Genetic characterization of dengue virus 4 complete genomes from East Java, Indonesia.

Dengue is an endemic arboviral disease with continuous transmission in Indonesia for more than five decades. A recent outbreak in Jember, East Java province, demonstrated the predominance of DENV-4, a serotype known for its low global spread and limited transmission. While epidemiological factors such as new serotype introduction and lacking herd immunity may explain its predominance, viral factors may also contribute. Using next-generation sequencing, we generated 13 representative complete genomes of DENV-4 responsible for the outbreak. Phylogenetic and evolutionary analyses on complete genomes were performed to understand the spatial and temporal dynamics of the viruses. Further analyses were done to study amino acid variations in DENV genes, as well as the potential events of recombination and selection pressure within the genomes. We revealed the DENV-4 genetic factors that may lead to its predominance in the 2019 Jember dengue outbreak. A combination of selection pressure and mutational genetic changes may contribute to the DENV-4 predominance in East Java, Indonesia. The possible intra-serotype recombination events involving the non-structural protein 5 (NS5) gene were also observed. Altogether, these genetic factors may act as additional factors behind the complex dengue outbreak mechanism.

Combining rapid diagnostic tests to estimate primary and post-primary dengue immune status at the point of care.

BACKGROUND: Characterising dengue virus (DENV) infection history at the point of care is challenging as it relies on intensive laboratory techniques. We investigated how combining different rapid diagnostic tests (RDTs) can be used to accurately determine the primary and post-primary DENV immune status of reporting patients during diagnosis. METHODS AND FINDINGS: Serum from cross-sectional surveys of acute suspected dengue patients in Indonesia (N:200) and Vietnam (N: 1,217) were assayed using dengue laboratory assays and RDTs. Using logistic regression modelling, we determined the probability of being DENV NS1, IgM and IgG RDT positive according to corresponding laboratory viremia, IgM and IgG ELISA metrics. Laboratory test thresholds for RDT positivity/negativity were calculated using Youden's J index and were utilized to estimate the RDT outcomes in patients from the Philippines, where only data for viremia, IgM and IgG were available (N:28,326). Lastly, the probabilities of being primary or post-primary according to every outcome using all RDTs, by day of fever, were calculated. Combining NS1, IgM and IgG RDTs captured 94.6% (52/55) and 95.4% (104/109) of laboratory-confirmed primary and post-primary DENV cases, respectively, during the first 5 days of fever. Laboratory test predicted, and actual, RDT outcomes had high agreement (79.5% (159/200)). Among patients from the Philippines, different combinations of estimated RDT outcomes were indicative of post-primary and primary immune status. Overall, IgG RDT positive results were confirmatory of post-primary infections. In contrast, IgG RDT negative results were suggestive of both primary and post-primary infections on days 1-2 of fever, yet were confirmatory of primary infections on days 3-5 of fever. CONCLUSION: We demonstrate how the primary and post-primary DENV immune status of reporting patients can be estimated at the point of care by combining NS1, IgM and IgG RDTs and considering the days since symptoms onset. This framework has the potential to strengthen surveillance operations and dengue prognosis, particularly in low resource settings.

Molecular epidemiology of dengue in a setting of low reported endemicity: Kupang, East Nusa Tenggara province, Indonesia.

BACKGROUND: Most regions in Indonesia experience annual dengue epidemics. However, the province of East Nusa Tenggara has consistently reported low incidence. We conducted a dengue molecular epidemiology study in Kupang, the capital of the province. METHODS: Dengue patients were recruited from May 2016 to September 2017. Dengue virus (DENV) screening was performed using NS1 and immunoglobulin G (IgG)/IgM detection. Serotype was determined using reverse transcription polymerase chain reaction and the envelope genes were sequenced to infer the genetic identity and phylogeny. RESULTS: From 119 patients, dengue was confirmed in 62 (52%). Compared with official data, underreporting of dengue incidence was observed. The majority (36%) of patients were children <10 y of age. Most patients (80%) experienced mild fever. All serotypes were detected, with DENV-3 as the predominant (57%). Kupang DENV-1 isolate was classified as genotype IV, an old and endemic strain, DENV-2 as cosmopolitan, DENV-3 as genotype I and DENV-4 as genotype II. Most isolates showed relatively low evolutionary rates and are closely related with strains from Bali and Timor Leste. CONCLUSIONS: The low dengue incidence was most likely caused by sustained local circulation of endemic viruses. This study provides information on the epidemiology of dengue in a low-endemicity setting that should help future mitigation and disease management.

Molecular epidemiology of dengue in North Kalimantan, a province with the highest incidence rates in Indonesia in 2019.

OBJECTIVES: Dengue is endemic to Indonesia, a country that has largely varied geographical and demographic conditions across different regions. In 2019, dengue epidemic occurred in North Kalimantan province and recorded as the highest incidence rate in Indonesia. This study aims to investigate the molecular epidemiology of dengue during outbreak in the province and compare the epidemiological characteristics between two cities/towns in North Kalimantan, namely Malinau, an inland town surrounded by a dense rainforest, and Tarakan, an island city. METHODS: A cross sectional study was conducted between September 2018 and July 2019. Dengue-like illness patients were recruited in hospitals and tested for dengue NS1 and IgG/IgM. Serological prevalence was measured using IgG ELISA, dengue virus (DENV) serotyping was conducted using RT-PCR and Envelope gene sequencing was performed to infer the virus origins and phylogeny. Clinical, demographical, and diagnostics data were also recorded and analyzed. RESULTS: We recruited 523 patients, 261 from Malinau and 262 from Tarakan. Among them, 349 patients were confirmed dengue. Cases in Malinau had a higher proportion of confirmed dengue (82.0%) compared to those in Tarakan (51.5%). Cases in Malinau were more likely to be dengue hemorrhagic fever with more severe hematological features compared to those in Tarakan. All four DENV serotypes were detected in both cities, the most prevalent serotype being DENV-2. The genetic characteristics of the viruses in the two towns was similar except for DENV-3. No sylvatic DENV was detected as well as alphaviruses and non-dengue flaviviruses during the outbreak. CONCLUSIONS: The molecular epidemiology of dengue in North Kalimantan revealed the similar virological characteristics but different clinical and demographic aspects in Malinau and Tarakan. The distinct dengue dynamics between different regions of Indonesia is prominent and this knowledge will be important for understanding future patterns of DENV transmission in the region.

Outbreak of severe dengue associated with DENV-3 in the city of Manado, North Sulawesi, Indonesia.

BACKGROUND: In early 2019, an outbreak of severe dengue was reported in Manado, North Sulawesi Province, Indonesia. This epidemic raised public concern and recorded the highest number of cases in the last 10 years. This study aimed to determine the clinical spectrum, disease aetiology and virological characteristics associated with this outbreak of severe dengue. METHODS: Dengue was diagnosed using non-structural protein 1 detection, reverse transcription polymerase chain reaction and immunoglobulin (Ig)G/IgM serology. Envelope gene sequencing was conducted to determine the phylogeny of the dengue virus (DENV). RESULTS: In total, 146 patients with a median age of 8 years (interquartile range IQR 5-11 years) were recruited. Most patients experienced expanded dengue syndrome, characterized by severe organ involvement including liver enlargement, stomach ache and coagulation problems. During the outbreak, DENV-3 was the dominant serotype (75.9%). Smaller numbers of DENV-1, -2 and -4 were also detected. Phylogenetically, the dominant DENV-3 strains were grouped in multiple clusters and were related to other Indonesian strains, suggesting the emergence of heterogenous local viruses. CONCLUSION: The occurrence of an outbreak of severe dengue in Manado was confirmed, and DENV-3 was found to be the dominant serotype during the outbreak. This study shows the benefits of virological surveillance in understanding the aetiological agents responsible for outbreaks of severe dengue.

Assessment of dengue and COVID-19 antibody rapid diagnostic tests cross-reactivity in Indonesia.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic remains ongoing around the world, including in areas where dengue is endemic. Dengue and COVID-19, to some extent, have similar clinical and laboratory features, which can lead to misdiagnosis, delayed treatment and patient's isolation. The use of rapid diagnostic tests (RDT) is easy and convenient for fast diagnosis, however there may be issues with cross-reactivity with antibodies for other pathogens. METHODS: We assessed the possibility of cross-reactivity between SARS-CoV-2 and dengue antibodies by: (1) testing five brands of COVID-19 IgG / IgM RDTs on 60 RT-PCR-confirmed dengue samples; (2) testing 95 RT-PCR-confirmed COVID-19 samples on dengue RDT; and (3) testing samples positive for COVID-19 IgG and/or IgM on dengue RDT. RESULTS: We observed a high specificity across all five brands of COVID-19 RDTs, ranging from 98.3 to 100%. Out of the confirmed COVID-19 samples, one patient tested positive for dengue IgM only, another tested positive for dengue IgG only. One patient tested positive for dengue IgG, IgM, and NS1, suggesting a co-infection. In COVID-19 IgG and/or IgM samples, 6.3% of COVID-19 IgG-positive samples also tested positive for dengue IgG, while 21.1% of COVID-19 IgM-positive samples also tested positive for dengue IgG. CONCLUSION: Despite the high specificity of the COVID-19 RDT, we observed cross-reactions and false-positive results between dengue and COVID-19. Dengue and COVID-19 co-infection was also found. Health practitioners in dengue endemic areas should be careful when using antibody RDT for the diagnosis of dengue during the COVID-19 pandemic to avoid misdiagnosis.

Serological cross-reaction and coinfection of dengue and COVID-19 in Asia: Experience from Indonesia.

Similar symptoms and laboratory findings between dengue and coronavirus disease 2019 (COVID-19) pose a diagnostic challenge in some dengue-endemic countries in Asia. In this study, we reported three cases of suspected COVID-19-dengue coinfection in hospitals of Bali, Indonesia. Serological data demonstrated that patients with positive results for dengue virus (DENV) NS1 antigen and anti-dengue IgM were also reactive to COVID-19 rapid antibody tests, suggesting dengue-COVID-19 coinfection. However, two patients were later confirmed negative for SARS-COV-2 by qRT-PCR, implying a plausible cross-reactivity of anti-dengue and anti-COVID-19 antibodies in the serological test. Coinfection of dengue and COVID-19 was evident in one patient, following confirmation of SARS-COV-2 by qRT-PCR and DENV infection using the NS1 antigen serology test. This case was the first case of dengue and COVID-19 coinfection in Indonesia and revealed possible cross-reactivity between SARS-COV-2 and DENV antibodies based on rapid serological tests. Our study indicates a public health concern regarding COVID-19 and dengue detection in Indonesia as well as in other dengue-endemic countries, and it is important for these nations to manage both pathogens concurrently.

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.

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

BACKGROUND: 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.

Dengue Virus Serotype 4 Is Responsible for the Outbreak of Dengue in East Java City of Jember, Indonesia.

Outbreaks of dengue virus (DENV) in Indonesia have been mainly caused by the DENV serotype-1; -2; or -3. The DENV-4 was the least-reported serotype in Indonesia during the last five decades. We recently conducted a molecular epidemiology study of dengue in the Jember regency, East Java province, Indonesia. Dengue is endemic in the region and outbreaks occur annually. We investigated the clinical characteristics and etiology of dengue-like febrile illness in this regency to understand the disease dynamics. A total of 191 patients with clinical symptoms similar to dengue were recruited during an 11-month study in 2019-2020. Children accounted for the majority of cases and dengue burden was estimated in 41.4% of the cases based on NS1 antigen, viral RNA, and IgG/IgM antibody detection with the majority (73.4%) being primary infections. Secondary infection was significantly associated with a higher risk of severe dengue manifestation. All four DENV serotypes were detected in Jember. Strikingly, we observed the predominance of DENV-4, followed by DENV-3, DENV-1, and DENV-2. Genotype determination using Envelope gene sequence revealed the classification into Genotype I, Cosmopolitan Genotype, Genotype I, and Genotype II for DENV-1, -2, -3, and -4, respectively. The predominance of DENV-4 in Jember may be associated with a new wave of DENV infections and spread in a non-immune population lacking a herd-immunity to this particular serotype.

Diagnostic accuracy of 5 different brands of dengue virus non-structural protein 1 (NS1) antigen rapid diagnostic tests (RDT) in Indonesia.

Indonesia is hyper-endemic for dengue. Dengue virus non-structural protein 1 (NS1) antigen detection is now increasingly used by clinicians in Indonesia to confirm dengue infection, but many available brands have not had any evaluation on their performance. We evaluated the diagnostic accuracy of 5 different brands of NS1 rapid tests against reference standards consisting of 100 virologically confirmed dengue samples covering all 4 serotypes and 49 non-dengue samples. These rapid tests had sensitivity ranging from 73% to 80%, and specificity of 100%. The tests had better sensitivity for detection during the first 4 days of fever, for DENV-3 serotype, and in primary infections. The evaluated tests can be easily used with adequate sensitivity, very good specificity, and no significant difference in performance between brands. However, certain characteristics such as age, fever day onset, serotype, and immunologic status may affect the accuracy of these tests and need to be taken into consideration.