Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.     

Terminology and quantification of environmental heterogeneity in species‐richness research

Spatial environmental heterogeneity (EH) is an important driver of species diversity, and its influence on species richness has been analysed for numerous taxa, in diverse ecological settings, and over a large range of spatial scales. The variety and ambiguity of concepts and terminology, however, have hampered comparisons among studies. Based on a systematic literature survey of 192 studies including 1148 data points, we provide an overview of terms and measures related to EH, and the mechanisms that relate EH to species richness of plants and animals in terrestrial systems. We identify 165 different measures used to quantify EH, referred to by more than 350 measure names. We classify these measures according to their calculation method and subject area, finding that most studies have analysed heterogeneity in land cover, topography, and vegetation, whereas comparatively few studies have focused on climatic or soil EH. Overall, elevation range emerged as the most frequent measure in our dataset. We find that there is no consensus in the literature about terms (such as ‘habitat diversity’ or ‘habitat complexity’), their meanings and associated quantification methods. More than 100 different terms have been used to denote EH, with largely imprecise delimitations. We reveal trends in use of terms and quantification with respect to spatial scales, study taxa, and locations. Finally, we discuss mechanisms involved in EH–richness relationships, differentiating between effects on species coexistence, persistence, and diversification. This review aims at guiding researchers in their selection of heterogeneity measures. At the same time, it shows the need for precise terminology and avoidance of ambiguous synonyms to enhance understanding and foster among‐study comparisons and synthesis.     

Convergence of EU nitrogen surplus,the RDP indicator of water quality

The EU Water Framework Directive (WFD), EU Nitrate Directive and EU Rural Development Policy (RDP) aim to improve water quality. The nutrient content of water can be decreased by reducing nitrogen emission. In this article a novel approach is applied to the evaluation of the impact of Agri Environmental Measures (AEM), which are part of axis 2 of the EU Rural Development Programme. The spending on AEM is linked to the reduction of nitrogen surplus, and hence, to the improvement of water quality. Reduction of nitrogen surplus is considered as a beta convergence process, in which the nitrogen surplus of EU member states converges to a steady state level. The convergence is tested, applying spatial econometrics on a panel data set of EU member states. The development over time of nitrogen surplus is explained applying the conditional beta convergence methodology. To allow for varying steady state nitrogen surpluses, structural variables are added to the analysis. RDP spending on AEM was added as structural variable to evaluate whether they affect the reduction of nitrogen surplus. The fixed effects panel data specification was tested to be the best model and preferred over spatial econometric specifications. A significantly negative effect is found between AEM expenditures and nitrogen surplus. Based on these estimation results it can be concluded that spending on AEM affects the convergence of nitrogen surplus towards a steady state level. A causal relationship cannot be tested with data on EU Member State level and additional analysis at smaller spatial level is warranted.     

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens

A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.     

Social-science Perspectives on Bioethics: Predictive Genetic Testing (PGT) in Asia

In this essay, I indicate how social-science approaches can throw light on predictive genetic testing (PGT) in various societal contexts. In the first section, I discuss definitions of various forms of PGT, and point out their inherent ambiguity and inappropriateness when taken out of an ideal–typical context. In section two, I argue further that an ethics approach proceeding from the point of view of the abstract individual in a given society should be supplemented by an approach that regards bioethics as inherently ambiguous, contested, changeable and context-dependent. In the last section, I place these bioethical discussions of PGT in the context of Asian communities. Here, a critical view of what constitutes a community and culture proves necessary to understand the role of bioethical debates and the empirical manifestations of PGT in Asian societies. A discussion of the concepts of family and kinship in relation to PGT indicates that any bioethical analysis has to take into account that bioethical values are not just reflections of a cultural community, but embody both bioethical ideals and prevalent political rhetoric which is exhibited, propagated and manipulated by individuals and collectives for a variety of purposes. I end by summarising the contributions that social science could make to the understanding of the bioethics of PGT.     

Immunochromatographic monoclonal test for detection of Helicobacter pylori antigen in stool is useful in children from high-prevalence developing country

BACKGROUND: Tests to detect Helicobacter pylori antigens in feces for diagnosis of infection in children demonstrate controversial results. One novel and fast monoclonal test improves diagnostic accuracy in adults, but clinical evidence of its usefulness at pediatric age is insufficient to date. The objective of this work was to evaluate the diagnostic accuracy of this test in a sample of Mexican children. METHODS: We conducted a transversal study in 150 selected children with digestive symptoms suggestive of organic disease in whom a clinical history was conducted in addition to a fast monoclonal test (ImmunoCardSTAT HpSA, Meridian Diagnostics) performed by immunochromatography. Patients were submitted to endoscopy and histopathologic study. RESULTS: Of the 150 children (mean age 7.8 +/- 4.7 years), 107 (71.3%) were positive for the test, and presence of H. pylori was confirmed histologically in 109 (72.7%) children, with sensitivity of 96.3% (95% CI = 95.8-96.8), specificity of 95.1% (95% CI = 93.9-96.4), and accuracy of 96.0% (95% CI, -95.6 to -96.3); pretest probability was 0.73, while post-test probability was 0.98. Infection rate and test accuracy increased with age. CONCLUSIONS: This test is useful for detecting H. pylori infection in children of all ages, and is a good alternative for screening studies in developing countries with elevated prevalence, due to its being fast, noninvasive, inexpensive, and easy to carry out.     

Developing molecular diagnostics for detection of red imported fire ants using two genes,Sinv11108 and Sinv11977

Aggressive red imported fire ants (RIFAs) are expanding their habitat due to active international trade and global warming. To prevent infestation and settlement, RIFAs must be removed during the quarantine process. Because RIFAs are social insects and have different morphological characteristics depending on their castes, non‐ant taxonomists have difficulty confirming RIFAs based on their morphological characteristics alone. The disadvantages of previously reported RIFA molecular diagnostics are that they require additional steps, such as restriction enzyme digestion followed by agarose gel electrophoresis separation or DNA sequence verification for polymerase chain reaction (PCR)‐amplified products. To overcome these drawbacks, two RIFA‐specific genes were selected and used to develop diverse PCR‐based RIFA molecular diagnostic techniques. We found that RIFAs could be confirmed by conventional PCR targeting of two RIFA‐specific genes followed by agarose electrophoresis separation. In addition, TaqMan probe real‐time PCR methods had the advantage of confirming RIFAs immediately after the reactions were completed by observing fluorescence indexes. Finally, multiplex PCRs enhanced RIFA specificity and sensitivity. The new molecular diagnostic methods developed in this study had the advantages of reducing false positive and negative results together with high specificity and sensitivity for RIFAs.