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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.

Mycobacterium genavense is a recently described agent which can induce disseminated infections in patients with AIDS. Up to now, no standard approach to treatment has been defined and patients have been treated empirically with antibiotics used for treating infections caused by other nontuberculous mycobacteria. In this study, we compared the effectiveness of ciprofloxacin, amikacin, ethambutol, clarithromycin and rifabutin in the treatment of an animal model of M. genavense infection in C57BL/6 mice. Antimycobacterial treatment was started 4 weeks after an intravenous bacterial challenge and was continued for 30 days. Treated and control mice were killed at days 15 and 30 of treatment and the number of viable bacteria in their spleens was counted. Treatment with clarithromycin (50 mg/kg/day sc) and rifabutin (20 mg/kg/day po) was found to decrease the bacterial counts in the spleens significantly as early as 15 days after the onset of treatment (P < 0.01). The effect of treatment was more pronounced after 30 days of treatment (P < 0.001). Amikacin (25 mg/kg/day sc) and ethambutol (50 mg/kg/day sc) were found to decrease significantly the cfu in the spleens only after 30 days of treatment (P < 0.01). Ciprofloxacin (25 mg/kg/day sc) was ineffective in the experimental conditions used here.

Previous studies have shown that gamma interferon (IFN-gamma) plays a major role in natural resistance to Salmonella typhimurium during the early phase of infection. To assess whether the level of natural resistance in mice is related to the level of IFN-gamma gene expression, we compared IFN-gamma mRNA levels by means of reverse transcriptase-PCR in the spleens of genetically susceptible Itys (C57BL/6 and BALB/c) and resistant Ityr (CBA and DBA/2) mice during the first 5 days of infection. The mRNA expression of interleukin-10 (IL-10), a cytokine which antagonizes IFN-gamma effects, was also investigated. Mice were infected with 10(3) CFU of the virulent strain S. typhimurium C5, a dose which is lethal within a week for susceptible mice only. IFN-gamma mRNA increased to similar levels in both susceptible and resistant mice, suggesting that susceptibility to S. typhimurium infection is not related to defective IFN-gamma gene expression. In contrast, IL-10 mRNA reached much higher levels in susceptible than in resistant mice. Similar results were found in Ity congenic mice, confirming a link between the presence of the Itys allele and a high level of IL-10 gene expression during infection. High levels of IL-10 mRNA in susceptible mice correlated with high IL-10 serum levels (on day 5), whereas IL-10 was not detectable in the sera of resistant mice. However, administration of neutralizing anti-IL-10 monoclonal antibodies did not modify the course of infection. To evaluate the influence of bacterial multiplication on IL-10 mRNA expression, susceptible mice were infected with an attenuated strain of S. typhimurium. This strain induced a low level of IL-10 mRNA expression. When susceptible mice were immunized with an attenuated strain and challenged with the virulent strain, they inhibited the growth of the challenge bacteria and exhibited a low level of IL-10 mRNA. In contrast, when resistant mice were infected with a high (lethal) dose of the virulent strain, they exhibited a high level of IL-10 mRNA. Taken together, these results indicate that the level of IL-10 gene expression correlates with the level of bacterial multiplication in the organs and that the high level of IL-10 mRNA in Itys mice is a consequence rather than the cause of their susceptibility to S. typhimurium infection.

We investigated the role of tumor necrosis factor alpha (TNF-alpha) in human peripheral monocytes infected with Legionella pneumophila in vitro. Exogenous TNF-alpha significantly inhibited the intracellular multiplication of the bacterium. This effect was concentration and time dependent and was abrogated by anti-TNF antibodies. TNF-alpha levels in the culture supernatants were low but were enhanced by the addition of gamma interferon. When monocytes were cultured and infected in the presence of pentoxyphilline, a potent inhibitor of TNF-alpha synthesis, the intracellular bacterial growth was enhanced. The effect of pentoxyphilline was concentration and time dependent and was due to the inhibition of TNF-alpha production, as shown by Northern (RNA) blot hybridization of total RNA. In addition, the pentoxyphilline partially abolished the inhibitory effect of gamma interferon on bacterial intracellular multiplication. These results suggest that gamma interferon inhibits, at least partially, the intracellular multiplication of L. pneumophila by enhancing TNF-alpha synthesis.

A randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular typing of Mycobacterium avium strains. This method was applied to epidemiologically unrelated M. avium strains isolated from the blood of 10 different AIDS patients and to strains that were considered epidemiologically related, as they had been isolated from the same patient but from different body locations (4 patients, 10 strains). Three oligonucleotide primers among the six tested were found to generate RAPD profiles with DNA from all M. avium strains and to successfully type them. This method for the typing of M. avium strains is rapid and easy to perform.

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.

Forty human clinical Mycobacterium avium-M. intracellulare complex strains isolated in Greece were characterized to the species level by PCR with three sets of primers specific for one or both species. M. avium predominated in both human immunodeficiency virus-positive and -negative patients, but the frequency of M. intracellulare isolation appeared to be higher in the latter.

A commercial kit (EnviroAmp) designed to detect the DNA of Legionella species in environmental water samples using PCR and reverse dot hybridization was applied to clinical specimens. Results correlated well with culture for bronchoalveolar lavages. In addition, this test was easy to perform and showed good sensitivity.

A polymerase chain reaction able to amplify specifically a 205-base-pair DNA fragment of Mycobacterium avium genome was used compared to a nonradioactive hybridization assay (AccuProbe) and to conventional biochemical identification. The PCR approach to diagnosis of M. avium infection is a valid diagnostic alternative to the more classical procedures. Copyright 1994, 1999 Academic Press

IS6100 is an insertion sequence of the IS3 family and it is present in multiple copies in the chromosome of Mycobacterium tuberculosis. Four to 15 copies are present in various strains of M. tuberculosis. In this study, the value of IS6110 as an epidemiological marker of tuberculosis was examined. Unrelated clinical strains from Greek patients presented, in restriction fragment length polymorphism analysis, a high degree of polymorphism, whereas patterns of related clinical strains from familial outbreaks were identical. Since RFLP analysis with acetylaminofluorene labeled IS6110 as the probe gave satisfactory results, it is suggested that this non-radioactive probe can be used in hospitals and health centres for the epidemiological survey of M. tuberculosis infections.