Biomedical subjects
Publications and source records attributed to J L Rodriguez-Tudela.
Metarrhizium anisopliae is a common insect pathogen that rarely causes infection in animals and humans. We report the first case of a disseminated skin infection in an immunocompromised adult patient. To date, only five cases of the disease in humans have been reported. There is no standard treatment for this infection.
BACKGROUND: To facilitate the design of strategies for prevention of invasive aspergillosis in solid-organ transplant recipients, this study investigates whether the development of early-onset and late-onset aspergillosis are related to different risk factors, thereby distinguishing 2 risk populations for this serious complication. METHODS: A retrospective case-control study was performed, including 156 cases of proven or probable invasive aspergillosis in patients recruited from 11 Spanish centers since the start of the centers' transplantation programs. RESULTS: Among all patients, 57% had early-onset IA (i.e., occurred during the first 3 months after transplantation). Risk factor analysis in this group identified as significantly associated risk factors a more complicated postoperative period, repeated bacterial infections or cytomegalovirus disease, and renal failure or the need for dialysis. Among patients with late-onset infections (i.e., occurred > 3 months after transplantation), who comprised 43% of cases, the patients at risk were older, were in an overimmunosuppressed state because of chronic transplant rejection or allograft dysfunction, and had posttransplantation renal failure. CONCLUSIONS: Risk factors in patients with early-onset cases and patients with late-onset cases of posttransplantation invasive aspergillosis are not the same, a fact that could have implications for the preventive approaches used for this infection.
The correlation between results obtained with the European Committee on Antibiotic Susceptibility Testing (EUCAST) antifungal susceptibility testing procedure (document 7.1) and four commercial systems was evaluated for a collection of 93 clinical isolates of Candida spp. Overall, agreement between the EUCAST procedure and the Sensititre YeastOne and Etest methods was 75% and 90.4%, respectively. The correlation indices (p < 0.01) between the EUCAST and commercial methods were 0.92 for Sensititre YeastOne, 0.89 for Etest, - 0.90 for Neo-Sensitabs, and 0.95 for Fungitest. Amphotericin B MICs obtained by Sensititre YeastOne were consistently higher than with the EUCAST method and, although very major errors were not observed, 91% of MICs were misclassified. Amphotericin B- and fluconazole-resistant isolates were identified correctly with Sensititre YeastOne, Etest and Fungitest. Neo-Sensitabs identified amphotericin B-resistant isolates, but misclassified > 5% of fluconazole-resistant isolates as susceptible. The commercial methods, particularly Etest and Fungitest, appeared to be suitable alternatives to the EUCAST procedure for antifungal susceptibility testing of clinical isolates of Candida.
Increased resistance to fluconazole has been reported in oral, oesophageal and urinary Candida isolates, but this has not been observed commonly in genital tract isolates. The rate of isolation of Candida spp. and their susceptibility to amphotericin B, flucytosine and azoles were determined in a number of clinical practices in the city of Ghent, Belgium. Patients with symptomatic vulvovaginal candidiasis (VVC) were treated with fluconazole, and the mycological and clinical outcomes were evaluated. Isolates were identified as Candida albicans (78.6%), Candida guilliermondii (17.3%), Candida glabrata (2.6%) and Candida dubliniensis (1.3%). The rates of mycological and clinical cures were 79.5% and 100%, respectively. Women with recurrent VVC were infected more frequently by non-albicans Candida spp., but no association was found between the use of antifungal agents and the presence of non-albicans spp. In-vitro resistance to fluconazole was not detected, even among subsequent Candida isolates from nine patients for whom mycological cure was not achieved.
The role of Aspergillus fumigatus 14alpha-sterol demethylase (Cyp51A) in azole drug susceptibility was assessed. Targeted disruption of cyp51A in azole-susceptible and -resistant strains decreased MICs from 2- to 40-fold. The cyp51A mutants were morphologically indistinguishable from the wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice.
The aim of this study was to compare MICs of fluconazole, itraconazole, posaconazole, and voriconazole obtained by the European Committee on Antibiotic Susceptibility Testing (EUCAST) and CLSI (formerly NCCLS) methods in each of six centers for 15 Candida albicans (5 fluconazole-resistant and 4 susceptible-dose-dependent [S-DD] isolates), 10 C. dubliniensis, 7 C. glabrata (2 fluconazole-resistant isolates), 5 C. guilliermondii (2 fluconazole-resistant isolates), 10 C. krusei, 9 C. lusitaniae, 10 C. parapsilosis, and 5 C. tropicalis (1 fluconazole-resistant isolate) isolates. CLSI MICs were obtained visually at 24 and 48 h and spectrophotometric EUCAST MICs at 24 h. The agreement (within a 3-dilution range) between the methods was species, drug, and incubation time dependent and due to lower EUCAST than CLSI MICs: overall, 94 to 95% with fluconazole and voriconazole and 90 to 91% with posaconazole and itraconazole when EUCAST MICs were compared against 24-h CLSI results. The agreement was lower (85 to 94%) against 48-h CLSI endpoints. The overall interlaboratory reproducibility by each method was > or =92%. When the comparison was based on CLSI breakpoint categorization, the agreement was 68 to 76% for three of the four species that included fluconazole-resistant and S-DD isolates; 9% very major discrepancies (< or =8 microg/ml versus > or =64 microg/ml) were observed among fluconazole-resistant isolates and 50% with voriconazole (< or =1 microg/ml versus > or =4 microg/ml). Similar results were observed with itraconazole for seven of the eight species evaluated (28 to 77% categorical agreement). Posaconazole EUCAST MICs were also substantially lower than CLSI MIC modes (0.008 to 1 microg/ml versus 1 to > or =8 microg/ml) for some of these isolates. Therefore, the CLSI breakpoints should not be used to interpret EUCAST MIC data.
A multicenter (six-center) study evaluated the performance (interlaboratory reproducibility, compatibility with reference methods, and categorical agreement) of 24-h visual and 48-h spectrophotometric MICs. MICs of fluconazole, itraconazole, voriconazole, and posaconazole were compared to reference 48-h microdilution broth visual MICs (CLSI [formerly NCCLS] M27-A2 document) for 71 isolates of Candida spp. that included 10 fluconazole-resistant strains. Twenty readings (5%) were reported as showing no growth at 24 h, mostly for Candida dubliniensis and from a single center. The overall interlaboratory agreement of 24-h visual readings and 48-h spectrophotometric MICs, as well their compatibility with reference values, were excellent with the four triazoles for most of the species (93 to 99%, within 3 dilutions). The categorical agreement between the investigational reading conditions and reference values was good with fluconazole and voriconazole (93 to 97%) but lower with itraconazole (86 to 88%), due primarily to minor errors. There were only 0 to 3% very major errors with these three triazoles; the number of substantial errors (more than three dilutions) was also low (<2%) with posaconazole. These data suggest that the performance of both investigational MIC readings gives results similar to those of reference MICs. Since spectrophotometric MICs are more objective and the 24-h time period would shorten the MIC determination of azoles, the description of either of these two reading conditions in the M27-A2 document should be considered by the CLSI subcommittee in addition to or instead of the longer, less practical, and more subjective 48-h visual MIC reading.
Five clinical isolates of Aspergillus fumigatus that exhibited similar patterns of reduced susceptibility to itraconazole and other triazole drugs were analyzed. Sequence analysis of genes (cyp51A and cyp51B) encoding the 14alpha-sterol demethylases revealed that all five strains harbored mutations in cyp51A resulting in the replacement of methionine at residue 220 by valine, lysine, or threonine. When the mutated cyp51A genes were introduced into an A. fumigatus wild-type strain, the transformants exhibited reduced susceptibility to all triazole agents, confirming that the mutations were responsible for the resistance phenotype.
Previous studies (Aufauvre-Brown et al., 1997; Mellado et al., 1996a,b ) have shown that only two genes of the Aspergillus fumigatus chitin synthase family, chsG and chsE, play a role in the morphogenesis of this fungal species. An A. fumigatus strain lacking both chsG (class III CHS) and chsE (class V CHS) genes was constructed by gene replacement of the chsE gene with a copy that has its conserved coding region interrupted by the hph resistance cassette in an A. fumigatus chsG- genetic background. Unexpectedly the double disruption was not lethal. The double mutant AfchsG-/chsE- strain (i) has reduced chitin synthase activity with or without trypsin stimulation, (ii) has a reduced colony radial growth rate, (iii) produces highly branched hyphae, (iv) exhibits aberrant features, such as periodic swellings along the length of the hyphae and a block in conidiation that can be partially restored by an osmotic stabilizer (v) shows alterations in the shape and germination capacity of the conidia, and (vi) has a cell wall that contains half the chitin of the parental strain and is, unexpectedly, highly enriched in alpha-(1-3) glucan.
The genes encoding 14alpha-sterol demethylases (cyp51A and cyp51B) were analyzed in 12 itraconazole (ITC)-resistant and three ITC-susceptible clinical isolates of Aspergillus fumigatus. Six ITC-resistant strains exhibited a substitution of another amino acid for glycine at position 54, which is located at a very conserved region of the Cyp51A protein. The cyp51A gene from the A. fumigatus wild-type strain (CM-237) was replaced with the mutated cyp51A gene copy of an ITC-resistant strain (AF-72). Two transformants exhibited resistance to ITC, both of which had incorporated the mutated copy of the cyp51A gene.
A three-laboratory study was performed to evaluate conidium counting in a hematocytometer as a technique of inoculum preparation for susceptibility testing of Aspergillus spp. In addition, inocula were quantified by colony counting and optical density determination. The agreement and correlation coefficient between conidium and colony quantifications were 89.2% and 0.73 (P < 0.01). Correlations with optical density determination were not significant.
Two different methods of inoculum preparation for susceptibility testing were analysed. The first method was adjustment of inoculum size by haemocytometer counting. The second method was spectrophotometric adjustment at 530 nm. The reliability of both methods was assessed by colony counting. The overall agreement between colony counting and haemocytometer counts was 93.6%, and the intraclass coefficient correlation was 0.71 (P < 0.05). Pearson's correlation index between colony counts and optical density values was -0.059 (P > 0.05). Optical densities ranged between 0.01 and 1.2, showing less reproducibility than expected by the NCCLS M 38-P standard. Haemocytometer counting is a more reliable method of inoculum preparation for antifungal susceptibility testing.
We describe six inpatients with acute non-lymphocytic leukaemia who developed invasive infection with Scedosporium prolificans resistant to amphotericin B, flucytosine, ketoconazole, fluconazole, and itraconazole. All six patients died. Phenotypic and genotypic assessment of samples from clinical material and ambient air from the isolation rooms where the patients were being treated showed that the epidemic was caused by a single strain. After implementation of aerial control measures, there were no further infections with this organism. We conclude that fatal multidrug-resistant S prolificans epidemics can be aerially transmitted and can be prevented with implementation of appropriate infection-control measures.
Scedosporium prolificans (SP) is an emerging opportunistic dematiaceous mould that causes serious infections in immunocompromised patients. Antifungal activities of human polymorphonuclear (PMN) and mononuclear (MNC) leukocytes against five SP isolates and Aspergillus fumigatus (AF) were evaluated. While monocyte-derived macrophages (MDM) phagocytosed conidia of both organisms comparably, they inhibited the germination of S. prolificans conidia less efficiently than those of A. fumigatus. Unopsonized hyphae of SP strains decreased the superoxide anion (O2-) produced by both PMN and MNC, whereas opsonized hyphae significantly stimulated it. In comparison to AF, phagocytes generally exhibited equal oxidative burst in response to SP. While PMN- and MNC-induced hyphal damage was similar among SP strains, phagocytes tended to damage SP hyphae to an equal or higher degree than AF hyphae. The susceptibility of SP to phagocytes contrasts with its high resistance to antifungal agents and may be related with the very low pathogenicity of the mould.
The activities of amphotericin B and itraconazole were studied in a temporarily neutropenic murine model of disseminated Absidia corymbifera infection, caused by two different strains. Amphotericin B MICs were 0.25 mg/L for both strains and itraconazole MICs were 1 and 2 mg/L. Amphotericin B was effective in vivo with both isolates. Itraconazole was less effective.
The in vitro susceptibility of 29 clinical isolates of fungi belonging to the division Zygomycota was reviewed. A broth microdilution test was performed by following the NCCLS reference method, with minor modifications. The species of Zygomycota tested were resistant in vitro to flucytosine, itraconazole and voriconazole. Amphotericin B showed the best activity against most of the strains evaluated.
Two cyp51-related genes (cyp51A and cyp51B) encoding 14-alpha sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-alpha sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-alpha sterol demethylase genes in other filamentous fungi.
OBJECTIVE: To study the epidemiologic aspects of a suspected outbreak of nosocomial invasive aspergillosis. METHODS: Sixteen Aspergillus fumigatus strains were isolated from bronchoalveolar washings or sputa of 10 patients during a 9-month period. Furthermore, two environmental samples, isolated in a microbiological screening of the hospital, were also available for analysis. Random amplified polymorphic DNA analysis (RAPD) was carried out. RESULTS: The analysis performed by RAPD clearly demonstrated substantial genetic variation among the isolates. Both of the two different primers selected for RAPD analysis (R-108 and AP12h) were able to demonstrate that the strains isolated from all patients infected with the same fungal species and the environmental samples were genotypically distinct. The results by RAPD typing demonstrated that this technique could detect variability among isolates of Aspergillus fumigatus from different patients and even from the same patient. CONCLUSIONS: RAPD genotyping proved that the outbreak of invasive aspergillosis consisted of a series of events, non-related, and probably not coming from the same source within the hospital. This type of analysis is an easy, quick and highly discriminatory technique that may help in planning epidemiologic studies of aspergillosis.