The burden of dengue and force of infection among children in Kerala, India; seroprevalence estimates from Government of Kerala-WHO Dengue study.

Background: Dengue shows high geographic heterogeneity within and across endemic countries. In the context of increasing burden and predicted outbreaks due to climate change, understanding the heterogeneity will enable us to develop region specific targeted interventions, including vaccination. World Health Organisation (WHO) suggests standard methodologies to study the burden and heterogeneity at national and subnational levels. Regional studies with robust and standard methodology to capture heterogeneity are scarce. We estimated the seroprevalence of dengue in children aged 9-12 years and the force of infection in Kerala, India, from where Zika cases also have been reported recently. Methods: We conducted a school-based cross-sectional survey in 38 clusters; selected by stratified random sampling, representing rural, urban, high burden and low-burden administrative units. Validation of Indirect IgG ELISA was done by Plaque Reduction Neutralization Test (PRNT90) using the local isolates of all four serotypes. Force of infection (FOI) was estimated using the WHO-FOI calculator. We conducted a follow-up survey among a subsample of seronegative children, to estimate the rate of sero-conversion. Results: Among 5236 children tested, 1521 were positive for anti-dengue IgG antibody. The overall seroprevalence in the state was 29% (95% CI 24.1-33.9). The validity corrected seroprevalence was 30.9% in the overall sample, 46.9% in Thiruvananthapuram, 26.9% in Kozhikkode and 24.9% in Kollam. Age-specific seroprevalence increased with age; 25.7% at 9 years, 29.5% at 10 years, 30.9% at 11 years and 33.9% at 12 years. Seroprevalence varied widely across clusters (16.1%-71.4%). The estimated force of infection was 3.3/100 person-years and the seroconversion rate was 4.8/100 person-years. 90% of children who tested positive were not aware of dengue infection. All the four serotypes were identified in PRNT and 40% of positive samples had antibodies against multiple serotypes. Interpretation: The study validates the WHO methodology for dengue serosurveys and confirms its feasibility in a community setting. The overall seroprevalence in the 9-12 year age group is low to moderate in Kerala; there are regional variations; high burden and low burden clusters co-exist in the same districts. The actual burden of dengue exceeds the reported numbers. Heterogeneity in prevalence, the high proportion of inapparent dengue and the hyperendemic situation suggest the need for region-specific and targeted interventions, including vaccination. Funding: World Health Organization.

Molecular Evidence of Wolbachia Species in Wild-Caught Aedes albopictus and Aedes aegypti Mosquitoes in Four States of Northeast India.

Wolbachia, a Gram-negative intracellular bacterium, naturally infects many arthropods, including mosquito vectors responsible for the spread of arboviral diseases such as Zika, chikungunya, and dengue fever. Certain Wolbachia strains are involved in inhibiting arbovirus replication in mosquitoes, and this phenomenon is currently being studied to combat disease vectors. A study was conducted in four states in north-eastern India to investigate the presence of natural Wolbachia infection in wild-caught Aedes albopictus and Aedes aegypti mosquitoes, the established vectors of dengue. The detection of a Wolbachia infection was confirmed by nested PCR and sequencing in the two mosquito species Ae. aegypti and Ae. albopictus. Positivity rates observed in Ae. aegypti and Ae. albopictus pools were 38% (44 of 115) and 85% (41 of 48), respectively, and the difference was significant (chi-square = 28.3174, p = 0.00000010). Sequencing revealed that all detected Wolbachia strains belonged to supergroup B. Although Wolbachia infection in Ae. aegypti has been previously reported from India, no such reports are available from north-eastern India. Data on naturally occurring Wolbachia strains are essential for selecting the optimal strain for the development of Wolbachia-based control measures. This information will be helpful for the future application of Wolbachia-based vector control measures in this part of the country.

Genetic differentiation among Aedes aegypti populations from different eco-geographical zones of India.

The present study explicitly evaluated the genetic structure of Aedes aegypti Linn, the vector of dengue, chikungunya, and Zika viruses, across different geo-climatic zones of India and also elucidated the impact of ecological and topographic factors. After data quality checks and removal of samples with excess null alleles, the final analysis was performed on 589 individual samples using 10 microsatellite markers. Overall findings of this study suggested that, Ae. aegypti populations are highly diverse with moderate genetic differentiation between them. Around half of the populations (13 out of 22) formed two genetic clusters roughly associated with geographical regions. The remaining nine populations shared genetic ancestries with either one or both of the clusters. A significant relationship between genetic and geographic distance was observed, indicating isolation by distance. However, spatial autocorrelation analysis predicted the signs of long-distance admixture. Post-hoc environmental association analysis showed that 52.7% of genetic variations were explained by a combination of climatic and topographic factors, with latitude and temperature being the best predictors. This study indicated that though overall genetic differentiation among Ae. aegypti populations across India is moderate (Fst = 0.099), the differences between the populations are developing due to the factors associated with geographic locations. This study improves the understanding of the Ae. aegypti population structure in India that may assist in predicting mosquito movements across the geo-climatic zones, enabling effective control strategies and assessing the risk of disease transmission.

Seasonal abundance of Leptotrombidium deliense, the vector of scrub typhus, in areas reporting acute encephalitis syndrome in Gorakhpur district, Uttar Pradesh, India.

The rickettsial pathogen Orientia tsutsugamushi, causing scrub typhus, has been implicated as a major cause of acute encephalitis syndrome (AES) in many places in India including Gorakhpur district of Uttar Pradesh. Seasonal abundance of the principal vector mite of the pathogen, Leptotrombidium deliense, its animal hosts, and prevalence of infection on them are important attributes in the assessment of outbreaks of the disease. Hence, these aspects were investigated, seasonally, in rural villages of Gorakhpur district, where peak incidence of AES cases were reported. A total of 903 animals (rodents/shrews) was collected using 6484 Sherman traps in eight study villages (14% overall trap rate). A sum of 5526 trombiculid mites comprising 12 species was collected from 676 live rodents/shrews screened. Suncus murinus, the Asian house shrew was the predominant species (67%). Among trombiculids, the principal vector mite, L. deliense, was predominant (64.7%) and its infestation index (i.e., average number of chiggers per host animal) was 5.3. The L. deliense infestation index was higher during July to November with a peak in October. Out of 401 animal sera samples screened, 68% were positive for antibodies against O. tsutsugamushi. Of 465 blood samples tested by nested PCR, seven were positive for the 56 kDa gene of O. tsutsugamushi. In conventional PCR, 41 out of 265 samples were positive for the 60 kDa groEL gene of O. tsutsugamushi. Among the 5526 mite samples, tested as 352 pools through nested PCR, four pools were positive for 56 kDa gene. Phylogenetic analysis of 56 and 60 kDa genes confirmed circulation of Karp and TA678 (rodents) and TA678 (mite) serotypes of O. tsutsugamushi in Gorakhpur. Peak incidence of AES in Gorakhpur district occurs during the rainy season (July-October), coinciding with the peak abundance of L. deliense. These results indicate involvement of L. deliense as the vector mite transmitting the scrub typhus pathogen O. tsutsugamushi to humans in the rural areas of Gorakhpur district, India.

Occurrence of Orientia tsutsugamushi, the Etiological Agent of Scrub Typhus in Animal Hosts and Mite Vectors in Areas Reporting Human Cases of Acute Encephalitis Syndrome in the Gorakhpur Region of Uttar Pradesh, India.

Outbreaks of acute encephalitis syndrome (AES) with high fatality and disability, are reported every year in the Gorakhpur region of Uttar Pradesh, India, with the etiology of >60% of the cases being attributed to scrub typhus. In the present study, the prevalence of Orientia tsutsugamushi, the etiological agent of scrub typhus, was investigated among animal hosts and their ectoparasitic trombiculid mites prevalent in AES-reported areas of Gorakhpur. A total of 154 rodents/shrews were collected using 777 Sherman traps set in 12 study villages, and the overall trap rate was 19.8%. In total, 2726 trombiculid mites belonging to 12 species were collected from 154 rodents/shrews trapped. The shrew mouse Suncus murinus was the predominant animal species (78.6%) collected. The principal vector mite Leptotrombidium deliense was the predominant species (82.7%), and its index was 14.6 per animal. Of 114 rodent/shrew sera samples screened through the Weil-Felix test, 57% were positive for antibodies against O. tsutsugamushi. Of 128 blood samples tested by polymerase chain reaction (PCR), one rodent sample was positive for the gene encoding 56 kDa protein and 25 for 60 kDa. Among 2726 mite samples tested as 315 pools through nested PCR, seven pools were positive for 56 kDa gene. Phylogenetic analysis confirmed circulation of Gilliam, Karp, and TA678 serotypes of O. tsutsugamushi in Gorakhpur. The study clearly demonstrated natural infection of O. tsutsugamushi in both small-animal hosts and vector mites in the AES-reporting villages of Gorakhpur, which confirms transmission of the scrub typhus pathogen in this region. The high infestation rate of L. deliense with O. tsutsugamushi infection indicates that the people living in the rural villages of Gorakhpur are at risk of infection with scrub typhus, which might lead to AES.

Detection of Chikungunya virus in wild populations of Aedes albopictus in Kerala State, India.

We detected Chikungunya virus (CHIKV) infection among wild populations of Aedes albopictus female specimens during the CHIKV outbreaks of 2009 and 2006 collected in different localities in Kerala State, India. The envelope 1 gene (E1) sequences of the virus isolate 2009 from the mosquito species showed close genetic relatedness (Kimura 2 Parameter genetic distance=0.0013) to CHIKV-positive isolates from human serum samples from the same area. E1 gene sequences from Ae. albopictus, as well as from human isolates, had the crucial non-synonymous C/T mutation at position 10670, leading to the A226V amino acid change. This natural inclination indicated the role of this mosquito species in the transmission of CHIKV during its recent outbreaks in Kerala State.

Chikungunya virus outbreak in Kerala, India, 2007: a seroprevalence study

India was affected by a major outbreak of chikungunya fever caused by Chikungunya virus (CHIKV) during 2006-2007. Kerala was the worst affected state during 2007 with a contribution of 55.8 percent suspected cases in the country. However, except for clinically reported case records, no systematic information is available on infection status of CHIKV in the region. Hence, we carried out a post-epidemic survey to estimate seroprevalence status [immunoglobulin G (IgG)] in the community using commercially available indirect immunofluorescence test. This methodology had been reported to be highly specific and sensitive for CHIKV infection. The study area selected was the worst affected mid-highlands region of Kerala which harbour vast area of rubber plantations. The study evidenced 68 percent of the population to be seropositive for CHIKV IgG. Males were found more affected than females (χ2 = 9.86; p = 0.002). Among males, prevalence was significantly higher in the age classes 21-30 (χ2 = 5.46; p = 0.019) and 31-40 (χ2 = 5.84; p = 0.016) years. This may be due to high occupational risk of the male population engaged in plantation activities exposed to infective bites of Aedes albopictus. The current study provides an insight into the magnitude of CHIKV outbreak in Kerala.

Chikungunya virus outbreak in Kerala, India, 2007: a seroprevalence study.

India was affected by a major outbreak of chikungunya fever caused by Chikungunya virus (CHIKV) during 2006-2007. Kerala was the worst affected state during 2007 with a contribution of 55.8% suspected cases in the country. However, except for clinically reported case records, no systematic information is available on infection status of CHIKV in the region. Hence, we carried out a post-epidemic survey to estimate seroprevalence status [immunoglobulin G (IgG)] in the community using commercially available indirect immunofluorescence test. This methodology had been reported to be highly specific and sensitive for CHIKV infection. The study area selected was the worst affected mid-highlands region of Kerala which harbour vast area of rubber plantations. The study evidenced 68% of the population to be seropositive for CHIKV IgG. Males were found more affected than females (χ2 = 9.86; p = 0.002). Among males, prevalence was significantly higher in the age classes 21-30 (χ2 = 5.46; p = 0.019) and 31-40 (χ2 = 5.84; p = 0.016) years. This may be due to high occupational risk of the male population engaged in plantation activities exposed to infective bites of Aedes albopictus. The current study provides an insight into the magnitude of CHIKV outbreak in Kerala.