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The outcome of SARS-CoV-2 infection is determined by multiple factors, including the viral, host genetics, age, and comorbidities. This study investigated the association between prognostic factors and disease outcomes of patients infected by SARS-CoV-2 with multiple S protein mutations. Fifty-one COVID-19 patients were recruited in this study. Whole-genome sequencing of 170 full-genomes of SARS-CoV-2 was conducted with the Illumina MiSeq sequencer. Most patients (47%) had mild symptoms of COVID-19 followed by moderate (19.6%), no symptoms (13.7%), severe (4%), and critical (2%). Mortality was found in 13.7% of the COVID-19 patients. There was a significant difference between the age of hospitalized patients (53.4 ± 18 years) and the age of non-hospitalized patients (34.6 ± 19) (p = 0.001). The patients’ hospitalization was strongly associated with hypertension, diabetes, and anticoagulant and were strongly significant with the OR of 17 (95% CI 2–144; p = 0.001), 4.47 (95% CI 1.07–18.58; p = 0.039), and 27.97 (95% CI 1.54–507.13; p = 0.02), respectively; while the patients’ mortality was significantly correlated with patients’ age, anticoagulant, steroid, and diabetes, with OR of 8.44 (95% CI 1.5–47.49; p = 0.016), 46.8 (95% CI 4.63–472.77; p = 0.001), 15.75 (95% CI 2–123.86; p = 0.009), and 8.5 (95% CI 1.43–50.66; p = 0.019), respectively. This study found the clade: L (2%), GH (84.3%), GR (11.7%), and O (2%). Besides the D614G mutation, we found L5F (18.8%), V213A (18.8%), and S689R (8.3%). No significant association between multiple S protein mutations and the patients’ hospitalization or mortality. Multivariate analysis revealed that hypertension and anticoagulant were the significant factors influencing the hospitalization and mortality of patients with COVID-19 with an OR of 17.06 (95% CI 2.02–144.36; p = 0.009) and 46.8 (95% CI 4.63–472.77; p = 0.001), respectively. Moreover, the multiple S protein mutations almost reached a strong association with patients’ hospitalization (p = 0.07). We concluded that hypertension and anticoagulant therapy have a significant impact on COVID-19 outcomes. This study also suggests that multiple S protein mutations may impact the COVID-19 outcomes. This further emphasized the significance of monitoring SARS-CoV-2 variants through genomic surveillance, particularly those that may impact the COVID-19 outcomes.

BackgroundThe SARS-CoV-2 Omicron variant has replaced the previously dominant Delta variant because of high transmissibility. It is responsible for the current increase in the COVID-19 infectivity rate worldwide. However, studies on the impact of the Omicron variant on the severity of COVID-19 are still limited in developing countries. Here, we compared the outcomes of patients infected with SARS-CoV-2 Omicron and Delta variants and associated with prognostic factors, including age, sex, comorbidities, and smoking.MethodsWe involved 352 patients, 139 with the Omicron variant and 213 with the Delta variant. The whole-genome sequences of SARS-CoV-2 were conducted using the Illumina MiSeq next-generation sequencer.ResultsCt value and mean age of COVID-19 patients were not significantly different between both groups (Delta: 20.35 ± 4.07 vs. Omicron: 20.62 ± 3.75; p=0.540; and Delta: 36.52 ± 21.24 vs. Omicron: 39.10 ± 21.24; p=0.266, respectively). Patients infected with Omicron and Delta variants showed similar hospitalization (p=0.433) and mortality rates (p=0.565). Multivariate analysis showed that older age (≥65 years) had higher risk for hospitalization (OR=3.67 [95% CI=1.22-10.94]; p=0.019) and fatalities (OR=3.93 [95% CI=1.35-11.42]; p=0.012). In addition, patients with cardiovascular disease had higher risk for hospitalization (OR=5.27 [95% CI=1.07-25.97]; p=0.041), whereas patients with diabetes revealed higher risk for fatalities (OR=9.39 [95% CI=3.30-26.72]; p=ConclusionsOur study shows that patients infected with Omicron and Delta variants reveal similar clinical outcomes, including hospitalization and mortality. In addition, our findings further confirm that older age, cardiovascular disease, and diabetes are strong prognostic factors for the outcomes of COVID-19 patients.

Growing evidence shows that viral co-infection is found repeatedly in patients with Coronavirus Disease-2019 (COVID-19). This is the first report of SARS-CoV-2 co-infection with viral respiratory pathogens in Indonesia.Over a one month period of April to May 2020, SARS-CoV-2 positive nasopharyngeal swabs in our COVID-19 referral laboratory in Yogyakarta, Indonesia, were tested for viral respiratory pathogens by real-time, reverse transcription polymerase chain reaction (RT-PCR). Proportion of co-infection reported in percentage.Fifty-nine samples were positive for other viral respiratory pathogens among a total of 125 samples. Influenza A virus was detected in 32 samples, Influenza B in 16 samples, Human metapneumovirus in 1 sample, and adenovirus in 10 samples. We did not detect any co-infection with respiratory syncytial virus. Nine (7.2%) patients had co-infection with more than two viruses.Viral co-infection with SARS-CoV-2 is common. These results will provide a helpful reference for diagnosis and clinical treatment of patients with COVID-19.

Rovik A, Daniwijaya EW, Supriyati E, Rahayu A, Kumalawati DA, Saraswati U, Handayaningsih AE, Rachman MP, Oktriani R, Kurniasari I, Candrasari DS, Nurhayati I, Sholeh R, Arianto B, Tantowijoyo W, Ahmad RA, Utarini A, Arguni E. 2022. Wolbachia genetic similarity in different insect host species: Drosophila melanogaster and Yogyakarta’s (Indonesia) Aedes aegypti as a novel host. Biodiversitas 23: 2321-2328. Wolbachia naturally presents in a large number of insects and other arthropod species. The Wolbachia strain wMel from Drosophila melanogaster has been stably transinfected into Aedes aegypti where it stops the mosquito host from being infected with medically important arbovirus like dengue. Consequently, Ae. aegypti infected with wMel have been released in Indonesia as a public health intervention against dengue. This study genetically compared wMel from Yogya field-caught D. melanogaster and the wMel in stably transfected Ae. aegypti used for field releases in Yogyakarta, Indonesia. The genetic similarity between wMel Wolbachia was evaluated by sequencing of Wolbachia surface protein (wsp) gene and some polymorphic genomic regions of insertion sites (IS) and variable number tandem repeats (VNTR) loci. The sequence of the Wolbachia surface protein (wsp) gene was 100% identical between hosts. There is no insertion sequence among specimens. The insertion sequence IS-WD1310 was identical between wMel from both hosts and among other strains, as well as the IS-WD516/7. The VNTR-141 period was identical within wMel from both hosts and among other strains, the VNTR-105 as well. Wolbachia Yogya field-caught D. melanogaster and Wolbachia strain wMel present in Ae. aegypti used for bio-control of dengue were genetically identical. These findings provide beneficial understanding to answer the public attention on safety issues, especially on the genetic similarity between Wolbachia strain in the natural and transfected hosts of this novel technology for dengue control.

Dengue has been endemic in Yogyakarta, Indonesia for decades. Here, we report the dengue epidemiology, entomology, and virology in Yogyakarta in 2016-2017, prior to the commencement of the Applying Wolbachia to Eliminate Dengue (AWED) randomized trial. Dengue epidemiological data were compiled and blood samples from dengue-suspected patients were tested for dengue virus (DENV). Ae. aegypti mosquito samples were caught from the field using BG-Sentinel traps and tested for the presence of DENV infection. Sequencing of the DENV E gene was used to determine the phylogeny and genotypes of circulating DENV. Within the last decade, the 2016-2017 dengue incidence was considered very high. Among the 649 plasma samples collected between March 2016-February 2017; and 36,910 mosquito samples collected between December 2016-May 2017, a total of 197 and 38 samples were DENV-positive by qRT-PCR, respectively. All four DENV serotypes were detected, with DENV-3 (n = 88; 44.67%) and DENV-1 (n = 87; 44.16%) as the predominant serotype, followed by DENV-4 (n = 12; 6.09%) and DENV-2 (n = 10; 5.08%). The Yogyakarta DENV-1 isolates were classified into Genotype I and IV, while DENV-2, DENV-3, and DENV-4 isolates were classified into the Cosmopolitan genotype, Genotype I, and Genotype II, respectively. Yogyakarta DENV isolates were closely related to Indonesian strains from neighboring Javanese cities, consistent with the endemic circulation of DENV on this highly populous island. Our study provides comprehensive baseline information on the DENV population genetic characteristics in Yogyakarta, which are useful as baseline data for the AWED trial and the future DENV surveillance in the city in the presence of a Wolbachia-infected Ae. aegypti population.

Background: Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) has been responsible for the current increase in Coronavirus disease 2019 (COVID-19) infectivity rate worldwide. We compared the impact of the Delta variant and non-Delta variant on the COVID-19 outcomes in patients from Yogyakarta and Central Java provinces, Indonesia.Methods: In this cross-sectional study, we ascertained 161 patients, 69 with the Delta variant and 92 with the non-Delta variant. The Illumina MiSeq next-generation sequencer was used to perform the whole-genome sequences of SARS-CoV-2.Results: The mean age of patients with the Delta variant and the non-Delta variant was 27.3 ± 20.0 and 43.0 ± 20.9 (p = 3 × 10−6). The patients with Delta variant consisted of 23 males and 46 females, while the patients with the non-Delta variant involved 56 males and 36 females (p = 0.001). The Ct value of the Delta variant (18.4 ± 2.9) was significantly lower than that of the non-Delta variant (19.5 ± 3.8) (p = 0.043). There was no significant difference in the hospitalization and mortality of patients with Delta and non-Delta variants (p = 0.80 and 0.29, respectively). None of the prognostic factors were associated with the hospitalization, except diabetes with an OR of 3.6 (95% CI = 1.02–12.5; p = 0.036). Moreover, the patients with the following factors have been associated with higher mortality rate than the patients without the factors: age ≥65 years, obesity, diabetes, hypertension, and cardiovascular disease with the OR of 11 (95% CI = 3.4–36; p = 8 × 10−5), 27 (95% CI = 6.1–118; p = 1 × 10−5), 15.6 (95% CI = 5.3–46; p = 6 × 10−7), 12 (95% CI = 4–35.3; p = 1.2 × 10−5), and 6.8 (95% CI = 2.1–22.1; p = 0.003), respectively. Multivariate analysis showed that age ≥65 years, obesity, diabetes, and hypertension were the strong prognostic factors for the mortality of COVID-19 patients with the OR of 3.6 (95% CI = 0.58–21.9; p = 0.028), 16.6 (95% CI = 2.5–107.1; p = 0.003), 5.5 (95% CI = 1.3–23.7; p = 0.021), and 5.8 (95% CI = 1.02–32.8; p = 0.047), respectively.Conclusions: We show that the patients infected by the SARS-CoV-2 Delta variant have a lower Ct value than the patients infected by the non-Delta variant, implying that the Delta variant has a higher viral load, which might cause a more transmissible virus among humans. However, the Delta variant does not affect the COVID-19 outcomes in our patients. Our study also confirms that older age and comorbidity increase the mortality rate of patients with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new virus responsible for the COVID-19 pandemic. The emergence of the new SARS-CoV-2 has been attributed to the possibility of evolutionary dynamics in the furin cleavage site (FCS) region. This study aimed to analyze the sequence of the FCS region in the spike protein of SARS-CoV-2 isolates that circulated in the Special Region of Yogyakarta and Central Java provinces in Indonesia. The RNA solution extracted from nasopharyngeal swab samples of confirmed COVID-19 patients were used and subjected to cDNA synthesis, PCR amplification, sequencing, and analysis of the FCS region. The sequence data from GISAID were also retrieved for further genome analysis. This study included 52 FCS region sequences. Several mutations were identified in the FCS region, i.e., D614G, Q675H, Q677H, S680P, and silent mutation in 235.57 C T. The most important mutation in the FCS region is D614G. This finding indicated the G614 variant was circulating from May 2020 in those two provinces. Eventually, the G614 variant totally replaced the D614 variant from September 2020. All Indonesian SARS-CoV-2 isolates during this study and those deposited in GISAID showed the formation of five clade clusters from the FCS region, in which the D614 variant is in one specific cluster, and the G614 variant is dispersed into four clusters. The data indicated there is evolutionary advantage of the D614G mutation in the FCS region of the spike protein of SARS-CoV-2 circulating in the Special Region of Yogyakarta and Central Java provinces in Indonesia.

Objectives Monitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome. Methods Six SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods. Results The results showed that the melting temperature (Tm) of the D614 variant was 79.39 ± 0.03 °C, which was slightly lower than the Tm of the G614 variant (79.62 ± 0.015 °C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing. Conclusions This study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation., SARS-CoV-2; D614G; Spike; qPCR; HRM analysis

Background: Recent studies focusing on the association of SARS-CoV-2 variants of concern (VOC) on COVID-19 outcomes have been reported. However, studies of the impact of multiple mutations within the spike (S) protein of SARS-CoV-2 on COVID-19 illness are limited. This study determined the association between multiple mutations within the S protein, prognosis factors, and the disease outcomes of SARS-CoV-2 infection. Methods: We included 51 COVID-19 patients from Yogyakarta and Central Java, Indonesia. Whole genome sequences of SARS-CoV-2 were determined by the Illumina MiSeq next-generation sequencer, followed by the phylogenetic analysis of 170 full-genomes of SARS-CoV-2 from different regions. We analyzed characteristics of COVID-19 patients and multiple mutations in association with different outcomes.Results: Among 51 patients, the clinical manifestations of COVID-19 were as follows: without any symptoms (13.7%), mild (47%), moderate (19.6%), severe (4%), critical (2%), and died (13.7%). The age of hospitalized patients (53.4 ± 18 years) was higher than non-hospitalized patients (34.6 ± 19) (p=0.001). A significant association between diabetes, hypertension, and anticoagulant and the hospitalization of patients was noted with p-value of 0.039 (OR=4.47 [95% CI=1.07-18.58]), 0.001 (OR=17 [95% CI=2-144]), and 0.02 (OR=27.97 [95% CI=1.54-507.13]), respectively; whereas a strong association between patients’ age, diabetes, anticoagulant, and steroid with the mortality of patients was revealed with p-value of 0.016 (OR=8.44 [95% CI=1.5-47.49]), 0.019 (OR=8.5 [95% CI=1.43-50.66]), 0.001 (46.8 [95% CI=4.63-472.77]), and 0.009 (OR=15.75 [95% CI=2-123.86]), respectively. All viruses contained the D614G variant, except one case. Accordingly, the samples were classified as the following clade: L (2%), GH (84.3%), GR (11.7%), and O (2%). Besides the D614G, the most common variants in the S protein were L5F (18.8%), V213A (18.8%), and S689R (8.3%). There was no significant association between multiple S protein variants with either hospitalization or mortality of COVID-19 (p=0.11 and 0.69, respectively). Multivariate analysis showed that hypertension and anticoagulant were the strong factors affecting the hospitalization and mortality of patients with COVID-19 with a p-value of 0.009 (OR=17.06 [95% CI=2.02-144.36]) and 0.001 (OR=46.8 (95% CI=4.63-472.77), respectively. Interestingly, the multiple S protein variants almost reached a significant level affecting the hospitalization of patients (p=0.07). Phylogenetic analysis showed that although most of the viruses from this study belonged to clade GH, none were detected as the variant of concern (VOC) and the variant of interest (VOI) of SARS-CoV-2.Conclusions: Here, we show for the first time the association between SARS-CoV-2 mutations within the S protein besides the VOC with the COVID-19 outcomes. Our findings suggest that multiple mutations in the S protein might affect the severity of COVID-19. Our study further suggests the importance of genomic surveillance to monitor SARS-CoV-2 variants, particularly those that might influence the outcomes of COVID-19 patients.

BACKGROUND: Aedes aegypti mosquitoes infected with the wMel strain of Wolbachia pipientis are less susceptible than wild-type A. aegypti to dengue virus infection. METHODS: We conducted a cluster-randomized trial involving releases of wMel-infected A. aegypti mosquitoes for the control of dengue in Yogyakarta, Indonesia. We randomly assigned 12 geographic clusters to receive deployments of wMel-infected A. aegypti (intervention clusters) and 12 clusters to receive no deployments (control clusters). All clusters practiced local mosquito-control measures as usual. A test-negative design was used to assess the efficacy of the intervention. Patients with acute undifferentiated fever who presented to local primary care clinics and were 3 to 45 years of age were recruited. Laboratory testing was used to identify participants who had virologically confirmed dengue (VCD) and those who were test-negative controls. The primary end point was symptomatic VCD of any severity caused by any dengue virus serotype. RESULTS: After successful introgression of wMel into the intervention clusters, 8144 participants were enrolled; 3721 lived in intervention clusters, and 4423 lived in control clusters. In the intention-to-treat analysis, VCD occurred in 67 of 2905 participants (2.3%) in the intervention clusters and in 318 of 3401 (9.4%) in the control clusters (aggregate odds ratio for VCD, 0.23; 95% confidence interval [CI], 0.15 to 0.35; P = 0.004). The protective efficacy of the intervention was 77.1% (95% CI, 65.3 to 84.9) and was similar against the four dengue virus serotypes. The incidence of hospitalization for VCD was lower among participants who lived in intervention clusters (13 of 2905 participants [0.4%]) than among those who lived in control clusters (102 of 3401 [3.0%]) (protective efficacy, 86.2%; 95% CI, 66.2 to 94.3). CONCLUSIONS: Introgression of wMel into A. aegypti populations was effective in reducing the incidence of symptomatic dengue and resulted in fewer hospitalizations for dengue among the participants. (Funded by the Tahija Foundation and others; AWED ClinicalTrials.gov number, NCT03055585; Indonesia Registry number, INA-A7OB6TW.).

Kumalawati DA, Supriyati E, Rachman MP. Oktriani R, Kurniasari I, Candrasari DS, Hidayati L, Handayaningsih AE, Probowati VC, Arianto B, Wardana DS, Pramuko NB, Utari A, Tantowijoyo W, Arguni E. 2020. Wolbachia infection prevalence as common insects’ endosymbiont in the rural area of Yogyakarta, Indonesia. Biodiversitas 21: 5608-5614. Control for mosquito-borne diseases such as dengue and chikungunya using vectors is urgently required. The World Mosquito Program, a multinational collaborative research program, is currently studying for the ability of Wolbachia-infected Aedes aegypti to control dengue. Community concerns on Wolbachia's natural existence in their surrounding living area. This study presents the field study of Wolbachia pipientis in insects commonly found in rural daily life. W. pipientis is an endosymbiotic bacterium commonly found in arthropods. Insects were collected from five villages in Sleman and Bantul District, Yogyakarta Province, Indonesia, from July to December 2012 and screened for Wolbachia infection using PCR. One hundred insects, including butterflies, moths, mosquitoes, flies, were collected. The results indicated that 44.9% of identified insect species were positive for Wolbachia pipientis, which support the existing data from other regions on the spread of Wolbachia infection in insects.

The successful establishment of the wMel strain of Wolbachia for the control of arbovirus transmission by Aedes aegypti has been proposed and is being implemented in a number of countries. Here we describe the successful establishment of the wMel strain of Wolbachia in four sites in Yogyakarta, Indonesia. We demonstrate that Wolbachia can be successfully introgressed after transient releases of wMel-infected eggs or adult mosquitoes. We demonstrate that the approach is acceptable to communities and that Wolbachia maintains itself in the mosquito population once deployed. Finally, our data show that spreading rates of Wolbachia in the Indonesian setting are slow which may reflect more limited dispersal of Aedes aegypti than seen in other sites such as Cairns, Australia., Author summary We show that the wMel strain of Wolbachia can be deployed successfully into mosquito populations in an area of intense dengue transmission in Yogyakarta, Indonesia. Deployment was achieved through either the release of eggs or adult mosquitoes with full community support. This represents the successful first step toward a large trial to evaluate the use of Wolbachia in Indonesia to disrupt transmission of arboviruses such as dengue, Zika and chikungunya.

Recent outbreaks of human coronaviruses, officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have put health authorities worldwide on a high alert. Firstly emerged in the city of Wuhan, China, SARS-CoV-2 infection is rapidly escalating into a global pandemic. It is first thought as the result of a zoonotic transmission event, similar to the previous epidemic of coronaviruses. However, a continuously increasing number of confirmed cases indicates that the virus gains capacity of efficient human-to-human transmission. Soon after the pandemic is arising, many efforts are focused on identifying the origin of SARS-CoV-2 infection in the human population. Current evidence suggests that the virus is probably derived from bat or pangolin coronaviruses as the natural host. Whether intermediate host(s) exist in the transmission cascade from bat or pangolin to humans is, to a great extent, elusive. This information is essential as the basis for infection prevention and control measures. In this review, we discuss our recent understanding of SARS-CoV-2 biology, highlighting its origin and molecular evolution.

BackgroundAe. aegypti mosquitoes stably transfected with the intracellular bacterium Wolbachia pipientis (wMel strain) have been deployed for the biocontrol of dengue and related arboviral diseases in multiple countries. Field releases in northern Australia have previously demonstrated near elimination of local dengue transmission from Wolbachia-treated communities, and pilot studies in Indonesia have demonstrated the feasibility and acceptability of the method. We conducted a quasi-experimental trial to evaluate the impact of scaled Wolbachia releases on dengue incidence in an endemic setting in Indonesia.Methods and findingsIn Yogyakarta City, Indonesia, following an extensive community engagement campaign, wMel Wolbachia-carrying mosquitoes were released every two weeks for 13–15 release rounds over seven months in 2016–17, in a contiguous 5 km2 area (population 65,000). A 3 km2 area (population 34,000) on the opposite side of the city was selected a priori as an untreated control area, on the basis of comparable socio-demographic characteristics and historical dengue incidence. Passive surveillance data on notified hospitalised dengue patients was used to evaluate the epidemiological impact of Wolbachia deployments, using controlled interrupted time series analysis. Rapid and sustained introgression of wMel Wolbachia into local Ae. aegypti populations was achieved. Thirty-four dengue cases were notified from the intervention area and 53 from the control area (incidence 26 vs 79 per 100,000 person-years) during the 24 months after Wolbachia was deployed. This corresponded in the regression model to a 73% reduction in dengue incidence (95% confidence interval 49%,86%) associated with the Wolbachia intervention. Exploratory analysis including an additional 6 months of post-intervention observations showed a small strengthening of this effect (30 vs 115 per 100,000 person-years; 76% reduction in incidence, 95%CI 60%,86%).ConclusionsThese findings demonstrate a significant reduction in dengue incidence following successful introgression of Wolbachia into local Ae. aegypti populations in an endemic setting in Indonesia. These results are consistent with previous field trials in northern Australia, and support the effectiveness of this novel approach for the control of dengue and other Aedes-borne diseases.

Background: Ae. aegypti mosquitoes stably transfected with the intracellular bacterium Wolbachia pipientis (wMel strain) have been deployed for the biocontrol of dengue and related arboviral diseases in multiple countries. Field releases in northern Australia have previously demonstrated near elimination of local dengue transmission from Wolbachia -treated communities. Methods: In Yogyakarta City, Indonesia, following an extensive community engagement campaign, wMel Wolbachia-carrying mosquitoes were released every two weeks for 13–15 rounds of release during a seven month period in 2016–17, in a contiguous 5 km2 area with population 65,000. A controlled interrupted time series (ITS) analysis of notified dengue cases was used to evaluate the epidemiological impact of Wolbachia deployments, in comparison to a predefined comparative untreated control area (3 km2 ; 34,000 population). Findings: Rapid and sustained introgression of wMel Wolbachia into local Ae. aegypti populations was achieved. Thirty-four dengue cases (hospitalised dengue hemorrhagic fever patients) were notified from the intervention area and 53 from the control area (incidence 26 vs 79 per 100,000 person-years) during the 24 months after Wolbachia was deployed. This corresponded in the ITS model to a 73% reduction in dengue incidence (95% confidence interval 49–86%) associated with the Wolbachia intervention. Exploratory analysis including an additional 6 months of post-intervention observations showed a small strengthening of this effect (30 vs 115 per 100,000 person-years; 76% reduction in incidence, 95%CI 60–86%). Interpretation: Our findings demonstrate that Wolbachia establishment in Ae. aegypti reduces dengue incidence in an endemic setting in Indonesia. Funding Statement: This work was funded by the Tahija Foundation, Indonesia. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Approval to release Wolbachia mosquitoes was obtained from the provincial and city governments of Yogyakarta prior to releases. Ethical approval was obtained from the Universitas Gadjah Mada Faculty of Medicine, Public Health and Nursing Ethics Committee to release Wolbachia mosquitoes, for blood-feeding the mosquito colony on human volunteers, and to access non-identifiable aggregate data on monthly notified DHF and DF case numbers from the Yogyakarta DHO. Written agreement to share dengue surveillance data and non-identifiable individual dengue RDT results from Puskesmas clinics was obtained from Yogyakarta DHO. Verbal and written consent was obtained from heads of households for hosting a BG trap or MRC, and BG hosts were compensated for the cost of powering traps.

Indonesia is one of the countries where dengue infection is prevalent. In this study we measure the prevalence and distribution of dengue virus (DENV) DENV-infected Aedes aegypti in Yogyakarta City, Indonesia, during the wet season when high dengue transmission period occurred, as baseline data before implementation of a Wolbachia-infected Aedes aegypti trial for dengue control. We applied One-Step Multiplex Real Time PCR (RT-PCR) for the type-specific-detection of dengue viruses in field-caught adult Aedes aegypti mosquitoes. In a prospective field study conducted from December 2015 to May 2016, adult female Aedes aegypti were caught from selected areas in Yogyakarta City, and then screened by using RT-PCR. During the survey period, 36 (0.12%) mosquitoes from amongst 29,252 female mosquitoes were positive for a DENV type. In total, 22.20% of dengue-positive mosquitoes were DENV-1, 25% were DENV-2, 17% were DENV-3, but none were positive for DENV-4. This study has provided dengue virus infection prevalence in field-caught Aedes aegypti and its circulating serotype in Yogyakarta City before deployment of Wolbachia-infected Aedes aegypti.

Indonesia is one of the countries where dengue infection is prevalent. In this study we measure the prevalence and distribution of dengue virus (DENV) DENV-infected Aedes aegypti in Yogyakarta City, Indonesia, during the wet season when high dengue transmission period occurred, as baseline data before implementation of a Wolbachia-infected Aedes aegypti trial for dengue control. We applied One-Step Multiplex Real Time PCR (RT-PCR) for the type-specific-detection of dengue viruses in field-caught adult Aedes aegypti mosquitoes. In a prospective field study conducted from December 2015 to May 2016, adult female Aedes aegypti were caught from selected areas in Yogyakarta City, and then screened by using RT-PCR. During the survey period, 36 (0.12%) mosquitoes from amongst 29,252 female mosquitoes were positive for a DENV type. In total, 22.20% of dengue-positive mosquitoes were DENV-1, 25% were DENV-2, 17% were DENV-3, but none were positive for DENV-4. This study has provided dengue virus infection prevalence in field-caught Aedes aegypti and its circulating serotype in Yogyakarta City before deployment of Wolbachia-infected Aedes aegypti.

Dengue is endemic in Indonesia. Here, we describe the epidemiology of dengue in the city of Yogyakarta, Central Java, as a prelude to implementation of a cluster-randomized trial of Wolbachia for the biocontrol of arboviral transmission. Surveillance records from 2006 to 2016 demonstrate seasonal oscillations of dengue incidence with varying magnitude. Two lines of evidence demonstrate a high force of infection; the hospitalized case burden of patients diagnosed with dengue hemorrhagic fever or dengue shock syndrome over the last decade consisted predominantly of children/adolescents, and a serosurvey of 314 healthy children aged 1–10 years found 68% possessed dengue virus–neutralizing antibodies. Finally, a mobility survey indicated children aged 1–10 years, and particularly 1–5 year-olds, spent most of their daytime hours at home. These findings inform the design of clinical trials to measure the impact of novel vector control methods such as Wolbachia introgression into Aedes aegypti mosquitoes, by providing baseline data on disease incidence and identifying subpopulations for recruitment into prospective studies of dengue virus infection and disease. The mobility survey findings indicate that in cluster trials of interventions applied at the community level, young children can reasonably be expected to spend most of their exposure time, in epidemiological terms, within the treatment arm to which they were randomized.