Cladophialophora Carrionii Research Articles

In vitroactivity of antifungal drugs againstCladophialophoraspecies associated with human chromoblastomycosis

The antifungal activity of six drugs was evaluated against 41 clinical and environmental isolates of Cladophialophora carrionii and its sister species C. yegresii. Drugs tested, including their ranges, were: 16-0.016 microg/ml for amphotericin B (AMB), itraconazole (ITZ) and voriconazole (VCZ), 8-0.008 microg/ml for terbinafine (TBF), and 64-0.063 microg/ml for flucytosin (5-FC) and fluconazole (FCZ). Strains were tested according to the CLSI guidelines (M38A). The MIC Gmeans for clinical strains in microg/ml were; 0.02 for TBF and ITZ, 0.07 microg/ml for VCZ, 0.49 for 5FC, 6.14 for FCZ and 9.42 for AMB. The MFC Gmeans in microg/ml were; 0.04 for TBF, 0.13 for ITZ, 0.72 for VCZ, 18.83 for 5FC, 36.16 for FCZ and 31.35 for AMB. The most active drugs against the fungi were TBF, ITZ, VCZ and 5FC. However, for all drugs more than two dilution steps were noted between the MIC and the MFC, indicating fungistatic activity. Despite in vitro susceptibility of C. carrionii to antifungal agents, the efficacy of therapy was shown to be not optimal.

Lipolytic activity of chromoblastomycosis agents measured by infrared spectroscopy and chemometric methods

The lipase activity of nine strains of six chromoblastomycosis agents (Fonsecaea pedrosoi, Phialophora verrucosa, Cladophialophora bantianum, Cladophialophora carrionii, Rhinocladiela aquaspersa and Exophiala jeanselmei) grown on solid medium was investigated using Fourier transform infrared spectroscopy and hierarchical clustering analysis. The data was quantified by p-nitrophenyl palmitate assay using partial least squares (PLS) regression. These methods allowed the correlation of six genera and species within the 1230-1650 and 2800+3000 cm(-1) spectral ranges among strains grown for 14 days from their respective lipolytic activity with RMSEV=0.048 and R2val=0.95 and ten latent variables. The lipolytic activity also was predicted using PLS models with 1230-1650 and 2800-3000 cm(-1) and 900-1450 cm(-1) spectral ranges for strains grown for 21 days. The separate analysis of F. pedrosoi strains yielded a prediction model for biomass at 21 days with RMSEV=0.065 and R2val=0.95 with eight latent variables using (1100-1300)+(1330-1460)+(1550-1650) cm(-1) spectral regions The best model obtained with the clustering of P. verrucosa, C. bantianum, C. carrionii, R. aquaspersa and E. jeanselmei strains was constructed with the same spectral ranges, but with RMSEV=0.074 and R2val=0.94 and ten latent variables. Infrared spectroscopy is suitable for the quantitation of extracellular lipase activity linked to the biomass of chromoblastomycosis agents.

Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment

Chromoblastomycosis is one of the most frequent infections caused by melanized fungi. It is a subcutaneous fungal infection, usually an occupational related disease, mainly affecting individuals in tropical and temperate regions. Although several species are etiologic agents, Fonsecaea pedrosoi and Cladophialophora carrionii are prevalent in the endemic areas. Chromoblastomycosis lesions are polymorphic and must be differentiated from those associated with many clinical conditions. Diagnosis is confirmed by the observation of muriform cells in tissue and the isolation and the identification of the causal agent in culture. Chromoblastomycosis still is a therapeutic challenge for clinicians due to the recalcitrant nature of the disease, especially in the severe clinical forms. There are three treatment modalities, i.e., physical treatment, chemotherapy and combination therapy but their success is related to the causative agent, the clinical form and severity of the chromoblastomycosis lesions. There is no treatment of choice for this neglected mycosis, but rather several treatment options. Most of the patients can be treated with itraconazole, terbinafine or a combination of both. It is also important to evaluate the patient's individual tolerance of the drugs and whether the antifungal will be provided for free or purchased, since antifungal therapy must be maintained in long-term regimens. In general, treatment should be guided according to clinical, mycological and histopathological criteria.

Cladophialophora carrionii: a rare agent of chromoblastomycosis in Rio de Janeiro State, Brazil

A 73 year-old male farm laborer from a rural area presented a 15 year history of extensive tumoral lesions over his left leg. Histological studies of skin biopsy showed pseudoepitheliomatous hyperplasia and granulomatous chronic inflammatory process with muriform cells, confirming chromoblastomycosis (CBM). Cladophialophora carrionii was isolated in culture. Treatment with itraconazole 400 mg/day for 12 months resulted in complete remission of lesions. As far we aware, this is the first case report of CBM caused by Cladophialophora carrionii in Rio de Janeiro State, Brazil.

In vitrointeraction of terbinafine with itraconazole and amphotericin B against fungi causing chromoblastomycosis in China

The combined effects of terbinafine with itraconazole and amphotericin B against Cladophialophora carrionii, Phialophora verrucosa and Fonsecaea pedrosoi were evaluated in vitro by the checker-board method and expressed as a fractional inhibitory concentration (FIC) index. Synergy was observed with the combination of terbinafine and itraconazole against one isolate of C. carrionii and no antagonism was observed. When amphotericin B was combined with terbinafine or itraconazole, no synergy or antagonism was noted with all isolates included in this investigation.

Chromoblastomycosis

Chromoblastomycosis is a chronic subcutaneous mycotic infection caused by pigmented or dematiaceous saprophytic moulds ubiquitous in the environment. The most common etiologic agents are Fonsecaea pedrosoi and Cladophialophora carrionii, both of which can be isolated from plant debris. The infection usually follows traumatic inoculation through penetrating thorn or splinter wounds. The fungal agents develop as small clusters of cells known as muriform bodies. Several months after the injury, painless papules or nodules appear in the affected area progressing to scaly and verrucose plaques. Direct examinations of skin scrapings or histopathologic study demonstrates the typical muriform bodies. Microbiologic culture is necessary for the correct determination of the etiologic agent. Itraconazole is the treatment of choice, often in combination with surgery. Even so, results are often unsatisfactory as patients present late to medical services because of lack of funds and the fact that the disease usually affects the main family earner.

Molecular analysis and pathogenicity of the Cladophialophora carrionii complex, with the description of a novel species.

Cladophialophora carrionii is one of the four major etiologic agents of human chromoblastomycosis in semi-arid climates. This species was studied using sequence data of the internal transcribed spacer region of rDNA, the partial β-tubulin gene and an intron in the translation elongation factor 1-alpha gene, in addition to morphology. With all genes a clear bipartition was observed, which corresponded with minute differences in conidiophore morphology. A new species, C. yegresii, was introduced, which appeared to be, in contrast to C. carrionii, associated with living cactus plants. All strains from humans, and a few isolates from dead cactus debris, belonged to C. carrionii, for which a lectotype was designated. Artificial inoculation of cactus plants grown from seeds in the greenhouse showed that both fungi are able to persist in cactus tissue. When reaching the spines they produce cells that morphologically resemble the muriform cells known as the “invasive form” in chromoblastomycosis. The tested clinical strain of C. carrionii proved to be more virulent in cactus than the environmental strain of C. yegresii that originated from the same species of cactus, Stenocereus griseus. The muriform cell expressed in cactus spines can be regarded as the extremotolerant survival phase, and is likely to play an essential role in the natural life cycle of these organisms.

ナノ銀粒子を静電吸着させたコラーゲン水解ペプチド（ＧＸ‐９５）の抗真菌活性

Silver has long been known to have an antimicrobial activity against bacteria and other microorganisms, and has been used as eating utensils, as dental fillings and so on. We developed a novel adduct, GX-95, of silver with nanometer-scale particles to peptidic hydrolysates from collagen. Antifungal activity of the adduct against pathogenic yeasts and filamentous fungi was examined in terms of minimal inhibitory concentrations (MICs). GX-95 was found to possess strong and broad antifungal activities against all fungi examined in the following MICs: 0.25 to 0.31 microg/ml for Candida albicans including resistant strains to fluconazole, itraconazole and flucytosine, 0.05 to 0.2 microg/ml for Cryptococcus neoformans strains, 0.025 to 0.4 microg/ml for Aspergillus fumigatus strains, 0.4 microg/ml for Trichophyton rubrum, and 0.05 microg/ml for Cladophialophora carrionii.

Human leukocyte antigen class I and MICA haplotypes in a multicase family withCladophialophora carrioniichromoblastomycosis

Previous studies carried out in an endemic semiarid region northwest of Venezuela at Falcon State have shown a prevalence of 15.4/1000 of chromoblastomycosis following traumatisms with xenophile vegetation infected with Cladophialophora carrionii. We performed high-resolution DNA typing of human leukocyte antigen (HLA)-A, -B and -C and major histocompatibility complex class I chain related gene A (MICA) alleles and segregation analysis in 49 members of one extended family with 12 affected individuals, who have lived for approximately 70 years in this endemic zone. None of the alleles, haplotypes or genotypes is shared by all the patients. No deviation from the expected HLA haplotype distribution or association of chromoblastomycosis with HLA-A, -B and -C haplotypes was observed. Further, a haplotype-sharing transmission/disequilibria testing of 11 nuclear families did not give enough evidence to claim linkage (P = 0.398), suggesting that genes located in the short arm of chromosome 6 may not be relevant in the immune response toward infection with C. carrionii in this Venezuelan endemic zone. Deleted MICA alleles on HLA-B*4802 haplotypes were present among several members of the extended family, but only two of them were affected.

Bacillus subtilis inhibits growth of Cladophialophora carrionii in vitro

The antifungal activity of Bacillus subtilis against a strain of Cladophialophora carrionii was evaluated using plate diffusion (PD) and sealed plate (SP) methods. For the PD test, the inoculum was obtained by homogenizing the log-phase fungus, adjusted to 0.5 McFarland and seeded (0.5 ml) on Sabouraud dextrose agar (SDA) plates; the B. subtilis extract was prepared from an 8-day culture, whose supernatant was lyophilized, concentrated 10 × and distributed (40 μl) in 4-mm holes previously punched in the seeded SDA plates. The plates were incubated at 30 °C for 72 hours. For the SP method, the inoculum was obtained from a 3-day fungal culture, seeded in the center of a SDA plate and placed over a SDA plate which had been inoculated with 0.2 ml of B. subtilis suspension. The two plates were sealed together and incubated for 7 days, either at room temperature or at 30 °C. With the PD method, a 20-mm diameter halo was observed after 72 h of incubation and the SP method showed 25% of fungal inhibition at both temperatures assayed. The results obtained with either method suggest the presence of metabolites from the bacterium that effectively inhibit the growth of C. carrionii. Although a single strain of C. carrionii was used here, these preliminary results provide grounds for further work to confirm the antifungal potential of B. subtilis against agents of chromoblastomycosis or to further characterize the chemical nature of the inhibitory substances involved.

Chromoblastomycosis in children and adolescents in the endemic area of the Falcón State, Venezuela

The present paper describes 22 cases of chromoblastomycosis (CBM) caused by Cladophialophora carrionii in children and adolescents (2-19 years old). The patients were seen between 1992 and 2004 and all resided in a CBM endemic area in the semi-arid zone of the Falcón state, Venezuela. Twelve of the 22 patients (54.55%) had close relatives who also had CBM and 19 (86.36%) were male. Lesions consisted of erythematous papules with desquamation or squamous plaques (0.12-14.19 cm in diameter), located primarily on the upper limbs (77.27% of patients). Thirteen of the patients were treated with topical 5-fluorouracil (5-FU; 1% cream), seven with topical ajoene (0.5% gel) and two had electrodesiccation and/or fulguration. Two patients who did not respond to 5-FU were treated with oral itraconazole (100 mg/day for 1 month). Complete clinical and mycological remission was achieved in 17/20 (85%) of the patients treated with 5-FC, ajoene and electrodesiccation and/or fulguration. In addition, similar results were obtained with the two patients who received itraconazole therapy. These cases emphasize the importance of early diagnosis in difficult-to-treat mycotic diseases such as CBM. By early intervention we were able to employ topical treatment with a minimum of adverse effects to achieve a high percentage of favorable therapeutic responses. The patients were thus able to avoid the evolution of the chronic, deforming and incapacitation clinical manifestations associated with CBM.

Inmunoanálisis enzimático (ELISA) en la evolución terapéutica de la cromoblastomicosis por Cladophialophora carrionii en el área endémica del Estado Falcón, Venezuela

The purpose of this research was to evaluate an ELISA indirect method in patients with chromoblastomycosis caused by Cladophialophora carrionii. Samples collected before, during and postreatment with ajoene or itraconazole, and those from apparently healthy people from the endemic area were evaluated with the ELISA test. 94 individuals were studied, 10 with chromoblastomycosis, and 84 apparently healthy subjects. All of them were evaluated by clinical-dermatological examinations. On those with lesions suggestive of chromoblastomycosis, mycological studies were carried out to confirm the disease. This approach was repeated during and at the end of therapy. Five patients with lesions < or = 5 cms were treated with ajoene and five with lesions > 5 cms, received itraconazole. Mycological cure (60%) was similar in both groups of patients and persisted three months after therapy. One hundred and fourteen sera were analyzed by ELISA, 30 from 10 patients with chromoblastomycosis, before, during and postreatment and 84 from apparently healthy people, using a somatic antigen of C. carrionii (AgSPP). All patients with chromoblastomycosis were positive before-treatment, two became negative on day 45 of treatment and a total of six patients were negative three months post-treatment. All sera from apparently healthy individuals were negative. The sensitivity and specificity was 100% and 98.9%, respectively. The relationship between clinical-mycological studies and the ELISA assay was 100% before and after treatment. In summary, ELISA could be a valuable tool for the diagnosis and evolution of the therapeutic efficacy in patients with chromomycosis (C. carrionii). The use of an ELISA test is therefore highly recommended to establish remission criteria in chromoblastomycosis caused by C. carrionii.

Chromoblastomycosis: An Unusual Diagnosis in Europe

Sir, Chromoblastomycosis is a chronic cutaneous and subcutaneous infection due to a group of dematiaceous fungi including Fonsecaea pedrosoi, Cladophialophora carrionii, Phialophora verrucosa, F. compacta and Rhinocladiella aquaspersa (1). This infection occurs in tropical and subtropical regions and results from traumatic implantation of material contaminated by the fungus (1, 2). We report here a case of chromoblastomycosis imported from French Polynesia which was diagnosed in metropolitan France and responded to itraconazole.

In vitroactivities of new and established triazoles against opportunistic filamentous and dimorphic fungi

The in vitro activities of three new triazoles were determined and compared to those of itraconazole and fluconazole against 306 clinical isolates of Blastomyces dermatitidis, Cladophialophora carrionii, Coccidioides immitis, Fonsecaea pedrosoi, Fusarium spp., Histoplasma capsulatum, Paecilomyces lilacinus, Pseudallescheria boydii and Sporothrix schenckii. Minimum inhibitory concentrations (MIC) were determined by a broth macrodilution method of the National Committee for Clinical Laboratory Standards M38-A procedure. Itraconazole (geometric mean MIC, 0.16-0.65 microg/ml), voriconazole (geometric mean MIC, 0.18-1.44 microg/ml), ravuconazole (geometric mean MIC, 0.18-1.09 microg/ml), and posaconazole (geometric mean MIC, 0.18-1.38 microg/ml), had relatively uniform values showing potent in vitro inhibitory activity against B. dermatitidis, C. carrionii, C. immitis, F. pedrosoi, H. capsulatum, and S. schenckii. The in vitro activity was variable with strains of P. boydii, P. lilacinus and Fusarium spp.

Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis

The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.

Black fungi: clinical and pathogenic approaches

Data are presented on the clinically relevant black yeasts and their relatives, i.e., members of the Ascomycete order Chaetothyriales. In order to understand the pathology of these fungi it is essential to know their natural ecological niche. From a relatively low degree of molecular variability of the black yeast Exophiala dermatitidis, potential agent of brain infections in patients from East Asia, it is concluded that this species is an emerging pathogen, currently going through a process of active speciation. It is found to be an oligotrophic fungus in hot, moist environments, such as steambaths. Cladophialophora-, Fonsecaea- and Ramichloridium-like strains, known in humans as agents of chromoblastomycosis, are frequently found on rotten plant material, but the fungal molecular diversity in the environment is much higher than that on the human patient, so that it is difficult to trace the etiological agents of the disease with precision. This approach has been successful with Cladophialophora carrionii, of which cells resembling muriform cells, the tissue form of chromoblastomycosis, were found to occur in drying spines of cacti. Phagocytosis assays provide a method to distinguish between pathogens and non-pathogens, as the killing rates of strict saprobes proved to be consistently higher than of those species frequently known as agents of disease. The therapeutic possibilities for patients with chromoblastomycosis are reviewed.

Dematiaceous fungal pathogens: analysis of ribosomal DNA gene polymorphism by polymerase chain reaction-restriction fragment length polymorphism.

Restriction fragment length polymorphisms (RFLP) of ribosomal gene small subunit (SSU rDNA) and internal transcribed spacer (ITS) regions was examined in 12 isolates of dematiaceous agents of chromoblastomycosis and phaeohyphomycosis. The amplicon length of the fragment ITS1-ITS4, comprising the 5.8 rDNA and ITS1-ITS2 spacers, ranged in size from 620 to 690 bp. This result indicated a polymorphism of size in this region. Additionally the RFLP profiles showed a high degree of inter- and intra-specific variability. In contrast, the SSU rDNA amplification, using NS1-NS2 primers, originated a fragment of approximately 570 bp and its restriction profile proved to be well conserved among the species studied and was clustered into only two genetically heterogeneous groups, the first one formed by Fonsecaea pedrosoi and Fonsecaea compacta and the second one formed by Cladophialophora (Cladosporium) carrionii, Cladophialophora (Xylohypha) bantiana, Phialophora verrucosa and Rhinocladiella species.

Partial chemical characterization of antigenic preparations of chromoblastomycosis agents.

Antigenic preparations (saline, methylic, metabolic and exoantigens) of four agents of chromoblastomycosis, Fonsecaea pedrosoi, Phialophora verrucosa, Cladophialophora (Cladosporium) carrionii and Rhinocladiella aquaspersa were obtained. Partial chemical characterization of these antigenic preparations was obtained by determination of the levels of total lipids, protein, and carbohydrates, and identification of the main sterols and carbohydrates. Methylic antigens presented the highest lipid contents, whereas metabolic antigens showed the highest carbohydrate content. Total lipid, protein, and carbohydrate levels were in the range of 2.33 to 2.00 mg/ml, 0.04 to 0.02 mg/ml and 0.10 to 0.02 mg/ml, respectively, in the methylic antigens and in the range of 0. 53 to 0.18 mg/ml, 0.44 to 0.26 mg/ml, and 1.82 to 1.02 mg/ml, respectively, in saline antigens. Total lipid, protein, and carbohydrate contents were in the range of 0.55 to 0.20 mg/ml, 0.69 to 0.57 mg/ml and 10.73 to 5.93 mg/ml, respectively, in the metabolic antigens, and in the range of 0.55 to 0.15 mg/ml, 0.62 to 0.20 mg/ml and 3.55 to 0.42 mg/ml, respectively, in the exoantigens. Phospholipids were not detected in the preparations. Saline and metabolic antigens and exoantigens presented hexose and the methylic antigen revealed additional pentose units in their composition. The UV light absorption spectra of the sterols revealed squalene and an ergosterol fraction in the antigens. The characterization of these antigenic preparations may be useful for serological evaluation of patients of chromoblastomycosis.

Mitochondrial DNA analysis of Phialophora verrucosa.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) was examined in 32 isolates of Phialophora verrucosa (eight isolates from Japan, 10 from China, four from the USA, six from Venezuela and four from Colombia) and in three of Phialophora americana using five restriction enzymes. P. verrucosa isolates were divided into 10 mtDNA types based on RFLP patterns. Phylogeny constructed on sequence divergence of mtDNA indicated that P. verrucosa is a single species and isolates are clustered into three groups. Japan and the USA contained Group A and Group B isolates, China Group B isolates and South America Group B and Group C isolates. RFLP patterns of P. americana mtDNA were identical to those of Type 1 or Type 4 of P. verrucosa mtDNA, suggesting that both are identical. RFLP patterns of P. verrucosa were distinct from those of other dematiaceous fungi including Exophiala jeanselmei, E. moniliae, E. dermatitidis, E. spinifera, Cladophialophora (Cladosporium) carrionii, Fonsecaea pedrosoi, and Hortaea werneckii. These results indicate that RFLP analysis of mtDNA is a useful method for the identification, taxonomy, typing, epidemiology and phylogeny of P. verrucosa.