Monitoring of pyrethroid resistance in Aedes aegypti: first report of double and triple kdr mutations in Buenos Aires Province

doi:10.21203/rs.3.rs-4681871/v1

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Monitoring of pyrethroid resistance in Aedes aegypti: first report of double and triple kdr mutations in Buenos Aires Province

https://doi.org/10.21203/rs.3.rs-4681871/v1

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published 09 Nov, 2024

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Background:

Dengue is an emerging disease in Argentina, due to the colonization of Aedes aegypti, the mosquito vector. Buenos Aires Province is the biggest and most populated Province in Argentina, suffering dengue outbreaks of growing magnitude. During epidemic periods, pyrethroid insecticides are used in this country for the control of adult mosquitoes. Pyrethroid resistance in dengue vectors has been reported worldwide, making necessary the implementation of resistance management strategies in the control campaigns. The voltage-gated sodium channel is the target site of pyrethroids. Mutations in the gene encoding this protein, called kdr mutations, are usually the molecular cause of pyrethroid resistance in insects. In Ae. aegypti from the Americas, three kdr substitutions were described: V410L, V1016I and F1534C. The diagnostic of kdr markers is recommended for the early detection of pyrethroid resistance, and the consequent planning of evidence-based control policies. The study presented here aimed to provide essential information on the detection of pyrethroid resistance, facilitating the planning and evaluation of initiatives to control dengue vector populations, and providing evidence of kdr evolution in Ae. aegypti around their Southern distribution range in Latin America.

Methodology and findings:

This was a multicenter study carried out through a joint effort of academic and governmental institutions that have formed the Argentine Network for Surveillance of Resistance to Pesticides Exclusively for Public Health. We distributed ovitraps across 16 localities in Buenos Aires Province, collecting a total of 22,123 eggs. A total of 522 mosquitoes were genotyped in positions 1016 and 1534 of voltage-gated channel, by using multiplex-High Resolution Melting and/or TaqMan probe methods. A subset of 449 samples was also genotyped by a singleplex High-Resolution Melting method developed ad hoc, and/or Sanger sequencing. We documented for the first time in the central region of Argentina the presence of the 1016I^kdr + 1534C^kdr allele, probably due to the selective pressure exerted by pyrethroids during recent dengue outbreaks. Additionally, our study reports the first identification of the V410L mutation in Argentina. These results underscore a growing trend of pyrethroid resistance in Ae. aegypti, fueled by the widespread use of these insecticides.

Conclusions and Significance:

Our results indicate that the pressure exerted by pyrethroids led to the emergence and expansion of 1016I^kdr + 1534C^kdr mutations in Ae. aegypti from Buenos Aires. We also detected V410L mutation in Argentina for the first time, indicating that pyrethroid resistance is a serious problem in the country, such as in other countries of the region. We also improve the processivity and accuracy of genotyping methods. The results are both a tool for resistance monitoring and a sign of alarm to direct efforts in the finding of sustainable methods for vector control, to complement or replace pyrethroids in the near future. Joined efforts between academia and authorities in public policies for vector control is a productive way for the transference of scientific results to its application in public health.

DENGUE

MOSQUITO

HIGH RESOLUTION MELTING

VECTOR MANAGEMENT

INSECTICIDE

Aedes aegypti, a vector of arboviruses such as dengue, Zika and chikungunya, has re-colonized Argentina northeast since 1987 [1]. The province of Buenos Aires (BAP), the biggest and most populated district in Argentina, was reinfested in 1991 [2], with high levels of mosquito dispersion found in private dwellings. Five dengue outbreaks of increasing magnitude occurred in 2009, 2016, 2020, 2023 and 2024. In the 2023 outbreak, the situation has been aggravated by the simultaneous circulation of two dengue virus serotypes (DENV-1 and DENV-2), whereas in 2024 DENV-1, DENV-2 and DENV-3 are circulating, augmenting the risk of severe cases. Nearly 10,000 dengue confirmed cases were reported in the 2022–2023 outbreak in Buenos Aires Province (BAP), whereas almost 100,000 confirmed cases are being reported for 2023–2024 until epidemiological week 20-2024 (https://www.gba.gob.ar/saludprovincia/boletines_epidemiologicos/1000?page=45). These numbers illustrate the extraordinary expansion of dengue in the region and are the cause of the serious concern for Ae. aegypti control.

Vector control is the method of choice for reducing transmission of arbovirus and other vector-borne diseases. Elimination of domestic breeding sites and the use of larvicides are measures that should be implemented throughout the year, to minimize arbovirus outbreaks. The use of adulticides is recommended to be restricted to epidemic periods [3]; for this, the neurotoxic pyrethroids are the most used compounds given their favorable toxicological properties. Even though the organophosphate malathion is used in cases of high pyrethroid resistance in the Americas, its application in Argentina was banned for their use in dwellings, being pyrethroids the only insecticide allowed in this country for public health applications [4]. This situation is challenging, given that the continued use of neurotoxics elicited the emergence of resistant vector populations worldwide. In the case of Ae. aegypti, pyrethroid resistance was detected in the whole distribution range of the species, making this group of insecticides obsolete in wide regions. For mosquito control, the World Health Organization (WHO) recommends an integrated management, that must incorporate resistance monitoring and management strategies [3].

Pyrethroid insecticides exert a toxic action through their target site, the voltage-gated sodium channel (VGSC), present in the membrane of excitable cells [5]. Insecticide resistance is a complex trait; one of the main causes of pyrethroid resistance is the existence of point mutations in the vgsc gene, called knockdown resistance mutations (kdr). These mutations are usually located in regions of the molecule that interact with pyrethroids (pyrethroid sites 1 and 2) [6]. Several kdr mutations have been described in Ae. aegypti [7]; in the Americas region, the more important ones, given their distribution range and association to resistance, are Val to Leu in position 410 (V410L), Val to Ile in position 1016 (V1016I) and Phe to Cys in position 1534 (F1534C) of VGSC protein (Fig. 1). In a recent work [8] we demonstrated for the first time the presence of 1534C^kdr mutation in BAP, whilst 410L^kdr or 1016I^kdr were not reported in the central region of Argentina to date.

Considering 1016 and 1534 positions, four kdr alleles are possible 1016V + 1534F (sensitive: S); 1016V + 1534C^kdr (resistant 1: R1); 1016I^kdr + 1534C ^kdr (resistant 2: R2); 1016I^kdr + 1534F (resistant 3: R3). Whereas R1 and R2 alleles are widely extended in Latin America, R3 was detected with a very low frequency (≤ 0.1% of the samples) in Brazil [9], México [10] and Florida [11], but not in Argentina to date. In our previous study analyzing samples collected in 2018–2019 [8], 1016I^kdr + 1534C ^kdr allele was detected in northern Argentina but not in BAP. This could be due to the more recent history of dengue in BAP, where the first outbreak was reported in 2016. From then, outbreaks of increasing magnitude occurred in this Province in 2020, 2023 and 2024. This scenario allows us to study the evolution of kdr genotypes in Ae. aegypti after two outbreaks in which pyrethroid-based adulticide campaigns were carried out.

Kdr genotyping of natural populations is relevant both for scientific and practical reasons, given that these mutations can be considered a molecular marker of pyrethroid resistance, providing information necessary for planning and evaluation of control interventions [12]. The use of TaqMan probes has been adopted as the gold-standard method for genotyping kdr in Ae. aegypti. Even though it is a reliable and robust method, probes are expensive, and multiplexing is not possible, making it necessary the use of a single reaction for each mutation. This makes TaqMan a sub-optimal method for implementation in wide regional control campaigns. We recently developed a multiplex High-Resolution Melting (mHRM) assay for rapid genotyping of both 1016 and 1534 positions in Ae. aegypti vgsc gene [8].

In the current context, it becomes imperative to develop integrated intervention strategies at the regional level, adapted to emerging and re-emerging diseases, particularly those caused by arboviruses. In response to this need, the Argentine Network for Surveillance of Resistance to Pesticides Exclusively for Public Health (RAReP) was formed in Argentina in 2021, comprising institutions from the National Ministry of Health and scientific-technical entities. RAReP mission is to contribute to the monitoring and management of pesticide resistance in arthropod vectors of pathogens by the generation of scientific evidence, to optimize efficiency in vector control. Within this framework, we present the findings of our collaborative multi-center study, which involved both scientific research and governmental teams. Our study aimed to provide essential information on the detection of insecticide resistance, facilitating the planning and evaluation of initiatives to control dengue vector populations. We documented for the first time in the Central region of Argentina the presence of the 1016I^kdr + 1534C^kdr allele, probably due to the selective pressure exerted by pyrethroids during recent dengue outbreaks. Additionally, our study reports the first identification of the V410L mutation in Argentina. These results underscore a growing trend of pyrethroid resistance in Ae. aegypti, fueled by the widespread use of these insecticides. Since pyrethroids are the only insecticides approved for domestic and sanitary use in Argentina, the perspectives of our study have critical implications for evidence-based public health policies of dengue vector control.

Study area

BAP is the biggest and most populated district in Argentina, considering both its surface (307.571 km²) and the number of inhabitants (around 17.5 million), representing 38% of the total population of the country and one-third of the national internal product. The sanitary system in BAP is organized into 12 Sanitary Regions (SR), each one composed of several Localities (“Municipios”). Each SR manages and monitors health policies at the local scale. The climate is temperate and humid to sub-humid, with a southwest-northeast gradient. In western areas, rainfall ranges between 400 and 500 mm annually, while in eastern areas, it exceeds 1,000 mm annually, being more abundant from October to March.

Sample collection:

Priorities proposed by BAP health authorities to include particular localities in the study were the registered outbreaks of dengue cases between 2016 and 2023, along with the consequent control of adult mosquitoes through spatial spraying. The samples were collected during 2023 in 16 localities of BAP, belonging to 5 different SR (Table 1): Avellaneda (34°39′45″S 58°22′04″W); Berisso (34°52′00″S 57°52′00″W); Berazategui (34°46′04″S 58°12′47″W); Escobar (34°21′00″S 58°46′00″W); Florencio Varela (34°49′00″S 58°17′00″W); Hurlingam (34°36′00″S 58°38′00″W); José C. Paz (34°31′S 58°47′W); Lanús (34°42′S 58°24′W); Lomas de Zamora (34°46′00″S 58°24′00″W); La Plata (34°55′00″S 57°57′00″W); Merlo (34°40′00″S 58°43′00″W); Moreno (34°39′00″S 58°47′00″W); Morón (34°39′00″S 58°37′00″W); Pilar (34°28′00″S 58°55′00″W); Pergamino (33°53′00″S 60°35′00″W) and San Fernando (34°27′00″S 58°34′00″W).

Ae. aegypti eggs were collected using 158 ovitraps during the peak oviposition activity period (February and March) in 2023; a total of 22,123 eggs were collected. Ovitraps were deposited outdoors in 79 primary health care centers and hospitals (between 3 and 8 ovitraps per site) by trained agents of BAP Ministry of Health. The ovitraps consisted of 500 ml black plastic containers filled with 200 ml of water, each equipped with five oviposition substrates. The substrates were exchanged weekly for six weeks. The collected eggs were transported either to CENDIE-ANLIS-Malbrán or to the Center for Parasitological and Vector Studies (CEPAVE-UNLP-CONICET). Eggs collected per locality were counted under stereoscopic microscopy. Under controlled laboratory conditions (temperature: 26º ± 2 ºC, humidity: 70 ± 5%, and photoperiod: 12:12 light:dark), oviposition substrates sourced from each location were immersed in plastic trays filled with dechlorinated water and yeast. Upon egg hatching, yeast was provided to the larvae until reaching the pupal stage. Subsequently, the pupae were transferred to plastic water containers. The adults were individually isolated and preserved at -80°C in absolute ethanol until DNA extraction.

The mosquito heads were removed and the remaining body parts were transferred into 1.5 ml tubes containing 100 µl of pre-warmed grinding buffer (5M NaCl, 0.5 M EDTA, 1M Tris base pH 7.5, 10% SDS). Samples were homogenized with sterile pistils and 3 µL of Proteinase K (20 µg/ml) were added, followed by incubation at 55º C for 15 min. Subsequently, 14 µL of 8 M potassium acetate were added to each tube and mixed thoroughly. The mixture was placed on ice for 15 min. before centrifugation at 13,000 rpm for 15 min. DNA from the supernatant was precipitated with absolute ethanol. The DNA concentration and quality extracted from each individual were estimated with a spectrophotometer and diluted until reaching a final concentration of 20 ng/µL. Samples with a 260:230 ratio higher than 1.5 and 260:280 ratio between 1.8 and 2.2 were used for genotyping.

mHRM, HRM, TaqMan and Sanger assays for genotyping 410, 1016 and 1534 positions

For genotyping 1016 and 1534 positions, mHRM assays were performed as described previously [8]. Briefly, 2 µl of individual genomic DNA were used for amplification in a final 12 µL reaction volume that included 6 µL of Brillant HRM Ultra-fast Loci Master Mix (Applied Biosystems) and 200 nM of each primer (see primer sequences in Table 2). The qPCR protocol comprised a 3-minute denaturation step at 95°C, followed by 40 cycles of 5-second denaturation at 95°C and a 30-second annealing extension at 62°C. Subsequently, the melting curves obtained were analyzed using AriaMX 1.5 software (Applied Biosystems). Results were determined with GENOMOS application (see below) and visualization of melting curves. For genotyping of 410 position, the reaction mix had a final reaction volume of 10 µL, including 5 µL of Brillant HRM Ultra-fast Loci Master Mix (Applied Biosystems) and 200 nM of each primer (see primer sequences in Table 2). The qPCR protocol was identical to the described above. Melting curves were visualized with AriaMX 1.5 software (Applied Biosystems).

For TaqMan-based genotyping, 5 µL of the Master Mix qPCR probe (Productos Bio-logicos), 2 µL of the DNA template (20ng/µL), and 0.25 µL of a mix of primers and probe (Thermo Fisher) were used in each reaction. Custom TaqMan SNP Genotyping Assays (ThermoFisher, AHS1DL6 for V1016I or AHUADFA for F1534C) were used. The reaction started with an activation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 90s with a signal acquisition in both, the FAM and HEX channels at the end of the annealing/extension step. For mHRM, HRM and TaqMan assays, samples of known genotypes were included into each plate in triplicates as standards for comparisons with unknown samples.

For Sanger sequencing, genomic DNAs were used as the template for PCR reactions with primers Ae410Fw and Ae410Rv (Table 2), and the polymerase GoTaq (Promega), according to manufacturing instructions. The cycling conditions included an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 30 seconds at 95°C (denaturation) and 30 seconds at 60°C, concluding with a final extension step at 72°C for 5 minutes. Samples were sequenced at Macrogen (Seoul, Korea) using the Ae410Rv primer.

GENOMOS application development:

We developed a desktop application (Genomos) to improve the processivity of the mHRM results analysis and interpretation, with a user-friendly graphical interface; it can be downloaded from https://github.com/gonzalohdominguez/GENOMOS. It has been developed in the Python programming language, designed with Qt Creator using the PySide2 module. GENOMOS application determines the absolute difference between melting temperature (MT) of the three standards (the two homozygous and the heterozygous) for 1016 and 1534 positions, and the MT of each sample. The lower difference will be determined as the probable genotype of the unknown sample. The application provides the results in percentages of allele and genotype distribution, and automatically generates pie charts for better visualization.

Allele and genotype frequencies of the kdr mutations V1016I and F1534C in BAP:

A total of 522 mosquitoes from 16 localities belonging to 5 SR were genotyped for 1016 and 1534 positions (Table 1; Fig. 2) using either mHRM and/or TaqMan probes. The genotypes of 274 samples were determined by using both techniques to compare the performance of both methods, finding a 100% agreement in the results.

All the populations under study presented 1016V + 1534F and 1016V + 1534C^kdr alleles (Fig. 2). We detected 1016I^kdr + 1534C^kdr in all the municipalities analyzed, except for Pergamino (Table 1, Fig. 2). Globally, the most abundant allele was 1016V + 1534C^kdr (48.27%) followed by 1016V + 1534F (28.66%) and finally 1016I^kdr + 1534C^kdr (23.07%). The frequency of the sensitive allele 1016V + 1534F varied significantly between localities, being the lowest in Berazategui (SR VI) with 8.3%, and the highest in La Plata (SR: XI) with 53.3%. The 1016V + 1534C^kdr allele showed the greatest diversity in frequency, where Berisso (SR: XI) presented the highest value with 63.3%, while La Plata (SR: XI) had the lowest frequency with 30%. In the case of 1016I^kdr + 1534C^kdr, Berazategui (SR: VI) was the locality with the highest frequency (56.7%), while it was not detected in Pergamino (SR: IV) (Table 1).

Genotype frequencies were determined, and the Hardy-Weimberg equilibrium hypothesis was tested in each population under study (Table 1). In every SR there was at least one locality where the assumption of Hardy–Weinberg equilibrium was rejected (Table 1) (SR IV: Pergamino p = 0.001; SR V: San Fernando p = 0.01; SR VI: Avellaneda p = 0.02, Lomas de Zamora p = 0.008; SR VII: Hurlingham p = 0.001, Merlo p = 0.03, Moreno p = 0.03; SR XI: La Plata p = 0.01). In most of these localities, except for Lomas de Zamora and Moreno, a deficit in the homozygous was the main cause of the disequilibrium (Table 1).

It was proposed that 1016I^kdr + 1534C^kdr allele emerges in Ae. aegypti populations in the presence of 1016V + 1534C^kdr genetic background, in response to pyrethroid selection pressure [13, 14]. In agreement, our present results indicate a recent emerging and rapid expansion of this double mutation in BAP, given that it was not detected in samples collected 4 to 5 years before [8]. We hypothesized that 1016I^kdr + 1534C^kdr allele emerged in BAP in a 1016V + 1534C^kdr genetic background, which would result in a higher frequency of double mutant allele in those regions under a more intense use of pyrethroids, also correlating with the number of dengue cases per inhabitant. To test this, Pearson’s correlation coefficient and its significance were calculated between dengue cases per inhabitant and 1016I^kdr + 1534C^kdr frequencies for all the localities under study, resulting in a positive and significant correlation (p < 0.005) (Fig. 3).

Presence of V410L kdr mutation in BAP:

V410L kdr mutation in Ae. aegypti has been reported in the Americas in recent years [15]. Given the existence of the double mutant allele in BAP reported here, we tested the presence of V410L, a mutation not described to date in Argentina. For this, we developed a HRM assay, whose representative results are shown in Fig. 4. As expected for substitution of guanine (in the wild type V410) by thymidine (in the 410L^kdr variant), the melting temperatures of the amplicons showed a higher value for the 410VV genotype (74.24°C +/- 0.001°C; N = 140) and a shift of 0.57°C for the genotype 410 L^kdrL^kdr (73.67°C +/- 0.008°C; N = 33). Interestingly, although the heterozygous 410 VL^kdr samples had a smaller shift of temperature (73.86°C +/- 0.001°C; N = 153), they also presented a different shape in their melting curves compared to both homozygotes (Fig. 4). Altogether, the shifts in temperature and shapes of the melting curves allowed a simple genotype determination for 410 position with HRM.

Four-hundred forty-nine samples were genotyped for 410 position (357 by HRM and 92 by Sanger sequencing). The kdr substitution V410L was detected, at different frequencies, in all the localities under study (Table 3). The three possible variants (VV410, VL410 and LL410) were found in all the localities analyzed, except for Pergamino, where the 410L^kdr mutation was not detected in homozygosis (Table 3). This mutation was found in lower frequency with respect to the wild-type V410 in all the localities, except for Berisso and Florencio Varela (both in SR VI) where 410L^kdr was the more abundant variant (Table 3). All the populations were found to be in a Hardy-Weimberg equilibrium, except Florencio Varela and Lomas de Zamora, both in SR VI.

When considering the 3 substitutions under analysis, a total of 19 genotypes were found (Fig. 5 and Supplementary information). Among the localities under study, Morón had the highest variety of genotypes (13), and Pergamino had the lowest (5). The remaining localities presented 7 (Escobar), 8 (La Plata), 9 (Pilar, Berazategui, Florencio Varela and Lomas de Zamora), 10 (San Fernando, Avellaneda, Hurlingham and Berisso) or 12 (Moreno) different genotypes (Fig. 5).

The triple kdr genotype (LL/II/CC) was detected at low frequencies (≤ 6.7%) in Pilar, San Fernando, Florencio Varela, Merlo, Moreno, Morón and Berisso), and at higher frequencies in two regions from SR VI: Berazategui (40%) and Lomas de Zamora (13.8%). Besides, the triple wild type (VV/VV/FF) was detected at low frequencies (≤ 10%) in Pergamino, Escobar, Lomas de Zamora, Morón, Moreno and La Plata), but not in the other localities analyzed. Considering all the samples studied, LL/II/CC was present in 5.84% and VV/VV/FF was detected in 2.5% (Fig. 5; Supplementary information). The more abundant genotype detected was VV/VV/CC (R1R1), in 17% of the samples from BAP. This genotype was followed in abundance by VL/VI/CC (R1R2; 15.4%), VV/VV/FC (R1S, 13.8%) VL/VV/FC (SR1; 13.7%), VL/VI/FC (SR2; 7.75%) and VL/VV/CC (R1R1;5.45%). The remaining genotypes were detected at frequencies lower than 5% (Fig. 5 and Supplementary information).

We formed a multi-center working group, to conduct an analysis of kdr mutations in Ae. aegypti from Buenos Aires, the most populous province of Argentina. We document, for the first time in the central region of the country, the presence of the 1016I^kdr; we also report for the first time in Argentina the circulation of 410L^kdr substitution. The combination of kdr mutations gave place to a total of 19 genotypes detected; from these, the more abundant was VV/VV/CC, followed by VL/VI/CC. Interestingly, this pattern of abundance was also observed in surveys from the Caribbean region in Colombia [16], Amazonian [17] and other regions of Brazil [9], with different antecedent of dengue outbreaks. This pattern could indicate a tendency of South American Ae. aegypti populations where dengue disease is an important health issue, which could be confirmed by further studies.

We observed a positive and significant correlation between dengue cases (a strong indicator of the use of pyrethroids), and the percentage of the double mutant 1016I^kdr + 1534C^kdr. Previous reports suggested a sequential selection of kdr mutations in Ae. aegypti, with an initial emergence of F1534C in response to DDT/pyrethroids provides a background for the emergence of the 1016I^kdr + 1534C^kdr double mutation in a context of a growing pressure with pyrethroids [14]. Our results reinforce the hypothesis of a sequential selection: given that the 1534C^kdr but not the double mutant was detected in samples from 2018–2019 [8], we hypothesized that the double mutant emerged recently in BAP, and that is being spread in response to the sustained use of pyrethroids during the recent dengue outbreaks.

The triple mutant 410L^kdr + 1016I^kdr + 1534C^kdr has a clear fitness disadvantage compared to the single mutant 1534C^kdr in the absence of pyrethroids; on the contrary, in the presence of a pyrethroid like deltamethrin, the fitness advantage of the triple mutant is enormous [18]. This opposite relationship between the level of protection during periods of insecticide pressure and the fitness cost in the absence of insecticides, predicts that the more fumigated localities (those with more dengue cases) will develop higher proportions of double and triple kdr individuals. This prediction is confirmed in our work, where Berazategui locality, with by far the higher number of dengue cases in 2023, was also the locality with a higher abundance of the double and triple mutant alleles.

Interestingly, the genotype frequencies for 1016 and 1534 positions of VGSC does not follow the assumptions of HWE for 50% of the populations studied, indicating that they are under selection pressure. Our results seem to corroborate that populations with intermediate to high levels of 1534C^kdr will develop higher resistance during the following 5 to 10 years if selection pressure is constant. This was attributed to the rapid emergence of mutations in 1016 and/or 410 positions in a 1534C^kdr genetic background [8, 13].

In a recent work [8], we showed a 100% agreement between Sanger sequencing and mHRM for position 1016 of Ae. aegypti VGSC, even though for position 1534 the accuracy of mHRM in comparison with Sanger was reduced to 85.7%. Here, we showed an improvement in mHRM, reaching 100% agreement with TaqMan probes for both positions, refining the purity of genomic DNA obtained from each mosquito; for this, we used an alternative method and removed the head of the insects before DNA purification. Also, we developed a simple application with a visual interface (GENOMOS), available for the rapid estimation and visualization of genotypic results for both 1016 and 1534 positions. For 410 position, we developed a singleplex HRM that allows the distinction between genotypes by both MT and the shape of the melting curve. These improvements make mHRM and HRM suitable tools for the timely management of the huge number of samples employed in country-wide determinations of genotype frequencies, during and after dengue outbreaks and insecticidal interventions. The simultaneous detection of three or more positions could be possible by using multiplex-HRM technique. Even though this three-sites multiplexing could require a careful setting of the assays, it could be a future improvement for the processivity of kdr genotyiping.

At the end of the austral summer season in 2024 (March 2024), Argentina, Brazil, and Paraguay concentrated 92% of the cases and 87% of the deaths caused by the dengue virus worldwide, according to the Pan American Health Organization (PAHO; https://www.paho.org/es/noticias/28-3-2024-ops-llama-accion-colectiva-ante-aumento-record-casos-dengue-americas). Particularly, arbovirus transmission in Argentina is a growing concern, given that Ae. aegypti populations are expanding across the central regions of the country, being reported in southern localities over time. The finding of new kdr mutations in the most densely populated province demands the inclusion of monitoring and management of insecticide resistance in control campaigns. These results highlight a growing trend of pyrethroid resistance in Ae. aegypti, driven by the widespread use of these insecticides. Since pyrethroids are the only insecticides approved for domestic and sanitary use in Argentina, the findings of our study have critical implications for evidence-based public health policies aimed at dengue control. The inclusion of alternative insecticidal and other control strategies should be considered, to reduce selection pressure on Ae. aegypti populations.

The work presented here is the consequence of a joint effort involving the local BAP government and scientific researchers, members of the Argentine Network for the Research of Plaguicide Resistance (RAReP). Our results indicate that the pressure exerted by pyrethroids led to the emergence and expansion of 1016I^kdr + 1534C^kdr mutations in Ae. aegypti from BAP. We also detected V410L mutation in Argentina for the first time, indicating that pyrethroid resistance is a serious problem in the country, such as in other countries of the region. We also improve the processivity and accuracy of genotyping methods. The results are both a tool for resistance monitoring and a sign of alarm to direct efforts in the finding of sustainable methods for vector control, to complement or replace pyrethroids shortly. Joined efforts between academia and authorities in public policies for vector control is a productive way for the transference of scientific results to its application in public health.

Availability of data and materials: All data generated or analyzed during this study are included in this published article.

Ethical approval: This study didn’t used vertebrate animal nor human subjects. Biosecurity considerations were in agreement with CONICET resolution 1619/2008, which is in accordance with the WHO Biosecurity Handbook (ISBN 92 4 354 6503). The collection of eggs and maintenance of mosquitoes was performed in agreement with the Argentinean National Health Ministry ethic requirements; an internal ethic board approved all the protocols.

Consent for publishing: All the authors of this manuscript have read and agreed to the content and publication. This article is original; authors consent to the BiomMed Central Copyright and License Agreement.

Competing interests: The authors declare that they have no competing interests

Funding: This work has been founded by a National Ministry of Health (Proyectos de investigación Beca Salud Investiga 2022-2023), ANLIS Malbrán Financial Resources and Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (ANPCyT) PICT2020-0518 to SO; Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (Idea Proyecto 2023 to SO). ANBI has a research fellowship from CONICET; SO and MVM are researchers of CONICET. IS has a post-doctoral research fellowship from ANPCyT.

Authors' contributions: ANBI: DNA sample preparation, genotyping assays, data analysis, contribution in manuscript writing; LL organized egg sampling, reared mosquitos to the adult stage, sample preparation and training of health agents; AAC: mosquito rearing, sample preparation and training of health agents; AB: mosquito rearing and sample preparation; CJT: organization of sampling, ovitraps and training of health agents; AHM: Sampling coordination, ovitraps and training of health agents; AJ; Coordination with the health regions of the province of Buenos Aires, organization of sampling, ovitraps, conceptualization; IS: sampling preparation, organization of sampling; MVM: Conceptualization, mosquito sampling, contributing in manuscript writing; IS: Mosquito sampling; MMA; Conceptualization, sampling coordination, contributing in manuscript writing, project funding coordination; SO: Conceptualization, data analysis, funding acquisition, writing of the manuscript. All the authors revised the manuscript.

Acknowledgements

The authors would like to thank to Government of Buenos Aires Province and the Municipalities involved for supporting health policies based on scientific evidence. Groups are members of the Argentinean Network for the Research in Pesticide Resistance (RAREP). Many thanks to Gonzalo Dominguez for his support in Genomos development and to sanitary agents from BAP who contributed to the sampling.

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Tables 1-3 are available in the Supplementary Files section.

No competing interests reported.

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Monitoring of pyrethroid resistance in Aedes aegypti: first report of double and triple kdr mutations in Buenos Aires Province

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Abstract

Background:

Methodology and findings:

Conclusions and Significance:

Figures

Background

Methods

Study area

mHRM, HRM, TaqMan and Sanger assays for genotyping 410, 1016 and 1534 positions

GENOMOS application development:

Results

Allele and genotype frequencies of the kdr mutations V1016I and F1534C in BAP:

Discussion

Conclusions

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1