Lab Matters Summer 2018 | Page 63

APHL 2018 Annual Meeting Poster Abstracts

level MOH representatives , as well as the clear goals and timelines for work achieved by each technical group . Collaborative efforts of all laboratory network stakeholders ( public , private , military , Universities , implementing partners ) will ensure ownership of the work featured in the document .

Presenter : Pat Sadate-Ngatchou , University of Washington , I-TECH , Seattle , WA , Email : psn @ uw . edu

Infectious Disease

Whole Genome MLST-based Typing and Strain Nomenclature for Clostridium difficile Isolates

S . Kiekens 1 , K . De Bryune 1 , K . Kingsley 2 ; 1 Applied Maths NV , Sint- Martens-Latem , Belgium , 2 Applied Maths , Inc ., Austin , TX

Background : Clostridium difficile is a bacterium that causes symptoms ranging from diarrhea to life-threatening inflammation of the colon , most commonly affecting older or immunocompromised adults in hospitals or long-term care facilities . In recent years , C . difficile infections have become more frequent , severe and difficult to treat . Rapid typing and characterization methods are essential epidemiological tools to prevent and control infection . Although bacterial WGS has become feasible in smaller clinical laboratories , non-standardized data analysis remains a bottleneck for routine surveillance . In this work , we assessed wgMLST and wgSNP for C . difficile typing .

Materials / methods : We created a core ( n = 1999 loci ) and pangenomic ( n = 6713 loci ) schema based on 259 reference sequences reflecting the known diversity of C . difficile . Also capturing the accessory loci greatly increased the discriminatory power of the schema . Adding MLST , CWP and loci associated with antibiotic resistance and virulence maximized consistency with classical typing methods . Assembly-free and BLAST-based algorithms determined locus presence and detected allelic variants . wgSNP further characterized defined clusters by mapping the reads to a reference chosen from within the cluster and filtering the variants . High-throughput data processing pipelines in BioNumerics ® implemented both methods on publicly available data .

Results : We ran wgMLST on + 1,500 samples to identify closely related clusters . We detected a wide diversity of samples and defined clusters at various allele difference cutoffs , allowing the creation of a stable strain nomenclature on the sample set . The defined thresholds determined 36 clusters for which additional resolution was obtained by running the wgSNP analysis , identifying linked clinical cases and linking additional metadata ( e . g ., date of isolation , geo information ) to the results .

Conclusions : WGS combined with automated analysis pipelines holds great promise for bacterial epidemiological surveillance . The pangenomic schema for C . difficile includes over 8000 loci and allows for the detection of subtype- or outbreak-specific markers . BioNumerics ® and integrated wgMLST and wgSNP functionality allows for accessible cluster analysis and typing of C . difficile isolates down to strain level .

Presenter : Kyle Kingsley , Applied Maths , Inc ., Austin , TX , Phone : 512.482.9700 , Email : kyle . kingsley @ biomerieux . com

Comparison of Automated Extraction Methods Using the CDC Human Influenza Virus Real-time RT-PCR Detection and Characterization Panels

L . S . Berman 1 , J . R . Murray 2 , J . Liu 3 , C . M . Warnes 1 , K . Wu 1 , S . Burke 2 , S . Lindstrom 1 ; 1 Centers for Disease Control and Prevention , Atlanta , GA , 2 CDC / Battelle , Atlanta , GA , 3 CDC / ARED , Atlanta , GA

Background : The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR ( rRT-PCR ) assays on the Applied Biosystems ® ( ABI ) 7500 Fast Dx Real-Time PCR system . The panel is configured in four separate kits . Each kit consists of oligonucleotide primers , fluorescently labeled hydrolysis probes and controls that are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness ( ILI ) or from virus culture . Six RNA extraction methods have been approved for use with the CDC rRT-PCR Flu Panel : the automated Roche MagNA Pure LC Total Nucleic Acid Isolation kit , Roche MagNA Pure Compact RNA Isolation kit I , Qiagen ® QIAamp Viral RNA manual extraction , Qiagen ® RNeasy RNA manual extraction , Qiagen ® QIAcube platform using Qiagen ® QIAamp Viral RNA kit and the BioMerieux NucliSENS easyMAG . In order to understand the performance of additional high and low throughput automated extraction methods used in domestic Public Health Laboratories ( PHLs ), we evaluated two additional automated extraction methods . The Qiagen EZ1 Advanced XL extraction platform using the EZ1 DSP Virus Kit and EZ1 RNA Tissue Mini Kit and the Roche MagNA Pure 96 using the DNA and Viral NA Small Volume Kit .

Method : A study was performed to assess the reproducibility of the Roche MagNA Pure 96 and Qiagen EZ1 Advanced XL instruments . The Roche MagNA Pure 96 was evaluated with the Roche DNA and Viral NA Small Volume Kit . The Qiagen EZ1 Advanced XL was evaluated with the Qiagen DSP Virus Kit and the Qiagen RNA Tissue Mini Kit . A blinded panel of contrived samples containing a background of beta-propiolactone ( BPL ) treated A549 cells in Viral Transport Medium ( VTM ) was assembled by adding a BPL inactivated influenza A ( H3N2 ) virus , A / Hong Kong / 4801 / 2014 . The samples included a moderate positive sample , a low positive sample near the established assay limit of detection for the CDC Influenza A Subtyping Kit and a negative sample consisting of background A549 cells and VTM . Three separate testing sites were selected for each extraction instrument platform . The sample panel was tested 5 times by two different analysts at each site over five different days . Analysts performed extractions with the investigational instrument and method and tested the extracted nucleic acids with the InfA , H3 and RP assay markers from the CDC Influenza A Subtyping Kit using Invitrogen SuperScript™ and utilizing the ABI 7500 Fast Dx real-time PCR system . Samples were analyzed using the ABI SDS software version 1.4 with 21CFR part 11 compliant module . Results were compared side-by-side and with data generated using approved methods .

Results : The Qiagen EZ1 Advanced XL and Roche MagNA Pure 96 platforms with their respective reagent kits and paired protocols demonstrated 100 % reproducibility ( 30 / 30 correct results per extraction platform ) across multiple sites , analysts and days . Both new platforms and methods performed comparably to methods previously qualified to be used with the CDC rRT-PCR Flu Panel assay .

Conclusions : Test results indicated that both the Qiagen EZ1 Advanced XL and Roche MagNA Pure 96 performed well under

Infectious Disease