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Seventy-seven strains of

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. Keywords: Mucormycosis, Malignant otitis externa, Saksenea vasiformis

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.

Aspergillus flavus 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.

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

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.

Typing is applied to highlight the genetic relationships between environmental and clinical fungal isolates involved in colonization or infection. A variety of techniques can be used to type fungi, depending on the epidemiological question and the available equipment. The use of typing techniques during clinical fungal outbreak investigations demonstrated patient-to-patient propagation in dermatophytoses outbreaks, patient-to-health-care worker and health-care worker-to-patient transmission in yeast infection outbreaks, airborne patient-to-patient transmission of the non-cultivable Pneumocystis jirovecii fungus involved in pneumonia outbreaks, and environmental sources of several mold outbreaks including aspergillosis or fusariosis keratitis. Typing was also useful to trace the source of invasive fungal disease outbreaks or epidemics, such as contaminated medical devices. More generally, typing provided important insights into fungal outbreak characteristics and helped to implement appropriate measures to control fungal infections, which are often severe, progress rapidly, and are difficult to diagnose and treat.

Purpose: Candida krusei strains are intrinsically resistant for the first choice antifungal. Fast identification of C. krusei as an infectious agent will decrease the risk of choice of not correct therapy. The aim of the present work was to study the epidemiology of Candida krusei infections during 10 years. We also attempt to study the phylogeny of these isolates by PCR- RFLP. Methods: Two hundred five cases of C. krusei candidiasis were referred to laboratory of parasitology mycology, UH Habib Bourguiba of Sfax-Tunisia during 10 years (2006 to 2016). Identification of our strains was performed by conventional methods and by PCR-ITS amplification followed by a digestion with three restriction enzymes MspI, HinfI and HincII. Result: The mean frequency of cases of C. krusei candidiasis was 17.08 per year. Invasive infection represented 10.24%. The superficial infections with C. krusei represented 89.76% of cases. Analysis of the phylogeny tree allowed us to deduce that there is a great diversity in C. krusei strains. No particular genotype has been associated with the sampling site, or department or year of infection. We noted that patient P4 was hosted by three strains with the same genotype. Conclusion: The modification in epidemiology of candidiasis emphasizes the necessity to monitor local incidence, species distribution and susceptibility in order to optimize therapy and outcome. Molecular methods are essential for correct identification of the Candida species in order to obtain clues regarding the source of infection and to apply the correct therapy for the infected individual.

Summary 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.

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.

There are limited data onin vitrosusceptibility testing of echinocandins againstAspergillusspecies. The objective of this study was to describe the phenotypes ofAspergillus flavusobserved on exposure to caspofunginin vitroand to test whether these phenotypes were associated withA. flavusgenotypes. The caspofungin MICs of 37A. flavusclinical 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−1and the MIC50and MIC90were 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 theseA. flavusisolates, the absence of a significant population structure or genetic differentiation indicated that trailing or paradoxical growth phenotypes were independent of microsatellite genotype.

Summary 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.

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.

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.

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.

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.

Whether in vitro antifungal susceptibility fi ndings 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 fl 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 classifi ed 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. fl avus 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. fl avus , respectively. Both infection with AMB-R A. fl avus ( P 0.014) strain and acute myelocytic leukaemia as the underlying primary disease ( P 0.036) were independent predictors of death. Our fi ndings indicate that in vitro resistance predicts a poor outcome in patients with A. fl avus 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.

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 0.25). 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

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 (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.

Les infections fongiques ont connu ces dernieres decennies une incidence croissante. Aspergillus fumigatus est un champignon filamenteux, saprophyte et sporulant. Il est responsable d’infections invasives chez les immunodeprimes. Sa virulence est multifactorielle. L’activite enzymatique et la capacite de former un biofilm des souches d’ A. fumigatus isolees chez 18 patients asthmatiques hospitalises a l’hopital Hedi Chaker de Sfax ont ete testees in vitro sur des milieux specifiques appropries afin de comparer la virulence de 22 souches collectees a partir des prelevements cliniques et 18 souches a partir des prelevements environnementaux. La production des hydrolases extracellulaires a montre que nos souches etaient productrices de phospholipases (67,5 %), de proteases (77,5 %), d’hemolysines (92,5 %) et d’elastases (75 %). Une production significativement plus elevee d’hydrolases extracellulaires a ete notee chez les souches cliniques ( p p A. fumigatus . L’etude de la formation de biofilm sur des plaques en polystyrene a montre que tous les isolats etaient aptes d’adherer a la surface de ce dispositif. Cependant, les souches cliniques ont montre une production plus elevee de biofilm ( p En conclusion, notre etude nous a permis de montrer que les souches cliniques ont une production plus elevees d’hydrolases extracellulaires et de biofilm. Ces facteurs de virulence expliquent bien le pouvoir pathogene d’ A. fumigatus dans le developpement des infections invasives.

Les levures du genre Malassezia sont des levures d’actualite ayant connu, dans cette derniere decennie, une evolution taxonomique importante et un interet accru en pathologie humaine et animale. L’identification de ces levures peut se faire par les techniques phenotypiques, biochimiques et physiologiques qui restent laborieuses et parfois imprecises vu que certaines especes presentent des caracteristiques tres proches. Nous avons mis au point une PCR multiplex en temps reel avec des sondes specifiques pour detecter les six especes de Malassezia : M. globosa, M. furfur, M. restricta, M. sympodialis, M. slooffiae et M. pachydermatis. Notre etude prospective a ete realisee sur 120 echantillons collectes a partir de 60 animaux domestiques (23 chevres, 10 chiens, 15 vaches, 3 chats, 8 lapins et 1 âne) et de 70 echantillons humains (28 patients atteints de pityriasis versicolor, 17 eleveurs et 25 personnes representant un groupe temoin). Quinze M. pachydermatis ont ete identifies chez les animaux. Nous avons identifie 61 isolats a partir des prelevements humains : M. globosa (28), M. furfur (15), M. restricta (6), M. sympodialis (8), M. slooffiae (2) et M. pachydermatis (2). La PCR en temps reel nous a permis aussi de detecter huit cas de co-infection chez 6 patients et 2 eleveurs. L’amplification a ete specifique pour chacune des especes de Malassezia : M. globosa, M. pachydermatis, M. sympodialis, M. furfur, M. slooffiae et M. restricta. En outre, aucun signal positif n’a ete retrouve par la PCR en temps reel pour les souches n’appartenant pas au genre Malassezia. Nos resultats montrent que l’analyse etait tres efficace pour identifier les especes de Malassezia. L’application de PCR multiplex en temps reel fournit un systeme d’identification sensible et rapide pour les especes de Malassezia, qui peut etre applique dans les enquetes epidemiologiques et la pratique courante.

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.

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.

La candidose vulvo-vaginale recidivante (CVVR) constitue un probleme majeur pour les femmes en âge de procreer. Il est actuellement admis que les femmes ayant une CVVR presentent un deficit immunitaire local. Nous avons evalue la secretion differentielle des anticorps de type IgG et IgAs anti- C. albicans, au niveau des secretions vaginales, de patientes atteintes de CVV, CVVR et de femmes asymptomatiques. Les prelevements, analyses par Elisa indirect, sont des secretions vaginales isolees chez 3 groupes de patientes : 14 patientes ayant une CVVR, 8 patientes ayant une CVV et 17 femmes temoins. L’analyse de l’ensemble des surnageants a revele une difference significative entre la prevalence des IgAs (73 %) et celle des IgG (33 %). La prevalence des IgAs et des IgG chez les patientes ayant une CVV (75 % et 37 %) et CVVR (86 % et 43 %) a ete superieure a celle observee chez les femmes temoins (61 % et 18 %, respectivement). La sensibilite et la specificite de la detection des IgAs par rapport a la culture mycologique des prelevements vaginaux, ont ete de l’ordre de 54 % et 83 %, respectivement. En conclusion, l’etude de l’immunite humorale au niveau des secretions vaginales a revele que la prevalence de detection des IgAs et des IgG chez les patientes est superieure a celle chez les femmes temoins. Ainsi, la CVVR ne peut pas etre attribuee uniquement a la deficience de l’immunite humorale locale et d’autres investigations proteomiques complementaires seront necessaires

Aspergillus flavus is second only to A. fumigatus in causing invasive aspergillosis and it is the major agent responsible for fungal sinusitis, keratitis and endophthalmitis in many countries in the Middle East, Africa and Southeast Asia. Despite the growing challenge due to A. flavus, data on the molecular epidemiology of this fungus remain scarce. The objective of the present study was to develop a new typing method based on the detection of VNTR (Variable number tandem repeat) markers. Eight VNTR markers located on 6 different chromosomes (1, 2, 3, 5, 7 and 8) of A. flavus were selected, combined by pairs for multiplex amplifications and tested on 30 unrelated isolates and six reference strains. The Simpson index for individual markers ranged from 0.398 to 0.818. A combined loci index calculated with all the markers yielded an index of 0.998. The MLVA (Multiple Locus VNTR Analysis) technique proved to be specific and reproducible. In a second time, a total of 55 isolates from Chinese avian farms and from a Tunisian hospital have been evaluated. One major cluster of genotypes could be defined by using the graphing algorithm termed Minimum Spanning Tree. This cluster comprised most of the isolates collected in an avian farm in southern China. The MLVA technique should be considered as an excellent and cost-effective typing method that could be used in many laboratories without the need for sophisticated equipment.

Candida albicans and Candida glabrata are the most common causative agents of both vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). Studying the population structure and genotype differentiation of Candida species that cause RVVC may lead to a significant improvement in clinical management. A total of 106 isolates were collected from 55 patients who were subdivided into three groups. Group I comprised 15 patients with RVVC (n = 50 isolates); group II comprised 16 patients, who had a history of at least two episodes of VVC in the last year (n = 32 isolates, two from each patient); and group III comprised 24 patients (n = 24 isolates) who had experienced a single episode of VVC in the previous 1 year period. C. albicans microsatellite markers CAI, CAIII and CAIV and C. glabrata RPM2, MTI and ERG3 microsatellites were amplified in a multiplex PCR. All isolates were subjected to population genetic analysis, which provided evidence that there is a predominantly clonal population structure of C. albicans in each group. However, recombination was detected to some degree in C. albicans isolates in group III. A genetic homogeneity between the different C. albicans groups was observed. Although, C. glabrata isolates showed an important genetic differentiation between group I and group III (FST = 0.207). Genotype analysis revealed that the dominant genotypes of C. glabrata and C. albicans strains were more prevalent in patients with RVVC. The frequent scenario for cases of recurrent infection in our study was strain replacement (53.3 %). In conclusion, the identification of recurrence-associated genotypes and a specific C. glabrata population structure in the RVVC group could be a significant marker for further investigations of virulence factors and RVVC management.

Late post-operative Aspergillus endocarditis is an under recognized life-threatening complication of heart valvular surgery. Optimal prevention and treatment need enhanced awareness. We report three cases of post-operative Aspergillus flavus endocarditis, including one with a 6-year long incubation period. Microsatellite typing incriminated a recurrent contamination of hospital environment.

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.

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.

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.

In Western Europe and the USA, the presence of anti-Saccharomyces cerevisiae antibodies (ASCAs) in Crohn's disease (CD) patients and their healthy relatives suggests that ASCAs may be influenced by genetic and/or environmental factors.To assess the prevalence of ASCAs in Tunisian patients with CD or ulcerative colitis (UC), and unaffected family members, in relation to clinical phenotype. Patients and methods. Seventy-seven patients (39 CD, 38 UC), 66 healthy relatives of CD patients, 16 relatives of UC patients and 70 healthy controls were studied. ASCAs were quantified with a new isotype-specific ELISA test involving an antigenic extract from S. cerevisiae strain W303 and by the original test which detects total immunoglobulins against S. cerevisiae Su1 mannan.The specificity of the two tests was identical (91%). The isotype-specific ASCA W303 test was more sensitive than the ASCA Su1 test for immunoglobulin detection, but some CD patients were positive only with this latter test. A high percentage of patients with CD (72%) and their unaffected family members (35%) were ASCA-positive in contrast to UC patients (16%) and their relatives (0%) and controls (8.6%). ASCAs were shown to be independent of rural or urban living, disease activity, but were associated with ileal location. The antigen of S. cerevisiae strain W303 discriminated patients depending on age at onset or location of the disease.This study confirms the antigenic heterogeneity of S. cerevisiae strains in their ability to detect ASCA. It suggests that ASCAs are markers of immunoregulatory disturbance in CD, independently of ethnic/cultural differences between Europe, the USA and North Africa.