Lab Matters Fall 2024 | Page 103

APHL 2024 POSTER ABSTRACTS

Genomic Characteristics of Long-term Infections in California COVIDNet Data

J . Bell 1 , E . Smith 2 , S . Gilliam 1 , M . Scribner 2 , D . Wadford 1 , California Department of Public Health 1 , Theiagen Genomics 2

Note : The findings and conclusions in this work are those of the authors and do not necessarily represent the views or opinions of the California Department of Public Health or the California Health and Human Services Agency .

While most COVID-19 infections resolve within 10-14 days , some are not cleared by the immune system within the usual two-week period , particularly among immunocompromised patients . It has been hypothesized that some of the most transmissible and immuneevasive COVID variants , including Alpha , Delta and Omicron , evolved some of their novel properties in immunocompromised patients before returning to community transmission . Thus , understanding the characteristics of long-term infections ( LTIs ) may contribute to our ability to better protect against SARS-CoV-2 as it becomes endemic in the human population .

The California COVIDNet sequencing initiative facilitated largescale whole genome sequencing of SARS-CoV-2 across the state . Robust genomic surveillance in conjunction with partnerships with medical providers such as Kaiser of Northern California allowed for the collection of multiple consecutive specimens from patients who were repeatedly positive for SARS-CoV-2 . Using COVIDNet databases as well as the California Department of Public Health ( CDPH ) Integrated Genomic Epi Database ( IGED ), which links SARS- CoV-2 sequences to epidemiological data derived from the California Reportable Disease Information Exchange ( CalREDIE ) electronic lab reports , we were able to analyze this data to identify and compare sequences of longitudinal SARS-CoV-2 specimens collected from the same individual .

We identified individuals with multiple sequenced SARS-CoV-2 specimens and then filtered the cases for possible LTIs by requiring a minimum of 21 days between the earliest and latest specimen collection dates and by requiring the sequences to have the same SARS-CoV-2 Nextclade assignment . In total , we found 220 possible LTIs , 164 of which had accessible FASTQ files that would allow us to investigate minor allele frequencies that might indicate intra-host evolution .

Manual analysis of the possible LTI sequence data , including quality metrics and phylogenetic placement , identified 93 specimen pairs as probable reinfections for one or more of the following reasons : 1 ) differences in Pango lineage , 2 ) large SNP distances to the most recent common ancestor of both samples , 3 ) genomic similarity of the more recent genome with contextual samples in the UShER subtrees ; and another 18 specimen pairs as complicated cases where we were unable to distinguish the most likely course of events with confidence , sometimes because of low data quality . After excluding those two categories , SARS-CoV-2 genomes from 53 patients were retained as probable LTIs .

Among the 53 probable LTIs , 31 had minimal intra-host evolution and did not justify further investigation , leaving 22 cases to be further examined . Of the 22 individuals remaining , 11 had two specimens sequenced , five had three specimens , three had four specimens , two had five specimens and one individual had six specimens sequenced over a 100-day period . Our results demonstrate significant intrahost evolution in some cases , including apparent oscillation between quasi-species . We discuss some of these LTI cases in detail as well as the variant evolution observed across patients in our data , including evolutionary convergence in LTIs vs . acute infections .

Presenter : John Bell , john . bell @ cdph . ca . gov

Genomic Characterization of Vibrio cholerae Genome : Insights into Sucrose Fermentation Phenotype Variation

J . Haan , M . Orth , H . Hwang , Minnesota Department of Health

Background : Vibrio mimicus shares a common ancestor with Vibrio cholerae despite notable differences , particularly in sucrose fermentation phenotype . A clinical isolate received at Minnesota Public Health Laboratory ( MDH PHL ) exhibited inconsistent identification results between conventional microbiological methods and advanced sequencing techniques , triggering further investigation .

Method : The isolate , obtained from an individual with a history of lake swimming and recent travel to Spain four months prior to developing tissue wound , recovered from a hip wound initially identified as V . cholerae at a clinical laboratory . The isolate was sent to MDH-PHL for species confirmation . Additional characterization was performed , including biochemical testing , MALDI-TOF , serum agglutination assay and shortread and long-read whole genome sequencing ( WGS ).

Results : Biochemical testing confirmed the isolate as V . mimicus , while Average Nucleotide Identity ( ANI ) analysis using WGS determined it as V . cholerae . The serum agglutination assay identified this isolate ’ s serotype as non-O1 / non-O139 . The absence of the ctx gene and the presence of V . cholerae toxR gene indicated this isolate is non-toxigenic V . cholerae . Comparative genomic analysis with other V . cholerae and V . mimicus isolates placed this isolate within the V . cholerae cluster , distant from V . mimicus . In addition , genes involved in sucrose fermentation in V . cholerae were further characterized to identify mutations that could explain the lack of sucrose fermentation in this isolate . Comparing srcR ( sucrose operon repressor ), fructokinase , sucrose-6-phasphatehydrolase and PTS system , sucrose-specific IIBC component genes from this isolate to the V . cholerae reference genome revealed several SNPs , including insertion and deletion that resulted in frame shifts . This finding suggests possible alteration in gene expression or enzyme structure changes that could contribute to the distinctive sucrose-negative fermentation phenotype observed in this isolate .

Conclusion : The isolate was identified as a sucrose negative V . cholerae . Hybrid assembly of short-read and long-read sequences through WGS offers a better understanding of genomic structures . The routine application of WGS in public health laboratories facilitates accurate identification and characterization of enteric organisms , enhancing the detection of anomalous organisms within our disease surveillance system .

Presenter : Jisun Haan , jisun . haan @ state . mn . us