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A cross-sectional and observational study was carried between December 2014 and June 2015 in the Clinical Hospital “Dr. Roberto Nettel Flores” located in the city of Tapachula (14°56’ N, 92°17’ W), Chiapas State, near 15km from the Mexico-Guatemala border. This hospital attends patients form Soconusco region, composed by Tapachula and other 15 municipalities: Metapa, Acapetahua, Acacoyagua, Escuintla, Frontera Hidalgo, Huehuetan, Huixtla, Union Juarez, Mazatan, Cacahoatan, Villa Comatitlan, Suchiate, Tuxtla Chico, Tuzantan and Mapastepec. This region has a population of 728,647, and dengue fever (DENF) has been historically endemic [24,25] in this area.
The study was approved by the Bioethical Committee of the National Institute of Public Health (Project #1312 and approval #1738). Patients were enrolled in the study during a CHIKV outbreak, and blood samples were collected as part of the routine laboratory analysis of patients admitted to Hospital Dr. Roberto Nettel (S1 File). Therefore, only an oral consent to participate in the study was obtained from each patient. All pertinent information about the study (purpose, procedures, risks, benefits, alternatives to participation, etc.) was explained to potential participants in the presence of an independent witness, and every patient who agreed to participate was recorded in an intake form by the hospital’s Epidemiology Department [26].
The Hospital “Dr. Roberto Nettel” attends only State workers and their immediate relatives who represent 4.5% of the total population of Soconusco Region [27]. Patients who were admitted in this hospital were included in the study if their clinical symptoms met the following inclusion criteria for suspicion of CHIKF: acute onset of fever >38.5°C accompanied by severe arthralgia not explained by other medical conditions in patients residing in the epidemic area [28]. Exclusion criteria were if patients disagree to participate in the study or were DENV-positive by RT-PCR or ELISA.
Clinical examination and blood collection: After enrollment, a clinical examination was performed following the epidemiological case study format for CHIKV provided by the General Direction of Epidemiology, Ministry of Health [26]. This format collects presence or absence of following symptoms: fever, polyarthralgia, arthritis, headache, myalgia, edema, exanthema, adenopathies, retroorbital pain, digestive alterations (vomiting, diarrhea, and nausea), etcetera. To avoid any confusion between polyarthralgia and arthritis, we differentiated them being the first a transient, intermittent, or persistent pain in multiple joints while the second is the inflammation of one or more joints. Additional epidemiological data such as patient age, gender and travel history is also included. After the clinical examination, the physician filled the format by patient and then collected three blood samples from each patient: one tube with EDTA for a complete blood cell count and one tube with citrate and one dry-tube for the remaining laboratory tests. Dry-tubes were centrifuged at 3,000 rpm, and serum was aliquoted and stored at −80°C for testing.
Hematological parameters including erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, white blood cells (WBC), mean platelet volume, and red cell distribution width were determined using a hematology analyzer Advia® 120 (Siemens Healthcare Diagnostics, Erlangen, Germany). Other parameters, including differential WBC (%), prothrombin time (PT), activated partial thromboplastin time (PTT), and fibrinogen, were determined using a coagulation analyzer IL ACL Elite PRO (Diamond® Diagnostics, Holliston, MA). Laboratory assessment of liver function was determined by measuring aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), γ-glutamyltransferase, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, direct bilirubin, and indirect bilirubin using an Integrated Chemistry System Dimension® RxL Max® (Siemens Healthcare Diagnostics, Erlangen, Germany). Others biochemical parameters, including glucose, urea, creatinine, uric acid, triglycerides, total cholesterol, serum electrolytes (sodium, chloride, and potassium), and inflammatory marker C-reactive protein (CRP) were measured using Dimension® RxL Max®.
RNA was extracted from 140μL of serum from each sample using the QIAmp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. For molecular diagnostics, one-step qRT-PCR was performed according to the Center of Disease Control (CDC) protocol using primers and probe designed for Caribbean lineage (Asian genotype) [29]: 1) we used the primers CHIK 856 5’-ACCATCGGTGTTCCATCTAAAG-3’ and CHIK 962c 5’-GCCTGGGCTCATCGTTATT-3’ and the TaqMan Probe CHIK 908 5’-FAM- ACAGTGGTTTCGTGTGAGGGCTAC-NFQ-3´; 2) the qRT-PCR was prepared with the QIAGEN QuantiTect Probe RT-PCR kit® (Qiagen, Venlo, Netherlands) in accordance with the manufacturer’s protocol, adding 10μL of RNA sample per reaction in duplicate; and 3) the samples were run in the 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA). Thermal cycling conditions were: one cycle of 50°C for 30 minutes and 95°C for 15 minutes followed by 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The cycle threshold (CT) value was 38: results were considered positive if less than 38 cycles were needed in both wells; negative if greater than 38 cycles were needed in both wells, and equivocal if less than 38 cycles were needed in one of the two wells. Serum samples were screened for anti-CHIKV IgM antibodies by enzyme-linked immunosorbent assay (ELISA) as previously described [30,31] using the CHIKjj Detect MAC-ELISA kit (InBios, Inc., Seattle, WA). All samples were tested in duplicate and any inconclusive samples were retested. For dengue diagnosis, the samples were separately sent to the State Laboratory of Public Health of Chiapas. Serum was tested with Panbio® Dengue Early ELISA and Panbio® Dengue IgM Capture ELISA (Alere, Waltham, MA) for detection of NS1 antigen and IgM antibody following the manufacturer’s protocol.
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