APHL 2024 POSTER ABSTRACTS
Next Generation Sequencing Validation and Analysis of HIV Genotypes and Drug Resistance Mutations Using Target Enrichment Methods
F . Kabir , E . Plaisance , C . Nwankwo , R . Watkins , E . Kirkscey , H . Kidane , M . Williams , D . Silva , V . Amadi , J . Stringer , L . Short , Dallas County Health and Human Services
Introduction : HIV / AIDS prevention and control present as one of the major global public health challenges . Due to its extremely high genetic variability and rapid evolution , the HIV genome can evade conventional laboratory detection , impede disease surveillance and targeted antiviral treatment and result in exacerbating outbreak risk . We aim to develop and validate a robust and scalable HIV next generation sequencing ( NGS ) method based on target enrichment hybrid capture protocols . This method could be valuable for genomic surveillance and clinical assessment and also extended to other STI panel analysis including HCV .
Methods : HIV serum samples were collected from patients originally requested for RNA NAAT ( Aptima HIV Quant Dx ) or antigenantibody testing ( Bioplex 2200 and Geenius HIV Ag / Ab assays ) at Dallas County Health and Human Services ( DCHHS ). Viral RNA was extracted using either manual ( GeneJET or Zymo Research ) or automated protocols ( MagMax viral pathogen kit on Kingfisher DuoPrime or EZ1 & 2 virus mini kit on Qiagen EZ1 Advanced XL ). HIV sequencing and validation were performed using Illumina based target enrichment hybrid capture protocol . The complete wet lab workflow includes RNA extraction , library prep by cDNA synthesis , tagmentation , indexing and enrichment steps and finally sequencing on MiSeq . The Bioinformatic analysis was performed by uploading and assembling fastq data on Illumina DRAGEN Microbial Enrichment application , CLC Genomics Workbench 23.0.5 and Stanford HIV Drug Resistance Database ( HIVDB ) tool .
Results : We successfully analyzed over 40 patient samples and 28 commercially available reference materials ( SeraCare / LGC ) as QC controls to validate HIV-1 subtypes A , B , C , D and HIV-2 A / B . Sequencing QC was assessed based on individual run metrics and parameters like > 85 % Q30 , > 90 % PF and optimal cluster density (~ 1000 ). We identified very high coverage depth of 1000-10,000X for controls and a range of 50- > 1500X depth for patient samples . Though sequencing depths varied in patient-derived samples because of their low viral loads , each sample was verified for their read counts of > 1 million and consensus sequences and identified for the respective HIV genotypes and subtypes . We also sequenced the targeted pol region of HIV-1 B genome as predesigned in a wild type and a recombinant construct harboring 49 drug resistance mutations analyzed on HIVDB tool .
Conclusion :. We have developed and optimized an NGS method at DCHHS that could be beneficial in accurately tracking HIV variants , detecting outbreaks and aiding patient management . Our validation strategy may strengthen public health monitoring of HIV infection with subtypes and strains with drug resistant mutations . This approach will not only allow the clinicians and surveillance teams in managing antiretroviral therapy , but also provide deeper insights into the transmission dynamics of the virus .
Presenter : Erin Plaisance , Erin . Plaisance @ dallascounty . org
Optimizing Outbreak Response : Harnessing Metagenomic and Whole Genome Sequencing Approaches for Bacterial Intoxication Investigations
M . Orth , H . Hwang , C . Glowac , A . Saupe , J . Haan , Minnesota Department of Health
Introduction : Clostridium perfringens is one of the most common foodborne pathogens , causing approximately one million illnesses annually in the United States . Public health laboratories face challenges in identifying and characterizing this organism due to its short incubation period and expensive , the limited shelf life of commercially available toxin kits .
Purpose : This study aims to explore the feasibility of utilizing metagenomic sequencing directly from stool specimens to enhance the detection of C . perfringens .
Methods : MDH received one food specimen and four stool specimens from individuals who fell ill after consuming improperly handled ground beef . All five raw specimens tested positive for C . perfringens toxin , with four isolates further characterized . Whole genome sequencing ( WGS ) and shotgun metagenomic analysis were conducted to assess the relatedness of the isolates and microbial composition , respectively , of C . perfringens .
Results : WGS results revealed close relatedness among all isolates based on SNP analysis ( 0-1 difference ). Metagenomic Intra-species Diversity Analysis System 2 ( MIDAS2 ) was run against the Genome Taxonomy Database ( GTDB ). Only one sample ( Specimen A ) had a Clostridium perfringens relative abundance of 0.01 . Taxonomic classification using Kraken2 against the PlusPF database identified Specimen A with the highest C . perfringens reads ( n = 111,432 ), while the remaining samples had fewer than 2000 C . perfringens reads . The number of contigs in Specimen A that aligned to the isolate sequence from ground beef was 3345 , with 64.4 % coverage .
Significance : WGS analysis on isolates is the best approach for evaluating relatedness among isolates from food and patients . Despite inconsistent metagenomic results across stool specimens , the detection of C . perfringens from metagenomic sequencing indicates the potential utility of metagenomic approaches using raw stools for investigating bacterial intoxication outbreaks . This approach also has the potential to decrease turnaround time and save resources in public health laboratories .
Presenter : Jisun Haan , jisun . haan @ state . mn . us
Optimizing Whole Genome Sequencing of Clinical RSV
A . Alford , D . Polanco , D . Mallal , A . Rossheim , S . Matzinger , Colorado Department of Public Health and Environment
Respiratory syncytial virus ( RSV ) causes infections in the lungs and respiratory tract , affecting as many as two million people in the United States every year , predominantly children and adults aged 60 years and older . RSV has two main subtypes , A and B , which are determined by antigenic drift and duplications in the G gene . During the 2022-2023 season , RSV activity was elevated and peaked early . In response to this increased burden on the public health system and impact on specific populations , RSV vaccines and antibody treatments were developed and implemented during the 2023- 2024 season . Conducting RSV surveillance through whole genome sequencing ( WGS ) tracks evolutionary change and helps to inform
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