Acuitas MDRO Gene Test

ICAAC
2014
D-892
Specificity, Reproducibility and Sensitivity of the Acuitas™ MDRO Gene Test for Direct Detection of
KPC, NDM, VIM, IMP, OXA, CTX-M and VanA Resistance Gene Families Associated with MultidrugResistant Organisms (MDROs) from a Single Peri-anal Swab
T. Rockweiler, R. Kersey, J. Quan, K. Frye, S. Mitchner, T. Wagner, D. Toal and T. Walker
OpGen Inc., Gaithersburg MD, USA
Introduction
Multi-drug resistant organisms (MDRO) are a major and
growing global concern.
Every year, more than two
million Americans are infected with antibiotic resistant
bacteria, resulting in 23,000 deaths.1
The CDC
recommend
screening
measures
for
carbapenemresistant Enterobacteriaceae (CREs) including point
prevalence surveys of patients and healthcare providers
in hospital and long-term care facilities, active
surveillance testing of pre-specified high-risk patients,
and epidemiological linkage of colonized carriers and
infected patients.2 Patients colonized with multidrugresistant organisms (MDROs) pose a risk for active
infection or transmission of MDROs to other patients.
OpGen provides Acuitas™ MDRO Gene Test results from
peri-anal swabs within 24 hours after sample receipt
through its CLIA laboratory for detection of antibiotic
resistance genes directly from peri-anal swab specimens
for CREs, carbapenem-resistant Gram-negative bacilli,
ESBLs and VREs. The Acuitas™ MDRO Gene Test detects
antibiotic resistance genes in actively infected patients or
subjects colonized with MDROs as an aid to patient
management and antibiotic stewardship for improved
patient outcomes, transmission prevention and control,
and better health economics.
Methods
The Acuitas™ MDRO Gene Test is a multiplex PCR test
that detects the antibiotic resistance gene families KPC,
NDM, VIM, IMP, OXA, CTX-M and VanA across hundreds
of gene subtypes. We evaluated the specificity of the
test by genotyping 243 clinical isolates with reported
antibiotic resistance genotypes and phenotypic antibiotic
susceptibility profiles.
Analytical sensitivity and
reproducibility were demonstrated using peri-anal swabs
spiked with antibiotic resistant culture isolates.
Conclusions
1. The Acuitas™ MDRO Gene Test is a sensitive and
specific test for direct detection of antibiotic resistance
genes from CREs, ESBLs and VREs using peri-anal swab
specimens and is suited for use as
a
rapid and
accurate MDRO screening test in healthcare facilities.
2. Specificity results from the Acuitas™ MDRO Gene Test
were 100% consistent with reported antibiotic
resistance genotypes or resolved genotypes using
independent PCR tests as confirmation.
3. Antibiotic resistance genotypes were consistent with
reported antibiotic susceptibility profiles. The test’s
limit-of-detection (LOD) ranges from 13 to 250 CFUs
per peri-anal swab for the individual target antibiotic
resistant genes.
4. The test exhibited excellent reproducibility over three
days, multiple laboratory operators and gene target
levels with replicate PCR cycle threshold values (Ct)
exhibiting intra- and inter-assay variation <12% CV.
5. The Acuitas MDRO Gene Test is more sensitive (100%)
than CRE culture (72%) for the detection of CREs.
References
1CDC.
Antibiotic Resistance Threats in the United States,
2013.1-1114 (2013).
2Guidance for Control of Carbapenem-resistant
Enterobacteriaceae (CRE). CRE Toolkit from the CDC
(2012) .
OpGen Inc.
708 Quince Orchard Road, Gaithersburg, Maryland 20878
301.869.9683
[email protected]
Results
Analytical Specificity
Table 1
Table 2
Table 3
(Tables 1 & 2)
Test specificity was demonstrated across a variety of gene
subtypes using 243 culture isolates with reported
genotypes for KPC, NDM, VIM, IMP, OXA, CTX-M and other
beta lactamase genes along with VanA. The specificity
panel included several common enteric species with a
variety of phenotypic antibiotic susceptibility profiles
including susceptible Gram-negative bacilli, carbapenemresistant Gram-negative bacilli, cephalosporin-resistant
(3rd
or
4th
generation)
Gram-negative
bacilli,
Vancomycin resistant Enterococci and Vancomycin
susceptible Enterococci.
Test specificity was 100% consistent with reported
antibiotic resistance genotypes or resolved genotypes
using independent PCR confirmation tests. The test did
not cross react with other antibiotic resistance genes such
as SHV, TEM, ACT/MIR, CMY-2, DHA-1, ACT-16 or VEB-1.
The antibiotic resistance genotypes were consistent with
reported phenotypes based on antibiotic susceptibility
testing.
Reproducibility
Table 4
(Table 3)
The gene test exhibited excellent reproducibility over
three days, multiple laboratory operators and gene target
levels with replicate PCR cycle threshold values (Ct)
exhibiting intra- and inter-assay variation <12% CV.
Analytical Sensitivity
(Table 4)
We
demonstrated
analytical
sensitivity
(limit-ofdetection, LOD) for each target antibiotic resistance gene
in the test using negative peri-anal swabs that were
quantitatively spiked with bacterial culture isolates
harboring reported antibiotic resistant genotypes. Spiked
culture levels were determined by parallel counting of
colony forming units (CFUs) on culture plates.
LODs for the target antibiotic resistance genes in the test
ranged from 13 to 250 CFUs per peri-anal swab.
Table 5
Gene Test Performance vs. CRE Culture
(Table 5)
Results from the Acuitas™ MDRO Gene Test were
compared to results from a conventional CRE screening
culture (Quest Diagnostics, Test Code 91669) using
negative
peri-anal
swab
specimens
that
were
quantitatively spiked with 16 clinical isolates that
included Enterobacteriaceae and carbapenem-resistant
Enterobacteriaceae (CRE) with a variety of phenotypic
antibiotic susceptibility profiles. Each isolate was tested
at approximately 200, 2000, 20,000 and 200,000 colony
forming units (CFUs) per peri-anal swab.
The Acuitas™ MDRO Gene Test was more sensitive than
the CRE screening culture with the gene test detecting 10
CRE samples that were negative by CRE culture including
some or all CFU levels of the following organisms with the
indicated carbapenemase genes families: K. pneumoniae
(VIM), K. pneumoniae (IMP), E. coli (NDM and CTX-M-1)
and S. marcescens (OXA-48).
Additionally, the Acuitas™ MDRO Gene Test detects other
carbapenem-resistant Gram-negative bacilli [e.g. A.
baumannii (OXA-23 and OXA-51) and P. aeruginosa
(VIM)] for which the CRE culture is not designed.
Detection of CREs by the Acuitas
MDRO Gene Test
A cuit as M D R O
Gene T est
Po sit ive f o r C R E
Gene
N eg at ive f o r C R E
Gene
C R E b y ID / A ST
Po sit ive
N eg at ive
36
0
0
28
Sensitivity of M DRO Gene Test
100%
Specificity of M DRO Gene Test
100%
Positive Predictive Value of M DRO Gene Test
100%
100%
Negative Predictive Value of M DRO Gene Test
CRE by ID/AST
Detection of CREs by CRE Culture
Positive
Negative
Po sit ive
26
0
N eg at ive
10
28
C R E C ult ur e
Sensitivity of CRE Culture
Specificity of CRE Culture
72%
100%
Positive Predictive Value of CRE Culture
100%
Negative Predictive Value of CRE Culture
74%