Antigen overlay technique for line immunoelectrophoresis and crossed-line immunoelectrophoresis of thermolabile antigens.

A simple antigen overlay technique for line immunoelectrophoresis and crossed-line immunoelectrophoresis is described. A blind gel is overlaid with antigen-impregnated filter paper instead to mix antigens with molten agarose and to pour them in a uniform sample gel. This makes it easier in line and crossed-line immunoelectrophoresis comparison of different antigen samples, simplifying technique as compared with the conventional method. Moreover, this technique is designated to obtain reproducible patterns from thermolabile proteins since they are unaffected by being heated.

A rapid and sensitive non-competitive avidin-biotin immuno-enzymatic assay for lysozyme.

A non-competitive avidin-biotin immuno-enzymatic assay (NABA) for lysozyme is described. The assay was found to be more sensitive than a competitive assay with biotinylated lysozyme. The lower detection limit of NABA was 0.1 ng lysozyme/ml compared to 1 ng/ml for the competitive assay. The intra-assay and inter-assay coefficients of variation for NABA were 5.9 and 9.1%, respectively. The total time for NABA can be decreased (to less than 1 h) without influencing the detection limit or the analytical range. Serum lysozyme levels measured by NABA and the enzymatic assay in 32 samples showed a correlation coefficient of r = 0.97.

Highly sensitive biotin-avidin sandwich ELISA for the rapid detection of pneumococcal capsular polysaccharide antigens.

The immunological detection of soluble pneumococcal polysaccharide antigens in pathological products is of importance in the direct diagnosis of meningitis or pulmonary infections. We have developed a double antibody sandwich ELISA method using a biotin-avidin system using antibodies constituted with a mixture of IgGs from pooled and/or monospecific antipneumococcal sera provided by the Danish Statens Seruminstitut. The sensitivity of this rapid ELISA method was optimized with purified capsular polysaccharides of the 24 main pneumococcal serotypes. With incubation steps of 30 min at 37 degrees C for the antigens and the conjugates, the detection limit was close to 1 ng/ml for 75% of the purified polysaccharides. A retrospective study of 46 CSF samples established the validity of the assay. This type of modified ELISA system represents a specific, sensitive and rapid procedure for the potential detection of capsular soluble antigens of all pneumococcal serotypes.

Rapid identification of medically important yeasts by electrophoretic protein patterns.

A simple electrophoretic method for yeast identification was evaluated. Whole cells were extracted by SDS and the protein profiles obtained in SDS-PAGE after Coomassie blue staining were compared for 52 strains from 9 species of yeast or yeast-like fungi commonly isolated from man (Candida albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, C. tropicalis, Geotrichum candidum, Saccharomyces cerevisiae). The corresponding patterns showed 30 to 45 polypeptides in the range 95-20 kDa and were clearly different for the 9 species. No differences could be detected between strains from the same species. The characteristic patterns were obtained within 24 h allowing rapid identification of the most commonly encountered clinical yeast isolates.

Survey of primary drug resistance of Mycobacterium tuberculosis in Casablanca, Morocco.

SETTING: In 1990, a 6-month short-course regimen (2 SHRZ/4 RH) was introduced for the treatment of tuberculosis in Morocco. OBJECTIVE: To assess the efficacy of the national tuberculosis control programme, a prospective study of primary drug resistance was conducted from April 1992 to July 1994 in Casablanca. DESIGN: A total of 402 strains isolated from 402 patients living in Casablanca with no previous history of tuberculosis was included in the study. RESULTS: The overall rate of primary drug resistance to at least one drug was 23.9%; it was 19.7% to streptomycin, 11.4% to isoniazid, and 8.2% to both streptomycin and isoniazid. The rates of resistance to rifampicin and ethambutol were both less than 1%. The survey was divided into two periods of 14 months each. The rates of primary drug resistance increased from 21.1% to 27.6% during these two periods (Odds Ratio [OR] 1.43; 95% Confidence Interval [CI] 0.88 to 2.32); this increase occurred only for streptomycin (15.9% to 24.7%, OR 1.73; 95% CI 1.02 to 2.93). CONCLUSION: The rate of primary drug resistance of Mycobacterium tuberculosis in Casablanca has risen in recent years to an ominous level. Urgent measures are needed in order to interrupt this trend.

[Study of an epidemic of Clostridium difficile-associated diarrhea]. / Etude d'une épidémie de diarrhées associées à Clostridium difficile.

Twenty three Clostridium difficile strains isolated from diarrheic faecal samples of 22 patients in a neurosurgical department of the Pellegrin Hospital in Bordeaux between January 1984 and May 1987 (15 isolated in 1984, 2 in 1985, 3 in 1986 and 3 in 1987) and 15 strains isolated from 15 patients in other departments of the same hospital in 1984 have been compared using their antimicrobial susceptibility, cytotoxin production and electrophoretic pattern. Twelve of the 15 strains isolated in the neurosurgical department and 4 of the 15 strains isolated in other departments in 1984 were phenotypically identical. These findings suggest that the origin of C. difficile associated-diarrheas can be nosocomial.

[Anti-Candida albicans IgM antibody: correlation between immunofluorescence and ultracentrifugation (author's transl)]. / Anticorps IgM anti-Candida albicans: corrélation entre l'immunofluorescence et l'ultracentrifugation.

The indirect fluorescent antibody (IFA) technique for specific IgM anti-Candida albicans was applied to 158 sera and compared with sucrose density gradient centrifugation. For detection of specific IgM results of both methods were in agreement for 157 sera (99.36%). Specific IgM titers determined with IFA on unfractionated sera followed those found on fractionated sera. The results show that specific IgM anti-C. albicans can be identified and titrated by the immunofluorescent technique.

Clostridium difficile and its cytotoxin in diarrhoeic stools of hospitalized patients. Toxigenic potential of the isolates.

Cytotoxin assay and culture for Clostridium difficile were performed on 303 diarrhoeic stools from 261 hospitalized patients. Specimens from 42 patients were positive by at least one of the methods, and 40 of them had an antibiotic-associated diarrhoea. The cytotoxin assay was positive in 5 of 7 patients with pseudomembranous colitis. Thirteen had an appropriate response to specific therapy and the remainder have resolved of diarrhoea without C. difficile directed chemotherapy. These findings show the lack of reliability of the cytotoxin assay for the diagnosis of C. difficile antibiotic-associated diarrhoea. The 6 strains isolated from patients with pseudomembranous colitis were examined for enterotoxin by the rabbit ileal loop test: 4 produced both toxins, 2 only enterotoxin. Both toxins could therefore not be essential for the clinical expression of the disease.

[Seroprevalence of Chlamydia trachomatis infection in STD consultants in Morocco]. / Séroprévalence de l'infection à Chlamydia trachomatis chez des consultants MST au Maroc.

We have conducted a seroepidemiological survey of Chlamydia trachomatis infection among 400 STD consultants in comparison with 400 blood donors. The study was performed by using the indirect microimmunofluorescence technique with Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae as antigens. The overall seroprevalences were 60% and 46% for STD consultants and blood donors respectively. The seroprevalences of Chlamydia trachomatis alone were 12.5% for STD consultants and 7.5% for blood donors. No differences were observed according to age in the two groups and people of 20-29 and 30-39 years old, of both sexes were the most concerned. We conclude that Chlamydia trachomatis infection remains an important problem in Morocco.

[Isolation of Chlamydia trachomatis in trachomatous Moroccan patients]. / Isolement de Chlamydia trachomatis chez des sujets trachomateux marocains.

PURPOSE: Isolation of Chlamydia trachomatis from ocular specimens of subjects living in trachomatous area in south Morocco. METHODS: One hundred and twenty ocular specimen of 60 subjects living in two provinces of a trachoma-endemic area (Errachidia and Ouarzazate) were tested by cells culture. The age range was 2 months to 85 years and the sex ratio was 1.06. RESULTS: The prevalence of positive cases was about 25% with a female predominance of 34% versus 16% for males. In our sample, 70% showed an active clinical trachoma. The most affected age ranges were children between 0 and 10 years old, with a very high frequency of isolation in children younger than 5 years. The intense inflammatory stage alone or associated with follicular stage was the most adequate for isolating Chlamydia trachomatis. CONCLUSION: Reducing or even eradicating trachoma can be realised only through a continuous treatment strategy associated with a sanitary education aiming at the development of hygienic conditions especially among children living in trachomatous communities.

[Pulmonary mucormycosis. Apropos of a case with serologic confirmation]. / Mucormycose pulmonaire. A propos d'un cas avec confirmation sérologique.

We report a new case of primary pulmonary mucormycosis. The radiographic evidence and anatomopathological facts demonstrate an intra-cavitary mycetoma of Fowler associated with a chronic pneumonia in the left basal segment. The patient was not immunosuppressed but had undergone a left upper lobectomy 25 years before. This appears to be the first case in which a serological study has confirmed the diagnosis.

[Fast identification of Streptococcus pyogenes serotype M.12 in throat swabs by an improvised coagglutination technique]. / Identification rapide de Streptococcus pyogenes sérotype M. 12 dans les prélèvements de gorge par coagglutination extemporanée.

A rapid procedure for identification of Streptococcus pyogenes serotype M. 12 directly in throat swabs, is reported and compared with standard culture method on blood agar plates and typing of group A Streptococci isolated, with double gel immuno-diffusion. This procedure consist of chlorhydric acid extraction of swabs and testing of the extract towards specific M. 12 protein serum using extemporaneous coagglutination technique. We have tested 1100 throat swabs, with this procedure and with standard culture procedure. Identification of group A Streptococci serotype M. 12 with reported method is obtainable within 30 to 45 minutes of receipt of the clinical specimen. This method is easy to perform, with a sensitivity and a specificity respectively: 89.7% and 98.8%.

[Comparative sensitivity of yeasts to antifungal agents using a standardized micromethod]. / Sensibilité comparée des levures aux antifongiques en microméthode standardisée.

The comparative susceptibility of 1 850 yeast strains belonging to 8 species was determined. The standardized micromethods used allows determinations of minimal inhibitory concentrations (MICs) or categorized sensitivities for two different concentrations (AB). Overall results showed that amphotericin B (AMB) is the most active agent, followed by the various imidazoles. 5-fluorocytosine (5FC) was the least effective drug, with 68% susceptible strains. However, results varied widely across species and drugs. For instance, among Candida albicans and Torulopsis glabrata strains, none were resistant to AMB and only 6% were resistant to 5-FC; in contrast, Candida albicans was highly susceptible to imidazoles (0.8 to 2.5% resistant strains) whereas Torulopsis glabrata showed much higher resistance rates (18% of strains for tioconazole and 70% for ketoconazole). Variations in susceptibility were also recorded across imidazoles: clotrimazole, tioconazole and ketoconazole were much more effective against Candida tropicalis and Candida parapsilosis than miconazole and econazole, whereas almost no strains were resistant to AMB and more than 50% of strains were resistant to 5-FC. Results obtained by AB (967 strains) and MIC (455 strains) were consistent for the 1 422 Candida albicans strains. Our results show that standardized micromethods should be used to determine the susceptibility of yeasts to antifungal agents.

Evaluation of the new Mycotube test-kit for yeast identification.

The performance of a modified Mycotube test for the identification of yeasts was evaluated using standard biochemical tests as reference. One hundred and eighty strains belonging to 12 medically important species (15 strains each) were tested. The overall rate of identification was 72%, and rose to 87.8% when macroscopic and microscopic morphologic features were also considered; a 100% rate was obtained with Candida albicans, Candida krusei, Candida parapsilosis, Candida pseudotropicalis, Candida tropicalis, Saccharomyces cerevisiae and Torulopsis glabrata. For the species Candida guilliermondii, Cryptococcus neoformans, Geotrichum candidum, Rhodotorula and Trichosporon cutaneum we propose a new evaluation chart and additional code numbers. Mycotube facilitates the routine identification of yeasts but recourse to macroscopic and microscopic examination may also be necessary.

[Sensitivity of Aspergillus to ketoconazole and itraconazole using a standardized micromethod]. / Sensibilité des Aspergillus au kétoconazole et à l'itraconazole en microméthode standardisée.

The standardized micromethod Mycototal is based on ready-to-use microtitration plates and the same culture medium for sensitivity testing of all antifungal agents. Since the method was very reproducible for yeasts it was applied for the determination of minimal inhibitory concentrations of Aspergillus to itraconazole and ketoconazole. Again the reproducibility for itraconazole was excellent since for 19 strains tested two times 18 showed no more than two dilutions different results and this was also observed for 6 strains over 7 tested 4 or 5 times. Over 31 strains tested belonging to different Aspergillus species, itraconazole was much more active than ketoconazole since for 2.5 mg/l all the strains were sensitive to itraconazole and only one to ketoconazole. Moreover no strains were sensitive to ketoconazole for 0.62 mg/l versus 9 strains sensitive to itraconazole. These results showed on one hand the reproducibility of the method and on the other hand the potency of itraconazole against Aspergillus according to the literature and let us hope to a better chemotherapy of aspergillosis.

[Comparative sensitivity of yeasts to ketoconazole, itraconazole and fluconazole using a liquid medium standardized micromethod]. / Sensibilité comparée des levures aux kétoconazole, itraconazole et fluconazole en microméthode standardisée en milieu liquide.

The minimal inhibitory concentrations of 200 yeast strains (48 reference strains and 152 recently isolated from pathological products) were evaluated with a new standardized micromethod using a liquid medium comparatively for ketoconazole, itraconazole and fluconazole. The ready-to-use microtitration plates contained the antifungal agents in concentrations ranging from 100 to 0.10 mg/l. The cumulative MIC curves clearly showed the superiority of ketoconazole, fluconazole being the less active product. These results were confirmed after categorization in sensitive (S: MIC less than 0.78 mg/l), intermediate (I: 0.78 less than MIC less than 6.25) or resistant (R: MIC greater than 6.25). These results are discussed particularly for fluconazole showing the largest half-life and for which higher plasmatic levels could be achieved. Important variations were observed depending on the species and as example for Candida tropicalis the sensitivities were ketoconazole 100%, itraconazole 55% and fluconazole 0%. The less sensitive species to the 3 azoles were Saccharomyces cerevisiae and Torulopsis glabrata. This new micromethod being very easy to use and allowing the determination of fungicidal activities should be introduced in routine.