Changes in bacterial composition and enzymatic activity in ileostomy and ileal reservoir during intermittent occlusion: a study using dogs.

Bacterial flora, activities of 10 potential mucus- and dietary polysaccharide-degrading enzymes, blood group antigenicity of the intestinal glycoproteins, and proteolytic activity in the output from experimentally colectomized dogs with conventional ileostomies and dogs with valveless ileal reservoirs (pouches) were determined. The ileostomies of dogs with conventional surgery (group II) and with pouches (group III) were occluded intermittently during a 6-week period. The duration of occlusion was progressively increased. Group I, five dogs with conventional ileostomies, served as a control group. After occlusion of the ileal pouch for 7 h, total numbers of bacteria increased threefold, glycosidase activity increased fivefold, and blood group antigenicity of the intestinal glycoproteins, which was high in the output from the nonoccluded pouch, was no longer detectable. Proteolytic activity was not influenced by occlusion of the pouch. Significantly lower numbers of bacteria, only minor glycosidase activity, high blood group antigenicities of the intestinal glycoproteins, and higher proteolytic activity were found in ileostomy effluents from groups I and II. Histopathological examination showed chronic inflammation and changes in crypt-villus ratio in all dogs with ileal reservoirs; the ileal mucosa from the dogs with conventional ileostomies did not show any abnormalities. Consequences of the flora-related enzyme activities for the ileal mucosa are discussed.     

Performance evaluation of Baermann techniques: The quest for developing a microscopy reference standard for the diagnosis of Strongyloides stercoralis

BackgroundSoil-transmitted helminths (STH) are common in low and middle income countries where there is lack of access to clean water and sanitation. Effective diagnosis and treatment are essential for the control of STH infections. However, among STH parasites, Strongyloides stercoralis is the most neglected species, both in diagnostics and control strategies. Diagnostic methods cover different approaches, each with different sensitivities and specificities, such as serology, molecular techniques and microscopy based techniques. Of the later, the Baermann technique is the most commonly used procedure. In the literature, several ways have been described to perform the Baermann method, which illustrates the overall lack of a ‘(gold) reference standard’ method for the diagnosis of S. stercoralis infection. In this study we have evaluated the performance of three Baermann techniques in order to improve the reference standard for the microscopic diagnosis of S. stercoralis infection thereby facilitating individual case detection, mapping of the disease and proper evaluation of treatment responses.Methods/Principal findingsA community based cross sectional study was conducted at Zenzelima, Bahir Dar Zuria Ethiopia. A total of 437 stool samples were collected and analyzed by the following procedures: conventional Baermann (CB), modified Baermann (MB), and modified Baermann with charcoal pre-incubation (MBCI). The diagnostic sensitivity and Negative Predictive Value (NPV) of each technique was calculated using the combination of all the three techniques as a composite reference standard. Our result indicated that larvae of S. stercoralis were detected in 151 (34.6%) stool samples. The prevalence of S. stercoralis infection based on the three diagnostic methods was 9.6%, 8.0%, and 31.3% by CB, MB, and MBCI respectively. The sensitivity and NPV for CB, MB, and MBCI were 26.7% and 70.8%, 22.1% and 69.6%, and 87.0% and 93.2%, respectively. The MBCI showed significant difference (P- value = <0.001) in the sensitivity and NPV values when compared with CB and MB values. The agreement between CB, MB, and MBCI with the composite reference standard was 31.8%, 26.7%, 89.6%, respectively.Conclusion/SignificanceOur results suggest the superior performance of MBCI. It is relatively easy to implement, simple to perform and comparatively cheaper. The CB is by far the commonly used method in routine diagnostic although this technique significantly underestimates the true burden of the disease and thereby contributing to the exclusion of S. stercoralis from the control strategies. Therefore, MBCI is recommended as a routine microscopy-based diagnostic test for S. stercoralis infection, particularly in settings where molecular procedures are not available.     

Heat stress but not inbreeding affects offensive sperm competitiveness in Callosobruchus maculatus

Environmental and genetic stress have well‐known detrimental effects on ejaculate quality, but their concomitant effect on male fitness remains poorly understood. We used competitive fertilization assays to expose the effects of stress on offensive sperm competitive ability in the beetle Callosobruchus maculatus, a species where ejaculates make up more than 5% of male body mass. To examine the effects of environmental and genetic stress, males derived from outcrosses or sib matings were heat shocked at 50°C for 50 min during the pupal stage, while their siblings were maintained at a standard rearing temperature of 28°C. Heat‐shocked males achieved only half the offensive paternity success of their siblings. While this population exhibited inbreeding depression in body size, sperm competitiveness was unaffected by inbreeding, nor did the effect of heat shock stress on sperm competitiveness depend on inbreeding status. In contrast, pupal emergence success was increased by 34% among heat‐stressed individuals, regardless of their inbreeding status. Heat‐shocked males' ejaculate size was 19% reduced, but they exhibited 25% increased mating duration in single mating trials. Our results highlight both the importance of stress in postcopulatory sexual selection, and the variability among stressors in affecting male fitness.     

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

Validation of the isothermal Schistosoma haematobium Recombinase Polymerase Amplification (RPA) assay,coupled with simplified sample preparation,for diagnosing female genital schistosomiasis using cervicovaginal lavage and vaginal self-swab samples

BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (QIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings.     

Process evaluation of a tailored intervention programme of cardiovascular risk management in general practices

Background

A tailored implementation programme to improve cardiovascular risk management (CVRM) in general practice had little impact on outcomes. The questions in this process evaluation concerned (1) impact on counselling skills and CVRM knowledge of practice nurses, (2) their use of the various components of the intervention programme and adoption of recommended practices and (3) patients’ perceptions of counselling for CVRM.

Methods

A mixed-methods process evaluation was conducted. We assessed practice nurses’ motivational interviewing skills on audio-taped consultations using Motivational Interviewing Treatment Integrity (MITI). They also completed a clinical knowledge test. Both practice nurses and patients reported on their experiences in a written questionnaire and interviews. A multilevel regression analysis and an independent sample t test were used to examine motivational interviewing skills and CVRM knowledge. Framework analysis was applied to analyse qualitative data.

Results

Data from 34 general practices were available, 19 intervention practices and 14 control practices. No improvements were measured on motivational interviewing skills in both groups. There appeared to be better knowledge of CVRM in the control group. On average half of the practice nurses indicated that they adopted the recommended interventions, but stated that they did not necessarily record this in patients’ medical files. The tailored programme was perceived as too large. Time, follow-up support and reminders were felt to be lacking. About 20% of patients in the intervention group visited the general practice during the intervention period, yet only a small number of these patients were referred to recommended options.

Conclusions

The tailored programme was only partly used by practice nurses and had little impact on either their clinical knowledge and communication skills or on patient reported healthcare. If the assumed logical model of change is valid, a more intensive programme is needed to have an impact on CVRM in general practice at all.

     

Long‐term vegetation changes in experimentally grazed and ungrazed back‐barrier marshes in the Wadden Sea

Abstract. Vegetation succession in three back‐barrier salt marshes in the Wadden Sea was studied using a data set comprising 25 years of vegetation development recorded at permanent quadrats. The effect of livestock grazing on succession was assessed by comparing quadrats where grazing was experimentally prevented or imposed. We studied changes at the species level as well as at the level of the plant community. Special attention is given to effects on plant species richness and community characteristics that are relevant for lagomorphs (hares and rabbits) and geese. Inundation frequency and grazing were most important in explaining the variation in species abundance data. The three marshes studied overlap in the occurrence of different plant communities and the observed patterns were consistent between them. Clear differences in frequency and abundance of plant species were observed related to grazing. Most plant species had a greater incidence in grazed treatments. Species richness increased with elevation, and was 1.5 to 2 × higher in the grazed salt marsh. Grazing negatively influenced Atriplex portulacoides and Elymus athericus, whereas Puccinellia maritima and Festuca rubra showed a positive response. The communities dominated by Elymus athericus, Artemisia maritima and Atriplex portulacoides were restricted to the ungrazed marsh. Communities dominated by Puccinellia maritima, Juncus gerardi and Festuca rubra predominantly occurred at grazed sites. As small vertebrate herbivores prefer these plants and communities for foraging, livestock grazing thus facilitates for them.     

Changes in bacterial composition and enzymatic activity in ileostomy and ileal reservoir during intermittent occlusion: a study using dogs.

Bacterial flora, activities of 10 potential mucus- and dietary polysaccharide-degrading enzymes, blood group antigenicity of the intestinal glycoproteins, and proteolytic activity in the output from experimentally colectomized dogs with conventional ileostomies and dogs with valveless ileal reservoirs (pouches) were determined. The ileostomies of dogs with conventional surgery (group II) and with pouches (group III) were occluded intermittently during a 6-week period. The duration of occlusion was progressively increased. Group I, five dogs with conventional ileostomies, served as a control group. After occlusion of the ileal pouch for 7 h, total numbers of bacteria increased threefold, glycosidase activity increased fivefold, and blood group antigenicity of the intestinal glycoproteins, which was high in the output from the nonoccluded pouch, was no longer detectable. Proteolytic activity was not influenced by occlusion of the pouch. Significantly lower numbers of bacteria, only minor glycosidase activity, high blood group antigenicities of the intestinal glycoproteins, and higher proteolytic activity were found in ileostomy effluents from groups I and II. Histopathological examination showed chronic inflammation and changes in crypt-villus ratio in all dogs with ileal reservoirs; the ileal mucosa from the dogs with conventional ileostomies did not show any abnormalities. Consequences of the flora-related enzyme activities for the ileal mucosa are discussed.