Detection of Zika Virus in April 2013 Patient Samples, Rio de Janeiro, Brazil

We tested 210 dengue virus‒negative samples collected from febrile patients during a dengue virus type 4 outbreak in Rio de Janeiro in April 2013 and found 3 samples positive for Zika virus. Our findings support previously published entomological data suggesting Zika virus was introduced into Brazil during October 2012–May 2013.

By August 2015, Zika virus infection had been confirmed in 13 states of Brazil (Bahia, Rio Grande do Norte, São Paulo, Alagoas, Pará, Roraima, Rio de Janeiro, Maranhão, Pernambuco, Ceará, Paraíba, Paraná, and Piauí), some of which were located >2,500 miles apart (4). Because Zika virus circulation can occur simultaneously with dengue virus (DENV) in regions plagued by Aedes aegypti mosquitoes, we used frozen serum samples previously collected during a DENV type 4 (DENV-4) outbreak to investigate whether cocirculation might have been occurring before reported cases.
We evaluated 210 samples collected from patients (median age 36.6 years) with acute febrile syndrome who visited an acute healthcare facility in Tijuca, a middle-class district in the northern zone of Rio de Janeiro, Brazil, during a DENV-4 outbreak occurring March-May 2013. All samples tested negative for DENV RNA by RT-PCR and DENV nonstructural protein 1 by Platelia Dengue NS1 Ag ELISA (Bio-Rad Laboratories, Marnes-la-Coquette, France) (5).
In June 2017, we performed a molecular test to rapidly detect Zika virus in previously frozen acute-phase samples. We extracted viral RNA from 200-µL samples by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. We performed quantitative RT-PCR (qRT-PCR) with the Quan-tiNova Probe RT-PCR Kit (QIAGEN) in a Rotor-Gene Q Sequence Detection System (QIAGEN) using 25-µL reaction mixtures containing 5 µL of RNA template. We used primers, probes, and cycling conditions for Zika virus detection recommended by the Centers for Disease Control and Prevention (6). Samples suspected positive (defined as having a cycle threshold <38) were retested in triplicate, and consistently positive samples were confirmed by repeating RNA extraction and qRT-PCR in duplicate.
Of the 210 samples, 21 tested positive by qRT-PCR and were thus suspected positive for Zika virus; 4 of 21 tested positive for Zika virus RNA in triplicate qRT-PCR reactions.

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However, 1 of the 4 also tested positive by Panbio Dengue IgM Capture ELISA (Standard Diagnostics Inc., Yongin, South Korea). We confirmed that the other 3 samples (2 from men and 1 from a woman) were positive for Zika virus genome after repetition of RNA extraction and qRT-PCR.
Zika virus-positive patients were young (18, 25, and 26 years of age), lived in Tijuca, had low-grade fever (1-2 days) during acute disease, and had no underlying conditions. Their travel histories were not available. All patients reported prostration, myalgia, arthralgia, headache, retroorbital pain, and nausea (Table). None reported rash or hemorrhages. Hematocrit levels were 40%-45%, platelet counts 2.19-3.53 × 10 5 /µL, and leukocyte counts 4.4-19.8 However, our findings suggest that Zika virus had already been circulating in Rio de Janeiro since April 2013, consistent with the report by Metsky et al. (9) stating that Zika virus had been circulating undetected in multiple regions for many months before the initial case reports. This view is also supported by entomological data from Ayllón et al. (10), who used a surveillance program involving field-trapped mosquitoes to perform genetic analyses of mosquitoborne viruses found in Rio de Janeiro during  Dr. Passos is a lead investigator at Evandro Chagas National Institute of Infectious Diseases in Rio de Janeiro, Brazil. Her research interests are clinical epidemiology of emerging infectious vectorborne diseases.