DOI: https://doi.org/10.21203/rs.3.rs-1979481/v1
Background: Since 2000, Burkina Faso has experienced regular dengue cases and outbreaks making dengue a health concern for the country. Previous studies in Burkina Faso reported the resistance of Aedes aegypti to pyrethroid insecticides associated with F1534C and V1016I kdr mutations. The current study reports high resistance of Ae. aegypti populations to pyrethroid insecticides supported by 410L/1016I/1534C kdr haplotypes; and a new multiplex PCR-based diagnostic of 1534C and 1016I kdr mutations is proposed.
Methods: Larvae of Ae. aegypti were collected from three health districts of Ouagadougou in 2018. The resistance status of Ae. aegypti to pyrethroid insecticides was tested using CDC-bottle bioassays, and to malathion using WHO tube tests. Bioassay results were interpreted according to used protocols.
Results: Females from each health district were strongly resistant to permethrin and deltamethrin (<20% mortality) but were fully susceptible to 5% malathion. The F1534C and V1016I kdr mutations were successfully detected using a newly-developed multiplex PCR, which was validated by comparison with fluorescent probe-based TaqMan assays for each mutation. The V410L kdr mutation was detected using an allele-specific-PCR, which was confirmed by TaqMan assays, and owing to novelty in local Ae. aegypti populations, also direct DNA sequencing. The 1534C kdr allele was near fixation, while V1016I and V410L kdr alleles were strongly correlated with allelic frequencies range from 0.5 to 0.7 across the three-health districts. The 1534C/1016I/410L haplotype was correlated with permethrin resistance (χ21=33.7; p<0.001) but not with deltamethrin resistance (χ21=0.03; p=0.86), however, the test power was limited by a low frequency of dead individuals.
Conclusions: The trio of kdr mutations (F1534C, V1016I and V410L) may explain the high resistance to pyrethroids, however lack of substantial resistance to malathion suggests that this remains a viable option for dengue vectors control in Ouagadougou.
Aedes aegypti is a prolific mosquito with a worldwide distribution, transmitting important arboviruses such as dengue, yellow fever, chikungunya and Zika. Dengue is by far the most important arbovirus with 390 million cases and 25,000 deaths recorded annually worldwide. The most important contemporary dengue outbreak in Burkina Faso occurred in 2017 with 14,455 recorded cases and 29 deaths [1] and involved three (DENV-I, DENV-II and DENV-III) of the four dengue virus serotypes [2]. With increasing urbanization and human densities, the West African region is particularly at risk of dengue [3]. Ae. aegypti is closely associated with urban environments and in West Africa, where they breed preferentially in man-made breeding sites such as plastic containers, drums, used tires, flower pots [4]. Insecticide-based vector control and breeding site reduction are the only effective dengue prevention methods [5–7] in the absence of an effective dengue vaccine which can protect against all dengue virus serotypes [5]. However, the intensive use of insecticides has led to worldwide development of resistance in Ae. aegypti populations [8] driven primarily by target site mutations and metabolic resistance mechanisms [8, 9].
Multiple target site knockdown resistance (kdr) mutations that affect pyrethroid susceptibility have been identified in Ae. aegypti of which the most common is the F1534C mutation. This mutation has a worldwide distribution [8] and is frequently found associated with the V1016G and S989P kdr mutations in Asia, and the V1016I kdr mutation in Latin America [10] and Africa [11, 12]. In addition to these kdr mutations, an important variant (V410L; also known as V419L using Ae. aegypti codon numbering) in the first domain of the voltage-gated sodium channel (Vgsc) has been detected more recently [13, 14]. Originally reported in a laboratory strain in Brazil [15], subsequent reports have shown high frequencies of this mutation in Colombia [16] and in Mexico, with steep increases seen from a first detection over 15 years ago [13, 17]. Most recently V410L has been reported in Central and West Africa [18–20]. Several of the Vgsc mutations are not associated with pyrethroid resistance when they occur alone [21], whilst V410L, V1016G and F1534C can confer resistance but with effects amplified by co-presence of additional mutants [22].
The current study reports high resistance to pyrethroid insecticides, at least partially underpinned by a trio of kdr mutations, a new diagnostic is proposed the 1534C and 1016I. Full susceptibility to malathion is recorded, an approved alternative for Ae. aegypti populations control in Ouagadougou.
Aedes aegypti larval collection and mosquito rearing
Ae. aegypti immature stages (larvae and pupae) were collected in Ouagadougou from three health districts (Baskuy, Bogodogo and Nongremassom). These health districts were selected based on variable dengue burden reported during the 2016 outbreak: Bogodogo and Nongremassom having recorded the highest numbers of dengue cases while Baskuy recorded few cases. Immatures were collected from tyres in Bogodogo and Nongremassom and from plastic containers in Baskuy. Signed informed consent was required from house owner when collected breeding sites are situated inside household. Collections were transported to the insectary of Université Joseph Ki-Zerbo at Ouagadougou and reared to adults using Tetramin®. Emerged adults were provided with 10% sugar solution. All life stages were maintained at 25–27°C temperature and 75–85% relative humidity.
Bioassays with pyrethroid insecticides were performed using 250 ml Wheaton bottles according to the CDC bioassay protocol [23]. A batch of 25 unfed females of Ae. aegypti mosquitoes of 3–5 day-old were exposed to insecticide in four bottles coated at the recommended concentrations: 15 µg/bottle for permethrin and 10 µg/bottle for deltamethrin dissolved in acetone. An additional bottle coated with acetone only used as negative control. Bioassays with malathion (5%) were carried out using WHO tubes bioassay [24]. All bioassays were carried out in parallel with susceptible Rockefeller laboratory strain, at 27°C with 82 ± 3% relative humidity. After 1 h exposure, mosquitoes were transferred to either cardboard cups covered in mesh following the CDC-Bottle bioassay, or to holding tubes following the WHO-tube bioassay. The number of dead mosquitoes was recorded at 24 h after bioassay. Mortality rates were calculated and adjusted with Abbott’s correction when the mortality in the control tube was between 5 and 20% [25]. Dead and alive mosquitoes were stored in 1.5 ml tubes over silica gel for molecular analysis.
DNA was extracted from 431 mosquitoes including 407 from pyrethroid-treated bottles and 24 from control bottles. For multiplex and AS-PCR, mosquito DNA was extracted following a previously described protocol [12], and the DNA pellet was suspended in 20 µl of TE buffer and stored in a freezer until use whilst for taqman method, extraction was performed as described in [26].
Multiplex PCR detection of the F1534C and V1016I kdr mutations detection
To simplify genotyping of two common kdr mutants, a multiplex PCR was developed using known genotypes from previously-sequenced Ae. aegypti DNA [12] as positive samples. F1534C detection primers [27] and V1016I detection primers [28] were mixed in a single tube for the detection of the two mutations (Additional file 1: Table S1). In total, seven primers were mixed in the single PCR reaction tube to simultaneously characterize the two SNPs (F1534C and V1016I).
The PCR reaction contained 1 µl of target DNA, 12.5 µl of DNA polymerase mix, (AmpliTaq, Thermo Fisher Scientific), 0.5 µl of c1534-f and c1534-r primers (1 µM), 0.125 µl of Ae1534F-r, Ae1534C-f, Iso1016f and Iso1016r primers (0.25 µM), 0.25 µl of Val1016f primer (0.5 µM). The PCR reaction volume was completed to 25 µl with distilled water. The PCR was performed on a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.) with the following program: 95°C/5 min for 1 cycle, (95°C/30 s, 55°C/30 s, 72°C/45 s) for 30 cycles, 72°C/10 min for 1 cycle, and maintained at 4°C after the PCR is completed. PCR products were mixed with 5 µl of 6x loading buffer and loaded on 3% TBE agarose gel. After 1 h of electrophoresis in 0.5% TBE buffer, the gel was stained for 10 min in 0.5 mg/ml ethidium bromide and de-stained in distilled water for 5 min for visualization of bands under UV light (Fig. 1).
V410L kdr mutation genotyping by Allele-specific (AS) -PCR
The V410L kdr mutation was screened using AS-PCR described by [16]. The wild-type and mutant alleles were detected in two different PCR reactions. Briefly, each PCR reaction contained 1 µl of target DNA, 6.25 µl of DNA polymerase mix (AmpliTaq, ThermoFisher Scientific), 0.3 µM of each primer (Additional file 1: Table S1). The PCR reaction volume was completed to 12.5 µl with distilled water. The PCR conditions were 95°C for 5 min and 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by a final extension step of 72°C for 7 min. PCR products were mixed with 6x loading buffer and 5 µl of the mixture was loaded on a 1.5% TAE agarose gel. After electrophoresis and ethidium bromide staining, bands sizes were interpreted according to [16].
Taqman detection of kdr mutations
A total of 134 mosquitoes were screened by taqman qPCR for the cross-validation of the V1016I and F1534C mutants detected in the novel multiplex assay and V410L Vgsc mutations [29]. Briefly, reactions were performed in 96 well plates by adding 5 µl of taqman gene expression SensiMix (Applied Biosystem, Foster city, USA), 0.125 µl of primer/probe, 3.875 µl of molecular grade sterile water and 1 µl of the DNA extract [26]. Reactions were run on an Agilent MX3000P qPCR thermal cycler using cycling conditions of an initial denaturation of 10 min at 95 ̊C, followed by 40 cycles of 92 ̊ C for 15 min and 60 ̊ C for 1 min.
DNA sequencing was used as a gold standard to validate the genotyping of the V410L kdr mutation. A partial sequence of domain I of the voltage-gated sodium channel was amplified by PCR in a 25 µl volume with iCycle thermal cycler (Bio-Rad Laboratories). Each reaction contained 12.5 µl of PrimeSTAR® Max DNA polymerase (TAKARA BIO INC), 1.5 µl (0.3 µM) of the reverse primer described by [16] and a newly designed forward primer to increase the amplification efficiency (Additional file 1: Table S1) and sterile water was added to make a final volume of 25 µl. Thermal cycling conditions were one cycle of denaturation at 98°C for 5 min, followed by 35 cycles of 98°C/10 s, 60°C/15 s, and 72°C/5 s, and ended by one cycle at 72°C during 7 min for the final extension. Ten microliters of the mixture of PCR products and 6x loading buffer were run on a 1.5% TAE agarose gel and the expected DNA band size was read at 688 bp. The DNA fragment was cut from the gel and purified from gel using a QIAEX® II Gel Extraction Kit according to manufacturer instructions. Purified PCR products were sequenced by Fasmac sequencing service (FASMAC Co., Ltd., Japan). Sequence alignment was performed with Snap Gene® Viewer 5.0.5 software.
Pyrethroid bioassay data were interpreted according to CDC guidelines: mosquitoes were considered susceptible if the mortality rates were over 98%, possibly resistant is mortality rates are between 80–97% and resistant if mortality rates were under 80% [23]. Malathion data were interpreted according to WHO guidelines with susceptibility as 90%. All statistical analyses were performed using R version 3.4.3 [30].
Chi-square tests were used to compare kdr allele frequencies between insecticide phenotypes (live or dead) and among Aedes larval collection sites.
Significance in the spatial variations in mortality of Ae. aegypti adults due to pyrethroid insecticide (permethrin and deltamethrin) were evaluated using a Generalised Linear Model (GLM) with binomial link function followed by Tukey’s test for mean separation. Linkage disequilibrium between the two-kdr mutations, V410L and V1016I was quantified (as r2) using Genetics package version 1.3.8.1.3 in R software. Data were pooled across the three districts to increase sample size to detect linkage disequilibrium and to test phenotype-kdr associations
Susceptibility status of Aedes aegypti to pyrethroid and malathion insecticides
Ae. aegypti populations from the three health districts were all strongly resistant to permethrin and deltamethrin at the recommended concentrations for both insecticides (< 20% mortality) (Fig. 2), while the Rockefeller strain was fully susceptible as expected (100% mortality). Whilst overall mortality did not vary between insecticides or localities, the significant insecticide x locality interaction term in the generalized linear model indicates some inconsistency, with permethrin mortality in Bogodogo and Nongremassom significantly higher than deltamethrin mortality in Baskuy (Table 1).
|
Predictors |
Estimate |
95%CL |
z-value |
Pr(>|z|) |
|---|---|---|---|---|
|
Intercept |
-2.47 |
[-3.00–1.94] |
-9.18 |
< 0.001 |
|
Insecticide [Deltamethrin] |
||||
|
Permethrin |
-0.54 |
[-1.39–0.31] |
-1.24 |
0.22 |
|
Locality [Baskuy] |
||||
|
Bogodogo |
-0.22 |
[-0.99–0.55] |
-0.56 |
0.58 |
|
Nongremassom |
-0.26 |
[-1.07–0.54] |
-0.64 |
0.52 |
|
Insecticide [Deltamethrin]: Locality [Baskuy] |
||||
|
Permethrin: Bogodogo |
1.68 |
[0.60–2.77] |
3.04 |
< 0.01 |
|
Permethrin: Nongremassom |
1.49 |
[0.36–2.62] |
2.58 |
0.01 |
Allele and genotype frequencies of V410L, V1016I and F1534C kdr mutations
Genotypes of samples from sequencing method were used as the gold standard to develop the one step multiplex PCR for a simultaneous detection of the 1016I and 1534C mutations. Of the 134 samples screened by the Taqman, 27, 59 and 47 were respectively identified as CCVV, CCVI and CCII genotypes. The multiplex PCR method returned the same genotypes in 133 of the samples. The final sample did not score successfully for F1534C with Taqman but which was genotyped as 1534CC by the multiplex method so a comparison was not possible.
Detection of the V410L kdr mutation in Ae. aegypti was confirmed by TaqMan in the same 134 mosquitoes with total agreement, and also by direct DNA sequencing in eight Ae. aegypti mosquitoes, confirming the expected substitution of G by T (GTA→TTA) (Fig. 3).
Genotyping of F1534C, V1016I, and V410L kdr mutations was carried out on 431 Ae. aegypti mosquitoes in total (407 pyrethroid exposed and 24 control mosquitoes); results are shown in Table 2. The overall mutant allelic frequencies were 0.999 (95% CL: 0.993-1.000) for 1534C; 0.636 (95% CL: 0.603–0.667) for 1016I; and 0.624 (95% CL: 0.591–0.656), for 410L. The 410L and 1016L kdr alleles displayed similar frequencies in Baskuy and Nongremassom but a significantly lower frequency in Bogodogo (Fig. 4). The V1016I and V410L kdr mutations were in near perfect linkage disequilibrium (r2 = 0.977). As a consequence, these two mutations were subsequently analysed together.
|
Locality |
V410L genotype |
Freq. of L allele |
V1016I genotype |
Freq. of I allele |
F1534C genotype |
Freq. of C allele |
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
LL |
VL |
VV |
II |
VI |
VV |
CC |
FC |
FF |
||||
|
Baskuy |
72 |
56 |
15 |
0.699 |
76 |
52 |
15 |
0.713 |
142 |
0 |
0 |
1.000 |
|
Bogodogo |
38 |
75 |
41 |
0.490 |
41 |
72 |
41 |
0.500 |
154 |
0 |
0 |
1.000 |
|
Nongremassom |
64 |
58 |
11 |
0.699 |
67 |
56 |
11 |
0.709 |
133 |
1 |
0 |
0.996 |
|
Total |
174 |
189 |
67 |
0.624 |
184 |
180 |
67 |
0.636 |
429 |
1 |
0 |
0.999 |
Kdr allele and genotype association with Aedes aegypti resistance phenotype
With only a single wild type allele specimen detected in all the samples, there was no evident difference in F1534C allele frequency between dead and alive females exposed to pyrethroids.
Only five genotypes were recorded across the three kdr mutations. The triple-homozygote mutant, CIL/CIL, was found at 24.7%, 47.7% and 50.0% in Bogodogo, Nongremassom and Baskuy, respectively (Table 3) and was significantly associated with permethrin resistance (χ21 = 33.72; p < 0.001) with a near 10-fold resistance advantage (4.6% mortality) compared to the single mutant CVV/CVV genotype (42.9% mortality). In contrast, for deltamethrin resistance, no association of CIL/CIL genotype to resistance was recorded (χ21 = 0.031; p = 0.861), though the very low number of dead individuals limited test power (Table 4).
|
Locality |
Genotype frequency |
||||
|---|---|---|---|---|---|
|
CIL/CIL |
CIV/CIL |
CVV/CIL |
CVV/CVV |
FIL/CIL |
|
|
Baskuy |
0.500 |
0.028 |
0.366 |
0.106 |
0.000 |
|
Bogodogo |
0.247 |
0.019 |
0.468 |
0.266 |
0.000 |
|
Nongremassom |
0.474 |
0.015 |
0.421 |
0.083 |
0.008 |
|
Genotypes |
Alive |
Dead |
Mortality (%) (CI95%) |
IL vs VV |
|---|---|---|---|---|
|
Permethrin |
χ2 ; p-value |
|||
|
IL/IL |
104 |
5 |
4.59 (1.98–10.29) |
χ21 = 33.72 p < 0.001 |
|
VV/IL |
97 |
16 |
14.169 (8.91–21.77) |
|
|
VV/VV |
24 |
18 |
42.86 (29.12–57.79) |
|
|
Deltamethrin |
||||
|
IL/IL |
60 |
3 |
4.76 (1.63–13.09) |
χ21 = 0.031 p = 0.861 |
|
VV/IL |
52 |
4 |
7.14 (2.81–16.98) |
|
|
VV/VV |
21 |
1 |
4.55 (0.81–21.80) |
I and L are the mutant alleles of V1016I and V410L while V are the wild-type alleles for both kdr mutations
Here, we developed a multiplex PCR for simultaneous detection of F1534C and V1016I kdr mutations in one reaction tube, useful to save time and resources, compared to individual detection of the two mutations. Results compared well with those from an established method to detect the mutants separately, with complete agreement where full genotypes were available from each method. Ae. aegypti populations from Baskuy, Bogodogo and Nongremassom health districts were susceptible to malathion at the dose tested as previously reported in some localities of Burkina Faso including elsewhere in Ouagadougou [26, 31]. Noting that the malathion dose used in the study was > 6-fold higher than the diagnostic dose of 0.8% for Aedes mosquitoes, the test result indicates a lack of substantial resistance rather than full susceptibility. These results reflect those from a previous review of Ae. aegypti malathion resistance across Africa [3], and more recent studies from Senegal, which found susceptibility to diagnostic doses of 0.8% and 5% [32], and to 5% malathion in Sudan [33] and Côte d’Ivoire [19]. This suggests that malathion remains a viable option for control of Ae. aegypti populations during dengue outbreaks in Africa, in contrast with at least some populations from America [34] and Asia [35].
In the three health districts involved in this study, Ae. aegypti populations were highly resistant to deltamethrin and permethrin as previously reported from elsewhere in Ouagadougou [12, 26]. This resistance was supported by near-fixation of the 1534C mutant and high frequency of the 1016I and 410L mutations. The 1016I kdr allelic frequency has increased rapidly in Ouagadougou within a two year period (χ21 = 97.22, p < 0.0001) from 0.20 and 0.47 in previous collections in 2016 [12, 26] to 0.64 in the current study (2018). The V410L kdr mutation was reported at high frequency (0.62) and in near-perfect linkage disequilibrium with V1016I suggesting that this mutation has been present, but undetected in Burkina Faso for some time. Toé et al. [20] detected V410L kdr mutation in Burkina Faso at frequencies ranging from 0.1 in Banfora to 0.36 in Ouagadougou using a real-time melting curve qPCR method. The 410L allele frequency reported for Ouagadougou is lower than that found in the current study, suggesting possible spatial and/or temporal fluctuation.
The V1016I, V410L, and F1534C kdr alleles co-occur in Ae. aegypti population from Burkina Faso and may have displayed similar dynamics as in Colombia [16] and Mexico [13] with positive selection for association of the three mutations. The V1016I and V410L kdr alleles varied significantly among sites (Fig. 4). Whilst we found significant association of the F1534C/V1016I/V410L haplotype with permethrin resistance. Lack of a significant association with deltamethrin resistance does not necessarily mean lack of any contribution of the kdr haplotype to phenotype, but rather, may reflect low power from the very low number of dead individuals. However, a contribution from other resistance mechanisms, perhaps including P450 enzyme overexpression [26], cannot be ruled out for either pyrethroid phenotype. The driver(s) of the increase in kdr genotype frequencies are unclear but recent studies in Ouagadougou recorded that 50% of households use pyrethroid insecticides as sprays or coils, and pyrethroid-treated ITNs were found in approximately 80% of households [4]. Such pressures could readily lead to the selection for Ae. aegypti carrying kdr alleles.
Ae. aegypti collected from three health districts in Ouagadougou, Burkina Faso were strongly resistant to deltamethrin and permethrin. Permethrin resistance was associated with kdr mutations, including the recently detected 410L mutant which was recorded at high frequencies. A multiplex PCR was developed and validated to detect simultaneously the F1534C and V1016I kdr mutations to complement the AS-PCR available for V410L for insecticide resistance surveillance.
Acknowledgments
The authors thank members and leaders of communities in the localities of 1200 Logements, Tabtenga and Goundry for their permission to perform the study and their cooperation throughout.
Funding
This work was supported by a WHO/ TDR grant (WHO/TDR/ RCS-KM 2015 ID235974), and the International Collaborative Research Program for Tackling the NTDs Challenges in African countries from Japan Agency for Medical Research and Development, AMED (JP17jm0510002h0003).
PJM’s research on peri-domestic behavior of Aedes aegypti receives support from MRC-UK (MR/T001267/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
The data of the current study are available from the corresponding author on request
Author Contributions
Conceptualization: Athanase Badolo, David Weetman, Philip J. McCall.
Data curation: Aboubacar Sombié, Athanase Badolo.
Formal analysis: Aboubacar Sombié, Athanase Badolo, David Weetman
Funding acquisition: Athanase Badolo, David Weetman, Philip J. McCall, Hirotaka Kanuka.
Investigation: Aboubacar Sombié, Mathias W. Ouédraogo, Manabu Oté, Erisha Saiki, Tatsuya Sakurai, Félix Yaméogo, Hirotaka Kanuka.
Methodology: Aboubacar Sombié, Athanase Badolo, David Weetman, Philip J. McCall, Hirotaka Kanuka.
Project administration: Athanase Badolo, Antoine Sanon, Hirotaka Kanuka.
Resources: Athanase Badolo, Antoine Sanon, Hirotaka Kanuka.
Software: Aboubacar Sombié, Athanase Badolo.
Supervision: Athanase Badolo, Hirotaka Kanuka.
Validation: Athanase Badolo, David Weetman, Hirotaka Kanuka.
Visualization: Aboubacar Sombié, Athanase Badolo.
Writing – original draft: Aboubacar Sombié, Athanase Badolo.
Writing – review & editing: David Weetman, Hirotaka Kanuka, Philip J. McCall.
Ethics approval and consent to participate
The study was approved by the National Ethical Research Committee of the Ministry of Health (No 2017-8-0126 of 02/08/2017). Signed informed consent was obtained from all householders included in the study before starting the field collection
Consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interests.
Author details
1Laboratoire d’Entomologie Fondamentale et Appliquée, Université Joseph Ki-Zerbo, Ouagadougou, Burkina Faso. 2Programme National de Lutte contre les Maladies Tropicales Négligées, Ministre de la Santé, Burkina Faso. 3Center for Medical Entomology, The Jikei University School of Medicine, Tokyo, Japan. 4Department of Tropical Medicine, The Jikei University School of Medicine, Tokyo, Japan. 5Laboratory Animal Facilities, The Jikei University School of Medicine, Tokyo, Japan. 6Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK