(41) Xylazine Test Strip Performance in Synthetic and Human Urine Biospecimens

Background & Introduction: Xylazine, a veterinary sedative not approved by the United States Food and Drug Administration for human use, is increasingly linked with adulteration of illicit opioids in the United States. Epidemiological studies demonstrate the growing prevalence of xylazine detection in street drug samples and in decedents in whom testing is performed.1,2 However, xylazine detection in clinical assessments of people who use opioids is limited to settings with access to gas chromatography/mass spectrometry (GC/MS) laboratory testing. Xylazine lateral flow chromatographic immunoassays (“test strips”) have been validated in drug testing with manufacturer-reported sensitivity up to 1000 ng/mL for use in assessing drug samples for the presence of xylazine. The contribution of these test strips to clinical practice is limited because most patients do not have drugs available for testing at sites of clinical care. GC/MS urine and serum xylazine tests have high specificity, but these tests are expensive and time intensive with delays in result reporting. We hypothesized that xylazine test strips in urine could serve as a rapid, point-of-care test to detect xylazine exposure. This could improve treatment for fentanyl-xylazine withdrawal and for xylazine-associated wound management.

Methods: Xylazine tests strips from BTNX (Ontario, Canada) were used in this study. We tested 6 synthetic urine samples spiked with xylazine standard to final concentrations of 0, 250, 500, 750, 1000 and 2000 ng/mL. We also evaluated 36 human urine samples submitted for xylazine testing to our hospital toxicology laboratory with GC/MS to estimate the sensitivity and specificity of test strip detection of xylazine in urine. Urine samples were tested within 1-13 days after collection and were stored at 5 °C until analyzed.

Results: Among the 6 synthetic urine samples mixed with xylazine standard, test strips detected xylazine in concentrations of 750-2000 ng/mL but did not detect xylazine in concentrations of 250-500 ng/mL. These results indicate that the cutoff level is more sensitive than that reported by the manufacturer of 1000 ng/mL.
Among 29 human urine samples with xylazine detected with GC/MS, 26 (89.6%) tested positive for xylazine using test strips. Semiquantitative evaluation of the 3 false negative samples demonstrated concentrations of less than 20 ng/mL which is significantly lower than the cutoff value of the xylazine strips. These results suggest that the false negatives were caused by the lower sensitivity of the xylazine strip at low xylazine concentrations in comparison with GC/MS.
For the 7 patient urine samples with GC/MS testing negative for xylazine, 5 (71%) tested negative and 2 (29%) tested positive for xylazine using test strips. Further investigation was carried out in the two samples (A and B) that gave false positive results. Sample A tested positive for cocaine metabolites and lidocaine. Lidocaine has been previously reported to yield false positive results.3,4 In contrast, sample B did not contain lidocaine but tested positive for cocaine metabolites, diphenhydramine, and nicotine metabolites. Cocaine, benzoylecgonine, cotinine, nicotine and diphenhydramine standards were spiked in synthetic urine up to a concentration of 20 000 ng/mL and did not exhibit false positive results. These results indicate that the origin of the interference for sample B is unknown and further investigation with a larger drug database is required to find more potential interfering candidates.

Conclusion & Discussion: In this pilot study, xylazine test strips detected xylazine at a concentration of >750 ng/mL in synthetic urine spiked with xylazine. These test strips had moderate sensitivity and lower specificity in detecting xylazine in urine collected in exposed patients. Testing urine with xylazine test strips could be a feasible approach to rapid, point-of-care testing for xylazine exposure in clinical settings and should be rigorously explored.

References: 1. Friedman J, Montero F, Bourgois P, Wahbi R, Dye D, Goodman-Meza D, Shover C. Xylazine spreads across the US: A growing component of the increasingly synthetic and polysubstance overdose crisis. Drug Alcohol Depend. 2022;233:109380. doi: 10.1016/j.drugalcdep.2022.109380.
2. Kariisa M, O’Donnell J, Kumar S, et al. Illicitly Manufactured Fentanyl–Involved Overdose Deaths with Detected Xylazine — United States, January 2019–June 2022. MMWR Morb Mortal Wkly Rep 2023;72:721–727. DOI: https://dx.doi.org/10.15585/mmwr.mm7226a4
3. Krotulski AJ, Shinefeld J, DeBord J, Teixera da Silva D, Logan BK: Evaluation of xylazine test strips for drug checking purposes. www.cfsre.org/images/content/reports/drug_checking/Evaluation_of_BTNX_Xylazine_Test_Strips_for_Drug_Checking_230322.pdf). Published March 22, 2023. Accessed December 22, 2023.
4. Sisco, E, Nestadt DF, Bloom MB, et al: Understanding sensitivity and cross-reactivity of xylazine lateral flow immunoassay test strips for drug checking applications. Drug Test Anal. 2023 Dec 3. doi: 10.1002/dta.3612