Unit: Cholera and Vibrios
Director: FOURNIER Jean-Michel
Cholera remains a major international health concern. In our Research Unit we are using several approaches to fight this scourge: (1) undertaking surveys and molecular epidemiology studies of cholera; (2) developing a rapid diagnostic test for cholera; (3) researching new, chemically defined conjugate vaccines. We are also actively engaged in the surveillance of infections due to non-cholera vibrios and the molecular characterization of these bacteria. The French Ministry of Health has designated our Research Unit the "National Reference Center for Vibrios and Cholera" because of our expertise in these particular fields.
1. Survey and molecular epidemiology of cholera (Marie-Laure QUILICI)
Cholera remains a major public and individual health problem. According to the WHO, the number of people susceptible to cholera due to socioeconomic problems has dramatically increased worldwide, creating favorable conditions for a global cholera crisis.
Cholera cases due to Vibrio cholerae O1 or O139 have been reported from all continents. France, like most developed countries, does not suffer directly from cholera epidemics. However, imported cases of cholera are regularly identified in travelers. Therefore, we collaborate closely with biologists from other countries in which cholera outbreaks occur, with members of the International Network of Pasteur Institutes and with non-governmental humanitarian organizations.
Analysis using molecular typing methods of 350 strains of cholera collected during a 30-year period since the arrival of cholera in Africa in 1970 has shown a high degree of genetic similarity among V. cholerae strains. These strains come from diverse geographical origins and have been isolated over a number of decades, and support evidence for the clonality of the seventh pandemic strains. The database that we built from the data from this study is now being actively used by us to to follow the evolution of cholera vibrio populations isolated in Africa and new cholera vibrio strains that may be imported into France. This year we have studied 114 V. cholerae O1 strains from eight African countries, in particular strains isolated from Cameroon and Senegal during the epidemics occurring there in2004. We have also studied more than 150 clinical V. cholerae O1 strains isolated in Mexico from 1991 to 2000.
2. Rapid Diagnostic Tests for cholera (Alain BOUTONNIER, Jean-Michel FOURNIER)
Our efforts in developing a conjugate vaccine that targets V. cholerae O1 and O139 has led to the development of monoclonal antibodies specific to the O1 or O139 lipopolysaccharides. 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, a rapid diagnostic test for V. cholerae O1 or O139. This diagnostic test is based on a one-step immunochromatographic technique and has been evaluated in Bangladesh and Madagascar, where it showed promising levels of sensitivity and specificity. The sensitivity and specificity of this test were evaluated in comparison with two other rapid diagnostic tests, at a diarrhea treatment center in Dhaka, Bangladesh. Our test showed the highest sensitivity of the three rapid tests evaluated, irrespective of the skill of the technician. However, these evaluations were carried out in sites having good research infrastructures. Therefore, our test was also evaluated in a setting with minimal facilities: the very places where rapid tests are most urgently needed. The International Vaccine Institute (Seoul, Korea), the WHO, and other health organizations, evaluated our test in a prospective study in Beira, Mozambique, during the 2004 cholera season. Our test performed well in the diagnostic of V. cholerae O1 in this urban setting typical of many areas where cholera is endemic.
3. 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 currently in the seventh pandemic, which began in India in 1961, invaded Africa in 1970 and spread to Latin America in 1991. The 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 definite indications that the incidence of V. cholerae O139 is rising in India and Bangladesh. Thus, this new cholera strain could well 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 less than five years of age.
We started our research by determining which antibodies protect against cholera. We have shown that immunoglobulin G (IgG) monoclonal antibodies directed against the polysaccharide moiety of the lipopolysaccharide of V. cholerae O1, expressing the serogroup or serotype determinants, are immunoprotective in an experimental model of cholera in neonatal mice. This 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, which should induce a long-lasting, thymus-dependent immune response. We have already prepared a conjugate composed of the polysaccharide of V. cholerae O139 and have shown that, in mice, this vaccine does indeed induce a protective thymus-dependent response.
This year, in preparing for a clinical evaluation of this V. cholerae O139 conjugate, we have continued to provide technical assistance to a pharmaceutical company concerning the production of a Good Manufactory Practice (GMP) batch of vaccine. For V. cholerae O1, we focused our efforts on the preparation of novel glycoconjugates, in which the immunogenicity of the antigenic determinant common to both the Ogawa and Inaba serotypes would be preserved. In particular, we exploited the presence of a unique free amino group carried by the D-glucosamine residue in the core of the lipopolysaccharide. We prepared a series of conjugates by varying both the sugar to protein ratio and the type and the length of the linker. Although the conjugates were antigenic, none was able to elicit a protective thymus-dependent response in mice. We have also continued investigating the structure of the antigenic determinant common to the Ogawa and Inaba serotypes of V. cholerae O1, using immunochemical and physicochemical methods. Determining this structure may allow us to rationally design a V. cholerae O1 conjugate.
4. Molecular characterization 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", which include isolates belonging to the serogroups O1 and O139 of Vibrio cholerae, and "non-cholera vibrios", which include (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, which include two species frequently isolated from human clinical samples: V. parahaemolyticus and V. vulnificus, and four other species less frequently isolated: V. alginolyticus, V. fluvialis, V. hollisae and V. mimicus.
Environmental and physiological modifications in certain coastal regions can provide vibrios with ideal conditions for proliferation. The development of international trade and increases in the consumption of raw or lightly cooked seafood, coupled with greater numbers of susceptible people, is causing concern that the incidence of infections in Europe may increase. Therefore, human vibrio infections are likely to spread and microbiological surveillance is needed to control public health risks.
The classic biochemical methods used for bacterial identification are not suitable for studying vibrio strains isolated from the environment. The increasing public health concerns emphasize the importance of developing molecular methods for the detection, identification and characterization of the pathogenic factors in vibrio strains isolated from humans, seawater or seafood. Therefore, we have been developing and using molecular methods for three Vibrio species of medical interest: V. parahaemolyticus, V. cholerae and V. vulnificus. Since 1996, the incidence of V. parahaemolyticus infections has increased dramatically in South-East Asia, Japan and North America. This increase appears to be related to the emergence of a new clone belonging to the O3:K6 serovar, which have pandemic potential.
The analysis, by PCR and molecular typing methods, including ribotyping, pulsed-field gel electrophoresis and arbitrarily primed PCR, of clinical isolates of V. parahaemolyticus collected in France between 1997 and 2004 showed, from 1997, the appearance of strains of the O3:K6 serovar which were indistinguishable from the O3:K6 clone isolated in Bangladesh in 1996 and were responsible for most outbreaks worldwide. Epidemiological studies showed that three patients had eaten local seafood harvested in uncontrolled areas, and moreover, had been harvested several years apart at the same place. This shows that pathogenic V. parahaemolyticus strains belonging to the new O3:K6 pandemic clone were introduced and are now present in the French coastal environment.
The detection of the newly emerged pathogenic V. parahaemolyticus O3:K6 in France, and previous results showing that pathogenic V. parahaemolyticus strains are present in French coastal areas at a higher frequency than usually reported in the literature, may provide an early warning to any possible pandemic. These results suggest that a long-term monitoring program should be started to detect pathogenic V. parahaemolyticus isolates present in French seafood and French 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, as in many other countries, cholera is a notifiable disease. Only a few cholera cases (<5) occur each year in France and most of them are imported. No cholera cases were identified in 2004. Moreover, as cholera epidemics do not respect national borders, we actively collaborate with biologists from other countries having cholera outbreaks and with non-governmental humanitarian organizations. As part of this collaboration, we have studied 114 V. cholerae O1 strains from eight African countries. These collaborations make it possible for us to study new cholera vibrio strains that may be imported into France.
The NRCVC is also responsible for identifying non-cholera vibrio strains from clinical laboratories. Human non-cholera vibrio infections can result in gastroenteritis, skin and soft tissue infections, septicemia, and other extraintestinal infections, such as ear infections. Patients with underlying immunosuppressive diseases are at high risk of rapid spread of infections due to these microorganisms. Most infections are associated with contact with seawater or the consumption of seafood, and their occurrence is correlated with the warmer months of the year.
In 2004, eight cases of human infections due to non-cholera vibrios were identified in France by the NRCVC. Four cases of septicemia and/or gastroenteritis were caused by V. cholerae non-O1/non-O139, and three cases of gastroenteritis and one case of septicemia were caused by V. parahaemolyticus. All of the V. parahaemolyticus strains had a hemolysin gene; however, none of the strains isolated in 2004 belonged to the new O3:K6 pandemic clone. None of the V. cholerae non-O1/non-O139 strains possessed the cholera toxin genes.
The NRCVC is also responsible for identifying non-cholera vibrio strains isolated from seafood imported into France. In 2004, among 198 strains studied, V. cholerae, V. parahaemolyticus and V. alginolyticus were the most frequently identified species. All seafood isolates of V. vulnificus had the hly gene, indicating that all strains of this species are potentially pathogenic. By 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 seafood samples possessed the cholera toxin genes.
The NRCVC has participated in post-graduate training programs and has given several lectures on cholera and other vibrios infections. It is also involved in the training of microbiologists and clinicians. We have run several 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, Cameroon, 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.
The size of vulnerable populations at risk for cholera is still increasing in many regions of the world where adequate sanitation is not available to all.
Keywords: cholera, vibrios, vaccination, conjugate, diagnosis, molecular epidemiology, seafood, public health