Background
Malaria is a major cause of mortality and morbidity in The Democratic Republic of the Congo (DRC), with over 40,000 deaths per year [
1]. It is estimated that 60% of the country’s population live in areas with an average
Plasmodium falciparum prevalence above 50% (hyper-endemic to holoendemic transmission), making the DRC one of the countries with the most intense transmission [
2]. Efforts to reduce the malaria burden are focusing on case management and treatment, and on vector control via the distribution of long-lasting insecticide-treated nets (LLINs) [
2]. Successful implementation of a vector control programme is reliant on knowledge of vector species and their resistance to insecticides. Few recent studies have been published on the vector species of the DRC or their insecticide resistance and those that exist are principally concerned with locations to the South and East of the country. Over 60 species of
Anopheles have been described in the DRC, with
Anopheles gambiae sensu lato (s.l.) and
Anopheles funestus thought to be the main malaria vectors, but other species, such as
Anopheles pharoensis, Anopheles moucheti, and
Anopheles coustani are potentially important for transmission (reviewed in [
2]). In the
An. gambiae s.l. species group,
An. gambiae sensu stricto (s.s.) was found to be the predominant vector in eastern DRC, whereas
Anopheles coluzzii was the main species found in Bandundu in the West. Both species were found in sympatry in several locations including Kinshasa, as well as Kisangani and Lodja (central West) and Kalemie (East). Only
An. gambiae s.s. was found in Equateur Province (North West) [
1,
3‐
5].
Contemporary data on insecticide resistance status in malaria vectors is improving but remains sparse for DRC and central Africa generally [
6]. A study in 2009 of four locations, Kingasani and Kimpese in the South West, Bolenge, in the West, and Katana located in the East demonstrated that all
An. gambiae s.s. populations were resistant to DDT, three were resistant to the pyrethroids deltamethrin, permethrin, lambda-cyhalothrin, and a single population was resistant to the organophosphate malathion [
3]. In 2012,
An. gambiae s.s. in the North East were found to be resistant to deltamethrin, DDT and bendiocarb. Pre-exposure to PBO (piperonyl butoxide), a synergist that inhibits the activity of cytochrome P450 enzymes and some esterases which may be involved in the detoxification of pyrethroids, significantly increased mortality in bioassays suggesting that metabolic enzymes were at least partly responsible for the resistance phenotype [
7]. A study in 2013 of two sites near Kinshasa found that
An. coluzzii were resistant to DDT and permethrin but fully susceptible to propoxur, bendiocarb and deltamethrin. Pre-exposure to PBO did not restore susceptibility in this population [
8]. A recent study carried out from 2013 to 2016 found
An. gambiae s.l. populations were resistant to permethrin in five of seven provinces studied in 2016 (Lodja and Kabondo located centrally, Mikalayi in the South, Kingasani in the West, and Kalemie in the East). A significant impact of PBO showed that metabolic resistance was involved in four of these sites. Resistance to deltamethrin was observed in Mikalayi and Kabondo, whilst resistance to DDT was observed in all six provinces where monitoring was carried out [
5]. A study in Kinshasa in 2015, found that
An. gambiae s.l. were resistant to DDT, four types of pyrethroid, dieldrin and bendiocarb, and that whilst P450 enzymes were involved, they were only partly responsible for the resistance observed [
4].
Point mutations in the voltage gated sodium channel (VGSC), the target for pyrethroids, at the L1014 locus (L995 using
An. gambiae codon nomenclature [
9]) typically cause knock down resistance (
kdr) to pyrethroids (and DDT), and are widespread in
An. gambiae s.l. [
10]. Two resistance alleles are found at this locus, resulting from the replacement of the wild type leucine allele with phenylalanine, L1014F [
11], or alternatively serine, L1014S [
12]. Both alleles can occur in the same population [
7,
13] and the frequency of both alleles appears to be increasing [
14‐
18].
In DRC the
Vgsc-1014F mutation was found at four locations in
An. gambiae s.s. specimens collected in 2009 with allele frequencies ranging from 0.13 to 0.95 [
3]. The L1014F
kdr mutation was also detected in
An. coluzzii at frequencies of 0.33 and 0.38 in two study sites near Kinshasa [
8]. The L1014F mutation was found in
An. gambiae s.l. in all five provinces studied in 2014 with extremely variable frequencies ranging from near fixation to near absence [
5]. The
Vgsc-1014S mutation has only been detected in one location in the North East where it occurred at high frequencies and co-occurred with both the
Vgsc-1014F mutation and the wild type allele [
7]. A study in Kinshasa, 2015, found both L1014S and L1014F, whilst the wild type allele was almost entirely absent [
4].
Detection of the
kdr mutations, which act as useful partial resistance diagnostics for pyrethroid resistance [
10], is important for monitoring the spread of resistance in areas were vector control is principally carried out using pyrethroid treated nets. A number of different assays exist for the detection of
kdr including allele specific PCR (AS-PCR) [
11,
12], Heated Oligonucleotide Ligation Assay (HOLA), [
19] Sequence Specific Oligonucleotide Probe Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) [
20], PCR-Dot Blot [
21], and the widely-used real-time TaqMan qPCR probe assay [
22]. However, all of these methods are reliant on performing two assays in order to detect both the
Vgsc-1014F and
Vgsc-1014S mutations. With the spread of both mutations across Africa [
9] there is a growing need for an assay that can detect both resistant alleles and the wild type allele in a single assay to aid interpretation, increase throughput, and reduce the cost per specimen. A newly-developed assay utilizing Locked Nucleic Acid (LNA) probes to simultaneously detect all three alleles whilst utilizing the same quantitative PCR platform used for TaqMan assays is presented. LNA probes incorporate modified RNA nucleotides that significantly increase target affinity and the melting temperature (Tm) of an allele-specific probe. This allows very short allele specific oligonucleotides to be produced that have a high difference in Tm between the target sequence and any mis-match sequence. Utilizing this technology, it was possible to design probes to detect all three alleles reliably in a single qPCR reaction and this assay was used to explore
kdr frequencies in samples from Nord Ubangi, DRC.
Discussion
The primary malaria vector species in the three study sites in Nord Ubangi was found to be
An. gambiae s.s., although
An. coluzzii was also found in low numbers. Data for this region of DRC are lacking, but previous studies in southern Equateur Province found only
An. gambiae s.s. [
1,
3]. The presence of
An. coluzzii in Nord Ubangi further extends the known range of this species.
Molecular analysis revealed the
kdr L1014F resistance mutation to be present at high frequency (over 0.79–0.90), and the
kdr L1014S mutation found to be present at moderate frequency (0.1–0.21) across all three study sites (Table
2). The wild-type susceptible allele was not detected in
An. gambiae s.s. but was found in the few
An. coluzzii specimens analysed. The N1575Y mutation, also located in the VGSC, has a synergistic effect on pyrethroid and DDT resistance when combined with the L1014F mutation [
27,
33]. Previously this mutation has been found in Burkina Faso, Ghana, Benin, Cameroon and Côte d’Ivoire [
27,
34‐
36]. It has been detected in both
An. gambiae s.s. and
An. coluzzii [
27,
34]. In this study the N1575Y was found at very low frequency in two of the three survey sites (Pambwa, 0.01 and Fiwa, 0.02) in
An. gambiae s.s. The mutation was found at a frequency of 0.25 (N = 4) in
An. coluzzii from Fiwa. This is first report of this mutation in DRC.
This study detected confirmed phenotypic resistance to deltamethrin, permethrin and DDT; the presence of both L1014 kdr resistance alleles at high frequency, the absence of the wild type L1014L mutation and the first detection of N1575Y. Such a resistance profile, mediated by both target site and metabolic mechanisms (indicated by PBO results) may provide challenges for LLIN-based vector control programmes in this region of DRC. However, susceptibility to bendiocarb and the absence of the Ace-1 G119S mutant suggest that indoor residual spraying (IRS) using non-pyrethroid formulations may not be compromised. Interestingly, the effect of PBO was not consistent across the two pyrethroids tested, with near-full susceptibility restored with deltamethrin, but not permethrin, suggesting a different balance in the contribution of different resistance mechanisms, with the latter perhaps more dependent upon target site resistance.
The LNA-kdr assay was found to perform as well as the TaqMan kdr assay in all populations of An. gambiae s.s. analysed, but with substantial time savings (67% reduction in run time) and since it utilizes non-proprietary probes also permits large cost savings (75%), which may be reduced further by use of different amplification master mixes. In addition, this single assay detection method may reduce the reporting of false positive wild-type alleles and permit the discovery of resistance alleles in places where they are not yet being screened for in single mutant detection assays. The development of a single assay for detection of both kdr alleles will allow rapid, reliable and low-cost screening of these important resistance mutations facilitating the monitoring of resistance across Africa.
Authors’ contributions
AL and DW conceived and designed the experiments. AO, AVN and DP made substantial contributions to the design and optimization of the LNA-kdr assay. Al, JM, KK and LBN were responsible for collecting mosquitoes and performing the experiments. TB provided resources and made contributions to conception and discussion of the study. AL, DW and MD conducted data analysis and wrote the manuscript. All authors read and approved the final manuscript.