Stability of Zika virus in urine: Specimen processing considerations and implications for the detection of RNA targets in urine

Background: Detection of Zika virus (ZIKV) RNA in urine is of increasing interest for the diagnosis of ZIKV infection. Pre-analytical variables can significantly impact the stability of RNA in urine. Methods: To determine optimal specimen processing protocols that would maximize detection of ZIKV RNA in urine by real-time, reverse transcriptase PCR, we investigated the effect of temperature, initial ZIKV concentration, use of nucleic acid stabilizers, and time on ZIKV RNA levels. Urine samples from healthy donors were spiked with ZIKV using the Exact Diagnostics ZIKV Verification Panel, a commercially available panel composed of heat-inactivated ZIKV, at concentrations of 5.0 log(10) copies/mL (ZIKV-high) and 4.0 log(10) copies/mL (ZIKV-low). Samples were stored at room temperature, 4 degrees C, or -80 degrees C and frozen aliquots were exposed to no stabilizer (urine), Buffer ATL (Qiagen, Germantown, MD), or DNA/RNA Shield (Zymo Research, Irvine, CA). Results: ZIKV RNA levels in urine declined steadily at room temperature, though was not significant by 48 h (ZIKV-high, p = 0.09; ZIKV-low, p = 0.20). ZIKV RNA titers were consistently higher when stored at 4 degrees C, suggesting that storage at 4 degrees C can slow the progression of RNA degradation. Freezing urine samples at -80 degrees C resulted in a significant loss of detectable ZIKV RNA in the ZIKV-low group. ZIKV RNA was detected in 5/6 replicates at 3 days, 1/6 replicates at 10 days, and 1/3 replicates at 30 days, with findings reproducible on repeat testing. Presence of either nucleic acid stabilizer in urine corrected this effect, and resulted in recovery of ZIKV RNA in all replicates. Use of a nucleic acid stabilizer in the ZIKV-high group did not add incremental benefit for the detection or quantitation of ZIKV RNA. Conclusions: ZIKV RNA is prone to degradation in urine with loss of detectable virus even when specimens are frozen at -80 degrees C for 10 days. Detection of ZIKV-positive urine samples, particularly those containing low ZIKV titers may be aided with the addition of a nucleic acid stabilizer during urine specimen processing.