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-
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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, [email protected]
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
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Presenter: Eric Ransom, AR Fellow, Centers for Disease Control and
Prevention, Atlanta, GA, [email protected]
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.