Publications by authors named "Simona Rondini"

29 Publications

  • Page 1 of 1

Development of FAcE (Formulated Alhydrogel competitive ELISA) method for direct quantification of OAg present in Shigella sonnei GMMA-based vaccine and its optimization using Design of Experiments approach.

J Immunol Methods 2019 08 27;471:11-17. Epub 2019 Apr 27.

GSK Vaccines Institute for Global Health (GVGH) S.r.l., Siena, Italy. Electronic address:

Many formulated vaccines, including 1790GAHB Shigella sonnei GMMA-based vaccine, contain Alhydrogel (aluminum hydroxide), consequently the antigen content must be determined in the formulated final vaccine product, as required by regulatory authorities. The direct quantification of antigens adsorbed on aluminum salts is difficult, and antigens may need to be extracted using laborious and often ineffective desorption procedures. To directly quantify the sugar vaccine target in the LPS of 1790GAHB, we have developed a new FAcE (Formulated Alhydrogel competitive ELISA) method. FAcE is an immunoassay based on the competition between S. sonnei LPS, coated on the ELISA plate, and the LPS in formulated S. sonnei GMMA, in binding a specific monoclonal antibody. To optimize the method, which is as easy to perform as a standard ELISA, we have applied a Design of Experiments (DOE) approach. A model was found to define the significant assay variables and to predict their impact on the output responses. Results obtained using the DOE optimized FAcE assay showed that the method is sensitive (0.02 μg/mL lower detection limit), precise, reproducible and can accurately quantify independently formulated drug products, making it a useful tool in routine tests of Alhydrogel-based vaccines. We are currently using this method to determine S. sonnei vaccine potency, stability and lot-to-lot variations, and are broadening its applicability to quantify active ingredients of other Alhydrogel GMMA-vaccines and in multivalent vaccines formulations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jim.2019.04.012DOI Listing
August 2019

Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal .

Proc Natl Acad Sci U S A 2018 10 27;115(41):10428-10433. Epub 2018 Sep 27.

Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom.

Nontyphoidal cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Typhimurium and Enteritidis vaccines. strains were chosen and deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1807655115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187145PMC
October 2018

Spontaneous point mutations in the capsule synthesis locus leading to structural and functional changes of the capsule in serogroup A meningococcal populations.

Virulence 2018 ;9(1):1138-1149

a Swiss Tropical and Public Health Institute , Molecular Immunology Unit , Basel , Switzerland.

Whole genome sequencing analysis of 100 Neisseria meningitidis serogroup A isolates has revealed that the csaABCD-ctrABCD-ctrEF capsule polysaccharide synthesis locus represents a spontaneous point mutation hotspot. Structural and functional properties of the capsule of 11 carriage and two disease isolates with non-synonymous point mutations or stop codons in capsule synthesis genes were analyzed for their capsular polysaccharide expression, recognition by antibodies and sensitivity to bactericidal killing. Eight of eleven carriage isolates presenting capsule locus mutations expressed no or reduced amounts of capsule. One isolate with a stop codon in the O-acetyltransferase gene expressed non-O-acetylated polysaccharide, and was not recognized by anti-capsule antibodies. Capsule and O-acetylation deficient mutants were resistant to complement deposition and killing mediated by anti-capsular antibodies, but not by anti-lipopolysaccharide antibodies. Two capsule polymerase mutants, one carriage and one case isolate, showed capsule over-expression and increased resistance against bactericidal activity of both capsule- and lipopolysaccharide-specific antibodies. Meningococci have developed multiple strategies for changing capsule expression and structure, which is relevant both for colonization and virulence. Here we show that point mutations in the capsule synthesis genes substantially contribute to the repertoire of genetic mechanisms in natural populations leading to variability in capsule expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/21505594.2018.1467710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086313PMC
October 2018

Setup of luminescence-based serum bactericidal assay against Salmonella Paratyphi A.

J Immunol Methods 2018 10 30;461:117-121. Epub 2018 Jun 30.

GSK Vaccines Institute for Global Health (GVGH) S.r.l., via Fiorentina 1, 53100 Siena, Italy.

Increasing awareness of Salmonella Paratyphi A's contribution to enteric fever episodes throughout Asia has led to the development of new S. Paratyphi A vaccines. Assays are needed to measure functional antibodies elicited by the new vaccine candidates to assess their immunogenicity and potential protective capacities. Serum bactericidal assay (SBA) is the method of choice to measure functional antibody titers against various bacterial pathogens, but it is rarely been used for large dataset and clinical samples because it is time consuming and labor-intensive. Recently we developed a high-throughput luminescence-based SBA method, against different pathogens, including Salmonella Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis. Here we further demonstrated the applicability of such method with invasive isolates of S. Paratyphi A to assess the complement-mediated antibody-dependent killing of both preclinical and clinical standard sera. As already found for other organisms, titers obtained by the luminescence-based SBA strongly correlated with those obtained by the conventional agar plate-based assay. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccines, with the potential of being applied to immunogenicity assessment in clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jim.2018.06.025DOI Listing
October 2018

Contribution of factor H-Binding protein sequence to the cross-reactivity of meningococcal native outer membrane vesicle vaccines with over-expressed fHbp variant group 1.

PLoS One 2017 25;12(7):e0181508. Epub 2017 Jul 25.

GSK Vaccines Institute for Global Health (GVGH), Siena, Italy.

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181508PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526518PMC
October 2017

Safety Profile and Immunologic Responses of a Novel Vaccine Against Shigella sonnei Administered Intramuscularly, Intradermally and Intranasally: Results From Two Parallel Randomized Phase 1 Clinical Studies in Healthy Adult Volunteers in Europe.

EBioMedicine 2017 Aug 15;22:164-172. Epub 2017 Jul 15.

GSK Vaccines Institute for Global Health, Siena, Italy. Electronic address:

Background: Approximately 164,000 deaths yearly are due to shigellosis, primarily in developing countries. Thus, a safe and affordable Shigella vaccine is an important public health priority. The GSK Vaccines Institute for Global Health (GVGH) developed a candidate Shigella sonnei vaccine (1790GAHB) using the Generalized Modules for Membrane Antigens (GMMA) technology. The paper reports results of 1790GAHB Phase 1 studies in healthy European adults.

Methods: To evaluate the safety and immunogenicity profiles of 1790GAHB, we performed two parallel, phase 1, observer-blind, randomized, placebo-controlled, dose escalation studies in France ("study 1") and the United Kingdom ("study 2") between February 2014 and April 2015 (ClinicalTrials.gov, number NCT02017899 and NCT02034500, respectively) in 18-45years old subjects (50 in study 1, 52 in study 2). Increasing doses of Alhydrogel adsorbed 1790, expressed by both O Antigen (OAg) and protein quantity, or placebo were given either by intramuscular route (0.059/1, 0.29/5, 1.5/25, 2.9/50, 5.9/100μg of OAg/μg of protein; study 1) or by intradermal (ID), intranasal (IN) or intramuscular (IM) route of immunization (0.0059/0.1, 0.059/1, 0.59/10μg ID, 0.29/5, 1.2/20, 4.8/80μg IN and 0.29/5μg IM, respectively; study 2). In absence of serologic correlates of protection for Shigella sonnei, vaccine induced immunogenicity was compared to anti-LPS antibody in a population naturally exposed to S. sonnei.

Findings: Vaccines were well tolerated in both studies and no death or vaccine related serious adverse events were reported. In study 1, doses ≥1.5/25μg elicited serum IgG median antibody greater than median level in convalescent subjects after the first dose. No vaccine group in study 2 achieved median antibody greater than the median convalescent antibody.

Interpretation: Intramuscularly administered Shigella sonnei GMMA vaccine is well tolerated, up to and including 5.9/100μg and induces antibody to the OAg of at least the same magnitude of those observed following natural exposure to the pathogen. Vaccine administered by ID or IN, although well tolerated, is poorly immunogenic at the doses delivered. The data support the use of the GMMA technology for the development of intramuscular multivalent Shigella vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2017.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552227PMC
August 2017

Immunogenicity of a Bivalent Adjuvanted Glycoconjugate Vaccine against Typhimurium and Enteritidis.

Front Immunol 2017 27;8:168. Epub 2017 Feb 27.

Laboratorio di Microbiologia Molecolare e Biotecnologia (LA.M.M.B.), Dipartimento di Biotecnologie Mediche, Università di Siena , Siena , Italy.

serovars Typhimurium and Enteritidis are the predominant causes of invasive non-typhoidal (iNTS) disease. Considering the co-endemicity of . Typhimurium and . Enteritidis, a bivalent vaccine formulation against both pathogens is necessary for protection against iNTS disease, thus investigation of glycoconjugate combination is required. In the present work, we investigated the immune responses induced by . Typhimurium and . Enteritidis monovalent and bivalent glycoconjugate vaccines adjuvanted with aluminum hydroxide (alum) only or in combination with cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG). Humoral and cellular, systemic and local, immune responses were characterized in two different mouse strains. All conjugate vaccines elicited high levels of serum IgG against the respective O-antigens (OAg) with bactericidal activity. The bivalent conjugate vaccine induced systemic production of antibodies against both . Typhimurium and . Enteritidis OAg. The presence of alum or alum + CpG adjuvants in vaccine formulations significantly increased the serum antigen-specific antibody production. The alum + CpG bivalent vaccine formulation triggered the highest systemic anti-OAg antibodies and also a significant increase of anti-OAg IgG in intestinal washes and fecal samples, with a positive correlation with serum levels. These data demonstrate the ability of monovalent and bivalent conjugate vaccines against . Typhimurium and . Enteritidis to induce systemic and local immune responses in different mouse strains, and highlight the suitability of a bivalent glycoconjugate formulation, especially when adjuvanted with alum + CpG, as a promising candidate vaccine against iNTS disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2017.00168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326758PMC
February 2017

Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout.

PLoS One 2017 13;12(2):e0172163. Epub 2017 Feb 13.

GSK Vaccines Institute for Global Health (GVGH) S.r.l., Siena, Italy.

Serum Bactericidal Activity (SBA) assay is the method of choice to evaluate the complement-mediated functional activity of both infection- and vaccine-induced antibodies. To perform a typical SBA assay, serial dilutions of sera are incubated with target bacterial strains and complement. The conventional SBA assay is based on plating on agar the SBA reaction mix and counting the surviving bacterial colony forming units (CFU) at each serum dilution. Even with automated colony counting, it is labor-intensive, time-consuming and not amenable for large-scale studies. Here, we have developed a luminescence-based SBA (L-SBA) method able to detect surviving bacteria by measuring their ATP. At the end of the SBA reaction, a single commercially available reagent is added to each well of the SBA plate, and the resulting luminescence signal is measured in a microplate reader. The signal obtained is proportional to the ATP present, which is directly proportional to the number of viable bacteria. Bactericidal activity is subsequently calculated. We demonstrated the applicability of L-SBA with multiple bacterial serovars, from 5 species: Citrobacter freundii, Salmonella enterica serovars Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis. Serum bactericidal titers obtained by the luminescence readout method strongly correlate with the data obtained by the conventional agar plate-based assay, and the new assay is highly reproducible. L-SBA considerably shortens assay time, facilitates data acquisition and analysis and reduces the operator dependency, avoiding the plating and counting of CFUs. Our results demonstrate that L-SBA is a useful high-throughput bactericidal assay.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172163PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305226PMC
August 2017

Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella enterica Serovars Typhimurium and Enteritidis.

Clin Vaccine Immunol 2016 Apr 4;23(4):304-14. Epub 2016 Apr 4.

Novartis Vaccines Institute for Global Health, S.r.l., Siena, Italy

Invasive nontyphoidal Salmonella (iNTS) disease is a neglected disease with high mortality in children and HIV-positive individuals in sub-Saharan Africa, caused primarily by Africa-specific strains of Salmonella enterica serovars Typhimurium and Enteritidis. A vaccine using GMMA (generalized modules for membrane antigens) fromS.Typhimurium andS.Enteritidis containing lipid A modifications to reduce potential in vivo reactogenicity is under development. GMMA with penta-acylated lipid A showed the greatest reduction in the level of cytokine release from human peripheral blood monocytes from that for GMMA with wild-type lipid A. Deletion of the lipid A modification genes msbB and pagP was required to achieve pure penta-acylation. Interestingly, ΔmsbBΔ pagP GMMA from S. Enteritidis had a slightly higher stimulatory potential than those from S. Typhimurium, a finding consistent with the higher lipopolysaccharide (LPS) content and Toll-like receptor 2 (TLR2) stimulatory potential of the former. Also, TLR5 ligand flagellin was found in Salmonella GMMA. No relevant contribution to the stimulatory potential of GMMA was detected even when the flagellin protein FliC from S. Typhimurium was added at a concentration as high as 10% of total protein, suggesting that flagellin impurities are not a major factor for GMMA-mediated immune stimulation. Overall, the stimulatory potential of S. Typhimurium and S. Enteritidis ΔmsbB ΔpagP GMMA was close to that of Shigella sonnei GMMA, which are currently in phase I clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00023-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820502PMC
April 2016

Strain Selection for Generation of O-Antigen-Based Glycoconjugate Vaccines against Invasive Nontyphoidal Salmonella Disease.

PLoS One 2015 7;10(10):e0139847. Epub 2015 Oct 7.

Sclavo Behring Vaccines Institute for Global Health S.r.l., a GSK company (formerly Novartis Vaccines Institute for Global Health S.r.l), Via Fiorentina 1, 53100, Siena, Italy.

Nontyphoidal Salmonellae, principally S. Typhimurium and S. Enteritidis, are a major cause of invasive bloodstream infections in sub-Saharan Africa with no vaccine currently available. Conjugation of lipopolysaccharide O-antigen to a carrier protein constitutes a promising vaccination strategy. Here we describe a rational process to select the most appropriate isolates of Salmonella as source of O-antigen for developing a bivalent glycoconjugate vaccine. We screened a library of 30 S. Typhimurium and 21 S. Enteritidis in order to identify the most suitable strains for large scale O-antigen production and generation of conjugate vaccines. Initial screening was based on growth characteristics, safety profile of the isolates, O-antigen production, and O-antigen characteristics in terms of molecular size, O-acetylation and glucosylation level and position, as determined by phenol sulfuric assay, NMR, HPLC-SEC and HPAEC-PAD. Three animal isolates for each serovar were identified and used to synthesize candidate glycoconjugate vaccines, using CRM197 as carrier protein. The immunogenicity of these conjugates and the functional activity of the induced antibodies was investigated by ELISA, serum bactericidal assay and flow cytometry. S. Typhimurium O-antigen showed high structural diversity, including O-acetylation of rhamnose in a Malawian invasive strain generating a specific immunodominant epitope. S. Typhimurium conjugates provoked an anti-O-antigen response primarily against the O:5 determinant. O-antigen from S. Enteritidis was structurally more homogeneous than from S. Typhimurium, and no idiosyncratic antibody responses were detected for the S. Enteritidis conjugates. Of the three initially selected isolates, two S. Typhimurium (1418 and 2189) and two S. Enteritidis (502 and 618) strains generated glycoconjugates able to induce high specific antibody levels with high breadth of serovar-specific strain coverage, and were selected for use in vaccine production. The strain selection approach described is potentially applicable to the development of glycoconjugate vaccines against other bacterial pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139847PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596569PMC
June 2016

Relationship between antibody susceptibility and lipopolysaccharide O-antigen characteristics of invasive and gastrointestinal nontyphoidal Salmonellae isolates from Kenya.

PLoS Negl Trop Dis 2015 Mar 4;9(3):e0003573. Epub 2015 Mar 4.

Novartis Vaccines Institute for Global Health (NVGH), Siena, Italy.

Background: Nontyphoidal Salmonellae (NTS) cause a large burden of invasive and gastrointestinal disease among young children in sub-Saharan Africa. No vaccine is currently available. Previous reports indicate the importance of the O-antigen of Salmonella lipopolysaccharide for virulence and resistance to antibody-mediated killing. We hypothesised that isolates with more O-antigen have increased resistance to antibody-mediated killing and are more likely to be invasive than gastrointestinal.

Methodology/principal Findings: We studied 192 NTS isolates (114 Typhimurium, 78 Enteritidis) from blood and stools, mostly from paediatric admissions in Kenya 2000-2011. Isolates were tested for susceptibility to antibody-mediated killing, using whole adult serum. O-antigen structural characteristics, including O-acetylation and glucosylation, were investigated. Overall, isolates were susceptible to antibody-mediated killing, but S. Enteritidis were less susceptible and expressed more O-antigen than Typhimurium (p<0.0001 for both comparisons). For S. Typhimurium, but not Enteritidis, O-antigen expression correlated with reduced sensitivity to killing (r = 0.29, 95% CI = 0.10-0.45, p = 0.002). Both serovars expressed O-antigen populations ranging 21-33 kDa average molecular weight. O-antigen from most Typhimurium were O-acetylated on rhamnose and abequose residues, while Enteritidis O-antigen had low or no O-acetylation. Both Typhimurium and Enteritidis O-antigen were approximately 20%-50% glucosylated. Amount of S. Typhimurium O-antigen and O-antigen glucosylation level were inversely related. There was no clear association between clinical presentation and antibody susceptibility, O-antigen level or other O-antigen features.

Conclusion/significance: Kenyan S. Typhimurium and Enteritidis clinical isolates are susceptible to antibody-mediated killing, with degree of susceptibility varying with level of O-antigen for S. Typhimurium. This supports the development of an antibody-inducing vaccine against NTS for Africa. No clear differences were found in the phenotype of isolates from blood and stool, suggesting that the same isolates can cause invasive disease and gastroenteritis. Genome studies are required to understand whether invasive and gastrointestinal isolates differ at the genotypic level.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0003573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352093PMC
March 2015

Structural analysis of O-polysaccharide chains extracted from different Salmonella Typhimurium strains.

Carbohydr Res 2014 Feb 11;385:1-8. Epub 2013 Dec 11.

Novartis Vaccines Institute for Global Health, Via Fiorentina 1, I-53100 Siena, Italy; Medical Research Council Centre for Immune Regulation, Institute of Biomedical Research, School of Immunity and Infection, College of Medicine and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK.

Salmonella Typhimurium is the major cause of invasive nontyphoidal Salmonella disease in Africa, with high mortality among children and HIV-infected individuals. Currently, no vaccine is available for use in humans. Antibodies directed against the O-polysaccharide of the lipopolysaccharide molecule of Salmonella mediate bacterial killing and are protective, and conjugation of the O-polysaccharide to a carrier protein represents a possible strategy for vaccine development. Here we have purified the O-polysaccharide from six different strains of S. Typhimurium and fully characterized them using analytical methods including HPLC-SEC, HPAEC-PAD, GC, GC-MS, 1D and 2D NMR spectroscopy. All the O-polysaccharide samples showed a similar bimodal molecular mass distribution, but differed with respect to the amount and position of O-acetylation and glucosylation. For some strains, O-acetyl groups were found not only on C-2 of abequose (factor 5 specificity), but also on C-2 and C-3 of rhamnose; glucose was found to be linked 1→4 or 1→6 to galactose in different amounts according to the strain of origin. This structural variability could have an impact on the immunogenicity of corresponding glycoconjugate vaccines and different strains need to be evaluated in order to identify the appropriate source of O-polysaccharide to use for the development of a candidate conjugate vaccine with broad coverage against S. Typhimurium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.carres.2013.12.003DOI Listing
February 2014

Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X.

Proc Natl Acad Sci U S A 2013 Nov 4;110(47):19077-82. Epub 2013 Nov 4.

Novartis Vaccines Institute for Global Health, and Research, Novartis Vaccines and Diagnostics, I-53100 Siena, Italy.

Neisseria meningitidis is a major cause of bacterial meningitis worldwide, especially in the African meningitis belt, and has a high associated mortality. The meningococcal serogroups A, W, and X have been responsible for epidemics and almost all cases of meningococcal meningitis in the meningitis belt over the past 12 y. Currently no vaccine is available against meningococcal X (MenX). Because the development of a new vaccine through to licensure takes many years, this leaves Africa vulnerable to new epidemics of MenX meningitis at a time when the epidemiology of meningococcal meningitis on the continent is changing rapidly, following the recent introduction of a glycoconjugate vaccine against serogroup A. Here, we report the development of candidate glycoconjugate vaccines against MenX and preclinical data from their use in animal studies. Following optimization of growth conditions of our seed MenX strain for polysaccharide (PS) production, a scalable purification process was developed yielding high amounts of pure MenX PS. Different glycoconjugates were synthesized by coupling MenX oligosaccharides of varying chain length to CRM197 as carrier protein. Analytical methods were developed for in-process control and determination of purity and consistency of the vaccines. All conjugates induced high anti-MenX PS IgG titers in mice. Antibodies were strongly bactericidal against African MenX isolates. These findings support the further development of glycoconjugate vaccines against MenX and their assessment in clinical trials to produce a vaccine against the one cause of epidemic meningococcal meningitis that currently cannot be prevented by available vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1314476110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839747PMC
November 2013

Differential epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu, Nepal: a matched case control investigation in a highly endemic enteric fever setting.

PLoS Negl Trop Dis 2013 22;7(8):e2391. Epub 2013 Aug 22.

Oxford University Clinical Research Unit, Patan Academy of Health Sciences, Kathmandu, Nepal.

Background: Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting.

Methodology And Principal Findings: A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection.

Conclusions And Significance: Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0002391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749961PMC
February 2014

Invasive African Salmonella Typhimurium induces bactericidal antibodies against O-antigens.

Microb Pathog 2013 Oct 10;63:19-23. Epub 2013 Jun 10.

Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy.

Nontyphoidal Salmonella are a major and emerging cause of fatal invasive disease in Africa, and are genetically distinct from those found elsewhere in the world. Understanding the targets of protective immunity to these African Salmonellae is key to vaccine development. We immunized mice and rabbits with heat-inactivated wild-type African invasive Salmonella Typhimurium D23580 and rough mutants lacking O-antigen. Wild-type Salmonella, unlike rough bacteria, induced a large bactericidal antibody response mainly against O-antigen. Bactericidal ability of anti-O-antigen antibodies was confirmed following purification by affinity chromatography. The current findings support the development of an O-antigen conjugate vaccine against invasive nontyphoidal Salmonellae for Africa.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.micpath.2013.05.014DOI Listing
October 2013

Characterization of Citrobacter sp. line 328 as a source of Vi for a Vi-CRM(197) glycoconjugate vaccine against Salmonella Typhi.

J Infect Dev Ctries 2012 Nov 26;6(11):763-73. Epub 2012 Nov 26.

Novartis Vaccines Institute for Global Health.

Introduction: Salmonella enterica serovar Typhi is the causative agent of typhoid fever with over 22 million cases and over 200,000 deaths reported annually. A vaccine is much needed for use in young children and the Novartis Vaccines Institute for Global Health (NVGH) is developing a conjugate vaccine which targets S. Typhi Vi capsular polysaccharide.

Methodology: Here we describe a method suitable for industrial scale production of the Vi antigen based on expression by a Citrobacter line. We optimized the production of Vi by selecting a suitable Citrobacter strain (Citrobacter 328) that yields high and stable expression of Vi in chemically defined medium under industrial-scale fermentation conditions.

Results: Vi-CRM197 made using Vi from Citrobacter 328 elicited high anti-Vi antibody levels in mice and rabbits.

Conclusions: Citrobacter 328 is a suitable strain for production of Vi for conjugate anti-Typhi vaccines. Being a BSL-1 organism, which grows in defined medium and stably produces high yields of Vi, it offers excellent potential for safe production of inexpensive vaccines for populations at risk of typhoid fever.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3855/jidc.2495DOI Listing
November 2012

O:2-CRM(197) conjugates against Salmonella Paratyphi A.

PLoS One 2012 7;7(11):e47039. Epub 2012 Nov 7.

Novartis Vaccines Institute for Global Health, Siena, Italy.

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM(197), using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM(197) as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047039PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492368PMC
April 2013

Immunization with the conjugate vaccine Vi-CRM₁₉₇ against Salmonella typhi induces Vi-specific mucosal and systemic immune responses in mice.

Vaccine 2012 Sep 15;30(43):6111-4. Epub 2012 Jun 15.

Laboratorio di Microbiologia Molecolare e Biotecnologia, Dipartimento di Biotecnologie, Università di Siena, Italy.

Typhoid fever is a public health problem, especially among young children in developing countries. To address this need, a glycoconjugate vaccine Vi-CRM₁₉₇, composed of the polysaccharide antigen Vi covalently conjugated to the non-toxic mutant of diphtheria toxin CRM₁₉₇, is under development. Here, we assessed the antibody and cellular responses, both local and systemic, following subcutaneous injection of Vi-CRM₁₉₇. The glycoconjugate elicited Vi-specific serum IgG titers significantly higher than unconjugated Vi, with prevalence of IgG1 that persisted for at least 60 days after immunization. Vi-specific IgG, but not IgA, were present in intestinal washes. Lymphocytes proliferation after restimulation with Vi-CRM₁₉₇ was observed in spleen and mesenteric lymph nodes. These data confirm the immunogenicity of Vi-CRM₁₉₇ and demonstrate that the vaccine-specific antibody and cellular immune responses are present also in the intestinal tract, thus strengthening the suitability of Vi-CRM₁₉₇ as a promising candidate vaccine against Salmonella Typhi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2012.05.081DOI Listing
September 2012

Safety, immunogenicity and dose ranging of a new Vi-CRM₁₉₇ conjugate vaccine against typhoid fever: randomized clinical testing in healthy adults.

PLoS One 2011 30;6(9):e25398. Epub 2011 Sep 30.

Center for the Evaluation of Vaccination, Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium.

Background: Typhoid fever causes more than 21 million cases of disease and 200,000 deaths yearly worldwide, with more than 90% of the disease burden being reported from Asia. Epidemiological data show high disease incidence in young children and suggest that immunization programs should target children below two years of age: this is not possible with available vaccines. The Novartis Vaccines Institute for Global Health developed a conjugate vaccine (Vi-CRM₁₉₇) for infant vaccination concomitantly with EPI vaccines, either starting at 6 weeks with DTP or at 9 months with measles vaccine. We report the results from a Phase 1 and a Phase 2 dose ranging trial with Vi-CRM₁₉₇ in European adults.

Methodology: Following randomized blinded comparison of single vaccination with either Vi-CRM₁₉₇ or licensed polysaccharide vaccines (both containing 25·0 µg of Vi antigen), a randomised observer blinded dose ranging trial was performed in the same center to compare three concentrations of Vi-CRM₁₉₇ (1·25 µg, 5·0 µg and 12·5 µg of Vi antigen) with the polysaccharide vaccine.

Principal Findings: All vaccines were well tolerated. Compared to the polysaccharide vaccine, Vi-CRM₁₉₇ induced a higher incidence of mild to moderate short lasting local pain. All Vi-CRM₁₉₇ formulations induced higher Vi antibody levels compared to licensed control, with clear dose response relationship.

Conclusions: Vi-CRM₁₉₇ did not elicit safety concerns, was highly immunogenic and is therefore suitable for further clinical testing in endemic populations of South Asia.

Trial Registration: ClinicalTrials.gov NCT01123941 NCT01193907.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025398PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184126PMC
January 2012

Evaluation of the immunogenicity and biological activity of the Citrobacter freundii Vi-CRM197 conjugate as a vaccine for Salmonella enterica serovar Typhi.

Clin Vaccine Immunol 2011 Mar 19;18(3):460-8. Epub 2011 Jan 19.

Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy.

Typhoid fever remains a major health problem in developing countries. Young children are at high risk, and a vaccine effective for this age group is urgently needed. Purified capsular polysaccharide from Salmonella enterica serovar Typhi (Vi) is licensed as a vaccine, providing 50 to 70% protection in individuals older than 5 years. However, this vaccine is ineffective in infants. Vi conjugated to a carrier protein (i.e., an exoprotein A mutant from Pseudomonas aeruginosa [rEPA]) is highly immunogenic, provides long-term protection, and shows more than 90% protective efficacy in children 2 to 5 years old. Here, we describe an alternative glycoconjugate vaccine for S. Typhi, Vi-CRM(197), where Vi was obtained from Citrobacter freundii WR7011 and CRM(197), the mutant diphtheria toxin protein, was used as the carrier. We investigated the optimization of growth conditions for Vi production from C. freundii WR7011 and the immunogenicity of Vi-CRM(197) conjugates in mice. The optimal saccharide/protein ratio of the glycoconjugates was identified for the best antibody production. We also demonstrated the ability of this new vaccine to protect mice against challenge with Vi-positive Salmonella enterica serovar Typhimurium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00387-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067394PMC
March 2011

Knowledge, attitude and practices study on reproductive health among secondary school students in Bolgatanga, upper east region, Ghana.

Afr J Reprod Health 2009 Dec;13(4):51-66

Novartis Vaccines Institute of Global Health, via Fiorentina 1, 53100 Siena, Italy.

This study was conducted within the secondary school student population of the Bolgatanga community, in Northern Ghana, to learn about knowledge, attitude and practices of reproductive health of this adolescent student population. Both quantitative and qualitative data were collected on adolescence perception, STIs and HIV/AIDS, family planning, male-female relationship, and vulnerability to sexual violence. The data collected show a concerning low familiarity of the student population with family planning methods and HIV/AIDS transmission, which, combined with minimal contraceptive use, pose them at high risk for unwanted pregnancies and sexual infections transmission. We argue that poor infrastructures and low accessibility of these rural areas in Northern Ghana may have led to uneven distribution of reproductive health educational programs in the country, urging more programs and interventions aimed particularly at these high risk groups.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2009

Ongoing genome reduction in Mycobacterium ulcerans.

Emerg Infect Dis 2007 Jul;13(7):1008-15

Swiss Tropical Institute, Basel, Switzerland.

Elucidation of the transmission, epidemiology, and evolution of Mycobacterium ulcerans, the causative agent of Buruli ulcer, is hampered by the striking lack of genetic diversity of this emerging pathogen. However, by using a prototype plasmid-based microarray that covered 10% of the genome, we found multiple genomic DNA deletions among 30 M. ulcerans clinical isolates of diverse geographic origins. Many of the changes appear to have been mediated by insertion sequence (IS) elements IS2404 and IS2606, which have high copy numbers. Classification of the deleted genes according to their biological functions supports the hypothesis that M. ulcerans has recently evolved from the generalist environmental M. marinum to become a niche-adapted specialist. The substantial genomic diversity, along with a prototype microarray that covered a small portion of the genome, suggests that a genome-wide microarray will make available a genetic fingerprinting method with the high resolution required for microepidemiologic studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878211PMC
http://dx.doi.org/10.3201/eid1307.060205DOI Listing
July 2007

Evolution of two distinct phylogenetic lineages of the emerging human pathogen Mycobacterium ulcerans.

BMC Evol Biol 2007 Sep 27;7:177. Epub 2007 Sep 27.

Swiss Tropical Institute, Socinstr, 57, 4002 Basel, Switzerland.

Background: Comparative genomics has greatly improved our understanding of the evolution of pathogenic mycobacteria such as Mycobacterium tuberculosis. Here we have used data from a genome microarray analysis to explore insertion-deletion (InDel) polymorphism among a diverse strain collection of Mycobacterium ulcerans, the causative agent of the devastating skin disease, Buruli ulcer. Detailed analysis of large sequence polymorphisms in twelve regions of difference (RDs), comprising irreversible genetic markers, enabled us to refine the phylogenetic succession within M. ulcerans, to define features of a hypothetical M. ulcerans most recent common ancestor and to confirm its origin from Mycobacterium marinum.

Results: M. ulcerans has evolved into five InDel haplotypes that separate into two distinct lineages: (i) the "classical" lineage including the most pathogenic genotypes - those that come from Africa, Australia and South East Asia; and (ii) an "ancestral" M. ulcerans lineage comprising strains from Asia (China/Japan), South America and Mexico. The ancestral lineage is genetically closer to the progenitor M. marinum in both RD composition and DNA sequence identity, whereas the classical lineage has undergone major genomic rearrangements.

Conclusion: Results of the InDel analysis are in complete accord with recent multi-locus sequence analysis and indicate that M. ulcerans has passed through at least two major evolutionary bottlenecks since divergence from M. marinum. The classical lineage shows more pronounced reductive evolution than the ancestral lineage, suggesting that there may be differences in the ecology between the two lineages. These findings improve the understanding of the adaptive evolution and virulence of M. ulcerans and pathogenic mycobacteria in general and will facilitate the development of new tools for improved diagnostics and molecular epidemiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2148-7-177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2098775PMC
September 2007

Local activation of the innate immune system in Buruli ulcer lesions.

J Invest Dermatol 2007 Mar 19;127(3):638-45. Epub 2006 Oct 19.

Swiss Tropical Institute, Department of Medical Parasitology and Molecular Immunology, Basel, Switzerland.

Buruli ulcer (BU) caused by Mycobacterium ulcerans is a chronic necrotizing disease of the skin and the underlying soft tissue. Fat tissue necrosis accompanied by minimal inflammation is considered the most reliable histopathologic feature of BU. There may be a constant influx of inflammatory cells to the sites of active infection but these are thought to be killed by mycolactone, a polyketide toxin produced by M. ulcerans, through apoptosis and necrosis. Here we describe the spatial correlations between mycobacterial load and the expression of dendritic cell (DC) surface markers (cluster of differentiation (CD)83, CD11c, and CD123), the Toll-like receptor (TLR) 9 and pro- and anti-inflammatory cytokines (IL-8, IL-6, tumor necrosis factor-alpha (TNF-alpha), IFN-alpha, IL-12p40, IL-10, and IFN-gamma) within BU lesions. Although IL-8, IL-6, and TNF-alpha messenger RNA (mRNA) was detectable by real-time PCR in all lesions, the expression of the other cytokines was only found as small foci in some lesions. Correlations of the distribution of mRNA encoding the activation marker CD83 and the DC subset markers CD123 and CD11c indicate that both activated plasmacytoid and myeloid dendritic cells were present in the lesions. Results suggest that M. ulcerans specific immune responses may develop once therapeutic interventions have limited the production of mycolactone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.jid.5700593DOI Listing
March 2007

Use of the immunodominant 18-kiloDalton small heat shock protein as a serological marker for exposure to Mycobacterium ulcerans.

Clin Vaccine Immunol 2006 Dec 4;13(12):1314-21. Epub 2006 Oct 4.

Molecular Immunology, Swiss Tropical Institute, CH 4002 Basel, Switzerland.

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00254-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1694454PMC
December 2006

What does detection of Mycobacterium ulcerans DNA in the margin of an excised Buruli ulcer lesion tell us?

J Clin Microbiol 2006 Nov 23;44(11):4273-5. Epub 2006 Aug 23.

Molecular Immunology, Swiss Tropical Institute, Basel, Switzerland.

We determined by real-time PCR the distribution of Mycobacterium ulcerans DNA in the excised lesion of a Buruli ulcer patient. A new lesion developed adjacent to the site of excision in the patient. The excised margin around the primary lesion contained a small amount of mycobacterial DNA in the area where the secondary lesion developed. These results suggest that a relatively small number of infiltrating mycobacteria can lead to the development of a recurrence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JCM.00970-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1698343PMC
November 2006

Genetic diversity in Mycobacterium ulcerans isolates from Ghana revealed by a newly identified locus containing a variable number of tandem repeats.

J Bacteriol 2006 Feb;188(4):1462-5

Swiss Tropical Institute, Socinstrasse 57, CH 4002 Basel, Switzerland.

The molecular typing methods used so far for Mycobacterium ulcerans isolates have not been able to identify genetic differences among isolates from Africa. This apparent lack of genetic diversity among M. ulcerans isolates is indicative of a clonal population structure. We analyzed the genetic diversity of 72 African isolates, including 57 strains from Ghana, by variable number of tandem repeat (VNTR) typing based on a newly identified polymorphic locus designated ST1 and the previously described locus MIRU 1. Three different genotypes were found in Ghana, demonstrating for the first time the genetic diversity of M. ulcerans in an African country. While the ST1/MIRU 1 allele combination BD/BAA seems to dominate in Africa, it was only rarely found in isolates from Ghana, where the combination BD/B was dominant and observed in all districts studied. A third variant genotype (C/BAA) was found only in the Amansie-West district. The results indicate that new genetic variants of M. ulcerans emerged and spread within Ghana and support the potential of VNTR-based typing for genotyping of M. ulcerans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.188.4.1462-1465.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1367230PMC
February 2006

Contiguous spread of Mycobacterium ulcerans in Buruli ulcer lesions analysed by histopathology and real-time PCR quantification of mycobacterial DNA.

J Pathol 2006 Jan;208(1):119-28

Molecular Immunology, Swiss Tropical Institute, Basel, Switzerland.

The distribution of M. ulcerans in Buruli ulcer lesions was analysed by IS2404 real-time PCR quantification of M. ulcerans DNA and by semi-quantitative microscopic assessment of the number of acid-fast bacilli (AFB). Mycobacterial burden was compared with histopathological changes. Focal distribution of tissue destruction extending into areas with high and low mycobacterial burden was a feature in all lesions analysed. Even where most of the mycobacteria were washed out of ulcerative lesions, peaks of mycobacterial DNA and AFB in the necrotic base of the ulcers still marked the position of the primary infection focus. Significant amounts of mycobacterial DNA and microcolonies were also present in samples from more peripheral regions and occasionally in excised margins of macroscopically and histologically healthy-appearing excised tissue margins. Additional peaks of mycobacterial DNA clearly marked sites where satellite lesions were developing. Even when granulomas provided evidence for the development of cell-mediated immunity, development of satellite lesions by contiguous spreading was not completely prevented. Areas free of mycobacterial DNA were found between primary and secondary infection foci and around scarring tissue of healing lesions. These results demonstrate that IS2404 real-time PCR analysis is a better tool than the less sensitive and only semi-quantitative microscopic enumeration of AFB for studying the dynamics of M. ulcerans infection in situ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.1864DOI Listing
January 2006

Buruli ulcer disease in Cameroon rediscovered.

Am J Trop Med Hyg 2004 May;70(5):520-6

Aide aux Lépreux Emmaüs-Suisse, Yaounde, Cameroon.

To assess the magnitude of the Buruli ulcer (BU) problem in Cameroon, we conducted a cross-sectional survey in the Nyong River basin and identified on clinical grounds a total of 436 cases of active or inactive BU (202 and 234, respectively). Swab specimens were taken from 162 active cases with ulcerative lesions and in 135 of these (83.3%) the clinical diagnosis was confirmed by the IS2404 polymerase chain reaction. Most lesions (93%) were located on the extremities, with lower limbs being twice as commonly involved as upper limbs. The age of patients with active BU ranged from 2 to 90 years with a median age of 14.5 years. Vaccination with bacilli Calmette-Guérin appeared to protect children against more severe forms of BU with multiple lesions. We conclude that in Cameroon BU is endemic, at least in the study area, and that a comprehensive control program for BU in Cameroon is urgently needed.
View Article and Find Full Text PDF

Download full-text PDF

Source
May 2004