Lab Matters Summer 2019 - Page 76

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 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. 74 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, 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- PublicHealthLabs @APHL 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.