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Table of Contents
RESEARCH ARTICLE
Year : 2017  |  Volume : 54  |  Issue : 4  |  Page : 334-340

Entomological determinants of malaria transmission in an epidemic prone area of District Nuh (Haryana state), India


ICMR–National Institute of Malaria Research, New Delhi, India

Date of Submission29-Aug-2017
Date of Acceptance29-Nov-2017
Date of Web Publication19-Feb-2018

Correspondence Address:
Nutan Nanda
Scientist ‘G’ (Retd.), 8/1 Block 41, Singh Sabha Road, Shakti Nagar, Delhi–110 007
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.225838

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  Abstract 

Background & objectives: Entomological investigations were carried out in highly malarious villages under Ujina PHC of District Nuh (Haryana state) which is an epidemic prone area in northwestern region of India. The study was aimed to have an in-depth understanding of the entomological parameters influencing malaria transmission in the study area.
Methods: The seasonal prevalence and biological attributes of vector mosquitoes were investigated during 2015 and 2016. Indoor resting vector mosquitoes were collected from human dwellings/cattle sheds and morphologically identified. Anopheles culicifacies were categorized to sibling species by species-specific inversions in polytene chromosomes and An. stephensi to ecological races on the basis of ridge number on egg float. The blood meal source analysis and incrimination studies of vectors were done by counter-current immunoelectrophoresis and enzyme-linked immunosorbent assay, respectively. Insecticide susceptibility test on vectors was performed as per WHO guidelines.
Results: Seasonal abundance of An. culicifacies and An. stephensi in the study area showed variation; the peak densities of both the vectors were observed during monsoon months which correlated well with the average monthly rainfall data. Though both vectors were found to be primarily zoophagic, the human blood index of An. culicifacies (HBI = 0.17) was significantly higher than that of An. stephensi (HBI= 0.02). Analysis of sibling species composition of An. culicifacies population showed that it comprised almost of sibling species A (>98%) which is an established malaria vector. Anopheles culicifacies was incriminated for Plasmodium vivax and P. falciparum circumsporozoite (CS) antigen during monsoon months in 2015 and 2016. Assessment of insecticide susceptibility status of malaria vectors against 0.5% deltamethrin revealed that An. culicifacies is more susceptible (95% mortality) than An. stephensi (85% mortality).
Interpretation & conclusion: The results suggest that An. culicifacies (species A) is playing a major role in malaria transmission in the study area and is almost susceptible to deltamethrin. Timely two rounds of indoor residual spray of synthetic pyrethroid with proper dosage and good coverage would be helpful in reducing vector population and consequently the malaria incidence. In addition, personal protection measures by the community would supplement the major intervention tool (IRS) in decreasing the man-vector contact.

Keywords: Anopheles culicifacies; An. stephensi; biological attributes; malaria vectors; malaria transmission; Nuh district


How to cite this article:
Nanda N, Singh S P, Prajapati B K, Ranjan K, Kar N P, Sharma S K, Valecha N. Entomological determinants of malaria transmission in an epidemic prone area of District Nuh (Haryana state), India. J Vector Borne Dis 2017;54:334-40

How to cite this URL:
Nanda N, Singh S P, Prajapati B K, Ranjan K, Kar N P, Sharma S K, Valecha N. Entomological determinants of malaria transmission in an epidemic prone area of District Nuh (Haryana state), India. J Vector Borne Dis [serial online] 2017 [cited 2018 May 21];54:334-40. Available from: http://www.jvbd.org/text.asp?2017/54/4/334/225838




  Introduction Top


The State of Haryana in northern India comprises of 21 districts and District Mewat[1] was carved out as the 20th district of Haryana from erstwhile Gurgaon and Faridabad districts in April 2005, which has now been renamed as District Nuh in 2016[1]. Since the major outbreak of malaria in Mewat region in 1996, several studies have been carried out enumerating various factors associated with malaria prevalence and persistence in the traditionally known epidemic belt of the northwestern plains of India. These included studies related to malaria vectors and parasite prevalence[2],[3]; geographic information systems (GIS) and remote sensing (RS) based surveys defining malaria paradigms and delineating high risk areas[4],[5], and trends of malaria incidence and infection over the years[6],[7]. Despite this, malaria still continues to be persistent in the region mainly because it lags far behind on almost all aspects of development as compared to the rest of Haryana and much remains to be achieved in terms of public health.

District Nuh is endemic for malaria and has three community health centres (CHCs), namely Nuh, Firozpur Jhirka and Punhana. Among these, Nuh CHC is highly malarious and contributes bulk of malaria cases reported from the district[6]. CHC Nuh comprises of five primary health centres (PHC), viz Nuh, Ujina, Ghasera, Taruru and Mohammadpur Ahir [Figure 1]a, [Figure 1]b, [Figure 1]c. As per epidemiological data available with the Chief Medical Officer (Nuh CHC); PHC Ujina has reported highest malaria cases from 2010–15 followed by PHC Nuh. Though An. culicifacies and An. stephensi have been reported as malaria vectors in this region, an in-depth study of their seasonal prevalence and biological characteristics is lacking. Therefore, a systematic longitudinal study was carried out to investigate the environment-dependent entomological factors that influence malaria transmission dynamics in the highly malarious villages under PHC Ujina. The findings of this study are presented in this communication.
Figure 1: (a) Map of India showing Haryana state; (b) District Nuh (earstwhile Mewat) showing Community Health Centres (CHCs); and (c) Primary Health Centres under Nuh CHC.

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  Material & Methods Top


Study area

District Nuh in Haryana state lies between 26° and 30° N latitude and 76° and 78° E longitude, and falls under the sub-tropical and semi-arid climatic zone. Though this district largely comprises of plains, there are patches of upland due to hills and hillock of the Aravali mountain. Nuh district is predominated by Muslims (Meos) whose main occupation is agriculture while animal husbandry is the secondary source of income.

Four highly malarious villages namely, Jaisinghpur, Karamchandpur, Chilawali and Gundbas falling under Ujina PHC were selected for this study on the basis of high annual parasite incidence (API) which ranged between 14.55 to 60.39 during 2010–14. These four villages in plain area are located within a radius of 3–5 km from each other and mainly have Pucca houses with adjacent cattlesheds. There are perennial ponds, and low-lying areas with seepage water due to high water table in the vicinity of these villages and majority of the houses have cemented tanks to store piped water supply.

Entomological investigations

Mosquito collection and processing: Indoor resting mosquitoes were collected from human dwellings and cattlesheds during early morning (0600 to 0800 hrs) in each village using a suction tube and torch light[8]. The collections were made almost every month during 2015 and during the transmission season (July–September) in 2016. The anophelines collected were morphologically identified to species following standard keys[9],[10]. The average monthly man hour densities of vectors An. culicifacies and An. stephensi were calculated using the formula: Number of mosquitoes of each vector species collected χ 60/Total collection time in minutes.

The dead and alive mosquitoes were brought to the laboratory for further processing to study various entomological parameters.

Anopheles culicifacies sibling species identification

In case of An. culicifacies, the blood-fed females were kept under ambient conditions in cloth cages and allowed to reach half gravid stage. From each individual half gravid female, ovaries were removed and preserved in modified Carnoy’s fixative (glacial acetic acid: methanol—1: 3). The preserved ovaries were processed for making polytene chromosome plates following method of Green and Hunt[11]. Anopheles culicifacies were identified to sibling species using diagnostic paracentric inversions in polytene chromosomes[12].

Identification of An. stephensi for ecological races

Single tubing of field collected An. stephensi females was carried out in insectary (maintained at 28 + 2°C and RH 60–70%) in order to obtain F1 progeny. Around 20–25 eggs of individual females were examined in binocular (Zeiss Axioplan, West Germany) for ridge number on the egg float. The individual single female was categorized as type form, intermediate and variety mysorensis as per the grading described by Subbarao et al[13].

Blood meal source identification of vector species

Anopheles culicifacies and An. stephensi collected from study villages were analysed for host feeding pattern. Midgut blood smears from fully-fed and half gravid females were subjected to blood meal source identification using human and bovine antisera by counter-current immunoelectrophoresis following the method described by Bray et al[14]. The human blood index (HBI) was calculated for each vector species.

Vector incrimination

From the field collected vector species, the head and thorax of individual An. culicifacies and An. stephensi were processed for detection of circumsporozoite (CS) antigen of malaria parasite species prevalent in the area. Homogenate of head and thorax of individual mosquito in grinding buffer was tested for the presence of CS proteins using P. vivax (210 and 247 variants) and P. falciparum -specific monoclonal antibodies by enzyme-linked immunosorbent assay (ELISA)[15],[16].

Insecticide susceptibility status of vectors

The susceptibility status of An. culicifacies and An. stephensi was determined against deltamethrin which is presently used as indoor residual spray (IRS) in District Nuh. Susceptibility test was performed following the standard procedure[17],[18]. Blood-fed An. culicifacies and An. stephensi were exposed to 0.05% deltamethrin-impregnated papers for 1 h along with parallel controls. Mortality was recorded after 24 h holding period. The percent corrected mortality was calculated and the mosquitoes of each vector species were categorized as resistant, susceptible and under ‘verification required category’ following the guidelines of WHO[18].

In addition, the average monthly rainfall data were obtained from Sadar Kanungo Department, District Nuh for the year 2015 for correlating with the seasonal variation in abundance of the malaria vectors.


  Results Top


Since perennial ponds and low-lying areas with seepage water were present in the vicinity of study villages and the inhabitants used cemented water storage tanks/ wells [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e, both An. culicifacies and An. stephensi, the primary vectors of malaria were prevalent in the villages almost all through the year, though their abundance varied with season. The average man hour density of primary malaria vectors in different months/seasons is shown in [Figure 3]. Extremely low densities were observed in winter months (December–January) and a build-up of population of both vector species was noticed during spring/early summer, which was more pronounced in case of An. stephensi, probably due to favourable temperature of the water in cemented storage tanks (the preferred breeding habitat of An. stephensi) and a short spell of rains during that period. The peak densities of both vectors were observed during monsoon months (July–September) with average man hour density ranging from 30 to >70 which later declined in post-monsoon months (October–November). The seasonal variation in the vector density correlated well with average monthly rainfall data [Figure 3]. Besides An. culicifacies and An. stephensi, the other anophelines, viz. An. subpictus, An. aconitus and An. annularis were also prevalent in the study villages.
Figure 2: (a–e): Breeding habitats of vector anophelines in study area—(a) Perennial pond; (b) Low-lying area with seepage water; and (c–e) Cemented water storage tanks.

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Figure 3: Average man hour density of An. culicifacies and An. stephensi in study villages of PHC Ujina, CHC Nuh and average monthly rainfall in 2015.

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For determining An. culicifacies sibling species composition in study villages, a total of264 females were examined during 2015–16. It revealed that species A was predominant in the study area, comprising 98.48% of total samples identified and was polymorphic for i[1] inversion [Figure 4]. However, the relative proportion of sibling species B and C was very low, comprising only 1.14 and 0.40% of the total identified samples, respectively. To know the ecological variants of An. stephensi, 50 isofemale lines were examined for ridge number on the egg float. The egg ridge number observed in single female culture ranged from 15–20 and the mode number of ridges was 16–17 which indicated that An. stephensi ‘type form’was mainly prevalent in study area.
Figure 4: Sibling species composition of An. culicifacies complex in study villages under Ujina PHC, District Nuh* (n=264). *Pooled data of all study villages (2015–2016).

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Regarding resting behaviour of An. culicifacies and An. stephensi, the preferred resting sites of both the species were cattle sheds. Out of 1410 An. culicifacies collected during study period 1311 (92.98%) were from cattle sheds and only 99 (7.02%) were found resting in human dwellings. Similarly, out of 1918 An. stephensi collected, 1806 (94.16%) were from cattle sheds and only 112 (5.84%) were found resting in human dwellings.

Feeding preference of vector species was also analysed in the study. Results of blood meal source identification revealed both An. culicifacies and An. stephensi to be primarily zoophagic. The overall HBI of An. culicifacies was 0.164 and the proportion of mixed positive samples was high [Table 1]. In case of An. stephensi, the HBI was comparatively much lower (0.019) and only a few mosquitoes were found exclusively feeding on human blood.
Table 1: Host preference of malaria vectors in study villages under Ujina PHC, District Nuh*

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Vector incrimination studies revealed two specimens of An. culicifacies, out of 742 tested, harbouring P. vivax 247 and P. falciparum sporozoites in the year 2015 and 2016, respectively. However, none of An. stephensi tested was found positive for CS antigen of Pv and Pf parasites [Table 2]. The sporozoite positive samples of An. culicifacies were retested to confirm the results.
Table 2: Incrimination of An. culicifacies and An. stephensi in study villages by ELISA*

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Results of susceptibility status of An. culicifacies and An. stephensi against 0.05% deltamethrin in study villages are summarized in [Table 3]. The percent mortality in An. culicifacies was 95.4, while in An. stephensi it was comparatively lower (85%). These observations indicate that An. culicifacies is more susceptible to deltamethrin as compared to An. stephensi.
Table 3: Susceptibility status of An. culicifacies and An. stephensi against 0.05% deltamethrin in study villages (PHC Ujina, District Nuh)*

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  Discussion Top


Malaria transmission is complex and dynamic process and various factors, viz environmental, entomological, parasitological and socioeconomic conditions of the community influence the transmission pattern[19]. These factors are strongly inter-linked and even minor change in one of the factors has its repercussion on others. Among entomological determinants of malaria, the prevalence and abundance (density) of vector species; the proportion of vector population biting humans (particularly in case of primarily zoophagic vectors) and longevity of vectors so as to enable them to support sporogonic development of malaria parasites are crucial in disease transmission. Seasonal variation in the prevalence of vector mosquito species and its biological attributes are very important in shaping malaria transmission pattern, especially in epidemic prone areas like Mewat region. Malaria transmission in Nuh is essentially seasonal and of the three CHCs in the district, highest malaria cases are reported from Nuh CHC. An upsurge in malaria cases was reported in this area[6] during 2011 and 2012. A hospital-based longitudinal study carried out by Naz et al[7] in Mewat has shown that almost 98% of the malaria cases were recorded between July and November and majority of the cases (80%) were reported during October and November. In such areas a spurt in malaria incidence is reported if vector control measures are not taken timely[3]. For appropriate vector control measures adequate information on seasonal prevalence and biological attributes of vector species are essential.

In highly malarious study villages under Ujina PHC, longitudinal study of malaria vectors revealed seasonal variation in their abundance and peak densities of An. culicifacies and An. stephensi were observed during monsoon months (July–September) which correlated well with good rainfall during this period. The rainfall not only creates numerous fresh water pools, puddles, temporary ponds etc conducive for vector breeding, but also aids in favourable temperature and humidity conditions that help in enhancing the longevity of vectors[20].

Analysis of An. culicifacies sibling species composition in study villages revealed predominance of species A (>98%) which is an established malaria vector in malaria endemic regions of India[12]. Additionally, An. stephensi population comprised of Type form, which is also an efficient malaria vector [21],[22].

Though, both the vector species were primarily zoophagic, the HBI in case of An. culicifacies was comparatively much higher than that of An. stephensi. Moreover, An. culicifacies was detected with plasmodial sporozoites for two consecutive years (2015–16). These observations suggest that An. culicifacies is playing a major role in malaria transmission in the study area. Anopheles culicifacies plays a significant role in malaria transmission in many other parts of India mainly due to high densities[23],[24].

Though there were indications of tolerance/resistance in malaria vectors against deltamethrin, which is presently used for IRS in the study area. Nevertheless, timely spray of two rounds of this insecticide with proper dosage and good coverage can significantly bring down the vector population and that will have an epidemiological impact, leading to substantial reduction in malaria incidence. There are such reports where IRS operation undertaken with proper dosage and good coverage, significantly brought down the malaria incidence in areas with prevalence of insecticide resistant An. culicifacies[25],[26],[27].

From the present study, it is evident that topography and rainfall greatly influence the abundance of malaria vectors. The potential environmental and entomological risk factors include long spell of monsoon season with intermittent rainfall, water accumulation in form of ponds, rain and seepage water collections, cemented tanks (for water storage) and prevalence of established malaria vectors like An. culicifacies (species A) and An. stephensi (Type form). In such a situation, microstratification of problematic areas, based on epidemiological data to identify the hot spots in epidemic prone area, and concerted vector control measures in such areas on priority basis assume greater importance to prevent an upsurge in vector population and consequently the malaria incidence. Periodic vector surveillance, timely procurement of insecticide for two rounds of IRS with proper dosage and good coverage, and arranging man power for execution and supervision of spray operations are essential components of vector control measures. Prior information to villagers regarding spray schedule and seeking their cooperation contribute to the success of IRS operation. In addition, the communities may be sensitized through IEC activities for personal protection measures like use of repellents, coils, vaporizers and bednets which will supplement the major intervention tool in decreasing the man vector contact. Though vector control is an integral component of malaria containment programme, the importance of early diagnosis and prompt proper treatment of malaria infected individuals cannot be underrated.

Conflict of interest

The authors declare that they have no competing interests.


  Acknowledgements Top


The authors acknowledge the technical assistance provided by the staff of Vector Biology and Control Division of the National Institute of Malaria Research, New Delhi in carrying out field and laboratory work for this study. The authors sincerely thank the Chief Medical Officer, Nuh CHC and Medical Officer, Ujina PHC for cooperation and providing requisite information. Authors are grateful to the Director, National Institute of Malaria Research for encouragement and providing necessary facilities. The study was conducted with intramural funds of the Institute



 
  References Top

1.
District Profile. Nuh (Haryana) India: Mewat Development Agency, 2017. Available from: www.mda.nicin/mewat-profile (Accessed on February 15, 2017).  Back to cited text no. 1
    
2.
Sharma RS, Lal S, Sharma SN, Joshi RD, Dhillon GP. Malaria outbreak in Mewat region Gurgaon district of Haryana state. J Commun Dis 1997; 29(3): 307-8.  Back to cited text no. 2
    
3.
Raghavendra K, Subbarao SK, Sharma V. An investigation into the recent malaria outbreak in District Gurgaon, Haryana, India. Curr Sci 1997; 73: 766-70.  Back to cited text no. 3
    
4.
Srivastava A, Nagpal BN, Saxena R, Wadhwa T, Mohan S, Siroha GP, et al. Malaria epidemicity of Mewat region, District Gurgaon, Haryana India: A GIS-based study. Curr Sci 2004; 86(9): 1297-303.  Back to cited text no. 4
    
5.
Kumari A, Kant R. Assessment of malaria high risk areas by using RS & GIS technologies in Rohtak and Mewat districts of Haryana. Int J Mosq Res 2016; 3(3): 51-3.  Back to cited text no. 5
    
6.
Kumari A, Kant R, Sharma PK, Chaudhary A. Trend of malaria incidence in Rohtak and Mewat districts of Haryana, India during 2008-2013. Indian J Health Sci Care 2015; 2: 36-40.  Back to cited text no. 6
    
7.
Naz R, Farooqui Mohammad Khalid, Girotra Ruchi, Malik AK. Pattern of malaria infection at tertiary care hospital of Haryana – A hospital based study. Int J Curr Microbiol App Sci 2016; 5(2): 330-7.  Back to cited text no. 7
    
8.
Manual on practical entomology in malaria. Pt II. Methods and techniques. Geneva: World Health Organization 1975; p. 141-7.  Back to cited text no. 8
    
9.
Christopher, SR. The Fauna of British India, including Ceylon and Burma, Diptera. New Delhi: Today and Tomorrow’s Printers and Publishers 1933; p.1-359.  Back to cited text no. 9
    
10.
Nagpal BN, Sharma VP. Indian Anophelines. New Delhi: Oxford IBH 1995; p. 1-416.  Back to cited text no. 10
    
11.
Green CA, Hunt RH. Interpretation of variation in ovarian polytene chromosomes of Anopheles funestus Giles, An. parensis Giles and An. aruni? Genetica 1980; 51: 187-95.  Back to cited text no. 11
    
12.
Subbarao SK. Anopheline species complexes in Southeast Asia. New Delhi: World Health Organization. Regional Office for Southeast Asia 1998; p. 82.  Back to cited text no. 12
    
13.
Subbarao SK, Vasantha K, Adak T, Sharma V, Curtis C. Egg–float ridge number in Anopheles stephensi: Ecological variation and genetic analysis. Med Vet Entomol 1987; 1: 265-71.  Back to cited text no. 13
    
14.
Bray RS, Gill GS, Killick-Kendrick R. Current and possible future technique for the identification of blood meals of vector haematophagous arthropods [Unpublished document, WHO/ MAL/84.1013 (WHO/VBC/84.905)]. Geneva, Switzerland: World Health Organization 1984; p. 1-5.  Back to cited text no. 14
    
15.
Wirtz RA, Burkot TR, Andre RG, Rosenberg R, Collins WE, Roberts DR. Identification of Plasmodium vivax sporozoites in mosquitoes using an enzyme-linked immunosorbent assay. Am J Trop Med Hyg 1985; 34(5): 1048-54.  Back to cited text no. 15
    
16.
Wirtz, RA, Duncan JF, Njeleeanji EK, Schneider I, Brown AE, Oster CN, et al. ELISA method for detecting Plasmodium falciparum circumsporozoite antibody. Bull World Health Organ 1989; 67(5): 535-42.  Back to cited text no. 16
    
17.
Instructions for determining the susceptibility or resistance of adult mosquitoes to organochlorine, organophosphate and carbamate insecticides—Diagnostic test (Unpublished docment, WHO/VBC/81.806). Geneva, Switzerland: World Health Organization 1981 (b); p. 1-7.  Back to cited text no. 17
    
18.
Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. II edn. Geneva, Switzerland: World Health Organization 2013; p. 1-47.  Back to cited text no. 18
    
19.
Kar NP, Kumar A, Singh OP, Carlton J, Nanda N. A review of malaria transmission dynamics in forest ecosystems. Parasit Vectors 2014; 7(1): 265.  Back to cited text no. 19
    
20.
Nanda N, Yadav RS, Subbarao SK, Joshi H, Sharma VP. Studies on Anopheles fluviatilis and Anopheles culicifacies sibling species in relation to malaria in forested hilly and riverine ecosystems in northern Orissa, India. J Am Mosq Control Assoc 2000; 16: 199-205.  Back to cited text no. 20
[PUBMED]    
21.
Rao B, Sweet W, Subba Rao A. Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. J Malar Inst India 1938; 1: 261-6.  Back to cited text no. 21
    
22.
Sharma S, Subbarao SK, Choudhury D, Pandey K. Role of An. culicifacies and An. stephensi in malaria transmission in urban Delhi. Indian J Malariol 1993; 30: 155-68.  Back to cited text no. 22
    
23.
Dash AP, Adak T, Raghavendra K, Singh OP. The biology and control of malaria vectors in India. Curr Sci 2007; 92: 1571-8.  Back to cited text no. 23
    
24.
Aktar N, Nagpal BN, Kapoor N, Srivastava A, Valecha N. Role of An. culicifacies as a vector of malaria in changing ecological scenario of Northeastern states of India. J Vector Borne Dis 2016; 53(3): 264-71.  Back to cited text no. 24
    
25.
Sharma VP, Uprety HC, Nanda Nutan, Raina VK, Parida SK, Gupta VK. Impact of DDT spraying on malaria transmission in villages with resistant An. culicifacies. Indian J Malariol 1982; 19: 5-12.  Back to cited text no. 25
    
26.
Raghavendra K, Velamuri PS, Verma V, Elamathi N, Barik TK, Bhatt RM, et al. Temporo-spatial distribution of insecticide-resistance in Indian malaria vectors in the last quarter-century: Need for regular resistance monitoring and management. J Vector Borne Dis 2017; 54(2): 111-30.  Back to cited text no. 26
    
27.
Sharma SK, Upadhyay AK, Haque Mohammed, Tyagi PK, Kindo K. Impact of changing over of insecticide from synthetic pyrethroids to DDT for indoor residual spray in a malaria endemic area of Orissa, India. Indian J Med Res 2012; 135(3): 382-8.  Back to cited text no. 27
    


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