Chikungunya Diagnosis

Blood and/or urine samples were collected at patient admission. Cerebrospinal fluid was also collected from patients with CNS IDD and stored at −80°C until further analysis. Biological samples were blindly tested for Zika (ZIKV), CHIKV, and Dengue (DENV) virus. Samples selected for exome analysis had their molecular and serological diagnosis confirmed as described below.

Virus RNA was obtained from serum, urine or CSF samples using the QIAmp MinElute Virus Spin Kit (Qiagen), according to manufacturer's instructions. RNA was eluted in 50 and 5 μL were used for virus genome detection using the Go-Taq Probe 1-step RT-qPCR System (Promega) and previously designed primers targeting CHIKV, DENV, and ZIKV (Lanciotti et al., 2007, 2008; Menting et al., 2011). Antibodies against CHIKV infection (IgG and IgM) were investigated by MAC-ELISA (Euroimmun) using serum samples, according to manufacturer's instructions. Plaque-reduction neutralization tests (PRNT) were also performed to detect neutralizing antibodies against CHIKV and to exclude cross-reaction with other arboviruses circulating in the same region. Briefly, plasma samples were incubated at 58°C for 30 min to inactivate CHIKV and limit complement effect. Serial dilutions (from 1:5 to 1:2,560) of heat-inactivated plasma were incubated with an equal volume containing 100 plaque forming units (PFU) of CHIKV isolate BHI3745/H804709 (Accession Number {"type":"entrez-nucleotide","attrs":{"text":"KP164570.1","term_id":"777861470","term_text":"KP164570.1"}}KP164570.1) for 1 h at 37° C. The virus-sera mixture was inoculated into confluent monolayers of VERO cells. After 1 h, inoculum was removed and semisolid medium (1.2% carboxymethylcellulose in alpha-MEM supplemented with 1% fetal bovine serum) was added. Cells were further incubated for 30 h and then fixed with 4% formaldehyde solution. Cells were stained with crystal violet dye solution. PRNT end-point titers are expressed as the reciprocal of the last serum dilution showing reduction in plaque counts ≥90%. PRNT₉₀ titer ≥20 was considered positive for the presence of neutralizing antibodies against CHIKV.