Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

Abstract

Zika virus (ZIKV), a flavivirus, and chikungunya virus (CHIKV), an alphavirus, are infectious RNA arboviruses transmitted to humans by the bite of Aedes species mosquitoes. Both viruses have only recently emerged in the Western Hemisphere (1, 2), and along with dengue virus (DENV), another flavivirus, now circulate widely in Brazil. The acute illness caused by these viruses, characterized by fever, rash, myalgia, arthralgia, and conjunctivitis, is nonspecific, and differential diagnosis on the basis of clinical findings alone is challenging. Later infectious sequelae include chronic arthritis for CHIKV (2) and encephalitis, immune-mediated syndromes, and stroke for DENV (3). Recently, the association between ZIKV infection and severe fetal complications such as microcephaly in pregnant women has been established (4), and the virus has also been linked to neurological complications such as Guillain-Barré syndrome (5). Thus, broadbased assays are needed for differential diagnosis of vectorborne febrile illnesses and to identify potential coinfections. Here we report the utility of metagenomic next-generation sequencing (mNGS) as a screening tool to identify coinfections and the use of genome recovery and phylogenetic analyses directly from patient serum samples in the context of the ongoing ZIKV outbreak. We also show that the clinical presentation of arboviral coinfections seems to favor the virus present at a higher titer in acutely infected individuals. MATERIALS AND METHODSZIKV serum sample collection, ZIKV RT-PCR, and DENV antibody testing. Written consent from patients was obtained under a study protocol approved by the Brazil Ministry of Health (Certificado de Apresentação para Apreciação Ética 45483115.0.0000.0046, no. 1159.184, Brazil). Serum samples were obtained from 15 patients seen at Aliança Hospital in Salvador, Bahia, Brazil, from April 2015 to January 2016 who were given a presumptive diagnosis of an acute viral illness by emergency department physicians and were found to be positive by qualitative reverse transcription-PCR (RT-PCR) testing for ZIKV. Serum samples Bahia01 to Bahia15 were subjected to RNA extraction using the QIAamp viral RNA minikit (Qiagen), and RNA was reverse transcribed using the Superscript II reverse transcriptase kit (Invitrogen), followed by qualitative RT-PCR testing for ZIKV using primers targeting the NS5 gene (6). Serum samples were also tested for DENV infection using an enzyme-linked immunosorbent assay (ELISA) specific for the NS1 antigen and anti-DENV IgG/IgM according to the manufacturer's instructions (Dengue Duo Test; Bioeasy Diagnostica, Brazil).Metagenomic next-generation sequencing. A separate serum aliquot was extracted for total nucleic acid using the Qiagen viral RNA minikit (Qiagen), followed by DNase treatment using a cocktail of Turbo DNase (Thermo Fisher Scientific) and Baseline-Zero DNase (Epicentre Biotechnologies), followed by NGS library construction using the NexteraXT kit (Illumina) as previously described (7,8). Runs of single-end, 160-base pair...

Prolonged shedding of Chikungunya virus in semen and urine: A new perspective for diagnosis and implications for transmission

“…The use of prednisolone may have contributed to the prolonged viral shedding and another factor that may, in part, have added to that was the possible interaction between Chikungunya and Dengue type 3. With the ongoing circulation of DENV, CHIKV and ZIKV in Brazil, such a finding of dual infections will be increasingly seen and reported, as we recently published for CHIKV and ZIKV [5]. In India, an estimate of co-infection with CHIKV and DENV using serological markers estimated point prevalences on the range of 5.7–9.5% [33].…”

“…In Brazil, acute exanthematous outbreaks related to Zika (ZIKV) and Chikungunya viruses (CHIKV) have been reported in many regions and sometimes as co-infections [2], [3], [4], [5].…”

Zika virus evolution and spread in the Americas

“…We initially pursued unbiased metagenomic sequencing to capture both ZIKV and other viruses known to be co-circulating with ZIKV 5 . In most of the 38 samples examined by this approach there proved to be insufficient ZIKV RNA for genome assembly, but it still proved valuable to verify results from other methods.…”

Toxoplasmosis cerebral congénita e infección por el virus del Zika y del chikunguña: reporte de un caso

“…Por otra parte, se ha reportado la circulación simultánea del CHIKV y el ZIKV en Nicaragua, Ecuador, Brasil y Colombia (25)(26)(27)(28). Específicamente en Nicaragua, un estudio serológico encontró que la infección simultánea se presentaba en el 4,6 % de las personas (25).…”