Whole genome sequencing of Indonesian dengue virus isolates using next-generation sequencing

Indonesia is a tropical country and hyperendemic for dengue. The disease prevalently affected Indonesian and it caused high morbidity and substantial economic burden. This vector-borne viral disease is caused by infection of dengue viruses (DENVs), which are the member of Flaviviridae family. While most of dengue studies in Indonesia focused on the epidemiology, the clinical aspects, the vectors, and to certain extent the virology, there were still gaps in the DENVs genomic aspects. Considering their high mutation rate, the DENVs were known for their high genetic diversity and it might affect the characteristics of the viruses. Comprehensive DENV genomic data were thus important for many aspects of disease management, including virus surveillance, pathogenesis, diagnostics, antiviral drug design, and vaccine development. We established in this study a method for DENV whole genome sequencing using the advanced Next-Generation Sequencing (NGS) and Nextera XT DNA library preparation kit, coupled with simplified bioinformatic analysis methods. The Indonesian DENVs from four serotypes were isolated from patients’ sera, while library was prepared from enriched templates and sequenced using Illumina NGS. Our study highlighted the potential of a robust NGS method in producing whole genome sequence of DENVs, which would be important for future dengue studies

An investig-ation into the epidemiology of chikungunya virus across neglected regions of Indonesia

Funder: US-CDCBackground: Chikungunya virus (CHIKV) is an important emerging and re-emerging public health problem worldwide. In Indonesia, where the virus is endemic, epidemiological information from outside of the main islands of Java and Bali is limited. Methodology/Principal Findings: Four hundred and seventy nine acutely febrile patients presenting between September 2017–2019 were recruited from three city hospitals situated in Ambon, Maluku; Banjarmasin, Kalimantan; and Batam, Batam Island as part of a multi-site observational study. CHIKV RNA was detected in a single serum sample while a separate sample was IgM positive. IgG seroprevalence was also low across all three sites, ranging from 1.4–3.2%. The single RT-PCR positive sample from this study and 24 archived samples collected during other recent outbreaks throughout Indonesia were subjected to complete coding region sequencing to assess the genetic diversity of Indonesian strains. Phylogenetic analysis revealed all to be of a single clade, which was distinct from CHIKV strains recently reported from neighbouring regions including the Philippines and the Pacific Islands. Conclusions/Significance: Chikungunya virus strains from recent outbreaks across Indonesia all belong to a single clade. However, low-level seroprevalence and molecular detection of CHIKV across the three study sites appears to contrast with the generally high seroprevalences that have been reported for non-outbreak settings in Java and Bali, and may account for the relative lack of CHIKV epidemiological data from other regions of Indonesia

An investig-ation into the epidemiology of chikungunya virus across neglected regions of Indonesia

Funder: US-CDCBackground: Chikungunya virus (CHIKV) is an important emerging and re-emerging public health problem worldwide. In Indonesia, where the virus is endemic, epidemiological information from outside of the main islands of Java and Bali is limited. Methodology/Principal Findings: Four hundred and seventy nine acutely febrile patients presenting between September 2017–2019 were recruited from three city hospitals situated in Ambon, Maluku; Banjarmasin, Kalimantan; and Batam, Batam Island as part of a multi-site observational study. CHIKV RNA was detected in a single serum sample while a separate sample was IgM positive. IgG seroprevalence was also low across all three sites, ranging from 1.4–3.2%. The single RT-PCR positive sample from this study and 24 archived samples collected during other recent outbreaks throughout Indonesia were subjected to complete coding region sequencing to assess the genetic diversity of Indonesian strains. Phylogenetic analysis revealed all to be of a single clade, which was distinct from CHIKV strains recently reported from neighbouring regions including the Philippines and the Pacific Islands. Conclusions/Significance: Chikungunya virus strains from recent outbreaks across Indonesia all belong to a single clade. However, low-level seroprevalence and molecular detection of CHIKV across the three study sites appears to contrast with the generally high seroprevalences that have been reported for non-outbreak settings in Java and Bali, and may account for the relative lack of CHIKV epidemiological data from other regions of Indonesia

Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia

Abstract: Background: Dengue virus (DENV) infects hundreds of thousands of people annually in Indonesia. However, DENV sequence data from the country are limited, as samples from outbreaks must be shipped across long-distances to suitably equipped laboratories to be sequenced. This approach is time-consuming, expensive, and frequently results in failure due to low viral load or degradation of the RNA genome. Methods: We evaluated a method designed to address this challenge, using the ‘Primal Scheme’ multiplex PCR tiling approach to rapidly generate short, overlapping amplicons covering the complete DENV coding-region, and sequencing the amplicons on the portable Nanopore MinION device. The resulting sequence data was assessed in terms of genome coverage, consensus sequence accuracy and by phylogenetic analysis. Results: The multiplex approach proved capable of producing near complete coding-region coverage from all samples tested (x¯ = 99.96%, n = 18), 61% of which could not be fully amplified using the current, long-amplicon PCR, approach. Nanopore-generated consensus sequences were found to be between 99.17–99.92% identical to those produced by high-coverage Illumina sequencing. Consensus accuracy could be improved by masking regions below 20X coverage depth (99.69–99.92%). However, coding-region coverage was reduced at this depth (x¯ = 93.48%). Nanopore and Illumina consensus sequences generated from the same samples formed monophyletic clades on phylogenetic analysis, and Indonesian consensus sequences accurately clustered by geographical origin. Conclusion: The multiplex, short-amplicon approach proved superior for amplifying DENV genomes from clinical samples, particularly when the virus was present at low concentrations. The accuracy of Nanopore-generated consensus sequences from these amplicons was sufficient for identifying the geographic origin of the samples, demonstrating that the approach can be a useful tool for identifying and monitoring DENV clades circulating in low-resource settings across Indonesia. However, the inaccuracies in Nanopore-generated consensus sequences mean that the approach may not be appropriate for higher resolution transmission studies, particularly when more accurate sequencing technologies are available

Distinct Dengue Disease Epidemiology, Clinical, and Diagnosis Features in Western, Central, and Eastern Regions of Indonesia, 2017–2019

The people of Indonesia have been afflicted by dengue, a mosquito-borne viral disease, for over 5 decades. The country is the world's largest archipelago with diverse geographic, climatic, and demographic conditions that may impact the dynamics of disease transmissions. A dengue epidemiology study was launched by us to compare and understand the dynamics of dengue and other arboviral diseases in three cities representing western, central, and eastern Indonesia, namely, Batam, Banjarmasin, and Ambon, respectively. A total of 732 febrile patients were recruited with dengue-like illness during September 2017–2019 and an analysis of their demographic, clinical, and virological features was performed. The seasonal patterns of dengue-like illness were found to be different in the three regions. Among all patients, 271 (37.0%) were virologically confirmed dengue, while 152 (20.8%) patients were diagnosed with probable dengue, giving a total number of 423 (57.8%) dengue patients. Patients' age and clinical manifestations also differed between cities. Mostly, mild dengue fever was observed in Batam, while more severe cases were prominent in Ambon. While all dengue virus (DENV) serotypes were detected, distinct serotypes dominated in different locations: DENV-1 in Batam and Ambon, and DENV-3 in Banjarmasin. We also assessed the diagnostic features in the study sites, which revealed different patterns of diagnostic agreements, particularly in Ambon. To detect the possibility of infection with other arboviruses, further testing on 461 DENV RT-PCR-negative samples was performed using pan-flavivirus and -alphavirus RT-PCRs; however, only one chikungunya infection was detected in Ambon. A diverse dengue epidemiology in western, central, and eastern Indonesia was observed, which is likely to be influenced by local geographic, climatic, and demographic conditions, as well as differences in the quality of healthcare providers and facilities. Our study adds a new understanding on dengue epidemiology in Indonesia

Distinct Dengue Disease Epidemiology, Clinical, and Diagnosis Features in Western, Central, and Eastern Regions of Indonesia, 2017–2019

The people of Indonesia have been afflicted by dengue, a mosquito-borne viral disease, for over 5 decades. The country is the world's largest archipelago with diverse geographic, climatic, and demographic conditions that may impact the dynamics of disease transmissions. A dengue epidemiology study was launched by us to compare and understand the dynamics of dengue and other arboviral diseases in three cities representing western, central, and eastern Indonesia, namely, Batam, Banjarmasin, and Ambon, respectively. A total of 732 febrile patients were recruited with dengue-like illness during September 2017–2019 and an analysis of their demographic, clinical, and virological features was performed. The seasonal patterns of dengue-like illness were found to be different in the three regions. Among all patients, 271 (37.0%) were virologically confirmed dengue, while 152 (20.8%) patients were diagnosed with probable dengue, giving a total number of 423 (57.8%) dengue patients. Patients' age and clinical manifestations also differed between cities. Mostly, mild dengue fever was observed in Batam, while more severe cases were prominent in Ambon. While all dengue virus (DENV) serotypes were detected, distinct serotypes dominated in different locations: DENV-1 in Batam and Ambon, and DENV-3 in Banjarmasin. We also assessed the diagnostic features in the study sites, which revealed different patterns of diagnostic agreements, particularly in Ambon. To detect the possibility of infection with other arboviruses, further testing on 461 DENV RT-PCR-negative samples was performed using pan-flavivirus and -alphavirus RT-PCRs; however, only one chikungunya infection was detected in Ambon. A diverse dengue epidemiology in western, central, and eastern Indonesia was observed, which is likely to be influenced by local geographic, climatic, and demographic conditions, as well as differences in the quality of healthcare providers and facilities. Our study adds a new understanding on dengue epidemiology in Indonesia

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Molecular characterization of dengue viruses isolated from patients in Central Java, Indonesia

Background: Dengue is hyper-endemic in Indonesia. Purwokerto city in Central Java province is routinely ravaged by the disease. Despite the endemicity of dengue in this city, there is still no data on the virological aspects of dengue in the city. We conducted a molecular surveillance study of the circulating dengue viruses (DENV) in Purwokerto city to gain information on the virus origin, serotype and genotype distribution, and phylogenetic characteristics of DENV. Methods: A cross-sectional dengue molecular surveillance study was conducted in Purwokerto. Sera were collected from dengue-suspected patients attending three hospitals in the city. Diagnosis was performed using dengue NS1 antigen and IgG/IgM antibodies detection. DENV serotyping was performed using Simplexa Dengue real-time RT-PCR. Sequencing was conducted to obtain full-length DENV Envelope (E) gene sequences, which were then used in phylogenetic and genotypic analyses. Patients’ clinical and demographic data were collected and analyzed. Results: A total of 105 dengue-suspected patients’ sera were collected, in which 80 (76.2%) were positive for IgM and/or IgG, and 57 (54.2%) were confirmed as dengue by NS1 antigen and/or DENV RNA detection using RT-PCR. Serotyping was successful for 47 isolates. All four serotypes circulated in the area with DENV-3 as the predominant serotype. Phylogenetic analyses grouped the isolates into Genotype I for DENV-1, Cosmopolitan genotype for DENV-2, and Genotype I and II for DENV-3 and -4, respectively. The analyses also revealed the close relatedness of Purwokerto isolates to other DENV strains from Indonesia and neighboring countries. Conclusion: We reveal the molecular and virological characteristics of DENV in Purwokerto, Banyumas regency, Central Java. The genotype and phylogenetic analyses indicate the endemicity of the circulating DENV in the city. Our serotype and genotype data provide references for future dengue molecular epidemiology studies and disease management in the region. Keywords: Dengue, Serotype, Genotype, Purwokerto, Indonesi

Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia.

BACKGROUND:Dengue virus (DENV) infects hundreds of thousands of people annually in Indonesia. However, DENV sequence data from the country are limited, as samples from outbreaks must be shipped across long-distances to suitably equipped laboratories to be sequenced. This approach is time-consuming, expensive, and frequently results in failure due to low viral load or degradation of the RNA genome. METHODS:We evaluated a method designed to address this challenge, using the 'Primal Scheme' multiplex PCR tiling approach to rapidly generate short, overlapping amplicons covering the complete DENV coding-region, and sequencing the amplicons on the portable Nanopore MinION device. The resulting sequence data was assessed in terms of genome coverage, consensus sequence accuracy and by phylogenetic analysis. RESULTS:The multiplex approach proved capable of producing near complete coding-region coverage from all samples tested ([Formula: see text] = 99.96%, n = 18), 61% of which could not be fully amplified using the current, long-amplicon PCR, approach. Nanopore-generated consensus sequences were found to be between 99.17-99.92% identical to those produced by high-coverage Illumina sequencing. Consensus accuracy could be improved by masking regions below 20X coverage depth (99.69-99.92%). However, coding-region coverage was reduced at this depth ([Formula: see text] = 93.48%). Nanopore and Illumina consensus sequences generated from the same samples formed monophyletic clades on phylogenetic analysis, and Indonesian consensus sequences accurately clustered by geographical origin. CONCLUSION:The multiplex, short-amplicon approach proved superior for amplifying DENV genomes from clinical samples, particularly when the virus was present at low concentrations. The accuracy of Nanopore-generated consensus sequences from these amplicons was sufficient for identifying the geographic origin of the samples, demonstrating that the approach can be a useful tool for identifying and monitoring DENV clades circulating in low-resource settings across Indonesia. However, the inaccuracies in Nanopore-generated consensus sequences mean that the approach may not be appropriate for higher resolution transmission studies, particularly when more accurate sequencing technologies are available

Continuous Circulation of Chikungunya Virus during COVID-19 Pandemic in Jambi, Sumatra, Indonesia.

Chikungunya fever is a self-limiting viral illness that is caused by the chikungunya virus (CHIKV). CHIKV is found in multiple provinces of Indonesia, with clustered local outbreaks. This case series investigates a local chikungunya outbreak during the COVID-19 pandemic, involving two virologically confirmed chikungunya cases found in Jambi, Sumatra, Indonesia in 2021 and the contact tracing of 65 people from the same neighborhood (one of which was also virologically confirmed with CHIKV). The two original cases were symptomatic with classic signs of chikungunya fever, while the CHIKV-positive neighbor was asymptomatic. Out of the 65 participants, chikungunya IgM was detected in seven (10.8%) people while chikungunya IgG was detected in six (9.2%) using capture ELISA. Dengue IgG was detected by rapid test in three (4.6%) of the participants, showcasing a history of dengue virus (DENV) infection along with the circulation of CHIKV in the area. A phylogenetic analysis demonstrates a close evolutionary relationship between all three 2021 Jambi CHIKV isolates and the 2015-2016 isolates from Jambi. This case series showcases the endemicity and persistent circulation of CHIKV in Jambi, leaving the area vulnerable to eminent outbreaks of chikungunya fever and doubling the burden of disease during the COVID-19 pandemic. Health staff training for case detection and notification, as well as an integrated vector surveillance should continue to be implemented to provide an early warning indicator of possible chikungunya outbreaks