Study design
The study area, period and characteristic of study population
All the 33 districts of Gujarat were considered as a site for sample collection from the human population during the year 2015, 2016 and 2017. Human serum samples were collected from Ahmedabad, Amreli, Patan, Aravalli, Kheda, Morbi, Kutch, Surendranagar, Mahesana, Jamnagar, Botad, Valsad, Anand, Rajkot, Panchmahal, Devbhoomi Dwarka, Banaskantha, Bharuch, Bhavnagar, Dahod, Gir-Somnath, Junagadh, Mahisagar, Narmada, Navsari, Sabarakantha, Surat, Tapi, Vadodara, Chhota Udepur, Porbandar, Gandhinagar and Dang districts of Gujarat State.
The transmission of CCHF infection to humans through contact with CCHFV infected individuals, animals and bite of ticks is well known. Since 2011, many sporadic CCHF cases were reported from Gujarat State making it an endemic state for CCHF [7, 9]. Based on available record history, individual CCHF survivors were identified, and their households were traced. The categories were designed based on socio-clinical data of the subject and the occupational risk to CCHF infection. Study population belonging to Gujarat State was categorized as Category A: CCHF affected person/CCHF affected house/caretaker of the patient, close contacts, person involved in transportation of CCHF cases to hospital; Category B: All the family members (except children aged ≤5 yrs.) of neighborhood (i.e. within 50–100 m of the radius of confirmed CCHF case house; Category C: Animal handlers (person living in close contact with livestock, persons grazing the livestock) Category D: General population (low-risk group); Category E: Farmers; Category F: Abattoir Worker; Category G: Veterinarians; Category H: Healthcare workers. Samples of pregnant women and critically ill patients were not collected. Stratified purposive sampling was planned from Category A and B category, and random sampling was done from categories C, D, E, F, G and H..
Sample collection and processing
The Institutional Human Ethics Committee of ICMR-NIV, Pune (IHEC No, NIV/IEC/2017/37/42) approved the study. Informed written consent was obtained from all the subjects and parent/guardian in case of a child (< 18 yrs) subjects from the study population. Blood sample (3–5 mL) was collected from the study population in clot activator tube and transported to ICMR-NIV, Pune in a cold chain. After the receipt of the samples at NIV, each sample was checked for its quality and condition. Serum was separated from the blood sample of each subject and further aliquoted as 50 μL, 250 μL, stock and stored at − 20 °C until further use. Each sample was registered in laboratory information management system software by giving a unique identification number/anonymous code. A demographic and socio-clinical detail of every subject was also recorded. The complete process of sampling from the transportation of specimen to the laboratory investigations was done with utmost precaution, which includes; collection of samples by trained personnel using personal protective equipment (apron, mask, and gloves) in a leak-proof container and transportation by maintaining cold chain conditions. Aliquoting and testing of samples were carried out in a BSL-2 facility.
Development of indigenous anti-CCHF human IgG ELISA, its validation and screening of human serum samples
The human serum samples were tested for the presence of anti-CCHFV IgG antibodies using in-house developed CCHF Human IgG ELISA as defined below. Micro-well plates were coated with Gamma-inactivated CCHFV infected Vero CCL81 cell lysate antigen strain number NIV11704 (Rows A-D) and uninfected Vero CCL81 cell lysate (Rows E-H) at 1:20 dilution in carbonate buffer (pH 9.2, 0.025 M) and kept overnight at 4 °C (100 μL/well). The plate was blocked with liquid plate sealer (LPS) (Candor Biosciences, Germany) (100 μL/well) and kept at room temperature for 2 h. Further, these blocked and dried plates were stored at 4 °C. Serum sample (diluted 1:600 in 1% BSA in 10 mM PBS containing 0.1% Tween) was added (100 μL/well) in respective positive as well as negative antigen-coated wells and incubated for 1 h at 37 °C. Subsequently, anti-human IgG HRP (1,30,000, 100 μL/well) (Pierce) was added to the wells and incubated for 1 h at 37 °C. After the incubation, TMB (3,3′, 5,5’-Tetramethylbenzidine) substrate (100 μL/well) was added to the wells and for 10 min incubated at room temperature. Finally, the reaction was terminated using 1 N H2SO4 and absorbance was measured at 450 nm. At the end of each step of this assay, all the wells were washed five times using ten mM PBS, pH 6.8, and 0.1% Tween-20 (Sigma, St. Louis, MO, USA). Appropriate controls were included in each test. For an unknown sample (test sample), when OD value of the sample with CCHF antigen was more than 0.2 and the P/N ratio more than 1.5, the sample considered as “Positive.” Less than 1.5 P/N ratio was considered as “Negative” for the presence of anti-CCHFV IgG antibodies.
For validation of the assay, 134 sera samples (5 CCHF IgG antibody positive and 129 CCHF IgG antibody negative samples) tested by Vector Best kit, Russia, were compared with NIV anti-CCHF Human IgG ELISA kit. The assay was also provided for external validation to three national level laboratories. Cross reactivity of the assay was checked with Ganjam virus of Family Nairoviridae, Rift valley fever virus of Phenuiviridae, Dengue virus, Kyasanur Forest Disease virus and Japanese Encephalitis virus of Flaviviridae, Chikungunya virus of Togaviridae and Hanta virus of Hantaviridae. A total of 4978 human serum samples were screened using this indigenous ELISA for the presence of anti-CCHFV IgG antibodies.
Data analysis of results of samples screened using anti-CCHF human IgG ELISA
To identify the risk factors, the result obtained by testing 4978 human serum samples by indigenous anti-CCHF Human IgG ELISA was analyzed by univariate regression analysis using Epi-info 7 (7.2.1.0, CDC, Atlanta, GA, USA) and MedCalc Software bvba, version 18 (1993–2018). For plotting state of Gujarat map, the svg file was downloaded from https://commons.wikimedia.org/wiki/File:India_Gujarat_location_map.svg and edited as required.