Prolonged detection of Zika virus RNA in urine samples during the ongoing Zika virus epidemic in Brazil.

Zika virus (ZIKV) is an emerging mosquito-borne virus that elongs to the genus Flavivirus. ZIKV may cause Zika virus disease ZVD) in humans that is characterized by fever, headache, myalgia nd rash. The first autochthonous transmission of ZIKV in Brazil was emonstrated in 2015 for patients from Rio Grande do Norte [1] and t was shown that the epidemic ZIKV strain in Brazil belong to the sian lineage [1,2]. Diagnosis of ZVD in humans is mainly based on NA detection in serum or plasma samples. Specific antibody detecion is mostly hampered due to strong serological cross-reactivity ith other circulating flaviviruses such as dengue virus or yellow ever virus [3–6]. Thus, there is an urgent need for a ZIKV diagostic protocol in Brazil that is effective in any ZIKV epidemic cenario, leading to a rapid, reliable and prolonged ZIKV RNA detecion. Paired samples of urine and serum from seven Brazilian ZVD atients were used for this study. For two additional ZVD patients nly urine samples were collected for a longer period after the onset f symptoms (ethical committee 80709). Viral RNA was extracted sing RTP® Pathogen Kit (Stratec, Birkenfeld—Germany), 400 l as used (for both, sera and urine samples), eluted in 50 l Elution uffer and ZIKV RT-PCR was performed according to Waehre et al. 7] using the Thermal Cycler peqLab—model peqstar 96X Univeral Gradient. Amplified fragments ∼200 pb were Sanger sequenced or confirmation. ZIKV RNA was detected in sera collected 2 days fter onset of symptoms, however urine samples from the same day ere tested negative. ZIKV RNA was first detectable in urine 4 days fter onset of symptoms, suggesting the beginning of renal excreion of ZIKV. For one patient ZIKV RNA excretion was observed until 4 days after onset of symptoms. In addition, our study demontrated higher levels of ZIKV RNA in urine when compared to serum. his was also observed by Gourinat et al. [8] during the ZVD epiemic in French Polynesia, reinforcing the evidence that the use f urine can increase the number of laboratory confirmed cases in n epidemic setting. The nucleotide sequences obtained with the mplicons confirmed the presence of the Asian lineage of ZIKV in io de Janeiro. In conclusion, urine samples should be considered s an important alternative to serum or plasma for the detection of IKV RNA because of a longer period of RNA detection, higher RNA evels, and less invasive sample collection.