A-272 Challenges in Designing an Antibiotic Gene Resistance Panel for Clinical Use
Authors: Dedhia L 
Published: September 2023

Abstract

Background
We faced 3 major challenges in our journey of designing a custom panel for antibiotic resistance genes (ABR). Challenge 1# Fast, robust, and affordable antimicrobial resistance testing (ABR) In general, qPCR-based methods are efficient, reliable, and fast with high specificity and sensitivity, and with the test results available within 2–3 h. We offer urinary tract infection identification by qPCR and wanted to supplement this test with detection of antibiotic resistance genes. we narrowed our choice of method to be qPCR based, Applied Biosystems™ OpenArray™ technology on the Applied Biosystems™ QuantStudio™ 12K Flex Real-Time PCR System. Challenge2# Genes to be tested. Antibiotic resistance occurs via various mechanism. Based on the literature review, we included the following gene in our custom panel 1. Aminoglycosides: aac(6′)-Ib-cr6, aac(6′)-Ib-cr, aac(6′)-Ib-cr4 2. Tetracycline: tet(M), tet(B) 3. Sulfonamides: sul1, sul2 4. Macrolide-lincosamide-streptogramin B-ketolides-oxazolidinones: erm C, erm A, erm B 5. Quinolone/(fluoroquinolones): qnrS , qnrA, aac(6′)-Ib-cr6, aac(6′)-Ib-cr, aac(6′)-Ib-cr4 6. Glycopeptides: VanA, VanB 7. Beta-lactam Class A: SHV, KPC, CTX-M 8. Beta-lactam Class B: NDM, VIM, IMP 9. Beta-lactam Class C: CMY, DHA, FOX, KPC 10. Beta-lactam Class D: OXA1, OXA-23, OXA- 48, 11. Methicillin resistance: mecA.

Results
Validation of the ABR panel Accuracy: 50 isolates were ordered from CDC AR isolate bank and were used to test the accuracy. The accuracy was detected to be 98% concordant. Sensitivity: The assay was sensitive to detect all urinary pathogens upto 10^3 CFU/mL. Precision: the inter and intra reproducibility was shown to be 99% concordant Specific: when tested against the CDC controls, the assays were 100% specific Challenge 3# Genotype—Phenotype correlation and prevalence of antibiotic resistance genes in USA. We compared 100 consecutive clinical specimens (urine) for genotype -phenotype co-relation. Data from the current qPCR panel and Microscan instrument was compared. It was observed for the pathogens that were phenotypically resistant to certain antibiotics, the corresponding gene association was not found in (2–50%) of antibiotic groups. This can be explained due to the limited number of genes in each drug class. Resistance to some antibiotics such as nitrofurantoin drug (100% missed) and sulphonamide drug resistance (50% missed) is induced by point mutations, which are not captured in open array genotyping qPCR assay. To overcome this gap between genotype and phenotype correlation and to understand the prevalence of antibiotic resistance genes in USA, we initiated a new project. We aim to test for 300–400 clinical samples which known resistance phenotypes and check for antibiotic resistance gene in a new expanded panel. We were successful in designing the new panel to include 88 genes (vs 30 genes in the old panel).

Conclusion
The current assay was approved by the CLIA authorities for clinical testing. In general, qPCR-based methods are efficient, reliable, and fast with high specificity and sensitivity, and with the test results available within 2–3 h.