Detailed evaluation of data analysis tools for subtyping of bacterial isolates based on whole genome sequencing : Neisseria meningitidis as a proof of concept

Whole genome sequencing is increasingly recognized as the most informative approach for characterization of bacterial isolates. Success of the routine use of this technology in public health laboratories depends on the availability of well-characterized and verified data analysis methods. However, multiple subtyping workflows are now often being used for a single organism, and differences between them are not always well described. Moreover, methodologies for comparison of subtyping workflows, and assessment of their performance are only beginning to emerge. Current work focuses on the detailed comparison of WGS-based subtyping workflows and evaluation of their suitability for the organism and the research context in question. We evaluated the performance of pipelines used for subtyping of Neisseria meningitidis, including the currently widely applied cgMLST approach and different SNP-based methods. In addition, the impact of the use of different tools for detection and filtering of recombinant regions and of different reference genomes were tested. Our benchmarking analysis included both assessment of technical performance of the pipelines and functional comparison of the generated genetic distance matrices and phylogenetic trees. It was carried out using replicate sequencing datasets of high- and low-coverage, consisting mainly of isolates belonging to the clonal complex 269. We demonstrated that cgMLST and some of the SNP-based subtyping workflows showed very good performance characteristics and highly similar genetic distance matrices and phylogenetic trees with isolates belonging to the same clonal complex. However, only two of the tested workflows demonstrated reproducible results for a group of more closely related isolates. Additionally, results of the SNP-based subtyping workflows were to some level dependent on the reference genome used. Interestingly, the use of recombination-filtering software generally reduced the similarity between the gene-by-gene and SNP-based methodologies for subtyping of N. meningitidis. Our study, where N. meningitidis was taken as an example, clearly highlights the need for more benchmarking comparative studies to eventually contribute to a justified use of a specific WGS data analysis workflow within an international public health laboratory context

A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium

Subtyping below the serovar level is essential for surveillance and outbreak detection and investigation of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant 1,4,[5],12:i:- (S. 1,4,[5],12:i:-), frequent causes of foodborne infections. In an attempt to overcome the intrinsic shortcomings of currently used subtyping techniques, a multiplex oligonucleotide ligation-PCR (MOL-PCR) assay was developed which combines different types of molecular markers in a high throughput microsphere suspension array. The 52 molecular markers include prophage genes, amplified fragment length polymorphism (AFLP) elements, Salmonella genomic island 1 (SGI1), allantoinase gene allB, MLVA locus STTR10, antibiotic resistance genes, single nucleotide polymorphisms (SNPs) and phase 2 flagellar gene fljB. The in vitro stability of these markers was confirmed in a serial passage experiment. The validation of the MOL-PCR assay for subtyping of S. Typhimurium and S. 1,4,[5],12:i:- on 519 isolates shows that the method is rapid, reproducible, flexible, accessible, easy to use and relatively inexpensive. Additionally, a 100 % typeability and a discriminatory power equivalent to that of phage typing were observed, and epidemiological concordance was assessed on isolates of 2 different outbreaks. Furthermore, a data analysis method is provided so that the MOL-PCR assay allows for objective, computerised data analysis and data interpretation of which the results can be easily exchanged between different laboratories in an international surveillance network

A genoserotyping system for a fast and objective identification of Salmonella serotypes commonly isolated from poultry and pork food sectors in Belgium

Humans are mostly contaminated by Salmonella through the consumption of pork- and poultry-derived food products. Therefore, a strict monitoring of Salmonella serotypes in food-producing animals is needed to limit the transmission of the pathogen to humans. Additionally, Salmonella can lead to economic loss in the food sector. Previously, a genoserotyping method using the MOL-PCR and Luminex technology was developed for the identification of the 6 Salmonella serotypes, and their variants, subjected to an official control in the Belgian food sector. In this study, 3 additional assays using the same technology were developed for the rapid and cost-effective detection of 13 dangerous highly invasive serotypes or other serotypes frequently isolated from the Belgian poultry and pork sector, i.e. Agona, Anatum, Brandenburg, Choleraesuis, Derby, Enteritidis vaccine strains, Gallinarum var. Gallinarum/Pullorum, Livingstone, Mbandaka, Minnesota, Ohio, Rissen and Senftenberg. Moreover, the previously developed first MOL-PCR assay was improved for S. Paratyphi B and serogroup O:3 detection. Finally, a Decision Support System hosted by a web application was created for an automatic and objective interpretation of the Luminex raw data. The 3 new assays and the modifications of the first assay were validated with a 100% accuracy, using 553 Salmonella and non-Salmonella strains in total

MLVA as a tool for public health surveillance of human Salmonella Typhimurium : prospective study in Belgium and evaluation of MLVA loci stability

Surveillance of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow earlier detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and the in Europe most commonly used 5-loci MLVA on 1,420 S. Typhimurium isolates collected between 2010 and 2012 in Belgium, was used to evaluate the added value of MLVA for public health surveillance. Phage types DT193, DT195, DT120, DT104, DT12 and U302 dominate the Belgian S. Typhimurium population. A combined resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) with or without additional resistances was observed for 42.5% of the isolates. 414 different MLVA profiles were detected, of which 14 frequent profiles included 44.4% of the S. Typhimurium population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, variations over time were observed for loci STTR6, STTR10, STTR5 and STTR9. This study demonstrates that MLVA improves public health surveillance of S. Typhimurium. However, the 5-loci MLVA should be complemented with other subtyping methods for investigation of possible outbreaks with frequent MLVA profiles. Also, variability in these MLVA loci should be taken into account when investigating extended outbreaks and studying dynamics over longer periods

How do participatory methods shape policy? Applying a realist approach to the formulation of a new tuberculosis policy in Georgia

From PubMed via Jisc Publications RouterThis paper presents the iterative process of participatory multistakeholder engagement that informed the development of a new national tuberculosis (TB) policy in Georgia, and the lessons learnt. Guided by realist evaluation methods, a multistakeholder dialogue was organised to elicit stakeholders' assumptions on challenges and possible solutions for better TB control. Two participatory workshops were conducted with key actors, interspersed by reflection meetings within the research team and discussions with policymakers. Using concept mapping and causal mapping techniques, and drawing causal loop diagrams, we visualised how actors understood TB service provision challenges and the potential means by which a results-based financing (RBF) policy could address these. The study was conducted in Tbilisi, Georgia. A total of 64 key actors from the Ministry of Labour, Health and Social Affairs, staff of the Global Fund to Fight AIDS, TB and Malaria Georgia Project, the National Centre for Disease Control and Public Health, the National TB programme, TB service providers and members of the research team were involved in the workshops. Findings showed that beyond provider incentives, additional policy components were necessary. These included broadening the incentive package to include institutional and organisational incentives, retraining service providers, clear redistribution of roles to support an integrated care model, and refinement of monitoring tools. Health system elements, such as effective referral systems and health information systems were highlighted as necessary for service improvement. Developing policies that address complex issues requires methods that facilitate linkages between multiple stakeholders and between theory and practice. Such participatory approaches can be informed by realist evaluation principles and visually facilitated by causal loop diagrams. This approach allowed us leverage stakeholders' knowledge and expertise on TB service delivery and RBF to codesign a new policy. [Abstract copyright: © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.]11pubpub

Cost and impact of policies to remove and reduce fees for obstetric care in Benin, Burkina Faso, Mali and Morocco

Background: Across the Africa region and beyond, the last decade has seen many countries introducing policies aimed at reducing financial barriers to obstetric care. This article provides evidence of the cost and effects of national policies focussed on improving financial access to caesarean and facility deliveries in Benin, Burkina Faso, Mali and Morocco. Methods: The study uses a comparative case study design with mixed methods, including realist evaluation components. This article presents results across 14 different data collection tools, used in 4-6 research sites in each of the four study countries over 2011-13. The methods included: document review; interviews with key informants; analysis of secondary data; structured extraction from medical files; cross-sectional surveys of patients and staff; interviews with patients and observation of care processes. Results: The article finds that the policies have contributed to continued increases in skilled birth attendance and caesarean sections and a narrowing of inequalities in all four countries, but these trends were already occurring so a shift cannot be attributed solely to the policies. It finds a significant reduction in financial burdens on households after the policy, suggesting that the financial protection objectives may have been met, at least in the short term, although none achieved total exemption of targeted costs. Policies are domestically financed and are potentially sustainable and efficient, and were relatively thoroughly implemented. Further, we find no evidence of negative effects on technical quality of care, or of unintended negative effects on untargeted services. Conclusions: We conclude that the policies were effective in meeting financial protection goals and probably health and equity goals, at sustainable cost, but that a range of measures could increase their effectiveness and equity. These include broadening the exempted package (especially for those countries which focused on caesarean sections alone), better calibrated payments, clearer information on policies, better stewardship of the local health system to deal with underlying systemic weaknesses, more robust implementation of exemptions for indigents, and paying more attention to quality of care, especially for newborns.sch_iih15pub4430pub

Bronchial or Laryngeal Obstruction Induced by Exercise?

A child suspected of exercise-induced laryngeal obstruction and asthma is examined by laryngoscopy and respiratory resistance (Rrs) after exercise challenge. Immediately at exercise cessation, the visualized adduction of the larynx in inspiration is reflected in a paroxystic increase in Rrs. While normal breathing has apparently resumed later on during recovery from exercise, the pattern of Rrs in inspiration is observed to reoccur following a deep breath or swallowing. The procedure may thus help diagnosing the site of exercise-induced obstruction when laryngoscopy is not available and identify re-inducers of laryngeal dysfunction

Studying complex interventions : reflections from the FEMHealth project on evaluating fee exemption policies in West Africa and Morocco

Peer reviewedPublisher PD

Efficient generation of CD34+ progenitorâ derived dendritic cells from Gâ CSFâ mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141647/1/jlb0957.pd