Yeast identification in the clinical microbiology laboratory: phenotypical methods.

Emerging yeast pathogens are favoured by increasing numbers of immunocompromised patients and by certain current medical practices. These yeasts differ in their antifungal drug susceptibilities, and rapid species identification is imperative. A large variety of methods have been developed with the aim of facilitating rapid, accurate yeast identification. Significant recent commercial introductions have included species-specific direct enzymatic colour tests, differential chromogenic isolation plates, direct immunological tests, and enhanced manual and automated biochemical and enzymatic panels. Chromogenic isolation media demonstrate better detection rates of yeasts in mixed cultures than traditional media, and allow the direct identification of Candida albicans by means of colony colour. Comparative evaluation of rapid methods for C. albicans identification, including the germ tube test, shows that chromogenic media may be economically advantageous. Accurate tests for single species include the Bichrolatex Albicans and Krusei Color tests, both immunologically based, as well as the Remel Rapid Trehalose Assimilation Broth for C. glabrata. Among broad-spectrum tests, the RapID Yeast Plus system gives same-day identification of clinical yeasts, but performance depends on inoculum density and geographic isolate source. The API 20 C AUX system is considered a reference method, but newer systems such as Auxacolor and Fungichrom are as accurate and are more convenient. Among automated systems, the ID 32 C strip, the Vitek Yeast Biochemical Card and the Vitek 2 ID-YST system correctly identify >93% of common yeasts, but the ID-YST is the most accurate with uncommon yeasts, including C. dubliniensis. Spectroscopic methods such as Fourier transformed-infrared spectroscopy offer potential advantages for the future. Overall, the advantages of rapid yeast identification methods include relative simplicity and low cost. For all rapid methods, meticulous, standardized multicenter comparisons are needed before tests are fully accepted.

Evaluation of six commercial systems for identification of medically important yeasts.

Six commercially available systems for the identification of yeasts were evaluated using 133 clinical isolates and four reference strains that had been previously identified by conventional methods and 19 recent clinical isolates that had been identified by the ID32C system (bioMérieux, France). The total identification rates (TIR) established for the total number of strains tested and the database identification rates (DBIR) established for the strains included in the respective manufacturer databases were both determined. After incubation for 4 h, the TIR and DBIR were 78% and 84%, respectively, for the RapID Yeast Plus system (Innovative Diagnostic Systems, USA). After incubation for 24 h, the TIR and DBIR were 32% and 32%, respectively, for the ID32C, 65% and 67% for the Auxacolor system (Sanofi Diagnostics Pasteur, France), 62% and 65% for the Fungichrom I system (International Microbio, France), 52% and 65% for the Fungifast I twin system (International Microbio), and 62% and 68% for the API Candida system (bioMérieux). The maximum TIR and DBIR (+/- 1%) obtained after incubation for 48 h were 86% and 88% for the Auxacolor, 85% and 89% for the Fungichrom I, 78% and 98% for the Fungifast I twin, and 82% and 91% for the API Candida. For the ID32C, the maximum TIR and DBIR were 98% and 98%, respectively, but these values were obtained only after 72 h of incubation. In addition, the six systems varied in their ease of use and readings. In conclusion, based on results obtained with 156 strains, the Auxacolor and Fungichrom systems seem the most appropriate for use in a clinical microbiology laboratory, due to their ease of use and reading, their rapidity, their cost per test, and their relatively high TIR results, which indicated acceptable performance with strains frequently isolated in our hospital. For a reference identification, the ID32C remains the sole system usable.

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.

[Chlamydia infection and female low fertility in Morocco]. / Chilamydiose et hypofertilité féminine au Maroc.

Chlamydia trachomatis infection is recognised as the most common asymptomatic sexually transmitted disease, and this may lead to severe complication including infertility. The purpose of this study was to evaluate the part that this pathology takes in the female hypofertility, using serologic, cell culture, and histopathologic tests. Some of the women had undergone biopsies during coelioscopic exam, the others during salpingectomy. Cervical specimens were carried from other women. They had as clinical signs: primary or secondary infertility, ectopic pregnancy, syndrome of synechie, hydrosalpinx, or pelvic pains. 128 of these women had undergone serologic exam, 57 a cell culture, and 47 an histopathologic test. The results showed that 26% had anti Chlamydia trachomatis antibodies and 46% from them with tubal problems confirmed, had anti Chlamydia trachomatis antibodies as well, only 7% had cell culture positive from cervix specimens, none from the biopsies, and 73% of them had inflammatory responses. All women with inflammatory responses had a serologic and/or cell culture positive tests. Our results allow us to conclude that this infection takes a good part in female hypofertility, there is a correlation between a previous Chlamydia trachomatis infection and a tubal histopathology. In front of the difficulties of isolation by cell culture the detection of the microorganism by molecular biology assays may resolve a lot of problems.

Evaluation of latex reagents for rapid identification of Candida albicans and C. krusei colonies.

A total of 322 yeast strains and yeastlike organisms belonging to the genera Candida, Cryptococcus, Geotrichum, Saccharomyces, and Trichosporon were tested with the new monoclonal antibody-based Bichro-latex albicans and Krusei color latex tests. Comparison of results with those obtained by conventional identification methods showed 100% sensitivity for both latex tests and 100% and 95% specificity for the Bichro-latex albicans and Krusei color tests, respectively. Because the test is easy to read and quick to perform, the Bichro-latex albicans test may be useful for rapid identification of Candida albicans colonies in the clinical laboratory.

[Chlamydial infections and male infertility in Morocco]. / Chlamydiose et infertilité masculine au Maroc.

Chlamydia trachomatis infection of the lower genital is recognised as the most common sexually transmitted disease, is role in male infertility is controversial, the objective of this study was to evaluate the part that this pathogen agent takes in male infertility among maroccan population, to compare serological tests, sperm abnormalities, antisperm-antibodies and DNA research in semen. Microimmunofluorescence (MIF) was done for 139 patients, 124 were checked for sperm abnormalities, 87 for antisperm-antibodies and 92 for DNA research in sperm. The results showed that MIF is positive in 24,5%, 11% of the subjects in antisperm antibodies, 8% of them simultaneously in anti-Chlamydia and antisperm antibodies and 5 of them had sperm abnormalities. Azoospermy was more observed in positives subjects in Chlamydia trachomatis antibodies. C. trachomatis DNA was found in 7,6% and there was no association between the detection of C. trachomatis in semen specimens and the presence of anti-Chlamydia trachomatis, antibodies in serum. We conclude that, because of the complexity of the Chlamydia's physiopathology, association between several tests is necessary in male infertility workup.

[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.

Production of a new monoclonal antibody specific to human des-gamma-carboxyprothrombin in the presence of calcium ions. Application to the development of a sensitive ELISA-test.

In order to explore pathologies possibly associated with vitamin K deficiency, several monoclonal antibodies (mAbs) were produced against human Desgamma-Carboxy-Prothrombin (DCP). One of these mAbs, designated C4B6, detected DCP forms in the presence of Calcium ions, confirmed by comparison with the patterns of two electrophoretic techniques: Affino-Immuno-Electrophoresis (CAIE) and Polyacrylamide Gel Electrophoresis followed by Electro-blotting (PAGE-Blot). An Enzyme-Linked-Imunosorbent Assay (ELISA) using mAb C4B6 has been developed, optimized and standardized. It has proven to be specific for DCP forms and has a minimum sensitivity of 0.156 A.U/ml.

[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.

[Serodiagnosis of Pseudomonas aeruginosa infections in mucoviscidosis: comparative study of Western blotting, ELISA exotoxin A and ELISA phospholipase C]. / Sérodiagnostic des infections à Pseudomonas aeruginosa dans la mucoviscidose: étude comparative du Western Blot, de l'ELISA exotoxine A et de l'ELISA phospholipase C.

Anti-Pseudomonas aeruginosa antibodies were studied by Western Blot, ELISA-exotoxin A and ELISA-phospholipase C for 91 serums from 31 patients with cystic fibrosis. More, for the two enzyme-linked immunosorbent assays, 44 serums from 44 healthy individuals were studied as controls. The study of these three parameters revealed the followings: with no infection by Pseudomonas aeruginosa all the results were negative, at the beginning of the infection, anti-exotoxin A antibodies appeared in first, followed in some cases by the reactions of Western Blot, anti-phospholipase C antibodies became positive at last and went on a par with the installation of the chronic characteristic of the infection, as soon as the chronicity were indisputable, the three methods revealed elevated serum antibodies amounts. Generally there was a correlation between detected antibodies and Pseudomonas aeruginosa isolation in sputum. Among these three methods, ELISA-exotoxin A appeared to be the most interesting because of its good reproducibility and its early positivity, before the others methods and sometimes before Pseudomonas aeruginosa isolation. It would be a significant argument to establish as soon as possible an antimicrobial therapy.

Evaluation of five commercial antifungal susceptibility testing systems.

Five commercial antifungal susceptibility testing systems were studied for repeatability and reproducibility as well as concordance of results with the MICs for ten reference strains belonging to six different species. Repeatability was determined by testing each strain in triplicate on the same day, and reproducibility by repeating this triple determination on three different days. On the basis of 630 yeast-antifungal agent results for Mycototal and Mycostandard, 540 for Candifast, and 450 for ATB Fungus and Diff Test, repeatability was consistently equal to or greater than 95%. Reproducibility was 80.07% for Candifast and greater than 95% for the other systems. The concordance with the reference MICs was 51.65% for Candifast, 75.33% for ATB Fungus, 80.89% for Diff Test, 90.16% for Mycostandard and 90.32% for Mycototal. Although the performance of Diff Test and ATB Fungus was satisfactory, Mycototal and Mycostandard gave notably better results with imidazoles. Mycostandard, which is easier to use and includes tests for fluconazole and itraconazole, would seem to be potentially the most useful antifungal susceptibility test available at present.

Purification and immunological studies of the cross-reaction between the 65-kilodalton gonococcal parietal lectin and an antigen common to a wide range of bacteria.

The 65-kDa gonococcal parietal lectin (GPL) has been purified and found to have a lectinlike activity exhibiting both structural and immunological similarities to the common antigen family. Ultrastructural localization of GPL was done by using anti-GPL monoclonal antibodies: GPL is a major component of the outer membrane and is also present in blebs formed by gonococci.

Three electrophoretic techniques comparison for des-gamma-carboxyprothrombin detection.

Des-gamma-carboxyprothrombin (DCP) is a marker that appears in the blood when modifications of vitamin K-dependent proteins carboxylation cycle occur. About 280 human plasma samples of diverse origins were tested by three different electrophoretic techniques for the evaluation of DCP: rocket immunoelectrophoresis (RIE) before and after barium carbonate adsorption, crossed affinoimmunoelectrophoresis (CAIE) and polyacrylamide gel electrophoresis in presence of calcium lactate followed by immunoblotting (PAGE-blot). A good correlation was found between CAIE and PAGE-blot in the CAIE detection limit, but not between RIE and the two other techniques. PAGE-blot was more sensitive than RIE and CAIE and allowed reliable quantification of abnormal prothrombin in plasma.

Protein A insensitive ELISA detection of staphylococcal enterotoxin B.

A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin B was developed using rat monoclonal antibodies as capture antibodies and as a biotinylated conjugate. This test was sensitive, less than 1 ng/ml of enterotoxin B was detected and interference by protein A was prevented by the use of rat monoclonal antibodies of the IgG2a isotype which were insensitive to protein A even at concentrations greater than 1000 ng/ml.

Des-gamma-carboxyprothrombin detection by immunoblotting after polyacrylamide gel affinoelectrophoresis in human plasmas.

In the absence of vitamin K or in the presence of the vitamin K antagonists, abnormal nonfunctional forms of prothrombin circulate in the blood. A reliable and reproducible technique, derived from traditional crossed affinoimmunoelectrophoresis in presence of calcium lactate, was developed and optimized. The technique is based on nondenaturing polyacrylamide gel affinoelectrophoresis, with calcium lactate, of plasma samples, followed by immunoblotting with rabbit anti-human prothrombin serum and detection with an anti-rabbit immunoglobulin peroxidase conjugate. Depending on the plasmas, one or two bands were visualized and quantified by densitometry of the immunoblots. The technique was able to detect abnormal des-gamma-carboxylated prothrombins at concentration of 0.1 microgram/mL.

Comparison of sandwich-ELISA and GM1-ELISA for the detection of Escherichia coli thermolabile enterotoxin.

Two different microtiter plate ELISA tests were devised for the detection of Escherichia coli thermolabile toxin (LTh) either free or extracted from isolated colonies. Both tests used as detection systems purified anti-LTh rabbit immunoglobulins conjugated to biotin, streptavidin peroxidase and TMB. The tests differed by their capture phase which was the GM1-ganglioside for GM1-ELISA and purified anti-LTh rabbit immunoglobulins for sandwich ELISA. The two methods were rapid since they could be performed in less than 2 hours. The detection limits for purified LT were 50 pg/ml and 1.3 ng/ml for sandwich ELISA and GM1-ELISA respectively. For the detection of toxinogenic isolates the extraction buffer containing Triton X-100 was always superior to polymyxin buffer. Using the polymyxin extraction buffer the sandwich ELISA was again more sensitive than the GM1-ELISA since a lower number of isolated colonies could be used for the detection of positive strains. With the Triton X-100 buffer both ELISAs could detect positive strains using a single colony but the sandwich ELISA gave the highest delta OD. We concluded that our sandwich ELISA can rapidly detect either the free Escherichia coli thermolabile toxin or LTh producing strains and could be applied routinely.

Rapid identification of Candida albicans by dot-enzyme immunoassay.

A highly sensitive and specific dot-enzyme immunoassay for the rapid identification of Candida albicans was developed using a murine monoclonal antibody (Mab), which adsorbed to cell surface-exposed determinants. This Mab reacted with 28 of the 28 C. albicans strains tested including the serotypes A and B and 2 C. stellatoidea. It did not react with 32 other isolates representing eight other Candida species commonly encountered in human materials. All the test could be performed in four steps in less than an hour. The yeasts were directly spotted on a strip of immunodyne membrane. Then the strip was incubated for 5 min. with the Mab, for 15 min. with a peroxidase-conjugate and for 30 min. with the enzyme substrate and 4-chloro 1 naphthol. This test proved useful for rapid and easy identification of C. albicans.