This study is a cross sectional research conducted in several health facilities in rural areas of South West, Nigeria.A cross section of the patients seeking diagnosis for malaria and typhoid were tested for Dengue Virus (DV) NS1,IgM, IgG, using ELISA and Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). The DV test was done independent of whether the patients are positive for malaria and typhoid or not.
This study involved all consenting out-patients that reported to health facilities for malaria and typhoid fever during the course of the research. Any sample testing positive to the different types of malaria parasite were included in the study and any sample testing positive to different causative agents of typhoid fever was included in the study or both diseases All collected sample must have been tested in the facility and result registered in the facility register. These samples were then tested for dengue NS1 used as a first line marker for dengue virus. All samples testing positive for dengue NSI were again tested for anti DV IgM, IgG and RT-PCR.
5 ml of blood was aseptically collected from patients (n=1074) seeking malaria and/or typhoid diagnosis in the health institutions in rural areas of South West Nigeria from October to September in the year sampled. The blood samples were collected into EDTA bottles from each participant by a trained phlebotomist using needle and syringe and were immediately transported in cold chain to the Microbiology Laboratory.Each bottle was labeled indicating their age,sex and location.Blood samples were shared into 2 EDTA bottles and one to be used for DV ELISA and the other for malaria and typhoid.
Test for Malaria
Malaria testing was done in the clinics using the Rapid Diagnostics Testing after which samples were immediately shipped to the laboratory and confirmed using the Giemsa staining technique.
Test for Typhoid Fever
Typhoid fever was tested in theclinical laboratory using the slide agglutination and typhoid RDT, samples were shipped to the laboratory for confirmation using the tube agglutination technique as adapted to the use of microtiter plate, all the patients were made to come back a week after the first test for a second sample collection used for paired sample testing .
Enzyme Linked Immunosorbent Assay
Blood samples for ELISA was immediately centrifuged at 3000 rpm (Beckman Microfuge centrifuge centrifuge) and sera separated from the whole blood and immediately used for ELISA. Sera stored in the refrigerator were brought out and allowed to attain room temperature as well as all the reagents. Sera were dispensed into the antibody impregnated ELISA microplate and the test carried out as described by the manufacturer of the ELISA test kits (Melsin Medicals, China). ELISA kits used included ELISA NS1, IgM and IgG for research. ELISA plates were loaded into the microplate reader (Molecular devices, USA) at optical density of 450nm, absorbance were generated and analyzed using the myassays software to generate the concentration of each sample in each of the parameters for analysis.
RNA was extracted using the Norgen Biotek total RNA extraction kit. 100 ml of non-coagulating whole blood was collected into well-labeled RNAse free microfuge tubes and 350 lysis buffer added to the blood in the microfuge tubes and the extraction procedure carried out as directed by the manufacturer (Norgen Biotek, Ontario Canada). All samples testing positive to DV NS1, IgM and IgG had their RNA extracted RT-PCR.
RT- PCR procedure
RT-qPCR was carried out on all samples testing positive to dengue NS1 by ELISA technique after RNA extraction. A PCR reaction mixture was set up using the MAXIMA SYBR green with ROX RT-PRCR master mix to achieve a total volume of 25ml using the hot start as described by the manufacturer. The primers used were obtained from already published research  which are universal primers targeting 3′ untranslated region of all complete genome sequences of dengue virus available in GenBank (n = 3,305)DENV_F- GCATATTGACGCTGGGARAGAC, DENV_R1-3 -TTCTGTGCCTGGAATGATGCTG, DENV_R4- YTCTGTGCCTGGATWGATGTTG) and probe (DENV_P- CAGAGATCCTGCTGTC). Hence all 4 types of DEV will be detected. The primers were sent to Inqaba Biotech, South Africa for synthesis. The PCR mixtures were put into the thermocycler (Biorad icycler, (Biorad USA). The PCR program was as follows- UDG pretreatment 50°C 2 mins for 1 cycle, initial denaturation 95°C for 10 mins 1 cycle, denaturation at 95°C for 15 sec 40 cycle, annealing at 60°C for 30 sec 40 cycle and extension at 72°C for 30 sec 40 cycles. Data acquisition was then done and analyzed with the icycler software for CT values.