Comparative evaluation of an improved test method for bioefficacy of insecticidal fabrics against dengue and malaria vectors.

BACKGROUND: Insecticidal fabrics are important personal protective measures against mosquitoes, ticks and other disease vectors. In the absence of internationally accepted guidelines, bioefficacy tests have been carried out using continuous exposure and three minutes exposure bioassay methods. Recently, we have reported an improved method for bioefficacy testing of insecticidal fabrics, which involves continuous exposure of mosquitoes to the test fabrics. The present paper reports the comparative evaluation of the outcomes of the continuous exposure bioassay and the three minutes bioassay on the same fabric samples. METHODS: Permethrin content in the treated fabric samples was determined through HPLC analysis and NMR studies were performed to establish the stability of the analyte. Bioefficacy tests were carried out against dengue vector Aedes aegypti and malaria vector Anopheles stephensi as per the improved test method and the three minutes bioassay method. RESULTS: The permethrin doses in the fabric samples ranged from 60 to 3000 mg/m2 and 36.2% of permethrin was retained after 10 washings. The extraction and chromatographic analysis were not found to affect the stability of permethrin. In continuous exposure, all fabric samples showed bioefficacy, as the mean complete knockdown time for both Ae. aegypti (10.5-34.5 min) and An. stephensi (14.5-36.8 min) was ≤ 71.5 min. The same samples were found to be not effective when tested using the three minutes bioassay method, since the knockdown and mortality percentages were well below the required bioefficacy values. The bioefficacy of the fabric samples in terms of complete knockdown time was significantly higher against Ae. aegypti in comparison to An. stephensi. The mean complete knockdown time of Ae. aegypti increased to 48.3 min after 10 washings indicating a significant reduction in bioefficacy. CONCLUSIONS: Bioefficacy testing of the insecticidal fabrics using the improved method resulted in outcomes, which could be correlated better with the permethrin content in the fabric samples. The improved method is more appropriate for the testing of insecticidal fabrics than the three minutes bioassay method. Further evaluation of the improved method using different test arthropods could help in the formulation of specific guidelines for the bioefficacy testing of insecticidal fabrics.

A review on test methods for insecticidal fabrics and the need for standardisation.

Insecticidal fabrics are effective personal protective measures against disease vectors and unlike bed nets, these fabrics can provide protection from day-biting mosquitoes and in outdoor environments. The rapid geographical expansion of day-biting mosquitoes and their role in disease transmission necessitate technological interventions, which can be effectively used during the daytime. There is a renewed interest in insecticidal fabrics mainly due to the recent outbreaks and geographical spread of dengue and chikungunya and with the emerging threat of Zika virus infection. Insecticidal fabrics are useful for protection from night-biting mosquitoes and also in situations were sleeping under a bed net is not possible. They are also effective against other biting arthropods like ticks, mites, tsetse flies, sand flies and body lice. Although long-lasting insecticidal fabrics factory-treated with permethrin are now commercially available for military and civilian use, there are no international guidelines for testing their efficacy. The different methods employed so far for testing bioefficacy, washing and quantification of permethrin are compiled in this review. The future prospects and challenges ahead for long-lasting insecticidal fabrics are discussed in the context of the increased threat from day-biting mosquitoes and the diseases transmitted by them. The review focuses on the need for standardisation of the test methods for ensuring adequate bioefficacy and safety to the user. The differences between long-lasting insecticidal nets and long-lasting insecticidal fabrics are elaborated, and the need for a separate registration and licencing procedure for long-lasting insecticidal fabrics is highlighted. A test procedure for insecticidal fabrics is described, which could be used until internationally accepted guidelines are available.

Rebound of Cotton leaf curl Multan virus and its exclusive detection in cotton leaf curl disease outbreak, Punjab (India), 2015.

Cotton leaf curl disease (CLCuD) outbreaks caused by CLCuD associated begomoviruses (CABs) significantly constrain cotton production in India and Pakistan. In comparison to the CABs circulating in Pakistan, molecular epidemiology, evolution and recombination patterns of CABs circulating in India are less studied. In this work, we characterized CAB complex sequences obtained from the most recent outbreak (Punjab, India, 2015), and rigorously analyzed them with reference to GenBank sequences, submitted from India, Pakistan and other neighbouring countries, using contemporary bioinformatics approaches. In this manuscript, we illustrate the detection of a recombinant, phylogenetically distinct clade of Cotton leaf curl Multan virus (CLCuMuV), suggesting rebound of CLCuMuV in this region. Interestingly, we could not detect Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bu), which was prevalent in this region, until now. Our study thus indicates substitution of the 'virulent resistance breaking' CLCuKoV-Bu by the re-emerging CLCuMuV recombinants. Our findings corroborate with that of a very recent study from Pakistan and we here discuss epidemiological links between the CAB complexes reported in these two studies. Taken together, these observations signify a shifting epidemiology of CABs, and seem to correlate with the recent prediction of the 'third epidemic' of CLCuD in the Indian subcontinent.

Phylogenetic Characterization of a Novel Insect-Specific Flavivirus Detected in a Culex Pool, Collected from Assam, India.

OBJECTIVE: We report the phylogenetic characterization of a unique flavivirus sequence detected in a wild Culex tritaeniorhynchus mosquito pool, collected from the northeast Indian state of Assam. METHODS: DNA and RNA were extracted from field-collected mosquito pools. Extracts were subjected to PCR and reverse transcriptase PCR amplification using universal and type-specific primers for direct detection of flavivirus-specific viral nucleic acids. An amplified flavivirus nonstructural protein 5 (NS5) genetic region was sequenced and BLAST searched, and phylogenetic analyses performed with reference sequences retrieved from GenBank. RESULTS: Phylogenetic analyses revealed the sequence to be related to insect-specific flaviviruses (ISFs) of the genus Flavivirus, family Flaviviridae. Despite being related to the Palm Creek virus (PCV; an ISF very recently reported from Northern Australia), the present sequence (provisionally named Assam virus) was found to be highly divergent from PCV and other ISF sequences available in GenBank. The partial NS5 sequence analysis demonstrated low nucleotide sequence identity (66-77%) with known ISFs reported from other parts of the globe. CONCLUSION: Findings of this study suggest the presence of a candidate novel ISF - the first to be reported from India.

Spatiotemporal distribution of dengue vectors & identification of high risk zones in district Sonitpur, Assam, India.

BACKGROUND & OBJECTIVES: Dengue is an arboviral disease of public health importance in many parts of India and recently many cases have been reported from northeastern India. Aedes mosquitoes, which are the vectors of dengue, are widely prevalent in the region. A study was initiated in Sonitpur district of Assam to understand the spatiotemporal distribution and seasonal prevalence of dengue vectors and to identify the high risk zones. METHODS: Ovitrap surveys were conducted in three randomly selected villages under each of the eight public health centres (PHC) in district Sonitpur of Assam, northeastern India during March 2011-February 2012. Three risk zones (high, medium and low) were identified on the basis of per trap density of Aedes mosquitoes. Meteorological data were collected to study the temporal distribution of dengue vectors. RESULTS: Aedes albopictus (99.3%) was the predominant dengue vector followed by Ae. aegypti (0.7%) recorded in the ovitraps. The highest vector density was observed during the post-monsoon (60.1±18 per trap) while the lowest during the winter (7.6±4.9 per trap) and the season-wise differences in the vector density were significant (P=0.005). Maximum temperature (correlation coefficient, r=0.45) and minimum temperature (r=0.408) showed the highest positive correlation with the vector density, whereas the number of rainy days showed high positive correlation (r=0.185) than the total rainfall (r=0.117). The high risk zone (Dekhiajuli, Behali, Bihaguri and Gohpur PHC) as indicated by the high larval densities of dengue vectors, 45.3±18, 42.1±22.3, 36.9±29.1, 35.3±22.6 per trap, respectively, was validated by dengue epidemiological data collected during 2012. INTERPRETATION & CONCLUSIONS: Yearlong monitoring of dengue vectors was done for the first time in this region. Monthly maximum temperature and the number of rainy days could be used for the prediction of larval density of Aedes mosquitoes. The identification high dengue risk zones would help in adopting targeted interventions for disease management in future.

Monitoring of malaria, Japanese encephalitis and filariasis vectors.

BACKGROUND: Vector monitoring in military stations would help in protecting the armed forces from vector borne diseases such as malaria, Japanese encephalitis and filariasis. METHODS: Adult mosquitoes were collected from four villages around a military station in India using light traps and the species composition was estimated. Insecticide susceptibility of disease vectors against DDT, deltamethrin and permethrin was established using WHO kits. RESULTS: The known malaria vectors constituted 4.9% of the total mosquito collections and Anopheles philippinensis/nivipes (2.05%) was the most abundant. Japanese encephalitis and dengue vectors constituted 25.3 and 0.05% whereas the known vectors of both Japanese encephalitis and filariasis formed 50.9%. The mean (±SEmean) of annual parasitic index, slide positivity and Plasmodium falciparum percentage among the civilian population during the study period were 1.46 ± 0.37, 1.65 ± 0.77 and 50.2 ± 10.7. The filariasis vector Culex quinquefasciatus was resistant to DDT with 65.4% mortality whereas the DDT resistance in the Japanese encephalitis vector Culex vishnui gr. with 91.9% mortality needs to be confirmed. All other species tested were susceptible to DDT, deltamethrin and permethrin. CONCLUSION: Targeted interventions are needed to reduce the disease burden and vector activity in the villages adjoining the military station. The use of insect repellents, bed nets and repellent impregnated uniforms by the troops should be ensured for protection from vector borne diseases.

Physicochemical characteristics of habitats in relation to the density of container-breeding mosquitoes in Asom, India.

BACKGROUND & OBJECTIVES: Container-breeding mosquitoes, especially Aedes spp are vectors of diseases such as dengue and chikungunya. The abundance of these disease vectors in an area depends on the availability of container habitats and their physicochemical characteristics. The species composition of container-breeding mosquitoes in Asom, India was studied and the larval density was correlated with the habitat characteristics. METHODS: Natural and man-made water-holding containers in Sonitpur district of Asom were surveyed for the presence of mosquito larvae. The percent composition of container-breeding mosquitoes and container index were calculated. The physicochemical characteristics of breeding water such as pH, conductivity, salinity, total dissolved solids, turbidity and dissolved oxygen were measured. RESULTS: Aedes albopictus (93.7%) was the predominant species in the container-breeding habitats whereas Culex quinquefasciatus (2.77%), Armigeres subalbatus (2.26%), Ae. aegypti (0.76%), Toxorhynchites sp (0.4%) and Lutzia sp (0.11%) were recorded in relatively low numbers. The larval density (mean ± SEmean) of the container breeding mosquitoes ranged from 4.4 ± 1.8 to 15.4 ± 8.2, while the container index ranged from 1.58 to 5.68%. The mean (± SEmean) pH, conductivity, salinity, total dissolved solids, turbidity, and dissolved oxygen of water in the container habitats were 7.15 ± 0.11; 396.1 ± 58.5 µS/cm; 0.24 ± 0.04 ppt; 207.1 ± 30.4 mg/l; 32.3 ± 5.1 NTU; and 1.42 ± 0.12% respectively. The mosquito larval density in the container habitats was having significant negative correlation with the conductivity of breeding water (r = - 0.89; p = 0.003). Salinity, total dissolved solids and turbidity of water in the habitats were negatively correlated, whereas pH and dissolved oxygen were positively correlated with the larval density. INTERPRETATION & CONCLUSION: The studies indicated the predominance of Ae. albopictus in the container-breeding habitats and reiterated its importance as a potential vector of dengue and chikungunya in the region. The spread of Ae. aegypti, the principal vector of dengue, in the semi-urban areas probably through road transport is a matter of public health concern. The use of conductivity of breeding water as an index for the proliferation of container breeding mosquitoes in the region could be explored further.

Malaria incidence among paramilitary personnel in an endemic area of Tripura.

BACKGROUND & OBJECTIVES: Paramilitary operations along the Indo-Bangladesh border are adversely affected by malaria induced morbidity and mortality. Villages surrounding the paramilitary installations often serve as disease reservoirs. Malaria incidence in Tripura State Rifles (TSR) units in Dhalai District of Tripura was studied and the role of the village population in disease transmission was also assessed. METHODS: Mass blood surveys were carried out among TSR personnel and villagers during 2007 to 2009. Malaria diagnosis through blood smear examination and rapid detection kits was done, and percentage parasitaemia was determined. Activity of malaria vectors was monitored using CDC light traps. RESULTS: Slide positivity rates (SPR) in the neighbouring villages (51.4%) was significantly higher than that in TSR (27.7%) (P<0.0001). Malaria incidence in villages did not show seasonal variability while it was lowest during post-monsoon season in TSR (P<0.325; OR = 0.74). Per cent Pf parasitaemia was high in TSR (0.29) as compared to villagers (0.20) (P<0.0001). Anopheles minimus and An. dirus were the major malaria vectors observed. INTERPRETATION & CONCLUSIONS: Paramilitary and public health authorities should adopt targeted measures to reduce the malaria incidence in the villages surrounding the paramilitary installations as the village populations play a major role in disease transmission.

Diversity and seasonal densities of vector anophelines in relation to forest fringe malaria in district Sonitpur, Assam (India).

The prevalence and bionomics of known Indian malaria vector anophelines was studied in three forest fringe villages in Sonitpur district of Assam, India. Anopheles philippinensis/nivipes (36.8%) and An. annularis (25.6%) were the most abundant of the seven vector anopheline species recorded. Densities of vector anophelines in general were high during monsoon season and low during winter months. An. dirus s.l. was the most anthropophagic (91.6%) of all the species collected whereas, An. annularis, An. fluviatilis s.l. and An. varuna were predominantly zoophagic. The highest and the lowest slide positivity rates (SPR) was recorded during monsoon (36%) and winter (12.5%) seasons, respectively. SPR was positively correlated with vector anopheline densities (r = 0.902), which itself was found to be correlated with mean minimum temperature and relative humidity (RH).

Malaria epidemiology along Indo-Bangladesh border in Tripura State, India.

Malaria epidemiological surveys were conducted in 16 villages along the Indo-Bangladesh border in Tripura, northeastern India. Insecticide resistance among malaria vectors and chloroquine resistance in the parasite were also studied along with monitoring of vector density using light traps. The epidemiological data indicated that malaria incidence was highest during June-July and lowest during November. Examination of blood smears collected through door to door surveys indicated slide positivity rate (SPR) of 25.2% and that Plasmodium falciparum was the predominant parasite (slide falciparum rate of 22.3%). The incidence rates of falciparum malaria varied significantly among the age groups (p<0.001) and 2-4 year olds were the most affected. Major malaria vectors recorded in light trap collections were An. dirus, An. minimus and An. philippinensis/nivipes. Chloroquine resistance studies indicated that treatment failure occurred in 35% of the cases and hence the use of artesunate combination therapy (ACT) was recommended for treatment of malaria in the area.