Intrahost Genetic Diversity of Dengue Virus in Human Hosts and Mosquito Vectors under Natural Conditions Which Impact Replicative Fitness In Vitro.

Dengue virus (DENV) is an arbovirus whose transmission cycle involves disparate hosts: humans and mosquitoes. The error-prone nature of viral RNA replication drives the high mutation rates, and the consequently high genetic diversity affects viral fitness over this transmission cycle. A few studies have been performed to investigate the intrahost genetic diversity between hosts, although their mosquito infections were performed artificially in the laboratory setting. Here, we performed whole-genome deep sequencing of DENV-1 (n = 11) and DENV-4 (n = 13) derived from clinical samples and field-caught mosquitoes from the houses of naturally infected patients, in order to analyze the intrahost genetic diversity of DENV between host types. Prominent differences in DENV intrahost diversity were observed in the viral population structure between DENV-1 and DENV-4, which appear to be associated with differing selection pressures. Interestingly, three single amino acid substitutions in the NS2A (K81R), NS3 (K107R), and NS5 (I563V) proteins in DENV-4 appear to be specifically acquired during infection in Ae. aegypti mosquitoes. Our in vitro study shows that the NS2A (K81R) mutant replicates similarly to the wild-type infectious clone-derived virus, while the NS3 (K107R), and NS5 (I563V) mutants have prolonged replication kinetics in the early phase in both Vero and C6/36 cells. These findings suggest that DENV is subjected to selection pressure in both mosquito and human hosts. The NS3 and NS5 genes may be specific targets of diversifying selection that play essential roles in early processing, RNA replication, and infectious particle production, and they are potentially adaptive at the population level during host switching.

Dengue virus in humans and mosquitoes and their molecular characteristics in northeastern Thailand 2016-2018.

Dengue is hyperendemic in most Southeast Asian countries including Thailand, where all four dengue virus serotypes (DENV-1 to -4) have circulated over different periods and regions. Despite dengue cases being annually reported in all regions of Thailand, there is limited data on the relationship of epidemic DENV infection between humans and mosquitoes, and about the dynamics of DENV during outbreaks in the northeastern region. The present study was conducted in this region to investigate the molecular epidemiology of DENV and explore the relationships of DENV infection in humans and in mosquitoes during 2016-2018. A total of 292 dengue suspected patients from 11 hospitals and 902 individual mosquitoes (at patient's houses and neighboring houses) were recruited and investigated for DENV serotypes infection using PCR. A total of 103 patients and 149 individual mosquitoes were DENV -positive. Among patients, the predominant DENV serotypes in 2016 and 2018 were DENV-4 (74%) and DENV-3 (53%) respectively, whereas in 2017, DENV-1, -3 and -4 had similar prevalence (38%). Additionally, only 19% of DENV infections in humans and mosquitoes at surrounding houses were serotypically matched, while 81% of infections were serotypically mismatched, suggesting that mosquitoes outside the residence may be an important factor of endemic dengue transmission. Phylogenetic analyses based on envelope gene sequences showed the genotype I of both DENV-1 and DENV-4, and co-circulation of the Cosmopolitan and Asian I genotypes of DENV-2. These strains were closely related to concurrent strains in other parts of Thailand and also similar to strains in previous epidemiological profiles in Thailand and elsewhere in Southeast Asia. These findings highlight genomic data of DENV in this region and suggest that people's movement in urban environments may result in mosquitoes far away from the residential area being key determinants of DENV epidemic dynamics.

Serological biomarker for assessing human exposure to Aedes mosquito bites during a randomized vector control intervention trial in northeastern Thailand.

BACKGROUND: Aedes mosquitoes are vectors for several major arboviruses of public health concern including dengue viruses. The relationships between Aedes infestation and disease transmission are complex wherein the epidemiological dynamics can be difficult to discern because of a lack of robust and sensitive indicators for predicting transmission risk. This study investigates the use of anti-Aedes saliva antibodies as a serological biomarker for Aedes mosquito bites to assess small scale variations in adult Aedes density and dengue virus (DENV) transmission risk in northeastern Thailand. Individual characteristics, behaviors/occupation and socio-demographics, climatic and epidemiological risk factors associated with human-mosquito exposure are also addressed. METHODS: The study was conducted within a randomized clustered control trial in Roi Et and Khon Kaen provinces over a consecutive 19 months period. Thirty-six (36) clusters were selected, each of ten houses. Serological and entomological surveys were conducted in all houses every four months and monthly in three sentinel households per cluster between September 2017 and April 2019 for blood spot collections and recording concurrent immature and adult Aedes indices. Additionally, the human exposure to Aedes mosquito bites (i.e., Mosquito Exposure Index or MEI) was estimated by ELISA measuring levels of human antibody response to the specific Nterm-34 kDa salivary antigen. The relationships between the MEI, vector infestation indices (adult and immature stages) and vector DENV infection were evaluated using a two-level (house and individual levels) mixed model analysis with one-month lag autoregressive correlation. RESULTS: There was a strong positive relationship between the MEI and adult Aedes (indoor and outdoor) density. Individuals from households with a medium mosquito density (mean difference: 0.091, p<0.001) and households with a high mosquito density (mean difference: 0.131, p<0.001) had higher MEI's compared to individuals from households without Aedes. On a similar trend, individuals from households with a low, medium or high indoor Aedes densities (mean difference: 0.021, p<0.007, 0.053, p<0.0001 and 0.037, p<0.0001 for low, medium and high levels of infestation, respectively) had higher MEI than individuals from houses without indoor Aedes. The MEI was driven by individual characteristics, such as gender, age and occupation/behaviors, and varied according to climatic, seasonal factors and vector control intervention (p<0.05). Nevertheless, the study did not demonstrate a clear correlation between MEI and the presence of DENV-infected Aedes. CONCLUSION: This study represents an important step toward the validation of the specific IgG response to the Aedes salivary peptide Nterm-34kDa as a proxy measure for Aedes infestation levels and human-mosquito exposure risk in a dengue endemic setting. The use of the IgG response to the Nterm-34 kDa peptide as a viable diagnostic tool for estimating dengue transmission requires further investigations and validation in other geographical and transmission settings.

Complex relationships between Aedes vectors, socio-economics and dengue transmission-Lessons learned from a case-control study in northeastern Thailand.

BACKGROUND/OBJECTIVES: Dengue fever is an important public health concern in most tropical and subtropical countries, and its prevention and control rest on vector surveillance and control. However, many aspects of dengue epidemiology remain unclear; in particular, the relationship between Aedes vector abundance and dengue transmission risk. This study aims to identify entomological and immunological indices capable of discriminating between dengue case and control (non-case) houses, based on the assessment of candidate indices, as well as individual and household characteristics, as potential risk factors for acquiring dengue infection. METHODS: This prospective, hospital-based, case-control study was conducted in northeastern Thailand between June 2016 and August 2019. Immature and adult stage Aedes were collected at the houses of case and control patients, recruited from district hospitals, and at patients' neighboring houses. Blood samples were tested by RDT and PCR to detect dengue cases, and were processed with the Nterm-34 kDa salivary peptide to measure the human immune response to Aedes bites. Socioeconomic status, and other individual and household characteristics were analyzed as potential risk factors for dengue. RESULTS: Study findings showed complex relationships between entomological indices and dengue risk. The presence of DENV-infected Aedes at the patient house was associated with 4.2-fold higher odds of dengue. On the other hand, Aedes presence (irrespective of infectious status) in the patient's house was negatively associated with dengue. In addition, the human immune response to Aedes bites, was higher in control than in case patients and Aedes adult abundance and immature indices were higher in control than in case houses at the household and the neighboring level. Multivariable analysis showed that children aged 10-14 years old and those aged 15-25 years old had respectively 4.5-fold and 2.9-fold higher odds of dengue infection than those older than 25 years. CONCLUSION: DENV infection in female Aedes at the house level was positively associated with dengue infection, while adult Aedes presence in the household was negatively associated. This study highlights the potential benefit of monitoring dengue viruses in Aedes vectors. Our findings suggest that monitoring the presence of DENV-infected Aedes mosquitoes could be a better indicator of dengue risk than the traditional immature entomological indices.

Interepidemic Detection of Chikungunya Virus Infection and Transmission in Northeastern Thailand.

Chikungunya fever is a viral mosquito-borne, acute febrile illness associated with rash, joint pain, and occasionally prolonged polyarthritis. Chikungunya outbreaks have been reported worldwide including many provinces of Thailand. Although chikungunya virus (CHIKV) occurs in Thailand, details on its epidemiology are lacking compared with dengue, a common mosquito-borne disease in the country. Therefore, study on CHIKV and its epidemiology in both humans and mosquitoes is required to better understand its importance clinically and dynamics in community settings. So a prospective examination of virus circulation in human and mosquito populations in northeastern Thailand using serological and molecular methods, including the genetic characterization of the virus, was undertaken. The study was conducted among febrile patients in eight district hospitals in northeastern Thailand from June 2016 to October 2017. Using real-time PCR on the conserved region of nonstructural protein 1 gene, CHIKV was detected in eight (4.9%) of 161 plasma samples. Only one strain yielded a sequence of sufficient size allowing for phylogenetic analysis. In addition, anti-CHIKV IgM and IgG were detected in six (3.7%) and 17 (10.6%) patient plasma samples. The single sequenced sample belonged to the East/Central/South Africa (ECSA) genotype and was phylogenetically similar to the Indian Ocean sub-lineage. Adult Aedes mosquitoes were collected indoors and within a 100-m radius from the index case house and four neighboring houses. CHIKV was detected in two of 70 (2.9%) female Aedes aegypti mosquito pools. This study clearly demonstrated the presence and local transmission of the ECSA genotype of CHIKV in the northeastern region of Thailand.

Spatial and temporal patterns of dengue incidence in northeastern Thailand 2006-2016.

BACKGROUND: Dengue, a viral disease transmitted by Aedes mosquitoes, is an important public health concern throughout Thailand. Climate variables are potential predictors of dengue transmission. Associations between climate variables and dengue have usually been performed on large-scale first-level national administrative divisions, i.e. provinces. Here we analyze data on a finer spatial resolution in one province, which is often more relevant for effective disease control design. The objective of this study was to investigate the effect of seasonal variations, monthly climate variability, and to identify local clusters of symptomatic disease at the sub-district level based on reported dengue cases. METHODS: Data on dengue cases were retrieved from the national communicable disease surveillance system in Thailand. Between 2006 and 2016, 15,167 cases were recorded in 199 sub-districts of Khon Kaen Province, northeastern Thailand. Descriptive analyses included demographic characteristics and temporal patterns of disease and climate variables. The association between monthly disease incidence and climate variations was analyzed at the sub-district level using Bayesian Poisson spatial regression. A hotspot analysis was used to assess the spatial patterns (clustered/dispersed/random) of dengue incidence. RESULTS: Dengue was predominant in the 5-14 year-old age group (51.1%). However, over time, dengue incidence in the older age groups (> 15 years) gradually increased and was the most affected group in 2013. Dengue outbreaks coincide with the rainy season. In the spatial regression model, maximum temperature was associated with higher incidence. The hotspot analysis showed clustering of cases around the urbanized area of Khon Kaen city and in rural areas in the southwestern portion of the province. CONCLUSIONS: There was an increase in the number of reported dengue cases in older age groups over the study period. Dengue incidence was highly seasonal and positively associated with maximum ambient temperature. However, climatic variables did not explain all the spatial variation of dengue in the province. Further analyses are needed to clarify the detailed effects of urbanization and other potential environmental risk factors. These results provide useful information for ongoing prediction modeling and developing of dengue early warning systems to guide vector control operations.

Correction to: Assessing dengue transmission risk and a vector control intervention using entomological and immunological indices in Thailand: study protocol for a cluster-randomized controlled trial.

In the original publication [1], the first of two objectives was to "Assess the effect of periodically treating water storage containers with a pyriproxyfen/spinosad combination on entomological and epidemiological outcomes".

Assessing dengue transmission risk and a vector control intervention using entomological and immunological indices in Thailand: study protocol for a cluster-randomized controlled trial.

BACKGROUND: Dengue fever is the most common and widespread mosquito-borne arboviral disease in the world. There is a compelling need for cost-effective approaches and practical tools that can reliably measure real-time dengue transmission dynamics that enable more accurate and useful predictions of incidence and outbreaks. Sensitive surveillance tools do not exist today, and only a small handful of new control strategies are available. Vector control remains at the forefront for combating dengue transmission. However, the effectiveness of many current vector control interventions is fraught with inherent weaknesses. No single vector control method is effective enough to control both vector populations and disease transmission. Evaluations of novel larval and adult control interventions are needed. METHODS/DESIGN: A cluster-randomized controlled trial will be carried out between 2017 and 2019 in urban community clusters in Khon Kaen and Roi Et cities, northeastern Thailand. The effectiveness of a pyriproxyfen/spinosad combination treatment of permanent water storage containers will be evaluated on epidemiological and entomological outcomes, including dengue incidence, number of female adult dengue vectors infected or not infected with dengue virus (DENV), human exposure to Aedes mosquito bites, and several other indices. These indices will also be used to develop predictive models for dengue transmission and impending outbreaks. Epidemiological and entomological data will be collected continuously for 2 years, with the intervention implemented after 1 year. DISCUSSION: The aims of the trial are to simultaneously evaluate the efficacy of an innovative dengue vector control intervention and developing predictive dengue models. Assessment of human exposure to mosquito bites by detecting antibodies generated against Aedes saliva proteins in human blood samples has, so far, not been applied in dengue epidemiological risk assessment and disease surveillance methodologies. Likewise, DENV detection in mosquitoes (adult and immature stages) has not been used in any practical way for routine disease surveillance strategies. The integration of multiple outcome measures will assist health authorities to better predict outbreaks for planning and applying focal and timely interventions. The trial outcomes will not only be important for Thailand, but also for the entire Southeast Asian region and further afield. TRIAL REGISTRATION: ISRCTN, ISRCTN73606171 . Registered on 23 June 2017.

Prevalence and risk factors for Opisthorchis viverrini infections in upper Northeast Thailand.

Opisthorchis viverrini is an ongoing public health problem in Northeast Thailand. Despite continuous efforts for decades by healthcare organizations to overcome this problem, infection rates remain high. To enable related personnel to identify and address the various issues effectively, a cross-sectional study was performed to investigate prevalence and risk factors for opisthorchiasis. The target group was 3,916 Thai residents of Northeast Thailand who were 15 or over. Participants were recruited using the 30 clusters sampling technique. The data were gathered through questionnaires, focus group discussions, in-depth interviews, and stool examinations for parasite eggs (using the Modified Kato Katz method). The data were analyzed using descriptive and inference statistics; in order to ascertain the risk factors and test them using the odds ratio and multiple logistic regressions. The prevalence of opisthorchiasis was 22.7% (95%CI: 0.26 to 0.24). The province with the highest prevalence was Nakhorn Phanom (40.9%; female to male ratio =1:1.2). The age group with the highest prevalence was 40-49 year olds. All age groups had a prevalence >20%. Four of seven provinces had a prevalence >20%. The factors related to opisthorchiasis were (a) sex, (b) age (especially > 50), (c) proximity and duration living near a water body, and (d) eating raw and/or fermented fish. In order to reduce the prevalence of opisthorchiasis, the focus in populations living in upper Northeast Thailand should be changing their eating behaviors as appropriate to their tradition and context.

Development of a community-based approach to opisthorchiasis control.

A liver fluke, Opisthorchis viverrrini (OV), is the major cause of the high incidence of cholangiocarcinoma in North-eastern Thailand. The prevalence of OV infection remains high in various parts of the country, especially in wetland rural areas where a large proportion of the community work in agriculture and continue the traditional practice of eating raw or uncooked cyprinoid fish products. The national control program seems to have had little impact in many of these areas, and it has been difficult to make precise assessments of the overall effectiveness of the program. Therefore there is a need for a community-based approach to prevent infection with the parasite, ideally involving as many players as possible. Here we document an attempt to assess the best means to prevention on the basis of a community intervention in three villages in north-east Thailand, with participation of representatives of Health Promotion Hospitals of the Ministry of Public Health with dedicated staff, but also school teachers, independent government sponsored village health volunteers, and housewives responsible for cooking and diet selection. An action plan was followed, allowing detailed discussions of practical proposals, their introduction and then repeated reflection and further proposals at the individual village level.

The surveillance system in health centers in northeastern Thailand.

In this study we assessed the existing communicable disease surveillance system in health centers of a province in northeastern Thailand. The first part of the study was an examination of medical records from 11 local health centers: 649 were examined for sensitivity, positive predictive value, and representativeness; and 433 were examined for timeliness and data quality. The second part of the study looked at 50 local officers from 11 local health centers, 1 district health office, 1 community hospital, and 8 sub-district administrative organizations. Quantitative data was collected through a review of medical records. Qualitative data was collected by focus groups and in-depth interviews. The reporting of suspected cases was 50.8%. Sensitivity was low for common diseases. Positive predictive value was lowest for fever of unknown origin (0%). Data quality for the date of onset and diagnosis was low. Case reporting was considered timely in only 45% of cases. Health officers perceived the surveillance system as not being up-to-date. They only collected data in a district without data analysis; the information provided by the system is not representative of the true epidemiological situation countrywide and cannot be used to help monitor disease patterns and trends. In conclusion, health officers should report diseases according to symptoms and analyze data continuously to identify urgent problem and elicit prompt responses.