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Objectives: The aim of our study was the molecular typing of 40 clinical isolates of Candida spp. obtained from patients with burns or trauma hospitalized in the intensive care unit of a general hospital. Methods: Isolates were recovered from blood, deep trauma, urine, sputum or from environment within a short period of time (4 months). The yeasts were identified using commercial yeast identification kits as C. albicans (17 isolates), C. tropicalis (16 isolates) and C. parapsilosis (10 isolates). The epidemiological relation of the isolates was tested with the Random Amplified Polymorphic DNA assay using three or four arbitrary chosen primers. Results: All C. albicans isolates presented distinct RAPD profiles, C. tropicalis isolates presented both the same and distinct RAPD patterns and the C. parapsilosis isolates presented the same RAPD pattern. All the environmental isolates were identified as C. parapsilosis and they had the same RAPD pattern as C. parapsilosis clinical isolates. Candida parapsilosis delineation was confirmed with PFGE. Conclusions: The colonization/infection with C. albicans was endogenous, the C. tropicalis colonization/infection was both endogenous and exogenous, and the C. parapsilosis colonization/infection had an environmental origin. Copyright 2001 The British Infection Society

Objectives:Sixteen Mycobacterium aviumstrains were isolated from the blood of eight AIDS patients over a period of months. All the patients were on combination therapies including clarithromycin, and all had treatment failure and relapses of M. aviumbacteremia. Paired clarithromycin-sensitive and resistant M. aviumstrains isolated at the beginning of treatment and at the first relapse of bacteremia were compared.

ABSTRACTArbitrarily primed PCR with three primers and pulsed-field gel electrophoresis were used to characterize a set of 75 clinicalLegionella pneumophilaserogroup 1 isolates, with no apparent epidemiological link, obtained from 24 hospitals in Paris, France, from 1987 to 1997. Unexpectedly, 25 clinical isolates from 15 hospitals had an identical profile (termed type A) by both methods. The same profile was subsequently found in 16 of 64 randomly selected environmental L. pneumophilaserogroup 1 isolates from 15 different sites in the Paris area. There was no evidence of geographic clustering or a peak incidence of type A isolation. Type A has not been found in France outside the Paris area, suggesting that a particular type of L. pneumophilaserogroup 1 is specifically present in the Paris water distribution network.

The rise of Mycobacterium genavenseinfections is making identification ever more important for diagnosis and treatment. Moreover, isolation and identification of M. genavenseare made difficult by the lack of growth on solid media and by its low generation rate in BACTEC liquid media. Thus, amplification by PCR or similar techniques represents the only possibility of detecting and identifying M. genavensefrom tissue samples. In order to set up a simple and species‐specific method based on the use of PCR and non‐radioactive hybridization technique, we decided to search for and clone a specific DNA fragment of this bacterial species. In the present study, a 1734‐bp fragment was isolated. This fragment was found to be highly specific for M. genavensestrains. A species‐specific pair of primers (MG22 and MG23) and two oligonucleotide probes (MG18 and MG19) were selected. They were successfully used to amplify and detect a 155‐bp DNA fragment from the 13 available strains of M. genavensewhich were isolated from clinical specimens or from birds. Conversely, the primers and probes did not hybridize with DNA from any of the 20 other mycobacterial species tested. It is worth noting that the chosen primers and probes did not hybridize with DNA of M. simiae, although it is closely related to M. genavense. The present PCR technique uses species‐specific primers for M. genavense. Followed by a non‐radioactive hybridization technique on microplates it is able to distinguish M. genavensefrom other mycobacteria in one step, without sequencing or restriction analysis. On the basis of the Southern blot hybridization, PCR and sandwich hybridization results, we concluded that the isolated 1.7‐kb sequence was specific for the M. genavensechromosome. The method developed here for M. genavenseidentification uses a simple methodology and commonly available reagents. Furthermore it can be easily automated.

There is a geographic distribution of Mycobacterium tuberculosis strains with various rpoB gene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify rpoB mutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detect rpoB mutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinct rpoB mutations in codons Ser531, His526, and Asp516 were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser531 and His526 accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related M. tuberculosis strains can have subclones bearing distinct mutant rpoB alleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in M. tuberculosis before the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.

ABSTRACTThere is a geographic distribution of Mycobacterium tuberculosisstrains with various rpoBgene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify rpoBmutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detectrpoBmutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinctrpoBmutations in codons Ser531, His526, and Asp516were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser531and His526accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related M. tuberculosisstrains can have subclones bearing distinct mutant rpoBalleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in M. tuberculosisbefore the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.

A panel of autoantigens (myosin, actin, myelin basic protein MBP, and thyroglobulin) was used to analyze antigen recognition by the peripheral blood leukocytes (PBL) of patients with active and stable multiple sclerosis (MS), patients with other neurological diseases (OND) and healthy individuals. The immune responsiveness was studied by examining the in vitro cell proliferation and the increase in the expression of two T-cell-surface activation markers (the interleukin-2 receptor IL-2R, and a late activation antigen recognized by the 19.2 monoclonal antibody). In MS, autoantigen recognition occured more frequently than in the other groups and it was manifested by moderate proliferation or marked elevation of the expression of the IL-2R, whereas autoantigen recognition in the other groups concerned essentially the expression of the late activation antigen. Results similar to those described above were obtained with enriched T lymphocytes either in the presence or absence of IL-2. Our results suggest that the peripheral immune system in MS patients may recognize and can be activated by different autoantigens and not only by MBP, and that this response is quantitatively and qualitatively different from that of PBL from OND patients and healthy individuals.