APHL 2019 POSTER ABSTRACTS
for A. baumannii infections, which are difficult to treat.
Methods: Whole genome sequencing (WGS) was performed on all
positive patient and environment samples submitted to the Utah
Public Health Laboratory (UPHL) from an Acinetobacter baumannii
outbreak at a long-term care faculty in Utah. WGS data was
analyzed using the reference free analysis pipeline developed at
UPHL (Oakeson et al.) to identify all shared homologous protein
coding genes in the isolates and build a phylogenetic tree. The
phylogenetic tree was then used to determine relatedness of the
isolates. Additionally, the WGS data for each isolate was searched
for the presence of know antimicrobial resistance genes.
Results: Phylogenetic analysis revealed that all the isolates are
closely related and form two monophyletic clades, indicating
multiple transmission events after a single contamination event.
All isolates sequenced contained a single copy of OXA-23, a
known carbapenemase that confers resistance to ampicillin and
cephalothin antibiotics.
Conclusions: WGS of hospital acquired infections can provide
invaluable information that can confirm to relatedness of isolate in
outbreak situations and antimicrobial resistance predictions that
can be used to inform treatment. Additionally, WGS can be used for
surveillance and early detection of clusters before outbreaks can
spread.
Harnessing Luminex Microsphere Technology to
Consolidate and Enhance Detection of Carbapenemase
and Mobile Colistin Resistance Genes
M. Precit 1 , S. Angeloni 2 , M. Tran 1 , R. Ruiz 1 , B.Hiatt 1 , R. Gautom 1 , W.
Glover 1 ; 1 Washington State Public Health Laboratories, 2 Luminex
Corporation
Background: Mechanisms conferring carbapenem or colistin
resistance, carbapenemase or mobile colistin resistance (mcr)
genes respectively, are of great concern to clinical and public health
officials. These mechanisms are typically plasmid-borne and can
disseminate rapidly by horizontal gene transfer making prompt
detection and subsequent response critical for infection control.
However, accurate detection remains a challenge due to numerous
genes and genetic variants that have been described. Real-time
PCR and other commonly available methods are often limited by
the number of targets detected requiring the use of multiple assays.
Thus, a more comprehensive multiplex genomic assay would
improve and streamline detection and response to carbapenemase
and mcr genes.
Objective: We sought to develop a consolidated multiplex genomic
assay to enhance detection of the known genetic variants of
carbapenemase and mcr genes from Gram-negative bacteria
including blaKPC, blaNDM, blaIMP, blaVIM, blaOXA¬-48/48-like,
blaOXA¬-24,40/24,40-like, serratia marcescens enzyme (sme), and
mcr-1-4 in a single assay via traditional PCR paired with Luminex
color-coded magnetic microsphere technology.
Methods: NCBI was used to collect and compile known genetic
variant sequences for the 11 targets. Variants were aligned,
and target-specific consensus sequences were generated with
Sequencher and used to design DNA oligonucleotides including
amplification primers and probes able to detect the known
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LAB MATTERS Summer 2019
Results: Preliminary findings showed that our laboratory developed
multiplex assay was able to amplify blaKPC 2-6, blaNDM 1, 4-7,
blaIMP 1,4,14,18,27, blaVIM 1-4, 11, 27, blaOXA¬-48,181,232,
blaOXA¬-24/40, sme 1,3, and mcr 1-4. Additionally, biotinylated
amplicons generated from a subset of 11 bacterial isolates (the
same strains used in PCR optimization) as well as the reverse-
compliment control capture oligonucleotides all hybridized to the
appropriate corresponding color-coded microsphere-probe pair, as
measured by the MagPix’s ratio of fluorescence over background.
Conclusions: This genomic assay shows promise as a possible
novel method able to consolidate and enhance detection of
carbapenemase and mcr genes present in Gram-negative bacteria.
Presenter: Mimi Precit, AR Fellow, Washington State Public Health
Laboratories, Shoreline, WA, [email protected]
High-Throughput Real-Time PCR for Candida auris
Colonization Testing
A. Bateman, T. Danz, A. Valley, A. Sterkel and D. Warshauer,
Wisconsin State Laboratory of Hygiene
Candida auris is rapidly emerging worldwide. Compared with
other Candida species, C. auris has higher levels of resistance
and an increased ability to colonize skin surfaces and survive in
the environment. The Wisconsin State Laboratory of Hygiene, as a
reference center for the Antibiotic Resistance Laboratory Network,
performs C. auris colonization testing to support health care
facilities control C. auris transmission. C. auris colonization was
initially performed using a culture-based method. For improved
turn-around-time, we adapted a published real-time PCR1 assay for
rapid results and high throughput. The PCR reagents and conditions
are unchanged, but the extraction was updated to the following: two
washes with phosphate-buffered saline followed by freezing at -20C
for 30 min and heating at 70C for 30 min. The updated extraction
is inexpensive, avoids bead beating and automated nucleic acid
extraction, and can be performed in a 96-well plate format for high-
throughput testing.
Initial studies used 56 archived samples from Wadsworth Center,
New York State Department of Health with Wadsworth’s results as
the gold standard. The sensitivity of culture-based colonization was
59%, while PCR was 85%. The specificity of both was 100%. In the
second phase, 51 prospectively-collected samples were tested in
parallel with culture and PCR, with 94% (48/51) agreement. The
limit of detection of the C. auris PCR was estimated to be 10 CFU/
reaction. The specificity of the PCR was assessed by testing 10
isolates of closely related Candida species; all results were negative,
as expected. To assess reproducibility, a panel of four specimens
was run by three different microbiologists on three different days,
with consistent results and Ct values.
PublicHealthLabs
@APHL
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Presenter: Erin Young, Bioinformatics Fellow, Utah Public Health
Laboratory, Taylorsville, UT, [email protected]
variants for each gene target of interest. Conditions for a multiplex
PCR generating biotinylated amplicon for targets of interest was
optimized using DNA extracted from 12 representative bacterial
isolates and preliminarily evaluated using an additional 20 strains.
Luminex MagPlex microspheres were coupled to respective capture
DNA oligonucleotide probes designed for each target, and tested for
hybridization using reverse-compliment capture oligonucleotides as
well as biotinylated amplicon generated from a subset of 11 isolates
to ensure target specificity measured via the MagPix fluorescence
based detection system.