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Citation style for this article: Pessôa R, Patriota JV, de Souza MdL, Abd El Wahed A, Sanabani SS. Detection of Zika virus in Brazilian patients during the first five days of infection – urine versus plasma. Euro Surveill. 2016;21(30):pii=30302. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2016.21.30.30302
Received:08 July 2016; Accepted:22 July 2016
Results of recent studies have suggested that after Zika virus (ZIKV) disease onset, the virus persists at higher levels and for a longer period in urine (up to ca 20 days) than in serum (up to ca 5 days) [1,2]. To provide further data, we tested for the presence of ZIKV in the urine and corresponding plasma specimens of 61 patients presenting symptoms of ZIKV disease in Brazil. The samples were collected between 1 and 5 days after symptom onset. During this phase of infection, the proportion of patients testing positive in plasma samples (46/61) appeared to be higher than those testing positive in urine samples (37/61).
Urine and corresponding plasma samples (i.e. from the same patient at the same date) that had been previously collected during the large outbreak in Tuparetama, Brazil, in 2015, were retrospectively analysed in this study. All samples had been obtained within the first 5 days of onset of symptoms, from a total of 61 individuals, who had been diagnosed as having ZIKV disease on clinical and epidemiological grounds. The main symptoms reported for the patients were rash (n=47), fever ≥38.5°C (n=42), headache (n=40), joint pain (n=39), and conjunctivitis (n=37). The median age of patients was 35 years (range: 1–80 years), with the majority being female (n=41). No patient was co-infected with another flavivirus such as dengue at the time of the sample collection, although previous infection with dengue was not known. None of the women were pregnant.
Specimens were investigated by real-time reverse-transcription polymerase chain reaction (RT-PCR), with a published primer set (FP: 5´-GAAGCCCTTGGATTCTTGAACGAGG-3´ and RP: 5´-CGACTCATCTCTTCTAGGACATATCC-3´)  and a fluorescein (FAM)/black hole quencher 1 (BHQ1)-labelled Taqman probe, targeting the non-structural protein (NS)5 genetic region of ZIKV (ZIKAp 5’-FAM- GGGAGAGAGAACTCAGGAGGTGG-BHQ1–3`). A sample was considered positive for ZIKV when the mean cycle threshold (Ct) value obtained from three parallel real-time RT-PCRs was ≤ 40 cycles.
Zika virus RNA in urine and plasma
Among the 61 patients, more tested positive for ZIKV RNA in plasma (n=46) than in urine (n=37) within the 5 days post-symptom onset, although the difference was not statistically significant (p=0.12; two-tailed test) (Table).
Real-time reverse transcription-polymerase chain reaction results of screening plasma and corresponding urine samples from patients in Brazil, 2015 (n=61 patients)
|2 days post-symptom onset
|3 days post-symptom onset
|4 days post-symptom onset
|5 days post-symptom onset
In 28 cases, ZIKV RNA was detected in both types of samples. Ct values from plasma and urine samples collected at the same time post-symptom onset did not seem to differ (Figure).
Comparison between the cycle threshold (Ct) values of plasma and corresponding urine samples from Zika virus infected patients testing positive for both types of samples in the first five days post-symptom onset, Brazil, 2015 (n=28 patients)
RT-PCR: reverse transcription-polymerase chain reaction; ZIKV: Zika virus.
A linear regression analysis was performed by plotting the Ct values of plasma and urine samples versus the time post onset of symptoms using PRISM (Graphpad SoJware Inc., San Diego, California). The Ct values of ZIKV RNA in plasma and urine samples appear similar.
Background and discussion
ZIKV belongs to the genus Flavivirus, a group of RNA viruses transmitted by arthropods. The virus name originates from the Zika Forest of Uganda, where it was first isolated in 1947 from an infected rhesus monkey . In 2007, ZIKV caused an outbreak in Yap State, Micronesia . The outbreak was characterised by relatively mild disease including symptoms such as rash, arthralgia, and conjunctivitis, which are commonly observed upon ZIKV infection [5,6]. Seven years later, the virus appeared in French Polynesia and larger outbreaks were reported in New Caledonia, the Cook Islands, and Easter Island, whereby coincident with the French Polynesia ZIKV outbreak, an increased incidence of neurological complications was observed . The first cases of ZIKV infection in the Americas were detected in Brazil in May 2015 [3,6,8] and, since, the virus spread to other countries in North and South America . In Brazil, concurrent with ZIKV infections, neurological complications were also reported, as well as congenital malformations including microcephaly .
Due to cross-reactivity between members of the Flavivirus genus, the serological diagnostic of ZIKV is difficult . Although there are molecular tests based on virus isolation and/or detection of ZIKV RNA during the acute phase of infection, the low-level and short period of viraemia remains a challenge . Recently, Gourinat et al  observed that ZIKV RNA in six patients from French Polynesia was detectable at higher levels and for longer periods after symptom onset in urine samples (up to ca 20 days) than in corresponding serum samples (up to ca 5 days). The larger time window post-disease onset of possible ZIKV detection in urine was confirmed by others who investigated returning travellers to the United States (US) . It was further noted that among 55 persons with urine and serum samples obtained within the first 5 days of symptom onset, a higher proportion tested positive for ZIKV RNA in urine than in serum . The findings opened the door for the use of urine for the diagnosis of ZIKV infection .
In our study, we focused on the detection of ZIKV in samples from 61 patients collected within the first 5 days of symptom onset. In contrast to the two previous studies mentioned above [1,2], patients in our study had an overall a higher ZIKV detection rate in plasma than in urine during active infection. Moreover, our data do not support the recent finding of higher level of ZIKV RNA in urine than in serum during the acute phase of the disease . The reasons for this discrepancy remain unclear, although this might have been due to the small number of patients investigated in all three studies. Another hypothesis could be that host immune and genetic factors might affect the viral load of ZIKV in distinct body fluids, whereby this may vary among individuals. A similar suggestion has been reported in cases of infection with dengue virus, another flavivirus . In this respect, patients in our study were Brazilian citizens, while the two other studies investigated patients from French Polynesia and travellers returning to the US. Previous infection with other flaviviruses might also alter and/or enhance the replication of ZIKV in specific organs . Information about past dengue infection was however not available for our patients. Finally, beside the small sample size, one limitation of our study was the cross-sectional design that only permitted obtaining data at single time points.
In conclusion, we recommend the simultaneous testing of blood and urine samples in ZIKV infected individuals with focus on plasma samples in the first 5 days of infection. Further investigations will be required to more fully determine factors influencing ZIKV pathogenesis.