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Stability of Zika virus in urine: Specimen processing considerations and implications for the detection of RNA targets in urine
Accepted Manuscript Title: Stability of Zika Virus in Urine: Specimen Processing Considerations and Implications for the Detection of RNA Targets in Urine Authors: Susanna K. Tan, Malaya K. Sahoo, Stephen Milligan, Nathaniel Taylor, Benjamin A. Pinsky PII: DOI: Reference:
S0166-0934(17)30077-0 http://dx.doi.org/doi:10.1016/j.jviromet.2017.04.018 VIRMET 13248
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
1-2-2017 28-4-2017 29-4-2017
Please cite this article as: Tan, Susanna K., Sahoo, Malaya K., Milligan, Stephen, Taylor, Nathaniel, Pinsky, Benjamin A., Stability of Zika Virus in Urine: Specimen Processing Considerations and Implications for the Detection of RNA Targets in Urine.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2017.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Stability of Zika Virus in Urine: Specimen Processing Considerations and Implications for the Detection of RNA Targets in Urine Susanna K. Tan1, Malaya K. Sahoo2, Stephen Milligan3, Nathaniel Taylor3 and Benjamin A. Pinsky1 ,2 ,3 ,* 1
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford
University School of Medicine, Stanford, California, United States of America 2
Department of Pathology, Stanford University School of Medicine, Stanford, California, United
States of America 3
Clinical Virology Laboratory, Stanford Health Care, Stanford, California, United States of
America *
Corresponding author: 3375 Hillview Ave, Room 2913, Palo Alto, CA 94304; Phone: +001
(650) 721-1896; Fax: +001 (650) 723-6918; E-mail: [email protected]
1
Highlights
Studied protocols to maximize detection of ZIKV RNA in urine
Evaluated temperature, time, ZIKV levels, and use of nucleic acid stabilizers
At low levels of viruria observed loss of detectable ZIKV RNA over time at -80C
Use of nucleic acid stabilizers resulted in recovery of ZIKV RNA
Summary 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 log10 copies/mL (ZIKV-high) and 4.0 log10 copies/mL (ZIKV-low). Samples were stored at room temperature, 4C, or -80C 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 hours (ZIKV-high, p=0.09; ZIKV-low, p=0.20). ZIKV RNA titers were consistently higher when stored at 4C, suggesting that storage at 4C can slow the progression of RNA degradation. Freezing urine samples at -80C 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.
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Conclusions: ZIKV RNA is prone to degradation in urine with loss of detectable virus even when specimens are frozen at -80C 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.
Keywords: Zika virus; Urine; RNA Stability; Pre-Analytical
1.0
Introduction
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that, in 2015, rapidly spread throughout the Americas and Caribbean. ZIKV disease is characterized by fever, myalgias, arthralgias, headache, conjunctivitis, and maculopapular rash, and is linked with devastating neurologic complications, such as fetal microcephaly and Guillain-Barre syndrome (Brasil et al., 2016; Russell et al., 2016; Waggoner and Pinsky, 2016). Complications from ZIKV reached epidemic proportions in 2016, leading the World Health Organization to declare a public health emergency of international concern (2016). The diagnosis of ZIKV infection has relied on the detection of ZIKV RNA or ZIKV IgM antibodies in serum, though duration of viremia is short and cross-reactivity of ZIKV antibodies with other flaviviruses, such as dengue virus (DENV), make the diagnosis of acute ZIKV infection challenging. Detection of ZIKV RNA in urine is of increasing interest, given reports of longer duration of virus shedding and at higher concentrations compared to serum. Several studies describe virus detection that persists >7 days longer in urine than in serum (Barzon et al., 2016; Campos Rde et al., 2016; Fourcade et al., 2016; Gourinat et al., 2015; Zhang et al., 2016). ZIKV RNA may also be detected in twice as many urine than serum specimens when both are tested concurrently in ZIKV-positive patients (Bingham et al., 2016). Though variability in these results has also been reported (Pessoa et al., 2016), all together, these studies emphasize the suitability of urine for ZIKV screening and diagnosis and highlight the potential utility of urine ZIKV RNA testing. However, conditions in urine pose unique challenges for nucleic acid detection and preanalytical variables can further impact the stability of viral RNA. While little is known about ZIKV RNA stability in urine, RNA in urine may be particularly sensitive to degradation. Urine is a suitable environment for nucleic acid hydrolyzing enzymes and contains several types of
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RNases with abundance and activity as much as 100-fold higher than in serum (Bryzgunova and Laktionov, 2015). In addition to matrix-specific effects, pre-analytical variables, such as specimen handling and storage, can further reduce the yield of ZIKV RNA in samples. Given the susceptibility of RNA degradation in urine, understanding pre-analytical specimen processing procedures that maximize the integrity of ZIKV RNA can optimize the utility of this specimen type for diagnostic testing. Here we evaluate the steps between urine collection and nucleic acid extraction that influence the yield of ZIKV RNA detected by real-time, reverse transcriptase-PCR (rRT-PCR). Specifically, we investigate the effect of storage temperature, freeze-thaw, and time on levels of ZIKV RNA in urine. We also evaluate the use of nucleic acid stabilizers and determine scenarios in which the addition of a nucleic acid stabilizer in urine may improve the detection of ZIKV RNA. Nucleic acid stabilizers may minimize RNA degradation by inactivating nucleases and preserving nucleic acids that are originally present in clinical samples (Anwar et al., 2009; Blow et al., 2008; Medeiros et al., 2003). Lastly, we study the effect of initial ZIKV concentrations on subsequent ZIKV RNA detection. This study aims to identify pre-analytical variables that impact the integrity of ZIKV RNA and specimen handling procedures that can maximize the detection and quantitation of ZIKV RNA in urine.
2.0
Materials and Methods
Ethics Statement. This study was reviewed and approved by the Institutional Review Board of Stanford University.
Study Samples. Urine samples from healthy donors were pooled and spiked with ZIKV using the Exact Diagnostics® ZIKV Verification Panel (Exact Diagnostics, Fort Worth, TX), a commercially available panel composed of several concentrations of heat-inactivated ZIKV formulated in EDTA plasma. Ten-fold serial dilutions of the top panel member (6.0 log10 copies/mL by digital droplet RT-PCR) were performed to obtain two concentrations of ZIKV in urine of 5.0 log10 copies/mL (ZIKV-high) and 4.0 log10 copies/mL (ZIKV-low), respectively.
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For plasma and urine comparisons, EDTA plasma (SeraCare, Milford, MA) was similarly spiked with ZIKV using Exact Diagnostics® ZIKV Verification Panel. Both plasma and urine stocks were confirmed negative for ZIKV RNA by rRT-PCR prior to use in the study.
Storage Conditions. To determine initial storage conditions that minimized degradation of ZIKV in urine, urine samples containing ZIKV at both concentrations were stored at room temperature (25C) or at 4C for 3, 6, 12, 24, and 48 hours prior to undergoing nucleic acid extraction and ZIKV RNA quantitation. Baseline (0 hours) samples proceeded directly to nucleic acid extraction and ZIKV RNA quantitation. All conditions were tested in triplicate.
Freeze-Thaw and Stabilizers. To determine the effect of storing specimens at -80C and the impact of nucleic acid stabilizers on ZIKV in urine, we exposed aliquots of virus to equal parts of no stabilizer (urine), Buffer ATL (Qiagen, Germantown, MD), or DNA/RNA Shield™ (Zymo Research, Irvine, CA). Samples were stored at -80C for 3 days, 10 days, and 30 days, and subsequently underwent thawing, nucleic acid extraction, and ZIKV RNA quantitation. Baseline (0 hours) samples did not undergo freeze-thawing and proceeded directly to nucleic acid extraction and ZIKV RNA quantitation. To account for the plasma (EDX panel matrix) content of the spiked urine, baseline, day 3, and day 10 experiments were performed a second time in a separate run with dilutions for ZIKV-low made from 5.0 log10 copies/mL panel member and dilutions for ZIKV-high made from the 6.0 log10 copies/mL panel member. All conditions were tested in triplicate. To further evaluate freeze-thaw effects, we subjected urine aliquots at both concentrations to overnight (18 hours) freezing at -80C, which was followed by extraction and rRT-PCR.
Nucleic Acid Extraction. Total nucleic acid was isolated from 200L of urine or plasma samples using the EZ1 Virus Mini Kit, v2.0 on the EZ1 Advanced XL instrument (Qiagen, Germantown, MD). All extractions were carried out according to the manufacturer’s recommendations. The purified nucleic acid was eluted into a final volume of 60L containing AVE buffer (Qiagen, Germantown, MD). rRT-PCR was performed immediately after extraction.
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ZIKV rRT-PCR Assay. Samples were tested for ZIKV using a single-reaction, multiplex rRTPCR assay that detects ZIKV, Chikungunya (CHIKV), and Dengue (DENV) viruses as previously described (Waggoner et al., 2016). Briefly, rRT-PCR were performed on the RotorGene Q (Qiagen, Germantown, MD) real-time PCR instrument and involved 25L reactions of the SuperScript III Platinum One-Step qRT-PCR kit (Life Technologies, Carlsbad, CA, USA) and 10L of RNA template. Cycling conditions for the ZCD assay were as follows: 52°C for 15 min; 94°C for 2 min; 45 cycles at 94°C for 15 sec, 55°C for 40 sec, and 68°C for 20 sec. Each run included a no-template control and positive controls for ZIKV. For quantitation, standard curves were prepared using quantitated ssDNA (Integrated DNA Technologies) containing the target sequences.
Statistical Analysis. ZIKV RNA concentrations in log10 copies/mL for each test condition were compared to baseline and between groups using unpaired t-tests. Statistical analysis was performed in Excel (Microsoft, Redmond, WA) and GraphPad software (GraphPad, San Diego, California). Figures were generated using Prism. A P value <0.05 was considered statistically significant.
3.0
Results
Room Temperature and 4C. We evaluated the effect of temperature (room temperature and 4C) at several time points (0, 3, 6, 12, 24, and 48 hours) on ZIKV RNA stability (Figure 1). Degradation of ZIKV displayed a steady decline at room temperature, though was not statistically significant at 48 hours when compared to baseline (ZIKV-high, p=0.09; ZIKV-low, p=0.20). The mean difference was -0.37 log10 copies/mL with a standard deviation (SD) of 0.25 for ZIKV-high, and -0.47 log10 copies/mL with SD of 0.33 for ZIKV-low. When urine was stored at 4C, the decline in ZIKV RNA load was marginal and ZIKV RNA loads were higher at each time point compared to room temperature, suggesting that storage at 4C can slow the progression of ZIKV degradation in urine and better maintain ZIKV titers.
Freeze-Thaw Effects. A single freeze-thaw of urine samples stored at -80C overnight (<24 hours) followed by extraction and rRT-PCR showed no effect on ZIKV RNA levels (ZIKV-high, 6
p=0.67; ZIKV-low, p=0.37). However, duration of freezing at -80C had a significant impact on both ZIKV RNA detection and quantity, particularly in the ZIKV-low group.
In the first
experiment, 2/3 replicates remained detectable at 3 days in the ZIKV-low group, 1/3 replicates at 10 days, and 1/3 replicates at 30 days of storage at -80C (Table 1). These findings were reproducible on repeat testing; all three replicates in the ZIKV-low group were negative for ZIKV RNA by day 10. These results confirmed our initial observations and demonstrated that the plasma matrix content of the urine dilutions did not substantially contribute to ZIKV-low stability. In the ZIKV-high group, the finding was less distinct, with ZIKV RNA remaining detectable in 5/6 replicates at 10 days across the two experiments.
Of the detectable replicates, there was a notable decline in ZIKV RNA load over time, most significant in the ZIKV-low group (Figure 2). For the ZIKV-low group, the mean difference was -0.49 log10 copies/mL with SD of 0.33 at 10 days (p=0.001) and -1.20 log10 copies/mL with SD of 0.53 at 30 days (p=0.001), though this represents 1/6 total replicates performed at 10 days and 1/3 total replicates at 30 days, as all other replicates no longer contained detectable ZIKV RNA. In the ZIKV-high group, there was a significant decline in ZIKV RNA levels by day 30 (p=0.002) with a mean difference of -0.55 log10 copies/mL and SD of 0.31. No changes in detection or levels of virus were seen in plasma frozen at -80C over time (ZIKV-high, p=0.71; ZIKV-low, p=0.51) indicating that the observed effects are urine matrix specific.
Nucleic Acid Stabilizers. To evaluate the effect of Buffer ATL (ATL) and DNA/RNA Shield (Shield) on ZIKV RNA stabilization in urine, urine aliquots containing equal volumes of nucleic acid stabilizer were tested in parallel with ZIKV-positive urine aliquots stored at -80C. Unlike urine without stabilizer, urine with ATL or Shield resulted in the detection of ZIKV RNA in all replicates, including those that were lost in the urine without stabilizer subgroup. Furthermore, the use of either ATL or Shield improved levels of ZIKV RNA recovered at 10 days (ATL, Shield, p<0.001) and 30 days (ATL, Shield, p=0.03) compared to the urine without stabilizer subgroup in the ZIKV-low group (Figure 2a). By contrast, no differences in ZIKV RNA levels were found at 3 days, 10 days, or 30 days between urine only, ATL, and Shield ZIKV-high subgroups (Figure 2b). Comparison of ATL and Shield at all time points did not reveal any differences in ZIKV RNA detection or levels.
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ZIKV RNA levels significantly declined by 30 days of freezing at -80C, regardless of starting viral load and use of a nucleic acid stabilizer, highlighting the effect of duration of freezing on ZIKV RNA levels. In the ZIKV-high group, the mean decline was approximately -0.5 log10 copies/mL by 30 days across all subgroups (Urine, -0.55 log10 copies/mL, SD 0.32, p=0.002; ATL, -0.56 log10 copies/mL, SD 0.29, p=0.001; Shield, -0.49 log10 copies/mL, SD 0.25, p=0.001). A similar decline was seen by 30 days of freezing at -80C in the ZIKV-low group, though the decline was greatest in the urine without stabilizer subgroup (Urine, -1.20 log10 copies/mL, SD 0.53, p=0.001; ATL, -0.30 log10 copies/mL, SD 0.17, p<0.001; Shield, -0.68 log10 copies/mL, SD 0.16, p<0.001).
Altogether, these findings suggest that use of nucleic acid stabilizers improves the recovery of ZIKV RNA, particularly when the abundance of ZIKV RNA is low and samples are frozen at 80C for as a short as 10 days.
4.0
Discussion
Pre-analytical variables can significantly impact the reliability of laboratory test results, and, in particular, influence the integrity of nucleic acids detected by molecular methods. In urine, proper specimen handling may be of increased importance given the several-fold higher RNAse activity and relative instability of RNA (Bryzgunova and Laktionov, 2015; Cherepanova et al., 2007). Here, we evaluated specimen processing conditions that optimize the recovery of ZIKV RNA in urine. We investigated the effect of temperature, initial ZIKV levels, nucleic acid stabilizers, and time between specimen collection and nucleic acid extraction on ZIKV RNA detected and quantified by rRT-PCR. As expected, ZIKV RNA levels declined when urine samples were stored at room temperature, though was not statistically significant at 48 hours. When samples were stored at 4C, the mean ZIKV RNA levels were improved and higher at each time point compared to room temperature. Detection of ZIKV RNA was preserved with freezing at -80C for up to 30 days, but only in urine specimens containing higher ZIKV titers (5.0 log10 copies/mL). Storing urine with lower ZIKV titers (4.0 log10 copies/mL) at -80C for as few as 10 days resulted in a 8
significant loss of detectable ZIKV RNA, findings that were present in multiple replicates and on repeat testing. The addition of either ATL buffer or DNA/RNA Shield to urine resulted in the detection of all ZIKV replicates lost with freezing at -80C, and also improved quantitative recovery of ZIKV RNA. At higher ZIKV levels, no difference in detection or quantitation of ZIKV RNA was found between urine with and without stabilizer. These findings are consistent with studies evaluating the stability of other flaviviruses in serum and urine specimens. Storage of serum and urine samples containing Hepatitis C virus (HCV) and dengue virus (DENV) at 4°C slowed the progression of viral RNA degradation (Halfon et al., 1996; Van den Bossche, Cnops and Van Esbroeck, 2015). Similarly, HCVpositive serum specimens stored at -20°C or -70°C resulted in decreased HCV levels, but remained detectable after storage for at least 6 months to several years (Baleriola et al., 2011; Halfon et al., 1996). DENV RNA in urine specimens also remained detectable at -20°C after one year (Van den Bossche, Cnops and Van Esbroeck, 2015). These findings are comparable to findings in the ZIKV-high group, but are in contrast to the ZIKV-low group where there was significant loss in detectable ZIKV RNA by 10 days. Use of a lysis buffer to stabilize viral RNA in serum has previously been demonstrated, extending the period in which DENV RNA remains detectable at room temperature and Venezuelan equine encephalitis and Rift Valley Fever virus RNA at 4°C and -20°C (Anwar et al., 2009; Blow et al., 2008). The utility of a nucleic acid stabilizer in urine specimens that are stored at -80°C to maintain ZIKV RNA detection has not been previously reported. All together, these findings indicate that urine specimens being evaluated for ZIKV can be stored at room temperature for 24-48 hours without significant impact on ZIKV RNA levels, and that storage of specimens at 4°C can further minimize nucleic acid degradation. Urine specimens may be frozen at -80°C for brief periods (within 3 days) without significant effect on ZIKV RNA detection; however, freezing at -80°C for longer periods, such as 10 days, may result in loss of detectable ZIKV RNA and a false negative test result, particularly if the ZIKV load in the urine specimen is low. To accurately detect ZIKV in urine specimens containing low levels of ZIKV, prompt nucleic acid extraction and testing would be optimal. However, if delays in testing are anticipated, the addition of a nucleic acid stabilizer, such as ATL or Shield, can maintain detection of ZIKV. Notably, adding a stabilizer to specimens before sending out for testing would need to be approved and validated by the receiving reference laboratory.
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The loss of low-level ZIKV RNA when urine is stored at -80°C is of uncertain clinical significance. Case reports of ZIKV-infected individuals document ZIKV titers in urine up to 8.0 log10 copies/mL (Lamb et al., 2016). This may reflect a clinical course consistent with high titer ZIKV shedding in urine or represent publication bias due to loss of lower ZIKV RNA concentrations from variations in specimen handling. Thus, a missed ZIKV diagnosis may occur only in patients who present particularly late after symptom onset. While titers are likely to drop with freezing at -80°C for extended duration, ZIKV RNA originally present at high levels in the urine should remain detectable. The significance of quantitative values for ZIKV in urine for diagnosis or prognosis has not yet been established. Limitations of this study include the narrow window of ZIKV concentrations tested and the extent of time points evaluated. The 1.0 log10 copies/mL gap between high-level and lowlevel ZIKV was necessitated by the relatively low stock concentration of the available reference material. It is also important to note that this reference material is comprised of heat inactivated virus, and additional experiments using split clinical specimens will be required to further evaluate the routine use of stabilizers in a clinical setting. This study evaluated the stability of ZIKV up to 48 hours at room temperature and 4°C, and 30 days at -80°C, and was not extended to determine time in which ZIKV RNA was no longer detectable. Notably, this study was performed at a clinical laboratory with validated, in-house ZIKV rRT-PCR capabilities. Time points were selected to assist with identifying optimal in-house protocols, where specimen processing and testing can occur relatively rapidly, a scenario that may not be present at other institutions or in research protocols where samples may be stored for months before testing. However, study findings can still be applied to institutions that send-out specimens for ZIKV RNA testing to reference or public health laboratories. Specimen handling should ideally involve refrigeration at 4°C or freezing specimens at -80°C, with minimal effect on ZIKV yield when urine specimens are transported frozen for several days, even when ZIKV titers are low in the urine sample. The addition of a nucleic acid stabilizer to urine specimens may be of benefit if longer time to testing is anticipated.
5.0
Conclusions
In summary, we determined pre-analytical factors that influence the detection and quantitation of ZIKV RNA in urine and identified specimen handling procedures that optimize the recovery of
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ZIKV RNA. Understanding pre-analytical factors that influence the integrity of RNA in urine specimens is of additional significance, particularly in emerging studies that evaluate RNA biomarkers of clinical importance in the fields of nephrology, oncology, transplantation, and infectious diseases (Lo, Kaplan and Kirk, 2014; Ralla et al., 2014; Traitanon, Poggio and Fairchild, 2014). Identifying key pre-analytical variables that contribute to differences in molecular profiles and specimen processing procedures that ensure the molecular integrity and clinical relevance of urine specimens is key to translation of RNA markers in urine into practice.
Financial support. This work was conducted with support from National Institutes of Health (NIH) training grant 5T32AI007502-20, Stanford Translational Research and Applied Medicine (TRAM) Pilot Grant Program, and a TL1 Clinical Research Training Program of the Stanford Clinical and Translational Science Award to Spectrum (NIH TL1 TR 001084) (to S.K.T).
Potential conflicts of interest. None.
Acknowledgments. We thank the staff of the Stanford Clinical Virology Laboratory for their diligent work and dedication to patient care. The authors also gratefully acknowledge Jesse Waggoner for his insightful comments and careful review of the manuscript.
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References
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Figure Legends Figure 1. Effect of High and Low Zika Virus Levels and Storage Temperature on Zika Virus RNA Levels in Urine
Figure 2. Comparison of Nucleic Acid Stabilizers, Buffer ATL (ATL) and DNA/RNA Shield (Shield), in Urine Stored at -80C on Quantitative Zika Virus RNA Levels. (A) At low titers, ATL or Shield improved levels of ZIKV RNA recovered at 10 days (ATL, Shield, p<0.001) and 30 days (ATL, Shield, p=0.03). (B) At high titers, differences in ZIKV RNA levels were found at 3 days, 10 days, or 30 days between groups.
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Fig. 1
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A ZIKV- Low 7
Urine ATL Shield
Log 10 copies/mL
6 5 4 3 2 1 0 0 3 10 30
0 3 10 30 Days
0 3 10 30
B ZIKV- High 7
Urine ATL Shield
Log 10 copies/mL
6 5 4 3 2 1 0
0 3 10 30
0 3 10 30 Days
0 3 10 30
Fig. 2
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Table 1. Urine Replicates Detected by Zika Virus (ZIKV) Concentration and Days Stored at -80°C.
Concentration
Baseline
3 Days
10 Days
30 Days
ZIKV-Higha
6/6
6/6
6/6
3/3
ZIKV-Lowb
6/6
5/6
1/6
1/3
a
5.0 log10 copies/mL
b
4.0 log10 copies/mL
17
S0166-0934(17)30077-0 http://dx.doi.org/doi:10.1016/j.jviromet.2017.04.018 VIRMET 13248
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
1-2-2017 28-4-2017 29-4-2017
Please cite this article as: Tan, Susanna K., Sahoo, Malaya K., Milligan, Stephen, Taylor, Nathaniel, Pinsky, Benjamin A., Stability of Zika Virus in Urine: Specimen Processing Considerations and Implications for the Detection of RNA Targets in Urine.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2017.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Stability of Zika Virus in Urine: Specimen Processing Considerations and Implications for the Detection of RNA Targets in Urine Susanna K. Tan1, Malaya K. Sahoo2, Stephen Milligan3, Nathaniel Taylor3 and Benjamin A. Pinsky1 ,2 ,3 ,* 1
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford
University School of Medicine, Stanford, California, United States of America 2
Department of Pathology, Stanford University School of Medicine, Stanford, California, United
States of America 3
Clinical Virology Laboratory, Stanford Health Care, Stanford, California, United States of
America *
Corresponding author: 3375 Hillview Ave, Room 2913, Palo Alto, CA 94304; Phone: +001
(650) 721-1896; Fax: +001 (650) 723-6918; E-mail: [email protected]
1
Highlights
Studied protocols to maximize detection of ZIKV RNA in urine
Evaluated temperature, time, ZIKV levels, and use of nucleic acid stabilizers
At low levels of viruria observed loss of detectable ZIKV RNA over time at -80C
Use of nucleic acid stabilizers resulted in recovery of ZIKV RNA
Summary 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 log10 copies/mL (ZIKV-high) and 4.0 log10 copies/mL (ZIKV-low). Samples were stored at room temperature, 4C, or -80C 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 hours (ZIKV-high, p=0.09; ZIKV-low, p=0.20). ZIKV RNA titers were consistently higher when stored at 4C, suggesting that storage at 4C can slow the progression of RNA degradation. Freezing urine samples at -80C 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.
2
Conclusions: ZIKV RNA is prone to degradation in urine with loss of detectable virus even when specimens are frozen at -80C 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.
Keywords: Zika virus; Urine; RNA Stability; Pre-Analytical
1.0
Introduction
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that, in 2015, rapidly spread throughout the Americas and Caribbean. ZIKV disease is characterized by fever, myalgias, arthralgias, headache, conjunctivitis, and maculopapular rash, and is linked with devastating neurologic complications, such as fetal microcephaly and Guillain-Barre syndrome (Brasil et al., 2016; Russell et al., 2016; Waggoner and Pinsky, 2016). Complications from ZIKV reached epidemic proportions in 2016, leading the World Health Organization to declare a public health emergency of international concern (2016). The diagnosis of ZIKV infection has relied on the detection of ZIKV RNA or ZIKV IgM antibodies in serum, though duration of viremia is short and cross-reactivity of ZIKV antibodies with other flaviviruses, such as dengue virus (DENV), make the diagnosis of acute ZIKV infection challenging. Detection of ZIKV RNA in urine is of increasing interest, given reports of longer duration of virus shedding and at higher concentrations compared to serum. Several studies describe virus detection that persists >7 days longer in urine than in serum (Barzon et al., 2016; Campos Rde et al., 2016; Fourcade et al., 2016; Gourinat et al., 2015; Zhang et al., 2016). ZIKV RNA may also be detected in twice as many urine than serum specimens when both are tested concurrently in ZIKV-positive patients (Bingham et al., 2016). Though variability in these results has also been reported (Pessoa et al., 2016), all together, these studies emphasize the suitability of urine for ZIKV screening and diagnosis and highlight the potential utility of urine ZIKV RNA testing. However, conditions in urine pose unique challenges for nucleic acid detection and preanalytical variables can further impact the stability of viral RNA. While little is known about ZIKV RNA stability in urine, RNA in urine may be particularly sensitive to degradation. Urine is a suitable environment for nucleic acid hydrolyzing enzymes and contains several types of
3
RNases with abundance and activity as much as 100-fold higher than in serum (Bryzgunova and Laktionov, 2015). In addition to matrix-specific effects, pre-analytical variables, such as specimen handling and storage, can further reduce the yield of ZIKV RNA in samples. Given the susceptibility of RNA degradation in urine, understanding pre-analytical specimen processing procedures that maximize the integrity of ZIKV RNA can optimize the utility of this specimen type for diagnostic testing. Here we evaluate the steps between urine collection and nucleic acid extraction that influence the yield of ZIKV RNA detected by real-time, reverse transcriptase-PCR (rRT-PCR). Specifically, we investigate the effect of storage temperature, freeze-thaw, and time on levels of ZIKV RNA in urine. We also evaluate the use of nucleic acid stabilizers and determine scenarios in which the addition of a nucleic acid stabilizer in urine may improve the detection of ZIKV RNA. Nucleic acid stabilizers may minimize RNA degradation by inactivating nucleases and preserving nucleic acids that are originally present in clinical samples (Anwar et al., 2009; Blow et al., 2008; Medeiros et al., 2003). Lastly, we study the effect of initial ZIKV concentrations on subsequent ZIKV RNA detection. This study aims to identify pre-analytical variables that impact the integrity of ZIKV RNA and specimen handling procedures that can maximize the detection and quantitation of ZIKV RNA in urine.
2.0
Materials and Methods
Ethics Statement. This study was reviewed and approved by the Institutional Review Board of Stanford University.
Study Samples. Urine samples from healthy donors were pooled and spiked with ZIKV using the Exact Diagnostics® ZIKV Verification Panel (Exact Diagnostics, Fort Worth, TX), a commercially available panel composed of several concentrations of heat-inactivated ZIKV formulated in EDTA plasma. Ten-fold serial dilutions of the top panel member (6.0 log10 copies/mL by digital droplet RT-PCR) were performed to obtain two concentrations of ZIKV in urine of 5.0 log10 copies/mL (ZIKV-high) and 4.0 log10 copies/mL (ZIKV-low), respectively.
4
For plasma and urine comparisons, EDTA plasma (SeraCare, Milford, MA) was similarly spiked with ZIKV using Exact Diagnostics® ZIKV Verification Panel. Both plasma and urine stocks were confirmed negative for ZIKV RNA by rRT-PCR prior to use in the study.
Storage Conditions. To determine initial storage conditions that minimized degradation of ZIKV in urine, urine samples containing ZIKV at both concentrations were stored at room temperature (25C) or at 4C for 3, 6, 12, 24, and 48 hours prior to undergoing nucleic acid extraction and ZIKV RNA quantitation. Baseline (0 hours) samples proceeded directly to nucleic acid extraction and ZIKV RNA quantitation. All conditions were tested in triplicate.
Freeze-Thaw and Stabilizers. To determine the effect of storing specimens at -80C and the impact of nucleic acid stabilizers on ZIKV in urine, we exposed aliquots of virus to equal parts of no stabilizer (urine), Buffer ATL (Qiagen, Germantown, MD), or DNA/RNA Shield™ (Zymo Research, Irvine, CA). Samples were stored at -80C for 3 days, 10 days, and 30 days, and subsequently underwent thawing, nucleic acid extraction, and ZIKV RNA quantitation. Baseline (0 hours) samples did not undergo freeze-thawing and proceeded directly to nucleic acid extraction and ZIKV RNA quantitation. To account for the plasma (EDX panel matrix) content of the spiked urine, baseline, day 3, and day 10 experiments were performed a second time in a separate run with dilutions for ZIKV-low made from 5.0 log10 copies/mL panel member and dilutions for ZIKV-high made from the 6.0 log10 copies/mL panel member. All conditions were tested in triplicate. To further evaluate freeze-thaw effects, we subjected urine aliquots at both concentrations to overnight (18 hours) freezing at -80C, which was followed by extraction and rRT-PCR.
Nucleic Acid Extraction. Total nucleic acid was isolated from 200L of urine or plasma samples using the EZ1 Virus Mini Kit, v2.0 on the EZ1 Advanced XL instrument (Qiagen, Germantown, MD). All extractions were carried out according to the manufacturer’s recommendations. The purified nucleic acid was eluted into a final volume of 60L containing AVE buffer (Qiagen, Germantown, MD). rRT-PCR was performed immediately after extraction.
5
ZIKV rRT-PCR Assay. Samples were tested for ZIKV using a single-reaction, multiplex rRTPCR assay that detects ZIKV, Chikungunya (CHIKV), and Dengue (DENV) viruses as previously described (Waggoner et al., 2016). Briefly, rRT-PCR were performed on the RotorGene Q (Qiagen, Germantown, MD) real-time PCR instrument and involved 25L reactions of the SuperScript III Platinum One-Step qRT-PCR kit (Life Technologies, Carlsbad, CA, USA) and 10L of RNA template. Cycling conditions for the ZCD assay were as follows: 52°C for 15 min; 94°C for 2 min; 45 cycles at 94°C for 15 sec, 55°C for 40 sec, and 68°C for 20 sec. Each run included a no-template control and positive controls for ZIKV. For quantitation, standard curves were prepared using quantitated ssDNA (Integrated DNA Technologies) containing the target sequences.
Statistical Analysis. ZIKV RNA concentrations in log10 copies/mL for each test condition were compared to baseline and between groups using unpaired t-tests. Statistical analysis was performed in Excel (Microsoft, Redmond, WA) and GraphPad software (GraphPad, San Diego, California). Figures were generated using Prism. A P value <0.05 was considered statistically significant.
3.0
Results
Room Temperature and 4C. We evaluated the effect of temperature (room temperature and 4C) at several time points (0, 3, 6, 12, 24, and 48 hours) on ZIKV RNA stability (Figure 1). Degradation of ZIKV displayed a steady decline at room temperature, though was not statistically significant at 48 hours when compared to baseline (ZIKV-high, p=0.09; ZIKV-low, p=0.20). The mean difference was -0.37 log10 copies/mL with a standard deviation (SD) of 0.25 for ZIKV-high, and -0.47 log10 copies/mL with SD of 0.33 for ZIKV-low. When urine was stored at 4C, the decline in ZIKV RNA load was marginal and ZIKV RNA loads were higher at each time point compared to room temperature, suggesting that storage at 4C can slow the progression of ZIKV degradation in urine and better maintain ZIKV titers.
Freeze-Thaw Effects. A single freeze-thaw of urine samples stored at -80C overnight (<24 hours) followed by extraction and rRT-PCR showed no effect on ZIKV RNA levels (ZIKV-high, 6
p=0.67; ZIKV-low, p=0.37). However, duration of freezing at -80C had a significant impact on both ZIKV RNA detection and quantity, particularly in the ZIKV-low group.
In the first
experiment, 2/3 replicates remained detectable at 3 days in the ZIKV-low group, 1/3 replicates at 10 days, and 1/3 replicates at 30 days of storage at -80C (Table 1). These findings were reproducible on repeat testing; all three replicates in the ZIKV-low group were negative for ZIKV RNA by day 10. These results confirmed our initial observations and demonstrated that the plasma matrix content of the urine dilutions did not substantially contribute to ZIKV-low stability. In the ZIKV-high group, the finding was less distinct, with ZIKV RNA remaining detectable in 5/6 replicates at 10 days across the two experiments.
Of the detectable replicates, there was a notable decline in ZIKV RNA load over time, most significant in the ZIKV-low group (Figure 2). For the ZIKV-low group, the mean difference was -0.49 log10 copies/mL with SD of 0.33 at 10 days (p=0.001) and -1.20 log10 copies/mL with SD of 0.53 at 30 days (p=0.001), though this represents 1/6 total replicates performed at 10 days and 1/3 total replicates at 30 days, as all other replicates no longer contained detectable ZIKV RNA. In the ZIKV-high group, there was a significant decline in ZIKV RNA levels by day 30 (p=0.002) with a mean difference of -0.55 log10 copies/mL and SD of 0.31. No changes in detection or levels of virus were seen in plasma frozen at -80C over time (ZIKV-high, p=0.71; ZIKV-low, p=0.51) indicating that the observed effects are urine matrix specific.
Nucleic Acid Stabilizers. To evaluate the effect of Buffer ATL (ATL) and DNA/RNA Shield (Shield) on ZIKV RNA stabilization in urine, urine aliquots containing equal volumes of nucleic acid stabilizer were tested in parallel with ZIKV-positive urine aliquots stored at -80C. Unlike urine without stabilizer, urine with ATL or Shield resulted in the detection of ZIKV RNA in all replicates, including those that were lost in the urine without stabilizer subgroup. Furthermore, the use of either ATL or Shield improved levels of ZIKV RNA recovered at 10 days (ATL, Shield, p<0.001) and 30 days (ATL, Shield, p=0.03) compared to the urine without stabilizer subgroup in the ZIKV-low group (Figure 2a). By contrast, no differences in ZIKV RNA levels were found at 3 days, 10 days, or 30 days between urine only, ATL, and Shield ZIKV-high subgroups (Figure 2b). Comparison of ATL and Shield at all time points did not reveal any differences in ZIKV RNA detection or levels.
7
ZIKV RNA levels significantly declined by 30 days of freezing at -80C, regardless of starting viral load and use of a nucleic acid stabilizer, highlighting the effect of duration of freezing on ZIKV RNA levels. In the ZIKV-high group, the mean decline was approximately -0.5 log10 copies/mL by 30 days across all subgroups (Urine, -0.55 log10 copies/mL, SD 0.32, p=0.002; ATL, -0.56 log10 copies/mL, SD 0.29, p=0.001; Shield, -0.49 log10 copies/mL, SD 0.25, p=0.001). A similar decline was seen by 30 days of freezing at -80C in the ZIKV-low group, though the decline was greatest in the urine without stabilizer subgroup (Urine, -1.20 log10 copies/mL, SD 0.53, p=0.001; ATL, -0.30 log10 copies/mL, SD 0.17, p<0.001; Shield, -0.68 log10 copies/mL, SD 0.16, p<0.001).
Altogether, these findings suggest that use of nucleic acid stabilizers improves the recovery of ZIKV RNA, particularly when the abundance of ZIKV RNA is low and samples are frozen at 80C for as a short as 10 days.
4.0
Discussion
Pre-analytical variables can significantly impact the reliability of laboratory test results, and, in particular, influence the integrity of nucleic acids detected by molecular methods. In urine, proper specimen handling may be of increased importance given the several-fold higher RNAse activity and relative instability of RNA (Bryzgunova and Laktionov, 2015; Cherepanova et al., 2007). Here, we evaluated specimen processing conditions that optimize the recovery of ZIKV RNA in urine. We investigated the effect of temperature, initial ZIKV levels, nucleic acid stabilizers, and time between specimen collection and nucleic acid extraction on ZIKV RNA detected and quantified by rRT-PCR. As expected, ZIKV RNA levels declined when urine samples were stored at room temperature, though was not statistically significant at 48 hours. When samples were stored at 4C, the mean ZIKV RNA levels were improved and higher at each time point compared to room temperature. Detection of ZIKV RNA was preserved with freezing at -80C for up to 30 days, but only in urine specimens containing higher ZIKV titers (5.0 log10 copies/mL). Storing urine with lower ZIKV titers (4.0 log10 copies/mL) at -80C for as few as 10 days resulted in a 8
significant loss of detectable ZIKV RNA, findings that were present in multiple replicates and on repeat testing. The addition of either ATL buffer or DNA/RNA Shield to urine resulted in the detection of all ZIKV replicates lost with freezing at -80C, and also improved quantitative recovery of ZIKV RNA. At higher ZIKV levels, no difference in detection or quantitation of ZIKV RNA was found between urine with and without stabilizer. These findings are consistent with studies evaluating the stability of other flaviviruses in serum and urine specimens. Storage of serum and urine samples containing Hepatitis C virus (HCV) and dengue virus (DENV) at 4°C slowed the progression of viral RNA degradation (Halfon et al., 1996; Van den Bossche, Cnops and Van Esbroeck, 2015). Similarly, HCVpositive serum specimens stored at -20°C or -70°C resulted in decreased HCV levels, but remained detectable after storage for at least 6 months to several years (Baleriola et al., 2011; Halfon et al., 1996). DENV RNA in urine specimens also remained detectable at -20°C after one year (Van den Bossche, Cnops and Van Esbroeck, 2015). These findings are comparable to findings in the ZIKV-high group, but are in contrast to the ZIKV-low group where there was significant loss in detectable ZIKV RNA by 10 days. Use of a lysis buffer to stabilize viral RNA in serum has previously been demonstrated, extending the period in which DENV RNA remains detectable at room temperature and Venezuelan equine encephalitis and Rift Valley Fever virus RNA at 4°C and -20°C (Anwar et al., 2009; Blow et al., 2008). The utility of a nucleic acid stabilizer in urine specimens that are stored at -80°C to maintain ZIKV RNA detection has not been previously reported. All together, these findings indicate that urine specimens being evaluated for ZIKV can be stored at room temperature for 24-48 hours without significant impact on ZIKV RNA levels, and that storage of specimens at 4°C can further minimize nucleic acid degradation. Urine specimens may be frozen at -80°C for brief periods (within 3 days) without significant effect on ZIKV RNA detection; however, freezing at -80°C for longer periods, such as 10 days, may result in loss of detectable ZIKV RNA and a false negative test result, particularly if the ZIKV load in the urine specimen is low. To accurately detect ZIKV in urine specimens containing low levels of ZIKV, prompt nucleic acid extraction and testing would be optimal. However, if delays in testing are anticipated, the addition of a nucleic acid stabilizer, such as ATL or Shield, can maintain detection of ZIKV. Notably, adding a stabilizer to specimens before sending out for testing would need to be approved and validated by the receiving reference laboratory.
9
The loss of low-level ZIKV RNA when urine is stored at -80°C is of uncertain clinical significance. Case reports of ZIKV-infected individuals document ZIKV titers in urine up to 8.0 log10 copies/mL (Lamb et al., 2016). This may reflect a clinical course consistent with high titer ZIKV shedding in urine or represent publication bias due to loss of lower ZIKV RNA concentrations from variations in specimen handling. Thus, a missed ZIKV diagnosis may occur only in patients who present particularly late after symptom onset. While titers are likely to drop with freezing at -80°C for extended duration, ZIKV RNA originally present at high levels in the urine should remain detectable. The significance of quantitative values for ZIKV in urine for diagnosis or prognosis has not yet been established. Limitations of this study include the narrow window of ZIKV concentrations tested and the extent of time points evaluated. The 1.0 log10 copies/mL gap between high-level and lowlevel ZIKV was necessitated by the relatively low stock concentration of the available reference material. It is also important to note that this reference material is comprised of heat inactivated virus, and additional experiments using split clinical specimens will be required to further evaluate the routine use of stabilizers in a clinical setting. This study evaluated the stability of ZIKV up to 48 hours at room temperature and 4°C, and 30 days at -80°C, and was not extended to determine time in which ZIKV RNA was no longer detectable. Notably, this study was performed at a clinical laboratory with validated, in-house ZIKV rRT-PCR capabilities. Time points were selected to assist with identifying optimal in-house protocols, where specimen processing and testing can occur relatively rapidly, a scenario that may not be present at other institutions or in research protocols where samples may be stored for months before testing. However, study findings can still be applied to institutions that send-out specimens for ZIKV RNA testing to reference or public health laboratories. Specimen handling should ideally involve refrigeration at 4°C or freezing specimens at -80°C, with minimal effect on ZIKV yield when urine specimens are transported frozen for several days, even when ZIKV titers are low in the urine sample. The addition of a nucleic acid stabilizer to urine specimens may be of benefit if longer time to testing is anticipated.
5.0
Conclusions
In summary, we determined pre-analytical factors that influence the detection and quantitation of ZIKV RNA in urine and identified specimen handling procedures that optimize the recovery of
10
ZIKV RNA. Understanding pre-analytical factors that influence the integrity of RNA in urine specimens is of additional significance, particularly in emerging studies that evaluate RNA biomarkers of clinical importance in the fields of nephrology, oncology, transplantation, and infectious diseases (Lo, Kaplan and Kirk, 2014; Ralla et al., 2014; Traitanon, Poggio and Fairchild, 2014). Identifying key pre-analytical variables that contribute to differences in molecular profiles and specimen processing procedures that ensure the molecular integrity and clinical relevance of urine specimens is key to translation of RNA markers in urine into practice.
Financial support. This work was conducted with support from National Institutes of Health (NIH) training grant 5T32AI007502-20, Stanford Translational Research and Applied Medicine (TRAM) Pilot Grant Program, and a TL1 Clinical Research Training Program of the Stanford Clinical and Translational Science Award to Spectrum (NIH TL1 TR 001084) (to S.K.T).
Potential conflicts of interest. None.
Acknowledgments. We thank the staff of the Stanford Clinical Virology Laboratory for their diligent work and dedication to patient care. The authors also gratefully acknowledge Jesse Waggoner for his insightful comments and careful review of the manuscript.
6.0
References
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shedding in a traveller returning from the Dominican Republic to Italy, January 2016. Euro Surveill 21, 30159. Bingham, A.M., Cone, M., Mock, V., Heberlein-Larson, L., Stanek, D., Blackmore, C. and Likos, A., 2016. Comparison of Test Results for Zika Virus RNA in Urine, Serum, and Saliva Specimens from Persons with Travel-Associated Zika Virus Disease - Florida, 2016. MMWR Morb Mortal Wkly Rep 65, 475-8. Blow, J.A., Mores, C.N., Dyer, J. and Dohm, D.J., 2008. Viral nucleic acid stabilization by RNA extraction reagent. J Virol Methods 150, 41-4. Brasil, P., Pereira, J.P., Jr., Moreira, M.E., Ribeiro Nogueira, R.M., Damasceno, L., Wakimoto, M., Rabello, R.S., Valderramos, S.G., Halai, U.A., Salles, T.S., Zin, A.A., Horovitz, D., Daltro, P., Boechat, M., Raja Gabaglia, C., Carvalho de Sequeira, P., Pilotto, J.H., Medialdea-Carrera, R., Cotrim da Cunha, D., Abreu de Carvalho, L.M., Pone, M., Machado Siqueira, A., Calvet, G.A., Rodrigues Baiao, A.E., Neves, E.S., Nassar de Carvalho, P.R., Hasue, R.H., Marschik, P.B., Einspieler, C., Janzen, C., Cherry, J.D., Bispo de Filippis, A.M. and Nielsen-Saines, K., 2016. Zika Virus Infection in Pregnant Women in Rio de Janeiro. N Engl J Med 375, 2321-2334. Bryzgunova, O.E. and Laktionov, P.P., 2015. Extracellular Nucleic Acids in Urine: Sources, Structure, Diagnostic Potential. Acta Naturae 7, 48-54. Campos Rde, M., Cirne-Santos, C., Meira, G.L., Santos, L.L., de Meneses, M.D., Friedrich, J., Jansen, S., Ribeiro, M.S., da Cruz, I.C., Schmidt-Chanasit, J. and Ferreira, D.F., 2016. Prolonged detection of Zika virus RNA in urine samples during the ongoing Zika virus epidemic in Brazil. J Clin Virol 77, 69-70. Cherepanova, A., Tamkovich, S., Pyshnyi, D., Kharkova, M., Vlassov, V. and Laktionov, P., 2007. Immunochemical assay for deoxyribonuclease activity in body fluids. J Immunol Methods 325, 96-103. Fourcade, C., Mansuy, J.M., Dutertre, M., Delpech, M., Marchou, B., Delobel, P., Izopet, J. and Martin-Blondel, G., 2016. Viral load kinetics of Zika virus in plasma, urine and saliva in a couple returning from Martinique, French West Indies. J Clin Virol 82, 1-4. Gourinat, A.C., O'Connor, O., Calvez, E., Goarant, C. and Dupont-Rouzeyrol, M., 2015. Detection of Zika virus in urine. Emerg Infect Dis 21, 84-6.
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Waggoner, J.J. and Pinsky, B.A., 2016. Zika Virus: Diagnostics for an Emerging Pandemic Threat. J Clin Microbiol 54, 860-7. Zhang, F.C., Li, X.F., Deng, Y.Q., Tong, Y.G. and Qin, C.F., 2016. Excretion of infectious Zika virus in urine. Lancet Infect Dis 16, 641-2.
Figure Legends Figure 1. Effect of High and Low Zika Virus Levels and Storage Temperature on Zika Virus RNA Levels in Urine
Figure 2. Comparison of Nucleic Acid Stabilizers, Buffer ATL (ATL) and DNA/RNA Shield (Shield), in Urine Stored at -80C on Quantitative Zika Virus RNA Levels. (A) At low titers, ATL or Shield improved levels of ZIKV RNA recovered at 10 days (ATL, Shield, p<0.001) and 30 days (ATL, Shield, p=0.03). (B) At high titers, differences in ZIKV RNA levels were found at 3 days, 10 days, or 30 days between groups.
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Fig. 1
15
A ZIKV- Low 7
Urine ATL Shield
Log 10 copies/mL
6 5 4 3 2 1 0 0 3 10 30
0 3 10 30 Days
0 3 10 30
B ZIKV- High 7
Urine ATL Shield
Log 10 copies/mL
6 5 4 3 2 1 0
0 3 10 30
0 3 10 30 Days
0 3 10 30
Fig. 2
16
Table 1. Urine Replicates Detected by Zika Virus (ZIKV) Concentration and Days Stored at -80°C.
Concentration
Baseline
3 Days
10 Days
30 Days
ZIKV-Higha
6/6
6/6
6/6
3/3
ZIKV-Lowb
6/6
5/6
1/6
1/3
a
5.0 log10 copies/mL
b
4.0 log10 copies/mL
17