A study of malaria vector surveillance as part of the Malaria Elimination Demonstration Project in Mandla, Madhya Pradesh

Understanding of malaria vector density, distribution, insecticide resistance, vector incrimination, infection status, and identification of sibling species are some of the essential components of vector control measures for achieving malaria elimination goals. As part of the malaria elimination demonstration project, entomological surveillance was carried out from October 2017 to October 2019 by collecting indoor resting mosquitoes using hand catch method. Susceptibility test was done for determining the insecticide resistance status of vector mosquito Anopheles culicifacies using standard protocols by the World Health Organization. The cone bioassay method was used for determining the efficacy and quality of insecticide sprayed. Mosquitoes collected from different ecotypes were identified and processed for parasite identification, vector incrimination and sibling species determination. The two known malaria vector species (Anopheles culicifacies and Anopheles fluviatilis) were found in the study area, which have been previously reported in this and adjoining areas of the State of Madhya Pradesh. The prevalence of An. culicifacies was significantly higher in all study villages with peak in July while lowest number was recorded in May. Proportion of vector density was observed to be low in foothill terrains. The other anopheline species viz, Anopheles subpictus, Anopheles annularis, Anopheles vagus, Anopheles splendidus, Anopheles pallidus, Anopheles nigerrimus and Anopheles barbirostris were also recorded in the study area, although their prevalence was significantly less compared to the An. culicifacies. In 2017, An. culicifacies was found to be resistant to dichloro-diphenyl-trichloroethane (DDT) and malathion, with possible resistance to alphacypermethrin and susceptible to deltamethrin. However, in 2019, the species was found to be resistant to alphacypermethrin, DDT, malathion, with possible resistance to deltamethrin. The bioassays revealed 82 to > 98% corrected % mortality of An. culicifacies on day-one post-spraying and 35 to 62% on follow-up day-30. Anopheles culicifacies sibling species C was most prevalent (38.5%) followed by A/D and E while B was least pre-dominant (11.9%). Anopheles fluviatilis sibling species T was most prevalent (74.6%) followed by U (25.4%) while species S was not recorded. One An.culicifacies (sibling species C) was found positive for Plasmodium falciparum by PCR tests in the mosquitoes sampled from the test areas. Based on the nine entomologic investigations conducted between 2017–2019, it was concluded that An. culicifacies was present throughout the year while An. fluviatilis had seasonal presence in the study areas. Anopheles culicifacies was resistant to alphacypermethrin and emerging resistance to deltamethrin was observed in this area. Anopheles culicifacies was confirmed as the malaria vector. This type of information on indigenous malaria vectors and insecticide resistance is important in implementation of vector control through indoor residual spraying (IRS) and use of insecticide-impregnated bed nets for achieving the malaria elimination goals.


Background
Malaria is a global public health problem with most of the morbidity and mortality in sub-Saharan Africa. Outside the African region, India has the highest burden of disease amongst the South-East Asian (SEAR) countries. It is a parasitic infectious disease transmitted by female Anopheles mosquitoes. More than one billion people are at risk of malaria [1] and despite of the significant reduction in malaria cases achieved in 2018, over 228 million malaria cases and 405,000 malaria-attributable deaths occurred worldwide in 2018 [2].
The In 2016, India's National Vector Borne Disease Control Programme (NVBDCP) launched a national frame-work to eliminate malaria by 2030 [3]. To complement the NVBDCP efforts in malaria elimination, the Malaria Elimination Demonstration Project (MEDP) was launched in 2017 in the tribal district of Mandla. This project is being undertaken in a public-privatepartnership (PPP) model between the Government of Madhya Pradesh, Foundation for Disease Elimination and Control of India (FDEC), established by Sun Pharmaceuticals Industries limited, and the Indian Council of Medical Research (ICMR) through ICMR-National Institute of Research in Tribal Health (ICMR-NIRTH). The main objective of the project is to demonstrate that malaria elimination is feasible using proven and fieldtested surveillance, case management and vector control strategies.
Government of India (GOI) introduced Indoor Residual Spraying and insecticide impregnated mosquito nets as the main intervention strategy for malaria vector control. Subsequently, it was observed that An. culicifacies have developed resistance to Dichloro-diphenyl-trichloroethane (DDT) [22,23], deltamethrin [24], Benzene Hexachloride (BHC) [25] and even to pyrethroids [26]. This entomologic surveillance study was conducted as part of the Malaria Elimination Demonstration Project (MEDP) project, with a goal to identify the vector species and to ascertain relative abundance, insecticide efficacy, role in malaria transmission and their behaviour.

Methods
Study area description: Mandla, a tribal district is in the centre of Madhya Pradesh marked with valleys, hills and thick dense forest at altitude of 450-950 M, (23° N latitude, 80° 10′ E longitude) is the site of the project. Narmada River flows through the district which provides the breeding sites for anophelines. The area of the district is about 8771 km 2 , consisting 9 blocks ( Fig. 1) with total 1.168 million population (projection for 2016 based on 2011 census data). The weather is categorized as monsoon (June-August), post monsoon (September-November), winter (December-February) and summer (March-May).
As per NVBDCP data, the malaria burden in 2015 was high (API: 3.52), while a significant reduction was observed in 2019 (API: 0.17), (Table 1). Since 2017, the district's malaria programme used alphacypermethrin 5% in IRS twice a year in areas with Annual Parasite Incidence (API) of 1 to 4.99. The LLINs were distributed in areas with API of 5 and above in 2017, and subsequently in areas with API of more than 2 in 2019. Neither IRS nor LLINs were provided in areas which had less than 1 API. In these areas, community was informed to use the personal protection measures, such as mosquito coils, anti-mosquito ointments. IRS and LLIN distributions were done as part of routine government vector control Conclusion: Based on the nine entomologic investigations conducted between 2017-2019, it was concluded that An. culicifacies was present throughout the year while An. fluviatilis had seasonal presence in the study areas. Anopheles culicifacies was resistant to alphacypermethrin and emerging resistance to deltamethrin was observed in this area. Anopheles culicifacies was confirmed as the malaria vector. This type of information on indigenous malaria vectors and insecticide resistance is important in implementation of vector control through indoor residual spraying (IRS) and use of insecticide-impregnated bed nets for achieving the malaria elimination goals. activity through the District Malaria Office with supportive supervision provided by the MEDP staff.
In this study, villages for entomological surveillance were selected in three category areas on the basis of API (2015) i.e. < 1(category A), 1.0 to 4.99 (category B), > 5 (category C). Three villages in each category encompassing different terrains viz., plain, forest and foothill were selected for mosquito sampling. The study was carried out between October 2017 to October 2019 by visiting study area once in every 3 months to cover all the seasons.

Mosquito sampling
Anopheline mosquitoes resting indoors and outdoors were collected during every field visit from all nine study villages (3 villages in each API category). The mosquito specimens were collected by a team of four insect collectors with a flashlight and mouth aspirators during early morning (0600-0800 h) from 4 human dwellings and 4 cattle sheds located in different parts of the villages [27]. The same team was deployed to catch mosquitoes in each study village. Mosquitoes collected from different localities were kept in separate test tubes and labeled with  location, village name, date and time of collection and brought to the field laboratory for identification and further processing for vector incrimination and sibling species determination.

Insecticide resistance status
Insecticide resistance status of An. culicifacies (major malaria vector) was ascertained during the study period. Susceptibility tests on adult An. culicifacies were conducted following standard WHO procedures [28]. Wild-caught mosquitoes, preferably blood-fed female mosquitoes, were collected from different resting sites (indoors-human dwellings/cattle sheds) and [27] and identified based on morphological characters [29]. The collected mosquitoes were brought by temperature controlled vehicle to the laboratory (about 10-20 km away from field) for testing in cloth cages wrapped with wet towel to maintain humidity. Female mosquitoes were exposed in replicates to the WHO impregnated papers with specified discriminating dosages of the insecticides (DDT: 4%, malathion: 5%, deltamethrin: 0.05% and alphacypermethrin 0.05%), respective insecticide controls for comparison (two replicates) for one hour and mortality was recorded after 24-h holding. The tests were repeated within 2 or 3 days in different villages and different terrains. Cartons with wet towels at the bottom were used to conduct the tests to maintain the ambient temperature of 25 ± 2 °C and RH of 80 ± 10% in the field laboratory [30]. Mortality after 24 h of holding period was recorded [31]. Percent mortality was calculated separately for the test and control replicates using the formula-If the mortality in control replicates is between 5 and 20%, the test mortality was corrected with the control mortality using Abbott's formula [32]. In case, the mortality in the controls exceeds 20%, the test was discarded.
According to the WHO criteria [31], if the mortality of mosquito species on exposure to the diagnostic dosage of a given insecticide is 98 to 100%, it is designated as 'susceptible' , if mortality is < 90%, it is designated as 'confirmed resistance' , and possible resistance if the mortality is between 90 and 98%. Cone bioassays were carried out during IRS months (October 2017, July, October 2018 and July and October in 2019) to assess the efficacy of insecticide used in IRS programme and also to assess the quality of IRS on different sprayed surfaces in the villages. The tests were done in 3 villages of 3 CHCs each on the basis of the availability of houses sprayed on day-one and on or after day-30 of the spray. The houses having different sprayed surfaces were selected for cone bioassays. Two unsprayed houses were selected for control. The bioassays were done on day-one and day-30 post-spraying with WHO cones [27,33] using field collected An. culicifacies from unsprayed area due to their availability in sufficient numbers. A total of 10 fed-female mosquitoes were exposed to sprayed surfaces for 30 min, and kept in paper cups covered with net. These mosquitoes were given 10% glucose solution soaked in cotton wool for the maintenance of 25 ± 2 °C temperature and 80 ± 10% moisture. Percent mortalities were calculated from the total number of live and dead mosquitoes by the Abbott's formula [32].

Processing for vector incrimination
Mosquito species of An. culicifacies and An. fluviatilis collected during the study period were separated into head thorax and abdomen parts and dried. These mosquitoes were kept in 1.5 ml micro tubes and sent to laboratory for molecular analysis of parasite using Polymerase Chain Reaction (PCR) method. They were categorized viz., species and habitats with respect to study villages. Genomic DNA was extracted from mosquito heads and thoraces by the method described by Coen et al. [34]. Pools of genomic DNA from 10 samples were prepared and Nested PCR was performed for detection of malaria parasites from the extracted DNA with the primers described by Snounou et al. [35].
Sibling species identification of An. culicifacies and An. fluviatilis mosquitoes was analysed using allele-specific PCR. The identification of An. culicifacies was done as reported by Singh et al. [36] and Goswami et al. [37]. For An. fluviatilis, it was done as reported by Singh et al. [38]. PCR reaction mixture was prepared using 200 μM of each dNTP, 1.5 mM MgCl2, 1 × PCR buffer and 1 unit of taq DNA polymerase. The cycling conditions included initial denaturation at 95 °C for 5 min, followed by 35 cycles each of denaturation for 30 s at 95 °C, annealing for 30 s at 50 °C and extension for 1 min at 72 °C, and then final extension at 72 °C for 7 min.

Ethical clearance
Ethical clearance was obtained from the Institutional Ethics committee (IEC) of ICMR-National Institute of Research in Tribal Health (ICMR-NIRTH) on 16th March 2017 bearing reference no. 201701/10. A verbal informed consent for mosquito collection was taken from the residents of the households.

Data management and analysis
The data was entered in data entry software designed on CS-Pro 7.0 platform and data analysis were done with Statistical Package for Social Sciences (SPSS) v20.0 by IBM.
No consistent pattern was seen in the number of anopheline mosquitoes collected throughout the year and month-to-month variation in the number of mosquitoes was common (Table 3). Anopheles culicifacies was found to be the predominant species followed by An. annularis and An. subpictus during ( Table 2) during 156man hours efforts. Outdoor resting mosquito collection were also undertaken, but we were unable to collect any mosquito and therefore, after one year of the study, the outdoor collection was discontinued.
The per man per hour density of An. culicifacies was lowest (3.8 ± 6.7) in May 2019 and highest (22.2 ± 1.93) in July 2018 which is statistically significant (p < 0.001). The density of An. fluviatilis, the other known vector of malaria, was highest in February 2018 (1.5), though small numbers were caught in every visit except in July 2018, May 2019 and July 2019. The overall An. culicifacies density in 2017 was 10.75 ± 1.6, which slightly increased in 2018 (13.4 ± 7.0) and declined significantly in the subsequent year 2019 (9.87 ± 7.97, p < 0.01). Anopheles fluviatilis density was 0.17, 1.03 and 0.9 in 2017, 2018 and 2019, respectively.
The study found that 9 anopheline species were present in all categories (category A, category B, category C) areas ( Table 2). The average per man hour anopheline density was slightly higher in category A (22.5). Similarly, the vector proportion was also found higher in villages of category A (63.4% An. culicifacies and 1.9% An. fluviatilis), as compared to villages of category B (An. culicifacies 53.3 and An. fluviatilis 0.9%) and category C (An. culicifacies 49.7% and An. fluviatilis 4.1%). Statistically, the proportion of An. culicifacies was found significantly higher in category A (63.4%) when compared to category B (53.3%, p < 0.001) and category C (49.7%, p < 0.001). However, the difference between the category B and C villages was not statistically significant.
Overall, the proportion An. culicifacies and An. fluviatilis varied significantly from category A to category B and C (chi sq for linear trend = 30.74; p < 0.0001, Table 3) with significant variation in vector density among three category villages (F 2,153 = 5.24; p < 0.01). The monthwise vector density was almost equal in all three areas throughout the year, except in January 2019 and July 2019 ( Table 3).
The ecotype analysis revealed that the per man-hour density of anophelines and An. culicifacies was found almost equal in villages of plains (21.1 and 11.3) and forest (23.1 and 12.74). However, it was slightly lower in foothill villages (17.7 and 10.6) (Fig. 2). The highest per man hour density of anophelines was found in the monsoon season (35.4) followed by post monsoon (24.4), winter (14.1) and summer (7.6) (Fig. 3).
The similar trend was found in An. culicifacies (21.9 in monsoon, 10.9 in post monsoon, 8.9 in winter and 4.8 in summer). However, An. fluviatilis slightly was higher in winter (1.0) as compared to post monsoon (0.7).
Anopheles fluviatilis was not found in monsoon and summer season. For mosquitoes caught resting, it was also observed that anopheline vector density was higher in cattle shed   20.7) as compared to the human dwelling (9.9) ( Table 4). Most of the An. culicifacies (77.5%) and An. fluviatilis (73.2%) were caught from outside of the houses i.e., in cattle sheds. This trend was almost similar in all category of villages.

Insecticide resistance status of Anopheles culicifacies
The susceptibility tests were carried out in the month of October 2017 and October 2019. Anopheles culicifacies specimens were found resistant to DDT and malathion with mortality 24.8 to 28.0% and 50.5 to 84.0%, respectively. Possible resistance to alphacypermethrin was observed in October 2017 with 95% mortality. In the same year, susceptible to deltamethrin with 98.3% mortality was observed. However, in 2019, An. culicifacies specimens were found to be resistant to alphacypermethrin (82.5% mortality) with possible resistance to deltamethrin with 95.6% mortality (Table 5).

Cone bioassays
The bioassays carried out on one-day post spraying revealed 98.7%, 96.7%, 87.3%, 82.1% and 81.2% average corrected % mortality of An. culicifacies in the month of October 2017, July and October 2018 and in July and October 2019, respectively (Fig. 4). The mortality data on day 30 after spraying was 40.6%, 61.7%, 44.4%, 41.4% and 35.0% for the same months/years.
Vector incrimination by PCR: A total of 1806 An. culicifacies (743 from category A, 590 from villages of category B, and 473 from category C) and 71 An. fluviatilis (22, 10, and 39 from villages of category A, B and C), collected from both the human dwellings and cattle shed were processed for molecular detection of malaria parasites. One An.culicifacies specimen collected in the month of October 2019 from category A village was found positive for Plasmodium falciparum malaria parasites by ribosomal DNA PCR tests.  Sibling species identification: A total of 1,806 An. culicifacies were collected during the study period from different category villages. They were tested for sibling species determination. The PCR revealed the AD vs BCE group (Fig. 5a). The proportion of An. culicifacies C was the highest (38.5%) followed by A/D and E, and lowest was subspecies B (11.9%) (Fig. 5b, Table 6). The sibling species distribution was found almost similar in each area. However, An. culicifacies B was slightly higher (13.3%) in villages of category A than other two areas, whereas, An. culicifacies C was slightly higher (42.1%) in villages of category C than other two areas. Anopheles culicifacies A/D and E were found equally distributed in all the three areas. The P. falciparum positive, one An. culicifacies specimen was identified as sibling species C.
The terrain wise data revealed that all sibling species were present in all three terrains viz. plain, forest and foothill. Season-wise observations revealed that in each category of villages, all sibling species of An. culicifacies were present in all seasons. More number of B (13.9%), C (35.4%), E (25.8%) and A/D (24.9%) were found during monsoon/rainy season than the summers and winters. Out of 71 specimens of An. fluviatilis 53 (74.6%) were of species T (Fig. 5c, Table 6). The highest number (34) of T subspecies were detected from villages of category C. Season wise data revealed that in winters, 31 (93.9%) were of T, whereas in summers and rains, the proportions of T and U were almost the same (Table 6).

Discussion
Malaria Elimination Demonstration Project (MEDP) is being undertaken in the Mandla district to demonstrate that malaria elimination is feasible using the existing case management and vector control tools and strategies. In the MEDP project, vector control is accomplished through the use of indoor residual spray and long-lasting insecticide treated nets and case management is accomplished by active surveillance combined with rapid diagnosis and prompt treatment [39].
This study has revealed that An. culicifacies and An. fluviatilis are present in the study area, which is in agreement with prior studies [9][10][11][12] in this region. The indoor resting densities of An. culicifacies was found throughout the year [9], possibly due to housing structures of tribal settlements. These structural characteristics of the houses, including cattle shed allow for easy mosquito ingress and egress and accordingly maintaining high densities.
However, in this study, less number of An. culicifacies were found inside the houses (human dwellings) as compared to the cattle sheds. This might be due to use of LLIN and application of IRS inside the houses. Prasad et al. [40] also reported a preference of mosquitoes to rest mainly in cattle sheds. With the increased distribution and enhanced use of LLIN in many areas, changes in vector behaviour from indoor resting to outdoor resting and from human dwelling to cattle sheds has also been observed in Odisha [41,42]. The behaviour change was observed from human dwellings to cattle sheds in the  study area. The An. culicifacies species transmit the disease mainly in rainy season i.e. July-October [43], which requires two rounds of IRS to be done to break malaria transmission cycle during the same period.
A prior study in the Balaghat district of Madhya Pradesh, which is adjacent to the Mandla district, has revealed that the insecticidal effect declined in one month after spraying of alphacypermethrin, which could be attributed to many factors such as improper spray, type of spray pumps, quality of insecticide, untrained man power and supervision [10]. In the present study, the results of the cone bioassay test also showed lower mortality of An. culicifacies one-month post spray during October 2017. Using the lesson learned from the Balaghat study [10] and present study observations in 2017, project implemented strict supervision program of the IRS campaigns in July 2018 using lessons learned from previous studies. Through the use of corrective protocols in IRS, study revealed a significant increase in mortality of An. culicifacies, which was remarkable, despite the observation of possible resistance against the used insecticides.
Subsequently in 2019, mortality of An. culicifacies declined in the study, which may be due to emergence of resistance against the alphacypermethrin insecticide. There is a need for a longitudinal entomologic study to get a full picture of the resistance pattern in the study area. The finding from this study that effective supervision of quality spray improves outcomes of spray further informs us that proper and well-supervised spray programmes would extend the life of insecticides and delay the emergence of resistance. Insecticide susceptibility status against the An. fluviatilis was not determined due to very low density in the study area for inadequate number of specimens. The lower vector density observed in the category C villages may be due to the impact of LLIN, because LLIN were introduced in these villages during the same time period.
Anopheles culicifacies have species complex of five sub-species with specific role in malaria transmission. In the present study, four species complex members of An. culicifacies species (B, C and E and A/D group) were found in the study area, of which C constituted about 38.5% in different collections followed by A/D, E and B. This observation is similar to the results of previous studies as the sub-species C considered efficient vector followed by A/D while sub-species B have least role in malaria transmission [6,11]. The present study also found one specimen of An. culicifacies (sub-species C) infected with P. falciparum malaria parasites. The low number of sporozoite positivity is probably because of significant reduction in malaria cases in the study area, which would have significantly reduced the number of gametocytaemia positive cases thus not enabling infection of mosquitoes.
The sibling species T of An. fluviatilis was prevalent (74.6%) in the study area which is almost similar to the earlier studies carried out in Madhya Pradesh [11], where 99% An. fluviatilis were identified as T. In a study published in 2015, An. culicifacies C was found sporozoite positive along with An. fluviatilis T, which was previously known as non-malaria vector [11]. In Mandla, An. culicifacies E and An. fluviatilis T were not found in the earlier studies [6], while An. culicifacies E is very efficient malaria vector in India and also globally [16,44,45].

Conclusion
Malaria vector control and elimination requires detailed knowledge of local vector species and their susceptibility to insecticides, as well as information on vector and human behaviours that may allow mosquitoes to avoid contact. Periodic collection of such data during elimination programmes is essential to inform vector control strategies and assess impact on malaria transmission.
The current study provides information on entomological data collected during nine investigations which may be helpful in national malaria control and elimination programme.