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.

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.

Regulation of Staphylococcus aureus capsular polysaccharide type 5: CO2 inhibition in vitro and in vivo.

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats, and in vitro using ELISA and IFA. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1%-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1%-5% of CP5-positive cells were detectable in pouch exudates. CF and pouch isolates, however, reexpressed CP5 (70%-90% of cells) when grown in vitro with air. Addition of > or = 1% CO2 to air or to O2/N2 gas mixtures reduced CP5 expression significantly (P < .001) in a dose-dependent manner (6%-1% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal that regulates CP5 expression.

[Regulation of Staphylococcus aureus capsular polysaccharide type 5: in vitro and in vivo inhibition by CO2]. / Regulation des Staphylococcus aureus-Kapselpolysaccharids vom Typ 5: Hemmung durch CO2 in vitro und in vivo.

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats and in vitro using ELISA and immunofluorescence. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1-5% CP5-positive cells were detectable in pouch exsudates. CF and pouch isolates, however, re-expressed CP5 (70-90% of cells) when grown in vitro with air. Addition of 1% CO2 or more to air or to O2/N2 gas mixtures reduced CP5 expression significantly (p < 0.001) in a dose-dependent manner (1-6% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal which regulates CP5 expression.

Epidemiology of Staphylococcus aureus in patients with cystic fibrosis.

Seven hundred and thirty-four isolates of Staphylococcus aureus, recovered from the sputum of 238 cystic fibrosis patients in six French hospitals, were characterized by esterase electrophoretic typing, capsular polysaccharide serotyping and phage typing and tested against 14 antibiotics for sensitivity. Thirty-four esterase electrophoretic types were found with a genotypic diversity coefficient of 0.91. Five hundred and forty-eight (78.7%) isolates produced capsular polysaccharide and 350 (50.3%) were type 8. Four hundred and sixty isolates (66.6%) were phage typable and 202 (28.2%) were lysed by group III bacteriophages. No esterase electrophoretic type, capsular type or phage type was specific to cystic fibrosis. Isolates belonged to a wide range of types, similar to strains acquired outside hospitals. Eighty-five patients had three or more consecutive isolates over at least 6 months. The ability of S. aureus to persist for long periods of time has been demonstrated in 73% of them. Methicillin-resistance was encountered among 73 strains (9.8%) which were also multiresistant. Two hundred and eighty-nine (39.9%) strains were sensitive to all antibiotics tested except to penicillin. Pristinamycin and co-trimoxazole were the most effective antibiotics. These results could contribute to the elaboration of a rational approach to the prophylaxis and therapy of respiratory staphylococcal infections in cystic fibrosis patients.

New latex reagent using monoclonal antibodies to capsular polysaccharide for reliable identification of both oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus.

A new latex agglutination test (Pastorex Staph-Plus, Sanofi Diagnostics Pasteur), consisting of a mixture of latex particles coated with fibrinogen and immunoglobulin G for the detection of clumping factor and protein A and latex particles sensitized with monoclonal antibodies directed to Staphylococcus aureus serotype 5 and 8 capsular polysaccharides, was compared with three commercially available rapid agglutination methods for the identification of 220 isolates of S. aureus (61 oxacillin resistant) and 128 isolates of coagulase-negative staphylococci. The sensitivity for identification of S. aureus was high with the Pastorex Staph-Plus test (98.6%) compared with those of the other tests, which ranged from 91.8 to 84.5%. Test sensitivities for the identification of oxacillin-resistant S. aureus were as follows: Pastorex Staph-Plus, 95.1%; Pastorex Staph, 73.8%; Staphyslide, 72.1%; and StaphAurex, 49.2%.

Typing of Staphylococcus aureus by pulsed-field gel electrophoresis, zymotyping, capsular typing, and phage typing: resolution of clonal relationships.

Sixty-nine Staphylococcus aureus isolates from two epidemiologically unrelated sources were typed by pulsed-field gel electrophoresis after SmaI digestion of chromosomal DNA (genome typing), and the results were compared with those obtained by other typing methods: phage typing with the international set of phages, capsular serotyping with monoclonal antibodies against capsular polysaccharides type 5 and 8, and zymotyping by polyacrylamide agarose electrophoresis for esterase polymorphism. A good correlation of S. aureus types was found by these four typing methods. Differentiation increased in the order capsular typing < zymotyping < phage typing < genome typing, yielding 2, 10, 20, and 26 different S. aureus types, respectively. Five of the 26 genome types were further divided into several subtypes revealing clonal relationships. When 36 French S. aureus isolates were compared with 33 German S. aureus isolates, 3 strains representing clonal populations were identical in both groups. S. aureus isolates from patients with cystic fibrosis were also typed at the beginning and the end of a 4-week summer camp for these patients. The results suggested a possible strain transmission during the summer camp. We conclude that genome typing by pulsed-field gel electrophoresis is a powerful tool not only for strain identification but also for the resolution of the clonal relationships of S. aureus strains.

Staphylococcal skin colonization in children with atopic dermatitis: prevalence, persistence, and transmission of toxigenic and nontoxigenic strains.

Staphylococcal skin colonization is a common feature of atopic dermatitis (AD) in adults. Little is known about prevalence and persistence of staphylococci in children. Forty-one AD children (mean age, 70 months) and 41 age-matched controls were studied. S. aureus was isolated from 38 AD patients (93%; 32% of controls, P less than .001) and 37% of AD patients (5% of controls, P less than .001) harbored toxigenic (enterotoxins, toxic shock syndrome toxin) S. aureus strains. No individual biotype prevailed. On follow-up (mean interval, 9 months), 70% of S. aureus strains were reisolated. Nasal and cutaneous S. aureus strains were identical in 73% of AD patients (7% of controls, P less than .001), reflecting increased self-contamination. Identical staphylococcal strains in AD children and their mothers were observed in 38% (S. aureus) and 16% (coagulase-negative strains; P less than .001). The prevalence of staphylococcal colonization in AD children is comparable to that in adults. High rates of self-contamination, transmission to contacts, and prevalence of toxigenic strains in AD children may have clinical and epidemiologic implications.

Diagnosing Staphylococcus aureus endocarditis by detecting antibodies against S. aureus capsular polysaccharide types 5 and 8.

Consecutive serum samples from patients with Staphylococcus aureus endocarditis or septicemia or non-S. aureus endocarditis and febrile nonsepticemic controls were tested for antibodies against S. aureus capsular polysaccharide (CP) types 5 and 8 by ELISA. The upper normal antibody levels were defined as the upper 99.5% confidence limits of the values from the febrile controls. All available patient isolates were tested for the presence of CP type 5 or 8 (85% of the isolates expressed either serotype), and all five patients with S. aureus endocarditis had positive antibody levels against the corresponding serotype within the first 10 days of infection. Three other endocarditis patients lacked isolates for CP testing but two of these were positive. Positive antibody levels were found in 0 of 28 septicemia patients, in 1 of 12 non-S. aureus endocarditis patients, and in 3 of 37 febrile controls. Thus, testing for anti-CP 5 or 8 antibodies, especially together with CP serotyping of the patient's isolate, seems to provide important information in the differential diagnosis of endocarditis in patients with S. aureus septicemia.

Correlation between esterase electrophoretic types and capsular polysaccharide types 5 and 8 among methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus.

The relationship between capsular polysaccharide types 5 and 8 and esterase electrophoretic types (zymotypes) in 160 French clinical isolates of Staphylococcus aureus was studied. Methicillin-susceptible strains of capsular types 5 and 8 were represented by 11 zymotypes, indicating a high polymorphism. Methicillin-resistant strains were mainly distributed in only two distinct populations. The predominant population was represented by strains of zymotype 6 and capsular type 5, and the second population was represented by strains of zymotype 14 and capsular type 8.

Staphylococcus aureus strains which are not identified by rapid agglutination methods are of capsular serotype 5.

A total of 183 recent Staphylococcus aureus clinical isolates were tested with three commercially available rapid agglutination methods. The capsular polysaccharide type and resistance to oxacillin of these isolates were also determined. Seven isolates were not identified correctly by agglutination methods. All isolates not identified by the rapid methods were of capsular serotype 5, and of these isolates, six were resistant to oxacillin. The results suggest that these agglutination kits can be improved by the use of antibodies reactive with S. aureus capsular polysaccharide.

Direct testing of blood culture for detection of the serotype 5 and 8 capsular polysaccharides of Staphylococcus aureus.

Monoclonal antibodies (MAbs) reactive with serotype 5 and 8 capsular polysaccharides of Staphylococcus aureus have been used to test, by enzyme-linked immunosorbent assay (ELISA), blood culture fluids for the presence of S. aureus. A total of 748 blood cultures from 665 patients yielding 706 bacterial isolates belonging to more than 26 bacterial species were studied. All blood cultures containing bacterial strains belonging to species other than S. aureus were negative in ELISA. All 23 blood cultures containing serotype 5 S. aureus were positive in ELISA with the corresponding MAb. Out of 20 blood cultures containing serotype 8 S. aureus, 19 were positive with the corresponding MAb. All 5 blood cultures containing nontypeable S. aureus were negative in ELISA with both MAbs. This method provides reliable identification of serotype 5 or serotype 8 S. aureus by direct testing of blood culture fluids with ELISA.

Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.

Chronic respiratory tract infections caused by Staphylococcus aureus are common in patients with cystic fibrosis (CF). Recently, it was shown in a few CF patients that S. aureus isolates produce capsular polysaccharides (CPs). However, it is not known whether this is a common feature and whether an immune response to CPs in CF is detectable. Therefore, we examined 170 S. aureus isolates from CF patients and healthy individuals for production of CP types 5 and 8 by using monoclonal antibodies. We found that CP-producing staphylococcal isolates were randomly distributed among CF patients and healthy carriers. Eighty-five percent of all isolates produced CPs, 77% of which were type 8. Examination of one sputum sample by an immunofluorescence technique suggested that production of CPs is not an in vitro phenomenon. S. aureus isolates from various sites of a single person often yielded more than one CP type. A random distribution of S. aureus strains with CP type 5 or 8 from the skin and respiratory tracts of patients and from the skin of healthy individuals was found. Antibody response to CP types 5 and 8, measured by enzyme-linked immunosorbent assay, was not elevated in CF patients with chronic S. aureus lung infection in comparison with healthy carriers. On the contrary, in CF patients the ratios of antibodies to CP 8 were significantly lower (P less than 0.005; alpha = 0.025). The ratios of antibodies to CP types did not change when monitored longitudinally over several months. This study suggests that the production of CPs is a universal property of S. aureus and that infected CF patients do not have elevated ratios of antibodies to these antigens.

Prevalence of capsular polysaccharide types 5 and 8 among Staphylococcus aureus isolates from cow, goat, and ewe milk.

Monoclonal antibodies to Staphylococcus aureus capsular polysaccharide types 5 and 8 were used to serotype by enzyme-linked immunosorbent assay 212, 54, and 33 isolates from cow, goat, and ewe milk, respectively. Capsular types 5 and 8 accounted for 69.4% of bovine isolates and 71.5 and 78.8% of goat and ewe isolates, respectively. Type 5 was predominant in strains from bovine sources (51.4%), whereas type 8 was prevalent in strains from caprine (68.5%) and ovine (75.8%) sources.

Predominance of capsular polysaccharide type 5 among oxacillin-resistant Staphylococcus aureus.

The relationship between capsular polysaccharide types 5 and 8 and resistance of Staphylococcus aureus to oxacillin was studied with a collection of 406 clinical isolates from six French hospitals. Of 175 type 5 isolates, 84 (48%) were resistant to oxacillin. In contrast, only 8 of 160 type 8 isolates (5%) and 5 of 71 nontypeable isolates (7%) were resistant to oxacillin. Therefore, capsular typing of clinical isolates of S. aureus may facilitate the choice of first-line antibiotic therapy.

Serotyping and electron microscopy studies of Staphylococcus aureus clinical isolates with monoclonal antibodies to capsular polysaccharide types 5 and 8.

Monoclonal antibodies to Staphylococcus aureus capsular polysaccharide types 5 and 8 were prepared and used to serotype 821 clinical isolates of S. aureus from four countries. The capsular polysaccharide-binding sites on the bacterial membrane were examined by transmission and scanning electron microscopy.

[Description of a new Aedes of the subgenus Ochlerotatus (Diptera: Culicidae) from Madagascar: Aedes ambreensis n.sp. and general observations on the females of the subgenus Ochlerotatus in the Afro-tropical region]. / Description d'un nouvel Aedes du sous-genre Ochlerotatus (Diptera: Culicidae) de Madagascar: Aedes ambreensis nova species et considérations générales sur les femelles du sous-genre Ochlerotatus dans la région afro-tropicale.

The authors describe the female of a new malagasy Aedes species belonging to the sub-genus Ochlerotatus, collected during epidemiological surveys on arboviroses: Ae. ambreensis n. sp. With this description, a review of Afro-tropical Ochlerotatus species is proposed, particularly on principal morphological female characters and geographical distributions. A key for identification of afro-tropical adult females of the sub-genus Ochlerotatus is annexed.