Susceptibility status of malaria vectors to insecticides commonly used for malaria control in Tanzania

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1 Tropical Medicine and International Health doi: /j x volume 17 no 6 pp june 2012 Susceptibility status of malaria vectors to insecticides commonly used for malaria control in Tanzania Bilali Kabula 1,2,3,4, Patrick Tungu 1, Johnson Matowo 2, Jovin Kitau 2, Clement Mweya 3, Basiliana Emidi 3, Denis Masue 1, Calvin Sindato 5, Robert Malima 1, Jubilate Minja 6, Shandala Msangi 7, Ritha Njau 4, Franklin Mosha 2, Stephen Magesa 1 and William Kisinza 1 1 National Institute for Medical Research, Amani Research Centre, Muheza, Tanzania 2 Kilimanjaro Christian Medical College of Tumaini University, Moshi, Tanzania 3 National Institute for Medical Research, Tukuyu Research Centre, Tukuyu, Tanzania 4 WHO Tanzania, Dar es Salaam, Tanzania 5 National Institute for Medical Research, Tabora Research Centre, Tabora, Tanzania 6 National Malaria Control Programme, Ministry of Health and Social Welfare, Dar es Salaam, Tanzania 7 Tropical Pesticides Research Institute, Arusha, Tanzania Abstract objective The aim of the study was to monitor the insecticide susceptibility status of malaria vectors in 12 sentinel districts of Tanzania. methods WHO standard methods were used to detect knock-down and mortality in the wild female Anopheles mosquitoes collected in sentinel districts. The WHO diagnostic doses of 0.05% deltamethrin, 0.05% lambdacyhalothrin, 0.75% permethrin and 4% DDT were used. results The major malaria vectors in Tanzania, Anopheles gambiae s.l., were susceptible (mortality rate of %) to permethrin, deltamethrin, lambdacyhalothrin and DDT in most of the surveyed sites. However, some sites recorded marginal susceptibility (mortality rate of 80 97%); Ilala showed resistance to DDT (mortality rate of 65% [, 54 74]), and Moshi showed resistance to lambdacyhalothrin (mortality rate of 73% [, 69 76]) and permethrin (mortality rate of 77% [95% CI, 73 80]). conclusions The sustained susceptibility of malaria vectors to pyrethroid in Tanzania is encouraging for successful malaria control with Insecticide-treated nets and IRS. However, the emergence of focal points with insecticide resistance is alarming. Continued monitoring is essential to ensure early containment of resistance, particularly in areas that recorded resistance or marginal susceptibility and those with heavy agricultural and public health use of insecticides. keywords malaria vectors, resistance, insecticides, susceptibility, Tanzania Introduction Vector control is a major component of the global strategy for malaria control, which aims to prevent parasite transmission mainly through interventions targeting adult anopheline vectors (WHO 2005). Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are the cornerstone of malaria control programmes (WHO 2008). High coverage with either of these interventions can result in a dramatic reduction in malaria-associated morbidity and mortality (Lengeler 2004; Pluess et al. 2010). The success of these tools has contributed towards the optimism that elimination of malaria as a public health problem in the African continent is a feasible objective (Roberts & Enserink 2007). However, the development of insecticide resistance to major malaria vectors has compromised these mosquito control efforts (Ranson et al. 2011). Successful implementation of these vector control strategies requires sound knowledge of vectors distributions, biology and changing trends on susceptibility status to available insecticide compounds. Malaria vector control is currently highly dependent on a single class of insecticides, the pyrethroids. These insecticides are the only class approved for use on insecticide-treated netting owing to their relatively low human toxicity, excito-repellent properties, rapid rate of knock-down and killing effects (Zaim et al. 2000), and are being increasingly deployed in indoor residual spraying 742 ª 2012 Blackwell Publishing Ltd

2 (IRS) programmes in Africa (Ranson et al. 2011). Pyrethroids are also widely used in the control of agricultural pests worldwide (Santolamazza et al. 2008). The extensive use of pyrethroids has increased the selection pressure on the major malaria vectors, which have inevitably developed resistance. In Kenya, use of permethrin-treated nets was associated with reduced permethrin susceptibility in A. gambiae s.s. (Vulule et al. 1994). Agricultural use of pyrethroids, primarily in cotton-growing areas, is thought to have contributed to selection for resistance in A. gambiae s.s. in Cote d Ivoire, Benin and Burkina Faso (Akogbeto & Yakoubou 1999; Chandre et al. 1999; Diabate et al. 2002). Comprehensive knowledge of the factors underlying resistance is needed for the implementation of efficient vector control programmes including resistance management strategies. This raises the need for countrywide and regular surveys for monitoring the insecticide susceptibility status of major vectors, detecting resistance genes and assessing their impact on vector control activities (Kelly-Hope et al. 2008). Anopheline mosquitoes exhibit two major mechanisms of pyrethroid resistance: increased insecticide detoxification (metabolic resistance) and target site insensitivity (Hemingway & Ranson 2000). Metabolic resistance to pyrethroids is usually associated with increased cytochrome P450-dependent monooxygenases (P450) (Müller et al. 2008) and esterases activity (Vulule et al. 1999) while target site insensitivity results from point mutations in the voltagegated sodium channel gene (Martinez-Torres et al. 1998; Ranson et al. 2000). The latter mechanism, also termed kdr (knock-down resistance), induces cross-resistance to DDT (Brooke et al. 1999). Two alternative kdr mutations have been described in A. gambiae s.l. populations, resulting in either a leucine-phenylalanine (L1014F) or a leucine-serine (L1014S) mutation at amino acid position 1014 of the sodium channel (Martinez-Torres et al. 1998; Ranson et al. 2000). The former is widely distributed in the M and S forms of A. gambiae in West Africa, and the latter, originally described from Kenya, has been found in the S form of A. gambiae from Central Africa (Santolamazza et al. 2008). Metabolic and target site resistances (alone or occurring together) of Anopheles arabiensis have recently been reported in north-eastern Tanzania (Kulkarni et al. 2006; Matowo et al. 2010). Similarly, the target site resistance has been reported in Uganda (Verhaeghen et al. 2006). Detection of the L1014F kdr mutation at low frequency in A. arabiensis in Lower Moshi, north-eastern Tanzania (Kulkarni et al. 2006) and the same L1014F kdr mutation in A. gambiae in Uganda (Verhaeghen et al. 2006) suggests the kdr alleles has spread across East Africa. DDT resistance can be due either to a specific detoxification mechanism (glutathione-s-transferase) or to a nerve insensitivity resulting from a modification of the target site (sodium channel). The latter, governed by the kdr gene, reduces both the knock-down and lethal effects of DDT. In West Africa, it induces a cross-resistance to pyrethroids, which also depends on kdr mutation (Martinez-Torres et al. 1998). In Zanzibar, however, DDT resistance induced by glutathione-s-transferase did not confer cross-resistance to pyrethroids (Prapanthadara et al. 1995). Pyrethroid insecticides are commonly used in Tanzania for agricultural purposes and for public health including malaria vector control through large-scale ITN LLINs distribution and IRS programmes. This study was carried out in 2009 and 2010 to monitor the insecticide susceptibility status of mosquito populations in 12 sentinel districts of Tanzania where ITNs LLINs coverage is high, where an IRS campaign is underway or where there is a relatively high use of pyrethroid insecticides for agricultural purposes. Mosquitoes belonging to the A. gambiae complex were the only malaria vectors included in the analysis. Materials and methods Study sites The study was carried out in 12 selected sentinel districts from different ecological zones of Tanzania in Coastal savannah, at m asl with temperatures ranging between 24 and 35 C and average annual rainfall of mm, was represented by Muheza and Ilala. Highlands with temperature range of C and average annual rainfall between 800 and 2000 mm were represented by Lushoto, Arumeru and Moshi; forest and grassland savannah with an average annual rainfall ranging between 900 and 1700 mm and temperatures of C was represented by Kilombero, Mvomero, Uyui (Tabora), Babati, Muleba and Kyela districts. Generally, the selection of sentinel districts was based on the WHOrecommended selection criteria: history of insecticide use by communities in the areas (in agricultural and public health), malaria endemicity (moderate to high malaria transmission area), high coverage with ITN LLINs, demographic settings (urban rural), and easy accessibility to the site. The distribution and the ITN coverage the demographic characteristics of these districts are shown in Figure 1 and Table 1 respectively. Mosquito collection Mosquitoes were collected using indoor resting catches (WHO 1975) in all locations. Freshly blood-fed, female ª 2012 Blackwell Publishing Ltd 743

3 N Muleba Mara Kigoma Kagera Mwanza Shinyanga Uyui Uyui Arusha Babati Arumeru Manyara Moshi Rural Kilimanjaro Lushoto Muheza Tanga Tabora Singida Dodoma Handeni Rukwa Mvomero Morogoro Dar es Salaam IIala IIala Pwani Mbeya Iringa Kilombero Kyela Lindi Legend Study site Ruvuma Mtwara Figure 1 Distribution of the study districts in Tanzania. Table 1 Insecticide-treated net (ITN) coverage and the demographic characteristic of the sentinel districts ITNs coverage * Other interventions using insecticides Demographic settings Tabora Agriculture, livestock Rural Babati Agriculture, livestock Rural Arumeru Floriculture, agriculture, livestock Semi-urban Kyela Agriculture Rural Kilombero Agriculture Rural Mvomero Agriculture, livestock Rural Dar es Salaam (Ilala) Horticulture Urban Handeni Agriculture, livestock Rural Muheza Agriculture Rural Moshi Agriculture Rural Lushoto Horticulture Rural Muleba IRS since 2006, agriculture Rural *Tanzania DHS THMS Anopheles mosquitoes were aspirated from their resting sites on the walls and other surfaces inside the houses. These wild collected mosquitoes were given fresh 10% glucose solution and transported to the field laboratory for susceptibility testing. Laboratory reared Kisumu-strain mosquitoes were exposed to insecticides as a reference susceptible population. Sampling of wild mosquitoes and testing were carried out almost at the same time (between 744 ª 2012 Blackwell Publishing Ltd

4 April and May) in all the sites with exception of Muleba district. Insecticide susceptibility resistance bioassay tests Insecticide susceptibility resistance bioassay tests were carried out using insecticide susceptibility test kits (WHO 1975) and standard procedures (WHO 1998) with four replicates of wild adult female mosquitoes per test tube. Mosquitoes were exposed to papers impregnated with the WHO-recommended discriminating concentrations (v w) of 0.05% deltamethrin, 0.05% lambdacyhalothrin, 0.75% permethrin and 4% DDT prepared at University Sains Malasyia (WHO 1998). The controls were exposed to clean paper impregnated with silicone oil (pyrethroids control) or risella oil (DDT control). During the exposure period, knock-down (KD) rates were recorded after 10, 15 20, 30, 40, 50 and 60 min. Mosquitoes were then transferred into the holding tube and fed on glucose solution. In case where less than 80% KD was observed after 60-min exposure period, KD was then noted after 80 min, that is, another 20 min in the holding tube. Mortality was scored after a 24-h holding period, during which time mosquitoes were provided with a 10% sugar solution. Resistance was defined according to WHO guidelines, which state that % mortality indicates susceptibility, 80 97% indicates the possibility of resistance that needs to be confirmed and <80% indicates resistance (WHO 1998). Morphological Identification of adult Anopheles mosquitoes After susceptibility tests, each Anopheles mosquito was morphologically identified (Gillies & de Meillon 1968; Gillies & Coetzee 1987) and then preserved dry over silica gel in a separate well-labelled Eppendorf tube for molecular analysis. Statistical analyses Abbott s formula was used to correct the observed mortality in adult susceptibility tests (Abbott 1925) when the mortality in control was between 5% and 20% (WHO 1998). The and KDT 95 were estimated by probit analysis (Finney 1971) provided from SPSS 13 for windows statistical software packages. The recorded from field-collected mosquitoes was compared with that of the A. gambiae Kisumu reference susceptible strain by estimates of ratios (RR). Results Mortalities Mortality rates of Anopheles mosquitoes 24 h postexposure to permethrin, deltamethrin, lambdacyhalothrin and DDT are shown in Tables 2 5, respectively. Permethrin (0.75%) susceptibility tests of Anopheles mosquitoes Table 2 Knock-down time and mortality rates of Anopheles mosquitoes exposed to 0.75% permethrin for a period of 60 min Number exposed (N) Number of replicates Mean corrected mortality (%) ± SD KDT 95 ratio (RR) Tabora ± Babati ± Arumeru ± Kyela ± Kilombero ± Mvomero ± Dar es Salaam ± (Ilala) Handeni ± Muheza ± Moshi ± Lushoto ± Muleba ± Susceptible Kisumu strain ± CI, confidence interval; SD, standard deviation; KDT, knock-down time;, time taken for 50% of the test mosquitoes to be knocked down; KDT 95, time taken for 95% of the test mosquitoes to be knocked down; RR, resistance ratio ( of the tested population of the susceptible Kisumu strain). ª 2012 Blackwell Publishing Ltd 745

5 Table 3 Knock-down time and mortality rates of Anopheles mosquitoes exposed to 0.05% deltamethrin for a period of 60 min Number exposed (N) Number of replicates Mean corrected mortality (%) ± SD KDT 95 ratio (RR) Tabora Babati ± Arumeru ± Kyela ± Kilombero ± Mvomero ± Dar es Salaam (Ilala) ± Muheza ± Moshi ± Lushoto ± Muleba ± Susceptible Kisumu strain ± CI, confidence interval; SD, standard deviation; KDT, knock-down time;, time taken for 50% of the test mosquitoes to be knocked down; KDT 95, time taken for 95% of the test mosquitoes to be knocked down; RR, resistance ratio ( of the tested population of the susceptible Kisumu strain). Table 4 Knock-down time and mortality rates of Anopheles mosquitoes exposed to 0.05% lambdacyhalothrin for a period of 60 min Number exposed (N) Number of replicates Mean corrected mortality (%) ± SD KDT 95 ratio (RR) Tabora ± Babati ± Arumeru ± Kyela ± Kilombero ± Mvomero ± Dar es Salaam (Ilala) ± Handeni ± Muheza ± Moshi ± Lushoto ± Muleba ± Susceptible Kisumu strain ± CI, confidence interval; SD, standard deviation; KDT, knock-down time;, time taken for 50% of the test mosquitoes to be knocked down; KDT 95, time taken for 95% of the test mosquitoes to be knocked down; RR, Resistance ratio ( of the tested population of the susceptible Kisumu strain). from six sentinel districts showed that they were highly susceptible in Tabora, Muleba, Babati, Kyela, Muheza and Lushoto (mortality rate range of %). Reduced susceptibility was recorded in Arumeru, Kilombero, Dar es Salaam and Handeni (mortality rates of %). Resistance to permethrin was recorded in Moshi (mortality rate of 77% [, 73 80]). Mortality in Anopheles mosquitoes owing to 0.05% deltamethrin ranged from 88% to 100%. Fully susceptibility to deltamethrin was recorded in Tabora, Babati, Kyela, Handeni, Muheza, Lushoto and Muleba (mortality rates of %). Reduced susceptibility was recorded in Arumeru, Kilombero, Dar es Salaam and Moshi (mortality rates of 88 97%). Mortality in Anopheles mosquitoes owing to 0.05% lambdacyhalothrin ranged from 73% to 100%. Full susceptibility to lambdacyhalothrin was recorded in Tabora, Babati, Lushoto and Muleba (mortality rates of %). Reduced susceptibility was recorded in Arumeru, Kyela, Kilombero, Dar es Salaam, Handeni, Muheza and 746 ª 2012 Blackwell Publishing Ltd

6 Table 5 Knock-down time and mortality rates of Anopheles mosquitoes exposed to 4% DDT for a period of 60 min Number exposed (N) Number of replicates Mean corrected mortality (%) ± SD KDT 95 ratio (RR) Tabora ± Babati ± Arumeru ± Kyela ± Kilombero ± Mvomero ± Dar es Salaam (Ilala) ± Muheza ± Moshi ± Lushoto ± Muleba ± Susceptible Kisumu strain ± CI, confidence interval, SD, standard deviation; KDT, knock-down time;, time taken for 50% of the test mosquitoes to be knocked down, KDT 95, time taken for 95% of the test mosquitoes to be knocked down; RR, resistance ratio ( of the tested population of the susceptible Kisumu strain). Moshi (mortality rates of 87 95%). As for permethrin, resistance to lambdacyhalothrin was recorded in Moshi (mortality rate of 73% [, 69 76]). Anopheles mosquitoes were highly susceptible to DDT in almost all sentinel districts except Lushoto and Kyela, which showed reduced susceptibility (mortality rates of 90 92%), and Dar es Salaam, which recorded resistance (mortality rate of 65% [, 54 74]). Trends of susceptibility status of Tanzanian populations of Anopheles mosquitoes to diagnostic concentrations of pyrethroids and DDT in 2004 and 2009 are shown in Table 6. The susceptibility status trend has remained the same in some sentinel districts, while falling in others. For example, the susceptibility status of Anopheles mosquitoes to deltamethrin in Morogoro dropped from full susceptibility in 2004 to 94.4% [, 90 97]) in Similar reduced susceptibility to deltamethrin was observed in Kilombero, Arumeru and Moshi (Table 6), to permethrin in Morogoro, Kilombero and Moshi, and to DDT in Mbeya. Knock-down The median knock-down time ( ) obtained from time-mortality regression using probit analysis ranged from 13.8 to 52.4 min while the KDT 95 ranged from 21.1 to 85.2 min for permethrin. The longest median knock-down times ( ) of 30.2 to 52.4 min were recorded in Arumeru, Dar es Salaam, Handeni and Moshi (Table 1). The for deltamethrin ranged from 9.4 to 35.6 min, for lambdacyhalothrin from 16.2 to 58.6 min and for DDT from 25.7 to 71.6 min (Tables 2 4). Considerably, it took a long time for mosquitoes to be knocked down by DDT compared with the other three insecticides. Arumeru district recorded higher KDT for all insecticide tested. The of the wild populations were compared with those obtained for the susceptible mosquitoes to obtain the resistance ratio (RR). The resistant ratio (RR) at level for permethrin ranged from 0.8 to 3.8, for deltamethrin from 0.7 to 2.8, for lambdacyhalothrin from 1.1 to 3.9, and for DDT from 1.1 to 3.1. Arumeru recorded the highest levels of RRs to all pyrethroids while the highest level for DDT was recorded in Dar es Salaam (Tables 1 4). Discussion This study demonstrates the continued susceptibility of the A. gambiae complex to three pyrethroids and DDT in areas of Tanzania where ITNs have been used for the past twenty years. These results are promising, given present malaria control strategies of scaling-up ITN LLINs use in Tanzania (Magesa et al. 2005), and suggest that with high ITN LLINs coverage, current insecticides used in the treatments of ITN LLINs can provide an effective means of malaria control. The situation has not dramatically deteriorated since an earlier study in Tanzania (Curtis et al. 2003). While the high susceptibility of Anopheles mosquitoes to diagnostic concentrations of pyrethroids and DDT is encouraging, in several areas mortalities less than 98% were observed. Continued monitoring is therefore ª 2012 Blackwell Publishing Ltd 747

7 Table 6 Trends of susceptibility status of Tanzanian populations of Anopheles mosquitoes to diagnostic concentrations of pyrethroids and DDT in 2004 and Mean percentage mortality in WHO susceptibility tests and number of mosquitoes exposed (N). Insecticide 2004* * N % Mortality N % Mortality Deltamethrin Morogoro Kilombero Mwanza Tabora Mbeya Arumeru Moshi Permethrin Morogoro Kilombero Tanga Mwanza Tabora Mbeya Arumeru Moshi DDT Morogoro Kilombero Tabora Mbeya Arumeru Moshi *WHO diagnostic concentrations used: deltamethrin 0.05%, permethrin 0.75%, DDT 4.0%. Kulkarni et al. (2007) required, particularly in the areas where marginal (i.e %) susceptibility is seen. In southern Tanzania, historical data suggest that while full susceptibility was observed during years of ITN scaling-up, there was a concurrent shift in knock-down time (Kulkarni et al. 2007). A similar shift has been observed in Moshi, where resistance to permethrin and lambdacyhalothrin was recorded. Such a shift correlates with early stages of resistance development in vector populations (Chandre et al. 1999), and thus, this area should be closely monitored. Permethrin resistance to malaria vectors has also been previously documented by Matowo et al. (2010). It is probably caused by the agricultural use of insecticides, especially in the rice fields (Matowo et al. 2010), as permethrin-treated bed nets have just been scaled up in Moshi. In Kenya, no evidence of decreased efficacy of ITNs was observed when dealing with permethrin-tolerant populations of A. gambiae s.s. (Vulule et al. 1994, 1996, 1999). The present level of resistance in Lower Moshi appears to not impair the effectiveness of permethrin-treated nets. A recent field trial in Moshi shows that while such treated nets kill relatively few host-seeking A. arabiensis that enter local houses, the nets continue to provide personal protection through the strong excito-repellent activity of permethrin (Mosha et al. 2008). Contrary to this, Stump et al. (2004) have reported pyrethroid resistance to malaria vectors in an area with long-term ITNs usage in western Kenya. Tanzania is scaling up the use of Olyset Ò -LLINs, which are permethrin-treated nets, and therefore, if the increased coverage selects further for permethrin resistance, the effectiveness of the LLINs strategy may ultimately be undermined. Interestingly, in Muheza district of coastal Tanzania, where ITNs have been distributed by research projects achieving high levels of coverage for over 20 years, similar high levels of susceptibility of Anopheles mosquitoes to all tested insecticides have been observed. These results are consistent with those of other studies conducted in Tanzania (Kulkarni et al. 2007). Similarly, Muleba district, where IRS operation started in (4 years before this survey), recorded high levels of susceptibility of Anopheles mosquitoes to all tested insecticides. However, the resistance to DDT in Dar es Salaam was unexpected because the insecticide is not yet officially widely used in the country. This resistance is linked to the informal local introduction and use of DDT to control public health pests in Dar es Salaam and rest of the country. This warrants further investigation. Permethrin and lambdacyhalothrin resistance in Moshi and DDT resistance in Dar es Salaam are strongly supported by increased median knock-down time ( ) as compared with the other sites where the Anopheles mosquitoes were susceptible to these insecticides. High were also recorded in areas with marginal susceptibility such as Handeni (for permethrin) and Arumeru (for all insecticides tested). For example, the for permethrin increased by 3.8-, 2.2- and 2.2-fold in Arumeru, Dar es Salaam and Handeni, respectively. Similar increase in the was recorded in sites with full susceptibility to lambdacyhalothrin such as Babati, Lushoto and Muleba. Higher values in the field population have been suggested to give an early indication of the involvement of kdr mechanism of resistance (Chandre et al. 1999). This may be consistent with the work carried out by Moshi by Kulkarni et al. (2006) who reported low frequency of kdr in A. arabiensis. This calls for further investigation to be carried out to determine the presence of the kdr alleles in our mosquito population. Although we have found no evidence of selection pressure for physiological resistance in malaria vector populations in most of the surveyed sites in Tanzania, the decreasing trend of susceptibility to some insecticides in some localities is quite alarming. We have recorded 748 ª 2012 Blackwell Publishing Ltd

8 decreased susceptibility of the Anopheles population to some insecticides in some study districts when comparing to the 2004 data. This may be due to increased selection pressure to these districts which might have contributed by the increased ITNs coverage or spreading of the resistance mosquito population. It should be noted that the areas where marginal susceptibility was observed are situated in regions with large-scale agricultural production, for example, floriculture in Arumeru region, and therefore, the potential contribution of agricultural insecticide use to resistance development should be considered in future studies. Conclusion and recommendations In most areas of the country, insecticides against malaria vectors were found to be effective. The emergence of focal points with insecticide resistance calls for close monitoring of insecticide resistance for early containment of the problem. Future studies should consider evaluating the factors influencing selection pressure in insecticide resistance in malaria vectors. Similarly, monitoring of insecticide susceptibility of the non-malaria vector nuisance mosquito populations may be needed to ensure public acceptability and sustained ITNs LLINs use. Further analysis is needed to determine the mode mechanism of resistance involved in areas where resistance have been recorded. Acknowledgements The study was conducted by the National Institute for Medical Research in Tanzania in collaboration with Kilimanjaro Christian Medical College (KCMC) and Tropical Pesticide Research Institute (TPRI) to support the National Malaria Control Programme (NMCP). The project was funded by The Wellcome Trust through Malaria Capacity Development Consortium (MCDC) and also by Bill and Melinda Gates foundation through WHO. The authors are grateful to Dr Martin Donnelley for reviewing the earlier version of the manuscript. 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