department of Health and Environment , shannon . matzinger @ state . co . us
Rapid Next-generation Sequencing of the Complete SARS- CoV-2 Genome Using the Ion AmpliSeq™ SARS-CoV-2 Research Panel
A . McGeachy , N . Li , W . Zhang , X . Duan , L . Pickle , J . Ni , Y . Fu , X . Meng , L . Guo , L . Zheng , Y . Zhong , A . Ewing , S . Roman , A . Shah and F . Hyland , Thermo Fisher Scientific , Carlsbad , CA
Whole genome sequencing of SARS-CoV-2 is essential to monitoring the emergence and spread of new viral variants such as the more transmissible B . 1.1.7 and 501 . V2 . The Ion AmpliSeq™ SARS-CoV-2 Research Panel is a targeted NGS solution that enables specific amplification , analysis and monitoring of the complete SARS-CoV-2 genome . We report the intelligent design for robust performance and utility in a wide variety of SARS-CoV-2 research applications . The design covers 99.6 % of the total bases of SARS-CoV-2 genome . Approximately 62 % of the viral genome is covered with two independent amplicons , giving the panel exceptionally robust performance given naturally occurring variation . Amplicons were designed to cover all serotypes of SARS-CoV-2 with high analytical specificity and demonstrate no crosstalk to other Coronaviruses . Panel performance was assayed using synthetic controls or heatkilled virus . Library preparation was performed manually or on the Ion Chef , sequenced on GeneStudio S5 or Genexus sequencers , and evaluated for sequencing uniformity , variant calling , and consensus sequence assembly with built-in plugins . Automated workflows reduce hands-on time to as little as 45 minutes using the Ion Chef ™ system or five minutes with the turnkey Ion Torrent™ Genexus™ sequencing system , reducing the risk of operator induced errors . With the 540 chip on Ion GeneStudio S5 , up to 80 samples can be multiplexed , allowing for the high throughput necessary for epidemiological studies . The panel exhibited strong targeted sequencing performance , with on-target reads above 95 % and coverage uniformity above 90 %. The panel also demonstrated high analytical sensitivity , with a limit of detection as low as 20 viral copies . The high sensitivity of the assay allows for sample inputs as low as 1 ng total RNA , increasing the eligibility of low viral load samples . Using the Ion AmpliSeq™ SARS-CoV-2 end-toend analysis solution , we successfully detected known variants in synthetic controls and assembled the genomes from heat-killed virus . The Ion AmpliSeq™ SARS-CoV-2 Research panel requires minimal sample input and as little as 24 hours turnaround time with the Genexus system for comprehensive genomic analysis , genome assembly , and variant annotation . Together , this uniquely positions the Ion AmpliSeq™ SARS-CoV-2 assay to provide rapid , high-quality results for tracking viral spread and evolution . To date , over 7,000 sequences have been deposited into GISAID using these workflows . The panel has proven to be robust to natural variation , making it usable in a wide array of applications including contacttracing and viral spread with a diversity of sample types such as nasopharyngeal swabs , wastewater , and post-mortem FFPE tissue . For Research Use Only . Not for use in diagnostic procedure .
Presenter : Anna McGeachy , Thermo Fisher Scientic , anna . mcgeachy @ thermofisher . com
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LAB MATTERS Summer 2021 |
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