Lab Matters Summer 2019 - Page 62

APHL 2019 POSTER ABSTRACTS key to improving patient outcomes and limiting transmission of tuberculosis. Here, we discuss the development of a new method that uses multiplex polymerase chain reaction (PCR) coupled with next-generation sequencing (NGS) to predict Mtb drug resistance. In this study, starting material was either Mtb isolates or processed sputum sediments positive by nucleic acid amplification test (NAAT) for Mtb. The assay was a three pool multiplex PCR that amplified 116 genomic regions spanning 19 loci associated with drug resistance. We initially optimized and evaluated the assay using high-quality genomic Mtb DNA. We achieved 100% coverage of the amplicons with a 17-fold depth variation from the depth mean (e.g., mean depth was 45,963 but lowest depth was 2,681). We also evaluated the assay on NAAT+ sputum sediments previously analyzed by Sanger sequencing. The percent and depth of coverage for each locus was similar for processed sputum sediments as compared to Mtb isolates. NGS results were 100% concordant with previous Sanger sequencing results for each sample. Preliminary findings using an Illumina Miniseq suggest this method can test 10 isolates on a single sequencing run and achieve 100% coverage across all 116 amplicons with a median coverage of ~1,000x. The anticipated decreased turn-around time, as compared to growth-based tests, could make this method a practical option for laboratories seeking to implement molecular testing. Distribution of Dual-mechanism Carbapenemase-producing Gram-negatives in the United States — AR Laboratory Network, 2017–2018 A. Wadhwa, J. Huang, R. Stanton, S. Malik, G. McAllister, A. Halpin and A. Brown, Centers for Disease Control and Prevention Introduction: Carbapenem resistance caused by extended-spectrum beta-lactamase producing Enterobacteriaceae is on the rise. Plasmid-mediated carbapenemase genes among healthcare- associated pathogens is a major public health concern because of the potential for rapid dissemination. CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to provide nationwide capacity for rapid detection of AR threats to facilitate their containment and prevention. Using AR Lab Network data, we describe the distribution and molecular epidemiology of carbapenem-resistant Enterobacteriaceae (CRE) and Pseudomonas aeruginosa (CRPA) isolates as well as colonization swabs that tested positive for two carbapenemase genes (dual-mechanism). Methods: In January 2017, AR Lab Network began testing CRE and CRPA isolates for organism identification, antimicrobial susceptibility testing (AST), carbapenemase production, and mechanism identification (KPC, NDM, OXA-48-like, VIM, and IMP genes). Colonization screening for carbapenemase-producing organisms (CPOs) was added to the activities in June 2017. We analyzed all monthly reports for dual-mechanism specimens to describe the distribution of carbapenemases, organisms, AST, and whole genome sequencing (WGS) data. Results: Through November 2018, CDC received 92 reports of dual-mechanism isolates; 89 CRE and 3 CRPA. Among CRE isolates, 76% were Klebsiella spp., 9% were Escherichia coli and 8% were Enterobacter spp. Seventy-four percent (n=57) of dual-mechanism isolates were New Delhi Metallo-beta-lactamase (NDM) with oxacillinase (OXA), 33 (58%) of which were detected in the Mid- 60 LAB MATTERS Summer 2019 Conclusion: The AR Lab Network is aiding detection of clinical isolates and specimens with multiple carbapenemases. Increased capacity, timely screening, and rapid AST can support increased containment and improved therapeutic success for infections caused by threats to patient safety. It is critical that all primary carbapenemase gene targets be included in routine public health laboratory workflows to ensure rapid identification of and response to isolates harboring multiple mechanisms of concern. Studies are underway to improve our understanding of transmission and evolution, and our strategies for detection and containment of these and other emerging AR threats. Presenter: Ashutosh Wadhwa, Centers for Disease Control and Prevention, Atlanta, GA, Towards Genomic Epidemiology of Bordetella pertussis with wgMLST and Culture-independent Sequencing M. Weigand 1 , Y. Peng 1 , D. Kania 1 , H. Pouseele 2 , A. Simon 1 , L. Xiaoli 1 , K. Bowden 1 , M. Williams 1 , L. Tondella 1 ; 1 Centers for Disease Control and Prevention, 2 Applied Maths, Inc. Whooping cough (‘pertussis’) remains a public health challenge. Although coverage with pertussis-containing vaccines remains high, cases have increased steadily in the United States since the late 1980s. The causative agent, Bordetella pertussis, exhibits little gene sequence variation rendering many standard molecular assays, such as multi-locus sequence typing (MLST), inadequate for supporting epidemiology. Study of pertussis disease resurgence is further limited by the declining use of diagnostic culture as fewer clinical isolates are available for whole-genome sequencing. To address these needs, we sought to improve the power of allele-based molecular typing and develop a culture-independent application for B. pertussis. A whole-genome MLST (wgMLST) scheme was developed on the BioNumerics platform from 214 reference-quality genome assemblies sequenced at CDC. The scheme was evaluated with Illumina HiSeq or MiSeq data from 2,200 isolates, representing cases of known outbreaks and state-wide epidemics characterized by existing molecular assays as well as replicates collected from individual patients. A targeted enrichment protocol with RNA baits was also evaluated for implementing genomic characterization of PCR-positive nasopharyngeal specimens. Scheme curation identified 3,507 protein-coding loci that cover >80% of the average B. pertussis genome. Allele typing with wgMLST demonstrated concordance with whole-genome SNP profiles, accurately resolved outbreak and sporadic cases in a retrospective comparison, and clustered replicate isolates collected from individual patients at a single time point. Reanalysis of isolates from two state-wide epidemics reconstructed the population structures of circulating disease with increased resolution to PublicHealthLabs @APHL Presenter: Eric Ransom, AR Fellow, Centers for Disease Control and Prevention, Atlanta, GA, Atlantic region. The next most common combination was NDM and Klebsiella pneumoniae carbapenemase (KPC) (19; 25%), which was detected in the Midwest region more often than elsewhere (8; 42%). Forty-eight percent (24/50) of isolates tested during 2018 showed resistance to ertapenem, imipenem, meropenem, ceftazidime, cefepime and aztreonam. Of ten CRE dual-mechanism isolates with WGS, the carbapenemase genes were found localized on separate plasmids. There were 86 dual-mechanism swabs from CPO screening, of which 34 (40%) were KPC/VIM and 33 (38%) were KPC/NDM.