The study was conducted in 39 locations corresponding to 39 districts/municipalities in 15 dengue endemics provinces in Indonesia (Figure 1). These provinces include Aceh, West Sumatra, Lampung, Bangka-Belitung, West Kalimantan, South Kalimantan, North Sulawesi, East Java, South-East Sulawesi, Maluku, West Nusa Tenggara, North Maluku. This study is part of the Indonesia national project, Rikhus Vektora that started in 2016
A mosquito survey was performed in all study sites from July to August 2016, during the rainy season. Single larval methods were performed randomly in at least 100 households in each study site during the study. Aedes larvae were collected and then reared in a field laboratory by using plastic trays with tap water and fish food for 3-4 days. Adult collection of Aedes mosquitoes were performed in the morning (morning resting) on mosquitoes resting inside houses using manual aspirators. Adult mosquitoes were also collected outside using standard procedures for all night human-landing collection methods from 6.00 pm to 6.00 am. Each adult collection method was performed in every study site. All methodologies used in this study have been previously described . Field data collections for larva and adult Aedes mosquitoes were performed by trained collectors in collaboration with local volunteers, local authorities and staff from district/municipality dengue control programs.
Single larva and rearing methods
The larval surveys were performed by collecting at least a single Aedes larva from each container. All larvae then were taken to the field laboratory located in the study sites and reared up to the fourth days until adult hatching. Emerged adult mosquitoes were then killed in a freezer (-20oC) or by using ethyl acetate for 5 to 10 minutes and immediately stored in 1.5 ml vial tubes with RNAlater (Qiagen, Hilden, Germany) by pools of 25 mosquitoes and kept refrigerated at 4°C prior to further analysis. Reared larvae and pupae that did not hatch to adult stage up to the fourth days were preserved under the same conditions for further analysis.
Adult mosquito collection methods
Three adult mosquito sampling methods were conducted simultaneously in all study sites, including: (1) morning resting collection, (2) human landing collection, (3) animal baited trap.
(1). Morning resting collections were made by eight collectors using hand nets and aspirators. Collections were conducted from 7.00 am to 9.00 am and included any resting locations within the house. All adults mosquitoes were placed into labelled paper caps and taken to the field laboratory for further analysis.
(2). Human landing collection were performed by eight local volunteers as collectors in three selected houses of each study sites for sampling adult mosquitoes using mouth aspirators. They were all trained before collecting mosquitoes. Three teams of two people sampled outdoors (up to 5 meters from the house) and indoors (inside the house). Each collector sat on chairs with exposing their legs for 50 of 60 minutes per hour. Sampling was conducted all night from 6.00 pm to 6.00 am. The team changed roles regularly every 2 hours with a 2 hour-break. Although, the targeted Aedes mosquitoes are diurnal, the Indonesia law does not allow human landing collections during day time. Therefore, collections had to be conducted at night. This introduces a strong bias in the sampling but since it is what surveillance teams do in accordance with the law, this method was nevertheless performed. Mosquitoes that have been collected per hour were then taken to the field laboratory for species identification and further analysis.
(3). Animal baited trap was conducted by using tame animal placed inside a net all night. Mosquito collections were carried out for 15 minutes per hour inside the nets by 3 collectors. Collected mosquitoes were then similarly preserved as for the human landing collection method.
All mosquitoes from these three collecting methods identified as Ae. aegypti and Ae. albopictus were then killed with ethyl acetate, pooled up to 25 mosquitoes in labelled 1.5 ml vial tubes with RNAlater (Qiagen, Hilden, Germany) and preserved based on the same cold chain management than the one above used for larvae.
Detection of Dengue virus from mosquitoes
The Ae. aegypti and Ae. albopictus mosquito pools were homogenized in 1.5ml tubes containing 200 µl PBS 1x by using pellet pestles. RNA was extracted using QIAamp®Viral RNA Mini Kit (Qiagen®, Courtaboeuf, France). RNAs were extracted from 200-µl homogenized samples following the manufacturer’s instructions.
All RNA extracted samples were analyzed for Dengue detection using Lanciotti’s protocol . The nested RT-PCR for Dengue was performed using SimpliAmp Thermal Cycler Applied Biosystems™ (ThermoFisher Scientific®, United States). Amplification of Dengue RNA was carried out with following specific primers : D1 (5’-TCA ATA TGC TGA AAC GCG CGA GAA ACC G-3’), D2 (5’-TTG CAC CAA CAG TCA ATG TCT TCA GGT TC-3’), TS1 (5’-CGT CTC AGT GAT CCG GGG G-3’), TS2 (5’-CGC CAC AAG GGC CAT GAA CAG-3’), TS3 (5’-TAA CAT CAT CAT GAG ACA GAG C- 3’), and TS4 (5’-CTC TGT TGT CTT AAA CAA GAG A-3’).
The first amplification of Dengue virus was performed using Superscript III one-step RT-PCR kit (Invitrogen, Carlsbad, CA). The cycling conditions consisted of initial 95°C denaturation step for 2 minutes, followed by 40 cycles of 95°C denaturation for 30 seconds, 60°C annealing for 1 minute, and 72°C extension for 1 minute 30 seconds, and a final extension step 72°C for 10 minutes. Samples were then stored at 4°C. First step PCR products were run on 2% agarose gel under 120 V current for 1 hour and followed by visualization using SYBR® safe DNA gel stain (Invitrogen, Carlsbad, CA, USA) under UV condition in GelDoc system and check for presence of the 511 bp control band corresponding to dengue virus (DENV) positive. Subsequent serotyping was conducted by using 1st step PCR product with thermal cycle setting as follow : initial denaturation step at 95°C for 2 minutes, followed by 10 cycles of denaturation step at 95°C for 30 seconds, 60°C annealing for 1 minute, and an extension step at 72°C for 1 minute and 30 seconds. The final extension step was conducted at 72°C for 10 minutes. Subsequently, samples were stored at 4°C. Amplification product on 2% agarose gel were then carried out under 80V current for 1 hour and check under UV condition. Multiplex serotyping reaction is expected to produce single-specific band with the size of 482bp for DEN-1 , 119bp for DENV-2, 290bp for DENV-3, and 389bp for DENV-4. All of field samples were tested for the presence of dengue virus after being pooled by 25 individuals of the same species.
A first Factor Analysis of Mixed Data (FAMD)  was conducted using the incidence data, the number of mosquitoes and the number of positive pools for each dengue serotype as quantitative parameters, and mosquito species, methods of collection and provinces as qualitative parameters. The effectiveness of the collection methods (qualitative data) against mosquito species (quantitative data) was assessed using a second FAMD. These analyses were performed using the R software with FactoMineR .