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.

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 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.

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. Competing Interest Statement The study was funded by a National Institute on Drug Abuse grant 1U44DA060264 to Ping Wang and Xiaofeng Xia. The other authors have no conflict of interest. Funding Statement The study was funded by a National Institute on Drug Abuse grant 1U44DA060264. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of University of Pennsylvania gave ethical approval for this work. IRB of Washington University gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present work are contained in the manuscript