Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions

The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time – calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional

Rational use of antibiotics by community health workers and caregivers for children with suspected pneumonia in Zambia: A cross-sectional mixed methods study

Background: Antibiotic resistance is an issue of growing global concern. One key strategy to minimise further development of resistance is the rational use of antibiotics, by providers and patients alike. Through integrated community case management (iCCM), children diagnosed with suspected pneumonia are treated with antibiotics; one component of an essential package to reduce child mortality and increase access to health care for remote populations. Through the use of clinical algorithms, supportive supervision and training, iCCM also offers the opportunity to improve the rational use of antibiotics and limit the spread of resistance in resource-poor contexts. This study provides evidence on antibiotic use by community health workers (CHWs) and caregivers to inform iCCM programmes, safeguarding current treatments whilst maximising access to care. Methods: 1497 CHW consultations were directly observed by non-clinical researchers, with measurement of respiratory rate by CHWs recorded by video. Videos were used to conduct a retrospective reference standard assessment of respiratory rate by experts. Fifty-five caregivers whose children were prescribed a 5-day course of antibiotics for suspected pneumonia were followed up on day six to assess adherence through structured interviews and pill counts. Six focus group discussions and nine in depth interviews were conducted with CHWs and caregivers to supplement quantitative findings. Results: The findings indicate that CHWs adhered to treatment guidelines for 92 % of children seen, prescribing treatment corresponding to their assessment. However, only 65 % of antibiotics prescribed were given for children with experts' confirmed fast breathing pneumonia. Qualitative data indicates that CHWs have a good understanding of pneumonia diagnosis, and although caregivers sometimes applied pressure to receive drugs, CHWs stated that treatment decisions were not influenced. 46 % of caregivers were fully adherent and gave their child the full 5-day course of dispersible amoxicillin. If caregivers who gave treatment for 3 to 5 days were considered, adherence increased to 76 %. Conclusions: CHWs are capable of prescribing treatment corresponding to their assessment of respiratory rate. However, rational use of antibiotics could be strengthened through improved respiratory rate assessment, and better diagnostic tools. Furthermore, a shorter course of dispersible amoxicillin could potentially improve caregiver adherence, reducing risk of resistance and cost

Diagnostic Testing of Pediatric Fevers: Meta-Analysis of 13 National Surveys Assessing Influences of Malaria Endemicity and Source of Care on Test Uptake for Febrile Children under Five Years.

In 2010, the World Health Organization revised guidelines to recommend diagnosis of all suspected malaria cases prior to treatment. There has been no systematic assessment of malaria test uptake for pediatric fevers at the population level as countries start implementing guidelines. We examined test use for pediatric fevers in relation to malaria endemicity and treatment-seeking behavior in multiple sub-Saharan African countries in initial years of implementation. We compiled data from national population-based surveys reporting fever prevalence, care-seeking and diagnostic use for children under five years in 13 sub-Saharan African countries in 2009-2011/12 (n = 105,791). Mixed-effects logistic regression models quantified the influence of source of care and malaria endemicity on test use after adjusting for socioeconomic covariates. Results were stratified by malaria endemicity categories: low (PfPR2-10<5%), moderate (PfPR2-10 5-40%), high (PfPR2-10>40%). Among febrile under-fives surveyed, 16.9% (95% CI: 11.8%-21.9%) were tested. Compared to hospitals, febrile children attending non-hospital sources (OR: 0.62, 95% CI: 0.56-0.69) and community health workers (OR: 0.31, 95% CI: 0.23-0.43) were less often tested. Febrile children in high-risk areas had reduced odds of testing compared to low-risk settings (OR: 0.51, 95% CI: 0.42-0.62). Febrile children in least poor households were more often tested than in poorest (OR: 1.63, 95% CI: 1.39-1.91), as were children with better-educated mothers compared to least educated (OR: 1.33, 95% CI: 1.16-1.54). Diagnostic testing of pediatric fevers was low and inequitable at the outset of new guidelines. Greater testing is needed at lower or less formal sources where pediatric fevers are commonly managed, particularly to reach the poorest. Lower test uptake in high-risk settings merits further investigation given potential implications for diagnostic scale-up in these areas. Findings could inform continued implementation of new guidelines to improve access to and equity in point-of-care diagnostics use for pediatric fevers

Natural infection by the protozoan Leptomonas wallacei impacts the morphology, physiology, reproduction, and lifespan of the insect Oncopeltus fasciatus

Trypanosomatids are protozoan parasites that infect thousands of globally dispersed hosts, potentially affecting their physiology. Several species of trypanosomatids are commonly found in phytophagous insects. Leptomonas wallacei is a gut-restricted insect trypanosomatid only retrieved from Oncopeltus fasciatus. The insects get infected by coprophagy and transovum transmission of L. wallacei cysts. The main goal of the present study was to investigate the effects of a natural infection by L. wallacei on the hemipteran insect O. fasciatus, by comparing infected and uninfected individuals in a controlled environment. The L. wallacei-infected individuals showed reduced lifespan and morphological alterations. Also, we demonstrated a higher infection burden in females than in males. The infection caused by L. wallacei reduced host reproductive fitness by negatively impacting egg load, oviposition, and eclosion, and promoting an increase in egg reabsorption. Moreover, we associated the egg reabsorption observed in infected females, with a decrease in the intersex gene expression. Finally, we suggest alterations in population dynamics induced by L. wallacei infection using a mathematical model. Collectively, our findings demonstrated that L. wallacei infection negatively affected the physiology of O. fasciatus, which suggests that L. wallacei potentially has a vast ecological impact on host population growth

Greenland ice sheet surface mass loss: recent developments in observation and modeling

Surface processes currently dominate Greenland ice sheet (GrIS) mass loss. We review recent developments in the observation and modelling of GrIS surface mass balance (SMB), published after the July 2012 deadline for the Fifth Assessment Report of the Intergovernmental Panel on Climate Change (IPCC AR5). Since IPCC AR5 our understanding of GrIS SMB has further improved, but new observational and model studies have also revealed that temporal and spatial variability of many processes are still poorly quantified and understood, e.g. bio-albedo, the formation of ice lenses and their impact on lateral meltwater transport, heterogeneous vertical meltwater transport (‘piping’), the impact of atmospheric circulation changes and mixed-phase clouds on the surface energy balance and the magnitude of turbulent heat exchange over rough ice surfaces. As a result, these processes are only schematically or not at all included in models that are currently used to assess and predict future GrIS surface mass loss

Improving access to health care for malaria in Africa: a review of literature on what attracts patients

BACKGROUND: Increasing access to health care services is considered central to improving the health of populations. Existing reviews to understand factors affecting access to health care have focused on attributes of patients and their communities that act as 'barriers' to access, such as education level, financial and cultural factors. This review addresses the need to learn about provider characteristics that encourage patients to attend their health services. METHODS: This literature review aims to describe research that has identified characteristics that clients are looking for in the providers they approach for their health care needs, specifically for malaria in Africa. Keywords of 'malaria' and 'treatment seek*' or 'health seek*' and 'Africa' were searched for in the following databases: Web of Science, IBSS and Medline. Reviews of each paper were undertaken by two members of the team. Factors attracting patients according to each paper were listed and the strength of evidence was assessed by evaluating the methods used and the richness of descriptions of findings. RESULTS: A total of 97 papers fulfilled the inclusion criteria and were included in the review. The review of these papers identified several characteristics that were reported to attract patients to providers of all types, including lower cost of services, close proximity to patients, positive manner of providers, medicines that patients believe will cure them, and timeliness of services. Additional categories of factors were noted to attract patients to either higher or lower-level providers. The strength of evidence reviewed varied, with limitations observed in the use of methods utilizing pre-defined questions and the uncritical use of concepts such as 'quality', 'costs' and 'access'. Although most papers (90%) were published since the year 2000, most categories of attributes had been described in earlier papers. CONCLUSION: This paper argues that improving access to services requires attention to factors that will attract patients, and recommends that public services are improved in the specific aspects identified in this review. It also argues that research into access should expand its lens to consider provider characteristics more broadly, especially using methods that enable open responses. Access must be reconceptualized beyond the notion of barriers to consider attributes of attraction if patients are to receive quality care quickly

DNA Barcode Sequence Identification Incorporating Taxonomic Hierarchy and within Taxon Variability

For DNA barcoding to succeed as a scientific endeavor an accurate and expeditious query sequence identification method is needed. Although a global multiple–sequence alignment can be generated for some barcoding markers (e.g. COI, rbcL), not all barcoding markers are as structurally conserved (e.g. matK). Thus, algorithms that depend on global multiple–sequence alignments are not universally applicable. Some sequence identification methods that use local pairwise alignments (e.g. BLAST) are unable to accurately differentiate between highly similar sequences and are not designed to cope with hierarchic phylogenetic relationships or within taxon variability. Here, I present a novel alignment–free sequence identification algorithm–BRONX–that accounts for observed within taxon variability and hierarchic relationships among taxa. BRONX identifies short variable segments and corresponding invariant flanking regions in reference sequences. These flanking regions are used to score variable regions in the query sequence without the production of a global multiple–sequence alignment. By incorporating observed within taxon variability into the scoring procedure, misidentifications arising from shared alleles/haplotypes are minimized. An explicit treatment of more inclusive terminals allows for separate identifications to be made for each taxonomic level and/or for user–defined terminals. BRONX performs better than all other methods when there is imperfect overlap between query and reference sequences (e.g. mini–barcode queries against a full–length barcode database). BRONX consistently produced better identifications at the genus–level for all query types

Climate simulations for 1880-2003 with GISS modelE

We carry out climate simulations for 1880-2003 with GISS modelE driven by ten measured or estimated climate forcings. An ensemble of climate model runs is carried out for each forcing acting individually and for all forcing mechanisms acting together. We compare side-by-side simulated climate change for each forcing, all forcings, observations, unforced variability among model ensemble members, and, if available, observed variability. Discrepancies between observations and simulations with all forcings are due to model deficiencies, inaccurate or incomplete forcings, and imperfect observations. Although there are notable discrepancies between model and observations, the fidelity is sufficient to encourage use of the model for simulations of future climate change. By using a fixed well-documented model and accurately defining the 1880-2003 forcings, we aim to provide a benchmark against which the effect of improvements in the model, climate forcings, and observations can be tested. Principal model deficiencies include unrealistically weak tropical El Nino-like variability and a poor distribution of sea ice, with too much sea ice in the Northern Hemisphere and too little in the Southern Hemisphere. The greatest uncertainties in the forcings are the temporal and spatial variations of anthropogenic aerosols and their indirect effects on clouds.Comment: 44 pages; 19 figures; Final text accepted by Climate Dynamic

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands