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.

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.

Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia.

Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eighty-four water samples were collected over a period of 4 months (September-December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial.

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.

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.

[Methods for studying the in vitro susceptibility of Candida spp. to antifungals]. / Méthodes d'étude de la sensibilité in vitro de Candida spp. aux antifongiques.

In recent years, an increase of systemic Candida infections was noted. Thus, identification and susceptibility testing to antifungals became of considerable importance. The technique of dilution in liquid medium developed by « National committee for clinical laboratory standards ¼ NCCLS or more recently named CLSI « Clinical and laboratory standards institute ¼ is the reference method most used. The European committee "European committee on antibiotic susceptibility testing" or EUCAST has made progress by determining the susceptibility of strains within a shorter time. However, the use of these techniques is limited especially in clinical microbiology laboratories. Other techniques for determining antifungal sensitivity have been developed such as those based on agar diffusion (E-test and disk diffusion), on microdilution (Sensititre yeastOne, Vitek 2 AST-YS01), on flow cytometry techniques and the MALDI-TOF.

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.