Type 2 Diabetic Education Benchmark Study

Evaluating the implementation of verbal and written education on diet for newly diagnosed type 2 diabetics

Behavioural and evolutionary responses of Anopheles gambiae S.S. to bednets and pyrethoids

Vector control by insecticides is fundamental to the global strategy foOr malaria prevention. Insecticide treated bednets (ITNs) are central to this approach but with only pyrethroids available for ITN use, resistance to this class of insecticides is a major threat. Knock-down resistance to pyrethroids is strongly associated with the presence of a single amino acid substitution (kdr) in the voltage-gated sodium channel in many insects, including African malaria vectors. With the long-term aim of extending the effective lifespan of pyrethroids and ITNs in particular, this study investigated knock-down resistance and behaviour at the bednet interface in Anopheles gambiae s.s. Specifically, the molecular studies aimed to improve the reliability of kdr detection methods, investigate the origins and spread of kdr mutations and determine the strength of the selection pressures acting on them; the behavioural studies investigated non-contact repellency by pyrethroids and arrival patterns ofhost-seeking mosquitoes at a human-baited bednet. Detection of kdr alleles is important in order to monitor the spread of resistance in the field, and a novel kdr detection method (Hot Oligonucleotide Ligation Assay or HOLA) was developed to improve on existing techniques. This method differentiated homozygous and heterozygous individuals for both kdr alleles and was transferred to a resource poor laboratory. HOLA was also used to confirm the first occurrence ofa phenylalanine heterozygous specimen. population genetic based approach was usedserine/ to investigate the relative age ofthe kdr mutations and selection pressures acting upon them. Sequencing ofthe voltage-gated sodium channel gene allowed the identification of 29 novel single nucleotide polymorphisms that were used to screen populations of field collected An. gambiae s.s. from seven locations in Africa. Analysis of the extended haplotypes revealed the signature of the selective sweeps associated with the kdr alleles, and suggested that the serine kdr mutation found in Kenya pre-dated the kdr mutations in West Africa, possibly the result of selection by the historic use of DDT rather than the more recent use of pyrethroids. Data indicated that the spread of the serine mutation in Gabon was recent, possibly due to a selective advantage conferred by co-expression with the phenylalanine kdr mutation and, confirming published data, that it had probably arisen at least twice through novel mutation events. The phenylalanine mutation in West and Central Africa is likely to have been the result of at least two separate mutation events, both ofwhich have been subjected to a strong selective sweep

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the φC31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces φC31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the φC31 integrase. We report two representative results of φC31mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. φC31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the φC31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors

Cytochrome P450associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.Fil: Balabanidou, Vasileia. Foundation for Research and Technology-Hellas; Grecia. Universidad de Creta; GreciaFil: Kampouraki, Anastasia. Universidad de Creta; GreciaFil: Mac Lean, Marina. University of Nevada; Estados UnidosFil: Blomquist, Gary J.. University of Nevada; Estados UnidosFil: Tittiger, Claus. University of Nevada; Estados UnidosFil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Mijailovsky, Sergio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Chalepakis, George. Universidad de Creta; GreciaFil: Anthousi, Amalia. Universidad de Creta; GreciaFil: Lynd, Amy. Liverpool School of Tropical Medicine; Reino UnidoFil: Antoine, Sanou. Liverpool School of Tropical Medicine; Reino UnidoFil: Hemingway, Janet. Liverpool School of Tropical Medicine; Reino UnidoFil: Ranson, Hilary. Liverpool School of Tropical Medicine; Reino UnidoFil: Lycett, Gareth J.. Liverpool School of Tropical Medicine; Reino UnidoFil: Vontas, John. Foundation for Research and Technology-Hellas; Grecia. Agricultural University of Athens; Greci

Demasculinization of the Anopheles gambiae X chromosome

Background: In a number of organisms sex-biased genes are non-randomly distributed between autosomes and the shared sex chromosome X (or Z). Studies on Anopheles gambiae have produced conflicting results regarding the underrepresentation of male-biased genes on the X chromosome and it is unclear to what extent sexual antagonism, dosage compensation or X-inactivation in the male germline, the evolutionary forces that have been suggested to affect the chromosomal distribution of sex-biased genes, are operational in Anopheles. Results: We performed a meta-analysis of sex-biased gene expression in Anopheles gambiae which provides evidence for a general underrepresentation of male-biased genes on the X-chromosome that increased in significance with the observed degree of sex-bias. A phylogenomic comparison between Drosophila melanogaster, Aedes aegypti and Culex quinquefasciatus also indicates that the Anopheles X chromosome strongly disfavours the evolutionary conservation of male-biased expression and that novel male-biased genes are more likely to arise on autosomes. Finally, we demonstrate experimentally that transgenes situated on the Anopheles gambiae X chromosome are transcriptionally silenced in the male germline. Conclusion: The data presented here support the hypothesis that the observed demasculinization of the Anopheles X chromosome is driven by X-chromosome inactivation in the male germline and by sexual antagonism. The demasculinization appears to be the consequence of a loss of male-biased expression, rather than a failure in the establishment or the extinction of male-biased genes

Widespread occurrence of copy number variants and fixation of pyrethroid target site resistance in Anopheles gambiae (s.l.) from southern Cote d’Ivoire

Resistance to pyrethroid and organophosphate insecticides in the malaria vector Anopheles gambiae (s.l.) is conferred by a variety of genetic mutations, including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). Knowledge of the distribution of these mutations in mosquito populations is a prerequisite for establishing better strategies for their management. In this study, a total of 755 Anopheles gambiae (s.l.) from southern Côte d’Ivoire were exposed to deltamethrin or pirimiphos-methyl insecticides and were screened to assess the distribution of SNPs and CNVs known or believed to confer resistance to one or other of the insecticide classes. Most individuals from the An. gambiae (s.l.) complex were identified by molecular tests as Anopheles coluzzii. Survival to deltamethrin (from 94% to 97%) was higher than to pirimiphos-methyl (from 10% to 49%). In An. gambiae (s.s.), the SNP in the Voltage Gated Sodium Channel (Vgsc) at the 995F locus (Vgsc-995F) was fixed, while other target site mutations were rare or absent (Vgsc-402L: 0%; Vgsc-1570Y: 0%, Acetylcholinesterase Acel-280S: 14%). In An. coluzzii, Vgsc-995F was the target site SNP found at highest frequency (65%) followed by other target site mutations (Vgsc-402L: 36%; Vgsc-1570Y: 0.33%; Acel-280S: 45%). The Vgsc-995S SNP was not present. The presence of the Ace1-280S SNP was found to be significantly linked to the presence of the Ace1-CNV, Ace1_AgDup. Significant association was found between the presence of the Ace1_AgDup and pirimiphos-methyl resistance in An. gambiae (s.s.) but not in An. coluzzii. The deletion Ace1_Del97 was found in one specimen of An. gambiae (s.s.). Four CNVs in the Cyp6aa/Cyp6p gene cluster, which contains genes of known importance for resistance, were detected in An. coluzzii, the most frequent being Dup 7 (42%) and Dup 14 (26%). While none of these individual CNV alleles were significantly associated with resistance, copy number in the Cyp6aa gene region in general was associated with increased resistance to deltamethrin. Elevated expression of Cyp6p3 was nearly associated with deltamethrin resistance, although there was no association of resistance with copy number. Use of alternative insecticides and control methods to arrest resistance spread in An. coluzzii populations is merited

LLIN Evaluation in Uganda Project (LLINEUP) - Impact of long-lasting insecticidal nets with, and without, piperonyl butoxide on malaria indicators in Uganda: study protocol for a cluster-randomised trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a key malaria control intervention, but their effectiveness is threatened by resistance to pyrethroid insecticides. Some new LLINs combine pyrethroids with piperonyl butoxide (PBO), a synergist that can overcome P450-based metabolic resistance to pyrethroids in mosquitoes. In 2017-2018, the Ugandan Ministry of Health distributed LLINs with and without PBO through a national mass-distribution campaign, providing a unique opportunity to rigorously evaluate PBO LLINs across different epidemiological settings. METHODS/DESIGN: Together with the Ministry of Health, we embedded a cluster-randomised trial to evaluate the impact of LLINs delivered in the 2017-2018 national campaign. A total of 104 clusters (health sub-districts) in Eastern and Western Uganda were involved, covering 48 of 121 (40%) districts. Using adaptive randomisation driven by the number of LLINs available, clusters were assigned to receive one of four types of LLINs, including two brands with PBO: 1) PermaNet 3.0 (n = 32) and 2) Olyset Plus (n = 20); and two without PBO: 3) PermaNet 2.0 (n = 37) and 4) Olyset Net (n = 15). We are conducting cross-sectional community surveys in 50 randomly selected households per cluster (5200 households per survey) and entomological surveillance for insecticide resistance in up to 10 randomly selected households enrolled in the community surveys per cluster (1040 households per survey) at baseline and 6, 12, and 18 months after LLIN distribution. Net durability and bio-efficacy will be assessed in 400 nets withdrawn from households with replacement at 12 months. The primary trial outcome is parasite prevalence as measured by microscopy in children aged 2-10 years in the follow-up surveys. DISCUSSION: PBO LLINs are a promising new tool to reduce the impact of pyrethroid resistance on malaria control. The World Health Organization has issued a preliminary endorsement of PBO LLINs, but additional epidemiological evidence of the effect of PBO LLINs is urgently needed. The results of this innovative, large-scale trial embedded within a routine national distribution campaign will make an important contribution to the malaria control policy in Uganda and throughout Africa, where pyrethroid resistance in malaria vectors has increased dramatically. This model of evaluation could be a paradigm for future assessment of malaria control interventions. TRIAL REGISTRATION: ISRCTN, ISRCTN17516395 . Registered on 14 February 2017. WORLD HEALTH ORGANIZATION TRIAL REGISTRATION DATA SET: See Additional file 1

Modelling spatiotemporal trends in the frequency of genetic mutations conferring insecticide target-site resistance in African mosquito malaria vector species

Background Resistance in malaria vectors to pyrethroids, the most widely used class of insecticides for malaria vector control, threatens the continued efficacy of vector control tools. Target-site resistance is an important genetic resistance mechanism caused by mutations in the voltage-gated sodium channel (Vgsc) gene that encodes the pyrethroid target-site. Understanding the geographic distribution of target-site resistance, and temporal trends across different vector species, can inform strategic deployment of vector control tools. Results We develop a Bayesian statistical spatiotemporal model to interpret species-specific trends in the frequency of the most common resistance mutations, Vgsc-995S and Vgsc- 995F, in three major malaria vector species Anopheles gambiae, An. coluzzii, and An. arabiensis over the period 2005-2017. The models are informed by 2418 observations of the frequency of each mutation in field sampled mosquitoes collected from 27 countries spanning western and eastern regions of Africa. For nine selected countries, we develop annual predictive maps which reveal geographically-structured patterns of spread of each mutation at regional and continental scales. The results show associations, as well as stark differences, in spread dynamics of the two mutations across the three vector species. The coverage of ITNs was an influential predictor of Vgsc allele frequencies, with modelled relationships between ITN coverage and allele frequencies varying across species and geographic regions. We found that our mapped Vgsc allele frequencies are a significant partial predictor of phenotypic resistance to the pyrethroid deltamethrin in An. gambiae complex populations. Conclusions Our predictive maps show how spatiotemporal trends in insecticide target-site resistance mechanisms in African An. gambiae vary across individual vector species and geographic regions. Molecular surveillance of resistance mechanisms will help to predict resistance phenotypes and track their spread

LLIN Evaluation in Uganda Project (LLINEUP): factors associated with ownership and use of long-lasting insecticidal nets in Uganda: a cross-sectional survey of 48 districts.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a key malaria control intervention. To investigate factors associated with ownership and use of LLINs in Uganda, a cross-sectional community survey was conducted in March-June 2017, approximately 3 years after a national Universal Coverage Campaign (UCC). METHODS: Households from 104 clusters (health sub-districts) in 48 districts were randomly selected using two-staged cluster sampling; 50 households were enrolled per cluster. Outcomes were household ownership of LLINs (at least one LLIN), adequate LLIN coverage (at least one LLIN per 2 residents), and use of LLINs (resident slept under a LLIN the previous night). Associations between variables of interest and outcomes were made using multivariate logistic regression. RESULTS: In total, 5196 households, with 29,627 residents and 6980 bed-nets, were included in the analysis. Overall, 65.0% of households owned at least one LLIN (down from 94% in 2014). In the adjusted analysis, factors most strongly associated with LLIN ownership were living in a wealthier household (highest tercile vs lowest; adjusted odds ratio [aOR] 1.94, 95% CI 1.66-2.28, p  15 years (44.1%) were more likely to use nets than children aged 5-15 years (30.7%;  15 years: aOR 1.37, 95% CI 1.29-1.45, p < 0.001). CONCLUSIONS: Long-lasting insecticidal net ownership and coverage have reduced markedly in Uganda since the last net distribution campaign in 2013/14. Houses with many residents, poorer households, and school-aged children should be targeted to improve LLIN coverage and use. Trial registration This study is registered with ISRCTN (17516395)

Characterizing pyrethroid resistance and mechanisms in Anopheles gambiae (s.s.) and Anopheles arabiensis from 11 districts in Uganda.

Insecticide resistance threatens recent progress on malaria control in Africa. To characterize pyrethroid resistance in Uganda, Anopheles gambiae (s.s.) and Anopheles arabiensis were analyzed from 11 sites with varied vector control strategies. Mosquito larvae were collected between May 2018 and December 2020. Sites were categorized as receiving no indoor-residual spraying ('no IRS', n ​= ​3); where IRS was delivered from 2009 to 2014 and in 2017 and then discontinued ('IRS stopped', n ​= ​4); and where IRS had been sustained since 2014 ('IRS active', n ​= ​4). IRS included bendiocarb, pirimiphos methyl and clothianidin. All sites received long-lasting insecticidal nets (LLINs) in 2017. Adult mosquitoes were exposed to pyrethroids; with or without piperonyl butoxide (PBO). Anopheles gambiae (s.s.) and An. arabiensis were identified using PCR. Anopheles gambiae (s.s.) were genotyped for Vgsc-995S/F, Cyp6aa1, Cyp6p4-I236M, ZZB-TE, Cyp4j5-L43F and Coeae1d, while An. arabiensis were examined for Vgsc-1014S/F. Overall, 2753 An. gambiae (s.l.), including 1105 An. gambiae (s.s.) and 1648 An. arabiensis were evaluated. Species composition varied by site; only nine An. gambiae (s.s.) were collected from 'IRS active' sites, precluding species-specific comparisons. Overall, mortality following exposure to permethrin and deltamethrin was 18.8% (148/788) in An. gambiae (s.s.) and 74.6% (912/1222) in An. arabiensis. Mortality was significantly lower in An. gambiae (s.s.) than in An. arabiensis in 'no IRS' sites (permethrin: 16.1 vs 67.7%, P ​< ​0.001; deltamethrin: 24.6 vs 83.7%, P ​< ​0.001) and in 'IRS stopped' sites (permethrin: 11.3 vs 63.6%, P ​< ​0.001; deltamethrin: 25.6 vs 88.9%, P ​< ​0.001). When PBO was added, mortality increased for An. gambiae (s.s.) and An. arabiensis. Most An. gambiae (s.s.) had the Vgsc-995S/F mutation (95% frequency) and the Cyp6p4-I236M resistance allele (87%), while the frequency of Cyp4j5 and Coeae1d were lower (52% and 55%, respectively). Resistance to pyrethroids was widespread and higher in An. gambiae (s.s.). Where IRS was active, An. arabiensis dominated. Addition of PBO to pyrethroids increased mortality, supporting deployment of PBO LLINs. Further surveillance of insecticide resistance and assessment of associations between genotypic markers and phenotypic outcomes are needed to better understand mechanisms of pyrethroid resistance and to guide vector control