Implication of efflux pumps and ERG11 genes in resistance of clinical Trichosporon asahii isolates to fluconazole.

Introduction. Trichosporon asahii has been recognized as an opportunistic agent having a limited sensitivity to antifungal treatment.Hypothesis/Gap Statement. Molecular mechanisms of azole resistance have been rarely reported for Trichosproron asahii. Similar to other fungi, we hypothesized that both ERG11 gene mutation and efflux pumps genes hyper-expression were implicated.Aim. The current work aimed to study the sensitivity of clinical T. asahii isolates to different antifungal agents and to explore their resistance mechanisms by molecular methods including real-time PCR and gene sequencing.Methods. The sensitivity of T. asahii isolates to fluconazole, amphotericin B and voriconazole was estimated by the Etest method. Real-time PCR was used to measure the relative expression of Pdr11, Mdr and ERG11 genes via the ACT1 housekeeping gene. Three pairs of primers were also chosen to sequence the ERG11 gene. This exploration was followed by statistical study including the receiver operating characteristic (ROC) curve analysis to identify a relationship between gene mean expression and the sensitivity of isolates.Results. In 31 clinical isolates, the resistance frequencies were 87, 16.1 and 3.2 %, respectively, for amphotericin B, fluconazole and voriconazole. Quantitative real-time PCR demonstrated that only Mdr over-expression was significantly associated with FCZ resistance confirmed by univariate statistical study and the ROC curve analysis (P <0.05). The ERG11 sequencing revealed two mutations H380G and S381A in TN325U11 (MIC FCZ=8 µg ml-1) and H437R in TN114U09 (MIC FCZ=256 µg ml-1) in highly conserved regions (close to the haem-binding domain) but their involvement in the resistance mechanism has not yet been assigned.Conclusion. T. asahii FCZ resistance mechanisms are proven to be much more complex and gene alteration sequence and/or expression can be involved. Only Mdr gene over-expression was significantly associated with FCZ resistance and no good correlation was observed between FCZ and VCZ MIC values and relative gene expression. ERG11 sequence alteration seems to play a major role in T. asahii FCZ resistance mechanism but their involvement needs further confirmation.

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.

Prevalence of free living amoeba in the domestic waters reservoirs in Sfax, Tunisia.

Free living amoebas (FLA) are opportunistic pathogen found in different water sources in the environment. The aim of this study was to investigate the prevalence of free living amoeba in different samples of domestic water reserves (DWR) in Sfax region from Tunisia. It was a prospective study dealing with 486 water samples collected from different DWR. After filtration through a cellulose acetate membrane samples were cultured on non-nutrient agar and the FLA were detected and strained with Giesma, Trichrome and red nuclear stain for morphological and morphotypic studies. FLA were found in 62% of samples. The Acanthopodial morphotype was detected in 43%, Polytactic (38%), Monotactic (28%), Fan-shaped (17%), Rugose (11%), Dactilopodial (10%) and Eruptive (9%). These results demonstrate that domestic water reserves are a significant source of the FLA and maintenance of DWR is recommended.

Isolation and molecular identification of Acanthamoeba spp from oasis water in Tunisia.

In the southern Tunisia Oasis, we conducted 211 water with drawals from various water traffic sites. This water is used for agriculture, swimming or various other human activities. Acanthamoeba genus was detected in 82% of collected samples. Sequencing of the amplification products with primers P892C/P892 has allowed us to detect genotypic variation with predominance of T4 genotype (51%) and presence of the genotypes T14, T5, T3, T16, T15, T10, T11, T9 and T7. They T4, T3, T5, T15, T11 and T10 genotypes have a high potential for pathogenicity and a very high degree of virulence due to their production of serine proteases and extracellular cysteine enzymes involved in tissue degradation of the host. T4 genotype was the most abundant in the environment as well as in infections caused by Acanthamoeba spp. T5 genotype was ranked second and T3 genotype was less abundant in the environment and its pathogenicity is discussed. Acanthamoeba strains with the genotypes T16, T9 and T7 were considered non pathogenic. In fact, they have been isolated only from the environment. However, for these strains, their role as a reservoir can be a real risk to human health.

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.

Molecular Identification of Malassezia Species in Patients with Malassezia folliculitis in Sfax, Tunisia.

AIM: Malassezia folliculitis is caused by the invasion of hair follicles by large numbers of Malassezia cells. Several Malassezia researches still use cultures, morphology and biochemical techniques. The aim of this study was to identify Malassezia species isolated from patients diagnosed with folliculitis, at the Parasitology and Mycology Laboratory of Sfax University Hospital, and to explore the genetic diversity of Malassezia by using PCR-RFLP and PCR-sequencing targeting the rDNA region of the Malassezia genome. PATIENTS AND METHODS: Specimens were taken from 27 patients with Malassezia folliculitis. For the molecular identification, PCR amplification of the 26S rDNAD1/D2 region was carried out using the Malup and Maldown primers and three restriction enzymes (BanI, MspI and HeaII) for RFLP analysis. The nucleotide sequences of each isolate were compared to those in the NCBI GenBank by using BLASTIN algorithm. RESULTS: Three species of Malassezia yeasts were identified among the 31 Malassezia strains isolated: M. globosa (83.9%), M. sympodialis (12. 9%) and M. furfur (3.2%). The sequence analysis of M. globosa showed six genotypes. CONCLUSION: There is a high genotypic variability of M. globosa colonizing patients with folliculitis.

Analysis of virulence factors and in vivo biofilm-forming capacity of Yarrowia lipolytica isolated from patients with fungemia.

Yarrowia lipolytica is ubiquitous in the environment, opportunistic, and might be considered as one of the causative agents of catheter-related candidemia. Our work aimed to study some virulence factors of Y. lipolytica such as hydrolases production and biofilm formation with comparison to the most frequent Candida specie in human disease. In sum, 58 clinical isolates of Y. lipolytica, 16 C. glabrata, and 12 C. albicans were collected from Intensive care unit (ICU). All were tested for enzymatic production and biofilm formation. All tested isolates of C. albicans and C. glabrata were able to degrade casein, and 98.2% of Y. lipolytica showed caseinase activity but no gelatinase activity was detected in all isolates. Y. lipolytica strains showed significantly lower (3.4%) in vitro phospholipase activity than C. albicans and C. glabrata (P < .05). No significant differences of the hemolytic activity were detected between the three species (P > .05). Concerning biofilm formation, and unlike the results obtained on polystyrene plate, the number of adhered and biofilm cultivable cells obtained by Y. lipolytica after 168 hours of catheter subcutaneous implantation is significantly greater and tends to be more compact and structured hyphal layer. Although C. albicans remains the most pathogenic yeast, development of selective ability of Y. lipolytica to adhere, to form a biofilm on catheter medical devices, and to produce phospholipase and hemolytic enzyme is of particular interest, and it is strongly recommended to be vigilant in the use of medical implanted medical devices, particularly in ICU.

Rhodotorula fungemia: Report of two cases in Sfax (Tunisia).

Rhodotorula is emerging as an important cause of nosocomial and opportunistic infections. We present two cases of Rhodotorula mucilaginosa fungemia diagnosed at our hospital during the last decade. The first case was of a term neonate who presented congenital heart disease (interventricular communication) and body dysmorphic disorder. He was admitted for respiratory failure and sepsis. The second case involved in a 33-year-old woman that had Hodgkinien lymphoma associated to tuberculosis. Identification was performed using commercial systems and confirmed by PCR sequencing of internal transcribed spacer, ITS1 and ITS2 regions of rDNA. Antifungal susceptibility tested by sensititre yeast revealed susceptibility to amphotericin B and resistance to fluconazole for the two strains. These cases emphasize the emerging importance of Rhodotorula sp. as a pathogen and it must be considered a potential pathogen in patients with immunosupression and with central venous catheters. Correct identification is mandatory for appropriate management, as Rhodotorula spp. are resistant to antifungal agents, such as fluconazole.

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.

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.

Geotrichum capitatum septicemia: case report and review of the literature.

Geotrichum capitatum is an uncommon cause of invasive infections in immunocompromised patients, particularly those with hematological malignancies and severe neutropenia. The aim of this study was to report the cases of invasive geotrichosis in our hospital. It is a retrospective study of invasive geotrichosis diagnosed in the Laboratory of Parasitology-Mycology of the UH Habib Bourguiba, Sfax, from January 2005 to August 2013. Six cases of invasive Geotrichum infections were diagnosed. There were three men and three women. The mean age was 35 years. Five patients have acute myeloid leukemia with a profound neutropenia, and one patient was hospitalized in the intensive care unit for polytraumatism. Clinically, the prolonged fever associated with pulmonary symptoms was the predominant symptom (n = 5). Geotrichum capitatum was isolated in one or more blood culture. Two patients had urinary tract infections documented by multiple urine cultures positive for G. capitatum. Five patients received conventional amphotericin B alone or associated with voriconazole. The outcome was fatal in four cases. Invasive geotrichosis is rare, but particularly fatal in immunocompromised patients. Approximately, 186 cases have been reported in the literature. The prognostic is poor with mortality over 50 %. So, early diagnosis and appropriate management are necessary to improve prognosis.

Fungemia caused by Yarrowia lipolytica.

Yarrowia lipolytica is weakly pathogenic yeast, which is rarely isolated from the blood. We report unusual cases of Y. lipolytica fungemia occurred between October 2012 and June 2014 in the intensive care unit (ICU) of the UH Habib Bourguiba Sfax. During this period, 55 cases of Y. lipolytica septicemia were diagnosed. There were 44 men and 11 women (sex ratio = 4).The median age was 43 years. The broad-spectrum antibiotics (100 %), the catheterization (96 %), and the prolonged hospitalization in ICU (91 %) were the main risk factors. Patients were hospitalized in ICU, mostly, for polytraumatism (45.4 %), pneumopathy (9 %), and post-operative complications (7 %). Fever unresponsive to broad-spectrum antibacterial therapy was the predominant sign of infection (83.6 %). Y. lipolytica was isolated in one or several blood cultures (14.5 %) and in the catheter tip culture of nine patients (16.3 %).Treatment was based on intravenous amphotericin B (58.2 %), fluconazole (45.4 %) and/or removal catheter (69 %). Apyrexia or blood cultures sterilization was obtained for 34 patients (61.8 %). Y. lipolytica candidemia is an opportunistic and emerging human yeast pathogen. It can reach to the bloodstream of immunocompromised or critically ill patients during hospitalization through intravascular catheterization. Further clinical data need to be evaluated for formulating management strategies of seriously ill patients infected with uncommon fungal agents.

Epidemiological profile of fungal keratitis in Sfax (Tunisia).

UNLABELLED: Fungal keratitis is responsible for a significant burden of blinding disease in the developing world. OBJECTIVE: The aim of this study was to determine the etiological agents, predisposing factors and therapy of keratomycosis in our region. METHODS: Retrospective study of 60 patients with clinically and cultured confirmed fungi keratitis, who were attended at department of mycology in Sfax (1995 to 2012). RESULTS: The mean age of patients was 47.2 years (sex ratio: 1.58). At least, one presumed predisposing factor was identified in 83.3% of cases. Corneal traumatism was established as the most common predisposing factor (61.6%) with vegetative matter (42.4%). Patients had corneal ulcer in 40% or abscess in 47.6%. All cases were positive on direct microscopy and 93% of cultures were positive. Filamentous fungi form the major etiologic agents (83%): Fusarium species (49% with F. solani [66%]), Aspergillus sp. (22%), Alternaria (5%), Scedosporium sp. (2%); and non-identified mold in (5%). Yeast were identified in 17% of cases. Topical agents were used in 97% of cases: ketoconazole 2%, amphotericin B (0.5%). Fluconazole per os was administrated for 11% of cases, itraconazole (2 cases) and voriconazole (one case). Keratoplasty was indicated for 27% of cases. The outcome was favorable in 16% of patients. Among the patients, 71% had persistent corneal deposit sequelae. Four patients lost the eyeball. CONCLUSION: Corneal traumatism was the principal risk factor for fungal keratitis in young and middle-aged farmers. Fusarium solani is the predominant cause in Sfax. Early diagnosis, coupled with appropriate treatment, is crucial for increasing the chance of complete recovery.

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.

Invasive fungal infections in renal transplant recipients: about 11 cases.

UNLABELLED: Invasive fungal infections are a major complication and an important cause of morbidity and mortality among solid organ transplant recipients. Their diagnosis is difficult and their prognosis is often pejorative. OBJECTIVE: The aim of this study was to report the cases of invasive fungal infections in renal transplant recipients in Habib Bourguiba Sfax university hospital and to identify the main fungal agents. MATERIALS AND METHODS: It is a retrospective study of invasive fungal infections in renal transplant recipient reported in our hospital from January 1995 to February 2013. RESULTS: Invasive fungal infections were diagnosed in 11 cases (3.4%) among 321 renal transplant recipients. These infections included four cases of pneumocystosis, two cases of candidiasis, two cases of aspergillosis, two cases of cryptococcosis and one case of mucormycosis. There were six men and five women. The mean age was 37 years. The infection was late in 63% of cases (>3 months after transplantation). The prolonged corticosteroid and immunosuppressive therapy were the main risk factors (100%) followed by renal failure (45%), graft rejection (45%), broad spectrum antibiotics (45%), CMV infection (36%), neutropenia (36%) and dialysis (18%). The evolution under treatment was favourable only in two cases (18%). CONCLUSION: Invasive fungal infections are not common among kidney transplant recipients. However, they remain an important cause of morbidity and mortality in this group of patients. Prevention, early diagnosis and appropriate management are necessary to improve prognosis and reduce mortality rate.

[Pneumocystis: epidemiology and molecular approaches]. / Pneumocystis : épidémiologie et approches moléculaires.

Pneumocystosis is a common opportunistic infection in immunocompromised patients, especially in AIDS patients. The diagnosis of this pneumonia has presented several difficulties due to the low sensitivity of conventional staining methods and the absence of culture system for Pneumocystis. The molecular biology techniques, especially the PCR, have improved the detection of DNA of this fungus in invasive and noninvasive samples, and in the environment which highlighted human transmission and the existence of environmental source of Pneumocystis. In addition, various molecular biology techniques were used for typing of Pneumocystis strains, especially P. jirovecii, which is characterized by a significant genetic biodiversity. Finally, the widespread use of cotrimoxazole for the treatment and prophylaxis of pneumocystosis has raised questions about possible resistance to sulfa drugs in P. jirovecii.