Wastewater-based epidemiology surveillance as an early warning system for SARS-CoV-2 in Indonesia.

BACKGROUND: Wastewater-based epidemiology (WBE) surveillance has been proposed as an early warning system (EWS) for community SARS-CoV-2 transmission. However, there is limited data from low-and middle-income countries (LMICs). This study aimed to assess the ability of WBE surveillance to detect SARS-CoV-2 in formal and informal environments in Indonesia using different methods of sample collection, to compare WBE data with patterns of clinical cases of COVID-19 within the relevant communities, and to assess the WBE potential to be used as an EWS for SARS-CoV-2 outbreaks within a community. MATERIALS AND METHODS: We conducted WBE surveillance in three districts in Yogyakarta province, Indonesia, over eleven months (27 July 2021 to 7 January 2022 [Delta wave]; 18 January to 3 June 2022 [Omicron wave]). Water samples using grab, and/or passive sampling methods and soil samples were collected either weekly or fortnightly. RNA was extracted from membrane filters from processed water samples and directly from soil. Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) was performed to detect the SARS-CoV-2 N and ORF1ab genes. RESULTS: A total of 1,582 samples were collected. Detection rates of SARS-CoV-2 in wastewater reflected the incidence of community cases, with rates of 85% at the peak to 2% at the end of the Delta wave and from 94% to 11% during the Omicron wave. A 2-week lag time was observed between the detection of SARS-CoV-2 in wastewater and increasing cases in the corresponding community. CONCLUSION: WBE surveillance for SARS-CoV-2 in Indonesia was effective in monitoring patterns of cases of COVID-19 and served as an early warning system, predicting the increasing incidence of COVID-19 cases in the community.

The association between high mobility group box 1 (HMGB1) and Interleukin-18 (IL-18) serum concentrations in COVID-19 inpatients.

Background: High mobility group box 1 (HMGB1) and interleukin-18 (IL-18) are involved in various non-coronavirus disease pathogenesis and are reported as potential biomarkers for coronavirus disease (COVID-19). However, their association with COVID-19 pathogenesis has not yet been explored. Aim: This study aimed to investigate the association between HMGB1 and IL-18 concentrations in the sera of COVID-19 patients versus non-COVID-19 patients. Material and methods: We used stored serum samples obtained from 30 COVID-19 patients and 30 non-COVID-19 patients. We collected data on age, gender, treatment status, principal diagnosis, and comorbidity from patient medical records. HMGB1 and IL-18 concentrations were analyzed in the serum by enzyme-linked immunosorbent assay (ELISA). The swab samples' RT-PCR cycle threshold (CT) values were obtained from the laboratory database. Results: HMGB1 concentrations were increased in the COVID-19 inpatients and non-COVID-19 inpatients compared to non-COVID-19 outpatients (COVID-19 inpatients vs. non-COVID-19 outpatients: 151.33 (90.27-192.38) vs. 80.75 (54.16-128.72) ng/ml; p = 0.0316; non-COVID-19 inpatients vs. non-COVID-19 outpatients: 152.66 (104.04-288.51) vs. 80.75 (54.16-128.72) ng/ml; p = 0.0199). IL-18 concentrations were also higher in the COVID-19 inpatients and non-COVID-19 inpatients compared to non-COVID-19 outpatients (COVID-19 inpatients vs. non-COVID-19 outpatients: 620.00 (461.50-849.6) vs. 403.10 (372.70-556.90) pg/ml; p = 0.0376; non-COVID-19 inpatients vs. non-COVID-19 outpatients: 835.70 (558.30-1602.00) vs. 403.10 (372.70-556.90) pg/ml; p = 0.0026). Moreover, HMGB1 was associated with IL-18 concentrations in the sera of COVID-19 inpatients (p = 0.0337; r = 0.5500). Conclusion: The association of HMGB1 and IL-18 in COVID-19 might indicate the potential for a dangerous cycle leading to a cytokine storm to occur.

Sequence analysis of the Spike, RNA-dependent RNA polymerase, and protease genes reveals a distinct evolutionary pattern of SARS-CoV-2 variants circulating in Yogyakarta and Central Java provinces, Indonesia.

During the Covid-19 pandemic, the resurgence of SARS-CoV-2 was due to the development of novel variants of concern (VOC). Thus, genomic surveillance is essential to monitor continuing evolution of SARS-CoV-2 and to track the emergence of novel variants. In this study, we performed phylogenetic, mutation, and selection pressure analyses of the Spike, nsp12, nsp3, and nsp5 genes of SARS-CoV-2 isolates circulating in Yogyakarta and Central Java provinces, Indonesia from May 2021 to February 2022. Various bioinformatics tools were employed to investigate the evolutionary dynamics of distinct SARS-CoV-2 isolates. During the study period, 213 and 139 isolates of Omicron and Delta variants were identified, respectively. Particularly in the Spike gene, mutations were significantly more abundant in Omicron than in Delta variants. Consistently, in all of four genes studied, the substitution rates of Omicron were higher than that of Delta variants, especially in the Spike and nsp12 genes. In addition, selective pressure analysis revealed several sites that were positively selected in particular genes, implying that these sites were functionally essential for virus evolution. In conclusion, our study demonstrated a distinct evolutionary pattern of SARS-CoV-2 variants circulating in Yogyakarta and Central Java provinces, Indonesia.

Prognostic factors for the outcomes of COVID-19 patients infected with SARS-CoV-2 Omicron and Delta variants.

BACKGROUND: The SARS-CoV-2 Omicron variant has replaced the previously dominant Delta variant because of high transmissibility. However, studies on the impact of the Omicron variant on the severity of COVID-19 are still limited in developing countries. Our study aimed to determine the prognostic factors for the outcomes of patients infected with SARS-CoV-2 Omicron and Delta variants, including age, sex, comorbidities, and smoking. METHODS: In this retrospective cross-sectional study, we involved 352 patients with COVID-19 from Yogyakarta and Central Java provinces, Indonesia, from May 2021 to February 2022, consisting of 164 males and 188 females. We included all patients with the PCR's Ct value of less than 30 for further whole-genome sequencing. RESULTS: Ct value and mean age of COVID-19 patients were not significantly different between both groups (p = 0.146 and 0.273, respectively). Patients infected with Omicron (n = 139) and Delta (n = 213) variants showed similar hospitalization (p = 0.396) and mortality rates (p = 0.565). Multivariate analysis of both groups showed that older age (≥ 65 years) had a higher risk for hospitalization (OR = 3.86 [95% CI = 1.29-11.5]; p = 0.015) and fatalities (OR = 3.91 [95% CI = 1.35-11.42]; p = 0.012). In both groups, patients with cardiovascular disease had a higher risk for hospitalization (OR = 5.36 [95% CI = 1.08-26.52]; p = 0.039), whereas patients with diabetes revealed a higher risk for fatalities (OR = 9.47 [95% CI = 3.23-27.01]; p = < 0.001). CONCLUSIONS: Our study shows that patients infected with Omicron and Delta variants reveal similar clinical outcomes, including hospitalization and mortality. Our findings further confirm that older age, cardiovascular disease, and diabetes are substantial prognostic factors for the outcomes of COVID-19 patients. Our findings imply that COVID-19 patients with older age, cardiovascular disease, or diabetes should be treated comprehensively and cautiously to prevent further morbidity and mortality. Furthermore, incomplete data on vaccination status hampered us from analyzing further its impact on hospitalization and mortality in our patients.

The high mutation rate at the D614G hotspot-furin cleavage site region increases the priming efficiency of the Spike protein by furin protease: analysis of Indonesian SARS-CoV-2 G614 variants obtained during the early COVID-19 pandemic.

D614G mutation plays a significant role in the transmissibility of SARS-CoV-2. Identification of other mutations related to D614G mutation within the Spike protein is pivotal as they might contribute to the pathogenicity of SARS-CoV-2. This study aims to analyze the mutation rate of furin cleavage site (FCS) region of Indonesian origin SARS-CoV-2 and to predict the effect of mutation against Spike priming efficiency by furin. A total of 375 sequences of Indonesian isolates obtained during the early pandemic were used for mutation analysis. Mutation analysis includes mutation pattern, variability, frequency of mutation, amino acid conservation, and mutation rate. The effect of mutation against Spike priming efficiency by furin protease from eight sequences with mutation in the FCS region was analyzed by protein-protein docking. We showed that mutations related to the G614 variant were increasing through time, in contrast to the D614 variant. The FCS region at the position 675-692 contained the most variable (66.67%) as well as the highest mutation frequency (85.92%) and has been observed to be the hotspot mutations linked to the D614G mutation. The D614G hotspot-FCS region (residue 600-700) had the highest amino acid change per site (20.8%) as well as the highest mutation rate as 1.34 × 10-2 substitution per site per year (95% CI 1.79 × 10-3-2.74 × 10-2), compared with other Spike protein regions. Mutations in the FCS region were the most common mutation found after the D614G mutation. These mutations were predicted to increase the Spike priming efficiency by furin. Thus, this study elucidates the importance of D614G mutation to other mutations located in the FCS region and their significance to Spike priming efficiency by furin. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00827-w.

The feasibility of SARS-CoV-2 surveillance using wastewater and environmental sampling in Indonesia.

BACKGROUND: Wastewater-based epidemiology (WBE) surveillance as an early warning system (EWS) for monitoring community transmission of SARS-CoV-2 in low- and middle-income country (LMIC) settings, where diagnostic testing capacity is limited, needs further exploration. We explored the feasibility to conduct a WBE surveillance in Indonesia, one of the global epicenters of the COVID-19 pandemic in the middle of 2021, with the fourth largest population in the world where sewer and non-sewered sewage systems are implemented. The feasibility and resource capacity to collect samples on a weekly or fortnightly basis with grab and/or passive sampling methods, as well as to conduct qualitative and quantitative identification of SARS-CoV-2 ribonucleic acid (RNA) using real-time RT-PCR (RT-qPCR) testing of environmental samples were explored. MATERIALS AND METHODS: We initiated a routine surveillance of wastewater and environmental sampling at three predetermined districts in Special Region of Yogyakarta Province. Water samples were collected from central and community wastewater treatment plants (WWTPs), including manholes flowing to the central WWTP, and additional soil samples were collected for the near source tracking (NST) locations (i.e., public spaces where people congregate). RESULTS: We began collecting samples in the Delta wave of the COVID-19 pandemic in Indonesia in July 2021. From a 10-week period, 54% (296/544) of wastewater and environmental samples were positive for SARS-CoV-2 RNA. The sample positivity rate decreased in proportion with the reported incidence of COVID-19 clinical cases in the community. The highest positivity rate of 77% in week 1, was obtained for samples collected in July 2021 and decreased to 25% in week 10 by the end of September 2021. CONCLUSION: A WBE surveillance system for SARS-CoV-2 in Indonesia is feasible to monitor the community burden of infections. Future studies testing the potential of WBE and EWS for signaling early outbreaks of SARS-CoV-2 transmissions in this setting are required.

Dengue virus population genetics in Yogyakarta, Indonesia prior to city-wide Wolbachia deployment.

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.

Co-infection of SARS-CoV-2 with other viral respiratory pathogens in Yogyakarta, Indonesia: A cross-sectional study.

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Aedes aegypti abundance and insecticide resistance profiles in the Applying Wolbachia to Eliminate Dengue trial.

The Applying Wolbachia to Eliminate Dengue (AWED) trial was a parallel cluster randomised trial that demonstrated Wolbachia (wMel) introgression into Ae. aegypti populations reduced dengue incidence. In this predefined substudy, we compared between treatment arms, the relative abundance of Ae. aegypti and Ae. albopictus before, during and after wMel-introgression. Between March 2015 and March 2020, 60,084 BG trap collections yielded 478,254 Ae. aegypti and 17,623 Ae. albopictus. Between treatment arms there was no measurable difference in Ae. aegypti relative abundance before or after wMel-deployments, with a count ratio of 0.96 (95% CI 0.76, 1.21) and 1.00 (95% CI 0.85, 1.17) respectively. More Ae. aegypti were caught per trap per week in the wMel-intervention arm compared to the control arm during wMel deployments (count ratio 1.23 (95% CI 1.03, 1.46)). Between treatment arms there was no measurable difference in the Ae. albopictus population size before, during or after wMel-deployment (overall count ratio 1.10 (95% CI 0.89, 1.35)). We also compared insecticide resistance phenotypes of Ae. aegypti in the first and second years after wMel-deployments. Ae. aegypti field populations from wMel-treated and untreated arms were similarly resistant to malathion (0.8%), permethrin (1.25%) and cyfluthrin (0.15%) in year 1 and year 2 of the trial. In summary, we found no between-arm differences in the relative abundance of Ae. aegypti or Ae. albopictus prior to or after wMel introgression, and no between-arm difference in Ae. aegypti insecticide resistance phenotypes. These data suggest neither Aedes abundance, nor insecticide resistance, confounded the epidemiological outcomes of the AWED trial.

Evolutionary dynamics of SARS-CoV-2 circulating in Yogyakarta and Central Java, Indonesia: sequence analysis covering furin cleavage site (FCS) region of the spike protein.

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.

Is the Infection of the SARS-CoV-2 Delta Variant Associated With the Outcomes of COVID-19 Patients?

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.

Association between prognostic factors and the outcomes of patients infected with SARS-CoV-2 harboring multiple spike protein mutations.

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.

Detection of SARS-CoV-2 spike protein D614G mutation by qPCR-HRM analysis.

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.

Efficacy of Wolbachia-Infected Mosquito Deployments for the Control of Dengue.

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.).

Molecular epidemiology of SARS-CoV-2 isolated from COVID-19 family clusters.

BACKGROUND: Transmission within families and multiple spike protein mutations have been associated with the rapid transmission of SARS-CoV-2. We aimed to: (1) describe full genome characterization of SARS-CoV-2 and correlate the sequences with epidemiological data within family clusters, and (2) conduct phylogenetic analysis of all samples from Yogyakarta and Central Java, Indonesia and other countries. METHODS: The study involved 17 patients with COVID-19, including two family clusters. We determined the full-genome sequences of SARS-CoV-2 using the Illumina MiSeq next-generation sequencer. Phylogenetic analysis was performed using a dataset of 142 full-genomes of SARS-CoV-2 from different regions. RESULTS: Ninety-four SNPs were detected throughout the open reading frame (ORF) of SARS-CoV-2 samples with 58% (54/94) of the nucleic acid changes resulting in amino acid mutations. About 94% (16/17) of the virus samples showed D614G on spike protein and 56% of these (9/16) showed other various amino acid mutations on this protein, including L5F, V83L, V213A, W258R, Q677H, and N811I. The virus samples from family cluster-1 (n = 3) belong to the same clade GH, in which two were collected from deceased patients, and the other from the survived patient. All samples from this family cluster revealed a combination of spike protein mutations of D614G and V213A. Virus samples from family cluster-2 (n = 3) also belonged to the clade GH and showed other spike protein mutations of L5F alongside the D614G mutation. CONCLUSIONS: Our study is the first comprehensive report associating the full-genome sequences of SARS-CoV-2 with the epidemiological data within family clusters. Phylogenetic analysis revealed that the three viruses from family cluster-1 formed a monophyletic group, whereas viruses from family cluster-2 formed a polyphyletic group indicating there is the possibility of different sources of infection. This study highlights how the same spike protein mutations among members of the same family might show different disease outcomes.

Reduced dengue incidence following deployments of Wolbachia-infected Aedes aegypti in Yogyakarta, Indonesia: a quasi-experimental trial using controlled interrupted time series analysis.

Background: Ae. aegypti mosquitoes stably transfected with the intracellular bacterium Wolbachia pipientis ( wMel strain) have been deployed for 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: In Yogyakarta City, Indonesia, following extensive community engagement, wMel Wolbachia-carrying mosquitoes were released every two weeks for 13-15 rounds over seven months in 2016-17, in a contiguous 5 km 2 area (population 65,000). A 3 km 2 area (population 34,000) on the opposite side of the city was selected a priori as an untreated control area. Passive surveillance data on notified hospitalised dengue patients was used to evaluate the epidemiological impact of Wolbachia deployments, using controlled interrupted time-series analysis. Results: 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 24 months following Wolbachia deployment. 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 6 months additional 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%). Conclusions: We demonstrate a significant reduction in dengue incidence following successful introgression of Wolbachia into local Ae. aegypti populations in an endemic setting in Indonesia. These findings are consistent with previous field trials in northern Australia, and support the effectiveness of this novel approach for dengue control.

Stable establishment of wMel Wolbachia in Aedes aegypti populations in Yogyakarta, Indonesia.

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.

Full-length genome characterization and phylogenetic analysis of SARS-CoV-2 virus strains from Yogyakarta and Central Java, Indonesia.

BACKGROUND: Recently, SARS-CoV-2 virus with the D614G mutation has become a public concern due to rapid dissemination of this variant across many countries. Our study aims were (1) to report full-length genome sequences of SARS-CoV-2 collected from four COVID-19 patients in the Special Region of Yogyakarta and Central Java provinces, Indonesia; (2) to compare the clade distribution of full-length genome sequences from Indonesia (n = 60) from March to September 2020 and (3) to perform phylogenetic analysis of SARS-CoV-2 complete genomes from different countries, including Indonesia. METHODS: Whole genome sequencing (WGS) was performed using next-generation sequencing (NGS) applied in the Illumina MiSeq instrument. Full-length virus genomes were annotated using the reference genome of hCoV-19/Wuhan/Hu-1/2019 (NC_045512.2) and then visualized in UGENE v. 1.30. For phylogenetic analysis, a dataset of 88 available SARS-CoV-2 complete genomes from different countries, including Indonesia, was retrieved from GISAID. RESULTS: All patients were hospitalized with various severities of COVID-19. Phylogenetic analysis revealed that one and three virus samples belong to clade L and GH. These three clade GH virus samples (EPI_ISL_525492, EPI_ISL_516800 and EPI_ISL_516829) were not only located in a cluster with SARS-CoV-2 genomes from Asia but also those from Europe, whereas the clade L virus sample (EPI_ISL_516806) was located amongst SARS-CoV-2 genomes from Asia. Using full-length sequences available in the GISAID EpiCoV Database, 39 of 60 SARS-CoV-2 (65%) from Indonesia harbor the D614G mutation. CONCLUSION: These findings indicate that SARS-CoV-2 with the D614G mutation appears to become the major circulating virus in Indonesia, concurrent with the COVID-19 situation worldwide.

Prevalence and Distribution of Dengue Virus in Aedes aegypti 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.

Baseline Characterization of Dengue Epidemiology in Yogyakarta City, Indonesia, before a Randomized Controlled Trial of Wolbachia for Arboviral Disease Control.

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.