APHL 2023 POSTER ABSTRACTS qPCR Assay for Identification of OXA-235 Variants Associated with Carbapenem-resistance in Enterobacteriaceae
S . Cossette , C . Connelly ; Streck
Background : Carbapenem-hydrolyzing class D ( CHDL ) enzymes , including members of the oxacillinase ( OXA ) family are resistant to antibiotics of last resort for potentially fatal infections . Organisms harboring these genes can acquire them through mobile genetic elements , including Carbapenem-resistant Acinetobacter spp ., classified as an urgent threat by the CDC in 2019 . Identification of new gene subfamilies , such as OXA-235 in Enterobacteriaceae , continues to increase the threat of carbapenem resistance ( CR ). As new AMR genes emerge , methods for AMR surveillance are imperative for outbreak prevention and antimicrobial stewardship . Therefore , this study describes the design of a qPCR-based assay that detects OXA-235 variants , expanding detection of OXA gene variants commonly detected in commercial PCR assays to include these medically relevant resistance mechanisms . Methods : Carbapenem-resistance OXA-235 subfamilies were identified through the β-Lactamase DataBase ( BLDB ). Sequence alignments for the OXA-235 variants were downloaded from the NIH Pathogen Detection Reference Gene Catalog and analyzed in Unipro ’ s UGENE software to facilitate primer design . Oligos were optimized by qPCR using a synthetic target sequence and then multiplexed with an internal control ( IC ) targeting a conserved region in Gram-negative bacteria to discriminate false negative results . Results : The OXA- 235 subfamily , associated with CRE and carbapenem resistant Acinetobacter spp ., consists of 9 A . schindleri variants including OXA-235 , OXA-236 , and OXA-237 . In silico analysis confirmed the primers and probe designed for OXA-235 variants could discriminate OXA-235 genes from the OXA-134-like subfamily . Additionally , this model predicts 100 % sensitivity and specificity for the identified OXA-235 variants . A 5-point serial dilution of the target sequence was analyzed by qPCR and demonstrates the assay is 100 % efficient with a correlation coefficient of 0.999 . Conclusions : As AMR spreads , it ’ s crucial to collaborate with the public health sector in monitoring emerging resistance to ensure tests are updated to detect these critical mechanisms . Assays that are not updated can lead to suboptimal rapid diagnostics and antimicrobial misuse , further contributing to the primary causes behind the spread of AMR . The development of the qPCR assay described above is aimed at supporting this need and the continued identification of AMR mechanisms associated with CR Enterobacteriaceae .
Presenter : Stephanie Cossette , scossette @ streck . com
Rapid Identification and Response of a Salmonella Typhimurium Outbreak Traced to Raw Alfalfa Sprouts , Nebraska , 2022
S . McGill 1 , A . Bartling 1 , A . Roden 1 , B . Loeck 2 , P . Iwen 1 ; 1 Nebraska Public Health Laboratory , 2 Nebraska Department of Health and Human Services
Background : On December 19 , 2022 , Douglas County Health Department ( DCHD ) identified a cluster of Salmonella cases reporting a common food item that contained raw alfalfa sprouts at a local restaurant . Nebraska Department of Health and Human Services ( NDHHS ) was notified about the cluster immediately and informed the Nebraska Public Health Laboratory ( NPHL ) to prioritize genetic analysis for isolates of interest . This report describes a collaborative effort among state and national partners to rapidly identify a salmonellosis outbreak . Methods : DNA was extracted from Salmonella isolates using the Qiagen EZ1 Advanced XL instrument with resulting concentration determined using a Qubit fluorometer . Whole genome sequencing ( WGS ) was performed using the Illumina DNA Prep Kit and run on a MiSeq 500 cycle v2 kit . Sequence results were assessed for quality using Illumina Sequence Analysis Viewer and BioNumerics Version 7.6 . Sequence comparisons were made using NDtree and BioNumerics software . Consequently , sequences were shared with the US Centers for Disease Control and Prevention ( CDC ) for comparison with other state WGS data to identify the extent of the outbreak . Results : Sequencing results were available on December 23 , 2022 . Due to technical difficulties with BioNumerics at the time of the outbreak , alternative comparison tools were used to define similarities among Salmonella strains . The outbreak strain was identified as Salmonella Typhimurium . Fastq files were shared with the CDC via ftp on December 23 , 2022 , who then prioritized the Nebraska outbreak isolate ’ s analysis and conducted a national comparison search . Eight Nebraska isolates and one Oklahoma isolate ( tested at the NPHL ) were indistinguishable at 0-1 allele difference by cgMLST . Subsequently , the CDC declared a multistate outbreak of Salmonella Typhimurium following potential contamination of raw alfalfa sprouts on December 27 , 2022 . Conclusion : The collaboration among DCHD , NDHHS , NPHL and the CDC resulted in quick identification and rapid response for early detection of a Salmonella Typhimurium outbreak traced to raw alfalfa sprouts . Despite issues analyzing the sequence data due to technical software problems , alternative means were utilized to determine genetic relatedness of isolates of interest . The combination of strong epidemiologic data , laboratory data and environmental traceback information successfully recognized a potential contamination of raw alfalfa sprouts leading to a voluntary recall of the affected products on December 29 , 2022 .
Presenter : Steve McGill , stmcgill @ unmc . edu
Syphilis and HIV Take a Road Trip !
L . Kimball 1 , S . Markos 1 , S . Delaney 1 , A . Muhit 1 ; M . Ellethy 1 , G . Anschuetz 2 , R . Siderits 1 , T . Kirn 1 , D . Woell 1 ; 1 New Jersey Department of Health Public Health and Environmental Laboratories , 2 New Jersey Department of Health Division of HIV , STD and TB Services
New Jersey ’ s Field Deployable Laboratory ( FDL ) unit was developed in response to the COVID-19 pandemic , to perform testing , aid in outbreaks , as well as perform surveillance testing throughout the state . As COVID-19 deployments slowed , NJ PHEL explored use-cases for mobile testing and decided , in combination with our Division of STD / HIV / TB , to develop a workflow for rapid , onsite confirmatory testing of Syphilis and HIV . To facilitate on-site testing , PHEL identified instrumentation that could perform a majority of testing on-site using minimally invasive procedures ( e . g ., fingerstick ). The Chembio ’ s DPP HIV-Syphilis instrument , which does combination antibody testing for HIV and treponemal antibody testing for syphilis , was chosen as the initial screening test . PHEL was then able to develop a testing algorithm for syphilis , incorporating RPR nontreponemal testing , and HIV 1 & 2 differentiation utilizing the Geenius platform if the initial specimen tested positive . In certain cases , involving discrepancies , confirmatory samples may need to be taken back to PHEL for further confirmation testing , but preliminary results would still be issued to patients at the point of care site . In this poster , we detail the
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Fall 2023 LAB MATTERS 105 |