Biomedical subjects
Publications and source records attributed to J L Rodriguez-Tudela.
Meningitis caused by Pseudallescheria boydii is an uncommon infection of the CNS that usually has a poor prognosis and a difficult treatment. We describe a case of chronic meningitis caused by P. boydii in an immunocompetent host that was successfully treated with voriconazole, a new antifungal agent.
In order to develop new approaches for the chemotherapy of invasive infections caused by Scedosporium prolificans, the in vitro interaction between itraconazole and terbinafine against 20 clinical isolates was studied using a checkerboard microdilution method. Itraconazole and terbinafine alone were inactive against most isolates, but the combination was synergistic against 95 and 85% of isolates after 48 and 72 h of incubation, respectively. Antagonism was not observed. The MICs obtained with the terbinafine-itraconazole combination were within levels that can be achieved in plasma.
A model of acute disseminated Candida albicans infection in New Zealand rabbits was developed to determine the sensitivity and accuracy of polymerase chain reaction (PCR) assay compared with the lysis-centrifugation blood culture method. Primers used amplify a DNA fragment from the multicopy gene coding for the small subunit rRNA, highly conserved in fungi. The sensitivity of PCR achieved in rabbit blood samples spiked with Candida albicans was 10-50 CFU/100 microL. A nested-PCR increased the limit of detection 10-fold. The sensitivity achieved exclusively with the lysis-centrifugation method (37.5%) was higher than that obtained with PCR (25%), but lower than nested PCR (52.5%). The combination of both techniques, lysis-centrifugation and nested PCR, increased the overall sensitivity rate to 62.5%. These results have demonstrated that, globally, the nested PCR was more sensitive than both single PCR and lysis-centrifugation culture in detecting C. albicans in blood from immunecompetent rabbits with acute disseminated candidosis. PCR could be a useful complementary technique to traditional methods in the early diagnosis of candidemia.
Six Candida dubliniensis isolates were recovered from two HIV-infected individuals in the course of a prospective study of recurrent oral candidosis among HIV-positive patients in Spain. Candida albicans strains as well as non-albicans strains were also obtained from these two patients. C. dubliniensis strains were germ-tube-positive and produced abundant chlamydospores. Fingerprinting the genomic DNAs of these six C. dubliniensis with the C. albicans-specific probe 27A as well as karyotyping was performed to confirm the identification of these isolates. Further analysis of their genomic DNAs was performed by PCR-fingerprinting with the core sequence of phage M13, and they exhibited species-specific multilocus band patterns, clearly distinct from those of C. albicans isolates analyzed in this study and in a previous one (Diaz-Guerra 1997). Intraspecies variation was also seen among PCR patterns yielded by C. dubliniensis isolates from different patients. Although few strains have been analyzed, the use of this PCR-fingerprinting procedure is a promising tool for further epidemiologic studies with C. dubliniensis. The isolation of C. dubliniensis from Spanish HIV-infected patients contributes to the idea of widespread geographic distribution of this species.
OBJECTIVE: To assess the genetic homogeneity or heterogeneity within each set of Candida albicans isolates colonizing/infecting the oral cavities of HIV-infected patients undergoing azole therapy when changes in susceptibility to fluconazole were detected. DESIGN: Fourteen HIV-positive patients suffering recurrent episodes of oral candidosis were prospectively followed from the first episode to the isolation of strains with decreased susceptibility to fluconazole. The strains of C. albicans isolated either from episodes or controls throughout the prospective study were analysed. METHODS: Electrophoretic karyotyping and hybridization with the repeated sequence probe 27A were used to delineate sequential isolates. In vitro susceptibility tests to fluconazole and ketoconazole were also performed. The results obtained by DNA fingerprinting with the probe combined with computer-assisted analysis were used to assess the genetic relationships amongst the strains. In addition, comparison with the genetic relatedness of a group of geographically unrelated strains was made. RESULTS: Isogenic populations of sequential isolates were observed only in two patients; 12 patients harboured heterogenic populations over time, although in 11 patients there was a predominant strain that was isolated more than once, and only one of these patients carried strains with a similarity index less than 80%. With the exception of two patients, each patient carried a major strain that became less susceptible to fluconazole. The similarity index for the unrelated strains was 59%. CONCLUSIONS: HIV-infected patients may carry a mixed population of strains, but the strains tend to be related to each other. The strains were maintained throughout the course of infection and at least one developed secondary resistance to fluconazole.
Nosocomial neonatal candidiasis is a major problem in infants requiring intensive therapy. The subjects of this retrospective study were nine preterm infants admitted to the neonatal intensive care unit of the Hospital Central de Asturias between March 1993 and August 1994. The infants were infected with or colonized by Candida albicans. Five patients developed C. albicans bloodstream infections. A total of 36 isolates (including isolates from catheters and parenteral nutrition) were examined for molecular relatedness by PCR fingerprinting and restriction fragment length polymorphism (RFLP) analysis. The core sequence of phage M13 was used as a single primer in the PCR-based fingerprinting procedure, and RFLP analysis was performed with C. albicans-specific DNA probe 27A. Both techniques were evaluated with a panel of eight C. albicans reference strains, and each technique showed eight different patterns. With the 36 isolates from neonates, each technique enabled us to identify by PCR and RFLP analysis seven and six different patterns, respectively. The combination of these two methods (composite DNA type) identified eight different profiles. A strain with one of these profiles was present in three patients and in their respective catheters. Patients infected with or colonized by this isolate profile were clustered in time. Among the other patients, each patient was infected over time and at multiple anatomic sites with a C. albicans strain with a distinct DNA type. We conclude that C. albicans was most commonly producing long-term colonizations, although horizontal transmission probably due to catheters also occurred.
Lipopeptide L-733,560 is a water-soluble derivative of pneumocandin B0 that exhibits enhanced anti-Candida activity. We investigated the in vitro activity of L-733,560 compared with those of amphotericin B, flucytosine, and itraconazole, against fluconazole-resistant (n = 44) and fluconazole-susceptible (n = 46) Candida albicans isolates. Tests were performed with a photometer-read broth microdilution method with RPMI-2% glucose and National Committee for Clinical Laboratory Standards reference strains. Except for those of itraconazole, MICs were not significantly different between the two groups of isolates, as expected for agents with different mechanisms of action. L-733,560 was the most active agent against C.albicans, with MICs for 50 and 90% of the strains tested of 0.01 and 0.06 microgram/ml, respectively.
We describe a patient with AIDS who simultaneously developed Candida meningitis with three positive cerebrospinal fluid cultures and oral candidiasis. This patient also had a history or recurrent episodes of oral candidiasis treated with fluconazole. The patient did not respond to this therapy but was cured with amphotericin B and flucytosine. In vitro susceptibility tests revealed that each infection was caused by fluconazole-resistant Candida albicans isolates. Strain delineation by karyotyping, NotI restriction pattern analysis, hybridization with the specific probe 27A, and PCR fingerprinting with the phage M13 core sequence clearly demonstrated that meningitis and oral thrush were caused by two genetically different isolates.
The MICs of fluconazole for four reference strains of Candida albicans were determined in one laboratory by the macrobroth dilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) and by a modified version of the method which incorporated microbroth dilution, RPMI 1640 medium supplemented with glucose and the reading of MIC endpoints by spectrophotometry. The results obtained by the two methods were within one two-fold dilution. In addition, the susceptibilities of 58 clinical isolates of C. albicans to fluconazole were determined in two laboratories by the modified NCCLS method. Close interlaboratory agreement was observed, 98.2% of MICs varying by no more than one two-fold dilution.
We have compared amphotericin B, flucytosine, ketoconazole and fluconazole susceptibilities of 40 clinical isolates of Candida albicans by broth microdilution in two different media: RPMI 1640 (RPMI) and the same medium supplemented with 18 g of glucose per L (RPMI-2% glucose). Preparation of media, drugs and inocula, as well as incubation temperature, followed the recommendations of the National Committee for Clinical Laboratory Standards (Villanova, PA, USA) antifungal agent working group for broth macrodilution tests with antifungal agents. Microtitre plates were agitated for 5 min before spectrophotometric readings were performed with an automatic plate reader. MIC endpoints were defined in three different ways: (i) MIC-100% for amphotericin B and flucytosine; (ii) MIC-80% for azole-drugs and also for flucytosine; (iii) IC1/2 for azole-drugs. The MIC endpoints were compared between the two different media in order to ascertain which was the best criterion. For amphotericin B, 100% of strains had a maximum difference of a twofold dilution in both media. For flucytosine, MIC values were very similar in both media, irrespective of the MIC endpoint chosen, MIC-100% or MIC-80%. For azole-drugs the (MIC-80%)50 and (MIC-80%)90 in RPMI were higher than those in RPMI-2% glucose. However, (IC1/2)50 and (IC1/2)90 were identical in both media as well as (MIC-80%)50 and (MIC-80%)90 in RPMI-2% glucose, The limited growth of yeasts in RPMI precludes the selection of an azole-MIC-80% endpoint, although the MIC determination was performed with an objective turbidimetric method (spectrophotometric reading plus mathematical MIC calculation). The use of RPMI-2% glucose produces the same MIC whatever methods was used, IC1/2 or MIC-80%. However, some minor discrepancies can be expected between IC1/2 and MIC-80% when strains with higher "trailing effect" are being tested. Therefore, we recommended IC1/2 in RPMI-2% glucose as the method of choice for MIC calculation, until more studies correlating in-vitro results with clinical outcome have been performed.
Two groups of recent clinical isolates of Candida albicans consisting of 101 isolates for which fluconazole MICs were < or = 0.5 microgram/ml (n = 50) and > or = 4.0 micrograms/ml (n = 51), respectively, were compared for their susceptibilities to fluconazole, clotrimazole, miconazole, ketoconazole, and itraconazole. Susceptibility tests were performed by a photometer-read broth microdilution method with an improved RPMI 1640 medium supplemented with 18 g of glucose per liter (RPMI-2% glucose; J. L. Rodríguez-Tudela and J. V. Martínez-Suárez, Antimicrob. Agents Chemother. 38:45-48, 1994). Preparation of drugs, basal medium, and inocula was done by the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h as the lowest drug concentration at which growth was just equal to or less than 20% of that in the positive control well (MIC 80%). In vitro susceptibility testing separated azole-susceptible strains from the strains with decreased susceptibilities to azoles if wide ranges of concentrations (20 doubling dilutions) were used for ketoconazole, miconazole, and clotrimazole. By comparison with isolates for which fluconazole MICs were < or = 0.5 microgram/ml, those isolates for which fluconazole MICs were > or = 4.0 micrograms/ml were in general less susceptible to other azole drugs, but different patterns of decreased susceptibility were found, including uniform increases in the MICs of all azole derivatives, higher MICs of several azoles but not others, and elevated MICs of fluconazole only. On the other hand, decreased susceptibility to any other azole drug was never found among strains for which MICs of fluconazole were lower.
Four cases of fatal disseminated Scedosporium prolificans (inflatum) infection occurring in neutropenic patients are reported. Because of hospital renovation, the patients were cared for in a temporary hematologic facility. S. prolificans (inflatum) was isolated from blood cultures of these four patients, two of whom underwent full necropsy, and revealed abundant vegetative hyphae and ovoid conida with truncate bases in many organs. In vitro susceptibility testing of fungal strains showed all isolates to be resistant to amphotericin B, flucytosine, miconazole, ketoconazole, fluconazole, and itraconazole, with MICs greater than 16 micrograms/ml. The reported infections, two in each of two rooms, occurred over a period of 1 month, with very similar clinical outcomes. Circumstancial evidence suggested a nosocomial outbreak, but the environmental samples collected from the rooms, corridors, and adjacent areas did not yield S. prolificans (inflatum). Nevertheless, circumstantial evidence suggested a nosocomial outbreak of S. prolificans (inflatum) infection.
We have compared fluconazole susceptibilities of 92 clinical isolates of Candida albicans by broth microdilution in two different media: standard RPMI 1640 (RPMI) and the same medium supplemented with 18 g of glucose per liter (RPMI-glucose). Preparation of media, drugs, and inocula, as well as incubation conditions, followed the preliminary recommendations of the National Committee for Clinical Laboratory Standards (Villanova, Pa.) antifungal agent working group for broth macrodilution tests with antifungal agents, adapted to microdilution. Microtiter plates were agitated for 5 min before spectrophotometric readings were performed with an automatic plate reader set at 405 mm. The MIC endpoint was defined as an inhibitory concentration calculated from the turbidimetric data as a function of the turbidity in the drug-free control wells. The mean absorbances in the drug-free wells in RPMI and RPMI-glucose were, respectively, 0.38 (41.6% transmission) and 0.99 (10.2% transmission) (P < 0.001; Student's t test). Despite the increased growth in RPMI-glucose, 98.9% of the C. albicans strains tested for fluconazole susceptibility yielded similar MICs (+/- 1 dilution) in both media. Moreover, strains with decreased susceptibility to fluconazole displaying similar MICs in both media are easier to detect in RPMI-glucose because of the greater differences between turbidimetric readings in wells with grown or fluconazole-inhibited cultures. This objective turbidimetric method, with an easy-to-read improved medium (RPMI with glucose), together with previous experience of the National Committee for Clinical Laboratory Standards antifungal agent subcommittee, could overcome some of the present problems associated with lack of reproducibility of azole susceptibility testing.
A case of CDC group IVc-2 peritonitis in a patient on continuous ambulatory peritoneal dialysis (CAPD) is described. To the authors' knowledge, this is the first case reported of CAPD peritonitis in which a member of this unusual group of bacteria was isolated as the sole microorganism. As this microorganism is usually resistant to most antibiotics commonly used to treat peritonitis in patients on CAPD, microbiological investigations with identification and antimicrobial susceptibility tests are mandatory.
Oral doses of norfloxacin (80 mg/kg of body weight per day) and ciprofloxacin (25 and 80 mg/kg/day) and intramuscular doses of teicoplanin (5 mg/kg/day), all administered once a day for 10 days, were evaluated as a means of preventing encrusted cystitis caused by Corynebacterium group D2. Zinc disks dipped into a 24-h broth culture of these microorganisms were inserted into the bladders of female Wistar rats, and treatment was started 14 days after bacterial challenge. The appearance of encrusted cystitis was directly related to a documented urinary tract infection by these coryneforms (71.7 and 0% for rats with positive and negative urine cultures, respectively). All rats that died between days 18 to 43 after bacterial challenge presented very severe encrusted cystitis, which was prevented by teicoplanin and high doses of ciprofloxacin. Rats surviving up to day 44 after bacterial challenge were sacrificed; they presented a lower incidence of encrusted cystitis which was also less severe, with teicoplanin and a high dose of ciprofloxacin being more active in reducing the rate of positive cultures (78.8 and 65.7% reduction, respectively). All antibiotics and doses used were active in vivo at preventing encrusted cystitis by Corynebacterium group D2, but the best therapeutic effect was obtained with teicoplanin.
A new method for the examination of molds that involves the use of a device that dispenses a thin layer of a transparent adhesive material over the surface of a coverslip is described. The advantages of this method over previous methods used for the microscopic examination of molds are delineated.
This paper describes two possibly related cases of hospital-acquired Mycobacterium fortuitum peritonitis associated with continuous ambulatory peritoneal dialysis (CAPD). To our knowledge these are the first reported cases of CAPD peritonitis with possible cross-contamination by such a microorganism. Investigations for mycobacteria should be performed in all cases of culture-negative peritonitis, especially those which do not respond to the usual antimicrobial therapy.
The in-vitro activity of amoxycillin/clavulanic acid, amoxycillin, erythromycin, doxycycline, chloramphenicol and cephalexin against 183 clinical isolates of Haemophilus influenzae and 61 of Branhamella catarrhalis was determined. Amoxycillin/clavulanic acid showed the greatest activity against H. influenzae. All isolates of B. catarrhalis were susceptible to amoxycillin/clavulanic acid, chloramphenicol, cephalexin and doxycycline.