Evaluation for West Nile Virus (WNV) RNA in Urine of Patients Within 5 Months of WNV Infection

Tothe Editor—Gibney et al recently reported finding no West Nile virus (WNV) RNA in urine samples collected from 40 patients at 6.5–6.7 years after acute WNV disease [1]. These findings were in contrast to Murray et al, who detected WNV RNA in urine samples collected from 5 of 25 patients (20%) at 1.6–6.7 years after their initial infections [2]. We present results from a prospective evaluation of WNV RNA in urine specimens collected from 63 persons within 5 months after their acute WNV infection. During the 2010 WNV outbreak in Maricopa County, Arizona, we identified persons with laboratory evidence of acute WNV infection, including detection of WNV immunoglobulin M antibodies in serum or cerebrospinal fluid samples from patients with a clinically compatible illness or WNV RNA in serum samples from asymptomatic blood donors. Information on demographic characteristics, medical history, current medications, and clinical illness was obtained by medical record review and interview with patients or their surrogate. A urine sample was collected during a site visit. The study was approved by the Centers for Disease Control and Prevention (CDC) and Arizona Department of Health Services (ADHS) human subjects review boards, and participants provided informed consent before enrollment. Immediately after collection, urine specimens were separated into 3 aliquots, frozen on dry ice, and transported at the end of each study day to ADHS, where they were stored for ≤7 weeks at −70°C before being batch-shipped to the CDC. Urine samples were tested for WNV RNA by using both reverse-transcription polymerase chain reaction (RT-PCR) and transcription-mediated amplification (TMA), as previously described [1, 3, 4]. In addition, we added an internal RNA control (Dengue virus type 2) to each urine specimen to assess for potential inhibitory components [1]. Of the 63 enrolled participants, 35 (56%) were male, and the median age was 52 years (range, 15–83 years) (Table 1). Thirty-two participants (51%) were classified as having WNV neuroinvasive disease (ie, meningitis, encephalitis, or acute flaccid paralysis), and 26 (41%) reported ≥1 underlying medical condition. The median time from onset of clinical signs for WNV disease cases or blood donation for viremic donors to urine collection was 90 days (range, 18–129 days). Demographic and Clinical Characteristics of Participants With Recent West Nile Virus (WNV) Infection Evaluated for WNV RNA in Their Urine (N = 63) Includes 20 (63%) patients with West Nile virus encephalitis, 10 (31%) with meningitis, and 2 (6%) with acute flaccid paralysis. Demographic and Clinical Characteristics of Participants With Recent West Nile Virus (WNV) Infection Evaluated for WNV RNA in Their Urine (N = 63) Includes 20 (63%) patients with West Nile virus encephalitis, 10 (31%) with meningitis, and 2 (6%) with acute flaccid paralysis. Urine was collected either as a clean-catch specimen (60 of 63 [95%]) or from an indwelling catheter (3 of 63 [5%]). Urine specimens from all 63 participants tested negative for WNV RNA by RT-PCR. One (1.6%) tested positive by TMA; urine culture was not performed. WNV RNA was detected by RT-PCR and TMA in 2 spiked control urine specimens, which had been frozen, stored, and thawed under the same conditions as the participant specimens. Testing of participants' urine samples following addition of the RNA control yielded positive Dengue virus RT-PCR results for every participant. The WNV RNA–positive urine sample was collected 29 days after symptom onset from a woman aged 83 years who had had WNV encephalitis. Her medical history was notable for ischemic heart disease, hypertension, and colon cancer. The sample was obtained from a urine collection bag connected to an indwelling urine catheter. The patient died at 57 days after symptom onset, and additional urine samples could not be collected for further testing. The finding of WNV RNA by TMA but not RT-PCR in 1 study participant’s urine sample suggests that a low level of RNA was present in the sample. TMA can detect as few as 9.8 copies/mL of WNV RNA in blood with 95% probability, whereas the lower limit of detection for RT-PCR is approximately 200 copies/mL [4, 5]. This study has certain limitations. Only 1 urine sample was collected from each participant; thus, if WNV shedding in urine is intermittent, a single sample might have failed to detect the presence of viral RNA. Repeat serial sampling might be needed to explore whether shedding is intermittent. The delay of weeks between sample collection and testing might result in a false-negative result, although this and previous studies indicate minimal inhibition or degradation of WNV RNA in urine specimens [1]. This is the first study to our knowledge to examine urine for presence of WNV RNA among a cohort of persons with recent diagnosis (<5 months) of acute WNV infection and ranging in clinical manifestations from asymptomatic infections to severe disease. Two previous studies evaluated cohorts 1–7 years after initial acute WNV neuroinvasive disease: 1 of the studies found WNV RNA in 5 of 25 participants (20%), but the other identified no WNV RNA in urine samples from 40 WNV-infected patients. We detected WNV RNA in urine samples from 1 of 63 persons (1.6%) tested <5 months after initial acute WNV infection. Our results indicate infrequent presence of WNV RNA in urine among persons within months after acute WNV infection. The findings and conclusions of this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.