Background & objectives: Diagnosis of dengue infection in acute phase is important for clinical care, implementing control measures, surveillance and research. Currently, dengue fever is diagnosed by means of virus isolation, reverse transcriptase PCR or IgM and IgG based ELISA. Given the limitations of all the existing diagnostic methods, there is a need for rapid, sensitive and high throughput methods for detection of dengue virus in early stages of the disease. The study was conducted with the objectives to evaluate a dengue virus NS1 antigen detection ELISA and a TaqMan based real time RT-PCR for detection of all four serotypes of dengue virus, as diagnostic tools for acute dengue virus infection. Methods: The acute phase serum samples of patients (n=153) presenting with dengue fever were subjected to NS1 antigen detection and real time RT-PCR. The results were compared to those of virus isolation in the C6/36 cell lines (n=55). Results: The efficiency, sensitivity, specificity, positive and negative predictive values of NS1 Ag detection ELISA were 83.6, 73.5, 100, 100 and 70% respectively while for real time RT-PCR these were 87.3, 79.4, 100, 100 and 75% respectively. Maximum sensitivity of NS1 antigen detection ELISA was seen in two days of fever and that of real time RT-PCR in three days of fever. Interpretation & conclusion: NS1 antigen detection ELISA and real time RT-PCR were found to be rapid, convenient and efficient tests for diagnosing of dengue fever in acute phase and the diagnosis could be made as early as within three days of onset of fever.

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Background & objectives: Description of severe vivax malaria and mixed species infection requires good clinical study. The present study was undertaken to evalute the characteristics of severe malaria patients in Bikaner, northwest India. Methods: This prospective study included 539 admitted adult patients of severe malaria (Plasmodium falciparum 274, P. vivax 221, and mixed infection of Pv + Pf 44). The diagnosis was confirmed by polymerase chain reaction. The categorization of severe malaria was done strictly as per WHO criteria. Results: The distribution of severe manifestation was similar in severe vivax, falciparum and mixed infections except more cases of thrombocytopenia in P. vivax (p=0.030) and in mixed infection (p=0.004). The risk of developing severe malaria was greatest in patients of mixed infection [53.01% (44/83)] in comparison to Plasmodium falciparum malaria [49.37% (274/555), RR= 1.135; p=0.616] and P. vivax malaria [45.38% (221/ 487), RR = 1.299, p=0.243]. Hepatic dysfunction was the commonest pernicious syndrome [P. falciparum 50% (137/274), P. vivax 43.89% (97/221), and mixed infections 54.55% (24/44)]. Multiorgan dysfunction was present in 40.26% (217/539) patients, the risk was greatest in mixed infection [90.90% (40/44)] in comparison to P. falciparum monoinfection [37.59% (103/274), RR = 12.238; p=0.0001] or P. vivax monoinfection [33.48% (74/ 221), RR = 13.25; p=0.0001]. The risk of mortality in severe malaria was 6.31% (34/539) in which mixed infection had greater risk [9.09% (4/44)] in comparison to P. falciparum [7.30% (20/274); OR = 1.270 (CI 0.347-4.217); p=0.757] or P. vivax [4.52% (10/221); 0R 2.110 (CI 0.527-7.826); p=0.260]. Interpretation & conclusion: Severe vivax or falciparum malaria had almost similar features and prognosis including mortality. Risk of developing severe malaria, multiorgan dysfunction and mortality was more in patients of mixed infection in comparison to P. falciparum or P. vivax monoinfection. A multicentric study on larger number of patients requires further confirmation.

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Background & objectives: Malaria and dengue fever are the most common mosquito-borne diseases in the Southeast Asia region (SEAR). We analysed a temporal record of annual cases of malaria and dengue fever from 1985-2009 in SEAR. Methods: Data of dengue and malaria cases were obtained from WHO website for the period from 1985-2009. El-Nino Southern Oscillation (ENSO) fluctuation data were obtained from NOAA Climate Prediction Centre, Maryland. The wavelet analysis was conducted to analyse the data. Results: Results showed that multiyear cycles of malaria outbreaks appeared in 1986 and 1996, concomitant with the timing of dengue cases at one year lag. The dynamics of both cases pronounce a regime shift in the 1999, when the coupling between dengue and ENSO is also stronger. The statistical significance of this coupling is evident from wavelet band-averaged cross power in 2-4 yr scale (95% confidence level). Interpretation & conclusion: The present analysis suggests that the dengue incidence patterns in SEAR are periodic. There is not much evidence of malaria and ENSO having periodic association in the region; however, dengue fever and ENSO shows statistical significant cross-coherence in the 2-4 yr wavelet band and the results are statistically significant in the last decade. This study also provides statistical evidence of geographical clustering which quantitatively demonstrate the cross-country and cross-epidemic situations that exist across SEAR.

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Background & objectives: The present study aimed to systematically quantify the well known risk of severe dengue during secondary infection in literature and to understand how epidemiological mechanisms of enhancement during the secondary infection influence the empirically estimated risk of severe dengue by means of mathematical modeling. Methods: Two conditional risks of severe dengue, i.e. symptomatic illness and dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), given secondary infection were explored based on systematically searched prospective studies. A two-strain epidemiological model was employed to simulate the transmission dynamics of dengue and to identify the relevant data gaps in empirical observations. Results: Using the variance-based weighting, the pooled relative risk (RR) of symptomatic illness during secondary infection was estimated at 9.4 [95% confidence interval (CI): 6.1-14.4], and similarly, RR of DHF/DSS was estimated to be 23.7 (95% CI: 15.3-36.9). A variation in the RR of DHF/DSS was observed among prospective studies. Using the mathematical modeling technique, we identified the duration of cross-protective immunity as an important modulator of the time-dependent behaviour of the RR of severe dengue. Different epidemiological mechanisms of enhancement during secondary infection yielded different RR of severe dengue. Interpretation & conclusion: Optimal design of prospective cohort study for dengue should be considered, accounting for the time-dependence in the RR during the course of dengue epidemic. It is critical to statistically infer the duration of cross-protective immunity and clarify how the enhancement influences the epidemiological dynamics during secondary infection.

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Background & objectives: An alarming rate of deforestation has been reported from Sonitpur district of Assam, India therefore, a study was initiated during 2009 using remote sensing (RS) to assess deforested areas in the district and to study the impact on malaria vectors in order to formulate appropriate control strategy. Methods: RS imageries of 2000 and 2009 were used to assess deforested areas in the selected district. Entomological data were collected in four surveys during 2009-2011. The data were analyzed statistically using test of single proportions (χ ² ) and pair-wise comparison. Vector incrimination was done using enzyme-linked immunosorbent assay (ELISA) and entomological inoculation rate (EIR) was calculated to estimate transmission intensity. Results: The deforested areas were identified in north-western parts of Sonitpur district falling in Dhekiajuli Primary Health Centre (PHC). The forest cover of the PHC decreased >50% during 2000-2009. Five species of anopheline vectors were collected. Anopheles minimus sensu lato (s.l.) was collected least abundantly while An. culicifacies s.l. prevailed most abundantly and significant difference was observed between proportions of the collected vector species. Pair-wise comparison between An. culicifacies s.l. and An. minimus s.l. was also found statistically significant indicating that An. culicifacies s.l. is establishing its population in deforested areas. An. culicifacies s.l. was found ELISA positive and EIR was measured as 4.8 during transmission season. Conclusion: An. culicifacies s.l. replaced An. minimus s.l., the vector of malaria in northeast India and was found ELISA positive, therefore could have possible role in malaria transmission in the deforested areas of the district.

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Background & objectives: Due to ever-growing insecticide resistance in mosquito vectors and environmental contamination by synthetic insecticides, plants may be a source of alternative agents for mosquito control. Therefore, the present investigation involved the determination of larvicidal and phytochemical properties of Callistemon rigidus leaf extracts against Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus. Methods: The standard protocol of WHO was used for larval tests. Twenty five IV instar larvae were exposed to various concentrations from 125-1000 ppm for methanol crude extract (MCE), hexane (HF), chloroform (CF), ethyl acetate (EAF) and methanol (MF) fractions, from 250-2000 ppm for aqueous extract (AE) and 2500 ppm for Diclorvos. The mortality was observed 24 h post-exposure. The LC50 and LC90 values were determined by Probit analysis. Results: The phytochemical analysis revealed that the presence of alkaloids, steroids, saponins, terpenoids, tannins and phenolic compounds, lipids, fats and fixed oils in MCE; terpenoids, steroids, lipids, fats and fixed oils in HF; terpenoids in CF; tannins and phenolic compounds in EAF and alkaloids, tannins, saponins and phenolic compounds in MF. Against Ae. aegypti, HF was the most active fraction with LC50 of 56.25 ppm. Against An. gambiae, HF demonstrated its potential mosquito larvicide killing relatively all exposed larvae at all concentrations with LC50 of 17.11 ppm. Against Cx. quinquefasciatus, only MCE and HF exhibited larvicidal activity with LC50 of 447.38 and 721.95 ppm, respectively. Interpretation & conclusion: Callistemon rigidus exhibited some promising larvicidal activity against medically important vector mosquitoes. Studies are indicated to identify the active compounds from this plant for developing mosquito larvicides

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Background & objectives: Japanese encephalitis (JE) is one of the dreaded mosquito-borne viral diseases mostly prevalent in south Asian countries including India. Early warning of the disease in terms of disease intensity is crucial for taking adequate and appropriate intervention measures. The present study was carried out in Dibrugarh district in the state of Assam located in the northeastern region of India to assess the accuracy of selected forecasting methods based on historical morbidity patterns of JE incidence during the past 22 years (1985-2006). Methods: Four selected forecasting methods, viz. seasonal average (SA), seasonal adjustment with last three observations (SAT), modified method adjusting long-term and cyclic trend (MSAT), and autoregressive integrated moving average (ARIMA) have been employed to assess the accuracy of each of the forecasting methods. The forecasting methods were validated for five consecutive years from 2007-2012 and accuracy of each method has been assessed. Results: The forecasting method utilising seasonal adjustment with long-term and cyclic trend emerged as best forecasting method among the four selected forecasting methods and outperformed the even statistically more advanced ARIMA method. Peak of the disease incidence could effectively be predicted with all the methods, but there are significant variations in magnitude of forecast errors among the selected methods. As expected, variation in forecasts at primary health centre (PHC) level is wide as compared to that of district level forecasts. Interpretation & conclusion: The study showed that adopted forecasting techniques could reasonably forecast the intensity of JE cases at PHC level without considering the external variables. The results indicate that the understanding of long-term and cyclic trend of the disease intensity will improve the accuracy of the forecasts, but there is a need for making the forecast models more robust to explain sudden variation in the disease intensity with detail analysis of parasite and host population dynamics.

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Background & objectives: The major malaria vector, Anopheles culicifacies Giles is reported to contribute ~ 65% of the malaria cases in India. This species developed resistance to DDT and later to HCH, malathion and also to pyrethroids in some states due to their use in the national malaria control programme. In the present study, insecticide susceptibility of this species was monitored in four states of India. Methods: To determine insecticide susceptibility status of the major malaria vector An. culicifacies, adult mosquitoes were collected from different localities of 32 tribal districts in the states of Andhra Pradesh, Odisha, Jharkhand and West Bengal during October/November 2009-10. Mosquitoes were collected from stratified ecotypes comprising a group of districts in West Bengal and individual districts in three other states. Mosquitoes were exposed to papers treated with WHO diagnostic dose: 4% DDT, 5% malathion and 0.05% deltamethrin following the WHO tube method. Results: Results provided the susceptibility status of An. culicifacies to different insecticides used in the public health programme in 32 districts in four states. An. culicifacies was found resistant to DDT (mortality range 0-36%) in all the 32 districts; to malathion it was resistant in 14 districts, verification required in 10 districts and susceptible in eight districts (mortality range 32.2-100%). It was resistant to deltamethrin in four districts, verification required in 11 districts and susceptible in 17 districts (mortality range 43.3-100%). Interpretation & conclusion: Development of widespread resistance to insecticides used in public health sprays for vector control including to pyrethroids in An. culicifacies in the surveyed districts is of great concern for the malaria control programme as the major interventions for vector control are heavily reliant on chemical insecticides, mainly synthetic pyrethroids used both for indoor residual spraying and for long-lasting insecticidal nets. Thus, there is a need to periodically monitor and update the susceptibility status of malaria vector(s) to suggest alternative vector control strategies for effective disease management.

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Background & objectives: Bedbug Cimex hemipterus lives in cracks and crevices, and shows aggregation activity in the harbourage containing its fecal matter. Limited information is available on the chemicals influencing this aggregation behaviour in C. hemipterus. Possible components responsible for aggregation, if identified and evaluated can be helpful in designing bedbug management. Methods: Study on the isolation and identification of chemicals from the excreta extract responsible for aggregation has been carried by GC-MS. The compounds identified were evaluated at different concentrations to C. hemipterus and their attraction index was determined. Results: The GC-MS analysis of the excreta extract resulted in identification of 33 different types of semiochemicals such as esters, carboxylic acids, alcohols, aldehydes, ketones and other hydrocarbons. Among these 33 compounds of various groups, the carboxylic acid (E)-2-hexenoic acid and the aldehyde (E)-2-hexenal found to elicit highest positive aggregation behavioural response in all the stages followed by hexenal. Interpretation & conclusion: The fecal matter influence the aggregation behaviour in C. hemipterus. (E)-2-hexenoic acid, hexanoic acid, (E)-2-hexenal and hexanal found to exhibit aggregation in various stages of bedbug life cycle. These chemical components can be further exploited for designing and development tool for management of bedbugs.

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Background & objectives: Diagnosis of lymphatic filariasis using serum has been established but the utility of hydrocele fluid for the purpose is not exactly known. Since, hydrocele is a chronic form of the disease manifestation in a variety of situations and often poses difficulty in diagnosing its origin, we have evaluated the usefulness usage of hydrocele fluid for diagnosis of filarial origin of hydrocele in this study. Methods: Paired samples of serum and hydrocele fluid from 51 individuals with hydrocele, living in an endemic area of Wuchereria bancrofti were assessed. Circulating filarial antigen, filarial specific antibody and cytokine assay were performed in both serum and hydrocele fluid of patients. Results: Og4C3 assay detected circulating filarial antigen (CFA) in serum and corresponding hydrocele fluids. The level of IgG, IFN-γ and IL-10 were found to be high in CFA-negative, while IgM and IgE were high in CFApositive hydrocele fluid and serum samples associated with hydrocele. On the other hand neither CFA-positive nor CFA-negative hydrocele fluid and serum samples associated with hydrocele showed any difference in IgG4 level. Interpretation & conclusion: This study showed that the filaria related antigens and antibodies found in serum can be detected with equal sensitivity in hydrocele fluid. Therefore, it can be used as an alternative to serum for immunodiagnosis of filariasis, and help monitoring the filarisis elimination programme.

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Background & objectives: In 2000, a dengue outbreak occurred in Bangladesh that included Dhaka City. Both dengue vectors, Aedes aegypti and Ae. albopictus are present in Bangladesh. Aedes aegypti mosquitoes mainly breed in and around houses and Ae. albopictus is an outside breeder. There are many old trees throughout Dhaka City in different parks, streets and the university campus which may have holes that can contribute as potential breeding habitat for the dengue vector. Therefore, a survey was conducted to investigate the presence of eggs of the dengue vector mosquitoes in treeholes during the dry season in February 2001 to know their contribution on dengue outbreaks. Methods: All treeholes in 10 different localities (parks, streets and university campus) of Dhaka City were surveyed. All trees were examined for treeholes up to the height of approximately 3 m and sampled. Debris were collected and packed in poly bags and brought to the laboratory for detailed studies. These were then soaked with tap water to observe egg hatching. The soaked materials were kept up to 20 days covered by a fine mosquito net. After 2-3 days, the eggs started hatching and larvae were separated from the sample for rearing up to IV instar. Results: A total of 245 treeholes were surveyed in 49 identified tree species and 18 unidentified trees. Altogether, 1365 Aedes larvae were found, of which 1096 were Aedes albopictus and 269 were other Aedes species. The largest number of larvae was observed in Delonix regia of Leguminosae family. The number of Aedes albopictus found in the treeholes have perfect positive correlation with the number of other Aedes species. Not a single egg of Aedes aegypti was found in this survey. Interpretation & conclusion: This information will inform public health workers as well as the national control programme to help to solve mosquito borne diseases specially that of dengue. This is critical in planning for vector control operations due to the diversity of dengue outbreak in the nature.

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