APHL 2019 POSTER ABSTRACTS
to detect small clusters of isolates which may be epidemiologically
important before the epidemiologists are even aware of them, and
allow them to intervene quickly to curb the spread of disease. Our
implementation of MASH (v2.0) is run on a cloud-based 8 CPU, 30
GB RAM linux virtual machine that costs about $0.27 per hour of
operation, and the current size of the quasi-database file which
contains information from 71 sequencing runs (comprising over
2000 samples) is 16.25 MB. The benefits of the size of this system
mean that there would be no need to pay for large storage spaces
or database curation. Therefore, this system could be implemented
in laboratories for a minimal cost (as the computational and storage
burdens are extremely low). Currently, work is being performed
to automate the entire system so that it would require very little
upkeep.
Presenter: Logan Fink, Colorado Department of Public Health and
Environment, Denver, CO, [email protected]
Validation of a New Platform for the Detection of Shiga-
toxin Producing Escherichia coli
J. Yeadon-Fagbohun, R. Genry, J. Lovchik and M. Glazier, Indiana
State Department of Health Laboratories
Background: The Indiana State Department of Health Laboratories
(ISDHL) perform testing on specimens suspected of being Shiga-
toxin producing Escherichia coli (STEC). At the end of the 2018
calendar year, the validated platform for testing, the Cepheid
SmartCycler II, was discontinued, including all products used with
the instrument. ISDHL contacted the CDC and the public health
laboratories in Michigan, Minnesota, and Wisconsin for information
about their STEC PCR methods. After some research on the
various protocols, a hybrid protocol was developed for the in-house
validation. Due to ease of use and availability in our laboratory, the
Applied Biosystems (AB) 7500 Fast DX instrument was the chosen
platform for the validation. This was a variation from all of the
protocols received from the contacted state public health labs. We
also chose to add in a marker for the detection of stx 2f that has
been known to cause illness in humans.
Methods: A total of 57 specimens were used in the validation, which
included a combination of negative, stx 1 positive, stx 2 positive,
stx 1 & 2 positive, and stx 2f positive specimens. Care was taken to
ensure a variety of specimen types and different serotypes of STEC
were chosen for the validation. DNA extraction was completed using
a plate sweep and heat block method. A real-time multiplex PCR
was run to detect stx 1 and stx 2 markers in the DNA. The validation
included operator variance and repeatability studies that compared
CT values between runs and analysts.
Results: There was 100% agreement in the 57 specimens tested
between the current method on the Cepheid SmartCycler II and the
new method on the AB 7500 Fast DX. Two specimens that had to be
re-grown due to discrepant results in the original testing. Agreement
of 100% was also achieved for the repeatability and operator
variance studies, with values that were no more than than 2 CTs
between runs. The method was approved for use at ISDHL. This
method has significant cost savings for ISDHL, which was an added
bonus to the validation.
Conclusions: ISDHL was able to collaborate with three other state
laboratories and the CDC to create a hybrid protocol for the testing
of STEC specimens. The method validated is for use on a platform
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that is common in most public health laboratories. This is a low
cost screening method that detects the presence of Shiga-toxins in
a culture. The new method also increased our testing capabilities
by adding stx 2f to the targets. ISDHL was able to successfully
complete the validation in time to use this new method for the busy
summer months.
Presenter: Jamie Yeadon-Fabohun, Indiana State Department of
Health Laboratories, Indianapolis, IN, [email protected]
Stool Culture Recovery of Shiga-Toxin Producing E. coli
Detected by PCR at a Tertiary Care Cancer Center
J. Hauser, L. Gomez and N.E. Babady, Memorial Sloan Kettering
Cancer Center
Introduction: Stx-producing Escherichia coli (STEC), including
O157:H7, is a food-borne pathogen that can cause bloody
diarrhea and in ~ 3-20% of infected individuals may also cause
the hemolytic uremic syndrome (HUS). PCR has allowed for the
increased detection of STEC in diarrheal samples tested in clinical
laboratories. In New York City (NYC) and New York State (NYS), STEC
is a reportable disease and laboratories are required to submit
isolates to both the NYC and NYS Department of Health (DOH). The
implementation of culture-independent diagnostic tests (CIDTs)
including multiplexed Gastrointestinal PCRs (GIPCR) has allowed
for the increased detection of STEC. However, the rate of recovery
of STEC isolates in the corresponding stool culture is unknown. We
performed a retrospective review of all GIPCR performed in 2018 at
our institution to evaluate the recovery in stool culture of STEC from
PCR positive stool samples.
Methods: GIPCRs were performed using the BioFire® FilmArray ®
Gastrointestinal (GI) Panel. All STEC positive stools were cultured
on Hektoen Enteric and MacConkey agar. E. coli isolates were
identified by VITEK MS (bioMérieux) and Stx production confirmed
using the SHIGA TOXIN QUIK CHEK™ rapid immunoassay (TechLab)
and PROLEX™ E. coli Latex Test Reagent Kit (Pro-Lab Diagnostics,
Round Rock, TX). All isolates were sent to NYCDOH and NYSDOH for
confirmatory testing. When recovery in culture failed, an aliquot of
the stool sample was sent. Medical records of positive patients were
reviewed to identify possible risk factors and confirm symptoms.
Results: 4521 GI PCRs were performed in 2018 with 29/4521
stools (0.6%) positive for STEC. E. coli was recovered from 25/29
(89%) stool samples from 23 unique patients. Stx production was
confirmed in 9/25 (%) isolates with 6/9 (67%) producing stx1 and
1/9 (11%) isolates producing both stx1 and stx2. Both NYCDOH
and NYSDOH laboratories confirmed the presence of toxin in all 9
isolates with additional serotyping including O157:H7 (1/9) and
non-O157 serotypes (3/9). 5/9 isolates were non-typeable. All
23 patients reported non-bloody, soft to watery diarrhea. 2/23
cases were possibly food related and 2/23 patients tested positive
following prophylaxis for dental procedures.
Conclusion: Detection of STEC by GIPCR in our patient population
was low with only a third recovered in stool culture. Our study
highlights both the advantages and limitations of CIDT methods to
increase detection of STEC but with limited recovery of the isolate
as needed for further public health surveillance work. Further
studies aimed at either improving recovery in culture or performing
surveillance directly on stool samples are needed.
Presenter: Jocelyn Hauser, CPEP Fellow, Memorial Sloan Kettering
Cancer Center, [email protected]
Summer 2019 LAB MATTERS
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