Development and Validation of a High-sensitivity Rapid Xylazine Dipstick for Clinical Urine Testing.

Abstract

Xylazine has been increasingly identified in human overdose deaths. Detection of xylazine in clinical urine samples has clinical utility when treating overdoses. No screening method for xylazine has been approved by the US Food and Drug Administration (FDA). We aim to develop and validate a rapid and high sensitivity xylazine test for clinical urine testing. Monoclonal antibodies with high sensitivity and specificity against xylazine were developed. The leading clone was used to develop a competitive lateral flow immunoassay. The analytical cutoff, specificity, and clinical performance of this test was characterized using standards in drug-free urine and clinical urine samples. The rapid xylazine dipstick test has a test time of 5 minutes and a cutoff of 10 ng/mL xylazine in drug-free urine. No cross reactivity with other commonly used drugs or endogenous metabolites were observed, except for 3% cross reactivity with clonidine. In 190 mass spectrometry confirmed clinical urine samples with xylazine concentrations ≥10 ng/mL and 168 urine samples with xylazine concentrations <10 ng/mL, the dipstick demonstrated a clinical sensitivity of 100% and a clinical specificity of 98%. All 4 false positives had combined xylazine and 4-hydroxy-xylazine concentrations in the 5-10 ng/mL range, with additional xylazine metabolites detected by mass spectrometry. When used with 10 ng/mL cutoff, the rapid xylazine dipstick demonstrates high clinical sensitivity and clinical specificity in urine samples, compared with gold standard mass spectrometry methods. This novel test has the potential to enable informed clinical decisions in cases with suspected xylazine exposure.