m2 against the malaria vectors in India

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Comparative efficacy of two rounds of indoor residual spraying of DDT 75% @ 1g/m2 with that of DDT 50% @ 1g/m2 against the malaria vectors in India

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Gunasekaran Kasinathana, Sudhansu Sekhar Sahua, , Krishnamoorthy Nallana, Vijayakumar Tharmalingama, Subramanian Swaminathana, Kali Prasad Beherab, Madan Mohan Pradhanb, Jambulingam Purusothamana a b

Indian Council of Medical Research-Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, India National Vector Borne Disease Control Programme, Odisha, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Anopheles fluviatilis Anopheles culicifacies Comparative efficacy DDT 50% DDT 75% India

While, dichlorodiphenyl-trichloroethane (DDT) water dispersible powder (WDP) 75% is considered as the high performance long lasting formulation for indoor residual spraying (IRS), no information is available regarding the comparative epidemiological effectiveness of the two DDT formulations when used for IRS in Indian conditions. The current study was undertaken to compare the effectiveness of IRS using DDT WDP 75% @ one g active ingredient (AI)/m2 with that using DDT WDP 50% @ one g AI/m2 in controlling Anopheles fluviatilis and An. culicifacies, the primary vectors of malaria in the selected endemic areas of Odisha State. Although, conebioassay mortality after 8 months of post spraying on DDT 75% sprayed surfaces as well as on sprayed but mud plastered surfaces was higher than DDT 50%, the six entomological parameters viz. resting density indoors and outdoors, trap density indoors, parous rate, human blood index and infection rate of An. fluviatilis and An. culicifacies did not show any statistically significant difference in reduction/ changes from pre- to post-spray period between the two DDT formulations.

1. Introduction

introduced two decades ago for IRS as well as for treating bed-nets. There are already reports of resistance in An. culicifacies to this insecticide family. In this situation and with no new insecticides at hand, there is a need to use the available insecticides judiciously. DDT is still the least expensive insecticide when compared to malathion and synthetic pyrethroids. DDT is available in two formulations, DDT water dispersible powder (WDP) 50% and DDT WDP 75%. In 1950, DDT water dispersible powder formulation, containing 50% or 75% technical DDT, was made available and the remarkable convenience in application led to its selection for the anti-malaria campaign (Raghavendra and Subbarao, 2002). Although, DDT WDP 75% is considered as the cost effective high performance formulation for IRS with longer residual activity, no information is available regarding the comparative epidemiological effectiveness of the two DDT formulations when used for IRS in Indian conditions. It is essential to have such operational information for an evidence-based selection of the optimal formulation of DDT.

In India, dichlorodiphenyl-trichloroethane (DDT) and lindane were introduced in public health programme in early 1950s (Bertram, 1950). In 1953, the Government of India launched National Malaria Control Programme with indoor residual spraying (IRS) of DDT (Sharma, 2003). While Benzene hexachloride (BHC) was banned from public health use in 1997, DDT is still being used for malaria and kala-azar control (Raghavendra and Subbarao, 2002; NVBDCP, 2016). Malathion, an organophosphate insecticide, was introduced in late 1960s in areas where vector resistance to DDT was reported. Though, development of resistance by malaria vectors, particularly Anopheles culicifacies, to these insecticides has been one of the major operational constraints, use of these insecticides does maintain malaria incidence at a low level with one million cases a year in India. Currently, under the National Vector Borne Disease Control Programme (NVBDCP), DDT, malathion and synthetic pyrethroids (SPs) are used for vector control. SPs were

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Corresponding author at: CMR-Vector Control Research Centre, Medical Complex, Indira Nagar, Puducherry, 605006, India. E-mail addresses: [email protected] (G. Kasinathan), [email protected] (S.S. Sahu), [email protected] (K. Nallan), [email protected] (V. Tharmalingam), [email protected] (S. Swaminathan), [email protected] (K.P. Behera), [email protected] (M.M. Pradhan), [email protected] (J. Purusothaman). https://doi.org/10.1016/j.actatropica.2019.03.028 Received 17 January 2019; Received in revised form 26 March 2019; Accepted 29 March 2019 Available online 31 March 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.

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randomized to receive either one of the two DDT formulations. Accordingly, Jogipaluru SC was sprayed with DDT WDP 75% (arm 1) and Kumbhari SC with DDT WDP 50% (arm 2), both @ one g/m2. In each arm, six villages (index villages) were randomly selected for mosquito collections to evaluate the impact of IRS with DDT 50% or 75%.

Hence, a study was undertaken to compare the effectiveness of IRS using DDT WDP 75% @ one g active ingredient (AI)/m2 with that using DDT WDP 50% @ one g AI/m2 in controlling An. fluviatilis and An. culicifacies, the major vectors of malaria (Sahu et al., 2015) in selected endemic areas of Odisha State in India. 2. Materials and methods 2.1. Study area

2.3. Intervention

The study was conducted during June 2013 to August 2014 in Narayanpatna community health centre (CHC), Koraput district of Odisha State, India (18°.8793779′ N latitude and 83°.1683564′ E longitude). The CHC had a population of 45,828 spread in 210 villages under 13 sub-centres (SCs). The annual parasite incidence (API) ranged from 72.5 to 148.4 during 2010–2014. The CHC has been endemic for Plasmodium falciparum malaria transmitted by An. fluviatilis and An. culicifacies (Sahu et al., 2015). Malaria transmission occurs perennially with two seasonal peaks; one during July-September and the other during November-December (Sahu et al., 2017). DDT WDP 50% was in use for IRS for malaria vector control (Sahu et al., 2015). The study was carried out in two SCs viz., Kumbhari (villages: 9; human dwellings: 605; Population: 3300) and Jogipaluru (villages: 10; human dwellings: 593; Population: 3082) of the CHC (Fig. 1). The villages included in the two SCs are situated on hill top.

Two rounds of IRS with DDT WDP 50% or 75% (trade name of DDT 50%: HIL DIT®-50 WDP and DDT 75%: HIL DIT®-75 WDP), 1st round during 16th to 25th July 2013 and the 2nd round during 6th to 15th November 2013, were carried out in each arm. Stirrup pumps were used for spraying by the district health department. The research team supervised the spraying to ensure quality and coverage. Villagers were motivated to refrain from mud-plastering the sprayed walls. Details on coverage, refusals, if any, and reasons for such refusals were recorded.

2.4. Spray coverage and quality Spray coverage (percentage of households/rooms sprayed) was monitored and recorded concurrently during each round of spraying by direct inspection of houses. Ease of handling spray equipment and the DDT formulations were also monitored. Spray quality was assessed by fixing filter papers (15 cm × 15 cm) before spraying on selected surfaces in each of the five randomly selected houses in six villages randomly selected in each arm for each round of spray. After spraying, filter paper samples were removed and sent to the Institute of Pesticide Formulation Technology, Gurgaon, Haryana for analysis for DDT content. Spray quality was expressed as percentage of houses (based on 3 sampling spots in each selected house) with adequate content of active ingredient per m2.

2.2. Study design This intervention study had two arms, one with indoor residual spraying of DDT WDP 50% and the other with DDT WDP 75%, both @ one g/m2. Since the study was conducted in an endemic area, where malaria control operations were ongoing, it was not possible or ethical to have an additional arm without any intervention. The two arms (two SCs) were comparable in terms of ecotypes, human population size and vector prevalence as the main parameter. The two arms were

Fig. 1. Map showing Jogipaluru and Kumbhari Sub-centres in Narayanpatna Community Health Centre, Koraput district, Odisha State. 124

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2.6.3. Residual effect of DDT on the sprayed surfaces Cone-bioassays were done fortnightly on sprayable surfaces (mud walls) before spraying and on sprayed surfaces after spraying, selecting randomly five houses in each of the six index villages and fixing three cones in each house. For the bioassays, An. fluviatilis females were collected from different villages (other than index villages) searching potential resting places inside human dwellings such as walls, roof, eaves and other dark areas using an oral aspirator and a flash light during the morning hours between 0600 and 0730 h. Five blood-fed (wild caught) female An. fluviatilis were exposed (in each cone) to the surfaces for 30 min and then held with access to sugar and mortality recorded at 24 h. The treated mortality was corrected to the control mortality. At the beginning, attempts were made to collect An. fluviatilis from the field for bioassays as An. stephensi colony was not available in the field site. Since, An. culicifacies was a DDT resistant species and An. fluviatilis was not available in adequate numbers in the field site after one month of 1st round of spraying, laboratory reared An. stephensi, which was susceptible to DDT, was brought from ICMR-VCRC Puducherry and used for the bioassays until end of the study. Bioassay with An. fluviatilis could be done only on two occasions i.e., 20 days and 3 months after 2nd round of spraying. In addition to cone-bioassays, scrapings from sprayed walls were analyzed for DDT content. From each arm, three villages and from each village five households were randomly selected. Scrapings from walls (each 10 cm × 10 cm) were collected from three sites in each household on six occasions; within 2–7 days, one month and two months after 1st round of spraying and 10, 140 and 200 days after 2nd round of spraying and sent to the Institute of Pesticide Formulation Technology, Gurgaon, Haryana for analysis.

2.5. Mud plastering of houses Plastering/smearing the walls of human dwellings using a mixture of cow dung, clay (mud) and water is a normal practice by tribal communities. During spraying, the villagers in both the arms were told about the purpose and benefit of the study and their co-operation was sought to prevent any tampering of sprayed walls including mud-plastering. After IRS, fortnightly survey was carried out in each of the 15 randomly selected villages to assess the percentage of houses/ rooms mud-plastered in each arm. A total of 90 households, six in each selected village, were visited in each arm. The sample size was calculated based on an expected mud-plastering of 60% of houses/ rooms in a fortnight, an error margin of 10% and 95% CI. The survey was discontinued as soon as 100% of the houses were found mud-plastered. 2.6. Entomological evaluation 2.6.1. Relative abundance of vector(s) resting indoors and outdoors Indoor-resting mosquitoes were collected fortnightly from nine fixed catching stations (6 human dwellings and 3 cattle sheds) using aspirators and flash lights, in each of the six selected villages, spending 10 min in each station in the morning. Mosquitoes resting outdoors were also collected, searching potential shelters in each village. Pit shelters have been reported to be the preferred outdoor resting site of An. fluviatilis (Das et al., 1990). Since, natural pit shelters were not available in adequate numbers in the villages, 12 pit shelters were dug (artificial creation) in shaded earth mounds around each village and the mosquitoes resting in those shelters were collected fortnightly. The number of vector mosquitoes collected per man-hour indoors (PMDI) and/or outdoors (PMDO) was the resting density of vectors. As light traps (LTs) were reported to be suitable for collections of anophelines in the study area (Gunasekaran et al., 1994), LTs were set fortnightly in three human dwellings (one LT in each dwelling) during dusk hours in each selected village of both the arms. The next morning, the trapped mosquitoes were collected, counted and recorded. The number collected per trap-night was the vector density (PTD).

2.6.4. Susceptibility of vectors to common insecticides Susceptibility/ resistance status of the vector species to DDT and deltamethrin was assessed in both the arms prior to each round of spraying and at the end of the study, using % corrected mortality as the indicator, using the World Health Organisation (WHO) tube test method (WHO, 2013). Fully fed, wild caught females of An. fluviatilis and An. culicifacies were exposed to insecticide impregnated papers at diagnostic concentration for 60 min and mortality recorded after 24 h of exposure.

2.6.2. Laboratory processing The anophelines collected indoors and outdoors were identified to species. Female mosquitoes were classified according to their gonotrophic stages. Based on ovariolar dilatations, the parity was determined. Blood meals of fully-fed females were analyzed for their source of feeding using agar gel diffusion method (Crans, 1969) and the proportion that fed on human was used to calculate human blood index (HBI) for each arm. The body of the identified mosquitoes was divided into two parts, abdomen and head + thorax and pooled separately for PCR assay for detection of parasite infection. Each pool comprised of maximum five head + thorax or five abdomen parts. DNA was extracted separately from the pools containing head + thorax and abdomen as per the manufacturers’ protocol (QIAamp DNA Mini Kit. Qiagen). Nested polymerase chain reaction (PCR) assay was carried out for amplification of the genus Plasmodium-specific fragment (PCR-1) and then the identification of P. falciparum and P. vivax (PCR-2) (Snounou et al., 1993). Every PCR assay had one negative (PCR mix without deoxyribonucleic acid (DNA)) and one positive control (PCR mix with DNA known to work in amplification). P. falciparum and P. vivax DNA extracted from human blood samples were used for positive control. After electrophoresis of PCR products, the gel was photographed using UVP GelDoc-It Imaging System, UVP, LCC Upland, CA 91786. PCR positives for P. falciparum (205 bp) and P. vivax (120 bp) were resolved by comparison with a standard 100bp DNA ladder. The parasite infection rate in An. fluviatilis and An. culicifacies was calculated using the Centers for Disease Control and Prevention (CDC) software PooledInfRate, Version 4.0 for pools of mosquitoes (Biggerstaff, 2009).

2.7. Sample blood survey and fever surveillance To assess malaria prevalence among the human population in the two arms, a sample blood survey (SBS) was carried out covering all age groups before (June 2013) and after (June 2014) spraying. All villages in both the SCs were covered for the blood survey. The required sample size for the blood survey with 95% confidence interval was worked out to be 750 for each SC assuming an average API of 20 and allowing 10% error margin. The sample size in each village was estimated based on proportion to the population. Informed consent was obtained from the village committee first and then from the persons from whom fingerprick blood samples were collected. In the case of children, informed consent was obtained from their parents/caregivers. Active case detection for malaria was carried out fortnightly by making house to house visits in all villages of the two arms from June 2013 to August 2014. Human blood samples (1 μl of blood for thin smear and 3–5 μl for thick smear from one person) were collected from finger pricks from the persons reported fever. Blood smears were Giemsa stained and checked for malaria parasites in 100 fields in each smear. As a quality control, all positive and 10% of the negative slides were cross-checked following the national guidelines (NMEP, 1995). Monthly (malaria) parasite incidence (MPI) was calculated for the two arms using the formula: Number of positives in a month/ Target population ×1000. Sample blood survey results were used to calculate parasite prevalence {slide positivity rate (SPR)} for each treatment group. SPR = Number of slides positive for malaria parasites/Total 125

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3. Results 3.1. Spray coverage During the 1st round, the room spray coverage was 83.4% (n = 1,425) in Kumbhari SC, DDT 50% arm (range: 74.7%–98.3%) and 86.9% (n = 1,473) in Jogipaluru SC, DDT 75% arm (range: 77.7%–99.1%). During the 2nd round, the spray coverage was 75.6% (n = 1467) in Kumbhari SC (range: 70.3%–87.9%) and 80.1% (n = 1572) in Jogipaluru SC (range: 64.9%–99.1%). 3.2. Mud plastering of sprayed rooms One month after 1st round of spraying, 22.6% (n = 1425) and 13.2% (n = 1473) of the sprayed rooms were found mud-plastered in DDT 50% arm and DDT 75% arm, respectively. After 2 months of spraying, the mud-plastered proportion increased in both DDT 50% (27.8%) and 75% (42.9%) arms. After 3 months, the proportion substantially increased and prior to 2nd round of spraying, > 90% of the sprayed rooms were found mud-plastered in both the arms. After the 2nd round of spraying, within a fortnight, 2.7% and 3.3% of the sprayed rooms were mud-plastered in the DDT 50% and 75% arm, respectively. Thereafter, mud-plastered proportion gradually increased, reaching 79.9% and 55.8%, respectively, by 5th month. Surprisingly, by five and half months post-spraying, 100% of the sprayed rooms were found mud-plastered in the DDT 50% arm and by sixth month, 100% mud-plastering was observed in the DDT 75% arm. 3.3. Entomological evaluation 3.3.1. Residual efficacy of DDT on the sprayed surfaces Cone-bioassays carried out on wall surfaces before spraying showed zero mortality of An. fluviatilis (n = 20) and An. culicifacies (n = 300) in both the arms confirming absence of any insecticide deposits. Two weeks after 1st round of spraying, mortality of An. culicifacies was 10.7% (n = 150) on wall surfaces sprayed with DDT 50% and 29.3% (n = 150) on surfaces sprayed with DDT 75%. Bioassays done with An. stephensi after one month of spraying showed that the mortality of An. stephensi was 98.4% (n = 190) against DDT 50% and 99.5% (n = 190) against DDT 75%. Three months after spraying, the mortality was 97.4% (n = 420) against DDT 75%, whereas, it decreased to 71.0% (n = 210) against DDT 50%. Bioassay was also done on sprayed but one time mud plastered surfaces. After 3 months of spraying, the mortality on mud-plastered surfaces was 80.8% (n = 255) against DDT 75% and against DDT 50% the mortality was only 47.9% (n = 390). Statistical analysis showed that up to one month post-spraying, mortality of An. stephensi on both sprayed and mud-plastered surfaces did not differ significantly between the two arms (p > 0.05by χ2test). At two and three months post-spraying, the mortality was significantly higher with DDT 75% than DDT 50% on sprayed and also on mud plastered surfaces (p < 0.001 by χ2test, for all comparisons) (Fig. 2) indicating a longer residual efficacy of DDT 75% than DDT 50%. After 2nd round of spraying, the cone-bioassay mortality on the sprayed surfaces was 100% (n = 450) up to 45 days with the two formulations. After 3.5 months of spraying, the mortality reduced to 81.3% (n = 450) with DDT 50% and 90.7% (n = 450) with DDT 75%. After 4.5 months of spraying, the mortality was 79.3% (n = 420) and 85.1% (n = 450) with DDT 50% and DDT 75%, respectively. Statistical analysis showed that up to 2.5 months post-spraying, mortality did not differ significantly between the two arms (p > 0.05 by χ2 test). At 3.5 and at 4.5 months post-spraying, the mortality against DDT 75% was significantly higher than that against 50% (p < 0.02 by χ2 test, for all comparisons). Beyond this period, bioassays could not be done on sprayed surfaces since all sprayed rooms were completely mud-plastered in both the arms. Bioassays were simultaneously carried out on sprayed but one time

Fig. 2. Mosquito mortality in bioassays done on sprayed surfaces (a) and on sprayed but mud-plastered surfaces (b) after 1st round of spraying.

slides collected and examined x 100.

2.8. Statistical analysis The number of An. culicifacies/ An. fluviatilis obtained from each house/ arm was recorded day wise and analyzed for variance and mean. Since, the variance was greater than the mean, the negative binomial regression model was used with number of females as dependent variable and arm and pre-post spray (pre-spray, post-spray 1 and postspray 2) as independent factors. The interaction effect between arm and pre-post spray period was used to compare relative changes in vector density between the two arms over the study period. For significant interaction effect, further comparison between the arms over pre and post-spray 1 or 2 was made from the 95% confidence intervals of the incidence rate ratio (IRR). The heterogeneity χ2-test was used to compare the relative changes in survival rate of the vector species between the two arms. To compare the malaria parasite prevalence between prior to and post-spraying in each arm and between two arms before and after spraying, χ2-test was used. The change in parasitic prevalence between the two arms from pre to post period was compared using logistic regression. The log-odds ratio interaction test was used to compare the incidence of malaria before and after intervention for each group of villages. All the analyses were carried out in STATA ver. 14.0. 126

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Fig. 3. Mosquito mortality in bioassays done on sprayed surfaces (a) and on sprayed but mud-plastered surfaces (b) after 2nd round of spraying. Fig. 4. Density of An. culicifacies prior to and after indoor residual spraying in DDT 50% and DDT 75% arms (a) Per man-hour resting density indoors (PMDI), (b) Per trap-night density indoors (PTD) and (c) Per man-hour resting density outdoors (PMDO).

mud-plastered surfaces from 15 days post-spraying. The mortality on mud plastered surfaces at 45 days post-spraying was 100% in both the arms and at 5.5 months post-spraying, the mortality was 44% (n = 450) against DDT 50% and 57.3% (n = 450) against DDT 75%. The mortality continued to decrease on mud-plastered surfaces over postspraying period and at 8 months post-spraying, the bioassays showed a mortality of 9.3% (n = 450) against DDT 50% and 16.2% (n = 450) against DDT 75%. Statistically, there was no significant difference in mortality on mud plastered surfaces between the two arms up to 4.5 months post-spraying (p > 0.05 by χ2 test for all comparisons). At 5.5–8 months post-spraying, the mortality was significantly higher with DDT 75% than that with 50% (p < 0.001 by χ2 test, for all comparisons (Fig. 3). Bioassays done with An. fluviatilis at 20 days and at 3 months post-spraying showed a mortality of 100% (n = 20) and 92.3% (n = 65) against DDT 50%, respectively and 100% (n = 20, 65) against DDT 75%, on both the occasions.

In the DDT 50% arm, prior to spraying the relative proportion of An. fluviatilis was 7.2% (n = 276) and after spraying it was reduced to 2.9% (n = 975). Similarly, in the DDT 75% arm the relative proportion of this species decreased from 15% (n = 334) to 6.2% (n = 1013). A reduction in the relative proportion of An. culicifacies (36.6%–20.1% and 37.4%–24.8%, respectively) was also observed in both the arms after spraying. Only six collections carried out during one month formed the baseline data (prior to spraying), whereas the post spraying data were from 27 collections over a period of 14 months. 3.3.3. Relative abundance of An. culicifacies resting indoors Prior to spraying, the per man hour density indoors (PMDI) of An. culicifacies in human dwellings of the six index villages varied from 0.2 to 5.2 (average ± SE = 2.7 ± 0.88) in DDT 50% arm and 0.0–5.0 (average ± SE = 3.3 ± 0.74) in DDT 75% arm (Fig. 4a). After 1st round of spraying, the average ( ± SE) PMDI was 2.4 ± 0.49 in the DDT 50% arm and 3.2 ± 1.02 in the 75% arm. After 2nd round of spraying, the PMDI was lower in both the arms, 0.6 ± 0.18 and 0.6 ± 0.19, respectively. The reductions/changes in the PMDI from pre- to post-spray period were not significantly different between the two arms (interaction effect, arm x pre-post spray, by negative binomial regression: χ2 = 0.95, p = 0.642). The dispersion parameter of the negative binomial model indicated that the number of females captured per man-hour was highly over-dispersed (alpha > 0 indicates over-

3.3.2. Species composition Totally, 2,598 anophelines belonging to 15 species viz., An. subpictus (45.3%), An. culicifacies (25.9%), An. vagus (15.7%), An. fluviatilis (6.2%), An. jeyporiensis (2.1%), An. maculatus (1.5%), An. tessellatus (1.0%), An. aconitus (0.6%), An. nigerrimus (0.5%), An. varuna (0.4%), An. pallidus (0.3%), An. splendidus (0.3%), An. jamesii (0.1%), An. theobaldi (0.1%) and An. ramsayi (0.04%), were collected in the two SCs during the study. Among them, An. fluviatilis and An. culicifacies are the recognized primary vectors of malaria and An. aconitus, An. jeyporiensis, An. maculatus and An. varuna are considered as vectors of secondary importance in India (Rao, 1984; Das et al., 1990; Sahu et al., 2017). 127

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Fig. 6. Density of An. fluviatilis prior to and after indoor residual spraying in DDT 50% and DDT 75% arms (a) Per man-hour resting density indoors (PMDI), (b) Per trap-night density indoors (PTD) and (c) Per man-hour resting density outdoors (PMDO).

Fig. 5. (a)Per man-hour resting density indoors (PMDI), (b) Per trap-night density indoors (PTD) and (c) Per man-hour resting density outdoors (PMDO) of An. culicifacies prior to and after 1st and 2nd round of spraying in 50% and 75% arms.

different between the two arms (interaction effect, arm x pre-post spray, by negative binomial regression: χ2 = 6.32, p = 0.09). The dispersion parameter of the negative binomial model indicated that the number of females captured per-trap was random (alpha = 6.2; 95% CI = 0.6–65.0, χ2 = 2.2, p = 0.07) (Fig. 5c).

dispersion = 1.58; 95% CI = 1.2–2.1; χ2 = 119.2, p < 0.0001) and thus justified the application of negative binomial regression over Poisson (Fig. 5a). Overall, in both arms, light trap catch of An. culicifacies was low in human dwellings. Prior to spraying, in DDT 50% arm, the average per trap-night density (PTD)+SE was 0.04+0.04 and after 1st round of spraying, it was 0.05+0.04. The corresponding values for DDT 75% arm were 0.06+0.02 and 0.11+0.07. After 2nd round of spraying, the PTD in both the arms was zero up to 59th week (Fig. 4b). The relative changes/ reduction in the PTD after spraying between the two arms was not significantly different (interaction effect, arm x pre-post spray, by negative binomial regression: χ2 = 0.16, p = 0.92). The dispersion parameter of the negative binomial model indicated that the number of females captured per man-hour was highly over-dispersed (alpha > 0 indicates over-dispersion = 6.84; 95% CI = 1.8–25.4; χ2 = 9.47, p < 0.0001) and thus justified the application of negative binomial regression over Poisson (Fig. 5b).

3.3.5. Relative abundance of An. fluviatilis resting indoors Before spraying, the average PMDI (in human dwellings) of An. fluviatilis was 0.17 ± 0.06 (range: 0.0 to 0.3) in DDT 50% arm and 0.18 ± 0.11 (range: 0.0 to 0.7) in 75% arm (Fig. 6a). After two rounds of spraying, the effectiveness of the two DDT formulations in bringing down the indoor resting density of the DDT susceptible vector, An. fluviatilis, was marked and comparable. The reductions/changes in the PMDI from pre- to post-spray period were not significantly different between the two arms (interaction effect, arm x pre-post spray, by negative binomial regression: χ2 = 3.48, p = 0.2). The dispersion parameter of the negative binomial model indicated that the number of females captured per man-hour was random (alpha = 5.4; 95% CI = 0.4–62.7, χ2 = 1.95, p = 0.08) (Fig. 7a). Both the DDT formulations significantly reduced the per trap-night density (PTD) of An. fluviatilis after spraying (Fig. 6b). The relative reduction of PTD during the post-spray period was significantly different between the two arms (interaction effect, arm x pre-post spray, by negative binomial regression: χ2 = 10.73, p = 0.004). Further, pairwise comparison showed that the PTD was significantly lower during post-1 and post-2 rounds in both arms compared to pre-spray PTD in DDT 50% arm (p < 0.05 for all comparisons). The dispersion parameter of the negative binomial model indicated that the number of females captured per-trap was significantly over dispersed (alpha =

3.3.4. Relative abundance of An. culicifacies resting outdoors In DDT 50% arm, there was a marginal reduction in the average per man hour density outdoors (PMDO)+SE of An. culicifacies after 1st round of spraying (0.05+0.03) compared to that prior to spraying (0.06+0.06), whereas in 75% arm, the density was relatively higher during post-spray period (0.21+0.1) than that prior to spraying (0.1+0.06). After 2nd round of spraying, the average PMDO was 0.07 ± 0.03 in DDT 50% arm and 0.05 ± 0.03 in 75% arm (Fig. 4c). The relative changes in the PMDO after spraying was not significantly 128

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Fig. 8. Parous rate in An. culicifacies before and after spraying in the two arms, DDT 50% and DDT 75%.

(p = 0.08) (Fig. 8). In the case of An. fluviatilis, the parous rate before spraying was 50% (n = 6) in both the arms. After spraying, parous rate could not be calculated as the numbers caught from indoors were very low in the study villages due to the effect of DDT spraying. 3.3.8. Human blood index (HBI) The human blood index (HBI) of An. culicifacies, before spraying, was low in both DDT 50% and DDT 75% arms, 0.15 (n = 55) and 0.08 (n = 76), respectively and not different from each other (Logistic regression, Wald statistic = 1.4, p = 0.23). After two rounds of spraying, there was no significant reduction in HBI in the two arms; 0.08 (n = 91) in the DDT 50% arm (Wald statistic = 1.69, p = 0.19) and 0.11(n = 104) in the DDT 75% arm (Wald statistic = 0.37, p = 0.54). The relative changes in HBI after two rounds of spraying was not significantly different between the two arms (logistic regression, Wald statistic = 1.84, p = 0.18). The very low sample size of An. fluviatilis obtained during the study did not permit to do any detailed analysis although there was a reduction in the number of this species after spraying compared to prior to start of spraying.

Fig. 7. (a) Per man-hour resting density indoors (PMDI), (b) Per trap-night density indoors (PTD) and (c) Per man-hour resting density outdoors (PMDO) of An. fluviatilis prior to and after 1st and 2nd round of spraying in 50% and 75% arms.

3.3.9. Vector infection and infectivity rate In DDT 50% arm, prior to spraying, the maximum likelihood estimate (MLE) of infection rate of An. culicifacies was 3.6% and after spraying, it was 1.07%. In DDT 75% arm, no infection was detected before spraying and during post-spray period, the infection rate was 1.2%. In both 50% and 75% arms, the infectivity rate during the postspray period was 1.2%. In outdoor collections, no infection was found in either arm (Table 1). The MLE of infection rate of An. fluviatilis in the 50% arm was 0% before spraying and 7.8% during post-spraying. The corresponding values in the 75% arm were 7.9% and 3.3% (Table 2). The infectivity rate of this vector species during post-spraying was 4% in the 50% arm and zero in the 75% arm. The corresponding values during pre-spraying were zero in both the species in two arms. Indoor collections did not yield any infective specimens in both the arms (Table 3).

2.3; 95% CI = 0.8–5.8, χ2 = 12.9, p < 0.05) and thus justified the application of negative binomial regression over Poisson (Fig. 7b). 3.3.6. Relative abundance of An. fluviatilis resting outdoors Prior to spraying, the PMDO of An. fluviatilis was 0.06+0.06 in 50% arm and 0.70+0.26 in 75% arm. The average PMDO after 1st round of spraying was 0.09+0.06 and 0.28+0.12 in DDT 50% and 75% arm, respectively. After 2nd round of spraying, the average PMDO was respectively 0.13 ± 0.03 and 0.31 ± 0.10 (Fig. 6c). The relative changes in the PMDO after spraying was not significantly different between the two arms (interaction effect, arm x pre-post spray, by negative binomial regression: χ2 = 7.04, p = 0.07). The dispersion parameter of the negative binomial model indicated that the number of females captured per-trap was significantly over dispersed (alpha = 2.26; 95% CI = 0.5–9.9, χ2 = 3.8, p = 0.26) and thus justified the application of negative binomial regression over Poisson (Fig. 7c).

3.3.10. Susceptibility of vectors to common insecticides Prior to spraying, the corrected mortality of An. culicifacies in filter paper bioassays was 9.8% against DDT and 76.1% against deltamethrin indicating resistance to these two insecticides. An. fluviatilis was susceptible to DDT with 100% mortality. After two rounds of spraying, the corrected mortality of An. culicifacies was 5.6% against DDT and 79.8% against deltamethrin, confirming its resistance to the two insecticides. During the post-spray period, susceptibility of An. fluviatilis could not be tested due to its very low density in the areas.

3.3.7. Parous rate In DDT 50% arm, the parous rate of An. culicifacies was 52.6% (n = 97) before spraying, 40% (n = 115) after 1st round and 25.4% (n = 67) after 2nd round of spraying. The corresponding values for DDT 75% arm were 54.6% (n = 119), 30.5% (n = 154) and 38.0% (n = 71). Logistic regression analysis showed that reduction of parous rates was significant between pre, and post 1st and post 2nd round spraying (p < 0.05, for both comparisons) in the DDT 75% arm, whereas in the 50% arm, it was significant only after 2nd round of spraying (p = 0.001). However, the relative reduction of parous rate after spraying did not differ significantly between the two arms,

3.4. Perceived side effects In total, 243 and 267 household adult members and 121 and 97 pregnant women in DDT 50% and 75% arm, respectively, were interviewed over a period of 8 months post-spraying, for their perceived 129

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Table 1 Maximum likelihood estimates (MLE) of malaria parasite infection rate in An. culicifacies before and after spraying. Test particulars

DDT 50% Before spray

No. of mosquitoes No. of pools No. of pools + ve Infection rate in % (95% CI)

59 13 2 3.6 (0.6–11.7)

DDT 75% After spray

Before spray

IDRC

ODRC

Total

180 35 2 (Pv) 1.1 (0.2–3.6)

9 3 0 0 (0.0–23.6)

189 38 2 1.07 (0.19–3.5)

72 15 0 0 (0.0–4.5)

After spray IDRC

ODRC

Total

233 46 3 (Pv) 1.3 (0.3–3.5)

15 3 0 0 (0.0–15.1)

248 49 3 1.2 (0.3–3.3)

Pv: Plasmodium vivax; IDRC: Indoor resting collection; ODRC: Outdoor resting collection.

3.7. Malaria prévalence

side-effects, if any, after spraying. In addition, 16 spray-men in each arm were interviewed during spraying and the next day after spraying for any side effects. Except the report of itching and headache by one spray-man in the 50% arm and nausea and headache by one pregnant woman in the 75% arm, no one reported any side effect. Moreover, the spray man and the pregnant woman who reported the side effects recovered within 12–24 h.

Before spraying, 802 and 804 blood smears were collected respectively from DDT 75% and DDT 50% arm and checked for malaria parasites. The average slide positivity rate (SPR) was 14.5% (range: 1.4–42.9) and 13.4% (range: 4.0–41.3), respectively and there was no significant difference (χ2 = 0.22, p > 0.05) between the two arms. At the end, i.e. post-two rounds of spraying, 791 (DDT 75%) and 772 blood smears (DDT 50%) were screened for malaria parasites and the average SPR was 7.2% (range: 1.5–24.5) in 75% arm and 14.4% (range: 5–43.6) in 50% arm. During the post-spraying period, the SPR in DDT 75% arm was significantly (χ2 = 17.45, p < 0.05) lower than that in 50% arm. Further, after spraying while there was no reduction of SPR in 50% arm compared to base-line, there was a significant (χ2 = 16.89, p < 0.05) reduction of SPR in 75% arm. The reduction in parasitic prevalence significantly differed between the two arms (odds ratio = 16.515; 95% CI: 11.242–24.261, p < 0.001; interaction effect: arm x pre-post).

3.5. Residue analysis In total, 72 filter papers, 36 for each arm, were fixed on the walls during 1st and 2nd round of spraying and analyzed for DDT content. The mean DDT content obtained for DDT 50% and 75% arm was 0.134+0.219 g/m2 (range: 0.003–1.194 g/m2) and 0.207+0.35 g/m2 (range: 0.006–1.436 g/m2), respectively during the 1st round, and 0.094+0.125 g/m2 (range: 0.007–0.561 g/m2) and 0.114+0.118 g/m2 (range: 0.012−0.444 g/m2) during the 2nd round. The mean DDT content in the filter paper samples did not differ significantly between the two arms during both the rounds (p > 0.05 by one way ANOVA). The observed DDT content in the scratched mud samples (the expected content was 1 g/m2) from the two arms over months postspraying are given in Table 4. Only at week one post-1st round spraying, the average DDT content was significantly different between the two DDT formulations and during the subsequent months of the 1st round and also during the 2nd round there was no significant difference between the two arms (DDT 75% and DDT 50%) (Table 4).

3.8. Malaria incidence Prior to first round of spraying, monthly (malaria) parasite incidence per 1000 population (MPI) in different villages ranged from 2.3–5.7 (average: 3.8+0.5) in DDT 50% arm and 2.2–10.3 (average: 4.8+1.2) in DDT 75% arm. After 1st round of spraying, there was a reduction of average MPI in both the arms, 1.4+0.2 (range: 0.0–3.3) in 50% and 2.1+0.4 (range: 0.0–6.2) in 75% arm. After 2nd round of spraying, the average MPI was further reduced to 0.9+0.22 (range: 0.0–2.4) and 1.2+0.32, (0.0–3.9), respectively. After two rounds of spraying, in both the arms, the average MPI was significantly reduced (log-odds ratio interaction test, p < 0.05) compared to that recorded before spraying, but the difference in the reduction of MPI between the two arms was not significant (p > 0.05) after spraying (Fig. 10).

3.6. Reduction of DDT content and bioassay mortality on sprayed surfaces Overall, there was a reduction in DDT content in the mud samples collected from sprayed walls in both the arms over the months postspraying. On mud-plastered surfaces the reduction was markedly higher resulting in lower mortality. Though, there was no significant difference in DDT content in the mud samples between the two arms, except at week one post 1st round spraying, the mortality obtained with DDT 75% was greater than 50% (Fig. 9), could be due to better bioavailability of the active ingredient.

3.9. Difference in ease of application In terms of ease of application, there was no difference between the two formulations except for preparation of mixture (spray solution) and requirement in quantity. To cover 5000 people, the requirement of DDT

Table 2 Maximum likelihood estimates (MLE) of malaria parasite infection rate in An. fluviatilis before and after spraying. Test particulars

DDT 50% Before spray

No. of mosquitoes No. of pools No. of pools + ve Infection rate in % (95% CI)

12 5 0 0 (0.0-20.6)

DDT 75% After spray

Before spray

IDRC

ODRC

Total

10 3 1 (Pf, Pv) 10.1 (0.3–45.3)

18 4 1 (Pf) 5.5 (0.3–25.9)

28 7 2 7.8 (1.4–24.9)

Pv: Plasmodium vivax; IDRC: Indoor resting collection; ODRC: Outdoor resting collection. 130

42 11 3 (Pf) 7.9 (2.1–20.6)

After spray IDRC

ODRC

Total

15 3 0 0 (0.0–14.9)

48 10 2 (Pf, Pv) 4.2 (0.8–13.4)

63 13 2 3.3 (0.6–10.8

Acta Tropica 194 (2019) 123–134 53 11 0 0 (0.0-5.9 43 9 0 0 (0.0–7.2) 10 2 0 0 (0.0–16.4)

4. Discussion DDT in the form of WDP has been the preferred choice for IRS. WDP formulation is made by mixing dry insecticide powder (active molecules) with non-insecticidal inert materials such as solvents, carriers, special additives and surface active agents, which facilitate dissolving the insecticide in water and its suspension. The WDP has been the most effective formulation as it is suitable for porous surfaces such as mud and brick walls which are the common sprayable surfaces in rural areas. Further, because of relatively larger size of insecticide particles, absorption is lesser. As a result, more active molecules remain available on the sprayed walls/ surfaces for the endophilic mosquitoes to pick up the lethal dose, and also there will be a longer residual effect of the insecticide (USAID, 2009). DDT 75% WDP and 50% WDP are the two DDT formulations used for IRS. The DDT 75% contains 75 g of DDT (active molecules) in every 100 g of the formulated product and the remaining 25 g are inert materials. Similarly, 100 g of DDT 50% contain 50 g of DDT and 50 g of inert molecules. DDT was introduced in 1944 as a residual insecticide for mosquito control on a trial basis in the army camps of Assam-Burma front and the results were very encouraging (Singh, 1962). Later, a few experiments were done in civilian areas of Odisha and Karnataka State (White, 1945; Vishwanathan and Parikh, 1946). In 1950, DDT (as 75% WDP and 50% WDP) was made available for public health use and the remarkable convenience in application led to its adoption for anti-malaria campaign. In India, DDT has been sprayed @ 1 g AI/m2 since 1953. However, gradually vectors developed resistance or changed their resting from indoors to outdoors. Later, spraying DDT even @ 2 g AI/m2 did not improve the impact on malaria transmission (Sharma, 2003). In 1971, there were reports of resistance in An. culicifacies to DDT 75% in some parts of Madhya Pradesh and consequently DDT was replaced by lindane in 1972 (Pacholi, 1993). Use of DDT for the control of disease vectors has been endorsed by Stockholm Convention. In India, DDT has been the insecticide of choice for IRS for the control of vectors of malaria and visceral leishmaniasis (UNEP, 2008). DDT is currently manufactured only by three countries viz., India, China and the Democratic People’s Republic of Korea (UNEP, 2008). Apart from the producer countries, only a few countries use DDT. Many malaria endemic countries now switch back to DDT for IRS (Sadasivaiah et al., 2007). In India, both DDT 50% and 75% WDP have been in use since 1953. However, DDT 75% was withdrawn from malaria control in 1972 without documenting any reason. Since then, only DDT 50% has been used for IRS. Despite the availability of DDT 75% in India and its good performance in malaria control in African countries (Pacholi, 1993), there is no documentation yet on its effectiveness in controlling malaria vectors in India. In the current study, due to the effect of DDT spraying, there was a reduction in the relative proportion of the two vector species, An. fluviatilis and An. culicifacies. Evidence generated by the control programme showed correct and timely implementation of indoor residual spraying can reduce malaria transmission by up to 90% (WHO, 2006). In the past, India was able to use DDT effectively for indoor residual spraying to reduce markedly the number of malaria cases and fatalities (Sharma, 2003). South Africa has re-introduced DDT for IRS to keep malaria incidence and fatality at all-time low levels and move towards malaria elimination. Currently, around 14 countries in the Sub-Saharan Africa are implementing IRS and among them 4–5 are using DDT for IRS. The primary objectives of the current study were to compare the effectiveness of two rounds of IRS using DDT 75% with

17 4 1 (Pf) 5.8 (0.3–27.3) 8 2 0 0 (0.0–26.4 239 47 3 1.2 (0.3–3.4)

Pv: Plasmodium vivax; IDRC: Indoor resting collection; ODRC: Outdoor resting collection.

13 3 0 0 (0.0–16.1 226 44 3 (Pv) 1.3 (0.3–3.6 167 32 2 1.2 (0.2–3.9

Total ODRC Total ODRC

9 3 0 0 (0.0–23.6 158 29 2 (Pv) 1.2 (0.2–4.1) No. of mosquitoes No. of pools No. of pools + ve MLE of Infectivity rate in % (95% CI)

IDRC IDRC

Test particulars

Table 3 Maximum likelihood estimation of malaria parasite infectivity rate in the vector species after spraying.

for one round of spraying would be 375 kg of 50% WDP or 250 kg of 75% WDP. At this rate, for one district, e.g. Koraput with a population of about 1.4 million, the requirement of DDT 50% would be 105 MT (2100 bags, each bag contains 50 kg DDT) and if it is DDT 75%, 70 MT (1400 bags) would be required, thereby reducing transportation cost by 30%, if one truck normally carries 200 bags or 10 MT.

25 6 1 4.0 (0.2–18.8)

Total ODRC IDRC

DDT 50% DDT 75% DDT 50%

IDRC

An. fluviatilis An. culicifacies

ODRC

Total

DDT 75%

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Table 4 DDT content in the scratched mud samples from the two arms over months post-spraying. Period post-spraying

Mean DDT content (g/m2) + SD DDT 75%

Bioassay mortality (%)

DDT 50%

I round of spraying (n = 108, 18 from each arm on every occasion) 2–7 days 0.414 + 0.338 0.124 2 months 0.043 + 0.041 0.042 3 months 0.112 + 0.111 0.047 II round of spraying (n = 108, 18 from each arm on every occasion) 10 days 0.075 + 0.064 0.075 4.5 months 0.112 + 0.138 0.051 # 6.5 months 0.006 + 0.008 0.003

p value* (75% vs 50%)

DDT 75%

DDT 50%

+ 0.214 + 0.051 + 0.095

< 0.05 > 0.05 > 0.05

99.5a 100 a 97.4a

98.4 a 76.7b 71.0b

+ 0.067 + 0.067 + 0.005

> 0.05 > 0.05 > 0.05

100a 85.1a 39.3a

100a 79.3b 22.5b

*By one way ANOVA; ND-Not done. Bioassay mortality: Numbers in the same row not sharing a letter differ significantly (p < 0.05). # Mud plastered surfaces.

Fig. 9. DDT content in the scratched mud samples and bioassay mortality during post-spray periods. 132

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sprayed rooms of their houses at a faster rate; after six months of spraying, all sprayed rooms were found mud-plastered and this was due to the number of festivals celebrated by people during this season. The current study was not without limitations. One of the limitations was that since adequate number of An. fluviatilis could not be collected from the study villages because of spraying, laboratory reared susceptible An. stephensi was used for bioassays at month 3 postspraying. Another limitation was that ‘before spray’ collections were limited, done during the low transmission season and there were relatively low numbers of mosquitoes. After spraying, there were many collections conducted, the collections were done during the rainy season, and higher numbers of mosquitoes were captured and subjecting them to PCR assay. This could be the reason for the 0% infection in the vector species before spray, but, from the data made available, it is clear that compared to before spray, there was a reduction of infection rate in An. culicifacies after spraying with DDT 50%; similarly, the infection rate was reduced in An. fluviatilis in DDT 75% arm after spraying. However, the strengths of the study were that it covered all the three seasons of the year during post-spraying and prior to implementation of the intervention the two arms were comparable in terms of vector density and malaria incidence.

Fig. 10. Malaria incidence in two arms before and after spraying.

that using DDT 50% @ 1 g AI/m2 in malaria vector control and to clarify a place for the actual formulation of DDT as a residual insecticide. DDT 75% WDP caused a higher mosquito mortality in conebioassays for a longer period (longer residual efficacy) on sprayed and on sprayed but mud-plastered surfaces than DDT 50%. At one month after 1st round of spraying, both formulations caused equal mortality on sprayed and on mud-plastered surfaces and at 2 and 3 months after spraying, the mortality was significantly higher with DDT 75% than DDT 50%. After 2nd round of spraying, up to 2.5 months, the mortality caused by the two formulations was nearly equal and at 3.5 and 4.5 months the mortality was significantly higher with DDT 75% than DDT 50%. Up to 4.5 months after spraying, the mortality on mud-plastered surfaces was almost equal with the two formulations; thereafter and up to 8 months, the mortality with DDT 75% was significantly higher than DDT 50% indicating a longer residual efficacy/ activity of DDT 75% than DDT 50%, although the mean DDT content in the filter paper samples did not differ significantly between the two formulations during both the rounds. This could be due to the lesser quantity, just 25%, of inert/filler materials used in DDT 75% WDP formulation, which might have permeated (through sorption/physical adherence) only a smaller quantity of DDT active molecules thereby increasing/ maintaining bio-availability unlike in DDT 50%, where the inert/filler materials form 50% and that, after spraying, might have increased the sorption rate of active molecules leading to decreased bio-availability. However, such longer residual activity (bio-availability on sprayed surfaces), interpreted from cone-bioassay mortality was not reflected from other entomological parameters as the reduction/ changes in resting density indoors and outdoors, trap density indoors, parous rate, human blood index and infection rate of the malaria vectors, An. fluviatilis and An. culicifacies, after spraying compared to before spraying did not differ significantly between the two DDT formulations. In conebioassays, mosquitoes were confined and forced to get exposed to the sprayed surfaces, whereas the other entomological parameters were studied from the natural free-flying mosquitoes. Therefore, the bioavailability on treated surfaces to the mosquitoes that were bio-assayed in cones might be different to the free-flying mosquitoes (Roberts and Alecrim, 1991); this needs to be studied in detail. There was a significant reduction of malaria prevalence in villages sprayed with DDT 75% compared to the villages sprayed with DDT 50%, whereas, the MPI was significantly reduced (log-odds ratio interaction test, p < 0.05) in both the arms after spraying compared to that recorded before spraying; the reduction/ relative changes in MPI from pre- to post-spraying period was not significantly different between the two arms. After the 2nd round of spraying, since there was also a seasonal decrease in incidence in the area, no difference in reduction could be seen between the two arms. Compared to the 1st round, spray coverage was lower during the 2nd round in both the arms, as spraying was delayed and coincided with paddy harvesting time. Some of the villagers, in spite of receiving advanced information, locked their houses and went to fields for harvesting when the spray team visited their houses. Also, after the 2nd round of spraying, in both the arms, the villagers mud-plastered the

5. Conclusions DDT has been known as the only insecticide that can be used for single application in areas where transmission period is about 6 months, as experienced by the control programme for decades (Sharma et al., 1986; Mnzava et al., 1993; Pluess et al., 2010; Zhou et al., 2010). In the current study, the relative reduction/ changes in resting density, trap density, parous rate, human blood index and infection rate of An. fluviatilis and An. culicifacies from pre- to post-spray period did not differ significantly between the two DDT formulations. However, DDT 75% WDP caused significantly higher mosquito mortality than DDT 50% for a longer period (8 months), even on the mud-plastered sprayed surfaces, as evident from the cone-bioassay results. As per the spray schedule, currently followed in India, the inter-spray period is 3 months between 1st (May–June) and 2nd round (September–October) and 7 months between 2nd and 1st round of the next year. From this point of view and from a significantly higher reduction of malaria prevalence (in terms of SPR) after two rounds of spraying, DDT 75% WDP may have an advantage over DDT 50% WDP. Authors' contributions SSS and KG designed and performed the study. SSM and KG compiled and analyzed the data. TV and NK involved in molecular analysis. KG and SSS drafted the manuscript. KPB, MMP and PJ critically reviewed the manuscript. All authors contributed to the writing of the manuscript and approved the final version. Ethics statement The study was approved by the Human Ethical Committee of Indian Council of Medical Research - Vector Control Research Centre (ICMR VCRC), Puducherry. Author summary The current study compared the effectiveness of IRS using DDT WDP 75% @ one g active ingredient (AI)/m2 with that using DDT WDP 50% @ one g AI/m2 in controlling Anopheles fluviatilis and An. culicifacies, the primary vectors of malaria in the selected endemic areas of Odisha State. The reduction/changes of resting density indoors and outdoors trap density indoors, parous rate, human blood index and infection rate of An. fluviatilis and An. culicifacies from pre to post-spray period were not significantly different between the two formulations. However, 133

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cone-bioassay mortality up to 8 months of post spraying on DDT 75% sprayed and mud plastered surfaces was higher than DDT 50%. Thus, DDT 75% has an advantage over DDT 50% in term of residual efficacy.

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Funding This trial was funded by Indian Council of Medical Research, New Delhi. Competing interests The authors declare that they have no competing interests. Acknowledgements The authors are highly thankful to Mr. Sachin R Yadav, Collector, Koraput district for his support and cooperation for conducting the study. The authors are also thankful to the Joint Director of Health Services (Malaria), the district VBD Consultant and the Medical Officer, MTS and other health staff of Narayanpatna CHC for their help in carrying out the study. The sincere and hard work of the technical staff of the ICMR-VCRC Field Station, Koraput are gratefully acknowledged. References Bertram, D.A., 1950. A critical evaluation of DDT and gammaxene in malaria control in upper Assam over five years with particular reference to effect on Anopheles minimus. Ann. Trop. Med. Parasitol. 44, 242. Biggerstaff, B.J., 2009. PooledInfRate, Version 4.0: A Microsoft Excel Add-in to Compute Prevalence Estimates From Pooled Samples. Centers for Disease Control and Prevention, Fort Collins, Colorado, U.S.A. Crans, W.J., 1969. An agar- gel diffusion method for identification of mosquito blood meal. Mosq. News 29, 563–566. Das, P.K., Gunasekaran, K., Sahu, S.S., Sadanandane, C., Jambulingam, P., 1990. Seasonal prevalence & resting behaviour of malaria vectors in Koraput District, Orissa. Indian J. Malariol. 27, 173–181. Gunasekaran, K., Jambulingam, P., Sadanandane, C., Sahu, S.S., Das, P.K., 1994. Reliability of light trap sampling for Anopheles fluviatilis, a vector of malaria. Acta Trop. 58, 1–11. Mnzava, A.E.P., Rwegoshora, R.T., Tanner, M., Msuya, F.H., Curtis, C.F., Irare, S.G., 1993. The effects of house spraying with DDT or lambda-cyhalothrin against Anopheles arabiensis on measures of malarial morbidity in children in Tanzania. Acta Trop. 54, 141–151. National Malaria Eradication Programme, 1995. Operational Manual for Malaria Action Programme (MAP). NMEP, Directorate General of Health Services. Ministry of Health and Family Welfare, Government of India, Delhi.

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