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

Background: Xylazine has been increasingly linked to human overdose deaths. No antidote has been identified and naloxone cannot reverse the effect of xylazine. Xylazine withdrawal is not alleviated by opioids. It is imperative to detect xylazine when treating overdoses. No screening method for xylazine has been approved by FDA. We aim to develop a rapid and high sensitivity xylazine test for clinical urine testing. Methods: 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. Results: 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 181 mass spectrometry confirmed clinical urine samples with xylazine concentrations > 10 ng/mL and 120 urine samples with xylazine concentrations <10 ng/mL, the dipstick demonstrated a clinical sensitivity of 100% and a clinical specificity of 97%. 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. Conclusions: When used with 10 ng/mL cutoff, the rapid xylazine dipstick demonstrates high clinical sensitivity and clinical specificity in urine samples, compared to gold standard mass spectrometry methods. This novel test has the potential to enable informed clinical decisions in suspected xylazine overdoses.