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  Director : FOURNIER Jean-Michel (fournier@pasteur.fr)


  abstract

 

For most developing countries, cholera is still a major problem. Our Unit is using an integrated approach to fight this problem. This approach involves: (1) a survey and molecular epidemiology studies of cholera; (2) the development of a rapid diagnostic test; (3) the design of a glycoconjugate cholera vaccine; (4) the study of clinical infections caused by non cholera vibrios; and (5) the study of the cholera and non cholera vibrios present in the environment and in seafood. The French Ministry of Health has designated our laboratory as the "National Reference Center for Vibrios", due to its expertise in the fields of cholera and non cholera vibrios.



  report

cale

Vibrios are gram-negative bacteria that naturally colonize the marine environment. Vibrios are divided into "cholera vibrios", including isolates belonging to serogroups O1 and O139 of Vibrio cholerae, and "non cholera vibrios", including (i) isolates belonging to serogroups other than O1 and O139 of V. cholerae – known as V. cholerae non-O1/non-O139 – and (ii) isolates belonging to other Vibrio species. Our aims are to improve our understanding of the propagation of cholera vibrios in countries in which cholera is endemic or epidemic, to develop a rapid diagnostic test for cholera, to design a new vaccine against cholera, and to improve the molecular characterization of cholera and non cholera vibrios isolated from seafood, to make it easier to evaluate the risk associated with these products.

1. Survey and molecular epidemiology of cholera (Marie-Laure QUILICI, Jean-Michel FOURNIER)

Cholera remains a major individual and public health problem. According to the WHO, the number of individuals susceptible to cholera due to socioeconomic problems has dramatically increased worldwide, creating conditions that are favorable for a global cholera crisis. In 2001, 58 countries officially notified the WHO of a total of 184,311 cases and 2,782 deaths. However, the actual global figures are much higher, due to underreporting and other limitations of surveillance systems.

France is not directly concerned by cholera epidemics as, like most developed countries, hygiene standards are good. However, imported cases of cholera are regularly identified in travelers. The WHO is notified about imported cholera cases. In 2001, one case of cholera was reported in France. This case was imported from Ivory Coast.

Cholera epidemics do not recognize national borders. We therefore collaborate with biologists from other countries in which cholera outbreaks occur, with members of the International Network of Pasteur Institutes and with nongovernmental humanitarian organizations. In 2001, we studied 58 V. cholerae O1 strains from five African countries. Our collaborations make it possible for us to carry out molecular epidemiology studies on cholera and on new cholera vibrio strains that might be imported into France and to study the spread of the cholera vibrio strains responsible for cholera outbreaks in Africa since the start of the seventh pandemic.

2. Development of a rapid diagnostic test for cholera (Alain BOUTONNIER, Jean-Michel FOURNIER)

We have developed and evaluated a rapid diagnostic test (RDT) for cholera based on immunochromatography in collaboration with the Antibody Engineering Laboratory from the Pasteur Institute, Paris, the Madagascar Pasteur Institute and the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). This test has been shown to be specific, sensitive and reliable both in the laboratory and in two cholera endemic areas (Madagascar and Bangladesh). Moreover, it should provide a simple tool for epidemiological surveys of cholera to improve our knowledge of the real situation in the world.

3. New vaccine against cholera (Alain BOUTONNIER, Bruno DASSY, Jean-Michel FOURNIER)

The long-term control of cholera depends on improving hygiene by providing safe drinking water and appropriate sewage disposal, and by changing personal hygiene behavior. However, these control measures are expensive and it is difficult to change people's hygiene and water-use behavior. Hence, there is an urgent need for cholera vaccines that confer reliable and long-term protection in all age groups, including children aged <5 years.

The starting point of our research, which began in 1991, was to determine which antibodies protect against cholera. We have shown that immunoglobulin G (IgG) monoclonal antibodies directed against the polysaccharide moiety of the lipopolysaccharide (LPS) of V. cholerae O1, are immunoprotective in an experimental model of cholera in neonatal mice. We have also shown that IgG antibodies injected intravenously into adult mice are transferred from blood vessels to the lumen of the intestine. These results encouraged us to start, in 1995, developing glycoconjugate vaccines composed of the polysaccharides of V. cholerae O1 and V. cholerae O139 covalently linked to a carrier protein to induce a long-lasting, thymus-dependent immune response. To optimize the preparation of these glycoconjugates, we used immunochemical and physicochemical methods to study the antigenic determinants expressed by the two serotypes - Ogawa and Inaba - of V. cholerae O1. We have also prepared a glycoconjugate composed of the polysaccharide of V. cholerae O139. In mice, this vaccine induces a protective thymus-dependent response.

This year, we began to prepare for a clinical evaluation of this V. cholerae O139 glycoconjugate. We have also prepared a glycoconjugate with the polysaccharide of the Inaba serotype of V. cholerae O1. The immunogenicity of this glycoconjugate has been studied in mice. A thymus-dependent immune response has been observed and the protective capacity of the antibodies is currently being evaluated.

4. Infections due to non cholera vibrios (Marie-Laure QUILICI, Jean-Michel FOURNIER)

The genus Vibrio contains more than 50 species, including three species that are frequently isolated from human clinical samples: V. cholerae, V. parahaemolyticus and V. vulnificus, and four other species that are isolated more rarely: V. alginolyticus, V. fluvialis, V. hollisae and V. mimicus. Human non cholera vibrio infections can result in gastroenteritis, skin and soft tissue infections, septicemia, and a number of extraintestinal infections, such as ear infections. Patients with underlying immunosuppressive diseases are at high risk of the rapid spread of infections with these microorganisms. Most of these infections are associated with contact with sea water or the consumption of seafood, and their occurrence is correlated with the warmer months of the year.

In 2002, 11 cases of infections due to non cholera vibrios were identified in France by the National Reference Center. Ten cases of septicemia, gastroenteritis or suppuration were caused by V. cholerae non-O1/non-O139. One case of septicemia and soft tissue infection was caused by V. vulnificus.

5. Molecular characterization of non cholera vibrios (Annick ROBERT- PILLOT, Marie-Laure QUILICI)

Ecological modifications to marine and estuarine environments, due to global climate change or local industrial activities, favor the proliferation and the dissemination of vibrios, which may then colonize humans either by direct contact with sea water or following the consumption of seafood. Increasing international trade and the consumption of raw seafood, together with an increase in the number of susceptible individuals with underlying immunosuppressive disease, are leading to concern that the incidence of infections due to cholera or non cholera vibrios may increase in developed countries. However, we have to be careful that developed countries do not respond inappropriately for example by restricting the importation of seafood products from developing countries facing cholera epidemics. This type of measure jeopardizes the economic situation of poor countries, dissuades these countries from notifying the authorities about cholera outbreaks and contributes to the propagation of cholera epidemics. Public health concern has stressed the importance of developing molecular methods for the detection of pathogenic isolates of two vibrio species, V. cholerae and V. parahaemolyticus, present both in sea water and in seafood, and causing foodborne infections. We have thus developed a method for the detection of V. cholerae in sea water, based on colony hybridization assays. We have also developed a PCR method, based on the R72H V. parahaemolyticus-specific sequence, to identify V. parahaemolyticus isolates in environmental samples.

This year, we have applied this PCR method to all bacterial isolates suspected of belonging to the V. parahaemolyticus species, which have been provided by food microbiological laboratories. This confirmed the value of this PCR for the identification of the V. parahaemolyticus species. However, it should be stressed that not all isolates of V. parahaemolyticus are pathogenic for humans. Only isolates that produce one or both of the two hemolysins - TDH and TRH - are pathogenic. Thus, we have developed a PCR method to detect the genes encoding these hemolysins. This PCR method has been applied to 11 clinical isolates, 130 strains isolated from imported seafood and 135 strains isolated from environmental samples collected in two French coastal areas. Most of the clinical isolates (9/11) possessed the hemolysin genes. Fewer isolates from environmental samples carried the hemolysin genes (3-11% depending on the geographic site) and only 1.5% of seafood isolates carried these genes. This suggests that the survey of imported seafood products should be continued and that a long-term monitoring program should be initiated to detect pathogenic V. parahaemolyticus isolates present in French seafood and coastal waters.

Photo: Rapid diagnosis of cholera by the immunochromatographic dipstick test. In the simplest setting, the dipstick can be held by a pair of forceps, dipped into the typical watery choleric stool for a short period of time and put back in a tube. Results are available after 2 to 15 minutes, usually within 5 minutes.

Keywords: cholera, vibrios, vaccination, glycoconjuguate, diagnosis, molecular epidemiology, seafood, ecosystem



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  BIDAULT Brigitte,bbidault@pasteur.fr DASSY Bruno,bdassy@pasteur.fr

FOURNIER Jean-Michel,fournier@pasteur.fr

QUILICI Marie-Laure,quilici@pasteur.fr

ROBERT-PILLOT Annick,arp@pasteur.fr BOUTONNIER Alain, Engineer IP,abouto@pasteur.fr

ROTGER Benoît, Quality Assurance Engineer,brotger@pasteur.fr

GUENOLE, Alain,Technician IP,aguenole@pasteur.fr

LEMEE Laure, Technician IP,llemee@pasteur.fr

Shared with the Laboratory of Listeria :

BERTEL Arnaud, Laboratory Assistant IP

DELAIRE Marie-Claire, Laboratory Agent IP

TESSAUD Nathalie, Laboratory Assistant IP


Activity Reports 2002 - Institut Pasteur
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