Unit: Cholera and Vibrios
Director: FOURNIER Jean-Michel
The scientific activity of the Unité du Choléra et des Vibrions focuses on two areas: cholera and non cholera vibrios. Cholera epidemics are still being reported throughout the world. Our contribution to the fight against this devastating disease includes: (1) research into new chemically defined conjugate vaccines; (2) the development of a rapid diagnostic test for cholera; (3) studies on the molecular epidemiology of cholera. We are also concerned by the study of other (non cholera) vibrios. Our expertise in cholera and other vibrio infections has led to our laboratory being designated as the National Reference Center for Vibrios and Cholera by the French Ministry of Health.
1. Research program on new chemically defined conjugate vaccines against cholera (Alain BOUTONNIER, Cyrille GRANDJEAN, Bruno DASSY, Jean-Michel FOURNIER)
Cholera is an epidemic diarrheal disease caused by two serogroups of a bacterium, Vibrio cholerae. We are now in the seventh pandemic, which began in India in 1961, invaded Africa in 1970 and spread to Latin America in 1991. This seventh pandemic is caused by V. cholerae serogroup O1, biotype El Tor, serotypes Ogawa or Inaba. A new V. cholerae serogroup, O139, appeared in India in 1992. There are definitive indications that the incidence of V. cholerae O139 is on the rise in India and Bangladesh. Thus, this new cholera strain may become a global threat. Hence there is an urgent need for cholera vaccines that confer reliable and long-term protection against V. cholerae O1 and V. cholerae O139 in all age groups, including children aged <5 years.
The starting point of our research 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, expressing the serogroup or serotype determinants, are immunoprotective in an experimental model of cholera in neonatal mice. These results encouraged us to start developing conjugate 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. We have prepared a conjugate composed of the polysaccharide of V. cholerae O139. In mice, this vaccine induces a protective thymus-dependent response.
This year, we continued the preparation of a clinical evaluation of this V. cholerae O139 conjugate. To this aim, we provided technical assistance to a pharmaceutical company concerning production of a Good Manufactory Practice (GMP) batch of O139 conjugate vaccine. We have also started an investigation of the structure of the V. cholerae O1 LPS. We have prepared new conjugates with the polysaccharide of the Inaba serotype of V. cholerae O1. The immunogenicity of these conjugates is currently being evaluated in mice.
The vibriocidal antibody titer reliably predicts resistance to cholera and is currently the only valid serologic assay that is correlated with protection. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for V. cholerae O1 and for V. cholerae O139. A cholera outbreak occurred in Madagascar in 1999, for the first time in its history. This assay has been used in the context of an epidemiological study on the cholera immunity of the population carried out in collaboration with the Institut Pasteur of Madagascar. This vibriocidal assay will also be used to screen serum samples obtained during the clinical trial planned with our O139 conjugate.
2. Development of Rapid Diagnostic Tests for cholera (Alain BOUTONNIER, Jean-Michel FOURNIER)
Our effort to develop a conjugate vaccine that targets V. cholerae O1 and O139, led to the development of monoclonal antibodies specific to O1 or O139 LPS. We used the specificity of these monoclonal antibodies to develop, in collaboration with the Laboratoire d'Ingénierie des Anticorps at the Institut Pasteur, Paris, and the Institut Pasteur of Madagascar, rapid diagnostic tests for V. cholerae O1 or O139. These tests are based on a one-step, vertical-flow immunochromatographic principle. The sensitivity and specificity of these rapid diagnostic tests have been tested in the laboratory setting and in two areas in which cholera is epidemic, namely in Madagascar and in Bangladesh, in collaboration with the International Centre for Diarrhoeal Diseases Research, Bangladesh (ICDDR,B). The specificity ranged between 84 and 100% and the sensitivity from 94.2 to 100%. These tests were also evaluated in Bangladesh for the rapid detection of V. cholerae O1 or O139 in rectal swabs collected from hospitalized diarrheal patients after enrichment for 4 hours in alkaline peptone water. The sensitivity and specificity were above 92% and 91% respectively. These are the first rapid tests that can successfully detect V. cholerae O1 and O139 in rectal swabs, and should immensely improve the surveillance of cholera, especially in remote locations.
3. Molecular studies of cholera vibrios (Marie-Laure QUILICI)
Cholera is a very unusual disease because the strains responsible for epidemics are highly homogeneous. Only two of the 200 or so O serogroups described in the V. cholerae species are responsible for current cholera epidemics: serogroup O1, responsible for the seventh cholera pandemic, and serogroup O139, which emerged in 1992. Nevertheless, the inexorable progress of the disease throughout the world since the first cholera pandemic, which started in India in 1817, has been accompanied by the diversification of cholera vibrio populations.
The development of molecular typing methods has enabled us to study the evolution of cholera vibrio populations isolated in Africa. We used ribotyping and pulsed-field gel electrophoresis (PFGE) to study 350 V. cholerae O1 strains isolated in African countries between 1970 and 2000. We differentiated 14 ribotype patterns and even more different but very similar PFGE patterns. Molecular typing data revealed a high degree of genetic similarity among V. cholerae strains from diverse geographic origins and sometimes isolated decades apart, and show that the 7th pandemic strains are clonal. Strains collected at the beginning of the seventh pandemic in Africa belonged to a ribotype that is still present nowadays. This ribotype is associated with endemic or epidemic strains, and is the most common of the 14 ribotypes detected in this continent over the past 30 years.
4. Molecular studies of non cholera vibrios (Annick ROBERT-PILLOT, Marie-Laure QUILICI)
Vibrios are gram-negative bacteria that are components of the normal bacterial flora of aquatic environments. 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 including two species that are frequently isolated from human clinical samples: V. parahaemolyticus and V. vulnificus, and four other species that are isolated more rarely: V. alginolyticus, V. fluvialis, V. hollisae and V. mimicus.
Modifications of environmental and physiological factors in certain coastal regions provide vibrios with ideal conditions for proliferation. Development of international trade and increases in the consumption of raw or lightly cooked seafood together with a larger number of susceptible persons is causing concern that the incidence of infections in Europe may increase. Thus, human vibrio infections are likely to spread and microbiological surveillance would help to control public health risks.
Classical biochemical methods for bacterial identification are not suitable for the study of vibrio strains isolated from the environment, and public health concerns stress the importance of developing molecular methods for the detection, identification and characterization of pathogenicity factors in vibrio strains isolated from humans, sea water or seafood. Thus, we pursued our program aimed at developing and using molecular methods (PCR-based) for three Vibrio species of medical interest: V. parahaemolyticus, V. cholerae and V. vulnificus.
An investigation of the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments was carried out by the Ifremer, Brest, France, in collaboration with our laboratory and with the Centre of Marine Biotechnology, University of Maryland, Biotechnology Institute, Baltimore, MD, USA. V. alginolyticus (99/189) was the predominant species isolated, followed by V. parahaemolyticus (41/189), V. vulnificus (20/189), and V. cholerae non-O1/non-O139 (3/189). Two of the 41 V. parahaemolyticus isolates were positive for the trh hemolysin gene. These results demonstrate the presence of pathogenic Vibrio species in French coastal waters, and suggest that a long-term monitoring program should be initiated to detect pathogenic V. parahaemolyticus isolates present in French seafood and coastal waters.
5. Activity of the National Reference Center for Vibrios and Cholera (Marie-Laure QUILICI, Jean-Michel FOURNIER)
The NRCVC is responsible for the identification of the cholera vibrio strains that cause clinical cases of cholera in France where cholera is a notifiable disease as in many other countries. Only a small number of cholera cases (0 to 5) occur each year in France and most of them are imported. No cholera cases have been identified in 2003. Moreover, as cholera epidemics do not recognize national borders, we collaborate with biologists from other countries reporting cholera outbreaks and with nongovernmental humanitarian organizations. These collaborations make it possible for us to study new cholera vibrio strains that might be imported into France.
The NRCVC is also responsible for the identification of non cholera vibrio strains from clinical laboratories. 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 2003, 11 cases of human infections due to non cholera vibrios were identified in France by the NRCVC. Six cases of septicemia, gastroenteritis or suppuration were caused by V. cholerae non-O1/non-O139, 2 cases of gastroenteritis and 1 case of soft tissue infection were caused by V. parahaemolyticus, and 2 cases of septicemia and soft tissue infection were caused by V. vulnificus.
The NRCVC is also responsible for the identification of non cholera vibrio strains isolated from seafood imported in France. In 2003, among 255 strains studied, V. cholerae, V. parahaemolyticus and V. alginolyticus were the most frequently identified species. All clinical or seafood isolates of V. vulnificus had the hly gene, indicating that all strains of this species are potentially pathogenic. Two of three V. parahaemolyticus strains isolated from human clinical cases had the tdh hemolysin gene. In contrast, a very low percentage (<1%) of strains of V. parahaemolyticus isolated from imported seafood had a hemolysin gene. None of the V. cholerae non-O1/non-O139 strains isolated from human or seafood samples possessed the cholera toxin genes.
The NRCVC has participated in post-graduate training programs by giving several lectures on cholera and other vibrios infections. It has also been involved in the training of microbiologists and clinicians. In this context, we have run courses on classical and molecular methods for the study of cholera and non cholera vibrios for microbiologists, technicians and students from several countries (France, Bangladesh, Democratic Republic of Congo, Madagascar, Mexico, Morocco). The NRCVC has also participated in national and international expert committees concerning cholera vaccination and risk assessment of Vibrio spp. in seafood.
Keywords: cholera, vibrios, vaccination, conjuguate, diagnosis, molecular epidemiology, seafood, public health