Identification and analysis of Single Nucleotide Polymorphisms (SNPs) in the mosquito Anopheles funestus, malaria vector

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most common source of genetic variation in eukaryotic species and have become an important marker for genetic studies. The mosquito Anopheles funestus is one of the major malaria vectors in Africa and yet, prior to this study, no SNPs have been described for this species. Here we report a genome-wide set of SNP markers for use in genetic studies on this important human disease vector. RESULTS: DNA fragments from 50 genes were amplified and sequenced from 21 specimens of An. funestus. A third of specimens were field collected in Malawi, a third from a colony of Mozambican origin and a third form a colony of Angolan origin. A total of 494 SNPs including 303 within the coding regions of genes and 5 indels were identified. The physical positions of these SNPs in the genome are known. There were on average 7 SNPs per kilobase similar to that observed in An. gambiae and Drosophila melanogaster. Transitions outnumbered transversions, at a ratio of 2:1. The increased frequency of transition substitutions in coding regions is likely due to the structure of the genetic code and selective constraints. Synonymous sites within coding regions showed a higher polymorphism rate than non-coding introns or 3' and 5'flanking DNA with most of the substitutions in coding regions being observed at the 3(rd )codon position. A positive correlation in the level of polymorphism was observed between coding and non-coding regions within a gene. By genotyping a subset of 30 SNPs, we confirmed the validity of the SNPs identified during this study. CONCLUSION: This set of SNP markers represents a useful tool for genetic studies in An. funestus, and will be useful in identifying candidate genes that affect diverse ranges of phenotypes that impact on vector control, such as resistance insecticide, mosquito behavior and vector competence

Field evaluation of a recombinant glutathione S-transferase-based pyrethroid quantification assay

A recombinant glutathione S-transferase (GST)-based pyrethroid quantification assay was field-tested in Ifakara, Tanzania. Initial laboratory tests suggested that all reagents used in the assay should be sufficiently stable for field use, provided that domestic refrigeration facilities were available. Insecticide-impregnated bednets were collected from a region where a social marketing programme was in progress. A total of 100 bednets were collected and the assay plus standard HPLC analysis was performed on the residues extracted from four replicate areas of each net. Insecticide residue estimations for assays performed on white and pale green bednet samples were accurate when compared with residue analysis by HPLC. However, for dark green or blue bednets, there was no correlation between the GST-based assay and HPLC pyrethroid quantification results. The assay failure with the dark coloured nets was caused by the extraction of the dyes along with the insecticide, which subsequently interfered with the GST assay. When the same samples were analysed by HPLC, the dyes were separated from the insecticide by reverse phase column chromatography and hence did not affect the result

Improved Assessment of Mass Drug Administration and Health District Management Performance to Eliminate Lymphatic Filariasis

Lymphatic filariasis (LF) elimination as a public health problem requires the interruption of transmission by administration of preventive mass drug administration (MDA) to the eligible population living in endemic districts. Suboptimal MDA coverage leads to persistent parasite transmission with consequential infection, disease and disability, and the need for continuing MDA rounds, requiring considerable investment. Routine coverage reports must be verified in each MDA implementation unit (IU) due to incorrect denominators and numerators used to calculate coverage estimates with administrative data. IU are usually the health districts. Coverage is verified so IU teams can evaluate their outreach and take appropriate action to improve performance. Mozambique and the Democratic Republic of Congo (DRC) have conducted MDA campaigns for LF since 2009 and 2014, respectively. To verify district reports and assess the achievement of the minimum 80% coverage of eligible people (or 65% of the total population), both countries conducted rapid probability surveys using Lot Quality Assurance Sampling (LQAS)(n=1102) in 2015 and 2016 in 58 IU in 49 districts. The surveys identified IU with suboptimal coverage, reasons for not residents did not take the medication, place where the medication was received, information sources, and knowledge about diseases prevented by the MDA. LQAS identified four inadequately covered IU triggering district team performance reviews with provincial and national teams and district retreatment. Provincial estimates using probability samples (weighted by populations sizes) were 10 and 17 percentage points lower than reported coverage in DRC and Mozambique. The surveys identified: absence from home during annual MDA rounds as the main reason for low performance and provided valuable information about pre-campaign and campaign activities resulting in improved strategies and continued progress towards elimination of LF and co-endemic Neglected Tropical Diseases

SMonitoring the operational impact of insecticide usage for malaria control on Anopheles funestus from Mozambique

<p>Abstract</p> <p>Background</p> <p>Indoor residual spraying (IRS) has again become popular for malaria control in Africa. This combined with the affirmation by WHO that DDT is appropriate for use in the absence of longer lasting insecticide formulations in some malaria endemic settings, has resulted in an increase in IRS with DDT as a major malaria vector control intervention in Africa. DDT was re-introduced into Mozambique's IRS programme in 2005 and is increasingly becoming the main insecticide used for malaria vector control in Mozambique. The selection of DDT as the insecticide of choice in Mozambique is evidence-based, taking account of the susceptibility of <it>Anopheles funestus </it>to all available insecticide choices, as well as operational costs of spraying.</p> <p>Previously lambda cyhalothrin had replaced DDT in Mozambique in 1993. However, resistance appeared quickly to this insecticide and, in 2000, the pyrethroid was phased out and the carbamate bendiocarb introduced. Low level resistance was detected by biochemical assay to bendiocarb in 1999 in both <it>An. funestus </it>and <it>Anopheles arabiensis</it>, although this was not evident in WHO bioassays of the same population.</p> <p>Methods</p> <p>Sentinel sites were established and monitored for insecticide resistance using WHO bioassays. These assays were conducted on 1–3 day old F1 offspring of field collected adult caught <it>An. funestus </it>females to determine levels of insecticide resistance in the malaria vector population. WHO biochemical assays were carried out to determine the frequency of insecticide resistance genes within the same population.</p> <p>Results</p> <p>In surveys conducted between 2002 and 2006, low levels of bendiocarb resistance were detected in <it>An. funestus</it>, populations using WHO bioassays. This is probably due to significantly elevated levels of Acetylcholinesterase levels found in the same populations. Pyrethroid resistance was also detected in populations and linked to elevated levels of p450 monooxygenase activity. One site had shown reduction in pyrethroid resistance since the base line in 1999.</p

The Anopheles gambiae glutathione transferase supergene family: annotation, phylogeny and expression profiles

BACKGROUND: Twenty-eight genes putatively encoding cytosolic glutathione transferases have been identified in the Anopheles gambiae genome. We manually annotated these genes and then confirmed the annotation by sequencing of A. gambiae cDNAs. Phylogenetic analysis with the 37 putative GST genes from Drosophila and representative GSTs from other taxa was undertaken to develop a nomenclature for insect GSTs. The epsilon class of insect GSTs has previously been implicated in conferring insecticide resistance in several insect species. We compared the expression level of all members of this GST class in two strains of A. gambiae to determine whether epsilon GST expression is correlated with insecticide resistance status. RESULTS: Two A. gambiae GSTs are alternatively spliced resulting in a maximum number of 32 transcripts encoding cytosolic GSTs. We detected cDNAs for 31 of these in adult mosquitoes. There are at least six different classes of GSTs in insects but 20 of the A. gambiae GSTs belong to the two insect specific classes, delta and epsilon. Members of these two GST classes are clustered on chromosome arms 2L and 3R respectively. Two members of the GST supergene family are intronless. Amongst the remainder, there are 13 unique introns positions but within the epsilon and delta class, there is considerable conservation of intron positions. Five of the eight epsilon GSTs are overexpressed in a DDT resistant strain of A. gambiae. CONCLUSIONS: The GST supergene family in A. gambiae is extensive and regulation of transcription of these genes is complex. Expression profiling of the epsilon class supports earlier predictions that this class is important in conferring insecticide resistance

Developing global maps of insecticide resistance risk to improve vector control.

Background Significant reductions in malaria transmission have been achieved over the last 15 years with elimination occurring in a small number of countries, however, increasing drug and insecticide resistance threatens these gains. Insecticide resistance has decreased the observed mortality to the most commonly used insecticide class, the pyrethroids, and the number of alternative classes approved for use in public health is limited. Disease prevention and elimination relies on operational control of Anopheles malaria vectors, which requires the deployment of effective insecticides. Resistance is a rapidly evolving phenomena and the resources and human capacity to continuously monitor vast numbers of mosquito populations in numerous locations simultaneously are not available. Methods Resistance data are obtained from published articles, by contacting authors and custodians of unpublished data sets. Where possible data is disaggregated to single sites and collection periods to give a fine spatial resolution. Results Currently the data set includes data from 1955 to October 2016 from 71 malaria endemic countries and 74 anopheline species. This includes data for all four classes of insecticides and associated resistance mechanisms. Conclusions Resistance is a rapidly evolving phenomena and the resources and human capacity to continuously monitor vast numbers of mosquito populations in numerous locations simultaneously are not available. The Malaria Atlas Project-Insecticide Resistance (MAP-IR) venture has been established to develop tools that will use available data to provide best estimates of the spatial distribution of insecticide resistance and help guide control programmes on this serious issue

Evolution of insecticide resistance and its mechanisms in Anopheles stephensi in the WHO Eastern Mediterranean Region

Background: While Iran is on the path to eliminating malaria, the disease with 4.9 million estimated cases and 9300 estimated deaths in 2018 remains a serious health problem in the World Health Organization (WHO) Eastern Mediterranean Region. Anopheles stephensi is the main malaria vector in Iran and its range extends from Iraq to western China. Recently, the vector invaded new territories in Sri Lanka and countries in the Horn of Africa. Insecticide resistance in An. stephensi is a potential issue in controlling the spread of this vector. Methods: Data were collated from national and international databases, including PubMed, Google Scholar, Scopus, ScienceDirect, SID, and IranMedex using appropriate search terms. Results: Indoor residual spaying (IRS) with DDT was piloted in Iran in 1945 and subsequently used in the malaria eradication programme. Resistance to DDT in An. stephensi was detected in Iran, Iraq, Pakistan, and Saudi Arabia in the late 1960s. Malathion was used for malaria control in Iran in 1967, then propoxur in 1978, followed by pirimiphosmethyl from 1992 to 1994. The pyrethroid insecticide lambda-cyhalothrin was used from 1994 to 2003 followed by deltamethrin IRS and long-lasting insecticidal nets (LLINs). Some of these insecticides with the same sequence were used in other malaria-endemic countries of the region. Pyrethroid resistance was detected in An. stephensi in Afghanistan in 2010, in 2011 in India and in 2012 in Iran. The newly invaded population of An. stephensi in Ethiopia was resistant to insecticides of all four major insecticide classes. Different mechanisms of insecticide resistance, including metabolic and insecticide target site insensitivity, have been developed in An. stephensi. Resistance to DDT was initially glutathione S-transferase based. Target site knockdown resistance was later selected by pyrethroids. Esterases and altered acetylcholinesterase are the underlying cause of organophosphate resistance and cytochrome p450s were involved in pyrethroid metabolic resistance. Conclusions: Anopheles stephensi is a major malaria vector in Iran and many countries in the region and beyond. The species is leading in terms of development of insecticide resistance as well as developing a variety of resistance mechanisms. Knowledge of the evolution of insecticide resistance and their underlying mechanisms, in particular, are important to Iran, considering the final steps the country is taking towards malaria elimination, but also to other countries in the region for their battle against malaria. This systematic review may also be of value to countries and territories newly invaded by this species, especially in the Horn of Africa, where the malaria situation is already dire

A new malaria vector in Africa: Predicting the expansion range of Anopheles stephensi and identifying the urban populations at risk

In 2012, an unusual outbreak of urban malaria was reported from Djibouti City in the Horn of Africa and increasingly severe outbreaks have been reported annually ever since. Subsequent investigations discovered the presence of an Asian mosquito species; Anopheles stephensi, a species known to thrive in urban environments. Since that first report, An. stephensi has been identified in Ethiopia and Sudan, and this worrying development has prompted the World Health Organization (WHO) to publish a vector alert calling for active mosquito surveillance in the region. Using an up-to-date database of published locational records for An. stephensi across its full range (Asia, Arabian Peninsula, Horn of Africa) and a set of spatial models that identify the environmental conditions that characterize a species’ preferred habitat, we provide evidence-based maps predicting the possible locations across Africa where An. stephensi could establish if allowed to spread unchecked. Unsurprisingly, due to this species’ close association with man-made habitats, our maps predict a high probability of presence within many urban cities across Africa where our estimates suggest that over 126 million people reside. Our results strongly support the WHO’s call for surveillance and targeted vector control and provide a basis for the prioritization of surveillance

Learning about the complexity of humanised care: findings from a longitudinal study of nurse students’ perceptions

Background, including underpinning literature and, wherever possible, the international relevance of the research The rise in elderly populations with complex long-term conditions is a global phenomenon (World Health Organisation, 2011). However evidence has emerged internationally around concerns about quality of care, particularly for older people (Katz 2011; Organisation for Economic Cooperation and Development, 2013). It has been argued that in target-driven care environments, there is the potential for care professionals to lose sight of the person receiving the care and focuses on task-completion, resulting in dehumanising care cultures (Galvin and Todres 2013). Educating nurse students to effectively as well as compassionately deliver complex care to vulnerable people represents considerable challenges. Following a number of high profile examples of poor quality care in the United Kingdom (UK) (Francis 2013), health profession education providers now follow professional body guidelines that mandate that values for compassionate practice clearly underpin curricula. Limited evidence exists however, concerning the impact of this initiative over time. This paper reports on a study that evidences the impact on students of an education programme based on a humanising care philosophy in shaping the students’ value base as they progress through their education programmes. Aim(s) and/or research question(s)/research hypothesis(es) . This paper reports on a five-phase longitudinal study exploring the impact of an undergraduate-nursing curriculum based on a humanising care philosophy on the values of individual students. Research methodology/research design, any ethical issues, and methods of data collection and analysis The study uses a qualitative longitudinal approach to understanding the beliefs and values of student nurses from the day of entry, through their education programme to completion. Focusing on two cohorts of students one year apart, data were collected by individual interview at commencement and completion and by focus groups at the end of their first placement and at the end of their first and second years. This presentation reports up to the end of the programme for the first cohort and end of second year for the second cohort. Ethics approval was gained for the entire project. At each stage of the process, students were formally invited to participate and on-going individual consent was gained. The audio-recordings were transcribed verbatim and analysed thematically. Key findings and recommendations By the end of their first placement, both groups were very similar in the expression of their values and to some extent presented a rather negative view of their experiences. However students from both groups became more articulate in expressing their values particularly in relation to their placement experience, as they moved through their programme. Four on-going and developing themes emerged from the data: personal journey, impact of the curriculum, impact of practice and values development. Overall, although each cohort followed two distinct curricula, there were few differences between the groups at each stage. Reasons for this will be discussed. Both groups felt they had become less judgmental, that the curriculum had empowered them to challenge practice and enhanced their confidence. Students also reported that their mentors (placement supervisors) role modelled authentic compassionate care in practice. Over time students’ insight into the complexity of caring developed and their understanding of the importance of practice that respected patient individuality deepened. This study has evidenced the strong link between curriculum and its impact on the evolving values of nursing students. Whilst this paper reports on a local study from one university in the UK, transferability of findings to other settings can be judged; it is argued that the insights will have resonance for nurse educators more widely as well as other health professional disciplines. References Commissioning Board Chief Nursing Officer and Department of Health Chief Nurse Advisor (2012). Compassion in Practice: Nursing, Midwifery and Care Staff. Our vision and strategy. Retrieved 22nd January 2016 from: http://www.england.nhs.uk/wp-content/uploads/2012/12/compassion-in-practice.pdf Francis Inquiry, (2013). Report of the Mid Staffordshire NHS Foundation Trust Public Inquiry: Executive summary. Crown Copyright: London. Galvin, K., Todres, L. (2013). Caring and Well-Being: A Lifeworld Approach. Routledge: Abingdon, Oxon. Katz, P.R. (2011). An International Perspective on Long Term Care: Focus on Nursing Homes. Journal of American Medical Directors Association. 12 (7): 487-492 OECD (2013) OECD Reviews of Health Care Quality: Denmark. Retrieved 7th Retrieved 22nd January 2016 from: http://www.oecd.org/els/health-systems/ReviewofHealthCareQualityDENMARK_ExecutiveSummary.pdf WHO (2011) Global Health and Ageing. Retrieved 22nd January 2016 from: http://www.who.int/ageing/publications/global_health.pdf Key words: (5) • Nurse education • Professional values • Humanising care • Curriculum • Longitudinal qualitative research 3 key points to indicate how your work contributes to knowledge development within the selected theme • Nursing students were able to clearly articulate values that underpin humanised care • This study provides evidence for the strong link between curriculum and its impact on the evolving values of nursing students • The curriculum is one factor in embedding humanised values in nursing students; further research is required