Towards the development of a DNA-sequence based approach to serotyping of Salmonella enterica

BACKGROUND: The fliC and fljB genes in Salmonella code for the phase 1 (H1) and phase 2 (H2) flagellin respectively, the rfb cluster encodes the majority of enzymes for polysaccharide (O) antigen biosynthesis, together they determine the antigenic profile by which Salmonella are identified. Sequencing and characterisation of fliC was performed in the development of a molecular serotyping technique. RESULTS: FliC sequencing of 106 strains revealed two groups; the g-complex included those exhibiting "g" or "m,t" antigenic factors, and the non-g strains which formed a second more diverse group. Variation in fliC was characterised and sero-specific motifs identified. Furthermore, it was possible to identify differences in certain H antigens that are not detected by traditional serotyping. A rapid short sequencing assay was developed to target serotype-specific sequence motifs in fliC. The assay was evaluated for identification of H1 antigens with a panel of 55 strains. CONCLUSION: FliC sequences were obtained for more than 100 strains comprising 29 different H1 alleles. Unique pyrosequencing profiles corresponding to the H1 component of the serotype were generated reproducibly for the 23 alleles represented in the evaluation panel. Short read sequence assays can now be used to identify fliC alleles in approximately 97% of the 50 medically most important Salmonella in England and Wales. Capability for high throughput testing and automation give these assays considerable advantages over traditional methods

Biannual versus annual mass azithromycin distribution and malaria seroepidemiology among preschool children in Niger: a sub-study of a cluster randomized trial.

BACKGROUND: Biannual mass azithromycin administration to preschool children reduces all-cause mortality, but the mechanism for the effect is not understood. Azithromycin has activity against malaria parasites, and malaria is a leading cause of child mortality in the Sahel. The effect of biannual versus annual azithromycin distribution for trachoma control on serological response to merozoite surface protein 1 (MSP-119), a surrogate for malaria incidence, was evaluated among children in Niger. METHODS: Markers of malaria exposure were measured in two arms of a factorial randomized controlled trial designed to evaluate targeted biannual azithromycin distribution to children under 12 years of age compared to annual azithromycin to the entire community for trachoma control (N = 12 communities per arm). Communities were treated for 36 months (6 versus 3 distributions). Dried blood spots were collected at 36 months among children ages 1-5 years, and MSP-119 antibody levels were assessed using a bead-based multiplex assay to measure malaria seroprevalence. RESULTS: Antibody results were available for 991 children. MSP-119 seropositivity was 62.7% in the biannual distribution arm compared to 68.7% in the annual arm (prevalence ratio 0.91, 95% CI 0.83 to 1.00). Mean semi-quantitative antibody levels were lower in the biannual distribution arm compared to the annual arm (mean difference - 0.39, 95% CI - 0.05 to - 0.72). CONCLUSIONS: Targeted biannual azithromycin distribution was associated with lower malaria seroprevalence compared to that in a population that received annual distribution. Trial Registration Clinicaltrials.gov NCT00792922

An assessment of the malaria-related knowledge and practices of Tanzania's drug retailers: exploring the impact of drug store accreditation.

BACKGROUND: Since 2003 Tanzania has upgraded its approximately 7000 drug stores to Accredited Drug Dispensing Outlets (ADDOs), involving dispenser training, introduction of record keeping and enhanced regulation. Prior to accreditation, drug stores could officially stock over-the-counter medicines only, though many stocked prescription-only antimalarials. ADDOs are permitted to stock 49 prescription-only medicines, including artemisinin combination therapies and one form of quinine injectable. Oral artemisinin monotherapies and other injectables were not permitted at any time. By late 2011 conversion was complete in 14 of 21 regions. We explored variation in malaria-related knowledge and practices of drug retailers in ADDO and non-ADDO regions. METHODS: Data were collected as part of the Independent Evaluation of the Affordable Medicines Facility - malaria (AMFm), involving a nationally representative survey of antimalarial retailers in October-December 2011. We randomly selected 49 wards and interviewed all drug stores stocking antimalarials. We compare ADDO and non-ADDO regions, excluding the largest city, Dar es Salaam, due to the unique characteristics of its market. RESULTS: Interviews were conducted in 133 drug stores in ADDO regions and 119 in non-ADDO regions. Staff qualifications were very similar in both areas. There was no significant difference in the availability of the first line antimalarial (68.9% in ADDO regions and 65.2% in non-ADDO regions); both areas had over 98% availability of non-artemisinin therapies and below 3.0% of artemisinin monotherapies. Staff in ADDO regions had better knowledge of the first line antimalarial than non-ADDO regions (99.5% and 91.5%, p = 0.001). There was weak evidence of a lower price and higher market share of the first line antimalarial in ADDO regions. Drug stores in ADDO regions were more likely to stock ADDO-certified injectables than those in non-ADDO regions (23.0% and 3.9%, p = 0.005). CONCLUSIONS: ADDO conversion is frequently cited as a model for improving retail sector drug provision. Drug stores in ADDO regions performed better on some indicators, possibly indicating some small benefits from ADDO conversion, but also weaknesses in ADDO regulation and high staff turnover. More evidence is needed on the value-added and value for money of the ADDO roll out to inform retail policy in Tanzania and elsewhere

Current ecotoxicity testing needs among selected U.S. federal agencies

U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards associated with substances that may be released into the environment, including but not limited to industrial chemicals, pharmaceuticals, pesticides, food additives, and color additives. These data are used to conduct hazard assessments and evaluate potential risks to aquatic life (e.g., invertebrates, fish), birds, wildlife species, or the environment. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for data from tests utilizing animals must first be clarified. Accordingly, the objective of this review was to identify the ecotoxicity test data relied upon by U.S. federal agencies. The standards, test guidelines, guidance documents, and/or endpoints that are used to address each of the agencies’ regulatory and research needs regarding ecotoxicity testing are described in the context of their application to decision-making. Testing and information use, needs, and/or requirements relevant to the regulatory or programmatic mandates of the agencies taking part in the Interagency Coordinating Committee on the Validation of Alternative Methods Ecotoxicology Workgroup are captured. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations

Infant mortality and growth failure after oral azithromycin among low birthweight and underweight neonates: A subgroup analysis of a randomized controlled trial.

BACKGROUND: Low birthweight (birthweight <2500 grams, g) and underweight (weight-for-age Z-score, WAZ, < -2) infants have higher risk of poor outcomes compared to their well-nourished peers. We evaluated the role of azithromycin for reducing mortality and improving growth outcomes in low birthweight and/or underweight infants. METHODS: Infants aged 8-27 days of age weighing ≥2500 g at enrollment in Burkina Faso were randomized 1:1 to a single, oral dose of azithromycin (20 mg/kg) or matching placebo. We evaluated mortality and anthropometric outcomes in four subgroups: 1) both low birthweight and underweight at enrollment; 2) low birthweight-only; 3) underweight-only; 4) neither low birthweight nor underweight. FINDINGS: Of 21,832 enrolled infants, 21,320 (98%) had birthweight measurements and included in this analysis. Of these, 747 (3%) were both low birthweight and underweight, 972 (5%) were low birthweight-only, 825 (4%) were underweight-only, and 18,776 (88%) were neither low birthweight nor underweight. Infants who were both low birthweight and underweight receiving azithromycin had lower odds of underweight at 6 months compared to placebo (OR 0.65, 95% CI 0.44 to 0.95), but the treatment group by subgroup interaction was not statistically significant (P = 0.06). We did not find evidence of a difference between groups for other outcomes in any subgroup. INTERPRETATION: Azithromycin may have some growth-promoting benefits for the highest risk infants, but we were unable to demonstrate a difference in most outcomes in low birthweight and underweight infants. As a secondary analysis of a trial, this study was underpowered for rare outcomes such as mortality. TRIAL REGISTRATION: ClinicalTrials.gov NCT03682653

PDE-4 inhibition rescues aberrant synaptic plasticity in Drosophila and mouse models of fragile X syndrome.

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS

Multispacer Sequence Typing for Mycobacterium tuberculosis Genotyping

Background: Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis. Methods and Findings: In M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing M<8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts <= 42 cycles in internal transcribed spacer-real time PCR. Conclusions: These results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTuberculosis/mst)

Clinical Outcomes and Patient-Matched Molecular Composition of Relapsed Medulloblastoma

PURPOSE We sought to investigate clinical outcomes of relapsed medulloblastoma and to compare molecular features between patient-matched diagnostic and relapsed tumors. METHODS Children and infants enrolled on either SJMB03 (NCT00085202) or SJYC07 (NCT00602667) trials who experienced medulloblastoma relapse were analyzed for clinical outcomes, including anatomic and temporal patterns of relapse and postrelapse survival. A largely independent, paired molecular cohort was analyzed by DNA methylation array and next-generation sequencing. RESULTS A total of 72 of 329 (22%) SJMB03 and 52 of 79 (66%) SJYC07 patients experienced relapse with significant representation of Group 3 and wingless tumors. Although most patients exhibited some distal disease (79%), 38% of patients with sonic hedgehog tumors experienced isolated local relapse. Time to relapse and postrelapse survival varied by molecular subgroup with longer latencies for patients with Group 4 tumors. Postrelapse radiation therapy among previously nonirradiated SJYC07 patients was associated with long-term survival. Reirradiation was only temporizing for SJMB03 patients. Among 127 patients with patient-matched tumor pairs, 9 (7%) experienced subsequent nonmedulloblastoma CNS malignancies. Subgroup (96%) and subtype (80%) stabilities were largely maintained among the remainder. Rare subgroup divergence was observed from Group 4 to Group 3 tumors, which is coincident with genetic alterations involving MYC, MYCN, and FBXW7. Subgroup-specific patterns of alteration were identified for driver genes and chromosome arms. CONCLUSION Clinical behavior of relapsed medulloblastoma must be contextualized in terms of up-front therapies and molecular classifications. Group 4 tumors exhibit slower biological progression. Utility of radiation at relapse is dependent on patient age and prior treatments. Degree and patterns of molecular conservation at relapse vary by subgroup. Relapse tissue enables verification of molecular targets and identification of occult secondary malignancies

Parenthood in survivors of Hodgkin lymphoma: an EORTC-GELA general population case-control study.

Contains fulltext : 108966.pdf (publisher's version ) (Open Access)PURPOSE: We investigated the impact of Hodgkin lymphoma (HL) on parenthood, including factors influencing parenthood probability, by comparing long-term HL survivors with matched general population controls. PATIENTS AND METHODS: A Life Situation Questionnaire was sent to 3,604 survivors treated from 1964 to 2004 in successive clinical trials. Responders were matched with controls (1:3 or 4) for sex, country, education, and year of birth (10-year groups). Controls were given an artificial date of start of treatment equal to that of their matched case. The main end point was presence of biologic children after treatment, which was evaluated by using conditional logistic regression analysis. Logistic regression analysis was used to analyze factors influencing spontaneous post-treatment parenthood. RESULTS: In all, 1,654 French and Dutch survivors were matched with 6,414 controls. Median follow-up was 14 years (range, 5 to 44 years). After treatment, the odds ratio (OR) for having children was 0.77 (95% CI, 0.68 to 0.87; P < .001) for survivors compared with controls. Of 898 survivors who were childless before treatment, 46.7% achieved post-treatment parenthood compared with 49.3% of 3,196 childless controls (OR, 0.87; P = .08). Among 756 survivors with children before treatment, 12.4% became parents after HL treatment compared with 22.2% of 3,218 controls with children before treatment (OR, 0.49; P < .001). Treatment with alkylating agents, second-line therapy, and age older than 35 years at treatment appeared to reduce the chances of spontaneous post-treatment parenthood. CONCLUSION: Survivors of HL had slightly but significantly fewer children after treatment than matched general population controls. The difference concerned only survivors who had children before treatment and appears to have more personal than biologic reasons. The chance of successful post-treatment parenthood was 76%