New advances in antifungal treatment.
Clinical aspects of treatment of invasive aspergillosis, infections caused by dematiaceous fungi, and mycoses caused by endemic, dimorphic fungi, are described in this review.
Antifungal Agents↗
Biomedical subjects
F C Odds
Publications and source records attributed to F C Odds.
New targets and delivery systems for antifungal therapy.
Development of new approaches for treatment of invasive fungal infections encompasses new delivery systems for approved and investigational compounds, as well as exploiting the cell membrane, cell wall and virulence factors as putative antifungal targets. Novel delivery systems consisting of cyclodextrins, cochleates, nanoparticles/nanospheres and long circulating ('stealth') liposomes, substantially modulate the pharmacokinetics of existing compounds, and may also be useful to enhance the delivery of antifungal agents to sites of infection. Further insights into the structure-activity relationship of the antifungal triazoles that target the biosynthesis of ergosterol in the fungal cell membrane have led to the development of highly potent broad spectrum agents, including posaconazole, ravuconazole and voriconazole. Similarly, a novel generation of cell-wall active semisynthetic echinocandin 1,3 beta-glucan inhibitors (caspofungin, FK463, and VER-002) has entered clinical development. These agents have potent and broad-spectrum activity against Candida spp, and potentially useful activity against Aspergillus spp. and Pneumocystis carinii. The ongoing convergence of the fields of molecular pathogenesis, antifungal pharmacology and vaccine development will afford the opportunity to develop novel targets to complement the existing antifungal armamentarium.
Antifungal Agents↗
Bioassays for itraconazole blood levels: an interlaboratory collaborative study.
Duplicate bioassays for itraconazole and hydroxy-itraconazole were run with 30 serum samples in five laboratories, each using a different method. Both itraconazole and hydroxy-itraconazole were used as standards. Despite quantitative variations, the results of the bioassays correlated sufficiently to indicate the relative level of antifungal activity in the test samples.
Antifungal Agents↗
Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans.
There is an immediate need for identification of new antifungal targets in opportunistic pathogenic fungi like Candida albicans. In the past, efforts have focused on synthesis of chitin and glucan, which confer mechanical strength and rigidity upon the cell wall. This paper describes the molecular analysis of CaMNT1, a gene involved in synthesis of mannoproteins, the third major class of macromolecule found in the cell wall. CaMNT1 encodes an alpha-1, 2-mannosyl transferase, which adds the second mannose residue in a tri-mannose oligosaccharide structure which represents O-linked mannan in C. albicans. The deduced amino acid sequence suggests that CaMnt1p is a type II membrane protein residing in a medial Golgi compartment. The absence of CaMnt1p reduced the ability of C. albicans cells to adhere to each other, to human buccal epithelial cells, and to rat vaginal epithelial cells. Both heterozygous and homozygous Camnt1 null mutants of C. albicans showed strong attenuation of virulence in guinea pig and mouse models of systemic candidosis, which, in guinea pigs, could be attributed to a decreased ability to reach and/or adhere internal organs. Therefore, correct CaMnt1p-mediated O-linked mannosylation of proteins is critical for adhesion and virulence of C. albicans.
Amino Acid Sequence↗
Functional aspects of secreted Candida proteinases.
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Animals↗
[What functions do six different genes for secretory proteinases have in Candida albicans?].
Secreted Aspartate Proteinases (Sap) are among those factors of the human pathogen Candida albicans, which promote infections in the immunocompromised host. Sap isoenzymes are encoded by at least nine different genes (SAP1-9), which are differentially regulated in vitro. RT-PCR analysis during experimental infections and from patient samples confirmed the expression of SAP genes in vivo. However, while Sap2 is the dominant isoenzyme under culture conditions, other SAP genes are also expressed during infections. In order to investigate the role of single isoenzymes during the pathogenesis of candidosis, mutants were produced which harbour deletions in SAP1, SAP2, SAP3 and SAP4-6. Although only SAP2 and SAP4-6 mutants showed a strong reduction of proteolytic activity in vitro, all SAP mutants were significantly attenuated in systemic infections. In addition, SAP2, SAP3 and SAP4-6 mutants were clearly more sensitive to neutrophilic leucocytes compared to the wild type SC5314. These investigations show that several proteinase isoenzymes are likely to be involved in the pathogenesis of candidosis.
Candida albicans↗
Evaluation of possible correlations between antifungal susceptibilities of filamentous fungi in vitro and antifungal treatment outcomes in animal infection models.
Nine isolates of filamentous fungi previously tested in 11 different laboratories for their susceptibilities to amphotericin B and itraconazole in vitro were injected intravenously into mice and guinea pigs, and responses to treatment with both agents were studied. The experiments were done in a single laboratory. Mean survival times, the percentages of animals surviving 12 days after infection, and culture results for samples of deep organs obtained postmortem were used as markers of antifungal efficacy. Because of variations in organism pathogenicity, interpretable test systems in vivo could not be established for Fusarium spp. in mice or guinea pigs or for Pseudallescheria boydii in mice, even with the use of immunosuppressive pretreatments. Among the infections that could be evaluated, some degree of response to the corresponding treatment in vivo was seen in animals infected with each of two Rhizopus arrhizus isolates susceptible to amphotericin B at < 0.5 microg/ml and Aspergillus spp. isolates susceptible to itraconazole at < 1.0 microg/ml. Conversely, no responses were apparent with infecting strains for which MICs were > or = 2 microg/ml (amphotericin B) or > or = 1 microg/ml (itraconazole). However, the limitations of the intravenous challenge systems studied mean that no firm conclusion relating MICs in vitro to the lowest effective doses in vivo could be drawn.
Amphotericin B↗
Prevalence of Candida dubliniensis isolates in a yeast stock collection.
To establish the historical prevalence of the novel yeast species Candida dubliniensis, a survey of 2,589 yeasts originally identified as Candida albicans and maintained in a stock collection dating back to the early 1970s was undertaken. A total of 590 yeasts, including 93 (18.5%) beta-glucosidase-negative isolates among 502 isolates that showed abnormal colony colors on a differential chromogenic agar and 497 other isolates, were subjected to DNA fingerprinting with the moderately repetitive sequence Ca3. On this basis, 53 yeasts were reidentified as C. dubliniensis (including the C. dubliniensis type strain, included as a blind control in the panel of yeasts). The 52 newly found isolates came from 36 different persons, and a further 3 C. dubliniensis isolates were detected by DNA fingerprinting of previously untested isolates from one of these individuals. The prevalence of C. dubliniensis among yeasts in oral and fecal samples was significantly higher than that among yeasts from other anatomical sites and was significantly higher among human immunodeficiency virus (HIV)-infected individuals than among known or presumed HIV-negative individuals. However, a single vaginal isolate and two oral isolates from healthy volunteers confirmed that the species is restricted neither to gastrointestinal sites nor to patients with overt disease. The oldest examples of C. dubliniensis were from oral samples of three patients in the United Kingdom in 1973 and 1975. In comparison with age-matched control isolates of C. albicans, the C. dubliniensis isolates showed slightly higher levels of susceptibility in vitro to amphotericin B and flucytosine and slightly lower levels of susceptibility to three azole antifungal agents.
Antifungal Agents↗
Reduction in the growth of non-dermatophyte moulds by itraconazole: evaluation by corneofungimetry assay.
The activity of itraconazole against fungi of dermatological importance was studied using corneofungimetry performed on four test organisms: Aspergillus flavus, Fusarium oxysporum, Scopulariopsis brevicaulis and Scytalidium dimidiatum. The human stratum corneum used in the bioassay was harvested from healthy volunteers and from patients undergoing treatment with itraconazole 200 mg daily for at least 2 weeks. A fungal mycelium grew on all untreated stratum corneum samples within 1 week. The pattern and extent of growth varied greatly among the test organisms. An inhibitory effect was observed on the itraconazole-treated samples. However, the level of inhibition was higher than that expected by calculating itraconazole minimum inhibitory concentrations in a reference susceptibility test. It is concluded that some non-dermatophyte moulds of medical importance can grow by forming hyphae on human stratum corneum. However, the process is partly inhibited by some unknown mechanisms originating from the stratum corneum itself. It is further inhibited by the oral administration of itraconazole. This global effect might be the result of the complementation inhibition due to the drug and/or some unknown components of the stratum corneum.
Antifungal Agents↗
Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients.
During a study of oral rinses of 130 HIV-infected individuals, both typical and atypical Candida albicans colonies were isolated from ten patients on a yeast differential medium. Typical Candida albicans colonies were light green; atypical colonies were dark green. Both types of colonies were germ tube-positive and produced chlamydospores. However, DNA fingerprinting of the atypical isolates with the Ca3 Candida albicans-specific probe showed that they belonged to the recently described species Candida dubliniensis. Candida dubliniensis colonies could also be differentiated from Candida albicans colonies on isolation plates by the absence of fluorescence of colonies on methyl blue-Sabouraud agar under Wood's light. Among other phenotypic characteristics, only the absence of intracellular beta-glucosidase activity reliably distinguished Candida albicans from Candida dubliniensis. Candida dubliniensis may be underreported in clinical samples because most currently used isolation and identification methods fail to recognize this yeast.
Acquired Immunodeficiency Syndrome↗
Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and candida infections. Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards.
The availability of reproducible antifungal susceptibility testing methods now permits analysis of data correlating susceptibility in vitro with outcome in vivo in order to define interpretive breakpoints. In this paper, we have examined the conceptual framework underlying interpretation of antimicrobial susceptibility testing results and then used these ideas to drive analysis of data packages developed by the respective manufacturers that correlate fluconazole and itraconazole MICs with outcome of candidal infections. Tentative fluconazole interpretive breakpoints for MICs determined by the National Committee for Clinical Laboratory Standards' M27-T broth macrodilution methodology are proposed: isolates for which MICs are < or = 8 microg/mL are susceptible to fluconazole, whereas those for which MICs are > or = 64 microg/mL appear resistant. Isolates for which the MIC of fluconazole is 16-32 microg/mL are considered susceptible dependent upon dose (S-DD), on the basis of data indicating clinical response when > 100 mg of fluconazole per day is given. These breakpoints do not, however, apply to Candida krusei, as it is considered inherently resistant to fluconazole. Tentative interpretive MIC breakpoints for itraconazole apply only to mucosal candidal infections and are as follows: susceptible, < or = 0.125 microg/mL; S-DD, 0.25-0.5 microg/mL; and resistant, > or = 1.0 microg/mL. These tentative breakpoints are now open for public commentary.
Animals↗
Molecular biological characterization of an azole-resistant Candida glabrata isolate.
Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were previously shown to differ in quantity and activity of the cytochrome P-450 14alpha-lanosterol demethylase which is the target for azole antifungals. The resistant isolate also had a lower intracellular level of fluconazole, but not of ketoconazole or itraconazole, than the susceptible isolate. In the present study a 3.7-fold increase in the copy number of the CYP51 gene, encoding the 14alpha-lanosterol demethylase, was found. The amount of CYP51 mRNA transcript in the resistant isolate was eight times greater than it was in the susceptible isolate. Hybridization experiments on chromosomal blots indicated that this increase in copy number was due to duplication of the entire chromosome containing the CYP51 gene. The phenotypic instability of the resistant isolate was demonstrated genotypically: a gradual loss of the duplicated chromosome was seen in successive subcultures of the isolate in fluconazole-free medium and correlated with reversion to susceptibility. The greater abundance of the amplified chromosome induced pronounced differences in the protein patterns of the susceptible and revertant isolates versus that of the resistant isolate, as demonstrated by two-dimensional gel electrophoresis (2D-GE). Densitometry of the 2D-GE product indicated upregulation of at least 25 proteins and downregulation of at least 76 proteins in the resistant isolate.
Antifungal Agents↗
Isolation of Candida species on media with and without added fluconazole reveals high variability in relative growth susceptibility phenotypes.
Mouthwashes from human immunodeficiency virus-positive individuals were sampled for yeasts by direct plating on a differential agar medium with and without added fluconazole and via enrichment broths with and without added fluconazole. The colonies of the yeasts isolated were tested for relative growth in the presence of single concentrations of itraconazole and fluconazole. Among 258 culture plates containing yeasts obtained via different isolation routes from 86 yeast-positive samples, 33 (12.7%) of the plates showed unexpectedly high colony-to-colony variation in relative growth. Intercolony variation was seen in 41 (47.7%) of the 86 isolates when relative growth data were analyzed for all colonies of an isolate tested, regardless of the medium used for isolation. The prevalence of relative growth variability with the azoles was highest for Candida glabrata (100% of 13 isolates), followed by Candida krusei (60% of 5 isolates) and Candida albicans (40% of 53 isolates), and the visual patterns of variability seen in scatter plots of the data showed species specificity. Relative growth phenotypes generally tended to be stable for each yeast colony in subcultures, whether or not the medium used for subculture contained antifungal agents. DNA fingerprinting of stable and variable C. albicans isolates showed changes in band patterns detected with the probe Ca3, suggesting that the variability may have resulted from selection of different subtypes of the yeasts during the isolation procedure. These findings suggest that the yeasts isolated from single clinical samples were often not clonal in nature. The relative growth test revealed colony variability more readily than conventional susceptibility testing.
Antifungal Agents↗
Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence.
Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null mutants were similar to those of the isogenic wild-type parental strain (SC5314) in complex and defined media. In medium with protein as the sole source of nitrogen, sap1 and sap3 mutants grew with reduced growth rates but reached optical densities similar to those measured for SC5314. In contrast, sap2 null mutants tended to clump, grew poorly in this medium, and produced the lowest proteolytic activity. Addition of ammonium ions reversed such growth defects. These results support the view that Sap2 is the dominant isoenzyme. When sap1, sap2, and sap3 mutants were injected intravenously in guinea pigs and mice, the animals had increased survival rates compared to those of control animals infected with SC5314. However, reduction of proteolytic activity in vitro did not correlate directly with the extent of attenuation of virulence observed for all Sap-deficient mutants. These data suggest that SAP1, SAP2, and SAP3 all contribute to the overall virulence of C. albicans and presumably all play important roles during disseminated infections.
Animals↗
A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence.
Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multigene family with at least nine members (SAP1 to SAP9) and are considered putative virulence factors important for the pathogenicity of this human pathogen. The role of Sap isoenzymes in the virulence of C. albicans has not yet been clearly established, and therefore, using recent progress in the genetics of this yeast, we have constructed a panel of isogenic yeasts, each with a disruption of one or several SAP genes. We focused on the construction of a C. albicans strain in which three related SAP genes (SAP4, SAP5, and SAP6) were disrupted. Growth of the delta sap4,5,6 triple homozygous null mutant DSY459 in complex medium was not affected, whereas, interestingly, growth in a medium containing protein as the sole nitrogen source was severely impaired compared to the growth of the wild-type parent strain SC5314. Since the presence of Sap2 is required for optimal growth on such medium, this suggests that Sap4, Sap5, or Sap6 plays an important role for the process of induction of SAP2. When guinea pigs and mice were injected intravenously with DSY459, their survival time was significantly longer than that of control animals infected with the wild-type SC5314. Attenuated virulence of DSY459 was followed by a significant reduction of yeast cells in infected organs. These data suggest that the group of Sap4, Sap5, and Sap6 isoenzymes is important for the normal progression of systemic infection by C. albicans in animals.
Animals↗
Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi.
A multicenter study was conducted to expand the generation and analysis of data that supports the proposal of a reference method for the antifungal susceptibility testing of filamentous fungi. Broth microdilution MICs of amphotericin B and itraconazole were determined in 11 centers against 30 coded duplicate pairs of Aspergillus spp., Fusarium spp., Pseudallescheria boydii, and Rhizopus arrhizus. The effect of inoculum density (approximately 10(3) and 10(4) CFU/ml), incubation time (24, 48, and 72 h), and procedure of MIC determination (conventional and colorimetric [Alamar Blue] evaluation of growth inhibition) on intra- and interlaboratory agreement was analyzed. Based on intra- (97 to 100%) and interlaboratory (94 to 95%) agreement for both drugs, the overall optimal testing conditions identified were determination of colorimetric MICs after 48 to 72 h of incubation with an inoculum density of approximately 10(4) CFU/ml. These testing conditions are proposed as guidelines for a reference broth microdilution method.
Antifungal Agents↗
Candida and Torulopsis: a blinded evaluation of use of pseudohypha formation as basis for identification of medically important yeasts.
Seventy yeast isolates representing species in the genera Candida and Torulopsis but excluding Candida albicans were examined in three laboratories for production of pseudohyphae in Dalmau cultures. The microscopic morphology of the isolates was scrutinized by four individuals experienced in yeast identification and three inexperienced persons, all of whom were blinded as to the putative identification of the yeasts. For 49 (70%) of the 70 isolates, the seven observers recorded comparable scores for morphology, but 5 (7%) of the isolates showed extreme variation in recorded morphologies, from true hyphae formed to no pseudohyphae formed. Isolates of Candida parapsilosis and Torulopsis glabrata consistently did and did not form pseudohyphae, respectively: however, other Candida and Torulopsis spp. did not always express their expected morphologies. In 48 (19%) of 252 readings (seven observers), 36 isolates of Candida spp. were scored as forming no pseudohyphae, and in 22 (9.2%) of 238 readings, 34 isolates of Torulopsis spp. were recorded as forming true hyphae or pseudohyphae. These results show that pseudohypha formation is not a reliable characteristic for identification of yeasts at the genus level; we suggest that the merger of Torulopsis spp. into the genus Candida should be finally accepted.
Candida↗
Mycology in oral pathology.
Candida species are responsible for all but exceptional examples of oral fungal infection. Oral Candida lesions are seen only in patients who are predisposed to such disease by physiological or immunological abnormalities, particularly by extremes of age and HIV infections. The infections can be acute or chronic, pseudomembranous ("thrush") or atrophic (erythemateous). In the AIDS patient, mixtures of clinical types may be seen. Diagnosis of oral Candida infection requires microscopic or culture proof of the involvement of a Candida species. Treatment depends on the type of patient and the type of infection. Topical antifungal agents, usually of the imidazole, triazole or polyene type, are commonly used, although non-specific antiseptics are recommended for denture cleansing in cases of denture-associated stomatitis.
AIDS-Related Opportunistic Infections↗