Unusual case of otomycosis caused by Saksenaea vasiformis.

Saksenaea vasiformis is a species of the order Mucorales rarely reported as a cause of human mucormycosis. We report an unusual case of S. vasiformis otitis occurring in a diabetic woman after penetration of an insect in the right ear. Direct microscopic examination of the clinical sample showed hyaline and non septate hyphae belonging to the order Mucorales. Fungal identification was performed by sequencing the ITS region of the rDNA. To our knowledge, this is the first report of S. vasiformis infection in Tunisia.

Microsatellite analysis of the population structure in Rhizopus arrhizus.

OBJECTIVES: Rhizopus arrhizus is recognized as an emergent agent of superficial and invasive mucormycosis. Despite an increasing number of these infections, the molecular epidemiology of Rhizopus species has not been well studied. MATERIALS AND METHODS: In this study, 43 R. arrhizus strains (25 environmental and 18 clinical isolates) were genotyped using six novel panels of microsatellite markers. RESULTS: Upon the analysis of 43 isolates, 4-8 distinct alleles were detected for each marker. The discriminatory power for the individual markers ranged from 0·522 to 0·830. The combination of all six markers yielded 33 different haplotypes with a high degree of discrimination (0·989 D value). A four-marker combination were selected as the most parsimonious panel achieving D > 0·95. One clinical isolate and one environmental isolate shared the same genotype suggesting the possible nosocomial outbreak of mucormycosis in hospitalized patients. We have noted that the strains isolated from cutaneous mucormycosis were different from the strains isolated from rhino-orbito-cerebral mucormycosis. Then, the hypothesis of particular tropism of infectious strains for a given site is not excluded. The standardized indices of association IA and rBarD were significantly different from zero (P < 0·01), suggesting a prevailing clonal reproduction. The environmental population was significantly differentiated from clinical populations (Fst = 0·2249). CONCLUSIONS: Microsatellite typing method described in our study showed an excellent degree of discriminatory power. It is a promising tool for illuminating the molecular epidemiology of R. arrhizus species, including strain relatedness and transmission pathways.

Detection of virulence factors and antifungal susceptibility of human and avian Aspergillusflavus isolates.

Aspergillusflavus is the second leading cause of invasive and non-invasive aspergillosis. Secretion of hydrolytic enzymes is considered as a virulence factor in this species. Our work aimed to study in vitro production of some virulence factors, to evaluate the biofilm production against human and avian A. flavus isolates and to investigate the antifungal susceptibility agents. Hydrolytic enzymes, biofilm production and molecular typing were studied for 62 human and 36 avian A. flavus isolates by specific solid media and six microsatellite markers. The susceptibility to antifungal agents was evaluated for 37 human isolates. All human and avian A. flavus isolates showed positive activities of extracellular hydrolase: phospholipase, protease and hemolysin. A positive elastase activity was seen in 64.51% of human A. flavus isolates and 86.1% of avian A. flavus isolates. All A. flavus in these two populations formed biofilms. Statistical significant difference was observed for the mean phospholipase activities (P=0.025) and biofilm quantification (P=0.0001) between human and avian A. flavus isolates. The in vitro susceptibility results showed a resistance in 83.7%, 81.08% and 16.21% of A. flavus isolates respectively to amphotericin B, itraconazole and posaconazole. No association was noted between all virulence factors and the genotypes of human and avian isolates. Our study allowed us to show that human strains have a higher production of extracellular hydrolases and biofilm then avian strains. These virulence factors appear to act synergistically to contribute to the virulence of A. flavus strains. Moreover, significant correlation between virulence patterns and antifungal susceptibility profiles was observed.

Contribution of the internal transcribed spacer regions to the detection and identification of human fungal pathogens.

Fungi are morphologically and phylogenetically diverse. There identification is largely based on phenotypic methods. Thus, related species, phenotypic variants and rare species may be unidentified. So, molecular methods have been introduced for identification of pathogenic molds to overcome these problems. In this study, we report the contribution of molecular tools (PCR sequencing) to identify fungal pathogens in both clinical and environmental samples. A total of 82 mold isolates were used (50 clinical samples and 32 environmental samples). PCR and direct sequencing, targeting the internal transcribed spacer (ITS) regions, were performed. We employed comparative sequence analysis to identify molds by using the GenBank database. 89% of isolates were identified by phenotypic methods. PCR- sequencing allowed the fungal identification in all cases. The concordance between molecular and morphological identification was obtained for 33 cases (40.2%). In 36 cases (43.9%), the molecular study gave the exact species identification. PCR sequencing allowed as revising mycological identification for 13 fungi strains (15.9%). The concordance of identification at species level by phenotypic method and by sequence analysis was obtained for 28% of clinical samples and for 59% of environmental samples. The phylogenetic tree for the ITS sequences showed six different clusters that are composed of isolates belonging to the same genus or species. PCR sequencing has been shown to be useful for the detection of the presence of fungal DNA in both environmental and clinical samples. It is rapid and more sensitive for the identification of medically important fungi.

Epidemiology of antifungal susceptibility: Review of literature.

Fungal infections are a major cause of morbidity and mortality despite the latest developments of diagnostic tools and therapeutic options. Early initiation of the appropriate antifungal therapy has been demonstrated to have a direct impact on the patient's outcome. Antifungal susceptibility testing methods are available to detect antifungal resistance and to determine the best treatment for a specific fungus. American and European standards have been developed, as well as equivalent commercial systems, which are more appropriate for clinical laboratories. These studies have allowed the development of interpretative breakpoints against the most frequent agents of fungal infections in the world. Surveillance of antifungal susceptibility patterns can provide the local drug resistance data to the clinicians, which can further aid better management of patients. Antifungal susceptibility tests have become essential tools to identify resistance to antifungals, to know the local and global disease epidemiology and to guide the treatment of fungal diseases. The distribution of species and the prevalence of antifungal resistance in fungi isolates varied among different areas. Here we summarize the epidemiology of antifungal susceptibility pattern of different fungal species.

Environmental and molecular study of fungal flora in asthmatic patients.

The aim of the present study was to investigate the epidemiological and fungal environmental profile in asthmatic patients. We conducted a prospective study involving 49 patients with allergic asthma. One hundred and forty-five clinical samples and 289 environmental samples were performed. Only 30 patients accepted to participate to the environmental study at their home. For specific IgE antibodies, ELISA assay was conducted for 21 patients. Molecular ITS sequencing was performed for 37 isolates. The frequency of attacks was significantly associated with the seasonality, which was closely related to climate (P=0.024), exposure to animals (cats, P=0.025), plants (olive, P=0.018), physical effort (P=0.04) and the number of permanent occupants in house (>6) (P=0.026). Fungal contaminants were detected from 78.6% of biological samples and 97.8% of environmental samples. Antibodies corresponding to the studied allergens were detected in 10 patients (10/21). PCR sequencing allowed as rectified morphological identification for 27.02% (10/37) strains of Aspergillus. The allergy in molds is an indisputable reality that is necessary to look for in front of any severe asthma. So, it is important to establish clearly a relationship between exposure to fungi and health disorders in order to set up specific and effective preventive measures.

Tinea manuum due to Trichophyton erinacei from Tunisia.

Trichophyton erinacei is a zoonotic fungus affecting hedgehogs. Although several human infections with this organism have been documented in the literature, it has rarely been isolated as a human pathogen. This paper reports on an erythematous lesion spotted on the hand of a 10-year-old girl. Based on the culture of the patient's skin scrapings, the pathogen was mycologically identified as T. erinacei, which was further confirmed by sequencing the internal transcribed spacers of the fungal nuclear ribosomal DNA using universal primer ITS1-ITS4. This is the first case of T. erinacei in a Tunisian patient. A survey was carried out on the environment of our patient, and the results revealed the presence of hedgehogs with suspect scaly lesions. The same fungus was isolated from the hair and scales of the hedgehog, which was confirmed by PCR sequencing. The frequency of T. erinacei has often been underestimated, which is attributed not only to the gaps of knowledge still existing in the current understanding of the dermatophyte but also to differential diagnosis problems. Molecular study offers a simple and rapid tool to identify the source of infection and, hence, avoid the risk of recurrence.

Polymorphisms in the ITS rDNA regions for differentiating strains of the Trichophyton mentagrophytes complex in Sfax-Tunisia.

The Trichophyton mentagrophytes complex is the main cause of superficial mycoses in humans and animals. Molecular research has provided useful insights into the taxonomy of this complex to overcome the challenges with conventional diagnostics. The aim of this study was to identify, type and differentiate anthropophilic and zoophilic species of the T. mentagrophytes complex. Sixty clinical samples identified as T. mentagrophytes by morphological characteristics were isolated using polymerase chain reaction-restriction fragment length polymorphism and sequence analysis of the internal transcribed spacer (ITS) regions. The identification of our strains by conventional methods was confirmed using polymerase chain reaction (PCR) sequencing in 93.34% of the cases. The strains under investigation were recategorised as T. rubrum (Tr2711). In addition, PCR products were independently digested with the restriction endonucleases, MvaI and HinfI, to produce a single dominant profile for T. interdigitale. ITS sequence analysis revealed a polymorphism in the ITS1 and 5.8S regions. Analysis of the consensus sequences distinguished four types of genotypes among our T. interdigitale species. Moreover, ITS type I was the dominant genotype characterising the anthropophilic variant of T. interdigitale. The phylogenetic study showed that only 5% of our strains were zoophilic. PCR sequencing was useful for distinguishing anthropophilic and zoophilic species of T. interdigitale, in which the differentiation is relevant because it helps to prescribe the correct treatment and to identify the surrounding source of infection.

Genetic structure of Aspergillus flavus populations in human and avian isolates.

Aspergillus flavus is the second leading cause of allergic, invasive, and colonizing fungal diseases in humans, and also the second most frequent organism associated with avian infections. Currently, it is not known whether there is a link between the environmental isolates and/or human isolates of A. flavus and those responsible for aspergillosis in birds. Microsatellite typing was used to analyze 29 A. flavus clinical and environmental avian isolates and 63 human clinical isolates collected from patients with a variety of aspergillosis diseases. The combination of all six markers yielded 77 different genotypes with a 0.98 D value. A. flavus genotypes obtained from avian isolates were compared with those obtained from human clinical and environmental samples. The standardized indices of association I (A) and rBarD were significantly different from zero (p < 0.01), suggesting a prevailing clonal reproduction. There was high genetic diversity between the hospital and poultry environments of A. flavus isolates. The human environmental population was significantly differentiated from environmental and clinical avian populations (F (st) > 0.25). The avian clinical subpopulation exchanged few strains with the environmental human (N (m) = 7.24) and avian (N (m) = 6.60) populations. The minimum spanning tree analysis identified three A. flavus genotype clusters that were highly structured according to the isolation source (p < 10(-4)).

Clinical utility and prognostic value of galactomannan in neutropenic patients with invasive aspergillosis.

UNLABELLED: Invasive aspergillosis (IA) is a major cause of morbidity and mortality in profoundly neutropenic patients. Delayed diagnosis and therapy may lead to poor outcomes. AIMS: The objective of this study was to assess the performance characteristics of the galactomannan (GM) assay in serum and bronchoalveolar lavage specimens for the diagnosis of IA in neutropenic patients with hematological malignancies. We also evaluated the prognostic outcome. PATIENTS AND METHODS: A total of 1198 serum samples and 42 BAL from 235 neutropenic patients were tested with a GM elisa platelia test. We used Cox modeling of time to 6- and 12-week mortality for GM level at the time of diagnosis (GM0) and GM decay in the week following diagnosis in proven and probable IA patients with more than two GM values. RESULTS: There were three proven, 55 probable, and four possible cases of IA. The sensitivity and specificity of the GM test were 96.8% and 82.4% respectively. In BAL samples, sensitivity was 86% and the specificity 93%. BAL GM was more sensitive than microscopy (22.2%) and BAL culture (38.9%). Among patients with proven/probable IA, serum and BAL GM were in agreement for 92.8% of paired samples. The hazard ratio (HR) of GM0 and 1-week GM decay per unit increase in Aspergillus enzyme immunoassay (EIA) was 1.044 (95% CI, 0.738 to 1.476) and 0.709 (95% CI, 0.236 to 2.130) respectively. CONCLUSION: We found good correlation between the GM0 and GM decay combination and outcome of IA patients. The GM is a useful tool for diagnosis and monitoring of IA.

Candida glabrata strain relatedness by new microsatellite markers.

We investigated six microsatellite markers to type 85 unrelated and 118 related isolates of Candida glabrata from 36 patients. Three new markers were selected from the complete sequence of CBS138 and three previously described markers, RPM2, MTI and ERG3 were used. We found a genetic diversity of 0.949 by combining four of them. By applying the new microsatellite markers GLM4, GLM5 and GLM6 we were able to discriminate 29 isolates, originally identified by the more established markers, RPM2, MTI and ERG3. When epidemiologically closely related isolates from 36 patients were typed, 25 patients (72%) exhibited identical or highly related multilocus genotypes. We noted a microvariation in 4 of the patients. This minor change of one locus could be explained by a single step mutation. Since one of these patients had not received antifungal treatment; thus, the relationship between genome variation and antifungal therapy remains controversial. We can conclude from our analysis of these new microsatellite markers that they are highly selective and therefore should be considered as a useful typing system for differentiating related and unrelated isolates of C. glabrata, as well as being able to detect microvariation.

Invasive aspergillosis: epidemiology and environmental study in haematology patients (Sfax, Tunisia).

Invasive aspergillosis (IA) is a major opportunistic infection in haematology patients. Preventive measures are important to control IA because diagnosis is difficult and the outcome of treatment is poor. We prospectively examined the environmental contamination by Aspergillus and other fungal species and evaluated the prevalence of invasive aspergillosis in the protect unit of haematology. A three-year prospective study (December 2004-September 2007) was carried out in the department of haematology of Hedi Chaker Hospital. Suspected invasive aspergillosis cases were reviewed and classified as proven, probable and possible invasive aspergillosis using the EORTC criteria. During the study period, we collected weekly environmental samples (patient's rooms, tables and acclimatisers) and clinical samples from each patient (nasal, expectoration and auricular). Among 105 neutropenic patients, 16 had probable and 13 had possible IA. A total of 1680 clinical samples were collected and A. flavus was most frequently isolated (79.2%). Analysis of 690 environmental samples revealed that Penicillium (44%) was the most frequent followed by Cladosporium (20%), Aspergillus spp. (18%) and Alternaria (13%). The PCR-sequencing of 30 A. flavus isolates detected from clinical and environmental samples confirmed the mycological identification. Our findings underline the importance of environmental surveillance and strict application of preventive measures.