Multiple insecticide resistance: an impediment to insecticide-based malaria vector control program

Background

Indoor Residual Spraying (IRS), insecticide-treated nets (ITNs) and long-lasting insecticidal nets (LLINs) are key components in malaria prevention and control strategy. However, the development of resistance by mosquitoes to insecticides recommended for IRS and/or ITNs/LLINs would affect insecticide-based malaria vector control. We assessed the susceptibility levels of Anopheles arabiensis to insecticides used in malaria control, characterized basic mechanisms underlying resistance, and evaluated the role of public health use of insecticides in resistance selection.

Methodology/Principal findings

Susceptibility status of An. arabiensis was assessed using WHO bioassay tests to DDT, permethrin, deltamethrin, malathion and propoxur in Ethiopia from August to September 2009. Mosquito specimens were screened for knockdown resistance (kdr) and insensitive acetylcholinesterase (ace-1^R) mutations using AS-PCR and PCR-RFLP, respectively. DDT residues level in soil from human dwellings and the surrounding environment were determined by Gas Chromatography with Electron Capture Detector. An. arabiensis was resistant to DDT, permethrin, deltamethrin and malathion, but susceptible to propoxur. The West African kdr allele was found in 280 specimens out of 284 with a frequency ranged from 95% to 100%. Ace-1^R mutation was not detected in all specimens scored for the allele. Moreover, DDT residues were found in soil samples from human dwellings but not in the surrounding environment.

Conclusion

The observed multiple-resistance coupled with the occurrence of high kdr frequency in populations of An. arabiensis could profoundly affect the malaria vector control programme in Ethiopia. This needs an urgent call for implementing rational resistance management strategies and integrated vector control intervention.     

Fitness costs of individual and combined pyrethroid resistance mechanisms,kdr and CYP-mediated detoxification,in Aedes aegypti

BackgroundAedes aegypti is an important vector of many human diseases and a serious threat to human health due to its wide geographic distribution and preference for human hosts. A. aegypti also has evolved widespread resistance to pyrethroids due to the extensive use of this insecticide class over the past decades. Mutations that cause insecticide resistance result in fitness costs in the absence of insecticides. The fitness costs of pyrethroid resistance mutations in A. aegypti are still poorly understood despite their implications for arbovirus transmission.Methodology/Principle findingsWe evaluated fitness based both on allele-competition and by measuring specific fitness components (i.e. life table and mating competition) to determine the costs of the different resistance mechanisms individually and in combination. We used four congenic A. aegypti strains: Rockefeller (ROCK) is susceptible to insecticides; KDR:ROCK (KR) contains only voltage-sensitive sodium channel (Vssc) mutations S989P+V1016G (kdr); CYP:ROCK (CR) contains only CYP-mediated resistance; and CYP+KDR:ROCK (CKR) contains both CYP-mediated resistance and kdr. The kdr allele frequency decreased over nine generations in the allele-competition study regardless of the presence of CYP-mediated resistance. Specific fitness costs were variable by strain and component measured. CR and CKR had a lower net reproductive rate (R₀) than ROCK or KR, and KR was not different than ROCK. There was no correlation between the level of permethrin resistance conferred by the different mechanisms and their fitness cost ratio. We also found that CKR males had a reduced mating success relative to ROCK males when attempting to mate with ROCK females.Conclusions/SignificanceBoth kdr and CYP-mediated resistance have a fitness cost affecting different physiological aspects of the mosquito. CYP-mediated resistance negatively affected adult longevity and mating competition, whereas the specific fitness costs of kdr remains elusive. Understanding fitness costs helps us determine whether and how quickly resistance will be lost after pesticide application has ceased.     

Combining Organophosphate Treated Wall Linings and Long-lasting Insecticidal Nets for Improved Control of Pyrethroid Resistant Anopheles gambiae

Background

New approaches to delivering insecticides need to be developed to improve malaria vector control. Insecticidal durable wall lining (DL) and net wall hangings (NWH) are novel alternatives to indoor residual spraying which can be produced in a long-lasting format. Non-pyrethroid versions could be used in combination with long-lasting insecticidal nets for improved control and management of insecticide resistant vector populations.

Methods

Experimental hut trials were carried out in Valley du Kou, Burkina Faso to evaluate the efficacy of pirimiphos methyl treated DL and NWH either alone or in combination with LLINs against pyrethroid resistant Anopheles gambiae ss. Comparison was made with pyrethroid DL. Mosquitoes were genotyped for kdr and ace-1R resistant genes to investigate the insecticide resistance management potential of the combination.

Results

The overall kdr and ace-1^R allele frequencies were 0.95 and 0.01 respectively. Mortality with p-methyl DL and NWH alone was higher than with pyrethroid DL alone (>95% vs 40%; P<0.001). Combining pyrethroid DL with LLINs did not show improvement in mortality (48%) compared to the LLIN alone (44%) (P>0.1). Combining p-methyl DL or NWH with LLINs reduced biting rates significantly (8–9%) compared to p-methyl DL and NWH alone (>40%) and killed all An gambiae that entered the huts. Mosquitoes bearing the ace-1^R gene were more likely to survive in huts with p-methyl DL alone (p<0.03) whereas all resistant and susceptible genotypes were killed by the combination.

Conclusion

P-methyl DL and NWH outperformed pyrethroid DL. Combining p-methyl DL and NWH with LLINs could provide significant epidemiological benefits against a vector population which is resistant to pyrethroids but susceptible to organophosphates. There was evidence that the single intervention would select kdr and ace-1^R resistance genes and the combination intervention might select less strongly. Technology to bind organophosphates to plastic wall lining would be worth developing.     

Insecticide susceptibility of Anopheles coluzzii and Anopheles gambiae mosquitoes in Ibadan,Southwest Nigeria

The emergence of insecticide resistance in Anopheles (Diptera: Culicidae) mosquitoes has great implications for malaria control in Nigeria. This study aimed to determine the dynamics of insecticide susceptibility levels and the frequency of knock‐down resistance (kdr) mutations (L1014F) in wild Anopheles coluzzii Coetzee & Wilkerson sp. n. and Anopheles gambiae Giles from the Ojoo and Bodija areas of Ibadan, in southwest Nigeria. Insecticide susceptibility to pyrethroids, organophosphates, carbamates and organochlorines was assessed using World Health Organization (WHO) bioassays. A subset of the mosquitoes exposed to pyrethroids and DDT was used for species and molecular form identification; kdr genotyping was determined using the TaqMan real‐time polymerase chain reaction assay. The mosquitoes were resistant to pyrethroids and DDT but completely susceptible to organophosphates and carbamates. Bodija samples (n = 186) consisted of An. gambiae (91.4%) and An. coluzzii (8.1%) and included one An. coluzzii/An. gambiae hybrid specimen. All mosquitoes screened in Ojoo (n = 26) were An. gambiae. The 1014F kdr mutation was detected at frequencies of 24.5 and 5.8% in Bodija and Ojoo, respectively. No correlation was observed between kdr genotypes and resistance phenotypes. The results indicate that metabolic resistance probably plays an important role in the development of resistance and highlight the need to implement insecticide resistance management strategies.     

Detection of the Nav channel kdr-like mutation and modeling of factors affecting survivorship of Culex quinquefasciatus mosquitoes from six areas of Harris County (Houston), Texas,after permethrin field-cage tests

Culex quinquefasciatus is one of the most important mosquito vectors of arboviruses. Currently, the fastest approach to control disease transmission is the application of synthetic adulticide insecticides. However, in highly populated urban centers the development of insecticide resistance in mosquito populations could impair insecticide efficacy and therefore, disease control. To assess the effect of resistance on vector control, females of Cx. quinquefasciatus collected from six mosquito control operational areas in Harris County, Texas, were treated in field cage tests at three different distances with the pyrethroid Permanone^® 31–66 applied at the operational rate. Females were analyzed by sequencing and/or diagnostic PCR using de novo designed primers for detecting the kdr-like mutation in the voltage-gated sodium channel (L982F; TTA to TTT) (house fly kdr canonical mutation L1014F). Females from the Cx. quinquefasciatus susceptible Sebring strain and those from the six operational areas placed at 30.4 m from the treatment source were killed in the tests, while 14% of field-collected mosquitoes survived at 60.8 m, and 35% at 91.2 m from the source. The diagnostic PCR had a with 97.5% accuracy to detect the kdr-like mutation. Pyrethroid resistant mosquitoes carrying the L982F mutation were broadly distributed in Harris County at high frequency. Among mosquitoes analyzed (n = 1,028), the kdr-kdr genotype was prevalent (81.2%), the kdr-s genotype was 18%, and s-s mosquitoes were less than 1% (n = 8). A logistic regression model estimated an equal probability of survival for the genotypes kdr-kdr and kdr-s in all areas analyzed. Altogether, our results point to a high-risk situation for the pyrethroid-based arboviral disease control in Harris County.     

Re-Visiting Insecticide Resistance Status in Anopheles gambiae from C?te d'Ivoire: A Nation-Wide Informative Survey

Insecticide resistance constitutes a major threat that may undermine current gain in malaria control in most endemic countries. National Malaria Control Programmes (NMCPs) need as much information as possible on the resistance status of malaria vectors and underlying mechanisms in order to implement the most relevant and efficient control strategy. Bioassays, biochemical and molecular analysis were performed on An. gambiae collected in six sentinel sites in Côte d''Ivoire. The sites were selected on the basis of their bioclimatic status and agricultural practices. An. gambiae populations across sites showed high levels of resistance to organochloride, pyrethroid and carbamate insecticides. The kdr and ace-1^R mutations were detected in almost all sentinel sites with mosquitoes on the coastal and cotton growing areas mostly affected by these mutations. At almost all sites, the levels of detoxifying enzymes (mixed-function oxidases (MFOs), non-specific esterases (NSE) and glutathione-S-transferases (GSTs)) in An. gambiae populations were significantly higher than the levels found in the susceptible strain Kisumu. Pre-exposure of mosquitoes to PBO, an inhibitor of MFOs and NSEs, significantly increased mortality rates to pyrethroids and carbamates in mosquitoes but resistance in most cases was not fully synergised by PBO, inferring a residual role of additional mechanisms, including kdr and ace-1 site insensitivity. The large distribution of resistance in Côte d''Ivoire raises an important question of whether to continue to deploy pyrethroid-based long-lasting insecticidal nets (LLINs) and insecticide residual spraying (IRS) towards which resistance continues to rise with no guarantee that the level of resistance would not compromise their efficacy. Innovative strategies that combine insecticide and synergists in LLINs or spatially LLIN and an effective non-pyrethroid insecticide for IRS could be in the short term the best practice for the NMCP to manage insecticide resistance in malaria vectors in Côte d''Ivoire and other endemic countries facing resistance.     

Lactobacillus rhamnosus Strain GG Reduces Aflatoxin B1 Transport, Metabolism, and Toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B₁ (AFB₁) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB₁ availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB₁ levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB₁ transport from the apical to the basolateral chamber was reduced from 11.1% ± 1.9% to 6.4% ± 2.5% (P = 0.019) and to 3.3% ± 1.8% (P = 0.002) within the first hour in monolayers coincubated with GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml, respectively). GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml) bound 40.1% ± 8.3% and 61.0% ± 6.0% of added AFB₁ after 1 h, respectively. AFB₁ caused significant reductions of 30.1% (P = 0.01), 49.4% (P = 0.004), and 64.4% (P < 0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1 × 10¹⁰ CFU/ml GG after 24 h protected against AFB₁-induced reductions in transepithelial resistance at both 24 h (P = 0.002) and 48 h (P = 0.04). DNA fragmentation was apparent in cells treated only with AFB₁ cells but not in cells coincubated with either 1 × 10¹⁰ or 5 × 10¹⁰ CFU/ml GG. GG reduced AFB₁ uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.     

Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali

In certain cases, a species may have access to important genetic variation present in a related species via adaptive introgression. These novel alleles may interact with their new genetic background, resulting in unexpected phenotypes. In this study, we describe a selective sweep on standing variation on the X chromosome in the mosquito Anopheles coluzzii, a principal malaria vector in West Africa. This event may have been influenced by the recent adaptive introgression of the insecticide resistance gene known as kdr from the sister species Anopheles gambiae. Individuals carrying both kdr and a nearly fixed X‐linked haplotype, encompassing at least four genes including the P450 gene CYP9K1 and the cuticular protein CPR125, have rapidly increased in relative frequency. In parallel, a reproductively isolated insecticide‐susceptible A. gambiae population (Bamako form) has been driven to local extinction, likely due to strong selection from increased insecticide‐treated bed net usage.     

Predictive Features of Severe Acquired ADAMTS13 Deficiency in Idiopathic Thrombotic Microangiopathies: The French TMA Reference Center Experience

Severe ADAMTS13 deficiency occurs in 13% to 75% of thrombotic microangiopathies (TMA). In this context, the early identification of a severe, antibody-mediated, ADAMTS13 deficiency may allow to start targeted therapies such as B-lymphocytes-depleting monoclonal antibodies. To date, assays exploring ADAMTS13 activity require skill and are limited to only some specialized reference laboratories, given the very low incidence of the disease. To identify clinical features which may allow to predict rapidly an acquired ADAMTS13 deficiency, we performed a cross-sectional analysis of our national registry from 2000 to 2007. The clinical presentation of 160 patients with TMA and acquired ADAMTS13 deficiency was compared with that of 54 patients with detectable ADAMTS13 activity. ADAMTS13 deficiency was associated with more relapses during treatment and with a good renal prognosis. Patients with acquired ADAMTS13 deficiency had platelet count <30×10⁹/L (adjusted odds ratio [OR] 9.1, 95% confidence interval [CI] 3.4–24.2, P<.001), serum creatinine level ≤200 µmol/L (OR 23.4, 95% CI 8.8–62.5, P<.001), and detectable antinuclear antibodies (OR 2.8, 95% CI 1.0–8.0, P<.05). When at least 1 criteria was met, patients with a severe acquired ADAMTS13 deficiency were identified with positive predictive value of 85%, negative predictive value of 93.3%, sensitivity of 98.8%, and specificity of 48.1%. Our criteria should be useful to identify rapidly newly diagnosed patients with an acquired ADAMTS13 deficiency to better tailor treatment for different pathophysiological groups.     

Resistance Evolution to Bt Crops: Predispersal Mating of European Corn Borers

Over the past decade, the high-dose refuge (HDR) strategy, aimed at delaying the evolution of pest resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops, became mandatory in the United States and is being discussed for Europe. However, precopulatory dispersal and the mating rate between resident and immigrant individuals, two features influencing the efficiency of this strategy, have seldom been quantified in pests targeted by these toxins. We combined mark-recapture and biogeochemical marking over three breeding seasons to quantify these features directly in natural populations of Ostrinia nubilalis, a major lepidopteran corn pest. At the local scale, resident females mated regardless of males having dispersed beforehand or not, as assumed in the HDR strategy. Accordingly, 0–67% of resident females mating before dispersal did so with resident males, this percentage depending on the local proportion of resident males (0% to 67.2%). However, resident males rarely mated with immigrant females (which mostly arrived mated), the fraction of females mating before dispersal was variable and sometimes substantial (4.8% to 56.8%), and there was no evidence for male premating dispersal being higher. Hence, O. nubilalis probably mates at a more restricted spatial scale than previously assumed, a feature that may decrease the efficiency of the HDR strategy under certain circumstances, depending for example on crop rotation practices.     

Experimental hut evaluation of bednets treated with an organophosphate (chlorpyrifos-methyl) or a pyrethroid (lambdacyhalothrin) alone and in combination against insecticide-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes

Background

Pyrethroid resistant mosquitoes are becoming increasingly common in parts of Africa. It is important to identify alternative insecticides which, if necessary, could be used to replace or supplement the pyrethroids for use on treated nets. Certain compounds of an earlier generation of insecticides, the organophosphates may have potential as net treatments.

Methods

Comparative studies of chlorpyrifos-methyl (CM), an organophosphate with low mammalian toxicity, and lambdacyhalothrin (L), a pyrethroid, were conducted in experimental huts in Côte d'Ivoire, West Africa. Anopheles gambiae and Culex quinquefasciatus mosquitoes from the area are resistant to pyrethroids and organophosphates (kdr and insensitive acetylcholinesterase Ace.1 ^R ). Several treatments and application rates on intact or holed nets were evaluated, including single treatments, mixtures, and differential wall/ceiling treatments.

Results and Conclusion

All of the treatments were effective in reducing blood feeding from sleepers under the nets and in killing both species of mosquito, despite the presence of the kdr and Ace.1 ^R genes at high frequency. In most cases, the effects of the various treatments did not differ significantly. Five washes of the nets in soap solution did not reduce the impact of the insecticides on A. gambiae mortality, but did lead to an increase in blood feeding. The three combinations performed no differently from the single insecticide treatments, but the low dose mixture performed encouragingly well indicating that such combinations might be used for controlling insecticide resistant mosquitoes. Mortality of mosquitoes that carried both Ace.1 ^R and Ace.1 ^S genes did not differ significantly from mosquitoes that carried only Ace.1 ^S genes on any of the treated nets, indicating that the Ace.1 ^R allele does not confer effective resistance to chlorpyrifos-methyl under the realistic conditions of an experimental hut.     

Food Deprivation Attenuates Seizures through CaMKII and EAG K+ Channels

Accumulated research has demonstrated the beneficial effects of dietary restriction on extending lifespan and increasing cellular stress resistance. However, reducing nutrient intake has also been shown to direct animal behaviors toward food acquisition. Under food-limiting conditions, behavioral changes suggest that neuronal and muscle activities in circuits that are not involved in nutrient acquisition are down-regulated. These dietary-regulated mechanisms, if understood better, might provide an approach to compensate for defects in molecules that regulate cell excitability. We previously reported that a neuromuscular circuit used in Caenorhabditis elegans male mating behavior is attenuated under food-limiting conditions. During periods between matings, sex-specific muscles that control movements of the male's copulatory spicules are kept inactive by UNC-103 ether-a-go-go–related gene (ERG)–like K⁺ channels. Deletion of unc-103 causes ~30%–40% of virgin males to display sex-muscle seizures; however, when food is deprived from males, the incidence of spontaneous muscle contractions drops to 9%–11%. In this work, we used genetics and pharmacology to address the mechanisms that act parallel with UNC-103 to suppress muscle seizures in males that lack ERG-like K⁺ channel function. We identify calcium/calmodulin-dependent protein kinase II as a regulator that uses different mechanisms in food and nonfood conditions to compensate for reduced ERG-like K⁺ channel activity. We found that in food-deprived conditions, calcium/calmodulin-dependent protein kinase II acts cell-autonomously with ether-a-go-go K⁺ channels to inhibit spontaneous muscle contractions. Our work suggests that upregulating mechanisms used by food deprivation can suppress muscle seizures.     

Presence of the knockdown resistance (kdr) mutations in the head lice (Pediculus humanus capitis) collected from primary school children of Thailand

Human head lice are blood-sucking insects causing an infestation in humans called pediculosis capitis. The infestation is more prevalent in the school-aged population. Scalp itching, a common presenting symptom, results in scratching and sleep disturbance. The condition can lead to social stigmatization which can lead to loss of self-esteem. Currently, the mainstay of treatment for pediculosis is chemical insecticides such as permethrin. The extended use of permethrin worldwide leads to growing pediculicide resistance. The aim of this study is to demonstrate the presence of the knockdown resistance (kdr) mutation in head lice populations from six different localities of Thailand. A total of 260 head lice samples in this study were collected from 15 provinces in the 6 regions of Thailand. Polymerase chain reaction (PCR) was used to amplify the α subunit of voltage-sensitive sodium channel (VSSC) gene, kdr mutation (C→T substitution). Restriction fragment length polymorphism (RFLP) patterns and sequencing were used to identify the kdr T917I mutation and demonstrated three genotypic forms including homozygous susceptible (SS), heterozygous genotype (RS), and homozygous resistant (RR). Of 260 samples from this study, 156 (60.00%) were SS, 58 (22.31%) were RS, and 46 (17.69%) were RR. The overall frequency of the kdr T917I mutation was 0.31. Genotypes frequencies determination using the exact test of Hardy-Weinberg equilibrium found that northern, central, northeastern, southern, and western region of Thailand differed from expectation. The five aforementioned localities had positive inbreeding coefficient value (F_is > 0) which indicated an excess of homozygotes. The nucleotide and amino acid sequences of RS and RR showed T917I and L920F point mutations. In conclusion, this is the first study detecting permethrin resistance among human head lice from Thailand. PCR-RFLP is an easy technique to demonstrate the kdr mutation in head louse. The data obtained from this study would increase awareness of increasing of the kdr mutation in head louse in Thailand.     

The genetic basis for mating-induced sex differences in starvation resistance in Drosophila melanogaster

Multiple genetic and environmental factors interact to influence starvation resistance, which is an important determinant of fitness in many organisms, including Drosophila melanogaster. Recent studies have revealed that mating can alter starvation resistance in female D. melanogaster, but little is known about the behavioral and physiological mechanisms underlying such mating-mediated changes in starvation resistance. In the present study, we first investigated whether the effect of mating on starvation resistance is sex-specific in D. melanogaster. As indicated by a significant sex × mating status interaction, mating increased starvation resistance in females but not in males. In female D. melanogaster, post-mating increase in starvation resistance was mainly attributed to increases in food intake and in the level of lipid storage relative to lean body weight. We then performed quantitative genetic analysis to estimate the proportion of the total phenotypic variance attributable to genetic differences (i.e., heritability) for starvation resistance in mated male and female D. melanogaster. The narrow-sense heritability (h²) of starvation resistance was 0.235 and 0.155 for males and females, respectively. Mated females were more resistant to starvation than males in all genotypes, but the degree of such sexual dimorphism varied substantially among genotypes, as indicated by a significant sex × genotype interaction for starvation resistance. Cross-sex genetic correlation was greater than 0 but less than l for starvation resistance, implying that the genetic architecture of this trait was partially shared between the two sexes. For both sexes, starvation resistance was positively correlated with longevity and lipid storage at genetic level. The present study suggests that sex differences in starvation resistance depend on mating status and have a genetic basis in D. melanogaster.     

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.     

Genetic Variability and Distribution of Mating Type Alleles in Field Populations of Leptosphaeria maculans from France

Leptosphaeria maculans is the most ubiquitous fungal pathogen of Brassica crops and causes the devastating stem canker disease of oilseed rape worldwide. We used minisatellite markers to determine the genetic structure of L. maculans in four field populations from France. Isolates were collected at three different spatial scales (leaf, 2-m² field plot, and field) enabling the evaluation of spatial distribution of the mating type alleles and of genetic variability within and among field populations. Within each field population, no gametic disequilibrium between the minisatellite loci was detected and the mating type alleles were present at equal frequencies. Both sexual and asexual reproduction occur in the field, but the genetic structure of these populations is consistent with annual cycles of randomly mating sexual reproduction. All L. maculans field populations had a high level of gene diversity (H = 0.68 to 0.75) and genotypic diversity. Within each field population, the number of genotypes often was very close to the number of isolates. Analysis of molecular variance indicated that >99.5% of the total genetic variability was distributed at a small spatial scale, i.e., within 2-m² field plots. Population differentiation among the four field populations was low (G_ST < 0.02), suggesting a high degree of gene exchange between these populations. The high gene flow evidenced here in French populations of L. maculans suggests a rapid countrywide diffusion of novel virulence alleles whenever novel resistance sources are used.     

Diagnostic Clinical and Laboratory Findings in Response to Predetermining Bacterial Pathogen: Data from the Meningitis Registry

Background

Childhood Meningitis continues to be an important cause of mortality in many countries. The search for rapid diagnosis of acute bacterial meningitis has lead to the further exploration of prognostic factors. This study was scheduled in an attempt to analyze various clinical symptoms as well as rapid laboratory results and provide an algorithm for the prediction of specific bacterial aetiology of childhood bacterial meningitis.

Methodology and Principal Findings

During the 32 year period, 2477 cases of probable bacterial meningitis (BM) were collected from the Meningitis Registry (MR). Analysis was performed on a total of 1331 confirmed bacterial meningitis cases of patients aged 1 month to 14 years. Data was analysed using EPI INFO (version 3.4.3-CDC-Atlanta) and SPSS (version 15.0 - Chicago) software. Statistically significant (p<0.05) variables were included in a conditional backward logistic regression model. A total of 838 (63.0%) attributed to Neisseria meningitidis, 252 (18.9%) to Haemophilus influenzae, 186 (14.0%) to Streptococcus pneumoniae and 55 (4.1%) due to other bacteria. For the diagnosis of Meningococcal Meningitis, the most significant group of diagnostic criteria identified included haemorrhagic rash (OR 22.36), absence of seizures (OR 2.51), headache (OR 1.83) and negative gram stain result (OR 1.55) with a Positive Predictive Value (PPV) of 96.4% (95%CI 87.7–99.6). For the diagnosis of Streptococcus pneumoniae, the most significant group of diagnostic criteria identified included absence of haemorrhagic rash (OR 13.62), positive gram stain (OR 2.10), coma (OR 3.11), seizures (OR 3.81) and peripheral WBC≥15000/µL (OR 2.19) with a PPV of 77.8% (95%CI 40.0–97.2). For the diagnosis of Haemophilus influenzae, the most significant group of diagnostic criteria included, absence of haemorrhagic rash (OR 13.61), age≥1year (OR 2.04), absence of headache (OR 3.01), CSF Glu<40 mg/dL (OR 3.62) and peripheral WBC<15000/µL (OR 1.74) with a PPV of 58.5% (95%CI 42.1–73.7).

Conclusions

The use of clinical and laboratory predictors for the assessment of the causative bacterial pathogen rather than just for predicting outcome of mortality seems to be a useful tool in the clinical management and specific treatment of BM. These findings should be further explored and studied.     

Preliminary report of knockdown resistance in Culex pipiens pallens and Aedes koreicus from Korea

Pyrethroid insecticides have been effective and powerful for controlling mosquitoes. However, abuse of these insecticides increases the number of resistant mosquitoes. In this study, Culex pipiens pallens and Aedes koreicus were collected from an artificial reservoir in the vicinity of a populated area in Korea, which is also a migratory bird catchment area. To monitor resistance to pyrethroid insecticides in mosquitoes, genomic DNA from the collected mosquitoes was sequenced for the kdr mutation in the voltage‐gated sodium channel (VGSC) gene. As a result, three samples with homozygous resistance (17.6%) and one with heterozygous resistance (5.9%) were found among 17 Cx. pipiens pallens specimens. One of the samples had a unique sequence at the amplified VGSC region. Of the 15 Ae. koreicus, no insecticide resistant individuals were found. In Korea, this is the first report of kdr genetic traits in Ae. koreicus and Cx. pipiens pallens and of a unique VGSC allele in Cx. pipiens pallens. Further investigation is needed to monitor the kdr resistance of these species in Korea and to determine how the unique sequence found in Cx. pipiens pallens is related to insecticide resistance.     

Mosquito population structure,pathogen surveillance and insecticide resistance monitoring in urban regions of Crete,Greece

BackgroundIn Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece’s major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority.MethodologyUnder the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018–2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species.Principal findingsImportant disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25–46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses.Conclusions/SignificanceThe findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.