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.

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.

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.

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.

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.

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.

Acanthamoeba T4 genotype associated with keratitis infections in Tunisia.

Acanthamoeba keratitis (AK) is a sight-threatening infection. We report five cases of AK diagnosed from 2005 to 2009 in the Laboratory of Parasitology-Mycology at Habib Bourguiba Sfax Hospital, Tunisia. All were associated with improper care of contact lenses (rinsing of contact lenses with tap water and inappropriate cleaning) and lens storage. The patients displayed different clinical presentations: corneal inflammation, corneal ulceration, and corneal abscess. The diagnosis was made after direct examination, culture, and polymerase chain reaction amplification with specific primers. The genotype classification was based on the highly variable DF3 region in the 18S rRNA gene. This is the first study characterizing Acanthamoeba genotype in Tunisia and North Africa. All Acanthamoeba isolates were associated to the T4 genotype. Three different DF3 sequence types were related to AK infections T4/10, T4/15, and T4/16.

Pathogenic free-living amoebae: epidemiology and clinical review.

Free-living amoebae are widely distributed in soil and water. Small number of them was implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Some of the infections were opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitis) while others are non opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis and some cases of Balamuthia encephalitis). Although, the number of infections caused by these amoebae is low, their diagnosis was still difficult to confirm and so there was a higher mortality, particularly, associated with encephalitis. In this review, we present some information about epidemiology, ecology and the types of diseases caused by these pathogens amoebae.

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.

Epidemiological survey of vulvovaginal candidosis in Sfax, Tunisia.

Vulvovaginal candidosis (VVC) is a common infection of the female genital tract affecting 75% women at least once in their lifetime. The aim of this study was to determine the incidence and potential risk factors associated with VVC and recurrent vulvovaginal candidosis (RVVC). A prospective study of women with vaginitis symptoms was conducted over 2 years in the regional clinic of population and family education in Sfax. A discriminant analysis was used to evaluate the association between the incidence of Candida vaginitis and potential risk factors. Sporadic and recurrent VVC were documented respectively in 48% and 6.1%. The most frequent factors associated with positive Candida culture were employed women, uncontrolled diabetes, history of genital infection and intrauterine device contraception. Increased episode numbers of VVC and condom/spermicidal contraception were positively associated with recurrences. Candida albicans was the predominantly isolated species (76.3%) followed by Candida glabrata (19.3%). Infection with C. glabrata occurred in 34% and 17.5% of patients with RVVC and VVC respectively. The discriminant investigation had provided further insights into the basis for prevention and control of RVVC. Increased prevalence of C. glabrata in patients with RVVC and observed risk factors should be taken into consideration to achieve success in the management of this infection.

Dihydropteroate synthase gene mutations in Pneumocystis jiroveci strains isolated from immunocompromised patients.

The emergence of Pneumocystis jiroveci drug resistance has been suggested recently by the mutations in the gene encoding dihydropteroate synthase (DHPS). The aim of the present study was to determine the prevalence of DHPS mutations in P. jiroveci strains isolates from bronchoalveolar lavages (BAL) and sputum samples of 21 immunocompromised patients. We used the touchdown-PCR for amplification of DHPS gene and the restriction fragment length polymorphism (RFLP) technique for discrimination of wild and mutant DHPS genotypes. The DHPS amplification was positive in 17 patients (81%). The association of wild genotype and mutant genotype was detected in two patients after the enzymatic digestion of the PCR products by AccI and HaeIII. No mutations in the DHPS gene were seen in 15 patients. In addition, no variation was observed in DHPS genotypes detected in the repeated specimens (BAL and sputum) from some patients. The touchdown PCR-RFLP technique is a simple and rapid method for revelation of DHPS gene mutations in P. jiroveci strains. It could be advantageously used in clinical laboratory to control the prevalence of mutations associated with sulfa resistance.

Antifungal susceptibility of bloodstream Candida isolates in Sfax hospital: Tunisia.

UNLABELLED: Invasive candidiasis has emerged as an important nosocomial infection, causing significant morbidity and mortality especially among critically ill patients. The aim of our study was to determine specie distribution and resistance profiles of Candida species isolated from blood cultures. MATERIALS AND METHODS: We conducted a retrospective study of all episodes of candidemia diagnosed in our laboratory from January 2006 to May 2009. The susceptibility to antifungal agents of all Candida isolates was tested by using a Sensititre(®) YeastOne panel. RESULTS: A total of 130 Candida isolates were recovered from blood cultures. Candida tropicalis was the most frequent specie (37.7%), followed by C. albicans (22.3%), C. glabrata (19.2%), and C. parapsilosis (12.2%). All the isolates were inhibited by ≤1 µg/ml of amphotericin B and ≤2 µg/ml of caspofungin. For fluconazole, 7.3% of clinical isolates were resistant. It was most active against C. parapsilosis (100% susceptible), C. albicans (95.8% susceptible), and C. tropicalis (94% susceptible). All of the fluconazole-susceptible isolates were susceptible to voriconazole, as were 83.3% of the fluconazole-susceptible-dose-dependent isolates. Among fluconazole-resistant isolates, 85.7% were susceptible to voriconazole. CONCLUSIONS: In our institution, C. tropicalis was the most frequent specie isolated from the bloodstream. Caspofungin had an excellent in vitro activity against Candida isolates and was the drug of choice among fluconazole-resistant isolates.

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.

Molecular diagnosis of Pneumocystis jiroveci pneumonia in immunocompromised patients.

Pneumocystis jiroveci is the major cause of pneumonia in immunocompromised patients. To evaluate the performance of single and nested-polymerase chain reaction (PCR) methods compared with immunofluorescent assay (IFA) and cytological staining for diagnosis of P. jiroveci infection, the bronchoalveolar lavage (BAL) and sputum samples from 60 immunocompromised patients were studied. Between January 2005 and March 2008, 75 respiratory specimens (41 BAL and 34 sputum samples) were examined for P. jiroveci identification. We used the clinical classification as our diagnostic standard and we considered true positive the definite or probable Pneumocystis pneumonia. Fourteen patients (23.3%) developed Pneumocystis pneumonia. Eleven patients had a positive IFA but only nine were positive by cytological staining. Sixteen patients had a positive detection of P. jiroveci by PCR and nested-PCR. Thirteen of these patients were considered as having a definite Pneumocystis pneumonia and one patient with a probable Pneumocystis pneumonia. Five other patients had a positive detection only by nested-PCR. These patients were classified as no Pneumocystis pneumonia. PCR detection of P. jiroveci is a very sensitive test and will offer a powerful technique in clinical laboratories for the routine diagnosis of Pneumocystis pneumonia. Using the nested-PCR, additional clinical cases can be diagnosed, but there is then an obvious risk of detecting subclinical colonisation by P. jiroveci.

First case of Trichophyton soudanense isolated in Tunisia.

This is a first case of Trichophyton soudanense isolated from Ivoiran student in Tunisia. A 24-year-old man was addressed for extensive erythematous, scaling lesions. Examination disclosed tinea capitis, tinea corporis, tinea pedis, and onychomycosis of toenails and fingernails. Isolates were identified as Trichophyton soudanense on the basis of macroscopy and microscopy colony characteristics. The patient was treated with fluconazole, topical econazole, and ciclopiroxolamine varnish. Although T. soudanense was identified since the late 1950s outside the African continent especially in the North America, Brazil, Australia, and many European countries, this is the first case reported in Tunisia. Accessibility to our universities for African students makes possible the emergence of this dermatophyte.

[Human demodicidosis in Sfax area (Tunisia)]. / La démodécidose humaine dans la région de Sfax (Tunisie).

Demodicidosis is an ectoparasitosis, common to humans and many mammals. It is caused by the proliferation of a mite Demodex sp in the pilosebaceous follicles. Its pathogenic role remains controversial. The aim of our study was to report epidemiological and clinical particularities of cases of demodicidosis diagnosed in our region. Over a period of nine years (January 2000 to December 2008), 427 cases of demodicidosis were diagnosed. 73.2% of cases were blepharitis and 26.8% of cases were facial dermatosis. The mean age was 44 years. Women were slightly more affected (56%) than men. Among 787 chronic blepharitis, 243 cases were due to Demodex sp (30.9%). They were treated with yellow oxide of mercury (Ophtergine® 1%). In the face, this mite has been isolated from erythematous and pruritic papulopustular lesions, and their distribution was as follows: cheeks (22.1%), forehead (13.4%), and nose (11.5%). The diagnosis was confirmed by parasitological examination of scales showing more than 5 Demodex sp/cm(2) and response to treatment with metronidazole (Flagyl®) for three months. Currently, there were a large number of arguments for the incrimination of Demodex sp in pathogenesis of dermatosis and blepharitis. Dermatologists and ophthalmologists must therefore think to this mite. The density of Demodex sp found by parasitological exam is a determining factor in establishing an anti-Demodex treatment whose effectiveness is a further argument for the diagnosis.

[Toxoplasmosis in Sfax, Tunisia]. / Etat actuel de la toxoplasmose dans la région de Sfax, Tunisie.

Abstract The frequency of toxoplasmosis depends on life-style and environment. Our objective was to study different epidemiological, clinical and biological aspects of toxoplasmosis in the Sfax area (Tunisia). This retrospective study has been performed on seria of 40,566 pregnant women in the Parasitology-Mycology Laboratory of Habib-Bourguiba Sfax hospital-Tunisia for 13 years from 1994 to 2006; 1,691 patients presenting with lymphadenopathy; 191 immunocompromised patients (78 HIV infected patients and 113 transplanted patients) and 21 patients presenting clinical signs of ocular toxoplasmosis. In pregnant women, the seroprevalence was 39.3% (15,952/40,567). Among 24,089 seronegative women, only 6,890 (28.6%) had been followed up during their pregnancy. An active toxoplasmosis possibly acquired during pregnancy was detected in 1.3% of cases. Sixteen congenital toxoplasmosis were detected. Toxoplasmosis was confirmed in 13.7% of the 169 patients with lymphadenitis. For HIV positive patients, 11.7% had cerebral toxoplasmosis. It revealed the HIV infection in four cases. Among transplant recipients, one case of active toxoplasmosis was diagnosed in a renal transplant recipient who received transplant from a seronegative donor. Twenty-one patients presenting toxoplasmic retinochoroiditis were treated by subconjonctival injections of clindamycin and systemic corticotherapy at a dose of 1 mg/kg per day. This clinical toxoplasmosis diversity explains the need for bioclinical confrontation to establish diagnosis.

[A fatal case of dermatomycoses with retropharyngeal abscess]. / Un cas de maladie dermatophytique fatale par abcès rétropharyngé

BACKGROUND: Dermatophytic disease is a rare chronic infection caused by dermatophytes and characterised by cutaneous and visceral invasion. It is observed in North Africa. It is associated with cellular immunodeficiency and refractoriness to drug treatment. CASE REPORT: We report a new case of dermatophytic disease in a 26-year-old Tunisian woman. The patient's parents were first cousins; no other family members had a similar disease state. At the age of 3 years, the patient developed extensive tinea corporis associated with onychomycosis resistant to the usual antifungal drugs. The patient was hospitalised for multiple subcutaneous vegetative and ulcerative lesions of the scalp, face and chest associated with multiple adenopathies, occasionally fistular, of the axillary, cervical, mammary and inguinal areas. Mycology and histology confirmed the presence of fungal hyphae. Trichophyton violaceum was isolated in cultures of various skin lesion and lymph node biopsy samples. Investigations showed no evidence of immunodeficiency. Although the patient initially responded well to itraconazole (Sporanox, 400 mg/day), her condition worsened with obstruction and dyspnoea due to retropharyngeal pus collection requiring repeated surgical evacuation, and ultimately leading to a fatal outcome. DISCUSSION: Dermatophytic disease remains a severe disease due to the possibility of potentially life-threatening visceral involvement and antifungal drug resistance.

First case of Microsporum ferrugineum from Tunisia.

This is the first case of Microsporum ferrugineum isolated from a Tunisian patient. A 60-year-old man was admitted for tinea sycosis associated with circinate herpes of the hand. Examination disclosed diffuse erythematic and perifollicular papules and pustules in the beard area. Typical ringworm vesiculo-pustular lesions involved skin of the hand. Isolates were identified as Microsporum sp on the basis of macroscopic and microscopic colony characteristics. The diagnosis of M. ferrugineum was confirmed by PCR sequencing of Chitin Synthase1 gene. The patient was treated successfully with Griseofulvin, which was administered for 4 weeks.