Characterizing the insecticide resistance of Anopheles gambiae in Mali

Recent evidences from survey data indicated that the scale-up of malaria interventions across sub-Saharan Africa has contributed to a reduction in under-five mortality [1]. The contribution of vector control measures, long-lasting insecticide nets (LLINs) and indoor residual spraying (IRS), to this effort cannot be overestimated, and should continue (assuming adequate resources) as long as vector populations remain susceptible to ‘public health’ insecticides [2, 3]. The susceptibility ‘condition’ for ongoing impact is by no means assured because of the small number of public health insecticide classes available. These include four classes for IRS (organochlorines (OCs), organophosphates (OPs), carbamates (CAs), and pyrethroids (PYs), and of even greater concern, only one, PYs, for use on nets.

The development and spread of insecticide resistance in the populations of Anopheles gambiae sensu lato (s.l.), a major vector of malaria in Africa, presents a serious threat to the progress made in malaria control. Extensive use of insecticides in agriculture and the scale-up of insecticide-based malaria vector control during the past decade appear to have played a pivotal role in the emergence and rapid spread of insecticide resistance on the continent [4, 5]. Resistance, especially to PY insecticides and dichlorodiphenyltrichloroethane (DDT), in An. gambiae s.l., occurs across Africa [2, 6‐8]. More recently, resistance to CA insecticides (bendiocarb and propoxur) and OPs (fenitrothion and malathion) has also been reported [2, 10, 11]. While few studies have assessed the public health impact of insecticide resistance, there is evidence of malaria vector control failure associated with pyrethroid resistance, [12, 13]. This threat may be more common than assumed since a recent systematic review and meta-analysis on the impact of pyrethroid resistance on the efficacy of LLINs points out that the heterogeneity of the studies masks relationships between resistance and control failure [14].

Anopheles gambiae s.l. populations show considerable heterogeneity in Mali. Anopheles coluzzii, Anopheles arabiensis, and Anopheles gambiae sensu stricto (s.s.) are present. Furthermore, there are at least two chromosomal forms of An. gambiae s.s.: Savanna and Bamako and a third one called Mopti that corresponds to An. coluzzi [15]. As early as 1987, the kdr allele was detected in the Savanna population from Bamako, and has increased in frequency over the years [16]. A more recent study on the spread of the kdr allele indicated a significant increase in frequency in the Savanna population and noted extension of the kdr allele to the Bamako chromosomal form for the first time [17].

The National Malaria Control Programme (NMCP) of Mali scaled up distribution of LLINs beginning in 2004, and is working towards universal coverage. Subsequently, IRS, also using PY class insecticides, was implemented in two districts: Bla and Koulikoro from 2008 to 2010, with a third district, Baraoueli, added in 2011, when CA insecticides were substituted for PY insecticides due to evidence of resistance in local vector populations [1]. Rotation to CAs (2011) was followed by another change in insecticide class, rotation to OPs in 2014 because of the short residual life of bendiocarb on muds walls. The increases in LLIN and IRS coverage in Mali, coupled with pesticide use in agriculture, have likely put selection pressure on malaria-transmitting mosquitoes, leading to an unfortunate emergence and spread of insecticide resistance [13].

The first step in managing resistance is to monitor its spread. Consequently, PMI has supported insecticide-resistance monitoring in Mali since 2007 and has documented the spread of resistance to DDT, PYs, and, most recently, CAs in An. gambiae s.l. in focal areas. Data from this effort form the basis of this report, which presents the current insecticide susceptibility/resistance status of An. gambiae s.l. populations at 13 sites across the central, south, and southwestern parts of the country. Additionally, we report on the presence and frequency of kdr and ace-1 ^R resistance mutations in An. gambiae sibling species, and assess the level and distribution of detoxifying enzymes, a second resistance strategy used by anopheline vectors. These results will help to mitigate the threat of resistance by informing a plan for resistance management and effective vector control interventions going forward.

To ensure the success of malaria vector control efforts and malaria elimination in Africa, it is critical that a strategic plan, informed by comprehensive monitoring and evaluation of resistance, be in place [27, 28]. The President’s Malaria Initiative (PMI) has supported this approach in Mali, focusing on areas where insecticide-based vector control measures (IRS and LLINs) have been deployed. One advantage of this ‘dual’ approach is that in addition to reducing transmission, and hence malaria burden, IRS, with its ability to draw on multiple classes of insecticide, can be used to manage the emergence of insecticide resistance, especially PY resistance that threatens the efficacy of LLINs [2, 3, 29].

There are two main reasons for ongoing support of vector insecticide resistance. First, information on malaria vector insecticide-resistance status is a key input to the decision process surrounding the choice of IRS insecticide. Therefore, PMI has supported vector insecticide-resistance surveillance to inform this issue, specifically the relative frequency of phenotypic resistance, by insecticide class. A second important programme issue informed by these data is the distribution and intensity of vector-pyrethroid resistance and its relationship to LLIN impact. There has been a universal coverage target for LLIN distribution since 2011. The spread and intensification of pyrethroid resistance threatens this strategy. Given the growing threat of insecticide resistance it is essential that up-to-date data on the magnitude and distribution of insecticide resistance be collected. Currently in Mali, PMI supports resistance monitoring annually in IRS target areas to inform the selection of an effective class of IRS insecticide. This study was conducted to expand resistance monitoring to 13 sites across the central, south, and southwestern parts of the country.

The utility of routine monitoring to update vector-insecticide resistance status can be seen by comparing recent (2009) data from WHO-AFRO-Mali [30] to these study results. Prior to our investigation, resistance to DDT, deltamethrin, and lambda-cyalothrin was reported from four, three, and seven out of eight sites, respectively, that were part of this study. However, for fenitrothion (OP) and bendiocarb (CA), all the vector populations tested were shown to be susceptible in 2009. The present results update this picture by showing that except in one site, Bougouni, where there was a possibility of resistance to deltamethrin, An. gambiae s.l. populations from all other tested sites were resistant to DDT, lambda-cyhalothrin, and deltamethrin. These results suggest cross-resistance between DDT and PY class insecticides exists, probably due to the kdr mutation. Evidence of fenitrothion resistance was seen in only one out of 13 sites in this study. While there was evidence of bendiocarb resistance at four of 13 sites, this is the first time that bendiocarb (CM) and fenitrothion (OP) resistance has been reported in Mali.

Insecticide selection pressure exerted on vector populations may explain the rapid spread of resistance. DDT is no longer officially sanctioned in Mali, neither for use in public health nor agriculture. Permethrin and deltamethrin, however, are used for malaria control through Ministry of Health (MOH)/NMCP distribution of LLINs, and lambda-cyhalothrin and deltamethrin were used for IRS in Koulikoro and Bla, two of the 13 sites studied from 2008 to 2009 and in 2010, respectively. From 2011 to 2013 bendiocarb was used for IRS in 3/13 districts, with the addition of Baraoueli to the IRS programme in 2011. In 2014, Bla and Baroueli were sprayed with pirimiphos-methyl, while Koulikoro was sprayed with bendiocarb.

All public health classes of insecticides are also used in agriculture, especially in cotton growing areas in Kita, Bougouni and Kadiolo, three of 13 sites described in this report. In this regard, it is interesting to note that there was An. gambiae s.l. resistance to bendiocarb (CA) observed in three places, and to fenitrothion (OP) in Kadiolo, where those insecticides were used for agriculture but not for public health activities. The intensive use of insecticide to control agriculture pests [31] may contaminate mosquitoes breeding sites, thus exerting significant and constant selection pressure on Anopheles larval populations. Such an effect might explain the emergence of insecticide resistance in malaria vector populations before they ‘see’ insecticide-based vector control interventions with any class of insecticides. The only way to detect this ‘stealth’ appearance of resistance is through monitoring. The emergence of resistance in populations of An. gambiae to common classes of insecticides used in public health has been reported in many countries in Africa, including Côte d’Ivoire [10, 32], Kenya [33], Benin [32, 34, 35], Niger [4], Burkina Faso [9], Mali [16], Nigeria [36, 37], South Africa [38] and Cameroon [39].

In addition to documenting phenotypic resistance, this study provides information on the frequency and distribution of common physiological resistance mechanisms such as the kdr-w mutation, probably one of the most important mechanisms for pyrethroid and DDT resistance. The significance of this finding is the identification of the kdr-w allele in An. arabiensis in Mali, in addition to An. coluzzii, An. gambiae s.s., previous reported from Mali. This finding is in agreement with previous reports from several other African countries that indicated the widespread of kdr-w mutations in the three major vector species of the An. gambiae complex. In a recent study conducted in Mali, Norris et al. [40] elucidated the dynamics of how An. coluzzii inherited the insecticide-resistance allele from the An. gambiae s.s., in areas of increased insecticide exposure due to high coverage of LLINs and the resistance genes subsequently spread in the population. In An. arabiensis kdr-w mutation is reported to have occurred through a de novo mutation event [41].

Increased selection pressure due to the increased (PY) LLIN coverage over time [42], the culture of using PY insecticides for crop protection in agriculture, IRS using PY class insecticides in three districts and even widespread use of pyrethrin-based aerosols, in combination, or alone might have been sufficient to drive kdr-w mutations to the high frequencies in An. gambiae s.l. Previous study results by Czeher et al. [4] indicated that large-scale countrywide distribution of LLINs led to an increased frequency of kdr-w mutations in Niger. Use of PYs at the household level and in small vegetable cultivation has also been reported to drive the kdr mutation to a higher frequency in Mali [16].

The ace-1 ^R allele that confers resistance to OPs and CAs [43] was present in four localities (Bla, Kita, Bougouni, and Kadiolo) at lower frequencies than kdr-w. Some mosquitoes were found carrying both resistant alleles simultaneously. OPs nor CAs have been deployed to Kita, Bougouni, and Kadiolo for malaria vector control but OPs are commonly used for crop protection. Indoor residual spraying with CAs was implemented in 2011 and 2012 in Bla before this study was conducted. This might explain the ace-1 ^R mutation observed in those sites. The frequency and distribution of the ace-1 ^R allele in the other study sites are unknown and further investigation is required to map the distribution and gain information on the frequency of the allele from nationally representative sites and further understand its linkage with use of pesticides for agriculture.

Although the data did not allow us to assess whether there was any association between kdr and ace-1 ^R mutations and phenotypic resistance, previous studies have established association between target site mutation and phenotypic resistance [5, 10, 44]. Apart from target site resistance, data on levels of metabolic resistance mechanisms suggest that these might have contributed to the overall profile of insecticide resistance observed in Mali. Elevated levels of GST activity were detected in eight of nine sites. GSTs breakdown DDT and catalyze PY induced lipid peroxidation [45, 46]. The widespread DDT and PY resistance observed in Mali might, therefore, be due to the complementary effect of overexpressed GST and high frequency kdr–w mutations.

An overall increase in cytochrome P450 monooxygenases and elevated levels of non-specific esterases (NSE) activity were also detected in two and four out of 9 sites, respectively. Elevated NSE activity has been found to play an important role in OP and CA resistance in a number of arthropod species, including mosquitoes [46]. Similarly, overexpressed cytochrome P450 monooxygenases has been reported to have an association with insect resistance to DDT and PYs [46]. Hence, these two enzymes, where overexpressed, might have contributed to the insecticide resistance frequency observed in Mali.

The results of this study revealed wide distribution of PY and DDT resistance in the population of An. gambiae s.l. in Mali. Resistance to CAs and OPs was also detected in some study sites. The study also demonstrated that multiple insecticide-resistance mechanisms have evolved in An. coluzzii, An. gambiae s.s. and An. arabiensis in Mali. The extent and variety of phenotypic resistance and the physiological mechanisms associated with it, serve as a ‘wake-up call’ for ongoing support of evidenced-based decision making around insecticide-based malaria control efforts. The results of this study highlight the importance of routine resistance monitoring to update the information base for rational deployment of the existing tools for effective control of malaria in Mali. The implications and operational impact of resistance to malaria control efforts needs to be urgently evaluated. Appropriate control strategies need to be put in place in a context of Insecticide Resistance Management. Innovative vector control tools that include new active ingredients for IRS and LLINs might be needed to complement or replace the existing strategies in areas of vector resistance.