How effective are population health surveys for estimating prevalence of chronic conditions compared to anonymised clinical data?

INTRODUCTION: Population health surveys are used to record person-reported outcome measures for chronic health conditions and provide a useful source of data when evaluating potential disease burdens. The reliability of survey-based prevalence estimates for chronic diseases is unclear nonetheless. This study applied methodological triangulation via a data linkage method to validate prevalence of selected chronic conditions (angina, myocardial infarction, heart failure, and asthma). METHODS: Linked healthcare records were used for a combined cohort of 11,323 adults from the 2013 and 2014 sweeps of the Welsh Health Survey (WHS). The approach utilised consented survey data linked to primary and secondary care electronic health record (EHR) data back to 2002 within the Secure Anonymised Information Linkage (SAIL) Databank. RESULTS: This descriptive study demonstrates validation of survey and clinical data using data linkage for selected chronic cardiovascular conditions and asthma with varied success. The results indicate that identifying cases for separate cardiovascular conditions was limited without specific medication codes for each condition, but more straightforward for asthma, where there was an extensive list of medications available. For asthma there was better agreement between prevalence estimates based on survey and clinical data as a result. CONCLUSION: Whilst the results provide external validity for the WHS as an instrument for estimating the burden of chronic disease, they also indicate that a data linkage appproach can be used to produce comparable prevalence estimates using clinical data if a defined condition-specific set of clinical codes are available.

A pre-enrichment step is essential for detection of Campylobacter sp. in turbid pond water.

This work aimed to detect Campylobacter species from naturally contaminated turbid pond water by PCR. A total of 16 water samples were collected from a turbid village pond. Four methods of DNA extraction were applied to centrifuge pellets from eight 100 ml pond water samples prior to attempted detection of Campylobacter by PCR without an enrichment step. These methods were (1) Tris-HCl and sodium dodecyl sulfate followed by phenol:chloroform:isoamylalcohol extraction followed by treatment with DNA clean up kit, (2) proteinase K, (3) Chelex® 100, and (4) boiling. The other eight pond water samples (10 ml and 100 ml) were filtered and filters were incubated overnight in Preston enrichment broth. The centrifuge pellets obtained from enrichment cultures were treated by proteinase K for DNA extraction. Primers CF03 and CF04 for the flagellin genes (flaA and flaB) of Campylobacter jejuni and Campylobacter coli were used for amplifying the extracted DNA. The DNA extracted from eight-100 ml pond water samples that were not subject to selective enrichment was never amplified with primers CF03 and CF04, hence Campylobacter was not detected. In contrast, the DNA that was from samples that were subjected to a selective enrichment step in Preston broth prior to PCR assay always gave amplified bands of 340-380 bp, therefore the presence of Campylobacter was confirmed. Detection of campylobacters from naturally contaminated, turbid, environmental water may not be feasible by direct PCR assay because of low numbers and the presence of high concentration of humic matter and other PCR inhibitors. The enrichment of water samples in selective broth, however, facilitated PCR detection of Campylobacter probably by increasing cell number and by diluting PCR inhibitors.

Bioactivity as an options value of sea cucumbers in the Egyptian Red Sea.

The utility of a species can be divided into its direct, indirect, and options values. In the marine environment, direct consumptive values predominate and often lead to overexploitation at the expense of significant options values derived through bioprospecting for natural products. We surveyed the waters of the Egyptian Red Sea coast (Gulf of Aqaba [north] and the Red Sea [south]) for species of sea cucumbers and analyzed extracts from species for a range of bioactivities with potential biomedical applications. All habitat types were surveyed within these regions. We found 22 species of sea cucumber of which two, Holothuria fuscogilva and Holothuria flavomaculata, were recorded in Egypt for the first time. Although none of the species identified were unique to the Gulf of Aqaba, 10 species were only found in the Red Sea sector. Bioassay results showed that although no species had antibacterial activity, most extracts exhibited activity against Candida and Leishmania but were most active against a LoVo mammalian carcinoma cell line. Our most significant finding was the intraspecific variation in bioactivity in individuals collected from different habitat types and sectors of the coast. This variation may reflect the effect of environment on secondary metabolite production or may indicate significant genetic diversity between populations within a species. Our results indicate a potentially significant options value to sea cucumbers through bioprospecting. Given the importance of economic development in countries such as Egypt and the perceived low conservation value of invertebrates such as sea cucumbers, the linking of these factors to conservation is vital for the maintenance and sustainable exploitation of these animals.

Two-photon excitation studies of m-THPC photosensitizer and photodynamic activity in an epithelial cell line.

A femtosecond laser delivering pulses of wavelength 800nm and 124fs duration at rates of 1kHz has been used to investigate the two-photon excited fluorescence in the photosensitizer m-THPC. The scaling of fluorescence amplitude with laser power and fluorescence sidelight imaging are found to support a predominantly two-photon excitation mechanism. A value for the two-photon cross-section of δ=1.8×10(-57)m(4)s is derived by comparing the fluorescence signals excited by wavelengths of 800 and 400nm. Preliminary results demonstrating the two-photon induced PDT activity of m-THPC in an epithelial cell line are also reported.

Recovery of thermophilic campylobacters from pond water and sediment and the problem of interference by background bacteria in enrichment culture.

The aim of this study was to address problems in the determination of thermophilic campylobacters in turbid pond water and sediment. Thirty sets of three samples of pond water (volumes 10, 100, 1000 ml) or sediment (0.1, 1.0, 5.0 ml) were examined for the presence of thermophilic campylobacters. The different volumes of pond water were processed by membrane filtration followed by selective enrichment. The samples of sediment were subjected directly to selective enrichment. Presumptive isolates were confirmed by Gram stain, cell morphology, presence of oxidase and catalase, growth under microaerobic but not aerobic conditions, and PCR. Confirmed Campylobacter species were recovered only from 10 and 100 ml samples of water and from 0.1 and 1.0 ml samples of sediments. The 1000 ml samples of water and 5.0 ml samples of sediment never gave positive isolates. PCR indicated that the confirmed isolates were all either Campylobacter jejuni or C. coli. Enrichment cultures from 1000 ml filtrations contained the highest number of background bacteria. It is suggested that the processing of large volumes of turbid environmental water samples or of sediment is counterproductive and may not yield positive Campylobacter cultures. This is probably due to antagonistic effects of large numbers of background bacteria out-competing campylobacters during the enrichment stage. Pilot studies to establish appropriate volumes of pond water or sediment samples should be undertaken before routine determination of campylobacters is begun.

Waterfowl and the bacteriological quality of amenity ponds.

This study investigated the impact of waterfowl on the bacteriological quality of village ponds in East Yorkshire, north-east England. Water and sediment samples were collected from ponds with and without resident ducks and geese; faecal indicator and potentially pathogenic bacteria were assayed by membrane filtration and by selective enrichment. Escherichia coli, faecal streptococci and, to a degree, Clostridium perfringens were more abundant in ponds with waterfowl; Salmonella was isolated in June-August from the sediment of a pond with waterfowl. The results suggested that the bacteriological quality of village ponds might be adversely affected by waterfowl. All water samples from ponds with waterfowl had faecal indicators at higher concentrations than EU requirements for bathing waters. Although these ponds are not bathing waters we suggest skin contact and accidental ingestion of water should be avoided.

Acanthamoeba can be differentiated by the polymerase chain reaction and simple plating assays.

Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells. Further, we tested nine isolates of Acanthamoeba for their ability to produce cytopathic effects (CPE) on corneal epithelial cells. On the basis of the results, Acanthamoeba were divided into pathogenic or nonpathogenic groups. However, because CPE assays are not available to every diagnostic laboratory, we developed a simple plating assay based on osmotolerance which correlated well with the CPE assays. Pathogenic Acanthamoeba showed growth on higher osmolarity (agar plates containing one molar mannitol), while growth of nonpathogens was inhibited on these plates. In conclusion, we have developed methods for the rapid identification and differentiation of Acanthamoeba.

Regulation of carbohydrate metabolism during Giardia encystment.

Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-D-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and MTZ's inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi1 is expressed during encystment. Glucosamine 6-P (GlcN6P), the synthetic product of GPI, activates UDP-N-acetylglucosamine (UDP-GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5-fold in the direction of UDP-GlcNAc synthesis. UDP-GlcNAc is epimerized to UDP-GalNAc and UDP-GalNAc is polymerized by "cyst wall synthase" (beta 1 --> 3 GalNAc transferase) into a highly insoluble beta 1,3-linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.

Reflective practice and clinical outcomes: practitioners' views on how reflective practice has influenced their clinical practice.

There has been a recent increasing interest in reflective practice in nursing. There is a wealth of literature about its apparent advantages and benefits, but very little empirical research into clinical outcomes consequent to reflective practice. This study attempts an initial exploration into this area. A retrospective, three-phase, multi-method study in a single department of nursing was conducted. The research sample comprised students and former students of the department who had previously participated in an assessed reflective practice course or module. Years of experience, speciality or academic level did not have a significant influence, but the effectiveness of the facilitator was an important factor. The results suggest that reflective practice is regarded highly and that most respondents could identify significant, long-term changes to clinical practice resulting from it.

Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba.

Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with Acanthamoeba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic Acanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.

Isolation of Acanthamoeba-specific antibodies from a bacteriophage display library.

Acanthamoeba causes opportunistic eye infections in humans, which can lead to severe keratitis and may ultimately result in blindness. Current methods for identifying this organism rely on culture and microscopy. In this paper, we describe the isolation of antibody fragments that can be used for the unequivocal identification of Acanthamoeba. A bacteriophage antibody display library was used to isolate antibody fragments that bind specifically to Acanthamoeba. Individual clones were studied by enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence. Four antibody clones that specifically bind to Acanthamoeba spp. were identified.

Use of phage antibodies to distinguish closely related species of protozoan parasites.

Acanthamoeba are typically identified in the laboratory using culture and microscopic observation. In this paper we describe the isolation and specificity of antibody fragments that can be used for the identification of Acanthamoeba. A phage library expressing a large repertoire (approx. 5 x 10(9)) of antibody fragments was used to generate two libraries one enriched for bacteriophage that exhibit genus specific binding and the other containing bacteriophage that bind specifically to pathogenic Acanthamoeba. Individual clones were isolated on the basis of binding by ELISA, and then flow cytometry and immunofluorescence were used for further characterisation. Four monoclonal antibodies were isolated, specific for Acanthamoeba at the generic level with clone HPPG6 exhibiting the highest level of binding. Furthermore clone HPPG55 was specific for pathogenic species of Acanthamoeba.

Metabolic changes in Giardia intestinalis during differentiation.

The oxygen uptake rate and metronidazole (MTZ) sensitivity in Giardia spp. cysts is greatly reduced from that in trophozoites. Thus, this project was undertaken to assess when in the encystation process these phenomena occur. Oxygen uptake rates approximately doubled (from approximately 4.9 to 8.3 microM O2 min(-1) 10(-6) cells) during the first 5 hr into encystation. This increase was followed by a marked decrease to 2.3 microM O2 min(-1) 10(-6) by 12 hr. By 50 hr into encystation, oxygen uptake was 0.7 microM O2 min(-1) 10(-6) cells. Glucose stimulated oxygen uptake by 89% in trophozoites but did not demonstrably stimulate oxygen uptake in cells after 12 hr into encystment. Deoxy-D-glucose uptake dropped by more than an order of magnitude in encysting cells compared to nonencysting cells. In contrast, aspartate uptake remained relatively constant regardless of whether cells were encysting or not. This suggests that there is a change in the parasite's ability to transport glucose during cyst formation; a similar change in the parasite's ability to transport aspartate was not observed after 40 hr into encystation. MTZ inhibited oxygen uptake by 77% in trophozoites, but there was no detectable inhibition of oxygen uptake 8 hr after trophozoites were transferred to encystation medium. We propose that this resistance to MTZ may be due to a change in metabolic flux away from the pyruvate ferredoxin oxidoreductase pathway. Oxygen uptake by noninduced cysts increased exponentially during the 30 min following the induction of excystation. Likewise, MTZ sensitivity returned within 15 min after the induction of excystation, and by 30 min into excystation full sensitivity had returned.

Oxygen uptake in cysts and trophozoites of Giardia lamblia.

Oxygen uptake in cysts and trophozoites of the parasitic protozoan Giardia lamblia was examined. Both showed oxygen uptake activity, but that of cysts was only 10% to 20% that of trophozoites. Oxygen dependence of oxygen uptake in cysts and trophozoites showed oxygen maxima above which oxygen uptake decreased. The oxygen concentration at which the oxygen uptake rate was greatest was higher for trophozoites than for cysts. The effect of various inhibitors on cyst and trophozoite oxygen uptake suggested that flavoproteins and quinones play some role in oxygen uptake. The substrate specificities and the effect of inhibitors on G. lamblia trophozoites were similar to those observed for G. muris. Metronidazole, the drug most commonly used in treatment of giardiasis, inhibited oxygen uptake and motility in trophozoites; however, it had no obvious effect on either oxygen uptake or excystation in cysts. Menadione, a redox cycling naphthaquinone, first stimulated, then completely inhibited, oxygen uptake in cysts and trophozoites; a complete loss of cyst viability and trophozoite motility was also observed. The effect of menadione on G. lamblia may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.

The effects of oxygen on fermentation in Giardia lamblia.

Detailed study of the effects of oxygen on the carbohydrate metabolism of Giardia lamblia revealed that low concentrations of oxygen (< 0.25 microM) produced profound alterations in the carbon balance of this organism. Although this concentration of oxygen could not be detected by mass spectrometry, a marked stimulation of ethanol production was observed. Associated with this was an inhibition of alanine production and oxidation of the intracellular NAD(P)H pool. Higher concentrations of oxygen inhibited ethanol production and further reduced levels of alanine. These results suggest that this stimulation is due to changes in carbon flux. Analysis of cell and medium hydrolysates after the growth of trophozoites in [U-14C]glucose suggests that G. lamblia does not synthesise detectable levels of labelled amino acids, except alanine and to a lesser extent valine, from this sugar. Trophozoites of G. lamblia have both glutamate dehydrogenase and alanine aminotransferase activity. As glutamate is taken up from the medium, it is suggested that glutamate dehydrogenase and alanine aminotransferase cooperate to convert pyruvate to alanine, with the concomitant oxidation of NAD(P)H.

Nippostrongylus brasiliensis and Ascaridia galli: characterization of peroxisomes.

A method is described for the isolation of peroxisomes from mitochondria-enriched fractions obtained from both species of nematodes. The distributions of these organelles are characterized after density gradient centrifugation in sucrose or Percoll by urate oxidase and catalase activities. The possession of peroxisomes may be part of an important defence mechanism in parasites.