Lab Matters Fall 2024 | Page 108

APHL 2024 POSTER ABSTRACTS
public health action , such as informing the composition of future RSV vaccine and treatment options .
In order to perform WGS and track RSV transmission and evolution in Colorado , the Colorado State Public Health Laboratory at the Colorado Department of Public Health and Environment ( CDPHE ) assessed available clinical RSV sequencing options on RSV positive clinical specimens obtained from Colorado Children ’ s Hospital from the 2021-2022 , 2022-2023 and 2023-2024 respiratory season . Prior to performing WGS , the clinical RSV samples were screened using a duplexed RT-qPCR RSV A and B assay . The screening process allowed us to differentiate the RSV subtype and detect the viral load for each sample . To assess RSV sequencing approaches of clinical specimens , we compared three different primers schemes : a 10-plex protocol from Centers for Disease Control and Prevention ( CDC ), duplexed protocol from the World Health Organization ( WHO ) and tiled amplicon approach from the University of Michigan . CDC protocol consists of 10 unique primer pools for RSV-A and 10 unique primer pools for RSV-B . WHO protocol uses two sets of pooled primers , regardless of subtype . The University of Michigan tiled amplicon approach consists of two amplicon pools for each subtype . To assess which of the three protocols generated the best data , we conducted a side-by-side comparison of the three schemes on six RSV-A clinical samples with Ct values < 28 and six RSV-B clinical samples with Ct values < 28 . The results were compared using genome percent coverage , mean depth of coverage , total number of raw reads , total number of reads mapped to the reference genome and NextClade lineage designation . As a result of these comparisons , we identified a RSV sequencing approach that is sufficient for the needs of CDPHE to implement clinical RSV genomic surveillance at this time . Future plans include running the primer comparison on wastewater samples that test positive for RSV on dPCR .
Presenter : Alexis Alford , lexi . alford @ state . co . us
Performance Evaluation of Four Multiplex PCR Assays for the Diagnosis of Genital Ulcer Disease
J . Thomas 1 , S . Lundy 1 , M . Koralur 1 , K . Pettus 1 , S . Cohen 2 , K . Workowski 3 , P . Naidu 4 , W . Cao 1 , A . Pillay 1 , Centers for Disease Control and Prevention 1 , San Francisco Department of Public Health 2 , Centers for Diseases Control and Prevention and Emory University 3 , Alberta Health Services 4
Objectives : Genital ulcer disease ( GUD ) is mainly caused by sexually transmitted infections . In the United States , the most common cause of GUD is herpes simplex virus and syphilis . The purpose of this study is to evaluate the performance of commercially available and CDC laboratory-developed multiplex real-time PCR ( MPCR ) assays for the detection of Herpes simplex virus ( HSV ) -1 and HSV-2 , Treponema pallidum and Haemophilus ducreyi in genital ulcers considering the limited molecular testing options for GUD in the US .
Methods : MPCR was conducted using 109 lesion swabs collected between 2004-2022 in the US and Canada . Specimens were included based on clinical suspicion for GUD , dark-field microscopy identification of T . pallidum , serology results ( nontreponemal / treponemal test reactive ), or DNA polymerase I or 47-kDa lipoprotein gene PCR results . Three commercial assays : PlexPCR VHS ( SpeeDx , NSW , Australia ; targets detected : HSV-1 / HSV-2 / T . pallidum / Varicella zoster virus ( VZV )), Fast-track Diagnostics Genital Ulcer ( FTD-19 ) RUO Assay ( Siemens , Germany ; targets detected : HSV-1 / HSV-
2 / T . pallidum ) and Allplex™ Genital Ulcer Assay ( Seegene , Korea targets detected : Cytomegalovirus ( CMV )/ HSV-1 / HSV-2 / H . ducreyi / Lymphogranuloma Venereum ( LGV )/ T . pallidum / VZV ) and a CDC laboratory-developed GUD MPCR that detects HSV1 / 2 , T . pallidum and H . ducreyi were evaluated in this study . All commercial assays were performed according to the manufacturers ’ instructions and specimens that tested positive for each DNA target by at least two assays were used as the gold standard to determine assay performance of each target .
Results : PlexPCR VHS detected T . pallidum in 49 specimens , HSV-1 in eight specimens , HSV-2 in 25 specimens and VZV in one specimen . Allplex detected T . pallidum in 50 specimens , HSV-1 in eight , HSV-2 in 24 and VZV in one . FTD-19 detected T . pallidum in 49 specimens , HSV-1 in eight and HSV-2 in 24 . The CDC MPCR detected T . pallidum in 50 specimens and HSV ( HSV-1 and HSV-2 undifferentiated ) in 31 specimens . CMV , LGV and H . ducreyi were not detected in any of the specimens . High analytical sensitivity ( 97 % -100 %) and specificity ( 98 % -100 %) was observed among evaluated assays for detection of T . pallidum , HSV-1 and HSV-2 . The positive predictive value ( PPV ) was between 96 % and 100 % and the negative predictive value ( NPV ) ranged from 98 % to 100 % for all assays . The MPCR assays had a strong agreeance for T . pallidum , HSV-1 and HSV-2 ( 99 % -100 %), with kappa index values of 0.99 , 1 and 0.99 , respectively .
Discussion : The four MPCR assays evaluated showed excellent performance and comparable results for the detection of HSV-1 , HSV-2 and T . pallidum in genital lesion swabs . These tests provide more diagnostic options until FDA cleared assays become available .
Presenter : Jeronay Thomas , Tqf2 @ cdc . gov
Performance Evaluation of the Average Nucleotide Identity ( ANI ) Test for the Identification of Enteric Bacterial Pathogens
K . Morey , Oregon State Public Health Laboratory
Background : Whole genome sequencing ( WGS ) offers the ability to perform advanced genomic characterization for microbial pathogens using customized bioinformatics pipelines . The Oregon State Public Health Laboratory ( OSPHL ) currently uses procedures and tools developed by PulseNet and has implemented WGS as the subtyping tool to monitor enteric diseases including the analysis of genomic data using BioNumerics v 7.6 software . The average nucleotide identity ( ANI ) test is housed within one of the organized workflow databases in BioNumerics . This bioinformatics tool can accurately identify bacterial species with well-characterized strain genomes available for comparison in a reference genome dataset validated for ANI in BioNumerics . Traditional laboratory methods to identify bacteria can be labor-intensive , time-consuming and limited in their ability to identify bacteria correctly and reliably . The purpose of this evaluation was to determine the performance of the ANI test for routine bacterial identification .
Methods : The ANI reference panel provided by CDC ’ s Enteric Diseases Laboratory Branch and historical sequencing data were evaluated by performing the established protocols for bacterial sequencing using the Illumina MiSeq sequencer . These two panels included bacterial pathogens commonly responsible for foodborne bacterial illnesses : Campylobacter spp ., Listeria spp ., Salmonella spp ., Shiga toxin-producing Escherichia coli , Shigella spp . and Vibrio
106 LAB MATTERS Fall 2024
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