Kdr Mutation Research Articles

Detection and population genetic analysis of Aedes albopictus (Diptera: Culicidae) based on knockdown resistance (kdr) mutations in the Yangtze River basin of China

BackgroundAedes albopictus is an important vector of chikungunya, dengue, yellow fever and Zika viruses. Insecticides are often the most effective tools for rapidly decreasing the density of vector populations, especially during arbovirus disease outbreaks. However, the intense use of insecticides, particularly pyrethroids, has led to the selection of resistant mosquito populations worldwide. Mutations in the voltage-gated sodium channel (VGSC) gene are one of the main drivers of insecticide resistance in Ae. albopictus and are also known as “knockdown resistance” (kdr) mutations. Knowledge about genetic mutations associated with insecticide resistance is a prerequisite for developing techniques for rapid resistance diagnosis. Here, we report studies on the origin and dispersion of kdr haplotypes in samples of Ae. albopictus from the Yangtze River Basin, China; MethodsHere, we report the results of PCR genotyping of kdr mutations in 541 Ae. albopictus specimens from 22 sampling sites in 7 provinces and municipalities in the Yangtze River Basin. Partial DNA sequences of domain II and domain III of the VGSC gene were amplified. These DNA fragments were subsequently sequenced to discover the possible genetic mutations mediating knockdown resistance (kdr) to pyrethroids. The frequency and distribution of kdr mutations were assessed in 22 Ae. albopictus populations. Phylogenetic relationships among the haplotypes were used to infer whether the kdr mutations had a single or multiple origins; ResultsThe kdr mutation at the 1016 locus had 2 alleles with 3 genotypes: V/V (73.38%), V/G (26.43%) and G/G (0.18%). The 1016G homozygous mutation was found in only one case in the CQSL strain in Chongqing, and no 1016G mutations were detected in the SHJD (Shanghai), NJDX (Jiangsu) or HBQN (Hubei) strains. A total of 1532 locus had two alleles and three genotypes, I/I (88.35%), I/T (8.50%) and T/T (3.14%). A total of 1534 locus had four alleles and six genotypes: F/F (49.35%), F/S (19.96%), F/C (1.48%) and F/L (0.18%); S/S (23.66%); and C/C (5.36%). Haplotypes with the F1534C mutation were found only in Ae. albopictus populations in Chongqing and Hubei, and C1534C was found only in three geographic strains in Chongqing. Haplotypes with the 1534S mutation were found only in Ae. albopictus populations in Sichuan and Shanghai. F1534L was found only in HBYC. The Ae. albopictus populations in Shanghai were more genetically differentiated from those in the other regions (except Sichuan), and the genetic differentiation between the populations in Chongqing and those in the middle-lower reaches of the Yangtze River (Huber, Jiangsu, Jiangxi, and Anhui) was lower. Shanghai and Sichuan displayed low haplotype diversity and low nucleotide diversity. Phylogenetic analysis and sequence comparison revealed that the 1016 locus was divided into three branches, with the Clade A and Clade B branches bearing the 1016 mutation occurring mostly in Jiangsu and the Clade C branch bearing the 1016 mutation occurring mostly in Chongqing, suggesting at least two origins for 1016G. IIIS6 phylogenetic analysis and sequence comparison revealed that F1534S, F1534C and I1532T can be divided into two branches, indicating that IIIS6 has two origins; ConclusionsCombined with the distribution of kdr mutations and the analysis of population genetics, we infer that besides the local selection of pyrethroid resistance mutations, dispersal and colonization of Ae. albopictus from other regions may explain why kdr mutations are present in some Ae. albopictus populations in the Yangtze River Basin.

LLIN Evaluation in Uganda Project (LLINEUP)–effects of a vector control trial on Plasmodium infection prevalence and genotypic markers of insecticide resistance in Anopheles vectors from 48 districts of Uganda

Pyrethroid bednets treated with the synergist piperonyl butoxide (PBO) offer the possibility of improved vector control in mosquito populations with metabolic resistance. In 2017–2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus PBO (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, we conducted cross-sectional household entomological surveys at baseline and then every 6 months for two years, which we use here to investigate longitudinal changes in mosquito infection rate and genetic markers of resistance. Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected (PCR-positive) with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s., but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Distribution of LLINs in Uganda was previously found to be associated with reductions in parasite prevalence and vector density, but here we show that the proportion of infective mosquitoes remained stable across both PBO and non-PBO LLINs, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies.Trial registration: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395.

Using QUASR-PCR as a field-based genotyping assay for a tick acaricide resistance marker

A novel, turnkey, field-based workflow was developed and validated using Rhipicephalus microplus DNA as a template to detect the presence of the voltage-gated sodium channel kdr mutation. The field-based compatible workflow comprises manual sample homogenization for DNA extraction, PCR amplification of the targets in a closed tube, and end-point detection of the PCR products. An R. microplus species-specific assay was also included to confirm species identity and ensure the validity of the kdr mutation assay. The assays were sensitive and specific to the targets, and the workflow resulted in a turnaround time of approximately 1 h at a low cost. The novel combination of PCR with closed-tube and end-point fluorescent detection allows for easy conversion of existing conventional lab-based PCR assays into field-based detection assays. The incorporation of custom-designed 3D-printed components in the workflow provides easy adaptability and modification of the components for diverse nucleic acid detection workflows.

Efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr nets on entomological indicators of malaria transmission: third year of a randomised controlled trial in Benin

The present cluster-randomised control trial aims to assess the entomological efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs compared to the standard pyrethroid-only LLINs, in their third year of community usage. Adult mosquito collections were performed every 3 months, in 4 randomly selected houses in each of the 60 trial clusters, using human landing catches. Adult mosquitoes were morphologically identified and Anopheles vectors were molecularly speciated and screened for the presence of the L1014F kdr mutation using PCR. Plasmodium falciparum sporozoite infection was assessed using ELISA. A subset of An. gambiae s.l. was also dissected to examine parity and fertility rates across study arms. There was no evidence of a significant reduction in indoor vector density and entomological inoculation rate by the pyrethroid-pyriproxyfen [DR 0.94 (95% CI 0.46–1.88), p = 0.8527; and RR 1.10 (95% CI 0.44–2.72), p = 0.8380], and pyrethroid-chlorfenapyr [DR 0.74 (95% CI 0.37–1.48), p = 0.3946; and RR 1.00 (95% CI 0.40–2.50), p = 0.9957] LLINs, respectively. The same trend was observed outdoors. Frequencies of the L1014F kdr mutation, as well as parous and fertility rates, were similar between study arms. In the third year after net distribution, entomological indicators show that the two dual active-ingredients nets performed similarly to the standard pyrethroid-only LLIN. To maintain malaria gains, it is crucial that net distribution cycles fit with their operational lifespan.

Aedes aegypti Knockdown Resistance Mutations and Dengue Virus Infection in Haiti.

Haiti is home to approximately 11 million people and has a high incidence of vector-borne disease, including more than 70,000 cases of dengue per year. Vector control is difficult in Haiti and adulticide spray of malathion is the main method of control employed during the outbreak of disease although pyrethroids are used in both bed net campaigns and in widely available aerosol cans for personal use. However, limited pathogen or insecticide resistance surveillance data are available for making operational decisions. In this study, we assessed Aedes aegypti from serial surveillance collections from 3 locations for the presence of dengue virus serotypes 1-3 (DENV1-3) by polymerase chain reaction and assessed, by melt curve analysis, samples from 10 locations in 2 departments for the presence of two mutations (V1016I and F1534C), that in combination, are linked to strong pyrethroid insecticide resistance. Only one of the 32 tested pools was positive for the presence of dengue virus. The two knockdown resistance (kdr) mutations were present in all locations. The 1016I mutation frequency varied from 0.29 to 0.91 and was in all sites lower than the 0.58-1.00 frequency of the 1534C mutation. We also observed that the genotype homozygous for both mutations (IICC), which has been linked to strong pyrethroid resistance, varied from 13 to 86% in each population. Notably, 3 locations - Ti Cousin and Christianville in Ouest department and Camp Coq in Nord department had more than 30% of the tested population without the presence of kdr mutations. These results indicate that the kdr markers of pyrethroid resistance are present in Haiti, at high frequency in several locations and, based on previous studies linking kdr genotypes and phenotypic resistance, that operational interventions with pyrethroids are not likely to be as effective as expected.

Physico-chemical characterization of Anopheles gambiae s.l. breeding sites and kdr mutations in urban areas of Cotonou and Natitingou, Benin

BackgroundThis study aimed to investigate the relationship between the physicochemical characteristics of An. gambiae s.s. and An. coluzzii breeding sites, the susceptibility profiles to commonly used insecticides in public health, and the underlying insecticide resistance mechanisms.MethodsAnopheles breeding sites surveys were conducted in Cotonou and Natitingou in September 2020, January and August 2021. Physicochemical properties and bacterial loads were determined in individual breeding sites. The WHO susceptibility assays were carried out using the female of the emerging adult mosquitoes. Anopheles species were identified through PCR techniques. Kdr L1014F/S, N1575Y and G119S mutations were investigated using TaqMan genotyping assays.ResultsMolecular analysis showed that all mosquitoes analyzed in Cotonou were Anopheles coluzzii, while those of Natitingou were Anopheles gambiae s.s. Fecal coliforms were identified as playing a role in this distribution through their significant influence on the presence of An. coluzzii larvae. WHO susceptibility assay indicated a high level of resistance to deltamethrin in the two cities. The resistance levels to deltamethrin were higher in Cotonou (X2 = 31.689; DF = 1; P < 0.0001). There was a suspected resistance to bendiocarb in Cotonou, whereas the mosquito population in Natitingou was resistant. The kdr L1014F mutation was highly observed in both mosquito populations (frequence: 86–91%), while the Ace-1 mutation was found in a small proportion of mosquitoes. In Cotonou, salinity was the only recorded physicochemical parameter that significantly correlated with the resistance of Anopheles mosquitoes to deltamethrin (P < 0.05). In Natitingou, significant correlations were observed between the allelic frequencies of the kdr L1014F mutation and pH, conductivity, and TDS.ConclusionThese results indicate a high level of pyrethroid resistance in the anopheles populations of both Cotonou and Natitingou. Moreover, this study report the involvement of abiotic factors influencing Anopheles susceptibility profile.

First report of V1016I, F1534C and V410L kdr mutations associated with pyrethroid resistance in Aedes aegypti populations from Niamey, Niger.

Ae. aegypti is the vector of important μ arboviruses, including dengue, Zika, chikungunya and yellow fever. Despite not being specifically targeted by insecticide-based control programs in West Africa, resistance to insecticides in Ae. aegypti has been reported in countries within this region. In this study, we investigated the status and mechanisms of Ae. aegypti resistance in Niamey, the capital of Niger. This research aims to provide baseline data necessary for arbovirus outbreak prevention and preparedness in the country. Ovitraps were used to collect Ae. aegypti eggs, which were subsequently hatched in the insectary for bioassay tests. The hatched larvae were then reared to 3-5-day-old adults for WHO tube and CDC bottle bioassays, including synergist tests. The kdr mutations F1534C, V1016I, and V410L were genotyped using allele-specific PCR and TaqMan qPCR methods. Ae. aegypti from Niamey exhibited moderate resistance to pyrethroids but susceptibility to organophosphates and carbamates. The kdr mutations, F1534C, V1016I and V410L were detected with the resistant tri-locus haplotype 1534C+1016L+410L associated with both permethrin and deltamethrin resistance. Whereas the homozygote tri-locus resistant genotype 1534CC+1016LL+410LL was linked only to permethrin resistance. The involvement of oxidase and esterase enzymes in resistance mechanisms was suggested by partial restoration of mosquitoes' susceptibility to pyrethroids in synergist bioassays. This study is the first report of Ae. aegypti resistance to pyrethroid insecticides in Niamey. The resistance is underpinned by target site mutations and potentially involves metabolic enzymes. The observed resistance to pyrethroids coupled with susceptibility to other insecticides, provides data to support evidence-based decision-making for Ae. aegypti control in Niger.

Discovery of knock-down resistance in the major African malaria vector Anopheles funestus.

A major mechanism of insecticide resistance in insect pests is knock-down resistance (kdr) caused by mutations in the voltage-gated sodium channel (Vgsc) gene. Despite being common in most malaria Anopheles vector species, kdr mutations have never been observed in Anopheles funestus, the principal malaria vector in Eastern and Southern Africa. While monitoring 10 populations of An. funestus in Tanzania, we unexpectedly found resistance to DDT, a banned insecticide, in one location. Through whole-genome sequencing of 333 An. funestus samples from these populations, we found 8 novel amino acid substitutions in the Vgsc gene, including the kdr variant, L976F (L1014F in An. gambiae), in tight linkage disequilibrium with another (P1842S). The mutants were found only at high frequency in one region, with a significant decline between 2017 and 2023. Notably, kdr L976F was strongly associated with survivorship to the exposure to DDT insecticide, while no clear association was noted with a pyrethroid insecticide (deltamethrin). Further study is necessary to identify the origin and spread of kdr in An. funestus, and the potential threat to current insecticide-based vector control in Africa.

Susceptibility Status of Several Field-Collected German Cockroaches (Blattella germanica) to a Pyrethroid Insecticide and Molecular Detection of Knockdown Resistance (kdr).

High frequency of insecticide used to control German cockroaches may lead to insecticide resistance development. We aimed to compare the level of insecticide resistance and heterogeneity in insecticide resistance of field-collected German cockroaches in eight selected zones of Mashhad City, Razavi Khorasan Province, Iran. The present study was conducted from October 2019 to May 2021. Adult and nymphs of German cockroaches were collected from infested restaurants or hotels in eight zones of Mashhad City and then colonized in an insectarium. The cockroaches were subjected to bioassay against cypermethrin insecticide. In addition, the genomic DNA of each cockroach population was analyzed in the region where the kdr mutations reside in the German cockroach. The LT50 values against cypermethrin in the susceptible strain (SS) and eight field-collected strains so varied from 17.52 to 95.36 min. The resistance ratio of the strains was also different in response to the insecticide. The similarity of multiple sequence alignments at the amino acid level was 97.5%-100%. An exon 20 transversion mutation and a nonsynonymous substitution were found. The L1014F substitution was detected in 83.4% of the cockroach samples. The field-collected strains were resistant to cypermethrin at different levels. Furthermore, the molecular study confirms the heterogeneity in the level of resistance among eight strains. Therefore, eight strains might have a different history of insecticide treatment.

Resistance to pyrethroids and the relationship between adult resistance and knockdown resistance (kdr) mutations in Aedes albopictus in dengue surveillance areas of Guizhou Province, China

The Ae. albopictus mosquito has gained global attention due to its ability to transmit viruses, including the dengue and zika. Mosquito control is the only effective way to manage dengue fever, as no effective treatments or vaccines are available. Insecticides are highly effective in controlling mosquito densities, which reduces the chances of virus transmission. However, Ae. albopictus has developed resistance to pyrethroids in several provinces in China. Pyrethroids target the voltage-gated sodium channel gene (VGSC), and mutations in this gene may result in knockdown resistance (kdr). Correlation studies between resistance and mutations can assist viruses in managing Ae. albopictus, which has not been studied in Guizhou province. Nine field populations of Ae. albopictus at the larval stage were collected from Guizhou Province in 2022 and reared to F1 to F2 generations. Resistance bioassays were conducted against permethrin, beta-cypermethrin, and deltamethrin for both larvae and adults of Ae. albopictus. Kdr mutations were characterized by PCR and sequencing. Additionally, the correlation between the kdr allele and pyrethroid resistance was analyzed. All nine populations of Ae. albopictus larvae and adults were found to be resistant to three pyrethroid insecticides. One kdr mutant allele at codon 1016, one at 1532 and three at 1534 were identified with frequencies of 13.86% (V1016G), 0.53% (I1532T), 58.02% (F1534S), 11.69% (F1534C), 0.06% (F1534L) and 0.99% (F1534P), respectively. Both V1016G and F1534S mutation mosquitoes were found in all populations. The kdr mutation F1534S was positively correlated with three pyrethroid resistance phenotypes (OR > 1, P < 0.05), V1016G with deltamethrin and beta-cypermethrin resistance (OR > 1, P < 0.05) and F1534C only with beta-cypermethrin resistance (OR > 1, P < 0.05). Current susceptibility status of wild populations of Ae. albopictus to insecticides and a higher frequency of kdr mutations from dengue-monitored areas in Guizhou Province are reported in this paper. Outcomes of this study can serve as data support for further research and development of effective insecticidal interventions against Ae. albopictus populations in Guizhou Province.

High Frequencies of kdr Mutation and Chikungunya Infection in Aedes aegypti Population from Minas Gerais, Brazil.

The Chikungunya virus (CHIKV) presents global health challenges, with Brazil experiencing outbreaks since its introduction in 2014. In 2023, following a CHIKV outbreak in Minas Gerais (MG), social media was used to optimize an entomological survey aimed at identifying vectors and viral lineages and assessing insecticide resistance. Following Instagram posts, residents with suspected CHIKV infection were able to schedule mosquito aspirations. In total, 421 mosquitoes (165 Aedes aegypti and 256 Culex quinquefasciatus) were captured from 40 households in Salinas city (MG) and tested for the Dengue, Zika, and Chikungunya viruses through RT-qPCR. Twelve of 57 pools (10 Ae. aegypti and two Cx. quinquefasciatus) tested positive for CHIKV RNA. Viral RNA was also detected in the heads of nine Ae. aegypti, indicating viral dissemination but not in Cx. quinquefasciatus. Genome sequencing yielded the first near-complete genome from the 2023 outbreak, unveiling that the CHIKV strain belonged to the East/Central/South African (ECSA) genotype. Additionally, genetic analyses revealed high frequencies of kdr alleles, including in CHIKV-infected mosquitoes, suggesting resistance to pyrethroid insecticides in this Ae. aegypti population. Social media was important for guiding mosquito-capture efforts in CHIKV transmission hotspots, thus optimizing the opportunity for viral detection. These findings emphasize the urgent need for innovative vector studies and control strategies, as well as interdisciplinary approaches in public health interventions.

Metabolic Resistance and Not Voltage-Gated Sodium Channel Gene Mutation Is Associated with Pyrethroid Resistance of Aedes albopictus (Skuse, 1894) from Cambodia.

(1) Background: In Cambodia, Aedes albopictus is an important vector of the dengue virus. Vector control using insecticides is a major strategy implemented in managing mosquito-borne diseases. Resistance, however, threatens to undermine the use of insecticides. In this study, we present the levels of insecticide resistance of Ae. albopictus in Cambodia and the mechanisms involved. (2) Methods: Two Ae. albopictus populations were collected from the capital, Phnom Penh city, and from rural Pailin province. Adults were tested with diagnostic doses of malathion (0.8%), deltamethrin (0.03%), permethrin (0.25%), and DDT (4%) using WHO tube assays. Synergist assays using piperonyl butoxide (PBO) were implemented before the pyrethroid assays to detect the potential involvement of metabolic resistance mechanisms. Adult female mosquitoes collected from Phnom Penh and Pailin were tested for voltage-gated sodium channel (VGSC) kdr (knockdown resistance) mutations commonly found in Aedes sp.-resistant populations throughout Asia (S989P, V1016G, and F1534C), as well as for other mutations (V410L, L982W, A1007G, I1011M, T1520I, and D1763Y). (3) Results: The two populations showed resistance against all the insecticides tested (<90% mortality). The use of PBO (an inhibitor of P450s) strongly restored the efficacy of deltamethrin and permethrin against the two resistant populations. Sequences of regions of the vgsc gene showed a lack of kdr mutations known to be associated with pyrethroid resistance. However, four novel non-synonymous mutations (L412P/S, C983S, Q1554STOP, and R1718L) and twenty-nine synonymous mutations were detected. It remains to be determined whether these mutations contribute to pyrethroid resistance. (4) Conclusions: Pyrethroid resistance is occurring in two Ae. albopictus populations originating from urban and rural areas of Cambodia. The resistance is likely due to metabolic resistance specifically involving P450s monooxygenases. The levels of resistance against different insecticide classes are a cause for concern in Cambodia. Alternative tools and insecticides for controlling dengue vectors should be used to minimize disease prevalence in the country.

Impact of the exposure of sublethal dose of mosquito coil on the development of insecticide resistance in Aedes aegypti (L.) (Diptera: Culicidae).

Mosquito coil is commonly used in many African households for protection against mosquito bites. The coil usually has semi-volatile pyrethroids as an active ingredient, which usually diffuse across open space, and the cloud either kills mosquitoes that are exposed, or mosquitoes can be exposed to sublethal doses of the insecticides. This study was conducted to assess the impact of sublethal doses of mosquito coil on the development of insecticide resistance in Aedes aegypti, a major vector for dengue fever and several other arboviral diseases. A laboratory colony of Ae. aegypti was exposed to sublethal doses of a meperfluthrin-based mosquito coil in a Peet-Grady chamber once per generation for 16 generations. The susceptibility of the exposed colony to a diagnostic dose of the mosquito coil as well as to three other insecticides was determined. Three different kdr mutations and five enzyme activities were evaluated in both the exposed and control colonies. After 16 generations of sublethal exposure to mosquito coils, the full diagnostic dose of the coil caused 68% mortality to the exposed colony compared to 100% mortality in the control colony. Mortality caused by deltamethrin (0.05%) was also significantly lower in the exposed colony. The frequency of 1016I kdr mutation as well as MFO and alpha esterase activities were higher in the exposed colony compared to the control colony. This study provides evidence of the development of pyrethroid resistance in an Ae. aegypti population due to sublethal exposure to mosquito coil for 16 generations. Given the large-scale use of mosquito coils in many African households, its role as a pyrethroid resistance selection source should be taken into consideration when designing resistance management strategies.

Insecticide resistance in Anopheles gambiae sensu lato (Diptera: Culicidae) across different agroecosystems in Niamey, Niger

Malaria vector control in Niger is currently based on the distribution of insecticide treated nets. However, vectors resistance to insecticides represents a major threat to the current national strategy against malaria. This study aims to characterize the impact of agroecosystems on insecticide resistance in Anopheles gambiae s.l. at Niamey. Larvae collected were reared until emergence. Adults aged 2-5 days were used to assess susceptibility to insecticides (pyrethroids, DDT and bendiocarb) after pre-exposure to piperonyl butoxide (PBO) synergist according to WHO protocols. PCRs were performed to identify the sibling species of An. gambiae complex and characterization resistance mutations (Kdr and ace-1). Overall, An. gambiae s.l. was resistance to pyrethroids and DDT (mortality rates from 1% to 55%) and susceptible to bendiocarb at most sites. Pre-exposure to the PBO synergist resulted in partial restoration of pyrethroid susceptibility. Two species of An. gambiae complex were found: An. arabiensis and An. coluzzii. The presence of An. coluzzii was strongly correlated with agricultural practices (99% in rice cultivation sites). Kdr mutations were found at all sites with kdr-w ranging from 45% to 70% in mosquitoes collected in unirrigated and rice field, respectively, and kdr-e found at 37% to 47% at each type of site, respectively. The ace-1 mutation was detected at low frequency (1%) and only from two rice cultivation sites. The high levels of pyrethroid and DDT resistance detected in Niamey had a strong link with rice cultivation, shown that agriculture is a driver of resistance that can compromise control malaria efforts. Keywords: Anopheles gambiae sensu lato, insecticide, resistance, agroecosystem, vector control, Niamey, Niger..

Clinicopathologic and molecular correlates to neoadjuvant chemotherapy-induced pathologic response in breast angiosarcoma.

Both primary and secondary breast angiosarcoma (AS) are characterized by multifocal presentation and aggressive behavior. Despite multimodality therapy, local and distant relapse rates remain high. Therefore, neoadjuvant chemotherapy (NACT) is employed to improve the R0 resection rates and survival, but its benefits remain controversial. Herein, we investigate pathologic and molecular correlates to NACT-induced histologic response in a group of 29 breast AS, 4 primary and 25 radiation-associated (RA). The two NACT regimens applied were anthracycline- and non-anthracycline-based. The pathologic response grade was defined as: I: ≤ 50%, II: 51%-90%, III: 91%-99%, and IV: 100%. An additional 45 primary AS and 102 RA-AS treated by surgery alone were included for survival comparison. The genomic landscape was analyzed in a subset of cases and compared to a cohort of AS without NACT on a paired tumor-normal targeted DNA NGS platform. All patients were females, with a median age of 31 years in primary AS and 68 years in RA-AS. All surgical margins were negative in NACT group. The NACT response was evenly divided between poor (Grades I-II; n = 15) and good responders (Grades III-IV; n = 14). Mitotic count >10/mm2 was the only factor inversely associated with pathologic response. By targeted NGS, all 10 post-NACT RA-AS demonstrated MYC amplification, while both primary AS harbored KDR mutations. TMB or other genomic alterations did not correlate with pathologic response. All four patients with Grade IV response remained free of disease. The good responders had a significantly better disease-specific survival (p = 0.04). There was no survival difference with NACT status or the NACT regimens applied. However, NACT patients with MYC-amplified tumors showed better disease-free survival (p = 0.04) compared to MYC-amplified patients without NACT. The overall survival of NACT group correlated with size >10 cm (p = 0.02), pathologic response (p = 0.04), and multifocality (p = 0.01) by univariate, while only size >10 cm (p = 0.03) remained significant by multivariate analysis.

Insecticide Resistance in Aedes aegypti Mosquitoes: Possible Detection of kdr F1534C, S989P, and V1016G Triple Mutation in Benin, West Africa.

Epidemics of arboviruses in general, and dengue fever in particular, are an increasing threat in areas where Aedes (Ae.) aegypti is present. The effectiveness of chemical control of Ae. aegypti is jeopardized by the increasing frequency of insecticide resistance. The aim of this study was to determine the susceptibility status of Ae. aegypti to public health insecticides and assess the underlying mechanisms driving insecticide resistance. Ae. aegypti eggs were collected in two study sites in the vicinity of houses for two weeks using gravid Aedes traps (GATs). After rearing the mosquitoes to adulthood, female Ae. aegypti were exposed to diagnostic doses of permethrin, deltamethrin and bendiocarb, using Centers for Disease Control and Prevention (CDC) bottle bioassays. Unexposed, un-engorged female Ae. aegypti were tested individually for mixed-function oxidase (MFO), glutathione-S-transferase (GST) and α and β esterase activities. Finally, allele-specific PCR (AS-PCR) was used to detect possible kdr mutations (F1534C, S989P, and V1016G) in the voltage-gated sodium channel gene in insecticide-exposed Ae. aegypti. Most traps were oviposition positive; 93.2% and 97% of traps contained Ae. aegypti eggs in the 10ème arrondissement of Cotonou and in Godomey-Togoudo, respectively. Insecticide bioassays detected resistance to permethrin and deltamethrin in both study sites and complete susceptibility to bendiocarb. By comparison to the insecticide-susceptible Rockefeller strain, field Ae. aegypti populations had significantly higher levels of GSTs and significantly lower levels of α and β esterases; there was no significant difference between levels of MFOs. AS-PCR genotyping revealed the possible presence of 3 kdr mutations (F1534C, S989P, and V1016G) at high frequencies; 80.9% (228/282) of the Ae. aegypti tested had at least 1 mutation, while the simultaneous presence of all 3 kdr mutations was identified in 13 resistant individuals. Study findings demonstrated phenotypic pyrethroid resistance, the over-expression of key detoxification enzymes, and the possible presence of several kdr mutations in Ae. aegypti populations, emphasizing the urgent need to implement vector control strategies targeting arbovirus vector species in Benin.

Clinical characteristics, proteins, and genes related to interstitial pneumonia-associated squamous cell carcinoma of the lungs

Squamous cell carcinoma (SCC) is a common histological type of lung carcinoma that is associated with interstitial pneumonia (IP). We hypothesized that identifying specific genetic alterations or molecular markers of SCC with IP may aid the development of novel therapeutic strategies for the same. Therefore, in the present study, we aimed to identify tumorigenic genetic alterations and molecular markers in cases of SCC with IP. We included 28 lung SCC cases (14 cases with IP and 14 cases without IP). We performed immunohistochemistry for STAT3, STAT5, and TLE1, and next-generation sequencing was performed using an iSeq 100 system. The panel used in this study targeted 50 cancer-associated genes. Immunohistochemically, the rate of TLE1 positivity was higher in the SCC without IP group (93 %) than in the SCC with IP group (29 %), while that of STAT5 was higher in the SCC with IP group (79 %) than in the SCC without IP group (14 %). STAT3 expression was high in both the groups (SCC with IP, 64 %; SCC without IP, 71 %). Eighteen genes were mutated in more than six samples, and FBXW7 mutation was mainly observed in the SCC with IP group (p < 0.01). Mechanisms underlying tumorigenesis in SCC with IP included STAT5 activation via inflammation, while that in SCC without IP included squamous TLE1-mediated metaplasia. These findings are based on smoking-induced STAT3 activation; therefore, patients with IP who smoke are more likely to have progressive SCC. We also found that FBXW7 mutations may be associated with SCC with IP and keratinization. ERBB4 and KDR mutations were observed in both with or without IP, and these genes may be tumor-related genes in SCC. These molecular markers may help determine the prognoses of patients with SCC with IP and direct the development of treatment approaches.

The L1014F Knockdown Resistance Mutation Is Not a Strong Correlate of Phenotypic Resistance to Pyrethroids in Florida Populations of Culex quinquefasciatus.

Culex quinquefasciatus is an important target for vector control because of its ability to transmit pathogens that cause disease. Most populations are resistant to pyrethroids and often to organophosphates, the two most common classes of active ingredients used by public health agencies. A knockdown resistance (kdr) mutation, resulting in an amino acid change from a leucine to phenylalanine in the voltage gated sodium channel, is one mechanism contributing to the pyrethroid resistant phenotype. Enzymatic resistance has also been shown to play a very important role. Recent studies have shown strong resistance in populations even when kdr is relatively low, which indicates that factors other than kdr may be larger contributors to resistance. In this study, we examined, on a statewide scale (over 70 populations), the strength of the correlation between resistance in the CDC bottle bioassay and the kdr genotypes and allele frequencies. Spearman correlation analysis showed only moderate (-0.51) or weak (-0.29) correlation between the kdr genotype and permethrin or deltamethrin resistance, respectively. The frequency of the kdr allele was an even weaker correlate than genotype. These results indicate that assessing kdr in populations of Culex quinquefasciatus is not a good surrogate for phenotypic resistance testing.

Monitoring Aedes populations for arboviruses, Wolbachia, insecticide resistance and its mechanisms in various agroecosystems in Benin

Aedes mosquitoes are the main vectors of arboviruses in Benin. Cases of dengue have been reported in Benin with all four serotypes of the virus actively circulating in this region. Some agricultural settings are known to harbor Aedes vectors responsible for the transmission of arboviruses. The massive use of certain insecticides in agricultural settings has probably contributed to insecticide resistance in these vectors. In Benin, the susceptibility of arbovirus vectors to insecticides is poorly studied. In addition, the distribution of Wolbachia spp., which is used against some arboviruses is unknown. Moreover, there is limited information regarding the vectors responsible for the transmission of arboviruses in Benin. This present study monitored the species composition, arboviruses, and Wolbachia symbiont status, as well as the phenotypic and molecular insecticide resistance profile of Aedes populations from three agroecosystems in Benin. Aedes species identification was performed morphologically and confirmed using qPCR. (RT)-qPCR assay was applied for monitoring the presence of DENV, CHIKV, ZIKV, and WNV pathogens as well as for naturally occurring Wolbachia symbionts. Insecticide resistance was assessed phenotypically, by permethrin (0.75%) exposure of Adults (F0) using World Health Organization (WHO) bioassay protocols, and at the molecular level, using TaqMan (RT)-qPCR assays for assessing knock-down resistance (kdr) mutations (F1534C, V1016G/I, and S989P) and the expression levels of eight detoxification genes (P450s from the CYP9 and CYP6 families, carboxylesterases and glutathione-S-transferases). Aedes aegypti (Ae. aegypti) mosquitoes were the most abundant (93.9%) in the three agroecosystems studied, followed by Aedes albopictus (Ae. albopictus) mosquitoes (6.1%). No arboviruses were detected in the study's mosquito populations. Naturally occurring Wolbachia symbionts were present in 7 pools out of 15 pools tested. This could influence the effectiveness of vector control strategies based on exogenously introduced Wolbachia, all present in the three agroecosystems. Full susceptibility to permethrin was observed in all tested populations of Ae. albopictus. On the contrary, Ae. aegypti were found to be resistant in all three agroecosystem sites except for banana plantation sites, where full susceptibility was observed. Molecular analysis revealed that individual target site resistance kdr mutations F1534C and V1016G/I were detected in most Ae. aegypti populations. Additionally, double mutant (F1534C + V1016G/I) mosquitoes were found in some populations, and in one case, triple mutant (F1534C + V1016G/I + S989P) mosquitoes were detected. Metabolic resistance, as reflected by overexpression of three P450 genes (CYP6BB2, CYP9J26, and CYP9J32), was also detected in Ae. aegypti mosquitoes. Our study provides information that could be used to strategize future vector control strategies and highlights the importance of continuing vector surveillance. Future studies should assess the effect of piperonyl butoxide (PBO) on metabolic resistance and identify the different strains of Wolbachia spp., to choose the best vector control strategies in Benin.

Molecular and entomological surveillance of malaria vectors in urban and rural communities of Benguela Province, Angola

BackgroundMalaria is a major public health problem in Angola, with Anopheles gambiae sensu lato (s.l.) and An. funestus s.l. being the primary vectors. This study aimed to clarify the information gaps concerning local Anopheles mosquito populations. Our objectives were to assess their abundance, geographical dispersion, and blood-feeding patterns. We also investigated their insecticide resistance. Molecular methods were used to identify sibling species, determine the origin of blood meals, measure Plasmodium falciparum infection rates, and detect the presence of knockdown resistance (kdr) mutations.MethodsAdult mosquitoes were collected indoors using CDC light traps from nine randomly selected households at two sentinel sites with distinct ecological characteristics. The samples were collected from 1 February to 30 June 2022. Anopheles mosquitoes were morphologically identified and subjected to molecular identification. Unfed Anopheles females were tested for the presence of P. falciparum DNA in head and thorax, and engorged females were screened for the source of the blood meals. Additionally, members of An. gambiae complex were genotyped for the presence of the L1014F and L1014S kdr mutations.ResultsIn total, 2226 adult mosquitoes were collected, including 733 Anopheles females. Molecular identification revealed the presence of Anopheles coluzzii, An. gambiae senso stricto (s.s.), An. arabiensis, and An. funestus s.s. Notably, there was the first record of An. coluzzii/An. gambiae s.s. hybrid and An. vaneedeni in Benguela Province. Plasmodium falciparum infection rates for An. coluzzii at the urban sentinel site and An. funestus s.s. at the rural site were 23.1% and 5.7%, respectively. The L1014F kdr mutation was discovered in both resistant and susceptible An. coluzzii mosquitoes, while the L1014S mutation was detected in An. gambiae s.s. for the first time in Benguela Province. No kdr mutations were found in An. arabiensis.ConclusionsThis study provides valuable insights into the molecular characteristics of malaria vectors from the province of Benguela, emphasising the need for continuous surveillance of local Anopheles populations regarding the establishment of both kdr mutations for tailoring vector control interventions.Graphical abstract