Triple insecticide resistance in Anopheles culicifacies: a practical impediment for malaria control in Odisha State, India

Use of insecticides for reducing populations of malaria vectors has been the main strategy for control of malaria in India. Currently, 12 insecticides belonging to four chemical classes are recommended by WHO Pesticide Evaluation Scheme (WHOPES) for indoor residual spraying (IRS)1. The current strategy to control malaria vectors in India mainly includes IRS with DDT/synthetic pyrethroids and use of long lasting insecticide nets (LLINs)23. Anopheles culicifacies (Diptera: Culicidae) is the widespread malaria vector species found throughout the rural and peri-urban areas, contributing to about 65 per cent of malaria cases in India4. In the beginning, indoor residual spraying with DDT was used for the control of this species. But, gradually An. culicifacies developed resistance against DDT and it was the first mosquito species to develop resistance against this insecticide5. Soon after the introduction of malathion in the country in 1969, especially in DDT resistant areas, double resistance was reported in An. culicifacies in Gujarat and Maharashtra States in 19776. In 1990s deltamethrin was introduced in the country for indoor residual spraying and treatment of mosquito nets. Consequently, reduced susceptibility to deltamethrin in An. culicifacies was reported for the first time from Ramnathapuram district, Tamil Nadu in 20027.

Studies have been carried out on the susceptibility status of An. culicifacies in different parts of Odisha State8. The studies carried out during 2004 in eight districts of the State reported that An. culicifacies was resistant to DDT in all the eight districts, to malathion in four districts (Mayurbhanj, Bolangir, Nuapada and Kalahandi) and showed a tendency of developing multiple resistance to DDT, malathion and deltamethrin in three districts (Bolangir, Nuapada and Kalahandi)9. An entomological study was conducted in ten southern districts of the State when LLINs were just distributed and it showed that in eight districts, the response of An. culicifacies to deltamethrin was under ‘verification required’ category indicating its tendency towards development of resistance to deltamethrin8. In view of this indication of tolerance of this species to the synthetic pyrethroid in the event of four to five years of field use of LLINs in the districts, a study was undertaken in five of the ten southern districts of Odisha State to verify the susceptibility/resistance status of An. culicifacies to deltamethrin and also to the other two commonly used insecticides i.e. DDT and malathion.

Material & Methods

The study was conducted in five southern districts of Odisha State viz. Rayagada, Nowrangpur, Kalahandi, Malkangiri and Koraput. An. culicifacies was tested against the diagnostic dosage of DDT, malathion and deltamethrin during April to June 2014. Dry summer (March-June), wet rainy (July-October) and dry winter (November-February) are the three prevailing seasons. The districts have been hyperendemic for malaria since many years8. Plasmodium falciparum is the predominant species, with a proportion of >90 per cent of the total malaria cases810. Anopheles fluviatilis and An. culicifacies are the two malaria vector species prevalent in the five districts811. The villages in the districts are situated on foothills or plains and are characterized by presence of perennial streams, rivulets, ponds and paddy fields, which are the major breeding habitats of the two vector species. Majority of the villagers live in huts made of mud walls with either thatched or tiled roofs. The walls of the hut are usually of seven feet in height, with a gap of 2 to 3 feet between the side walls and roof. The cattle sheds are kept close by human dwellings. Human dwellings are sprayed with two rounds of DDT or synthetic pyrethroids every year. In addition, long lasting synthetic pyrethroid (deltamethrin) treated mosquito nets (LLINs) have been distributed in these districts since 2009 in a phased manner.

One community health centre (CHC) from each of the five districts was randomly selected. In each CHC, three villages, representing the respective CHC with reference to the density of An. culicifacies, were randomly selected for collection of An. culicifacies to determine its susceptibility/resistance status8.

Mosquito collections and susceptibility test: Female Anopheles mosquitoes were collected from cattle sheds and human dwellings in the study villages during early morning hours using mouth aspirator and flash light. The collected mosquitoes were transported to the laboratory at Vector Control Research Centre, Field Station, Koraput, Odisha, in a mosquito cage (one cubic foot) covered with a wet towel, identified to species based on morphological characters using a standard key12 and separated according to their gonotrophic conditions. WHO kits were used to conduct the susceptibility test3. The temperature and relative humidity (RH) in the laboratory was maintained at 25 ± 2 °C and 70-85 per cent RH. Papers impregnated with DDT 4 per cent, malathion 5 per cent and deltamethrin 0.05 per cent were obtained from the University Sains Malaysia, Penang, Malaysia. The fully fed mosquitoes were exposed to the diagnostic dosage of the insecticides for one hour in 5 to 6 replicates, each replicate with 15 to 25 mosquitoes. Parallel controls were maintained for comparison (i.e. 2 to 3 replicates each with 15 to 25 mosquitoes)3. Number knocked down was recorded after one hour of exposure and after the exposure the mosquitoes were maintained in holding tubes with access to glucose food for 24 h at the same temperature and relative humidity. Mortality was scored after 24 h of holding. Since the control mortality in all the tests remained below 5 per cent, the test mortality was not corrected using Abbott's formula13. According to the WHO criteria3, a corrected mortality of >98 per cent is ‘susceptible’, <90 per cent is ‘resistant’ and 90-98 per cent is ‘verification required’.

Results & Discussion

A total of 135, 130 and 131 female An. culicifacies mosquitoes in Rayagada, 105, 110 and 113 in Nawarangapur, 105, 111 and 131 in Kalahandi, 111, 105 and 119 in Malkangiri and 111,111 and 112 in Koraput district were exposed to DDT 4 per cent, malathion 5 per cent and deltamethrin 0.05 per cent, respectively. The test mortality of this vector species against the three insecticides are given in the Table. The test mortality ranged between 11.4 and 15.3 per cent against DDT 4 per cent, 60.4 and 76.2 per cent against malathion 5 per cent, and 72.6 and 84.0 per cent against deltamethrin 0.05 per cent in these districts. The results showed that An. culicifacies was resistant to DDT, malathion and deltamethrin in all the five districts.

Table Response of An. culicifacies to DDT, malathion and deltamethrin in the five southern districts of Odisha State

Under the modified plan of operation implemented in 1977, the study districts continued to receive DDT indoor residual spraying, since annual parasite incidence (API) was >2 in all the districts14. From 2001 onwards, DDT was replaced with synthetic pyrethroids (deltamethrin/alphacypermethrin/lambdacyhalothrin)for indoor residual spraying in five of the 11 CHCs in Rayagada district and in seven of the 13 CHCs (where API >10) in Kalahandi district. (State National Vector Borne Diseases Control Programme Office, Bhubaneshwar, personal communication). Insecticide treated mosquito nets (ITMNs) were introduced during 2001 to 2008 in some of the CHCs of the five districts. In addition, long lasting synthetic pyrethroid (deltamethrin) treated mosquito nets (LLINs) were distributed in these districts from 2009 to 2013 in a phased manner.

An. culicifacies was reported to be resistant to DDT but susceptible to malathion and deltamethrin15 during 1995 in Koraput district. Subsequently, during 2010-2011 the response of this species to deltamethrin in Rayagada, Kalahandi and Malkangiri districts was found to be resistant, in Nawarangpur district was under the ‘verification required’ category and in Koraput district was susceptible8 as per the guidelines of WHO, 20133. Resistance to DDT and malathion in An. culicifacies was confirmed during the current study. In addition, the current study confirmed the development of resistance in this species to deltamethrin in the five southern districts of the State. An. fluviatilis, the other malaria vector in the study area was susceptible to DDT, malathion and deltamethrin815. However, the susceptibility status of An. fluviatilis could not be assessed during the current study due to non-availability of adequate number of this species from the study area.

During the early part of 20^th century, indoor residual spraying of insecticides, particularly DDT, was the key component of malaria control and was responsible for the spectacular reduction in malaria incidence16. Over time and with widespread use in agriculture, mosquito resistance to DDT had emerged17. Between 1961 and 1966, the malaria rate in India increased three-fold; DDT resistance was a partial cause for this1718. Several studies have been conducted on vector susceptibility to insecticides in different parts of the country1819. Resistance to DDT in An. culicifacies is widespread in the country202122 and to malathion in the States of Maharashtra, Gujarat, Tamil Nadu and Uttar Pradesh6232425. There are a few reports of decreased susceptibility in this vector to synthetic pyrethroids7926. This species has developed resistance to DDT in 286 districts, to malathion in 81 and to pyrethroids in two districts in India27. A recent study conducted in four States (Andhra Pradesh, Odisha, Jharkhand and West Bengal) of India showed that An. culicifacies was resistant to DDT in 32 districts and to malathion in 14 districts, under verification required category in 10 districts and susceptible in eight districts. To deltamethrin this species was resistant in four districts, under verification required category in 11 districts and susceptible in 17 districts28.

One of the limitations of the study was that parallel data could not be generated for An. fluviatilis due to its non-availability in adequate number. Considering operational implication, generation of data was not done up to sibling species level in An. culicifacies, which might be another limitation. The spread of insecticide resistance, especially pyrethroid resistance as indicated by the current study and also the studies conducted in other parts of the country, is a major threat for the vector control programmes, as it is being extensively used for IRS and impregnation of bed nets in India. In this context, preservation of pyrethroid susceptibility in target vector population should be the key priority in the choice of vector control methods at this hour. Insecticide resistance management needs to be considered for vector control, including the selection of insecticides for IRS. Using the same insecticide for multiple successive IRS cycles may not be recommended; it is preferable to use a system of rotation with different groups of insecticides including carbamates in the area where An. culicifacies is resistant to deltamethrin29. Rotations should start with the insecticides to which there is the lowest frequency of resistance. In the areas of high coverage with LLINs, pyrethroids may not be a good option for IRS, as this will add to selection pressure. Further, using nets impregnated with a synthetic pyrethroid together with a synergist would be a better option against pyrethroid resistant malaria vectors. Mixture nets such as this may have application against resistant mosquitoes, particularly those whose resistance is based on oxidative metabolism30. Since, monitoring insecticide resistance is a necessary element of the implementation of insecticide-based vector control interventions, regular monitoring of susceptibility/resistance status of the malaria vectors to commonly used insecticides, at least once a year or preferably every six months3 is warranted particularly in An. culicifacies predominant areas to strengthen the evidence base for the effectiveness of ongoing vector control interventions.