Genomic history of the seventh pandemic of cholera in Africa.

The seventh cholera pandemic has heavily affected Africa, although the origin and continental spread of the disease remain undefined. We used genomic data from 1070 Vibrio cholerae O1 isolates, across 45 African countries and over a 49-year period, to show that past epidemics were attributable to a single expanded lineage. This lineage was introduced at least 11 times since 1970, into two main regions, West Africa and East/Southern Africa, causing epidemics that lasted up to 28 years. The last five introductions into Africa, all from Asia, involved multidrug-resistant sublineages that replaced antibiotic-susceptible sublineages after 2000. This phylogenetic framework describes the periodicity of lineage introduction and the stable routes of cholera spread, which should inform the rational design of control measures for cholera in Africa.

Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of Vibrio cholerae O1, serotype Inaba.

A free amino group present on the acid-detoxified lipopolysaccharide (pmLPS) of V. cholerae O1 serotype Inaba was investigated for site-specific conjugation. Chemoselective pmLPS biotinylation afforded the corresponding mono-functionalized derivative, which retained antigenicity. Thus, pmLPS was bound to carrier proteins using thioether conjugation chemistry. Induction of an anti-LPS antibody (Ab) response in BALB/c mice was observed for all conjugates. Interestingly, the sera had vibriocidal activity against both Ogawa and Inaba strains opening the way to a possible bivalent vaccine. However, the level of this Ab response was strongly affected by both the nature of the linker and of the carrier. Furthermore, no switch from IgM to IgG, i.e. from a T cell-independent to a T cell-dependent immune response was detected, a result tentatively explained by the possible presence of free polysaccharide in the formulation. Taken together, these results encourage further investigation towards the development of potent pmLPS-based neoglycoconjugate immunogens, fully aware of the challenge faced in the development of a cholera vaccine that will provide efficient serogroup coverage.

A cluster of Vibrio cholerae O1 infections in French travelers to Rajasthan (India), May 2006.

A woman aged 60 years was hospitalized for Vibrio cholerae serogroup O1 cholera. Twenty-six fellow travelers and 48 health care workers who cared for the patient were individually traced and contacted. Of the 23/27 travelers with diarrhea during the trip, 4 presented antibodies. There was no person-to-person transmission.

Retrospective analysis of the cholera cases imported to France from 1973 to 2005.

BACKGROUND: The manners of traveling and travelers' vulnerability to infection are changing: increasing numbers of travelers, travels at the extreme ages of life, "backpacker" tourism in close contact with local populations. What is the epidemiologic situation and what are the trends of imported cholera to Metropolitan France? METHOD: A descriptive retrospective study was undertaken on all the confirmed cases of cholera imported to France, and notified from January 1, 1973, to December 31, 2005, using compulsory notification data from local health departments and information from the National Reference Centre. RESULTS: A total of 129 imported cases of cholera were notified between 1973 and 2005 (3.9 cases/y on average). The geographical sources of infection have changed with time: in the 1980s, 94% of the patients were infected in Maghreb (Morocco and Algeria) but none were in 2000. On the other hand, Asia and West Africa progressively emerged and now predominate. In spite of certain poorly informed data and possible underdetection, the number of cases of importation appears to be low and falling. CONCLUSIONS: The patient profile seems to have evolved and increasingly concerns people at the extreme ages of life, living elsewhere than the principal basins of immigration in France, and diagnosis is increasingly made in nonteaching hospitals. The lessons likely to help clinicians will be discussed.

[Cholera]. / Choléra.

Cholera is an acute intestinal infection that has reached pandemic proportions and presents a major international health concern. Every year, more than 100000 cholera cases and 2000-3000 deaths are officially reported to WHO. The real figures for cholera are thought to be much higher, however, due to underreporting and other limitations of surveillance systems. Cholera is caused by two serogroups (O1 and O139) of a gram-negative bacterium, Vibrio cholerae. Cholera toxins cause a massive outpouring of electrolyte-rich isotonic fluid into the bowel and can lead to volume depletion and shock. In poor sanitary and individual hygiene conditions, the massive release of cholera vibrios into the environment intensifies and exacerbates cholera epidemics, which thus serve as clear markers of poverty and lack of basic sanitation. Rehydration therapy, either intravenous or oral, considerably decreases the number of deaths. The WHO recommends antibiotics for cholera cases with severe dehydration. If left untreated, cholera has a 25-50% mortality rate. Treatment reduces this to less than 1%. Bacteriological diagnosis of cholera is reasonably easy because cholera bacteria are abundant in stool. Epidemics, however, often occur in areas with either limited or no laboratory facilities. A rapid and accurate diagnosis of cholera is essential to mobilize resources for treatment and containment of the epidemic. Therefore, the Pasteur Institute has developed a rapid diagnostic test based on a one-step immunochromatographic technique, which should be commercially available soon. To date, two oral cholera vaccines have been shown to offer good (more than 70%) short-term (one year) protection. WHO recommends these vaccines as an additional public health tool to be implemented with the standard cholera control measures, including provision of safe drinking water and adequate sanitation. Nonetheless, a cholera vaccine that can offer a long-term protection for all age groups, including children younger than five years, is still needed.

Field evaluation of a rapid immunochromatographic dipstick test for the diagnosis of cholera in a high-risk population.

BACKGROUND: Early detection of cholera outbreaks is crucial for the implementation of the most appropriate control strategies. METHODS: The performance of an immunochromatographic dipstick test (Institute Pasteur, Paris, France) specific for Vibrio cholerae O1 was evaluated in a prospective study in Beira, Mozambique, during the 2004 cholera season (January-May). Fecal specimens were collected from 391 patients with acute watery nonbloody diarrhea and tested by dipstick and conventional culture. RESULTS: The overall sensitivity and specificity of the rapid test compared to culture were 95% (95% confidence interval [CI]: 91%-99%) and 89% (95% CI: 86%-93%), respectively. After stratification by type of sample (rectal swab/bulk stool) and severity of diarrhea, the sensitivity ranged between 85% and 98% and specificity between 77% and 97%. CONCLUSION: This one-step dipstick test performed well in the diagnosis of V. cholerae O1 in a setting with seasonal outbreaks where rapid tests are most urgently needed.

Evaluation of three rapid diagnostic tests for cholera: does the skill level of the technician matter?

OBJECTIVE: To evaluate SMART, Medicos Dip Stick and an Institut Pasteur (IP) cholera dipstick tests for accuracy and ease of use. METHOD: Every 50th patient presenting with diarrhoea at ICDDR,B between 1 April 2003 and 30 November 2003 was enrolled. The rapid diagnostic tests were performed by field and laboratory technicians, and sensitivity (Se), specificity (Sp), positive (PPV) and negative (NPV) predictive values calculated. RESULTS: We isolated Vibrio cholerae O1 from 116 (38%) of 304 patients. The Se, Sp, PPV and NPV of the SMART test were 58%, 95%, 84% and 84% for field technicians, and 83%, 88%, 83% and 88% for laboratory technicians. The Se, Sp, PPV and NPV of the IP dipstick test were 93%, 67%, 63% and 94% for field technicians, and 94%, 76%, 70% and 95% for laboratory technicians. The Se, Sp, PPV and NPV of the Medicos test were 84%, 79%, 71% and 90% for field technicians, and 88%, 80%, 72% and 92% for laboratory technicians. A high proportion of indeterminates (30%) hampered the performance of the SMART test. The IP dipstick had the highest Se, irrespective of technician skill level. CONCLUSION: The IP dipstick is the most appropriate rapid diagnostic assay for the detection of V. cholerae O1 in locations where the skill level of personnel may be low, such as remote areas or refugee camp settings. High cost may limit the utility of any diagnostic test in the developing world.

On the preparation of carbohydrate-protein conjugates using the traceless Staudinger ligation.

[reaction: see text] The nature of a linker used for preparing glycoconjugate vaccines is of utmost importance as it may lead to immunogenic biomolecules. We report the conjugation of carbohydrate haptens to protein carriers leading to potential vaccines using the traceless Staudinger ligation. The ligation relies on the selective transfer of a phosphane substituent to an azide to form a native amide bond in the final product upon release of an oxidized phosphane byproduct. We designed new phosphino-functionalized cross-linkers suitable for protein carrier derivatization. We evaluated their utility in preparing conjugates using both synthetic and purified bacterial carbohydrates. The use of a borane-protected phosphane which is deprotected at the time of the ligation reaction led to the best results observed thus far in terms of stability toward oxidation and reactivity.

Occurrence of the tdh and trh genes in Vibrio parahaemolyticus isolates from waters and raw shellfish collected in two French coastal areas and from seafood imported into France.

The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected in two French coastal areas, clinical samples, and seafood products imported into France was studied. Polymerase chain reaction (PCR) with two sets of primers was used to detect the hemolysin genes. Most of the clinical isolates (91%) and 1.5% of the isolates from seafood possessed the hemolysin genes. Three and fifteen percent, respectively, of the two groups of environmental strains carried the hemolysin genes depending on the geographic site. The tdh and trh genes play important roles in virulence. Thus, our results indicate that pathogenic V. parahaemolyticus isolates are present in French coastal areas and in seafood imported into France. Furthermore, they may also be present in French seafood products.

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139.

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.

The binding of synthetic analogs of the upstream, terminal residue of the O-polysaccharides (O-PS) of Vibrio cholerae O:1 serotypes Ogawa and Inaba to two murine monoclonal antibodies (MAbs) specific for the Ogawa lipopolysaccharide (LPS).

The binding of nineteen analogues of the upstream, terminal, monosaccharide residue of each of the O-polysaccharide (O-PS) of Vibrio cholerae O:1, serotype Ogawa and Inaba, with two murine monoclonal IgG antibodies both specific for the Ogawa LPS were measured using fluorescence spectroscopy. The use of the deoxy and the deoxyfluoro analogs allowed further refinement of the hydrogen-bonding pattern involved in the binding. Based on the binding characteristics observed for some of the ligands in the Inaba series, the binding of the monosaccharide that represents the upstream, terminal unit of the O-PS of V. cholerae O:1 serotype Inaba was redefined. We show for the first time that the upstream, terminal monosaccharide of the Inaba O-PS shows weak binding with these two anti-Ogawa antibodies. The results obtained allow further rationalization of the structural basis for the binding of V. cholerae O:1 antigens to their homologous antibodies.

Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA-DNA hybridization.

We compared the efficiencies of biochemical methods and polymerase chain reaction (PCR) for the identification of Vibrio parahaemolyticus strains. The 122 isolates studied, identified by biochemical tests as V. parahaemolyticus or Vibrio alginolyticus, were tested by R72H PCR assay. The results obtained with the two methods were consistent for 90% of the strains studied. PCR amplification of the R72H fragment generated two unique amplicons, 387 bp and 320 bp in length. For 11% of the strains from seawater, the results of biochemical identification did not correlate with PCR results. DNA-DNA hybridization experiments provided evidence that some strains identified as V. alginolyticus in biochemical tests should be considered members of the V. parahaemolyticus species. We therefore suggest that biochemical tests are not accurate enough for the identification of V. parahaemolyticus isolates and we demonstrate that amplification of the R72H fragment, whether the amplicon is 320 bp or 387 bp long, is a powerful tool for the reliable identification of V. parahaemolyticus.

Induction of protective immunity by synthetic Vibrio cholerae hexasaccharide derived from V. cholerae O1 Ogawa lipopolysaccharide bound to a protein carrier.

Synthetic antigens that mimic the terminal hexasaccharide epitope of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, were conjugated to bovine serum albumin (BSA). Conjugates with carbohydrate-to-carrier molar ratios of 15.5:1, 9.2:1, and 4.6:1 were tested for immunogenicity and efficacy in mice. The role of preimmunity to BSA and the use of adjuvant in the generation of the serologic response to the O-specific polysaccharide and protection against virulent V. cholerae was examined. Preimmunity to BSA did not affect the anti-Ogawa titers but seemed to enhance the protective capacity of antiserum. All 3 conjugates were immunogenic, but adjuvant was effective at inducing higher and earlier antibody responses. In tertiary serum samples, a correlation was found between vibriocidal activity and protection. The protective capacity of antiserum was evident in serum from mice immunized with all conjugates, but it was highest in the groups that received the conjugate with the lowest level of substitution. Further studies are required to increase understanding of the reason for differential protection.

Improved specific detection of Vibrio cholerae in environmental water samples by culture on selective medium and colony hybridization assay with an oligonucleotide probe.

We developed a rapid and efficient method based on culture on selective medium and colony hybridization assay for the detection of Vibrio cholerae in estuarine water samples. A 22-oligonucleotide sequence of the 16S-23S rDNA intergenic spacer region was labeled with digoxigenin and evaluated for specificity and sensitivity by dot blot and colony hybridization with collection strains and environmental and clinical isolates. No isolates of species other than V. cholerae hybridized with the oligonucleotide probe. Colony hybridization was then performed with mixed microbial populations from brackish and sea water samples isolated, after an enrichment step, on selective culture media. Plating on alkaline nutrient agar without added NaCl (modified alkaline nutrient agar, mANA) resulted in higher V. cholerae colony counts than did plating on other frequently used selective media, and favored direct detection by colony hybridization. The combined use of mANA agar and an oligonucleotide probe resulted in the specific recovery of V. cholerae and could be used for confirmation of the most-probable-number procedure usually used to count this bacterium in environmental samples.

Immunochemical characterization of an Ogawa-Inaba common antigenic determinant of Vibrio cholerae O1.

Cholera remains an important public health problem in many parts of the world and the availability of an effective cholera vaccine is important for the prevention of cholera in the countries affected by this disease. Despite the appearance in 1992 of a new serogroup, 0139, of Vibrio cholerae, most of the cholera outbreaks are still caused by V. cholerae O1 biotype El Tor. Vaccine trials in Asia from 1968 to 1971, and studies of the production of serotype-specific antiserum in rabbits and of the protective activity of monoclonal antibodies against diarrhoeal disease in neonatal mice, have led to the conclusion that the Ogawa serotype contains a specific antigenic determinant whereas the Inaba serotype contains a different antigenic determinant that cross-reacts with the Ogawa serotype. By studying the binding of anti-Ogawa monoclonal antibodies to synthetic oligosaccharide fragments mimicking the Ogawa O-specific polysaccharide, it has been shown that the terminal monosaccharide, bearing the 2-O-methyl group in the O-specific polysaccharide, is most probably the serotype-specific determinant for the Ogawa strain. However, study of the binding of a monoclonal antibody recognizing both Ogawa and Inaba serotypes suggested partial recognition of the core as well as of the O-specific polysaccharide of the LPS of V. cholerae O1. To further characterize this antigenic determinant that is common to the Ogawa and Inaba serotypes, the core and the O-specific polysaccharide linked to the core of V. cholerae O1 LPS were purified by preparative electrophoresis. The O-specific polysaccharide linked to the core was subjected to periodate oxidation to destroy sugars from the core. Binding studies of these purified saccharide fragments to a monoclonal antibody which is protective in mice and specific to the antigenic determinant common to Ogawa and Inaba serotypes showed that both the core and the O-specific polysaccharide are involved in this common antigenic determinant. This explains how the presence or the absence of the Ogawa-specific antigenic determinant would lead to the expression of two independent antigenic determinants of V. cholerae O1, one specific to the Ogawa serotype and the other common to both Ogawa and Inaba serotypes.