Lab Matters Summer 2019 - Page 55

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, logan.fink@state.co.us 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 PublicHealthLabs @APHL APHL.org 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, jhadley@isdh.in.gov 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, hauserj@mskcc.org Summer 2019 LAB MATTERS 53