2501. Real-Time PCR Targeting Mosaic penA XXXIV for Prediction of Extended-Spectrum Cephalosporins Susceptibility in Clinical Neisseria gonorrhoeae Isolates
Session: Oral Abstract Session: STIs - Diagnostics and Therapy
Saturday, October 7, 2017: 2:45 PM
Room: 07AB
Background: Antimicrobial resistant Neisseria gonorrhoeae (NG) is a global public health problem, resulting in limited empirical treatment options. Due to increasing minimum inhibitory concentrations (MICs) of ESCs against NG in the US, it is critical that susceptibility to ESCs be monitored. Since few laboratories routinely perform culture & susceptibility testing for NG, there is a need for a rapid test to predict susceptibility to ESCs. More than 98% of isolates with decreased susceptibility to cefixime (CFM) in the US carry mosaic penA XXXIV. In this study, we developed a multiplex real-time PCR for mosaic penA XXXIV & previously validated gyrA to predict ESCs MICs & ciprofloxacin (CIP) susceptibility.
Methods: 150 NG isolates with known cefpodoxime (CPD), CFM, ceftriaxone (CRO) & CIP MICs were obtained from Neisseria Reference Laboratory at University of Washington & CDC Antimicrobial Resistance Bank. DNA extracted from culture was used in multiplex HybProbe real-time PCR on Lightcycler 480. gyrA was genotyped by melt curve and served as internal control, while presence of mosaic penA XXXIV was detected by selective amplification.
Results: All 32 (100%) CIP-susceptible & 118 (100%) CIP-resistant isolates, as determined by Clinical and Laboratory Standards Institute breakpoints, demonstrated wild-type & Ser91 mutant gyrA genotype, respectively. Melt curve genotyping demonstrated mosaic penA XXXIV melt patterns in 66/68 (97%) isolates with at least one ESC MIC above alert value set forth by the CDC (CPD and CFM MICs ≥0.25 µg/ml; CRO MIC ≥0.125), while all 82 (100%) isolates with ESC MICs under alert values did not amplify. The first of the 2 false-negative isolates had MICs above alert values for all ESCs tested and harbored IX mosaic type, while the second one had CRO MIC above alert value and harbored XII mosaic type. Both of these mosaic types did not share homology with mosaic penA XXXIV in the region targeted by the assay.
Conclusion: The mosaic penA XXXIV assay demonstrated 97% sensitivity and 100% specificity in predicting alert ESCs MIC values among clinical isolates tested, and was successfully multiplexed with gyrA assay. Clinical utility of this assay may be limited due to false negativity in isolates with non-XXXIV mosaic types, but it could serve as a useful surveillance tool for XXXIV mosaic.
Peera Hemarajata, MD, PhD, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, Lisa Wong, MPH, Emory University School of Medicine, Atlanta, GA, Olusegun Soge, PhD, Neisseria Reference Laboratory, University of Washington, Harborview Medical Center, Seattle, WA, Romney Humphries, PhD, UCLA David Geffen School of Medicine, Los Angeles, CA and Jeffrey Klausner, MD MPH, Division of Infectious Diseases, Department of Medicine, University of California, Los Angeles, Los Angeles, CA


P. Hemarajata, None

L. Wong, None

O. Soge, None

R. Humphries, Roche: Consultant , Consulting fee

J. Klausner, None

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