Application of a recombinase polymerase amplification (RPA) assay and pilot field testing for Giardia duodenalis at Lake Albert, Uganda.

BACKGROUND: Giardia duodenalis is a gastrointestinal protozoan causing 184 million cases of giardiasis worldwide annually. Detection is by microscopy or coproantigen assays, although sensitivity is often compromised by intermittent shedding of cysts or trophozoites, or operator expertise. Therefore, for enhanced surveillance field-applicable, point-of-care (POC), molecular assays are needed. Our aims were to: (i) optimise the recombinase polymerase amplification (RPA) assay for the isothermal amplification of the G. duodenalis β-giardin gene from trophozoites and cysts, using published primer and probes; and (ii) perform a pilot field validation of RPA at a field station in a resource-poor setting, on DNA extracted from stool samples from schoolchildren in villages around Lake Albert, Uganda. Results were compared to an established laboratory small subunit ribosomal RNA (SSU rDNA) qPCR assay with additional testing using a qPCR targeting the triose phosphate isomerase (tpi) DNA regions that can distinguish G. duodenalis of two different assemblages (A and B), which are human-specific. RESULTS: Initial optimisation resulted in the successful amplification of predicted RPA products from G. duodenalis-purified gDNA, producing a double-labelled amplicon detected using lateral flow strips. In the field setting, of 129 stool samples, 49 (37.9%) were positive using the Giardia/Cryptosporidium QuikChek coproantigen test; however, the RPA assay when conducted in the field was positive for a single stool sample. Subsequent molecular screening in the laboratory on a subset (n = 73) of the samples demonstrated better results with 21 (28.8%) RPA positive. The SSU rDNA qPCR assay resulted in 30/129 (23.3%) positive samples; 18 out of 73 (24.7%) were assemblage typed (9 assemblage A; 5 assemblage B; and 4 mixed A+B). Compared with the SSU rDNA qPCR, QuikChek was more sensitive than RPA (85.7 vs 61.9%), but with similar specificities (80.8 vs 84.6%). In comparison to QuikChek, RPA had 46.4% sensitivity and 82.2% specificity. CONCLUSIONS: To the best of our knowledge, this is the first in-field and comparative laboratory validation of RPA for giardiasis in low resource settings. Further refinement and technology transfer, specifically in relation to stool sample preparation, will be needed to implement this assay in the field, which could assist better detection of asymptomatic Giardia infections

Comparison of routine health management information system versus enhanced inpatient malaria surveillance for estimating the burden of malaria among children admitted to four hospitals in Uganda.

The primary source of malaria surveillance data in Uganda is the Health Management Information System (HMIS), which does not require laboratory confirmation of reported malaria cases. To improve data quality, an enhanced inpatient malaria surveillance system (EIMSS) was implemented with emphasis on malaria testing of all children admitted in select hospitals. Data were compared between the HMIS and the EIMSS at four hospitals over a period of 12 months. After the implementation of the EIMSS, over 96% of admitted children under 5 years of age underwent laboratory testing for malaria. The HMIS significantly overreported the proportion of children under 5 years of age admitted with malaria (average absolute difference = 19%, range = 8-27% across the four hospitals) compared with the EIMSS. To improve the quality of the HMIS data for malaria surveillance, the National Malaria Control Program should, in addition to increasing malaria testing rates, focus on linking laboratory test results to reported malaria cases

Strategic sourcing supplier selection misalignment with critical success factors:findings from multiple case studies in Germany and the United Kingdom

Strategic sourcing plays an important role in organisations' performance. Strategic sourcing has been researched extensively using empirical studies as well as review work, such as strategic sourcing importance, issues and challenges, processes, source selection criteria and framework. However, there is no research on critical success factors for strategic sourcing specific to industry and country. This research aims to qualitatively evaluate and understand the current role of strategic sourcing, the critical success factors for business performance and its relationship with strategic sourcing, and strategic supplier evaluation criteria from multiple stakeholders' perspectives specific to industry and country. This research studies twenty organisations from Germany and the United Kingdom (UK) covering two industry sectors - electronics manufacturing and construction. We consider five organisations from each industry sector and each country. The findings from twenty case studies reveal comparative analysis of strategic sourcing practices of two countries and two industries

Observations and models of inertial waves in the deep ocean

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February, 1980The structure of the inertial peak in deep ocean kinetic energy spectra is studied here. Records were obtained from Polymode arrays deployed in the Western North Atlantic Ocean (40°W to 70°W, 15°N to 42°N). The results are interpreted both in terms of local sources and of turning point effects on internal waves generated at lower latitudes. In most of the data, there is a prominent inertial peak slightly above f; however, the peak height above the background continuum varies with depth and geographical environment. Three classes of environment and their corresponding spectra emerge from peak height variations: class 1 is the 1500 m level near the Mid-Atlantic Ridge, with the greatest peak height of 18 db; class 2 includes (a) the upper ocean (depth less than 2000 m), (b) the deep ocean (depth greater than 2000 m) over rough topography, and (c) the deep ocean underneath the Gulf Stream, with intermediate peak height of 11.5 db; class 3 is the deep ocean over smooth topography, with the lowest peak height of 7.5 db. Near f, the horizontal coherence scale is 0(60 km) at depths from 200 m to 600 m, and the vertical coherence scale is O(200 m) just below the main thermocline. A one turning point model is developed to describe inertial waves at mid-latitudes, based on the assumption that inertial waves are randomly generated at lower latitudes (global generation) where their frequency-wavenumber spectrum is given by the model of Garrett and Munk (1972 a, 1975). Using the globally valid wave functions obtained by Munk and Phillips (1968), various frequency spectra near f are calculated numerically. The model yields a prominent inertial peak of 7 db in the horizontal velocity spectrum but no peaks in the temperature spectrum. The model is latitudinally dependent: the frequency shift and bandwidth of the inertial peak decrease with latitude; energy level near f is minimum at about 30° and higher at low and high latitudes. The observations of class 3 can be well-described by the model; a low zonal wavenumber cutoff is required to produce the observed frequency shift of the inertial peak. The differences between the global generation model and the observations of class 1 and class 2 are interpreted as the effects of local sources. A locally forced model is developed based on the latitudinal modal decomposition of a localized source function. Asymptotic eigensolutions of the Laplace's tidal equation are therefore derived and used as a set of expansion functions. The forcing is through a vertical velocity field specified at the top or bottom boundaries of the ocean. For white noise forcing, the horizontal velocity spectrum of the response has an inertial peak which diminishes in the far-field. With the forcing located at either the surface or the bottom, several properties of the class 2 observations can be described qualitatively by a combination of the global and local models. The reflection of inertial waves from a turbulent benthic boundary layer is studied by a slab model of given depth. Frictional effects are confined to the boundary layer and modelled by a quadratic drag law. For given incident waves, reflection coefficients are found to be greater than 0.9 for the long waves which contain most of the energy. This result suggests that energy-containing inertial waves can propagate over great distance as is required by the validity of the model of global generation.This work was supported by the National Science Foundation through grant OCE 76-80210 and its continuation OEE 78-19833

Development and initial validation of a measure of metacognitive beliefs in health anxiety: The MCQ-HA

Metacognitive beliefs have been shown to correlate with emotional disorders and more recently have been implicated in health anxiety. Research exploring these beliefs have tended to use the Metacognition Questionnaire (MCQ), which is a general measure. To facilitate research on the metacognitive model applied to health anxiety the present study reports on the development and initial evaluation of a new specific metacognitive measure of health anxiety, the Metacognitions Questionnaire-Health Anxiety (MCQ-HA). Principal components analysis identified 14 suitable items to be explored. Subsequent exploratory factor analysis of the MCQ-HA identified three factors: “Beliefs that Thoughts can cause Illness”, “Beliefs about Biased thinking”, and “Beliefs that Thoughts are Uncontrollable”. Confirmatory factor analysis supported the three factor model with all selected goodness-of-fit statistics equivalent to or better than recommended values. Preliminary evidence suggests good internal-consistency, incremental, convergent and discriminant validity in relation to associated measures. The MCQ-HA appears to be a potentially useful predictor of health anxiet

Consequences of Eukaryotic Enhancer Architecture for Gene Expression Dynamics, Development, and Fitness

The regulatory logic of time- and tissue-specific gene expression has mostly been dissected in the context of the smallest DNA fragments that, when isolated, recapitulate native expression in reporter assays. It is not known if the genomic sequences surrounding such fragments, often evolutionarily conserved, have any biological function or not. Using an enhancer of the even-skipped gene of Drosophila as a model, we investigate the functional significance of the genomic sequences surrounding empirically identified enhancers. A 480 bp long “minimal stripe element” is able to drive even-skipped expression in the second of seven stripes but is embedded in a larger region of 800 bp containing evolutionarily conserved binding sites for required transcription factors. To assess the overall fitness contribution made by these binding sites in the native genomic context, we employed a gene-replacement strategy in which whole-locus transgenes, capable of rescuing even-skipped- lethality to adulthood, were substituted for the native gene. The molecular phenotypes were characterized by tagging Even-skipped with a fluorescent protein and monitoring gene expression dynamics in living embryos. We used recombineering to excise the sequences surrounding the minimal enhancer and site-specific transgenesis to create co-isogenic strains differing only in their stripe 2 sequences. Remarkably, the flanking sequences were dispensable for viability, proving the sufficiency of the minimal element for biological function under normal conditions. These sequences are required for robustness to genetic and environmental perturbation instead. The mutant enhancers had measurable sex- and dose-dependent effects on viability. At the molecular level, the mutants showed a destabilization of stripe placement and improper activation of downstream genes. Finally, we demonstrate through live measurements that the peripheral sequences are required for temperature compensation. These results imply that seemingly redundant regulatory sequences beyond the minimal enhancer are necessary for robust gene expression and that “robustness” itself must be an evolved characteristic of the wild-type enhancer