Lab Matters Fall 2023 | Page 76

APHL 2023 POSTER ABSTRACTS

ID / AMR Panel ( UPIP ) and Viral Surveillance Panel ( VSP ) -two precision metagenomics assays using biotinylated DNA probes to enrich microbial target sequences . UPIP targets 174 genitourinary pathogens and > 3700 AMR markers , while VSP targets 66 viruses . Shotgun metatranscriptomics confirmed viruses known to be abundant in wastewater , such as hCoV-OC43 and Rotavirus A . Precision metagenomics with UPIP and VSP allowed for more indepth strain identification as well as discovery of a greater number of less abundant pathogens , such as various noroviruses and enteroviruses . The results from these studies provide important guidance on how collection and concentration methods can impact the types and quantities of pathogens detected in wastewater samples and highlight the benefits of NGS approaches for advancing the potential of wastewater surveillance .

Presenter : Kayley Janssen , kayley . janssen @ slh . wisc . edu

Performance Evaluation of Genomic Analysis Tools for Shigella and Enteroinvasive Eschericia coli Species Identification and Serotype Prediction

L . Li , F . Arroyo , L . Lai , A . Escoto , H . Duran , Y . Zhao , M . Shiozaki , G . Inami , S . Abromaitis ; California Department of Public Health

Shigella and enteroinvasive Escherichia coli ( EIEC ) are genetically and phenotypically closely related . They also have similarities in the pathology of infection and can cause diarrhea , fever and stomach cramps . Commercially available or laboratory-developed culture independent diagnostic tests that cannot differentiate between Shigella and EIEC are commonly used ; additionally , isolation and differentiation of the two organisms can be challenging due to biochemical similarities and O-antigen cross-reactivity . Correct identification at the species and serotype levels is essential for public health laboratory surveillance and epidemiological investigations . In this study , we assessed the performance of ShigaTyper , ShigEiFinder and Mykrobe genomic analysis tools for species identification and serotype predication of Shigella and EIEC . Whole genome sequencing data of 42 reference strains and more than 300 clinical strains of Shigella and EIEC from our collection were included in the analyses , covering the predominant serotype submissions to the California Department of Public Health Microbial Diseases Laboratory ( CDPH-MDL ). Additionally , all strains also had species and serotype designations determined through conventional testing schemes by our laboratory . Shigella is classified into four species and more than 50 serotypes based on biochemical tests and lipopolysaccharide O-antigen serotyping . Currently , the CDPH- MDL performs conventional Shigella serotyping for diagnostic reporting and conducts whole genome sequencing of Shigella for surveillance and epidemiological investigation . Challenges associated with conventional Shigella serotyping include subjectivity of interpretation , inability to type rough strains and recognize newly emerged serotypes , potential cross-reactivity and safety concerns due to manipulation of highly concentrated Shigella suspensions for which the infectious dose can be as low as 10 – 100 organisms . For these reasons molecular serotyping was evaluated as a potential replacement for traditional methods . In our study , the serotyping prediction generated by ShigaTyper , ShigEiFinder and Mykrobe provided robust and discriminatory typing of diverse Shigella serotypes , demonstrating that the evaluated analysis workflow would be acceptable for surveillance of Shigella infections . In addition , the combination of the three genotyping analysis programs demonstrated accurate identification and differentiation of Shigella and EIEC . An analysis of the potential cost savings of transitioning to molecular serotyping for diagnostic reporting was conducted to further support future implementation of these genomic analysis tools .

Presenter : Linlin Li , linlin . li @ cdph . ca . gov

Rapid Implementation of Mpox Virus Whole Genome Sequencing Protocol , Using Multiple Sequencing Platforms , Allowed for Real-Time Surveillance of Outbreak

N . Palmateer , B . Schwem , B . Jeong , K . Patel , H . Shah , L . Schlitt , A . Kowalsky , E . Acheampong , D . Woell , R . Siderits , T . Kirn ; New Jersey Department of Health Public Health and Environmental Laboratories

Mpox virus is a member of the orthopox virus genus . The virus is spread by close skin-to-skin contact , and infection with the virus can lead to symptoms that include fever , myalgia , and skin lesions . The WHO declared a global outbreak of mpox virus in the summer of 2022 . The first patient specimen that tested positive with mpox virus in New Jersey was identified at the New Jersey Public Health and Environmental Laboratories ( PHEL ) on June 16th , 2022 . To test for mpox virus , DNA was extracted from skin lesion swabs in a Biosafety Level 3 laboratory with either a manual protocol ( Qiagen column ) or automated approach ( Qiagen EZ1 ), using CDC protocols . Whole genome amplicon-based sequencing was applied to positive specimens ( Ct value < 35 ) using both short-read sequencing ( Illumina MiSeq V2 / 500 cycles ) and long-read sequencing ( Oxford Nanopore Technologies ). Amplicons ranged in length from 1587 bp - 2497 bp . A total of 56 specimens were mpox positive using qPCR at PHEL , and all of which were sequenced using short-read sequencing technology . The range of Ct values for the positive specimens sequenced was 15.0 - 31.4 cycles . Additionally , 14 specimens were also sequenced using a long-read sequencing method . Using assemblies generated from each short-read sequencing dataset , a whole genome alignment was conducted to generate a phylogenetic tree . Quality Control ( QC ) metrics were also compared in these two approaches . The QC analysis shows that the percentage of the reference genome covered and the mean depth of coverage were greater , on average , when performing short-read sequencing . The subsequent Nextclade analysis showed that all specimen sequences were identified as Clade IIb , which was the main circulating lineage of the outbreak , and associated with less severe symptoms than Clade I . There were consensus lineage calls between sequencing approaches for 12 / 14 specimens , while one specimen had discrepant lineage calls and one specimen was only identified to the lineage level using long-read sequencing , but the sub-lineage was identified with short-read sequencing . Genome sequence data were submitted to the National Center for Biotechnology Information ( NCBI ) as Biosamples and raw sequence data ( fastq files ). Whole genome sequence assemblies that met internal QC thresholds were also submitted to Genbank . Specifically , the short-read sequencing data and resulting assemblies from 47 specimens were submitted . While Mpox infection levels in the community dropped off drastically in the fall of 2022 and remain low in early 2023 , PHEL has demonstrated preparedness to implement whole genome sequencing of Mpox . Furthermore , the laboratory ’ s expanded next-generation sequencing infrastructure