Malaria Vector Bionomics: Countrywide Surveillance Study on Implications for Malaria Elimination in India.

BACKGROUND: The biological characteristics of mosquito vectors vary, impacting their response to control measures. Thus, having up-to-date information on vector bionomics is essential to maintain the effectiveness of existing control strategies and tools, particularly as India aims for malaria elimination by 2030. OBJECTIVE: This study aims to assess the proportions of vector species resting indoors and outdoors, determine their preference for host biting/feeding, identify transmission sites, and evaluate the susceptibility of vectors to insecticides used in public health programs. METHODS: Mosquito collections were conducted in 13 districts across 8 Indian states from 2017 to 2020 using various methods to estimate their densities. Following morphological identification in the field, sibling species of Anopheles mosquitoes were identified molecularly using polymerase chain reaction (PCR)-specific alleles. Plasmodium falciparum and Plasmodium vivax infections in the vectors were detected using enzyme-linked immunosorbent assay (ELISA) and PCR assays. In addition, we assessed the insecticide susceptibility status of primary malaria vectors following the World Health Organization (WHO) protocol. RESULTS: Anopheles culicifacies, a primary malaria vector, was collected (with a man-hour density ranging from 3.1 to 15.9) from all states of India except those in the northeastern region. Anopheles fluviatilis, another primary vector, was collected from the states of Madhya Pradesh, Maharashtra, Karnataka, and Odisha. In Haryana and Karnataka, An. culicifacies sibling species A predominated, whereas species C and E were predominant in Madhya Pradesh and Maharashtra. An. culicifacies displayed mainly endophilic behavior across all states, except in Madhya Pradesh, where the proportion of semigravid and gravid mosquitoes was nearly half of that of unfed mosquitoes. The human blood index of An. culicifacies ranged from 0.001 to 0.220 across all study sites. The sporozoite rate of An. culicifacies ranged from 0.06 to 4.24, except in Madhya Pradesh, where none of the vector mosquitoes were found to be infected with the Plasmodium parasite. In the study area, An. culicifacies exhibited resistance to DDT (dichlorodiphenyltrichloroethane; with <39% mortality). Moreover, it showed resistance to malathion (with mortality rates ranging from 49% to 78%) in all districts except Angul in Odisha and Palwal in Haryana. In addition, resistance to deltamethrin was observed in districts of Maharashtra, Gujarat, Haryana, and Karnataka. CONCLUSIONS: Our study offers vital insights into the prevalence, resting behavior, and sibling species composition of malaria vectors in India. It is evident from our findings that resistance development in An. culicifacies, the primary vector, to synthetic pyrethroids is on the rise in the country. Furthermore, the results of our study suggest a potential change in the resting behavior of An. culicifacies in Madhya Pradesh, although further studies are required to confirm this shift definitively. These findings are essential for the development of effective vector control strategies in India, aligning with the goal of malaria elimination by 2030.

Evaluation of intron-1 of odorant-binding protein-1 of Anopheles stephensi as a marker for the identification of biological forms or putative sibling species.

BACKGROUND: Anopheles stephensi, an invasive malaria vector, has been reported to have three biological forms identifiable mainly based on the number of ridges present on the egg's floats. Recently, the first intron of the odorant-binding protein-1 (AsteObp1) has been introduced as a molecular marker for the identification of these forms, and based on this marker, the presence of three putative sibling species (designated as species A, B and C) has been proposed. However, there is no data on the association of proposed markers with biological form or putative species on field populations. METHODS: Field collected and laboratory-reared An. stephensi were characterized for biological forms based on the number of ridges on the egg's float. DNA sequencing of the partial AsteObp1 gene of An. stephensi individuals were performed by Sanger's method, either directly or after cloning with a plasmid vector. Additionally, AsteObp1 sequences of various laboratory lines of An. stephensi were retrieved from a public sequence database. RESULTS: AsteObp1 intron-1 in Indian An. stephensi populations are highly polymorphic with the presence of more than 13 haplotypes exhibiting nucleotides as well as length-polymorphism (90-to-121 bp). No specific haplotype or a group of closely related haplotypes of intron-1 was found associated with any biological form identified morphologically. High heterozygosity for this marker with a low inbreeding coefficient in field and laboratory populations indicates that this marker is not suitable for the delimitation of putative sibling species, at least in Indian populations. CONCLUSIONS: AsteObp1 cannot serve as a marker for identifying biological forms of An. stephensi or putative sibling species in Indian populations.

Durability, household usage and washing pattern of DuraNet© and Interceptor® long-lasting insecticidal nets in long-term field trials in India.

BACKGROUND & OBJECTIVES: Universal coverage of population with long-lasting insecticidal nets (LLINs) living in endemic areas inhabited by ethnic tribal communities or in difficult to reach areas was found effective for disease control where other interventions such as indoor residual spraying have limited success because of operational issues. Evaluation of different LLINs with varied insecticides and fabrics are being evaluated to meet the demand of new products. This study was undertaken on two brands of LLINs, DuraNet© and Interceptor® that varied in fabric and manufacturing technologies to assess the usability in field conditions for atleast three years. METHODS: In large-scale field trials DuraNet©, alpha cypermethrin incorporated polyethylene net, was evaluated in Odisha state while, Interceptor® net, alpha cypermethrin coated polyester net was evaluated in Chhattisgarh and Gujarat states for a period of three years following WHO guidelines. Durability, usage pattern and washing behavior were monitored through periodic surveys and physical examination of nets. RESULTS: Survivorship of both the nets was 84-100% and every night usage rate was >62% in all seasons. Washing frequency was largely within the prescribed limits. The proportion of DuraNet© with holes was 26.7% in year one 74% in year three. In Gujarat, proportion of Interceptor® nets with holes at six months was 33.3% increased to 87% in year three and in Chhattisgarh, 6.7% after six months to 93.3% after three years of use respectively. INTERPRETATION & CONCLUSION: Both the nets revealed a useful life of three years under the field condition.

Small-scale (Phase II) evaluation of the efficacy and residual activity of SumiShield® 50 WG (clothianidin 50%, w/w) for indoor residual spraying in comparison to deltamethrin, bendiocarb and pirimiphos-methyl for malaria vector control in Karnataka state, India.

BACKGROUND & OBJECTIVES: There is an urgent need of introducing new insecticide molecules with novel modes of action to counter the ever growing insecticide-resistance in mosquito vectors. In the present study, a new insecticide molecule, SumiShield 50 WG (clothianidin 50%, w/w) was investigated for its efficacy as an indoor residual spray along with its residual action in comparison to deltamethrin, pirimiphos-methyl and bendiocarb. METHODS: The study area included three villages in Almatti Dam catchment area in Bagalkot district, Karnataka, India. Spraying was done using Hudson sprayers with the following dosages-Clothianidin, 300 mg AI/m2; deltamethrin, 25 mg AI/m2; bendiocarb, 400 mg AI/m2; and pirimiphos-methyl, 1 g AI/m2. Cone bioassays were conducted on cement and mud plastered surfaces at fortnightly intervals to assess the bioefficacy and residual activity. Mosquito densities in the sprayed houses were recorded at regular intervals for assessment of the insecticidal efficacy. Filter paper samples collected from the sprayed houses were analyzed for insecticide content sprayed on different wall surfaces at the Walloon Agricultural Research Institute, Gembloux, Belgium. RESULTS: Chemical content analysis of filter paper samples revealed that the applied to target ratios were in the acceptable range (1 + 0.5) for all the treatment types. Duration of persistence of effectiveness of bendiocarb (≥80% mortality in cone bioassays) was 19 to 21 wk on cement plastered surfaces and 15 to 19 wk on mud plastered surfaces. Duration of persistence of effectiveness of deltamethrin was 17 to 21 wk on both mud and cement plastered surfaces and that of pirimiphos-methyl was 15 to 19 wk. For SumiShield, it was 17 to 25 wk on both types of surfaces, indicating slow action of SumiShield. The densities of Anopheles culicifacies were lower in bendiocarb sprayed houses throughout the observation period, followed by pirimiphos methyl, deltamethrin and clothianidin sprayed houses. In case of other mosquitoes also, similar trend was observed. INTERPRETATION & CONCLUSION: Considering the persistence of effectiveness of SumiShield on sprayed surfaces, effectiveness in reducing the density of mosquitoes, operational feasibility, safety and community acceptance, the formulation of clothianidin is a better option for IRS for the control of insecticide-resistant mosquito vectors.

Chlorfenapyr: irritant effect compared to other insecticides and its intrinsic toxicity in multiple-insecticide-susceptible and -resistant Anopheles stephensi (Diptera: Culicidae).

BACKGROUND & OBJECTIVES: For effective management of vector resistance there is a need for new insecticide molecules with novel modes of action. For desired toxic effect of an insecticide, apart from other behavioural aspects, toxicity and chemical nature of the molecule are important that may cause irritability in the mosquito to the insecticide affecting the uptake. In this study, a pyrrole class insecticide, chlorfenapyr (a late acting insecticide) was tested for its irritability against multiple-insecticide-susceptible and -resistant strains of Anopheles stephensi Liston 1901 (Diptera: Culicidae). METHODS: Studies were conducted to assess the irritability due to chlorfenapyr, DDT, malathion, deltamethrin and permethrin and intrinsic toxicity of chlorfenapyr in multiple-insecticide-susceptible and -resistant laboratory strains of An. stephensi following standard WHO methods. RESULTS: Chlorfenapyr molecule has shown least irritant effect against susceptible and resistant strains among all the insecticides tested allowing more landing time to the vector species on the impregnated surfaces to pick-up lethal dose. CONCLUSION: Chlorfenapyr could be an ideal insecticide for management of multiple-insecticide-resistance including pyrethroids.

Evaluation of indoor residual spray and insecticide treated bed nets in a malaria endemic area of Santhal Pargana, Dumka district (Jharkhand).

The study was carried out for evaluation of various activities of malaria control programme in five different tribal and malaria endemic Primary Health Centres of Dumka district (Jharkhand) during 2007-08. A total of 321 houses of 18 villages were surveyed on use of indoor residual spray (IRS) and insecticide-treated bed nets (ITN) and other activities as tool for vector control and interrupting the transmission of malaria. Out of 690 living rooms and 343 verandahs examined, IRS with Dichlorodiphenyl-trichloro-ethane (DDT) was done only in 16.23% living rooms and 64.72% verandahs. Refusal rate of IRS in living rooms was 81.93% due to lack of knowledge regarding the importance of IRS, no prior information to villagers, houses locked, reluctance to remove domestic articles, dislike of smell of DDT spray. Compliance rate of ITN uses was 71.66% during the night, which might be a factor for decline in malaria cases in the study area. Various important components of the programme, viz. surveillance and compliance to treatment activities, use of rapid diagnostic test kits (RDKs), involvement of accredited social health activist (ASHA's) and fever treatment depots (FTDs), laboratory activities, adult mosquito collection, other activities like constitution of village health sanitation committee, information education and communication activities, capacity building, use of larvivorous fishes, supervision of IRS etc. require much strengthening. However, 100% community acceptance was recorded for ITN in the villages surveyed. In addition, an entomological study was carried out for information on prevalence of mosquito species in this area to find out effectiveness of IRS activities. Eleven anopheline species, including three malaria vectors i.e., An. culicifacies, An. stephensi, An. fluviatilis, An. annularis, An. subpictus, An. nigerrimus, An. pallidus, An. aconitus, An. vagus, An. jamsii and An. splendidus were collected from cattle and human dwellings.

Evaluation of the impact of ZeroFly®, an insecticide incorporated plastic sheeting on malaria incidence in two temporary labour shelters in India.

BACKGROUND & OBJECTIVES: Prevention of malaria is a major technical and operational problem in displaced and mobile populations such as refugee camps and temporary labour settlements. Insecticide incorporated plastic sheeting is a new technology to control mosquitoes in emergency shelters and also temporary habitations at different locations. In view of this, efficacy of ZeroFly® , an insecticide incorporated plastic sheeting (factory treated with deltamethrin 2.0 g/kg or 265 mg/m2 ) was evaluated for its efficacy against malaria vectors and its impact on malaria incidence in temporary labour settlements in two urban areas in India. METHODS: This trial was conducted in two labour settlements in two urban areas, Delhi and Noida (U.P.), India with ~ 250 populations. In an area, two localities were selected for intervention with ZeroFly and untreated plastic sheets (control). Entomological and epidemiological data were collected using standard methods for one year. RESULTS: Baseline studies on the susceptibility of mosquitoes in Delhi and Noida areas revealed 100% susceptibility of the malaria vector species Anopheles culicifacies and An. stephensi to deltamethrin. Cone bioassay tests performed against An. culicifacies and An. stephensi to determine the efficacy of ZeroFly sheets showed 100% mortality against An. culicifacies and An. stephensi with 3 min exposure and after 24 h recovery period. Against Culex quinquefasciatus and housefly 100% mortality was obtained after 30 min of exposure period. Intervention with the ZeroFly plastic sheets resulted in almost complete reduction in the resting density of An. culicifacies and An. stephensi, the two major malaria vectors and also in the reduction of malaria cases in ZeroFly camps as compared to control camps. The ZeroFly plastic sheeting was found to be safe for human. Barring some complaints of skin irritation and itching, which were temporary in nature, no adverse health effects were reported by the users. The community acceptance was high. CONCLUSION: Results of the present study revealed that ZeroFly® plastic sheeting is highly effective in reducing the indoor resting density of mosquitoes, man -vector contact and malaria incidences in labour populations living in temporary shelters.

Efficacy of Advanced Odomos repellent cream (N, N-diethyl-benzamide) against mosquito vectors.

BACKGROUND & OBJECTIVES: Repellents are commonly used personal protection measures to avoid mosquito bites. In the present study, Advanced Odomos cream (12% N, N-diethyl-benzamide) was tested for its efficacy against mosquitoes in comparison to DEET (N,N-diethyl-3-methyl benzamide). METHODS: Bioassays were conducted to assess the repellency of Advanced Odomos and DEET creams against Anopheles stephensi and Aedes aegypti. Their efficacy was tested on human volunteers applied with different concentrations of test creams ranging from 1 to 12 mg/cm 2 and by exposing them to mosquitoes at hourly intervals. Field evaluation was also carried out to test the duration of protection of the test creams against Anopheles and Aedes mosquitoes during whole night and day time collections, respectively on human volunteers. Mosquito collections were done using torch light and aspirator. RESULTS: Complete (100%) protection was achieved at 10 mg/cm 2 cream formulation of Advanced Odomos (1.2 mg a.i/cm 2 ) dose against An. stephensi and 12 mg/cm 2 (1.44 mg a.i./cm 2 ) against Ae. aegypti on human baits. There was no statistically significant differences in per cent protection against mosquito bites between Advanced Odomos and DEET cream (P>0.05) in respective doses. Complete protection up to 11 h was observed against Anopheles mosquitoes during whole night collections and up to 6 h against Ae. aegypti in day time collections. No adverse reactions such as itching, irritation, vomiting, nausea, etc. were reported by the volunteers. INTERPRETATION & CONCLUSIONS: Advanced Odomos cream applied at 10 mg/cm 2 concentration provided 100% protection from Anopheles mosquitoes up to 11 h whereas about 6 h protection was recorded against Ae. aegypti. The laboratory and field trials indicate that for longer protection against Anopheles mosquitoes 10 mg/cm 2 will be appropriate and in case of Ae. aegypti more than 10 mg/cm 2 application is required for complete protection. In conclusion, the Advanced Odomos cream was comparable to the known repellent cream DEET for prolonged protection against malaria and dengue vectors.

Field evaluation of lambda-cyhalothrin (ICON 10 CS) indoor residual spraying against Anopheles culicifacies in India.

BACKGROUND & OBJECTIVES: Field trials of lambda-cyhalothrin 10 CS (ICON 10 CS) in indoor residual spraying (IRS) with 25 mg a.i./m2 against Anopheles culicifacies was undertaken vs malathion IRS (25% WP-2 g a.i./m2) in Tumkur district, Karnataka; vs deltamethrin IRS (2.5% WP-20 mg a.i./m2) in Dharmapuri district; and vs lambda-cyhalothrin (10 WP-25 mg a.i./m2) in Ramanathapuram district, Tamil Nadu, India. METHODS: Spray operations in the experimental villages were done by the National Institute of Malaria Research (NIMR) and in the control villages by the respective State Health Department staff. Persistence of efficacy of insecticide sprayed in villages was assessed by contact bioassays against vector mosquitoes. Entomological indicators such as per structure density, parity rates of vector mosquitoes and sporozoite rates were measured in all the three study areas using standard procedures. Mass blood surveys and active fever case detections were carried out in experimental and control villages to study the impact of IRS on malaria transmission. RESULTS: Persistence of effectiveness of ICON 10 CS was observed up to 2-3 months in all the three study areas. ICON 10 CS was found effective at par with or better than the insecticides used in the national programme in reducing the mosquito densities and in interrupting malaria transmission in the study villages. Vector density, parity rates and malaria cases considerably reduced in the ICON 10 CS-sprayed villages. CONCLUSION: Field trials at three sites have established that ICON 10 CS formulation was relatively more effective than malathion 25% WP, deltamethrin 2.5% WP and lambda-cyhalothrin 10% WP in some evaluation parameters like indoor resting mosquitoes, parity rates in vector mosquitoes and persistence of effectiveness. It can be used for IRS for malaria vector control with two rounds of spray at an interval of 3 months for curtailing the malaria transmission and an additional round is recommended in perennial malaria transmission areas.

Evaluation of the pyrrole insecticide chlorfenapyr for the control of Culex quinquefasciatus Say.

Culex quinquefasciatus Say (Diptera: Culicidae) is a widely distributed mosquito vector species in India and also in other tropical regions of the world. This species is implicated in the transmission of lymphatic filariasis in many countries. This species is reported to be widely resistant to insecticides of different classes in current use. In the present study, bio-efficacy of chlorfenapyr, an insecticide of pyrrole class with a novel mode of action was tested for the control of Cx. quinquefasciatus. Studies were performed to determine the diagnostic dosage; residual efficacy on different artificially fabricated substrates, namely wood, mud, mud+lime, cement and cement+distemper; to assess cross-resistance with different insecticides; and synergism/antagonism using piperonyl butoxide (PBO). A dosage of 5.0% chlorfenapyr was determined as diagnostic dosage with 2 h exposure and 48 h holding period for assessing the susceptibility of mosquitoes. The residual efficacy was observed up to 34 weeks on wood and mud+lime substrates while on other substrates, it was about 15 weeks at a dosage of 400mg a.i./m(2). Laboratory-reared strains of Cx. quinquefasciatus showed cross-resistance, whereas field-collected mosquitoes showed absence of cross-resistance to chlorfenapyr. Potentiation bioassays showed antagonistic effect of PBO to chlorfenapyr toxicity owing to the involvement of oxidases in the initial step of a conversion of pro-insecticide chlorfenapyr to toxic form CL 303268. The present study results have shown that chlorfenapyr can be a potential insecticide for the control of multiple insecticide resistant strains of Cx. quinquefasciatus. However, in countries where indoor residual spray (IRS) is not targeted for the control of this species, like in India, chlorfenapyr used in IRS for the control of malaria vectors in rural and peri-urban areas can additionally provide control of Cx. quinquefasciatus also.

Malaria in seasonal migrant population in Southern Gujarat, India.

Malaria in migrant workers is always a major problem to control due to their temporary stay in shelters, and other operational constraints. Hence, a study was undertaken in brick kilns in Bharuch district, Gujarat state, India to study the problem of malaria in the work force. Mass blood surveys were carried out in 15 brick kilns. Blood slides were collected from both febrile and afebrile cases. Positive cases were treated as per the national drug policy and were followed up. Mosquito collections were carried out by pyrethrum spray collection in early morning hours. Human blood index and sporozoite rates were determined as per standard procedures. All age groups were found affected with malaria at brick kilns. Prevalence of malaria was significantly higher in ≤ 14 years of age-group as compared to adults. Post treatment follow up examination of patients revealed high malaria infection due to non-compliance of chloroquine. The appearance of parasitaemia among Plasmodium falciparum treated cases indicate the possibility of chloroquine resistance. The proportion of P. falciparum was >50% in migrant population. In stable population in villages, overall decline in malaria cases was observed in 2008-2010. The sporozoite rate of 4.2% in Anopheles culicifacies indicates active malaria transmission at brick kilns. The investigation demonstrated that suitable microclimatic conditions for malaria transmission exist in these areas during hottest period. The district health department should consider these factors in planning malaria surveillance and control. As current magnitude and diversity of population movements in rural as well as in urban areas are unprecedented, this issue is worthy of attention.

Malaria in seasonal migrant population in Southern Gujarat, India

Malaria in migrant workers is always a major problem to control due to their temporary stay in shelters, and other operational constraints. Hence, a study was undertaken in brick kilns in Bharuch district, Gujarat state, India to study the problem of malaria in the work force. Mass blood surveys were carried out in 15 brick kilns. Blood slides were collected from both febrile and afebrile cases. Positive cases were treated as per the national drug policy and were followed up. Mosquito collections were carried out by pyrethrum spray collection in early morning hours. Human blood index and sporozoite rates were determined as per standard procedures. All age groups were found affected with malaria at brick kilns. Prevalence of malaria was significantly higher in 50% in migrant population. In stable population in villages, overall decline in malaria cases was observed in 2008-2010. The sporozoite rate of 4.2% in Anopheles culicifacies indicates active malaria transmission at brick kilns. The investigation demonstrated that suitable microclimatic conditions for malaria transmission exist in these areas during hottest period. The district health department should consider these factors in planning malaria surveillance and control. As current magnitude and diversity of population movements in rural as well as in urban areas are unprecedented, this issue is worthy of attention.

Persistence of DDT, malathion & deltamethrin resistance in Anopheles culicifacies after their sequential withdrawal from indoor residual spraying in Surat district, India.

BACKGROUND & OBJECTIVES: Under the national antimalaria programme DDT was introduced in early 1950s for vector control and later hexachloro cyclohexane (HCH) followed by malathion and recently synthetic pyrethroids in 1990s to manage the insecticide resistance in Anopheles culicifacies. Subsequent replacement led to development of multiple resistances in An. culicifacies in Surat district in Gujarat State. Indoor residual spray (IRS) was completely withdrawn in southern villages in Surat in 2002. This study was undertaken in these areas to study the persistence of resistance to DDT, malathion and deltamethrin after sequential withdrawal of IRS with these insecticides at different times. METHODS: Susceptibility tests on An. culicifacies were conducted using standard WHO methods and kits. Mortality, knockdown time and lethal times were calculated for An. culicifacies exposed to WHO prescribed diagnostic concentrations of different insecticide impregnated papers. RESULTS: Persistence of DDT-resistance was observed even after 30 yr of its withdrawal from IRS. Similarly, persistence of malathion resistance was also observed after 9 yr of its withdrawal from IRS, while reversal of deltamethrin-resistance was observed very fast within 2-3 yr after its withdrawal from IRS in 2002. INTERPRETATION & CONCLUSION: Present data indicate that the quantum of reversion of insecticide resistance in a population is relative and depends on the genetic stability of the respective resistance genes in the mosquitoes. In the present study withdrawal of pyrethroid-IRS resulted in increased susceptibility against pyrethroids alone and was independent of existence of resistance to insecticides of other groups. This study emphasizes that appropriate rotation of different insecticides; including carbamates may prevent or delay the onset of resistance.

Impact of etofenprox (Vectron 20 WP) indoor residual spray on malaria transmission.

BACKGROUND & OBJECTIVE: A longitudinal study was carried out to assess the impact of indoor residual spray with Vectron 20 WP (etofenprox) against Anopheles culicifacies and on malaria transmission in a selected village of Dadri primary health centre (PHC), District Gautam Budh Nagar, Uttar Pradesh, India. METHODS: Two villages, namely Patadi and Anandpur in District Gautam Budh Nagar, Uttar Pradesh, with similar malaria incidence and vector prevalence were selected for the present evaluation. In one village two rounds of indoor spraying of etofenprox (0.1 g/m(2)) were done at an interval of 12 wk and the other village was kept as control where no intervention except intensive active surveillance for early detection and prompt treatment (EDPT) was undertaken during the study period. Entomological and epidemiological data were collected using standard procedures. RESULTS: Persistence of the effectiveness of etofenprox against An. culicifacies was observed up to 12 wk. Spraying of etofenprox significantly reduced the density (P<0.001) and proportion of parous An. culicifacies mosquitoes (P<0.05) in the experimental village. There was a significant reduction in malaria cases in the experimental village during the post-spray period when compared to the control village (P<0.05). No adverse effect was reported by the spraymen and inhabitants during and after the spray. INTERPRETATION & CONCLUSION: Indoor residual spray of etofenprox (0.1 g/m(2)) with an interval of three months in between two rounds of spray produced the desired impact in reducing the indoor resting density of vector mosquitoes and also in curtailing malaria transmission in the sprayed village when compared with the control village without spray.

Wash resistance and efficacy of three long-lasting insecticidal nets assessed from bioassays on Anopheles culicifacies and Anopheles stephensi.

OBJECTIVE: To test the wash resistance and efficacy of long-lasting insecticidal nets (LLINs), namely Olyset Net and PermaNet 2.0; and a long-lasting treatment kit, K-O Tab 1-2-3, on Anopheles culicifacies and An. stephensi, major malaria vectors in India, by bioassays. Conventionally treated deltamethrin net (CTDN with K-O Tab) was used for comparison. METHOD: Mortality and median time for knockdown (MTKD) of mosquitoes were determined using contact bioassays and ball frame bioassays respectively. Hand washing and machine washing were used. RESULTS: LLINs showed good bio-efficacy against An. culicifacies and An. stephensi. The mortality of mosquitoes remained >80% after up to 20 hand washes and up to 15 machine washes on all LLINs tested. No significant differences were observed in mortalities between the An. culicifacies and An. stephensi in cone bioassays (P > 0.05). MTKD increased progressively with successive washes and there was a significant difference in median time for knockdown of test mosquitoes and between hand-washed and machine-washed nets (P < 0.05). CONCLUSION: LLINs are more efficacious and last longer when washed by hand than by machine.