Tissue invasiveness and non-acidic pH in human candidiasis correlate with "in vivo" expression by Candida albicans of the carbohydrate epitope recognised by new monoclonal antibody 1H4.

BACKGROUND: The morphogenetic conversion between yeast and hyphal growth forms appears to be crucial in the pathogenesis of invasive candidiasis, and can be regulated by environmental signals such as extracellular pH. AIMS: To characterise the epitope recognised by monoclonal antibody 1H4, and to evaluate the expression of its corresponding epitope in Candida albicans cells under different conditions of pH and temperature, and "in vivo", in tissue samples from patients with human candidiasis. METHODS: Monoclonal antibody 1H4 was generated against the 58 kDa cell wall mannoprotein of C albicans (mp58), and was further characterised by immunoblot analysis, periodate treatment of the antigenic preparations, and agglutination experiments of C albicans strains 3153A, SC5314, and 412, cultured under different environmental conditions (growth media and pH). An immunohistochemical study was performed in 24 human tissue samples from patients with mucocutaneous and systemic candidiasis. RESULTS: 1H4 recognises a pH sensitive carbohydrate epitope on the surface of C albicans cells, and this epitope is not restricted to mp58, but is shared with other cell wall mannoproteins. Immunohistochemical findings indicated that expression of the 1H4 epitope on C albicans cells in tissue sections from human candidiasis correlates with tissue invasion and pH of the niche. 1H4 immunoreactivity was also found in candida remnants within macrophages. CONCLUSIONS: The fact that 1H4 epitope expression selectively identifies invasive forms of C albicans, in addition to candida remnants within macrophages, supports its potential value in the diagnosis and management of human candidiasis.

Candidemia at a tertiary-care hospital: epidemiology, treatment, clinical outcome and risk factors for death.

The demographic, clinical and microbiological data of patients with candidemia at the "Hopital Universitario La Fe", a tertiary-care hospital in Valencia, Spain, from 1995 to 1997 was analyzed retrospectively. Candida spp. were isolated in blood cultures from 145 patients, 32% of whom were children (25% of these were neonates). The most common species isolated was Candida albicans, followed by Candida parapsilosis, Candida krusei and Candida tropicalis. Risk factors for candidemia included underlying disease, therapy with broad-spectrum antibiotics and the presence of a central venous catheter. The majority of children were treated with amphotericin B, whereas 52% of adults received fluconazole. Overall mortality was 44% (30% in children and 50% in adults), and attributable mortality was 30% (24% in children and 33% in adults). Multivariate analysis indicated that neutropenia, corticosteroid therapy, lack of antifungal treatment, and failure to replace the central venous catheter were factors associated with candidemia-related death. Among the adult population, an APACHE II score greater than 15 predicted candidemia-related death.

Two cases of fungemia due to Candida lusitaniae and a literature review.

Reported here are two cases of candidemia caused by Candida lusitaniae that occurred in two immunocompromised patients at Hospital Universitario "La Fe" in Valencia, Spain. Case 1 involved a low-birth-weight premature infant with congenital nephrotic syndrome who was successfully treated with amphotericin B, and case 2 involved a 50-year old woman with a high-grade malignancy lymphoma who succumbed to the infection. Antifungal susceptibility testing of the Candida lusitaniae isolates recovered from both patients revealed sensitivity to amphotericin, 5-flucytosine and fluconazole. Results are presented and discussed together with a comprehensive review of the literature, covering all previously reported cases of fungemia caused by this emerging pathogen.

Correlación entre las pruebas de sensibilidad in vitro a los antifúngicos y la evolución clínica de los pacientes con candidiasis y criptococosis / Correlation between in vitro susceptibility to antifungal drugs and the clinical evolution of patients with candidiasis and cryptococcosis

El aumento del número de infecciones fúngicas, así como la descripción de resistencias frente a los diversos antifúngicos, ha puesto de manifiesto la necesidad de realizar estudios de sensibilidad in vitro que sean útiles para predecir la evolución clínica de los pacientes con este tipo de infecciones. Recientemente, el National Committee for Clinical Laboratory Standards (NCCLS) aprobó un método de referencia para las pruebas de sensibilidad a los antifúngicos que reflejó en el documento M27-A. Este avance importante en la estandarización de las pruebas de sensibilidad in vitro ha permitido también comparar los resultados obtenidos en el laboratorio con la evolución de los pacientes. En este artículo se revisan los estudios de correlación de la sensibilidad in vitro con la evolución clínica de los pacientes con infecciones fúngicas. En general se puede predecir la evolución clínica, sobre todo en los pacientes infectados por el VIH con candidiasis orofaríngea tratados con fluconazol. Sin embargo, en otros grupos más heterogéneos de pacientes no es fácil relacionar la sensibilidad in vitro con la evolución clínica (AU)

Heterogeneous mechanisms of azole resistance in Candida albicans clinical isolates from an HIV-infected patient on continuous fluconazole therapy for oropharyngeal candidosis.

Molecular mechanisms of azole resistance in Candida albicans include alterations in the target enzyme and increased efflux of drug, but the impact of specific treatment regimens on resistance has not been established. A patient with advanced AIDS was enrolled in a longitudinal study to receive continuous oral fluconazole (FLU) 200 mg/day for the treatment of oropharyngeal candidosis (OPC). Oral cultures were obtained at time of enrollment, during episodes of OPC and quarterly for surveillance. The patient had five symptomatic relapses on continuous FLU during 43 months. All OPC episodes were successfully treated with increasing doses of FLU although increased FLU MICs were detected for C. albicans isolates with progression of time. DNA-typing techniques demonstrated that resistance developed in a persistent strain of C. albicans. Both FLU-resistant and isogenic isolates with reduced susceptibility were detected in the same clinical samples through multiple episodes. Analysis of molecular mechanisms of resistance revealed overexpression of MDR and CDR genes encoding efflux pumps (but not ERG11) in isolates with decreased FLU susceptibility. In addition, the presence of the G464S amino acid substitution in their lanosterol demethylase, affecting its affinity for FLU, was also detected. However, other isogenic, but FLU-susceptible isolates recovered from the same samples did not harbour the mutation, indicating microevolution of yeast populations within the oral cavity. In this patient, the continuous antifungal pressure exerted by FLU resulted in development of resistance of multifactorial nature. Despite their clonal origin, different subpopulations of C. albicans demonstrated distinct resistance mechanisms, including concomitant presence and absence of functional point mutations in ERG11 genes.

[Correlation between in vitro susceptibility to antifungal drugs and the clinical evolution of patients with candidiasis and cryptococcosis]. / Correlación entre las pruebas de sensibilidad in vitro a los antifúngicos y la evolución clínica de los pacientes con candidiasis y criptococosis.

The increase in the incidence of fungal infections and the emergence of resistance call for the development of techniques for measuring in vitro antifungal susceptibility that are useful for predicting clinical outcome in patients suffering from these infections. In the past, the lack of standardized testing techniques led to poor intra- and interlaboratory reproducibility. Recently, the National Committee for Clinical Laboratory Standards (NCCLS) has developed a reference method for antifungal susceptibility testing, document M27A. This document is a necessary and important step towards the standardization of antifungal susceptibility testing, which has important implications in the analysis of clinical and microbiological data. This article provides a comprehensive review of studies correlating in vitro antifungal susceptibility testing and clinical outcome. In general, it is possible to predict the therapeutic outcome, especially in HIV infected patients with oropharyngeal candidiasis treated with fluconazole. However, in other more heterogeneous groups of patients it is more difficult to correlate the in vitro and in vivo data.

Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients.

Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles in C. albicans strains displaying high-level fluconazole resistance (MICs, > or =64 microg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14alpha-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.

Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.

Candida albicans is implicated in many biomaterial-related infections. Typically, these infections are associated with biofilm formation. Cells in biofilms display phenotypic traits that are dramatically different from those of their free-floating planktonic counterparts and are notoriously resistant to antimicrobial agents. Consequently, biofilm-related infections are inherently difficult to treat and to fully eradicate with normal treatment regimens. Here, we report a rapid and highly reproducible microtiter-based colorimetric assay for the susceptibility testing of fungal biofilms, based on the measurement of metabolic activities of the sessile cells by using a formazan salt reduction assay. The assay was used for in vitro antifungal susceptibility testing of several C. albicans strains grown as biofilms against amphotericin B and fluconazole and the increased resistance of C. albicans biofilms against these antifungal agents was demonstrated. Because of its simplicity, compatibility with a widely available 96-well microplate platform, high throughput, and automation potential, we believe this assay represents a promising tool for the standardization of in vitro antifungal susceptibility testing of fungal biofilms.

Biofilm formation by Candida dubliniensis.

Candida dubliniensis is an opportunistic yeast closely related to Candida albicans that has been recently implicated in oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Most manifestations of candidiasis are associated with biofilm formation, with cells in biofilms displaying properties dramatically different from free-living cells grown under normal laboratory conditions. Here, we report on the development of in vitro models of C. dubliniensis biofilms on the surfaces of biomaterials (polystyrene and acrylic) and on the characteristics associated with biofilm formation by this newly described species. Time course analysis using a formazan salt reduction assay to monitor metabolic activities of cells within the biofilm, together with microscopy studies, revealed that biofilm formation by C. dubliniensis occurred after initial focal adherence, followed by growth, proliferation, and maturation over 24 to 48 h. Serum and saliva preconditioning films enhanced the initial attachment of C. dubliniensis and subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to further characterize C. dubliniensis biofilms. Mature C. dubliniensis biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material. C. dubliniensis biofilms displayed spatial heterogeneity and an architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile C. dubliniensis cells, including the type strain and eight different clinical isolates, against fluconazole and amphotericin B compared to their planktonic counterparts. C. dubliniensis biofilm formation may allow this species to maintain its ecological niche as a commensal and during infection with important clinical repercussions.

Candida dubliniensis in radiation-induced oropharyngeal candidiasis.

Candida dubliniensis is a recently described species that has been shown to cause oropharyngeal candidiasis in patients with HIV. We present a detailed evaluation of a patient undergoing head and neck radiation for oral cancer who developed oropharyngeal candidiasis from a mixed infection of C dubliniensis and Candida albicans. To our knowledge, this is the first described case of C dubliniensis contributing to oropharyngeal candidiasis in this patient population.

Identification of continuous B-cell epitopes on the protein moiety of the 58-kiloDalton cell wall mannoprotein of Candida albicans belonging to a family of immunodominant fungal antigens.

The 58-kiloDalton mannoprotein (mp58) on the surface of Candida albicans is highly immunogenic, is expressed by all C. albicans isolates tested, and elicits strong antibody responses during candidiasis. It belongs to a family of immunodominant fungal antigens with representatives also in different species of Aspergillus. The amino acid sequence of the protein portion of mp58 as deduced from the DNA sequence of its encoding gene (FBP1/PRA1) was used to synthesize a complete set of overlapping dodecapeptides (overlap, 7; offset, 5) covalently attached to the surface of derivatized polyethylene pins. The pin-coupled peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by a number of antiserum preparations containing anti-mp58 antibodies. This comprehensive epitope-scanning study revealed the presence of multiple immunoreactive continuous B-cell epitopes within the protein sequence. Regions of increased reactivity included both the amino and carboxy termini of the mature protein (encompassing amino acid residues 16 to 50 and 286 to 299, respectively) and four internal regions spanning amino acids at positions 66 to 92, 121 to 142, 148 to 192, and 211 to 232. Further delineation of epitopic regions and identification of the boundaries of the antigenic sites was performed upon ELISA testing with a second Pepset consisting of completely overlapping 8-mer peptides spanning these reactive regions in the protein moiety of mp58. The highly reactive epitopic region at the C terminus of the protein was further evaluated using both window net and replacement net analyses. A synthetic peptide corresponding to the last 10 amino acid residues at the C terminus of the protein was immunogenic when injected into mice after being coupled to a carrier protein. Moreover, antibodies in the resulting sera specifically recognized the homologous mp58 in ELISAs and immunoblot assays. Delineation of the antibody responses to mp58 could provide the basis for the development of novel immunity-based prophylactic, therapeutic, and diagnostic techniques for the management of candidiasis.

Characteristics of biofilm formation by Candida albicans.

A variety of manifestations of Candida albicans infections are associated with the formation of biofilms on the surface of biomaterials. Cells in biofilms display phenotypic traits that are dramatically different from their free-floating planktonic counterparts, such as increased resistance to anti-microbial agents and protection form host defenses. Here, we describe the characteristics of C. albicans biofilm development using a 96 well microtitre plate model, microscopic observations and a colorimetric method based on the use of a modified tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide, XTT) to monitor metabolic activities of cells within the biofilm. C. albicans biofilm formation was characterized by initial adherence of yeast cells (0-2 h), followed by germination and micro-colony formation (2-4 h), filamentation (4-6 h), monolayer development (6-8 h), proliferation (8-24 h) and maturation (24-48 h). The XTT-reduction assay showed a linear relationship between cellular density of the biofilm and metabolic activity. Serum and saliva pre-conditioning films increased the initial attachment of C. albicans, but had minimal effect on subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize C. albicans biofilms. Mature C. albicans biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material. C. albicans biofilms displayed a complex three dimensional structure which demonstrated spatial heterogeneity and a typical architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile C. albicans cells against clinically used fluconazole and amphotericin B as compared to their planktonic counterparts.

Growth competition between Candida dubliniensis and Candida albicans under broth and biofilm growing conditions.

Seven isolates each of Candida albicans and Candida dubliniensis were paired (11 pairs) and examined for competitive interaction. Equal numbers of CFU of each competitor were inoculated into Sabouraud dextrose broth and incubated at 37 degrees C with vigorous shaking under conditions favorable to either broth or biofilm growth. Surviving proportions of each competitor were calculated from the broth culture at 24 and 96 h and the biofilm culture at 96 h, with species differentiation done on CHROMagar Candida medium. C. albicans had a competitive advantage over C. dubliniensis in broth culture and under biofilm growing conditions; however, with the presence of a supporting structure for biofilm formation, C. dubliniensis was able to better withstand the competitive pressures from C. albicans.

Immunodetection of CD45 epitopes on the surface of Candida albicans cells in culture and infected human tissues.

Candida albicans is a leading cause of disseminated fungal infection in immunocompromised patients. Candida-host cell interactions are mediated at the cell surface. Since blood-group I epitopes have been detected on the surface of C albicans cells, we investigated whether CD45, the molecule that carries the I antigen on human lymphocytes, is present on the C albicans cell surface, in culture and in human tissue specimens of human candidiasis. By using monoclonal antibodies to CD45, CD45RO, and CD45RA, we found a strong immunoreactivity at the cell surface of blastoconidia bearing germ tubes but weak or no immunostaining of the germ tubes themselves. In human tissues, immunostaining of C albicans yeast cells was detected, whereas pseudohyphae were mostly negative. CD45 epitopes on the surface of C albicans might have a role in tissue invasion and dissemination of the fungus. On the other hand, its detection may disturb quantitative non-morphology-based determinations of lymphoid cell populations in infected tissues.

Comparison of DNA-based typing methods to assess genetic diversity and relatedness among Candida albicans clinical isolates.

Three serial isolates of Candida albicans were obtained from each of five HIV infected patients with recurrent oropharyngeal candidiasis from the same geographical area. Isolates from one patient remained susceptible to fluconazole whereas serial isolates from the other four patients showed decreasing susceptibilities to the drug. Strain identity was investigated by pulse-field gel electrophoretic (PFGE) separation of chromosomes, restriction fragment length polymorphism (RFLP) of chromosomal DNA, Southern blot analysis with the moderately repetitive probe Ca3 of the materials present in the RFLP gels after transfer to nylon membranes, and random amplification of polymorphic DNA (RAPD). All techniques were able to group isolates obtained from the same patient. Techniques resulting in more complex banding profiles exhibited increased discriminatory power allowing detection of strain variants. Methods resulting in less complex banding patterns, especially Southern hybridization of SfiI digested chromosomal DNA with the moderately repetitive probe Ca3, were more helpful to determine isogenicity among isolates obtained from the same patient. The combination of results from methods with high discriminatory power (to maximize detection of strain variants) and methods resulting in less complex banding patterns (to allow determination of isogenic isolates) should facilitate the delineation of the epidemiology of C. albicans infection.

Epidemiology of oropharyngeal Candida colonization and infection in patients receiving radiation for head and neck cancer.

Oral mucosal colonization and infection with Candida are common in patients receiving radiation therapy for head and neck cancer. Infection is marked by oral pain and/or burning and can lead to significant patient morbidity. The purpose of this study was to identify Candida strain diversity in this population by using a chromogenic medium, subculturing, molecular typing, and antifungal susceptibility testing of clinical isolates. These results were then correlated with clinical outcome in patients treated with fluconazole for infection. Specimens from 30 patients receiving radiation therapy for head and neck cancer were cultured weekly for Candida. Patients exhibiting clinical infection were treated with oral fluconazole. All isolates were plated on CHROMagar Candida and RPMI medium, subcultured, and submitted for antifungal susceptibility testing and molecular typing. Infections occurred in 27% of the patients and were predominantly due to Candida albicans (78%). Candida carriage occurred in 73% of patients and at 51% of patient visits. Yeasts other than C. albicans predominated in carriage, as they were isolated from 59% of patients and at 52% of patient visits. All infections responded clinically, and all isolates were susceptible to fluconazole. Molecular typing showed that most patients had similar strains throughout their radiation treatment. One patient, however, did show the acquisition of a new strain. With this high rate of infection (27%), prophylaxis to prevent infection should be evaluated for these patients.

Interactions between Candida albicans and the human extracellular matrix component tenascin-C.

Tenascins are large multimeric proteins that contain repeated structural motifs that include epidermal growth factor (EGF)-like repeats, fibronectin type III repeats and a globular fibrinogen-like domain, and are involved in tissue and organ morphogenesis, as well as in adhesion and migration of cells. C. albicans germ-tubes, but not blastospores, were able to bind to soluble human tenascin-C as revealed by an indirect immunofluorescence assay. However, materials present in cell wall extracts from both morphologies attached to tenascin-C immobilized in wells of a microtiter plate. The binding specificity was demonstrated by the inhibitory effect of antibodies against C. albicans cell wall components and an anti-tenascin antibody, but not anti-laminin antibody. Fibronectin, but not fibrinogen, inhibited binding, thus indicating a role of the fibronectin type III repeats in the interaction between the fungus and tenascin-C. Binding of C. albicans cell wall materials to tenascin was RGD- and divalent cation-independent.

Low levels of antigenic variability in fluconazole-susceptible and -resistant Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis.

Three serial isolates of Candida albicans were obtained by direct swab or by oral saline rinses from each of five human immunodeficiency virus-infected patients with recurrent oropharyngeal candidiasis. Genotyping techniques confirmed the presence of a persistent strain in multiple episodes from the same patient, which was different from the strains isolated from other patients. Fluconazole susceptibility was determined by both an agar dilution method and the National Committee for Clinical Laboratory Standards macrobroth procedure. In four of these patients the strains developed fluconazole resistance, and in one patient the strain remained susceptible. The different isolates were propagated as yeast cells on a synthetic medium, and their cell wall proteinaceous components were extracted by treatment with beta-mercaptoethanol. Protein and mannoprotein components present in the extracts were analyzed by electrophoresis, immunoblotting, and lectin-blotting techniques. The analysis showed a similar composition, with only minor qualitative and quantitative differences in the polypeptidic and antigenic patterns associated with the cell wall extracts from serial isolates from the same patient, as well as those from different strains isolated from different patients. Use of monospecific antibodies generated against two immunodominant antigens during candidiasis (enolase and the 58-kDa fibrinogen-binding mannoprotein) demonstrated their expression in all isolates tested. Overall, the antigenic makeup of C. albicans strains remained constant during the course of infection and was not affected by development of fluconazole resistance. In contrast to previous reports, the low degree of antigenic variability observed in this study may be due to the fact that the isolates were obtained from a highly homogeneous population of patients and to the uniformity in techniques used for the isolation, storage, and culture of the different strains, as well as extraction methodologies.