Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates.

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp51A and cyp51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp51A and cyp51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp51A and cyp51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.

Genotypic Analysis of the Population Structure in Malassezia globosa and Malassezia restricta.

The molecular characterization of Malassezia spp. isolates from animals and humans has not been thoroughly studied. Although a range of molecular methods has been developed for diagnosing Malassezia species, they have several drawbacks, such as inefficiency in differentiating all the species, high cost and questionable reproducibility. The present study aimed to develop VNTR markers for genotyping Malassezia isolated from clinical and animal samples. A total of 44 M. globosa and 24 M. restricta isolates were analyzed. Twelve VNTR markers were selected on seven different chromosomes (I, II, III, IV, V, VII and IX), six for each Malassezia species. The highest discriminatory power for a single locus was obtained with the STR-MG1 marker (0.829) and STR-MR2 marker (0.818) for M. globosa and M. restricta, respectively. After the analysis of multiple loci, 24 genotypes were noted among 44 isolates in M. globosa, with a discrimination index D of 0.943 and 15 genotypes were noted among 24 isolates in M. restricta, with a discrimination index D of 0.967. An endogenous infection was detected in two patients. Different genotypes of M. globosa strains colonized one patient. Interestingly, VNTR markers analysis revealed a carriage between a breeder and his dog in three cases for M. globosa and two for M. restricta. The FST (0.018 to 0.057) values indicate a low differentiation between the three populations of M. globosa. These results suggest a dominant clonal mode of reproduction in M. globosa. The typing of M. restricta showed a genotypic diversity of the strains, which can cause various skin pathologies. However, patient five was colonized with strains having the same genotype collected from different body parts (back, shoulder). VNTR analysis was capable of identifying species with high accuracy and reliability. More importantly, the method would facilitate monitoring Malassezia colonization in domestic animals and humans. It was shown that the patterns are stable and the method is discriminant, making it a powerful tool for epidemiological purposes.

Production and Quantification of Virulence Factors in Malassezia Species.

Seventy-seven strains of Malassezia were included in this study. Biofilm and hydrolytic enzyme production were studied by using specific solid media. The Real-Time reverse transcriptase qPCR method was applied to determine the overexpression of genes encoding the extracellular enzymes. All included Malassezia species produced biofilms. No statistically significant difference was observed between Malassezia species in biofilm formation (p = 0.567). All Malassezia species produced lipase, and 95% of Malassezia globosa showed a strong enzymatic activity (Pz = 0.55 ± 0.02). A statistically significant difference was observed between the mean keratinase indices of Malassezia slooffiae and the other Malassezia species (p = 0.005). The overexpression of one or more genes was observed in 100% of strains isolated from patients with folliculitis, 87.5% - with pityriasis versicolor, and 57.14% of the control group isolates. A statistically significant difference in the lipase gene expression (p = 0.042) was between the strains from patients with folliculitis and the control group. This investigation provides more information about the frequency of the production of the major enzymes considered virulence factors of Malassezia species. Interestingly, the overexpression of one or more genes was observed in strains isolated from patients with Malassezia disorders.

Molecular Genotyping of Candida parapsilosis Species Complex.

BACKGROUND: The Candida parapsilosis complex species has emerged as an important cause of human disease. The molecular identification of C. parapsilosis isolates at the species level can be helpful for epidemiological studies and then for the establishment of appropriate therapies and prophylactic measures. METHODS: The present study was undertaken to analyze 13 short tandem repeat (STR) markers (7 minisatellites and 6 microsatellites) in a global set of 182 C. parapsilosis complex isolates from different origins including invasive and superficial clinical sites. RESULTS: Upon the analysis of 182 strains of C. parapsilosis complex species, 10-17 haplotypes were detected for each minisatellite marker. The combination of 7 minisatellite markers yielded 121 different genotypes with a 0.995 D value. Upon the analysis of 114 isolates (68 from invasive infections and 46 from superficial infections), 21-32 genotypes were detected for each microsatellite marker. The combination of all 13 markers yielded 96 different genotypes among 114 isolates with a high degree of discrimination (0.997 D value). The same multilocus genotype was shared by isolates recovered from some patients and from the hand of theirs correspondent healthcare worker. For another patient, the same multilocus genotype of C. metapsilosis was detected in blood and skin confirming that candidemia usually arises as an endogenous infection following prior colonization. CONCLUSIONS: These STR markers are a valuable tool for the differentiation of C. parapsilosis complex strains, to support epidemiological investigations especially studies of strain relatedness and pathways of transmission.

Virulence factors, antifungal susceptibility and molecular mechanisms of azole resistance among Candida parapsilosis complex isolates recovered from clinical specimens.

BACKGROUND: The aim of this study was to determine the biofilm formation, the extracellular enzymatic activities of 182 clinical isolates of the Candida parapsilosis complex. METHODS: Molecular identification of the C. parapsilosis species complex was performed using PCR RFLP of SADH gene and PCR sequencing of ITS region. The susceptibility of ours isolates to antifungal agents and molecular mechanisms underlying azole resistance were evaluated. RESULTS: 63.5% of C. parapsilosis were phospholipase positive with moderate activity for the majority of strains. None of the C. metapsilosis or C. orthopsilosis isolates was able to produce phospholipase. Higher caseinase activities were detected in C. parapsilosis (Pz = 0.5 ± 0.18) and C. orthopsilosis (Pz = 0.49 ± 0.07) than in C. metapsilosis isolates (Pz = 0.72 ± 0.1). 96.5% of C. parapsilosis strains and all isolates of C. metapsilosis and C. orthopsilosis produced gelatinase. All the strains possessed the ability to show haemolysis on blood agar. C. metapsilosis exhibited the low haemolysin production with statistical significant differences compared to C. parapsilosis and C. orthopsilosis. The biofilm forming ability of C. parapsilosis was highly strain dependent with important heterogeneity, which was less evident with both C. orthopsilosis and C. metapsilosis. Some C. parapsilosis isolates met the criterion for susceptible dose dependent to fluconazole (10.91%), itraconazole (16.36%) and voriconazole (7.27%). Moreover, 5.45% and 1.82% of C. parapsilosis isolates were respectively resistant to fluconazole and voriconazole. All strains of C. metapsilosis and C. orthopsilosis were susceptible to azoles; and isolates of all three species exhibited 100% of susceptibility to caspofungin, amphotericin B and 5-flucytosine. CONCLUSIONS: A combination of molecular mechanisms, including the overexpression of ERG11, and genes encoding efflux pumps (CDR1, MDR1, and MRR1) were involved in azole resistance in C. parapsilosis.

Real-Time PCR Identification of Six Malassezia Species.

Lipophilic yeast Malassezia species is widely found on the skin surface of humans and other animals. This fungus can cause pityriasis versicolor, Malassezia folliculitis, and seborrheic dermatitis. Still now, there is a problem with species identification of Malassezia with conventional methods. We developed a real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting M. globosa, M. furfur, M. restricta, M. sympodialis, M. slooffiae, and M. pachydermatis. The amplification curves and specific melting peaks of the probes hybridized with real-time PCR product were used for species identifications. The assay was further evaluated on 120 samples which were performed by swabbing from 60 domestic animals (23 goats, 10 dogs, 15 cows, 3 cats, 8 rabbits, and 1 donkey) and in 70 human samples (28 patients with pityriasis versicolor, 17 breeders, and 25 control group). Fifteen M. pachydermatis were identified from animals. From human, 61 isolates were identified as M. globosa (28), M. furfur (15), M. restricta (6), M. sympodialis (8), M. slooffiae (2), and M. pachydermatis (2). Eight cases of co-detection from 6 patients and 2 breeders were revealed. Our findings show that the assay was highly effective in identifying Malassezia species. The application of multiplex real-time PCR provides a sensitive and rapid identification system for Malassezia species, which may be applied in further epidemiological surveys from clinical samples.

First genotype identification of Trichosporon asahii in Sfax, Tunisia.

PURPOSE: The objectives of our study were species identification and genotyping of Trichosporon isolates collected at the Parasitology and Mycology Laboratory in Sfax, Tunisia. METHODOLOGY: Molecular identification was carried out by analysing the IGS1 regions of the rDNA of 30 Trichosporon isolates. RESULTS: Trichosporon asahii was the most frequent species detected. Furthermore, four genotypes were identified in Tunisia: 1 (46.4 %), 4 (35.7 %), 7 (14.3 %) and 3 (3.6 %). In vitro antifungal susceptibility testing of the isolates showed that voriconazole exhibited the highest activity. CONCLUSION: This is the first reported study of genotype identification of T. asahii in Tunisia and even in the African continent.

Molecular study of the Candida parapsilosis complex in Sfax, Tunisia.

Candida parapsilosis, which was previously considered to be a complex of three genetically distinct groups, has emerged as a significant agent of nosocomial infections. Recently, this complex was separated into three species: C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis In Tunisia, data pertaining to these fungi are limited. Thus, the purpose of our study was to determine by BanI PCR-RFLP and ITS sequencing, the occurrence of Candida parapsilosis complex among 182 isolates identified as C. parapsilosis by phenotypical methods. C. parapsilosis sensu stricto represented 94.5% of all isolates, while C. metapsilosis and. C. orthopsilosis were identified in 3.3% and 2.2%, respectively. Sequence analysis of internal transcribed spacer region confirmed and revealed only one genotype among the C. parapsilosis sensu stricto strains, three genotypes among six C. metapsilosis strains and two genotypes among four C. orthopsilosis strains.

Molecular characterization of strains of the Trichophyton verrucosum complex from Tunisia.

Trichophyton verrucosum is the most frequent etiologic agent of cattle dermatophytosis. Throughout the world, it was the second most common agent of zoophilic dermatophytes in human. The aim of our study was to evaluate the efficacy of the PCR- RFLP and PCR-sequencing methods for the identification and differentiation of T. verrucosum strains.Thirty-six clinical strains identified by morphological characteristics as T. verrucosum were isolated from patients referred to parasitology-mycology laboratory of Sfax University Hospital. Identification of our strains by conventional methods was confirmed by molecular methods in 94.4% of cases. Two strains were reclassified as T. violaceum PCR products digested with HinfI produced three profiles and two patterns with MvaI. Sequence analysis revealed a polymorphism in the ITS1and 5.8S regions. Analysis and alignment of consensus sequences has distinguished two types of genotypes among our T. verrucosum strains. The ITS type I was the dominant genotype (93.7%). Phylogenetic study showed that one cluster comprised T. verrucosum strains with ITS type I and species of T. mentagrophytes complex. It was related to Arthroderma vanbreuseghemii complex. The other cluster contained the two T. verrucosum strains with ITS type II, and was related to Arthroderma benhamiae complex. In this study, most of T. verrucosum isolates were type I, dissimilar to others rare studies where type II has been the most common. Specie and strain differentiation is relevant because it helps in prescribing the correct treatment and determining the source of the infection.

Changes in genotype and fluconazole susceptibility of isolates from patients with Candida glabrata in Tunisia.

UNLABELLED: Candida glabrata has emerged as an opportunistic pathogen of considerable importance in invasive and superficial infections. AIMS: To analyze the development of fluconazole resistance in patients under treatment through epidemiological survey in our hospital. PATIENTS AND METHODS: Twenty two patients (89 clinical strains) were collected. Molecular typing of isolates was performed by polymorphic markers. Analysis of gene expression was realized by reverse transcriptase-real time polymerase chain reactions (RT-qPCR). RESULTS: Genetic analysis showed that 63% persists with apparently unchanged strains (n=14). Among them, four showed fluconazole resistance development. A strain replacement was observed in 6 patients and two patients selected more resistant isolates during the course of treatment. An analysis of Candida glabrata cerebellar degeneration-related protein 1 (CgCDR1), Candida glabrata cerebellar degeneration-related protein 2 (CgCDR2) and Candida glabrata sterol 14 alpha-demetylase Erg 11 (CgERG11) expression revealed an over-expression in 10 resistant isolates. CONCLUSION: This study demonstrated that C. glabrata strain undergo frequent changes in vivo. The increase in CgCDR1 and CgCDR2 expression was the most mechanism associated with fluconazole resistance.

Trailing or paradoxical growth of Aspergillus flavus exposed to caspofungin is independent of genotype.

There are limited data on in vitro susceptibility testing of echinocandins against Aspergillus species. The objective of this study was to describe the phenotypes of Aspergillus flavus observed on exposure to caspofungin in vitro and to test whether these phenotypes were associated with A. flavus genotypes. The caspofungin MICs of 37 A. flavus clinical isolates collected from 14 patients with invasive aspergillosis were determined using Etest assays. Caspofungin MICs ranged from 0.012 to 0.064 mg l(-1); the modal MIC was 0.023 mg l(-1) and the MIC50 and MIC90 were 0.032 and 0.064 mg l(-1), respectively. A clear end point was noted in 24 (65 %) isolates, whereas seven (19 %) displayed a trailing effect and six (16 %) showed paradoxical growth when exposed to caspofungin. In these A. flavus isolates, the absence of a significant population structure or genetic differentiation indicated that trailing or paradoxical growth phenotypes were independent of microsatellite genotype.

Highly discriminatory variable-number tandem-repeat markers for genotyping of Trichophyton interdigitale strains.

Trichophyton interdigitale is the second most frequent cause of superficial fungal infections of various parts of the human body. Studying the population structure and genotype differentiation of T. interdigitale strains may lead to significant improvements in clinical practice. The present study aimed to develop and select suitable variable-number tandem-repeat (VNTR) markers for 92 clinical strains of T. interdigitale. On the basis of an analysis of four VNTR markers, four to eight distinct alleles were detected for each marker. The marker with the highest discriminatory power had eight alleles and a D value of 0.802. The combination of all four markers yielded a D value of 0.969 with 29 distinct multilocus genotypes. VNTR typing revealed the genetic diversity of the strains, identifying three populations according to their colonization sites. A correlation between phenotypic characteristics and multilocus genotypes was observed. Seven patients harbored T. interdigitale strains with different genotypes. Typing of clinical T. interdigitale samples by VNTR markers displayed excellent discriminatory power and 100% reproducibility.

Microsatellite typing of Aspergillus flavus in patients with various clinical presentations of aspergillosis.

Aspergillus flavus is the second most important Aspergillus species associated with aspergillosis and the incidence of infections caused by it are increasing in the immunocompromised population. This species is of major epidemiological importance in regions with a dry and hot climate. Despite the growing clinical significance of A. flavus, data on its molecular epidemiology are scarce. This study was aimed at examining whether isolates from distinct genotypes were involved in distinct clinical forms of aspergillosis. Sixty-three clinical isolates of A. flavus recovered from 35 patients with various clinical presentations of aspergillosis were characterized by microsatellite typing. The highest discriminatory power for a single locus was obtained with the AFLA1 marker, which had 14 distinct alleles and a 0.903 D value. The combination of all six markers yielded 48 different genotypes with a 0.994 D value. There was a considerable genetic diversity in the isolates and patients with invasive aspergillosis were usually colonized by multiples genotypes. There was no evidence that a given genotype was associated with a particular clinical presentation of A. flavus aspergillosis. The occurrence of more than one genotype in clinical samples indicates that a patient may be infected by multiple genotypes and that any particular isolate from a clinical specimen may not necessarily be the one causing aspergillosis.

Externa otitis caused by the Graphium stage of Pseudallescheria apiosperma.

We report a case of otomycosis caused by the Graphium stage of Pseudallescheria apiosperma in an immunocompetent 32 years old man who was suffering from hypoacusia and purulent otorrhea. Isolates were identified as Graphium stage of Pseudallescheria sp. on the basis of macroscopic and microscopic characteristics. Pseudallescheria apiosperma was correctly identified by PCR sequencing of ITS regions and ß-tubulin gene. In this case the contamination could be due to intensive activity of gardening with poor hygiene.

Microsatellite typing of Aspergillus flavus from clinical and environmental avian isolates.

Aspergillosis is one of the most common causes of death in captive birds. Aspergillus fumigatus accounts for approximately 95 % of aspergillosis cases and Aspergillus flavus is the second most frequent organism associated with avian infections. In the present study, the fungi were grown from avian clinical samples (post-mortem lung material) and environmental samples (eggs, food and litter). Microsatellite markers were used to type seven clinical avian isolates and 22 environmental isolates of A. flavus. A. flavus was the only species (28 % prevalence) detected in the avian clinical isolates, whereas this species ranked third (19 %) after members of the genera Penicillium (39 %) and Cladosporium (21 %) in the environmental samples. Upon microsatellite analysis, five to eight distinct alleles were detected for each marker. The marker with the highest discriminatory power had eight alleles and a 0.852 D value. The combination of all six markers yielded a 0.991 D value with 25 distinct genotypes. One clinical avian isolate (lung biopsy) and one environmental isolate (egg) shared the same genotype. Microsatellite typing of A. flavus grown from avian and environmental samples displayed an excellent discriminatory power and 100 % reproducibility. This study showed a clustering of clinical and environmental isolates, which were clearly separated. Based upon these results, aspergillosis in birds may be induced by a great diversity of isolates.

Amphotericin B in vitro resistance is associated with fatal Aspergillus flavus infection.

Whether in vitro antifungal susceptibility findings correlate with the outcome of patients with invasive aspergillosis (IA) remains debated. This study aimed to test whether IA patients' outcomes were associated with in vitro susceptibility results. To do so, we determined the in vitro susceptibility to amphotericin B (AMB) of 37 Aspergillus flavus isolates from 14 patients with haematological malignancies diagnosed with proven or probable IA, of which 13 were treated with AMB deoxycholate. Minimal inhibitory concentrations (MICs) were determined by Etest with the isolates classified as in vitro sensitive (AMB-S) or resistant (AMB-R) if their MICs were < 2 or ≥ 2 mg/l, respectively. The association of the patients' death with primary disease, administered antifungal treatment, and infection with AMB-R A. flavus was tested using generalized estimating equations logistic regression. We assessed AMB-R in 31/37 (84%) isolates. In the patients treated with AMB, the survival rate was 2/3 (67%) and 2/9 (22%) for those infected with AMB-S or AMB-R A. flavus, respectively. Both infection with AMB-R A. flavus (P = 0.014) strain and acute myelocytic leukaemia as the underlying primary disease (P = 0.036) were independent predictors of death. Our findings indicate that in vitro resistance predicts a poor outcome in patients with A. flavus invasive disease treated with AMB. Recent advances in non-culture-based microbiological methods should not discourage efforts to obtain in vitro antifungal susceptibility results, which are critical for the choice of antifungal therapy in patients with IA.

Invasive aspergillosis: resistance to antifungal drugs.

Although the arsenal of agents with anti-Aspergillus activity has expanded over the last decade, mortality due to invasive aspergillosis remains unacceptably high. Resistance of the Aspergillus spp. species to antifungal drugs increased in the last 20 years with the increase in antifungal drugs use and might partially account for treatment failures. Recent advances in our understanding of mechanisms of antifungal drug action in Aspergillus, along with the standardization of in vitro susceptibility testing methods, have brought resistance testing to the forefront of clinical mycology. Recent modifications in taxonomy and understanding of the acquired resistance mechanisms of Aspergilli to drugs should support a better management of Aspergillus infections. In this paper, we review the current knowledge on epidemiology and underlying mechanisms involved in antifungal resistance in Aspergillus.

A review molecular typing methods for Aspergillus flavus isolates.

Aspergillus flavus is the second most important Aspergillus species causing human infections. The importance of this fungus increases in regions with a dry and hot climate. Small phylogenetic studies in Aspergillus flavus indicate that the morphological species contains several genetically isolated species. Different genotyping methods have been developed and employed in order to better understand the genetic and epidemiological relationships between environmental and clinical isolates. Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. Typing techniques can also give us a deeper understanding of the colonization pattern in patients. Most of these studies focused on Aspergillus fumigatus because it is medically the most isolated species. To date, there has not been any publication exclusively reviewing the molecular typing techniques for Aspergillus flavus in the literature. This article reviews all these different available methods for this organism.

Epidemiology of dermatophytoses in Sfax, Tunisia.

The distribution of dermatophytes varies in different countries and geographical areas depending on several factors. To determine the frequency of aetiological agents and the clinical variants of dermatophytoses, we carried out a study between 1998 and 2007. Out of 25 432 subjects suspected to have superficial mycoses, 9960 (39.2%) were affected with dermatophytoses; 14957 positive samples were obtained. The mean age was 35.7 years (range: 21 days to 97 years). Sex ratio was 0.9. Our patients were from urban regions in 81.9% of cases. The most common type of infection was onychomycosis (30.3%), followed by tinea pedis (24.8%), intertrigo (21.7%), tinea corporis (11.4%) and tinea capitis (9.6%). Fifteen patients had generalised dermatophytosis. Hadida and Schousboe disease was diagnosed in one case with lethal evolution. The most isolated dermatophyte was Trichophyton rubrum (74.5%), followed by T. violaceum (7.9%), T. mentagrophytes (7.5%), Microsporum canis (3.8%), Epidermophyton floccosum (0.7%) and T. verrucosum (0.54%). Other species were occasionally isolated: T. schoenleinii, T. tonsurans, M. audouinii and M. ferrugineum. The prevalence of dermatophytoses remains high in our country (996 cases/year). Trichophyton rubrum is the predominant causal agent. However, zoophilic agents become more prevalent. Epidemiological surveys are an essential tool for developing strategies for infection control.