APHL 2019 POSTER ABSTRACTS
TB (including children with HIV associated TB). Drug resistance
to the standard antimicrobials has emerged to be a serious
health challenge. Since babies and young children are unable to
expectorate sputum, aspiration or lavage of gastric fluid is the usual
and more difficult procedure for obtaining a pediatric specimen to
test for M tuberculosis and drug susceptibility. Currently, there is no
standard laboratory procedure available in the literature for culture
of pediatric gastric fluid specimens with conflicting information on
processing, value of neutralization and sample handling.
Our study, which is in its second year and funded by the NIH NIAID
contract number HHSN272201700001C for Mycobacterium
Tuberculosis (Mtb) Quality Assessment Program (TBQA), evaluated
laboratory procedures for detection of M tuberculosis in gastric fluid.
The NIAID-funded AIDS Clinical Trials group (ACTG) and International
Maternal Pediatric Adolescent AIDS Clinical Trials Network
(IMPAACT) are coordinating a large scale clinical trial on protecting
household contacts on exposure to Newly Diagnosed Index Multi-
drug Resistant Tuberculosis Patients. (PHOENIx). Household
contracts, particularly children will be evaluated for exposure to and
infection with TB via analysis of gastric fluid.
Presenter: Erin Tacheny, MRIGlobal, Kansas City, MO
[email protected]
Molecular Detection of Four Common Etiologies of Genital
Ulcer Disease Using Sequential and Parallel Testing
C.L. McGowin, S. McCune, J. Engstrom-Melnyk, Roche Diagnostics Corp
Introduction: Genital ulcers (GUs) remain a frequent chief complaint
of men and women attending US sexual health clinics, but
presumptive clinical diagnoses based solely on lesion characteristics
is insensitive for the differentiation between common sources of
GUs, which include Treponema pallidum (Syphilis), HSV1/2, and
VZV. Adding to the complexity of diagnosis, tests that directly detect
T. pallidum in lesions are not widely available and no current blood
tests exist for the diagnosis of active infections. Clinically, the ability
to accurately differentiate and identify the underlying etiology of
GUs would lead to more timely and appropriate treatment decisions,
thereby improving patient care, expanding antibiotic stewardship
impact, positively impacting public health initiatives, and ultimately
curtailing transmission. Considering the incidence of syphilis has
increased by an alarming 76% since 2013, urgent public health pleas
and published “calls to action” now exist that aim to stem this re-
emerging healthcare burden.
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LAB MATTERS Summer 2019
Method: Commercially available control material for HSV1, HSV2,
VZV, and T. pallidum were spiked into MSwab medium at varying
concentrations; eight (8) swabs from the MSwab TM System were
dipped into each of the spiked specimen vials and transferred to
their respective collection tubes. Initial testing was split between
two runs performed by the cobas ® HSV1 and 2 Test on the cobas ®
4800 System. The extracted nucleic acid remaining in the deep-well
plate was subsequently used for VZV and T. pallidum testing on the
cobas ® 4800 System User Defined Function (UDF) channel using
published primer/probe sequences.
Results: Detection of VZV and T. pallidum on the UDF channel
using nucleic acid extracts obtained following HSV1/2 testing
on the cobas ® 4800 System was possible, demonstrating high
reproducibility and precision across several replicas.
Conclusion: Novel solutions that aim to reduce empiric therapy,
or shorten the interval to treatment success, are critical for
both diagnostic and antibiotic stewardship. Through the use
of a sequential diagnostic testing algorithm, more accurate
discrimination between GU etiologies may provide valuable clinical
insight for accurate patient care.
Presenter: Chris McGowin, Roche Diagnostics, Indianapolis, IN,
[email protected]
Detection of Plasmid-mediated Colistin Resistance Genes
(mcr) by Multiplex Real-Time PCR: Improving Surveillance
of an Emerging Global Threat
E. Alao, S. Cossette, M. Torres and C. Connelly, Streck
Background: Recent increases in colistin-resistant infections led
the CDC to launch an urgent public health response for mcr gene
surveillance. Colistin is a last-resort antibiotic that is more utilized
due to increases in carbapenem-resistant infections. Since mcr-1
was first reported, more mcr gene variants have been identified,
but few screening tools have been developed to rapidly detect
mcr-positive samples. To improve surveillance for mcr genes, we
describe a multiplex real-time PCR assay that detects mcr gene
variants 1 through 5 in less than 45 minutes.
Materials/methods: This study utilizes sequence-specific primers
and probes for the real-time PCR-based detection of colistin
resistance (mcr) gene variants. An internal control (IC), targeting a
conserved region in Gram-negative bacteria, is also included in the
multiplex mix to discriminate false negatives samples. Positive DNA
controls are included with the multiplex assay. Data were generated
using the Bio-Rad CFX96 Touch™ Real-time PCR Detection System.
Results: The mcr real-time PCR multiplex assay is optimized to
amplify mcr genetic variants 1 through 5. Amplification of serial
dilutions of target controls generated PCR efficiencies ≥ 90% and
a correlation coefficient of ≥ 0.999 for all variants tested. The
specificity of the assay was tested using 80 clinical isolates and
determined to be 100%. Internal control DNA were detected in 100%
of samples and within 20 PCR cycles.
Conclusions: The mcr real time PCR assay provides a rapid
amplification and detection strategy to monitor plasmid-
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Several laboratory conditions and methods of neutralization were
assessed including time of neutralization and sample handling
conditions prior to laboratory processing. Our previous results
showed there was a benefit to neutralization of the gastric fluid,
based on increased organism recovery in paired samples. In
addition, though most samples were processed following the
Association of Public Health Laboratories (APHL) sample processing
method, our team also compared that method of sample processing
to another published method. Six rounds of experiments were
completed in all using commercially available simulated gastric
fluid inoculated with M tuberculosis H37Ra. Sample sediments post
processing were inoculated in duplicate or triplicate onto Lowenstein
Jensen agar bottles and incubated for 6 weeks, checking
periodically for growth and counting colonies. Results of these
studies indicate sample handling and processing are as important
as neutralization of fluid in organism recovery.
Here, we describe a diagnostic workflow that allows for sequential
and parallel testing to characterize GUs, relying on a combination
of IVD and LDT NAAT-based solutions performed on the cobas ®
4800 System, to detect HSV1/2, VZV, and T. pallidum from single
specimens.