**3. History, distribution and current status of insecticide resistance in East Africa**

Insecticides (chemicals) which have been used for the control of vector-borne diseases and crop protection is believed to enhance the evolution of resistance in insects [49]. The intensive use of DDT in agriculture and public health programs and the introduction of pyrethroids in 1970s and its increased utilization since 1990s caused resistance to have been detected in malaria vectors from different sites in different countries of East Africa. Moreover, the longterm use of a single class of insecticide or combination of different classes of insecticides have led to the emergence of single resistance mechanism or multiple resistance mechanisms in different areas of East Africa [50]. Thus, insecticide resistance intensity and its distribution are increasing in East Africa (Figure 1).

**Figure 1.** Distribution of DDT and pyrethroid resistance in East Africa. Note: Numbers in orange spots indicate the number of sampling sites.

#### **3.1. Kenya**

The major malaria vectors in Kenya are *An. gambiae* complex (*An. gambiae* s.s., *An. arabiensis, An. merus*) and *An. funestus* while other vector species in the country include A*n. melas, An.* *nili, An. paludis, An. pharoensis* and *An. coustani* [20]. From peer-reviewed sources, other anopheline species documented included: *An. christyi, An. demeilloni, An. gibbinsi, An. harperi, An. implexus, An. maculipalpis, An. marshalli, An. pretoriensis, An. rufipes, An. squamosus, An. swahilicus, An. theileri, An. wilsoniand An. ziemanni*, none of which are considered as important or primary vectors in Africa [51]. The malaria vector distribution in the country is not uniform due to variation in climatic factors, particularly temperature and rainfall.

In Kenya, the first reported case of resistance was in the context of insecticide-treated net use in Western Kenya where reduced knockdown rates have been observed [1]. Complete susceptibility of populations of *An. arabiensis* to DDT, fenitrothion, bendiocarb, lambdacyha‐ lothrin and permethrin was documented from Mwea rice irrigation scheme, Central Kenya [52]. Widespread resistance against pyrethroids and DDT was observed across western Kenya [53]. *An. gambiae s.l.* showed different levels of resistance to deltamethrin, lambdacyhalothrin and bendiocarb Kilifi, Malindi and Taveta districts in coastal Kenya. Pyrethroid resistance has been reported in *An. gambiae s.s* and *An. arabiensis* from four districts of Western Kenya. Stump and others also found significant differences in *kdr* gene frequency between the large-scale insecticides treated nets [54].

Kamau and Vulule reported that *An. gambiae* s.l. and *An. funestus* from western, coastal and central Kenya were susceptible to DDT, fenitrothion, bendiocarb, lambdacyhalothrin and permethrin [52]. The same study also showed the presence of Leucine-Serine (East African) *kdr* mutation in *An. gambiae* s.s. of western Kenya, but the leucine–phenylalanine (West African) mutation was absent in this mosquito population. Though the East African *kdr* mutation was detected from west Kenyan populations of *An. gambiae*, it has never occurred at homozygous state. The frequency of the L1014S *kdr* allele doubled in the ITN test village and its nearest neighbor from 1987 to 2001, but not outside of this area. This suggests that ITN use has further selected for the *kdr* mutation in the population.

#### **3.2. Uganda**

use of DDT in agriculture and public health programs and the introduction of pyrethroids in 1970s and its increased utilization since 1990s caused resistance to have been detected in malaria vectors from different sites in different countries of East Africa. Moreover, the longterm use of a single class of insecticide or combination of different classes of insecticides have led to the emergence of single resistance mechanism or multiple resistance mechanisms in different areas of East Africa [50]. Thus, insecticide resistance intensity and its distribution are

**Figure 1.** Distribution of DDT and pyrethroid resistance in East Africa. Note: Numbers in orange spots indicate the

The major malaria vectors in Kenya are *An. gambiae* complex (*An. gambiae* s.s., *An. arabiensis, An. merus*) and *An. funestus* while other vector species in the country include A*n. melas, An.*

increasing in East Africa (Figure 1).

192 Insecticides Resistance

number of sampling sites.

**3.1. Kenya**

The main malaria vectors in Uganda are *An. gambiae* and *An. funestus*, with *An. arabiensis* involved in local transmission. Recent study also showed that *An. funestus* and *An. gambiae* are the widely distributed vectors in Uganda. Other less dominant anophelines which were implicated in malaria transmission in the country include: *An. coustani*, *An. listeri*, *An. mar‐ shalli* and *An. kingi* [55, 56].

There is widespread insecticide resistance in the main malaria vectors, *An. gambiae*, *An. funestus* and *An. arabiensis*. In Uganda, resistance to pyrethroid insecticides has been reported in the three main malaria vectors, *An. gambiae, An. arabiensis* [55, 57, 58] and *An. funestus* [59]. A reduced susceptibility by *An. gambiae* s.l. to three pyrethroid insecticides, deltamethrin, cyfluthrin and cypermethrin, has been observed [60]. *An.gambiae* s.l. was DDT- and pyrethroidresistant in central and eastern Uganda [58]. There are currently no reports of organophosphate resistance, but resistance to carbamates including propoxur has been documented. Mawejje and co-workers observed high pyrethroid resistance in *An. gambiae* and *An. Arabiensis*, but both species were fully susceptible to bendiocarb and fenitrothion from eastern Uganda [55]. Resistance to DDT and deltamethrin has also been reported in populations of *An. funestus* and *An. gambiae* s.l. from southwestern Uganda [56]. *An. funestus* in Tororo, eastern Uganda, was resistant to pyrethroids, permethrin and deltamethrin. Suspected DDT resistance was also observed in *An. funestus*. However, this population was completely susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin. Recently, widespread resistance against pyrethroids and DDT was observed across Uganda [53, 61]. Mutations which confer resistance to DDT and pyrethroids, West African (L1014F) and East African (L1014S) muta‐ tions, have been reported from the Ugandan *An. gambiae*. Increased esterase activity was also detected in pyrethroid- and DDT-resistant *An. gambiae* populations. The presence of the East African *kdr* mutation (L1014S) is shown for the first time in *An. arabiensis* from Uganda [62]. The resistance in this species was due to both target site (*kdr*) and metabolic mechanisms and there was also cross-resistance between DDT and pyrethroids. Resistance to pyrethroids is present, and apparently increasing, in *An. arabiensis* from Jinja, eastern Uganda [55], but it is not mediated by known 'knockdown resistance' target-site mechanisms (L1014F and L1014S) in the voltage-gated sodium channel, which are extremely rare in this species in this area [55]. In the absence of a known target-site mechanism, metabolic mechanisms are strongly impli‐ cated in the resistance phenotype. However, knockdown resistance mutation conferring pyrethroid/DDT resistance has also been suggested to occur in other axons of the sodium channel gene in *An. gambiae*. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA. The low frequency of L1014S and L1014F mutations and complete restoration of susceptibility to permethrin and deltamethrin by the two species after synergist assay using PBO indicate involvement of other mechanisms such as P450s in the same study. Populations of *An. gambiae* s.l. from eastern Uganda tested for the presence of knockdown resistance (*kdr*) and altered acetylcholinesterase (ace-1R) alleles showed the presence of *kdr* L1014S allele, while ace-1R and *kdr* L1014F alleles were absent [57]. All populations from the same area remain highly susceptible to carbamate, organophosphate and dieldrin insecticides. Metabolic resistance through elevated expression of cytochrome P450s has been implicated in these mosquito populations.

#### **3.3. Ethiopia**

Forty-two anopheline species have been recorded in Ethiopia [63]. There are only four anopheline mosquito species reported as malaria vectors. *Anopheles arabiensis* is the primary vector of malaria and it is widely distributed throughout the country [64], while *An. funestus, An. pharoensis* and *An. nili* are secondary vectors with localized distribution [65]. *An. arabien‐ sis* belongs to the *An. gambiae* complex of sibling species. Only two member species of the *An. gambiae* complex, *An. arabiensis* and *An. Amharicus* (formerly known as *An. quadriannulatus* B), are reported to exist in Ethiopia. *An. quadriannulatus* species B had been described as a new species from southwestern Ethiopia [66]. This species was reported to be zoophilic and exophilic and is assumed to have no role in malaria transmission in Ethiopia [67]. *Anopheles arabiensis* is responsible for most of malaria infections in Ethiopia. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are pillars in malaria prevention and control strategy in Ethiopia. For over five decades, the main vector control strategy by the national malaria control program has been indoor residual spraying (IRS), using DDT with a limited application of malathion as an alternate insecticide. However, DDT use for IRS was replaced in favor of deltamethrin in 2009, and after 2 years of use deltamethrin was also replaced with bendiocarb in 2011 due to the reduced susceptibility of the principal vector to the mentioned insecticides. Insecticide susceptibility tests carried out in different parts of the country have shown different levels of resistance by the principal vector to insecticides in use for IRS and/or to treat LLINs. Insecticide resistance by *An. arabiensis* to DDT was reported during the early 1990s [64, 68]. Balkew and others reported resistance by *An. arabiensis* to permethrin and DDT [69]. Another study by Yewhalaw and his colleagues from southwestern Ethiopia indicated that *An. arabiensis* developed resistance to DDT, permethrin, deltamethrin and malathion. In contrast, *An. arabiensis* was susceptible to bendiocarb and Propoxur [70] and primiphos methyl (PMI/USID unpublished data). Abate and Haddis also reported high level of DDT and pyrethroid resistance in populations of *An. gambiae* s.l, presumably *An. arabien‐ sis* from different parts of the country [71]. Another recent report by Massebo and others showed that populations of *An. arabiensis* from southwest Ethiopia developed resistance against lambda-cyhalothrin, alpha-cypermethrin, cyfluthrin, deltamethrin and DDT [33]. Moreover, high knockdown resistance mutation (West African *kdr*) was detected in popula‐ tions of *An. arabiensis* from northwestern, central and southwestern Ethiopia [70, 72]. Bottle bioassay studies using synergists also revealed possible involvement of metabolic resistance in addition to *kdr* mutations in these populations of *An. Arabiensis*, which could further complicate the current malaria vector control program in the country [73]. The development of resistance by malaria vectors against insecticides used for public health could potentially jeopardize the malaria vector control strategy in Ethiopia, and hence it is imperative to monitor the level and distribution of insecticide resistance to develop new effective vector control tool and/or plan sound insecticide resistance management (IRM) strategy in the country.

#### **3.4. Tanzania**

*An. gambiae* s.l. from southwestern Uganda [56]. *An. funestus* in Tororo, eastern Uganda, was resistant to pyrethroids, permethrin and deltamethrin. Suspected DDT resistance was also observed in *An. funestus*. However, this population was completely susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin. Recently, widespread resistance against pyrethroids and DDT was observed across Uganda [53, 61]. Mutations which confer resistance to DDT and pyrethroids, West African (L1014F) and East African (L1014S) muta‐ tions, have been reported from the Ugandan *An. gambiae*. Increased esterase activity was also detected in pyrethroid- and DDT-resistant *An. gambiae* populations. The presence of the East African *kdr* mutation (L1014S) is shown for the first time in *An. arabiensis* from Uganda [62]. The resistance in this species was due to both target site (*kdr*) and metabolic mechanisms and there was also cross-resistance between DDT and pyrethroids. Resistance to pyrethroids is present, and apparently increasing, in *An. arabiensis* from Jinja, eastern Uganda [55], but it is not mediated by known 'knockdown resistance' target-site mechanisms (L1014F and L1014S) in the voltage-gated sodium channel, which are extremely rare in this species in this area [55]. In the absence of a known target-site mechanism, metabolic mechanisms are strongly impli‐ cated in the resistance phenotype. However, knockdown resistance mutation conferring pyrethroid/DDT resistance has also been suggested to occur in other axons of the sodium channel gene in *An. gambiae*. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA. The low frequency of L1014S and L1014F mutations and complete restoration of susceptibility to permethrin and deltamethrin by the two species after synergist assay using PBO indicate involvement of other mechanisms such as P450s in the same study. Populations of *An. gambiae* s.l. from eastern Uganda tested for the presence of knockdown resistance (*kdr*) and altered acetylcholinesterase (ace-1R) alleles showed the presence of *kdr* L1014S allele, while ace-1R and *kdr* L1014F alleles were absent [57]. All populations from the same area remain highly susceptible to carbamate, organophosphate and dieldrin insecticides. Metabolic resistance through elevated expression of cytochrome P450s has been implicated in these

Forty-two anopheline species have been recorded in Ethiopia [63]. There are only four anopheline mosquito species reported as malaria vectors. *Anopheles arabiensis* is the primary vector of malaria and it is widely distributed throughout the country [64], while *An. funestus, An. pharoensis* and *An. nili* are secondary vectors with localized distribution [65]. *An. arabien‐ sis* belongs to the *An. gambiae* complex of sibling species. Only two member species of the *An. gambiae* complex, *An. arabiensis* and *An. Amharicus* (formerly known as *An. quadriannulatus* B), are reported to exist in Ethiopia. *An. quadriannulatus* species B had been described as a new species from southwestern Ethiopia [66]. This species was reported to be zoophilic and exophilic and is assumed to have no role in malaria transmission in Ethiopia [67]. *Anopheles arabiensis* is responsible for most of malaria infections in Ethiopia. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are pillars in malaria prevention and control strategy in Ethiopia. For over five decades, the main vector control strategy by the national malaria control program has been indoor residual spraying (IRS), using DDT with a limited

mosquito populations.

**3.3. Ethiopia**

194 Insecticides Resistance

The principal vectors of malaria in Tanzania are mosquitoes of the *An. gambiae* s.s, *An. arabiensis* and *An. funestus*. Other vectors which have limited role in malaria transmission include: *A. merus, A. nili, A. paludis, A. pharoensis, An. coustani. An. leesoni, An. parensis, An. merus, An. marshallii* and *An. rivulorum* [74, 75]. Recent entomological data indicate that *An. funestus* is prevalent on the mainland as well, particularly in the Kagera Region. Moreover, in coastal areas of north-eastern Tanzania and Zanzibar, high coverage of ITNs and IRS has resulted in a shift in the malaria vector population from *An. gambiae* to *An. arabiensis*. Resistance to pyrethroids by *An. gambiae* s.s. and *An. arabiensis* has been reported from several districts of the mainland of Tanzania [76–78]. Okumu and his colleague reported that *An. arabiensis* from southeastern Tanzania showed 100% susceptibility to DDT but 95.8% to deltamethrin, 90.2% to lambda cyhalothrin and 95.2% to permethrin [79]. In Zanzibar, *An. arabiensis* was resistance to pyrethroids (lambda-cyhalothrin, deltamethrin and permethrin), but was susceptible to carbamates (bendiocarb) and organochlorides (DDT). Moreover, in a similar study, resistance was documented in *An. gambiae s.s* to the same pyrethroid insecticides but was susceptible to bendiocarb, DDT and malathion [80]. In Pemba, resistance was detected in sites monitored for lambdacyhalothrin, permethrin, deltamethrin and DDT, but no resistance was detected for bendiocarb and pirimiphos-methyl CS. Similarly in Unguja, lambda-cyhalothrin resistance was detected in four of the five sites tested and permethrin resistance in one of the two sites tested. However, insecticide resistance was not detected for bendiocarb, pirimiphos-methyl CS and DDT. *Anopheles gambiae* s.s showed reduced susceptibility to the carbamate insecticide, bendiocarb [81]. *An. arabiensis* collected from Lower Moshi showed complete susceptibility to pirimiphos-methyl and malathion, but reduced susceptibility to permethrin [82, 83]. In northwestern Tanzania, there was cross-resistance between pyrethroids and DDT. In Zanzibar, resistance is not homogeneously expressed across islands, and pyrethroid resistance is stronger in Pemba than Unguja.

West African leucine phenylalanine *kdr* mutation was detected in two heterozygous individ‐ uals field-collected *An. arabiensis* from Tanzania [84]. A study also showed that a low frequency of permethrin resistance mediated by mixed function oxidases and esterases are present in *An. arabiensis* from Lower Moshi. The permethrin resistance is probably caused by the agricultural use of insecticides, especially in the rice fields, as permethrin-treated nets were not widely used in Lower Moshi [76]. The *kdr*-eastern variant was present in homozygous form in 97% of *An. gambiae* s.s but was absent in *An. arabiensis*. Synergist assays with PBO showed to restore susceptibility to pyrethroids, indicating that the resistance is in part due to an oxidase enzyme mechanism. Knockdown resistance mutation (target site insensitivity) was also detected in Pemba [84, 85].

#### **3.5. Burundi**

The primary vector of malaria in Burundi is *Anopheles gambiae* s.s, while secondary vectors *An. funestus, An. arabiensis* and *An. nili*. The most predominant members of vector species complex in the highlands of Burundi are *An. gambiae* s.s and *An. funestus* s.s [86, 87]. Insecticide susceptibility study in Karusi for *An. gambiae* s.l. showed reduced mortality to permethrin, DDT and deltamethrin. There was complete susceptibility of *An. funestus* to DDT and pyreth‐ roids. A high frequency of East African *kdr* allele was detected in *An. gambiae* s.l., leading to cross resistance between DDT and permethrin in mosquito population. As there is little information on the frequency and distribution of insecticide resistance and the status of the susceptibility level of malaria vectors to insecticides used for vector control in the country, there is an urgent need for a nationwide and systematic evaluation of vector susceptibility level to current WHOPES-approved insecticides for malaria vector control, to inform ongoing interventions and control program.

#### **3.6. Rwanda**

Earlier entomological studies indicate that *Anopheles gambiae* s.l. and *An. funestus* are the main vectors responsible for malaria transmission in Rwanda. *An. arabiensis* is also a locally important vector of malaria. The main malaria foci are in the east and southeast areas where the altitude is generally below 1,500 m and surrounded by marshy plains.

Insecticide susceptibility studies conducted in 2012 in several sites indicated signs of resistance to DDT in some areas, possible emergence of resistance to some pyrethroid compounds, and complete susceptibility to bendiocarb and fenitrothion. A similar insecticide susceptibility study conducted by the national malaria control program in the same year showed established resistance to pyrethroids in Mimuri, a sentinel site in the country. A high frequency of the *kdr* gene in *An. gambiae* s.l. has been attributed to explain the new established resistance to pyrethroids in one district. A countrywide resistance monitoring also showed resistance to pyrethroids, DDT and bendiocarb and higher resistance was reported from eastern province, southern province and Kigali city. A continuous monitoring of resistance and resistance mechanisms is required in order to guide program for the best strategies to prevent the development and spread of resistance in the country.
