[Cases of imported cholera in France, summer 2005. A. Tarantola for the incident management teams]. / Cas de choléra d'importation en France, été 2005. A. Tarantola pour les groupes de gestion des incidents.

Cholera is a bacterial infection, which causes digestive symptoms and massive diarrhoea. It may lead to dehydration and death if appropriate medical management is not rapidly initiated. Most cases of infection by choleric vibrio, however, remain symptom-free or may mimic common gastroenteritis. A review of two cases of imported cholera in France in the summer of 2005 and the community- and hospital-based investigation, which they triggered, enabled the incident management teams to assess risks of transmission. There were no secondary cases among 58 hospital contacts and 15 family contacts of the cases. Clinicians will find a discussion of possible clinical presentations and the risk of secondary transmission, in the context of progressing epidemics in countries, which have maintained close ties with France.

Use of dipsticks for rapid diagnosis of cholera caused by Vibrio cholerae O1 and O139 from rectal swabs.

We evaluated the recently developed dipsticks for the rapid detection of Vibrio cholerae serotypes O1 and O139 from rectal swabs of hospitalized diarrheal patients after enrichment for 4 h in alkaline peptone water. The sensitivity and specificity of the dipsticks were above 92 and 91%, respectively. The dipsticks represent the first rapid test which has been successfully used to diagnose cholera from rectal swabs, and this would immensely improve surveillance for cholera, especially in remote settings.

[Vibrio vulnificus septicemia transmitted through a wound caused by a crustacea]. / Septicémie à Vibrio vulnificus secondaire à une blessure par écrevisse.

INTRODUCTION: Vibrio vulnificus proliferates during the summer in salt water where it infects the crustaceans. Expression of its pathogenicity depends on the underlying condition and mode of contamination. OBSERVATION: A 65 year-old man presented with a Vibrio vulnificus septicaemia of cutaneous origin, transmitted when he cut himself with a crawfish. The severity of the infection was enhanced by severe immuno-depression and haemochromatosis. The infection regressed with appropriate antibiotherapy. COMMENTS: Severe V. vulnificus infections are rare. Depending on the underlying condition and mode of contamination, one can distinguish between benign gastro-enteritis, local occasionally devastating infections and usually fatal septicaemia. CONCLUSION: Even the most severe forms of V. vulnificus infections may be cured with early and well adapted anti-infectious treatment.

One-step immunochromatographic dipstick tests for rapid detection of Vibrio cholerae O1 and O139 in stool samples.

We describe the development and evaluation of a rapid diagnostic test for Vibrio cholerae O1 and O139 based on lipopolysaccharide detection using gold particles. The specificity ranged between 84 and 100%. The sensitivity of the dipsticks ranged from 94.2 to 100% when evaluated with stool samples obtained in Madagascar and Bangladesh. The dipstick can provide a simple tool for epidemiological surveys.

Occurrence of pathogenic vibrios in coastal areas of France.

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.

Regulation of Staphylococcus aureus type 5 and type 8 capsular polysaccharides by CO(2).

Staphylococcus aureus expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO(2). Here we show that CO(2) reduces CP5 expression at the transcriptional level and that CO(2) regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO(2) caused a significant decrease in CP8 expression in four S. aureus strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine S. aureus strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of cap5 and cap8. To test whether these sequence variations are responsible for the different responses to CO(2), promoter regions from selected strains were fused to the reporter gene xylE in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO(2) in all derivatives of strain Newman and was always positively regulated by CO(2) in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO(2) response. trans-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive S. aureus strains.

[Isolation of Vibrio strains in French coastal waters and infection with Vibrio cholerae non-O1/non-O139]. / Isolement de vibrions dans les eaux côtières françaises et infection à Vibrio cholerae non-O1/non-O139.

OBJECTIVE: Although the incidence in France of V. cholerae non-O1/non-O139 infection in man has increased since 1996, it remains low (7 cases in 1999). After the death in 1994 of an immunodepressed patient presenting a skin lesion showing superinfection by a strain of non-O1/non-O139 V. cholerae following exposure to seawater, we examined 22 samples of sea-water collected from 20 French coastal areas (Mediterranean coast). METHODS: The sea-water samples were filtered and enriched with alkaline peptone water (APW), and the strains of Vibrio were isolated on TCBS, SS and BCP media and identified using the API 20 E system (bioMérieux, France). RESULTS: We isolated 6 strains belonging to 3 species of Vibrio: 2 V. cholerae (non-O1/non-O139), 3 V. parahaemolyticus and 1 V. alginolyticus. One of the V. cholerae strains was isolated from sea-water sampled at the coastal town in which the patient had been staying. The seawater strains exhibited high sensitivity (MIC determined by agar dilution) to the following antibiotics: aminoglycosides, tetracyclines, azithromycin, cotrimoxazole, rifampicin and fluoroquinolones. The beta-lactams were very active against strains of V. cholerae isolated from seawater, while the strain isolated from this patient presented a new carbenicillinase (CARB-6) recently described. CONCLUSION: The presence of Vibrio in seawater along the French coast-line constitutes a risk for immunocompromised patients, and the severity of Vibrio infections warrants improved monitoring both of these organisms and of the marine environment. In addition, awareness on the part of doctors would allow patients at risk to be warned against these dangers.

Preparation, immunogenicity, and protective efficacy, in a murine model, of a conjugate vaccine composed of the polysaccharide moiety of the lipopolysaccharide of Vibrio cholerae O139 bound to tetanus toxoid.

The epidemic and pandemic potential of Vibrio cholerae O139 is such that a vaccine against this newly emerged serogroup of V. cholerae is required. A conjugate made of the polysaccharide moiety (O-specific polysaccharide plus core) of the lipopolysaccharide (LPS) of V. cholerae O139 (pmLPS) was prepared by derivatization of the pmLPS with adipic acid dihydrazide and coupling to tetanus toxoid (TT) by carbodiimide-mediated condensation. The immunologic properties of the conjugate were tested using BALB/c mice injected subcutaneously three times at 2 weeks interval and then a fourth time 4 weeks later. Mice were bled 7 days after each injection and then once each month for the following 6 months. LPS and TT antibody levels were determined by enzyme-linked immunosorbent assay using immunoplates coated with either O139 LPS or TT. Both pmLPS and pmLPS-TT conjugate elicited low levels of immunoglobulin M (IgM), peaking 5 weeks after the first immunization. The conjugate elicited high levels of IgG antibodies, peaking 3 months after the first immunization and declining slowly during the following 5 months. TT alone, or as a component of conjugate, induced mostly IgG antibodies. Antibodies elicited by the conjugate recognized both capsular polysaccharide and LPS from V. cholerae O139 and were vibriocidal. They were also protective in the neonatal mouse model of cholera infection. The conjugation of the O139 pmLPS, therefore, enhanced its immunogenicity and conferred T-dependent properties to this polysaccharide.

Glucose-induced activation of rubidium transport and water flux in sunflower root systems.

Excised 20-d-old sunflower roots (Helianthus annuus L. cv. Sun-Gro 393) were used to study the effect of different sugars on rubidium and water fluxes. The roots sensed and absorbed glucose from the external medium inducing the activation of rubidium accumulated in the root (Rb(+) root), the flux of exuded rubidium (J(Rb)) and, to a lesser degree, the exudation rate (J(v)). These effects were also triggered by fructose, but not by 6-deoxyglucose (6-dG), a glucose analogue which is not a substrate for hexokinase (HXK). The effect of 2-deoxyglucose (2-dG), an analogue that is phosphorylated but not further metabolized, was complex, suggesting an inhibitory effect on solute transport to the xylem. The amounts of glucose required to activate rubidium and water fluxes were similar to those previously reported to regulate different processes in other plants (0.5--10 mM). When sorbitol was used instead of glucose, neither rubidium uptake (Rb(+) root plus J(Rb)) nor J(v) was activated. It is proposed that glucose present in the root plays an important signalling role in the regulation of Rb(+) (K(+)) and water transport in plant roots.

Adherence of Staphylococcus aureus to endothelial cells: influence of capsular polysaccharide, global regulator agr, and bacterial growth phase.

The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase on S. aureus adherence to EC. Whereas S. aureus Newman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in the agr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC. S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P<0.005). Complementation of the cap5O mutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant and cap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: molecular basis for serotype specificity.

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab-Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Nonlinear identification of NMR spin systems by adaptive filtering.

In this paper, we present two new methods for identifying NMR spin systems. These methods are based on nonlinear adaptive filtering. The spin system is assumed to be time-invariant with memory. The first method uses a truncated discrete Volterra series to describe the nonlinear relationship between excitation (input) and system response (output). First-, second-, and third-order kernels of this series are estimated employing the least mean square (LMS) algorithm. Three parallel filters can then model the NMR spin system so that its output is no more than simple sum of three convolution products between combinations of the input signal and filters coefficients. It is also shown that the contribution of the Volterra second-order term to the total system response is neglected compared with the contributions of the first- and the third-order terms. In the second identification method, the output signal is related to the input signal through a recursive nonlinear difference equation with constant coefficients. The LMS algorithm is used again to estimate the equation coefficients. The two methods are validated with a simulated NMR system model based on Bloch equations. The results and the performances of these methods are analyzed and compared. It is shown that our methods permit a simple identification of NMR spin systems. The field of applications of this study is promising in the optimization of NMR signal detection, especially in the cases of low signal-to-noise ratios where optimum signal filtering and analysis must be performed.

Genetic analysis of the cap5 locus of Staphylococcus aureus.

Staphylococcus aureus expresses at least eight distinct serotypes of capsular polysaccharide (CP). Gene clusters involved in the expression of serotypes 1, 5 and 8 have been cloned and sequenced. In this report we describe the isolation and analysis of serotype 5 capsular polysaccharide-defective mutants. A naturally occurring cap mutation in the laboratory strains 8325-4 and RN4220 was mapped to the cap5E gene by genetic complementation. The cap5H-K genes were shown to be responsible for CP5 serotype specificity by transduction and complementation.

[Cholera update and vaccination problems]. / Actualité du choléra et problématique vaccinale.

Cholera remains an important public health problem. The long-term control of cholera depends on good personal hygiene, uncontaminated water supply and appropriate sewage disposal. However, the improvement of hygiene is distant goal for many countries. Thus the availability of an effective cholera vaccine is important for the prevention of cholera in these countries. Research on new cholera vaccines has mainly focused on oral formulations that stimulate the mucosal secretory immune system. Two oral cholera vaccines were experimented on large scale in human. The first vaccine, containing inactivated bacterial cells and B-subunit of cholera toxin, has been tested in Bangladesh from 1985 to 1989. This vaccine, according to WHO, may prove useful in the stable phase of refugee/displaced person crises, especially when given preventively. The second vaccine is a live attenuated vaccine containing the genetically manipulated Vibrio cholerae O1 strain CVD 103-HgR. Despite its efficacy in adult volunteers, results of a large-scale field trial carried-out in Indonesia for 4 years have shown a surprisingly low protection. Moreover, one of the safety concerns associated with live cholera vaccine is a possible horizontal gene transfer and recombination event leading to reversion to virulence. A new vaccine development program for cholera is based upon the hypothesis that immunoglobulins G directed to the O-specific polysaccharide of Vibrio cholerae O1 could confer protective immunity to cholera by inactivating the inoculum on intestinal mucosal surface. This program may lead to the development of cholera conjugate vaccines to elicit protection in infants.

[Cholera in a Paris resident with no history of travel. A case report]. / Choléra autochtone à Paris. Une observation.

BACKGROUND: Cases of imported cholera are frequently observed, but cholera almost never occurs in subjects who have never travelled to an endemic area. In the last 30 years, 4 cases have been reported. We report an indigenous case diagnosed in Paris in September 1996. CASE REPORT: The patient was hospitalized for severe dehydration and acute renal failure. Cultures of a fecal specimen grew Vibrio cholerae O1 Ogawa serotype. An epidemiological study was conducted to identify the vehicle and mode of contamination and suggested that this case was associated with the consumption of fresh sorrel imported from West Africa. No other cases were identified in contacts of the patient. DISCUSSION: Asymptomatic carriage of V. cholerae is rare. However, air travel has allowed people to arrive in non-endemic areas during the incubation period. The agent may also be transported in contaminated foods. Cholera should be suspected in all adults presenting acute watery diarrhea with severe dehydration. History taking should also look for risk exposure and these patients should be systematically asked about possible exposures.

On the antigenic determinants of the lipopolysaccharides of Vibrio cholerae O:1, serotypes Ogawa and Inaba.

Monoclonal, murine IgG1s S-20-4, A-20-6, and IgA 2D6, directed against Vibrio cholerae O:1 Ogawa-lipopolysaccharide exhibited the same fine specificities and similar affinities for the synthetic methyl alpha-glycosides of the (oligo)saccharide fragments mimicking the Ogawa O-polysaccharide (O-PS). They did not react with the corresponding synthetic fragments of Inaba O-PS. IgG1s S-20-4 and A-20-6 have absolute affinity constants for synthetic Ogawa mono- to hexasaccharides of from approximately 10(5) to approximately 10(6) M-1. For IgG1s S-20-4, A-20-6, and IgA 2D6, the nonreducing terminal residue of Ogawa O-PS is the dominant determinant, accounting for approximately 90% of the maximal binding energy shown by these antibodies. Binding studies of derivatives of the Ogawa monosaccharide and IgGs S-20-4 and A-20-6 revealed that the C-2 O-methyl group fits into a somewhat flexible antibody cavity and that hydrogen bonds involving the oxygen and, respectively, the OH at the 2- and 3-position of the sugar moiety as well as the 2'-position in the amide side chain are required. Monoclonal IgA ZAC-3 and IgG3 I-24-2 are specific for V. cholerae O:1 serotypes Ogawa/Inaba-LPS.1 The former did not show binding with members of either series of the synthetic ligands related to the O-antigens of the Ogawa or Inaba serotypes, in agreement with its reported specificity for the lipid/core region (1). Inhibition studies revealed that the binding of purified IgG3 I-24-2 to Ogawa-LPS might be mediated by a region in the junction of the OPS to the lipid-core region of the LPS. cDNA cloning and analysis of the anti-Ogawa antibodies S-20-4, A-20-6, and 2D6 revealed a very high degree of homology among the heavy chains. Among the light chains, no such homology between S-20-4 and A-20-6 on the one hand, and 2D6 on the other hand, exists. For the anti-Inaba/Ogawa antibodies I-24-2 and ZAC-3, their heavy chains are completely different, with some homology among the light chains.

[The current status of research on a cholera vaccine]. / Etat actuel des recherches sur le vaccin anticholérique.

Cholera remains today a major health problem in most developing countries. The long-term control of cholera depends on the improvement of hygiene but this is a distant goal for many countries. The availability of an effective cholera vaccine is thus important for the prevention of cholera in such countries. More than a century after the first attempt to vaccinate against cholera by Ferran in Spain, there is still no truly effective cholera vaccine. A bacterial fraction vaccine, referred to as CH1 +2 was prepared by Professor A. Dodin. A field trial of this vaccine was carried out in Zaire in 1983. Significant protection was observed but this vaccine was not evaluated in additional trials. Two other oral cholera vaccines, developed in Sweden and in the USA, were widely experimented on human beings: a combination of cholera toxin B-subunit and inactivated bacterial cells, and a live attenuated vaccine containing the genetically manipulated Vibrio cholerae O1 strain CVD 103-HgR. Despite their efficiency as evaluated in field trials (inactivated vaccine) or on volunteers (live vaccine), these vaccines have drawbacks that may limit their usefulness as practical vaccines. Protection induced by the inactivated vaccine was transient in young children, lasting only approximately for six months. One of the safety concerns associated with live vaccines is a possible reversion to virulence. Efforts should be continued to find a better cholera vaccine. A new vaccine development program based upon the hypothesis that immunoglobulin G directed to the O-specific polysaccharide of Vibrio cholerae O1 could confer protective immunity to cholera. This program may lead to the development of a cholera conjugate vaccine to elicit protection in infants.