Background
Most countries in Africa depend heavily on two vector control interventions in their battle against malaria: long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). These tools use insecticides from four chemical classes: organochlorines, pyrethroids, carbamates and organophosphates. Whereas 14 formulations belonging to these classes are approved by the World Health Organization (WHO) for use in IRS [
1], only pyrethroids are approved for use in LLINs [
2] because of their low mammalian toxicity, excito-repellent properties and rapid knock-down and killing effect [
3]. It has been estimated that since 2000 more than 670 million cases of malaria have been averted by combining IRS and LLINs with case management and community education [
4,
5]. In Rwanda, the national scale-up of vector control interventions has contributed to a steady reduction of malaria cases from 1.6 million in 2005 to 472,000 cases in 2012 [
6,
7]. This reduction has been attributed to the combined effects of universal coverage with LLINs [
7] and targeted IRS operations in districts with the highest malaria endemicity (5 out of 30 districts) based on epidemiologic and entomologic data. The Rwanda Malaria Indicator Survey carried out in 2013 showed that overall LLIN coverage is high with 83% of the households owning at least one LLIN and 74% reported to have slept under a LLIN the previous night for pregnant women and children under five [
8]. From 2005 to 2013, the National Malaria Control Programme (NMCP) of the Rwanda Ministry of Health has distributed approximately 11.2 million LLINs to a population of an estimated 11 million people [
9].
From 2007 to 2012, nationwide distributions of LLIN have been conducted in conjunction with annual IRS applications of pyrethroids in high malaria transmission districts either in focal sectors or district-wide by blanket spraying, covering an estimated 98% of the targeted structures. In 2013, the NMCP shifted from pyrethroids to the use of carbamates (Bendiocarb 80% WP) for IRS as part of an insecticide resistance management strategy. This switch was because of confirmed pyrethroid resistance and following the WHO guidance of using active ingredients with different modes of action in rotation [
6,
10].
The main mechanisms by which mosquitoes display resistance to insecticides are the expression of elevated levels of detoxifying enzymes (metabolic resistance) and target site insensitivity (knock-down mutations or altered acetylcholinesterase) [
10,
11]. Two point mutations in the voltage-gated sodium channel are associated with knock down resistance (
kdr) to DDT and pyrethroids in the malaria mosquito
Anopheles gambiae s.s. [
12]. One mutation involves a leucine (TTA) to phenylalanine (TTT) substitution at residue 1014 of the gene (L1014F). This mutation is mainly found in West Africa and hence named
kdr-west [
13]. The other mutation involves a leucine (TTA) to serine (TCA) substitution at the same residue (L1014S) and is mostly found in East Africa (
kdr-east) [
14], although both mutations co-occur in some parts of Africa [
15].
In 2012, the WHO reported that insecticide resistance in malaria vectors had already been found in more than 64 malaria endemic countries worldwide, with the majority reporting resistance to pyrethroids [
10]. This spread is alarming as it poses serious threats to the efficacy of vector control interventions and the gains made in malaria control over the last 10 years. Therefore, it is concerning that most national malaria control programmes (NMCPs) continue to use pyrethroid insecticides for vector control. The situation is also compounded by the extensive use of pyrethroids in agriculture, which poses an additional selection pressure on malaria vectors, for example via insecticide-contaminated ground water that permeates to mosquito larval habitats [
14,
16,
17].
The WHO calls for all countries to develop and implement insecticide resistance management strategies in their malaria control programmes in order to curb the spread of resistance as well as preserve the effectiveness of LLINs [
10]. Many African countries have now implemented entomological monitoring and susceptibility testing. To inform decisions in the control of malaria in Rwanda, the NMCP has been conducting entomological monitoring of malaria vectors in 12 sentinel sites throughout the country since 2010. Both
An. gambiae s.l. (94.3%) and
Anopheles funestus (5%) were the dominant
Anopheles species.
Anopheles arabiensis was the predominant sibling species of the
An. gambiae complex (E.H., unpublished data). Mosquito susceptibility to the WHO recommended classes of insecticides are included annually in this routine survey to monitor resistance to organochlorines, organophosphates, pyrethroids and carbamates [
18]. Here, findings of susceptibility tests conducted in 2011 and 2013 to detect knock-down mutations and mortality rates in female
An. gambiae s.l. mosquitoes are described. This is the first report presenting nationwide results on the composition of the
An. gambiae complex and its susceptibility to insecticides used for malaria control.
Discussion
In Rwanda, integrated malaria control interventions (artemisinin-based combination therapy (ACT), LLINs and targeted IRS) have been in use since 2006. This has contributed to a significant reduction in clinical malaria cases in the country [
7]. However, the gains made are fragile due to the decrease of efficacy of interventions, partially as a result of insecticide resistance development that has spread throughout Africa [
4,
23,
24]. Rwanda achieved universal coverage with LLINs in 2011, but the major challenge is to maintain this coverage and use with effective mosquito nets, especially after it was recently reported for Rwanda that LLIN effectiveness lasts less than 3 years due to the rapid loss of insecticidal activity and physical deterioration in the field [
25]. LLIN deterioration problems were also shown in recent findings from Senegal where damaged nets provided less protection from malaria compared to intact ones [
26].
The fact that millions of nets may have lost their effectiveness and thus continue to expose mosquitoes to a sub-lethal dose of pyrethroids is of major concern, because this may contribute to the further development of resistance. Similarly, annual consecutive use of pyrethroids in IRS, combined with extensive use of pyrethroids in agriculture has also implications for emerging insecticide resistance [
27]. Hence, Rwanda with the support of PMI and the Global Fund has been keen to monitor insecticide resistance so that it can take action to mitigate the emergence of resistance. In 2010, an initial susceptibility survey was conducted in eight sites in which mosquitoes were tested with deltamethrin, permethrin, bendiocarb, malathion and DDT using the CDC bottle assay [
28,
29]. These mosquitoes were found to be fully susceptible, except to DDT in two sites (E.H., unpublished data).
The data collected in the current surveys (2011 and 2013) confirm that resistance to DDT has been on the rise since 2011. In addition, the results show resistance to the pyrethroids (deltamethrin, permethrin, and lambda-cyhalothrin) being present in 2011 and further increasing in 2013. Although DDT was banned for usage in Rwanda in 1989, high levels of resistance are of concern because resistance to DDT confers cross-resistance to pyrethroids [
10]. Mosquitoes were fully susceptible to fenitrothion in all the sites, while possible emergence of resistance to bendiocarb was reported in the present study. Nevertheless, a switch to IRS with bendiocarb was made from September 2013 onwards so that the two main vector control interventions (LLINs and IRS) employed different classes of insecticide to delay resistance development.
The presence of
kdr mutations recorded in some sites can be explained by intense indoor interventions with IRS and LLINs, as was reported in Burundi and Tanzania [
30,
31]. However, it should be noted that the frequency of
kdr in the vector population may not be a reliable marker for actual resistance [
32] and that care should be taken when interpreting such data as other resistance mechanisms can play a role as well. In current study, metabolic resistance involving oxidases was proven by using piperonyl butoxide (PBO). Susceptibility was fully restored in the six selected sites where resistance to pyrethroids had been identified. This suggests that metabolic resistance may even account for all observed resistance in bioassays with malaria vectors from Rwanda.
The resistance level in Mimuri (Nyagatare District) could explain the high number of malaria cases occurring in this district: about 42% of all malaria cases of the 30 districts in 2011 were found here [
33] before the introduction of bendiocarb for IRS. This site, as well as Mashesha, Mareba, and Kibogora, is characterized by rice growing in which agricultural pesticides, and pyrethroids in particular, are extensively used.
In response to these findings, Rwanda developed and implemented an insecticide resistance management (IRM) plan in 2013 that recommended transitioning to non-pyrethroid IRS to mitigate pyrethroid resistance and to lengthen the effectiveness of LLINs [
6]. Rwanda transitioned to carbamate (bendiocarb) for IRS and the country will switch to a long-lasting organophosphate (Actellic–pirimiphos-methyl CS) in 2016 due to the reported trends of suggestive resistance to bendiocarb. The question remains, however, whether Rwanda should continue with blanket IRS treatment in an entire district when data on resistance is reported from only one sentinel site per district. To answer this question, it is recommended that additional sites should be identified in the targeted districts in order to determine whether or not the spread of resistance is homogeneous throughout the district. Similar to a recent report from Malawi, the use of long-lasting IRS formulations, such as pirimiphos-methyl, may be costly initially, but cost-effective in the longer term in managing insecticide resistance [
34].
In this study, characterization of
An. gambiae s.l. from 10 sentinel sites revealed that the predominant sibling species is
An. arabiensis (83%). This is contrary to a study conducted in one site near Kigali City in 2007 by PMI-Rwanda, in which it was reported that
An. gambiae s.s. accounted for 93.6% of the total 157
An. gambiae s.l. examined by PCR while
An. arabiensis accounted for only 6.4% [
35]. Although the earlier sampling was carried out in one site only, the results suggest that
An. gambiae s.s. was the predominant species before the scale-up of interventions with LLINs and IRS. Such a shift in species composition has been reported in neighbouring countries, for instance in Kenya, Uganda and Tanzania [
36‐
38]. This phenomenon has important implications for malaria epidemiology and control given that
An. arabiensis is an opportunistic feeder which has a tendency to rest and feed on humans outdoors [
39]. Outdoor-biting mosquitoes are less susceptible to indoor interventions and therefore outdoor interventions that supplement LLINs and IRS will need to be instituted in the context of an integrated approach to vector management [
38,
40]. Although
An. arabiensis is the dominant vector in Rwanda and resistance levels are high in
An. gambiae s.l. populations, future studies should include testing of resistance by sibling species to assess differences in susceptibility levels.
With the goal of reaching the pre-elimination phase of malaria by 2018, the Ministry of Health developed an integrated vector management (IVM) strategy which aims at improving the efficiency, effectiveness, ecological soundness and sustainability of vector control interventions in Rwanda [
41]. Entomological monitoring, including testing for insecticide resistance, is now a major part of the NMCP in Rwanda and a rotation strategy for management of insecticide resistance has been adopted in line with IVM.
Meanwhile, with spreading insecticide resistance and behavioural change of malaria vectors, there is an interest to integrate other innovative interventions which do not rely on insecticides [
42,
43]. Larval control interventions have proven cost-effective across a range of different settings and include application of environmental management, insect growth regulators, and biological control [
44]. Recently, successful field experiments have been carried out with microbial larvicides in Tanzania, Kenya, the Gambia and Benin. These showed a substantial impact on malaria disease [
45‐
47]. It was demonstrated that for a sustainable solution, a horizontally organized community-based programme that takes the needs and wishes of people into account has to be established and technically empowered [
48,
49]. Currently, Rwanda is supporting a research project in Ruhuha (South East Rwanda) that aims to involve communities in the application of the microbial larvicide
Bacillus thuringiensis var.
israelensis (Bti) (E.H., unpublished data). This may form the basis for the integration of alternative vector control interventions for the management of insecticide resistance [
42].
Authors’ contributions
EH designed the study, trained and supervised technical staff, performed resistance and laboratory tests, compiled and analysed data and drafted the manuscript. DM trained and supervised technical staff involved in mosquito rearing and bio-assays, performed laboratory tests and reviewed the manuscript. GI supervised technical staff involved in mosquito rearing and bio-assays, performed bioassays tests and reviewed the manuscript. JET contributed to the data analysis and review of the manuscript. JG contributed to the data analysis, draft of the manuscript, technical guidance and review of the manuscript. CK contributed to the design and implementation of the study, mobilized funds for monitoring activities and reviewed the manuscript. WT contributed to the design of manuscript outlines, data analysis and critical review of the manuscript. AB contributed to the design of the study, and review of the manuscript. CJMK contributed to the data analysis, writing and review of manuscript. All authors read and approved the final manuscript.