Schistosome vaccine testing: lessons from the baboon model

The high level of protection elicited in rodents and primates by the radiation-attenuated schistosome vaccine gives hope that a human vaccine relying on equivalent mechanisms is feasible. In humans, a vaccine would be undoubtedly administered to previously or currently infected individuals. We have therefore used the olive baboon to investigate whether vaccine-induced immunity is compromised by a schistosome infection. We showed that neither a preceding infection, terminated by chemotherapy, nor an ongoing chronic infection affected the level of protection. Whilst IgM responses to vaccination or infection were short-lived, IgG responses rose with each successive exposure to the vaccine. Such a rise was obscured by responses to egg deposition in already-infected animals. In human trials it would be necessary to use indirect estimates of infection intensity to determine vaccine efficacy. Using worm burden as the definitive criterion, we demonstrated that the surrogate measures, fecal eggs, and circulating antigens, consistently overestimated protection. Regression analysis of the surrogate parameters on worm burden revealed that the principal reason for overestimation was the threshold sensitivity of the assays. If we extrapolate our findings to human schistosomiasis mansoni, it is clear that more sensitive indirect measures of infection intensity are required for future vaccine trials.

The utility of rhesus monkey (Macaca mulatta) and other non-human primate models for preclinical testing of Leishmania candidate vaccines

Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.

Assessing patient's belief on HIV testing before elective surgery: room for improvements in Switzerland !

Background: ln Switzerland no HIV test is performed without the patient's consent based on a Voluntary Counseling and Testing policy (VCT). We hypothesized that a substantial proportion of patients going through an elective surgery falsely believed that an HIV test was performed on a routine basis and that the lack of transmission of result was interpreted as being HIV negative. Method: All patients with elective orthopedic surgery during 2007 were contacted by phone in 2008. A structured questionnaire assessed their belief about routine preoperative blood analysis (diabetes, coagulation function, HIV test and cholesterol level) as well as result awareness and interpretation. Variables included age and gender. Analysis were conducted using the software JMP 6.0.3. Results: 1123 patients were included. 130 (12 %) were excluded (Le. unreachable, unable to communicate on the phone, not operated). 993 completed the survey (89 %). Median age was 51 (16-79). 50 % were female. 376 (38 %) patients thought they had an HIV test performed before surgery but none of them had one. 298 (79 %) interpreted the absence of result as a negative HIV test. A predictive factor to believe an HIV test had been done was an age below 50 years old (45 % vs 33 % for 16-49 years old and 50-79 years old respectively, p < 0.001). No difference was observed between genders. Conclusion: ln Switzerland, nearly 40 % of the patients falsely thought an HIV test had been performed on a routine basis before surgery and were erroneously reassured about their HIV status. These results should either improve the information given to the patient regarding preoperative exams, or motivate public health policy to consider HIV opt-out screening instead of VCT strategy.

Testing Delivery Systems in Transnational virtual learning: the vocational management training for the european tourism industry (VocMat) case study

This article discusses the lessons learned from developing and delivering the Vocational Management Training for the European Tourism Industry (VocMat) online training programme, which was aimed at providing flexible, online distance learning for the European tourism industry. The programme was designed to address managers ‘need for flexible, senior management level training which they could access at a time and place which fitted in with their working and non-work commitments. The authors present two main approaches to using the Virtual Learning Environment, the feedback from the participants, and the implications of online Technology in extending tourism training opportunities

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Consensus statement of the European guidelines on clinical management of HIV-1 tropism testing

Introduction: Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. To date, in most European countries HIV tropism is determined using a phenotypic test. Recently, new data have emerged supporting the use of a genotypic HIV V3-loop sequence analysis as the basis for tropism determination. The European guidelines group on clinical management of HIV-1 tropism testing was established to make recommendations to clinicians and virologists. Methods: We searched online databases for articles from Jan 2006 until March 2010 with the terms: tropism or CCR5-antagonist or CCR5 antagonist or maraviroc or vicriviroc. Additional articles and/or conference abstracts were identified by hand searching. This strategy identified 712 potential articles and 1240 abstracts. All were reviewed and finally 57 papers and 42 abstracts were included and used by the panel to reach a consensus statement. Results: The panel recommends HIV-tropism testing for the following indications: i) drug-naïve patients in whom toxicity or limited therapeutic options are foreseen; ii) patients experiencing therapy failure whenever a treatment change is considered. Both the phenotypic Enhanced Trofile assay (ESTA) and genotypic population sequencing of the V3-loop are recommended for use in clinical practice. Although the panel does not recommend one methodology over another it is anticipated that genotypic testing will be used more frequently because of its greater accessibility, lower cost and shorter turnaround time. The panel also provides guidance on technical aspects and interpretation issues. If using genotypic methods, triplicate PCR amplification and sequencing testing is advised using the G2P interpretation tool (clonal model) with an FPR of 10%. If the viral load is below the level of reliable amplification, proviral DNA can be used, and the panel recommends performing triplicate testing and use of an FPR of 10%. If genotypic DNA testing is not performed in triplicate the FPR should be increased to 20%. Conclusions: The European guidelines on clinical management of HIV-1 tropism testing provide an overview of current literature, evidence-based recommendations for the clinical use of tropism testing and expert guidance on unresolved issues and current developments. Current data support both the use of genotypic population sequencing and ESTA for co-receptor tropism determination. For practical reasons genotypic population sequencing is the preferred method in Europe.

In vitro activity of fosfomycin against extended-spectrum-beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: comparison of susceptibility testing procedures

The agar dilution, broth microdilution, and disk diffusion methods were compared to determine the in vitro susceptibility of 428 extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae to fosfomycin. Fosfomycin showed very high activity against all ESBL-producing strains. Excellent agreement between the three susceptibility methods was found for E. coli, whereas marked discrepancies were observed for K. pneumoniae.

Postmortem circulation: a new model for testing endovascular devices and training clinicians in their use.

The development of new medical devices, such as aortic valves, requires numerous preliminary studies on animals and training of personnel on cadavers before the devices can be used in patients. Postmortem circulation, a technique used for postmortem angiography, allows the vascular system to be reperfused in a way similar to that in living persons. This technique is used for postmortem investigations to visualize the human vascular system and to make vascular diagnoses. Specific material for reperfusing a human body was developed recently. Our aim was to investigate whether postmortem circulation that imitates in vivo conditions allows for the testing of medical materials on cadavers. We did this by delivering an aortic valve using minimally invasive methods. Postmortem circulation was established in eight corpses to recreate an environment as close as possible to in vivo conditions. Mobile fluoroscopy and a percutaneous catheterization technique were used to deliver the material to the correct place. Once the valve was implanted, the heart and primary vessels were extracted to confirm its position. Postmortem circulation proved to be essential in several of the cadavers because it helped the clinicians to deliver the material and improve their implantation techniques. Due to the intravascular circulation, sites with substantial arteriosclerotic stenosis could be bypassed, which would have been impossible without perfusion. Although originally developed for postmortem investigations, this reperfusion technique could be useful for testing new medical devices intended for living patients.

Testing of Diagnostic Methods for Detection of Influenza Virusfor Optimal Performance in the Context of an Influenza Surveillance Network

Influenza surveillance networks must detect early the viruses that will cause the forthcoming annual epidemics and isolate the strains for further characterization. We obtained the highest sensitivity (95.4%) with a diagnostic tool that combined a shell-vial assay and reverse transcription-PCR on cell culture supernatants at 48 h, and indeed, recovered the strain

The BENEFIT trial: testing the hypothesis that trypanocidal therapy is beneficial for patients with chronic Chagas heart disease

Among the pathophysiological derangements operating in the chronic phase of Chagas disease, parasite persistence is likely to constitute the main mechanism of myocardial injury in patients with chronic chagasic cardiomyopathy. The presence of Trypanosoma cruzi in the heart causes a low-grade, but relentless, inflammatory process and induces myocardial autoimmune injury. These facts suggest that trypanocidal therapy may positively impact the clinical course of patients with chronic Chagas heart disease. However, the experimental and clinical evidence currently available is insufficient to support the routine use of etiologic treatment in these patients. The BENEFIT project - Benznidazole Evaluation for Interrupting Trypanosomiasis - is an international, multicenter, double-blind, placebo-controlled trial of trypanocidal treatment with benznidazole in patients with chronic Chagas heart disease. This project is actually comprised of two studies. The pilot study investigates whether etiologic treatment significantly reduces parasite burden, as assessed by polymerase chain reaction-based techniques and also determines the safety and tolerability profile of the trypanocidal drug in this type of chagasic population. The full-scale study determines whether antitrypanosomal therapy with benznidazole reduces mortality and other major cardiovascular clinical outcomes in patients with chronic Chagas heart disease.

Compositional hypotheses of subcompositional stability and specific perturbation change and their testing

In standard multivariate statistical analysis common hypotheses of interest concern changes in mean vectors and subvectors. In compositional data analysis it is now well established that compositional change is most readily described in terms of the simplicial operation of perturbation and that subcompositions replace the marginal concept of subvectors. To motivate the statistical developments of this paper we present two challenging compositional problems from food production processes.Against this background the relevance of perturbations and subcompositions can beclearly seen. Moreover we can identify a number of hypotheses of interest involvingthe specification of particular perturbations or differences between perturbations and also hypotheses of subcompositional stability. We identify the two problems as being the counterpart of the analysis of paired comparison or split plot experiments and of separate sample comparative experiments in the jargon of standard multivariate analysis. We then develop appropriate estimation and testing procedures for a complete lattice of relevant compositional hypotheses

Methods for testing association between uncertain genotypes and quantitative traits.

Interpretability and power of genome-wide association studies can be increased by imputing unobserved genotypes, using a reference panel of individuals genotyped at higher marker density. For many markers, genotypes cannot be imputed with complete certainty, and the uncertainty needs to be taken into account when testing for association with a given phenotype. In this paper, we compare currently available methods for testing association between uncertain genotypes and quantitative traits. We show that some previously described methods offer poor control of the false-positive rate (FPR), and that satisfactory performance of these methods is obtained only by using ad hoc filtering rules or by using a harsh transformation of the trait under study. We propose new methods that are based on exact maximum likelihood estimation and use a mixture model to accommodate nonnormal trait distributions when necessary. The new methods adequately control the FPR and also have equal or better power compared to all previously described methods. We provide a fast software implementation of all the methods studied here; our new method requires computation time of less than one computer-day for a typical genome-wide scan, with 2.5 M single nucleotide polymorphisms and 5000 individuals.

Testing the validity of a set of diagnostic criteria for sensory neuronopathies: a francophone collaborative study.

There are no validated criteria for the diagnosis of sensory neuronopathy (SNN) yet. In a preliminary monocenter study a set of criteria relying on clinical and electrophysiological data showed good sensitivity and specificity for a diagnosis of probable SNN. The aim of this study was to test these criteria on a French multicenter study. 210 patients with sensory neuropathies from 15 francophone reference centers for neuromuscular diseases were included in the study with an expert diagnosis of non-SNN, SNN or suspected SNN according to the investigations performed in these centers. Diagnosis was obtained independently from the set of criteria to be tested. The expert diagnosis was taken as the reference against which the proposed SNN criteria were tested. The set relied on clinical and electrophysiological data easily obtainable with routine investigations. 9/61 (16.4 %) of non-SNN patients, 23/36 (63.9 %) of suspected SNN, and 102/113 (90.3 %) of SNN patients according to the expert diagnosis were classified as SNN by the criteria. The SNN criteria tested against the expert diagnosis in the SNN and non-SNN groups had 90.3 % (102/113) sensitivity, 85.2 % (52/61) specificity, 91.9 % (102/111) positive predictive value, and 82.5 % (52/63) negative predictive value. Discordance between the expert diagnosis and the SNN criteria occurred in 20 cases. After analysis of these cases, 11 could be reallocated to a correct diagnosis in accordance with the SNN criteria. The proposed criteria may be useful for the diagnosis of probable SNN in patients with sensory neuropathy. They can be reached with simple clinical and paraclinical investigations.

Application of Molecular Diagnostic Techniques for Viral Testing.

Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory.