Lab Matters Fall 2024 | Page 92

APHL 2024 POSTER ABSTRACTS

peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing . The three-day inter / intraday analysis generated great reproducibility with CV 0.994 . this study shows the successful protein extraction from DBS and top-down analysis for structural and quantitative Hb variant analysis .

Presenter : Jingshu Guo , jingshu . guo @ thermofisher . com

NEXT GENERATION / LONG-READ SEQUENCING , METAGENOMICS AND BIOINFORMATICS

A Database of Mutations is Not Enough ! Tailoring a Next Generation Sequencing Workflow for TB Drug Resistance Detection

M . Sylvester 1 , S . Wright 2 , F . Ambrosio 2 , T . Holbrook 1 , I . Mendes 2 , E . Smith 2 , Z . Berrada 2 , V . Kozyreva 1 , California Department of Public Health 1 , Theiagen Genomics 2

The findings and conclusions in this article 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 .

The California Department of Public Health ’ s Microbial Diseases Laboratory ( MDL ) serves as the reference laboratory for tuberculosis ( TB ) testing for the state of California , as well as the CDC National TB Drug Susceptibility Testing ( DST ) Reference Center . We recently implemented next generation sequencing ( NGS ) for TB genotyping and molecular prediction of drug resistance in Mycobacterium tuberculosis complex ( MTBC ). Utilization of NGS for drug resistance prediction may reduce turnaround times compared to phenotypic DST , resulting in timely and appropriate patient treatment . Additionally , the generated sequence data have a dual use to determine TB genotype for TB control programs to aid in detection and investigation of possible outbreaks .

Here we share our overall testing NGS workflow , with a focus on creating a bioinformatics pipeline that permits automated end-toend analysis . The interpretation script that we developed assigns predicted resistance based on well-characterized variants and by application of “ expert rules ”. The latter allows for automated interpretation of novel mutations that have not been previously described in the WHO catalogue , while ensuring standardization of final interpretations . The bioinformatics pipeline also enables CLIA-compliant diagnostic use of the NGS assay by providing built-in quality control checks and required traceability of analysis . Finally , unambiguous reporting language is key for making NGSbased DST predictions useful to submitters . The pipeline we developed automatically generates reports with clinician-oriented final reporting language ready for ingestion into our laboratory information management system ( LIMS ).

Presenter : Matthew Sylvester , matthew . sylvester @ cdph . ca . gov

A Workflow for Successful NGS Candida auris Sequencing

A . Lemon , M . Spann , N . West , N . Anderson , L . Thomas , A . Burks , X . Qian , S . Cheng , R . Fowler , K . Levinson , Tennessee Department of Health , Division of Laboratory Services

Introduction : Candida auris is an emerging and previously understudied threat to public health . The first clinical cases of C . auris were reported in the United States in 2016 and became nationally notifiable in 2018 . In that time , the CDC rated C . auris as an “ urgent threat ,” at the highest level of concern as C . auris cases increased by 59 % from 2019-2020 and further increased 95 % in 2021 . In addition to the rapid proliferation of C . auris , at least 90 % of clinical isolates are resistant to at least one antifungal and the number of isolates that were resistant to the last line of antifungals , echinocandins , increased over 300 % in 2021 from 2019-2020 . The Tennessee Department of Health is the Southeast ( SE ) regional lab for the CDC ’ s Antimicrobial Resistance Laboratory Network ( AR Lab Network ) and has routinely been testing C . auris colonization and isolates for antifungal susceptibility for the SE region since early 2018 . However , further molecular characterization is now needed for epidemiologic investigation of potential outbreaks and spread of C . auris . The advances in next-generation sequencing ( NGS ) and bioinformatics make it possible to accurately determine clades and antifungal resistant genes . As states begin NGS testing for C . auris , multiple challenges have been identified . Here , Tennessee presents a workflow for sequencing C . auris by NGS , detailing how C . auris isolates are extracted and sequenced with Illumina ’ s shortread sequencing-by-synthesis technology to provide a blueprint for easier onboarding of this testing platform to other public health laboratories .

Methods : The Tennessee AR Lab Network department works closely with southeastern state epidemiologists to determine which C . auris samples should be submitted for NGS-based surveillance . The sequencing team is alerted once C . auris isolates received by the Tennessee Public Health Laboratory for NGS-based surveillance are plated and cultured . The sequencing team extracts C . auris using the Illumina Flex Lysis kit in a modified heat lysis protocol developed by the Utah Department of Public Health , completing extraction in less than an hour . DNA extracts are prepared using the Illumina DNA Prep kit through an automated process on the Hamilton STAR instrument . Libraries are loaded onto an Illumina MiSeq for sequencing .

Results : Once sequencing is complete , . fastq files are sent to the bioinformatics department , where they are run through the MycoSNP-nf1.5 pipeline . Clade ID results are reported to sequencing scientists for entry into LIMS . The bioinformatics department reports clade ID and a neighbor-joining phylogenetic tree back to ARLN for entry into REDCap to be shared with the CDC .

Discussion : C . auris sequencing brings multiple departments together to characterize and survey this urgent threat to public health . This complex test has many moving pieces and the Tennessee Department of Health has developed a workflow to successfully sequence this eukaryote in a high-throughput manner .

Presenter : Athena Lemon , athena . lemon @ tn . gov