Relationship Between Phenotypic and Genotypic Characteristics of Trichophyton mentagrophytes Strains Isolated from Patients with Dermatophytosis.

According to epidemiological, clinical and mycological criteria, it has long been admitted that the Trichophyton mentagrophytes species includes two varieties: a zoophilic variety (var. mentagrophytes) and an anthropophilic variety (var. interdigitale) that involve the upper and the lower part of the body, respectively. The further application of molecular techniques to the characterization of dermatophyte strains showed that this classification is unreliable. The aim of our study was to assess the usefulness of PCR-RFLP (restriction fragment length polymorphism) and sequencing in the characterization of T. mentagrophytes strains taken from Tunisian patients. The study was carried out in 2008 in the laboratory of Parasitology-Mycology of Farhat Hached University Hospital, Sousse, Tunisia. A total of 133 strains were isolated from 133 patients addressed to the laboratory for dermatological lesions very evocative of dermatomycosis. Eighty strains were isolated from lesions located on the lower part of the body (onychomycosis, tinea pedis) and 53 strains from the upper part of the body (tinea capitis, tinea corporis). All strains were submitted to mycological examination (direct microscopic examination and culture on Sabouraud medium) and further investigated by using RFLP analysis of the PCR-amplified ITS1-5.8 s-ITS2 region of the ribosomal DNA and the MvaI restriction enzyme. In addition, 62 strains were further submitted to a sequencing of the ITS1-5.8 s-ITS2 region. On the basis of mycological criteria, all strains were diagnosed as T. mentagrophytes. All strains produced the same RFLP pattern and were identified as T. mentagrophytes interdigitale regardless of the location of lesions. Out of the 62 sequenced strains, 16 were found anthropophilic and 46 were zoophilic. In conclusion, all strains provisionally diagnosed as T. mentagrophytes on the basis of mycological criteria were shown to belong to T. interdigitale by using PCR-RFLP and sequencing irrespective of the site of lesions. The predominance of zoophilic strains needs further investigation.

Multiplex PCR assay for the detection of common dermatophyte nail infections.

Onychomycosis is one of the most prevalent dermatophytic diseases. Mycological methods used in the conventional diagnosis may not be optimal. Multiplex (MX) PCR was reported as a reliable alternative. Dermatophyte gene sequence records were used to design a MX PCR for detection and identification of dermatophytes in nail specimens. A MX PCR method based on the amplification of the chitin synthase 1 and internal transcribed spacer genes was developed. The study included 93 strains of dermatophytes and non-dermatophytic fungi, six dermatophytic reference strains and 201 nail specimens from patients with dermatophytic onyxis. DNA extraction directly from nail samples was carried out by using the QIAamp DNA extraction kit (Quiagen). A set of primers was designed and their specificity was assessed. MX PCR detected the causal agent in specimens from which Trichophyton rubrum and T. interdigitale grew in culture and also identified a dermatophyte species in an additional 32 specimens that were negative in microscopy and culture. None of the investigated non-dermatophytic strains was positive. Sensitivity of MX PCR was higher as compared to mycological examination (97% vs. 81.1%). MX PCR for direct detection of dermatophytes from nail samples yielded mixed flora in 32.8% of samples. MX PCR proved sensitive and adequate for the diagnosis of dermatophytic onychomycosis. It is much adapted to cases where culture is negative or contaminated by overgrowing moulds, which makes the identification of the causal agent problematic.

Clinical and mycological features of onychomycosis in central Tunisia: a 22 years retrospective study (1986-2007).

Onychomycosis is the most frequently encountered nail disease and may be difficult to diagnose and treat. The objective of this study was to determine the prevalence, the clinical and mycological characteristics of onychomycosis in central Tunisia. It is a retrospective study performed over a 22-year period (1986-2007). It included 7151 patients (4709 women and 2442 men) with suspected fingernails and/or toenails onychomycosis. The patients were referred to the Mycology-Parasitology Laboratory of Farhat Hached hospital in Sousse for mycological examination. Both direct microscopy and culture of the nail material were performed to diagnose and identify the causative fungal species. Onychomycosis was confirmed in 78.6% of investigated patients (5624/7151). The positivity rate was higher in women as compared with men. In both men and women, fingernails were most frequently involved than toenails. No significant relation was found between gender and toenails onychomycosis, whereas fingernails were frequently involved in women. As far as aetiological agents are considered, dermatophytes, yeast and moulds were responsible for 49.9%, 47.4% and 2.7% of onyxis cases respectively. In fingernail infections, yeast were the most frequent fungi (83.6%), Candida albicans being the leading species (51.6%). In contrast, in toenail infections, dermatophytes were more frequent (74.1%). Trichophyton rubrum was by far the dominant species (88.1%). Yeast were observed more frequently in women whereas dermatophytes were more common in men. Moulds were involved in 4.2% of cases. The most frequent species were Aspergillus sp. and Chrysosporium sp. Onychomycosis is a frequent disease in central Tunisia. T. rubrum is the predominant agent in toenails infection and yeast, mainly C. albicans, in fingernails onychomycosis.

Neonatal invasive candidiasis in Tunisian hospital: incidence, risk factors, distribution of species and antifungal susceptibility.

The aim of our study was to assess epidemiological features of neonatal invasive candidiasis in Farhat Hached hospital of Sousse, Tunisia, including incidence, risk factors, mortality, species distribution and antifungal susceptibility. Laboratory data from 1995 to 2010 and medical records of 127 invasive candidiasis cases were reviewed. We tested the susceptibility of 100 Candida sp isolates by using ATB fungus(®) 3 and to fluconazole by using E-test(®) strips. A total of 252 cases of neonatal invasive candidiasis occurred over the study period. The incidence increased 1.8-fold from 1995 to 2006 and decreased fourfold from 2007 to 2010. Candida albicans was the predominant species up to 2006 and a shift in the species spectrum was observed with increase of the non-albicans species mainly C. parapsilosis. The agreement between the ATB Fungus(®) and the E-test(®) for determining fluconazole susceptibility was high. All tested isolates were susceptible to fluconazole, flucytosine, amphotéricine B and voriconazole and the itraconazole resistance rate was 5%. The mortality rate was 63%. The invasive candidiasis incidence increased from 1995 to 2006 and decreased from 2007 to 2010. The spectrum of Candida species and the lack of fluconazole-resistant strains argue for the usefulness of fluconazole as an empiric treatment.

Asymptomatic carriage of Leishmania in family members of patients with visceral leishmaniasis in Central Tunisia.

INTRODUCTION: In Tunisia, asymptomatic carriage of Leishmania is poorly documented. OBJECTIVE: The aim of the present study was to estimate the frequency of asymptomatic infection among the family members of patients with patent visceral leishmaniasis by using the Western blotting kit based on 14 and 16kDa bands. MATERIAL AND METHODS: We tested 94 sera collected from 24 patients with patent visceral leishmaniasis and 70 from their families' members. RESULTS: The rate of seropositivity was 100% in the group of patients and 54.3% in the group of families' members. The analysis of the Western blotting patterns showed that the 33kDa, 24kDa and to a lesser extent the 22kDa band were very indicative of patent visceral leishmaniasis in contrast to asymptomatic infection where these bands were very rarely detected. CONCLUSION: The results reported herein showed the high frequency of asymptomatic carriers of Leishmania among the families' members of visceral leishmaniasis cases and the usefulness of the Western blotting as a screening technique and in distinguishing between patent visceral leishmaniasis and the asymptomatic carriage of Leishmania.

[Mycological and epidemiological aspects of tinea capitis in the Sousse region of Tunisia]. / Aspects mycologiques et épidémiologiques des teignes du cuir chevelu dans la région de Sousse (Tunisie).

AIM: Tinea capitis continues to be considered a public health problem in Tunisia. The purpose of our study was to investigate trends in the incidence and the mycological and epidemiological aspects of tinea capitis in the Sousse region (Central Tunisia). METHOD: Our work is a retrospective study concerning all scalp samples taken by the parasitology laboratory of the Farhat Hached Hospital in Sousse, Tunisia, over a 26-year period (1983-2008). RESULTS: A total of 10,505 specimens were examined. Of these, 5593 were positive with positive direct examination and/or positive culture. The average incidence was 215 cases per year. Patients were aged under 12 years in 89.3% of cases. A total of 175 cases of tinea capitis in adults were diagnosed. Ten dermatophyte species were isolated: Trichophyton (T.) violaceum (66.7%), Microsporum (M.) canis (29.3%), T. schoenleinii (1.6%), T. mentagrophytes (1.1%), T. verrucosum (0.6%), T. tonsurans (0.2%), T. rubrum (0.2%), M. gypseum (0.1%), M. audouinii (0.03%) and M. nanum (0.01%). CONCLUSION: Our study showed a decrease in the annual incidence of tinea capitis over the study period with an evident decrease in trichophytic tinea and disappearance of favus giving way to microsporic and inflammatory tinea.

Systemic neonatal candidosis: the karyotyping of Candida albicans strains isolated from neonates and health-workers.

Candida albicans has become an important cause of nosocomial infections in neonatal intensive care units (NICUs). The aim of the present study was to compare C. albicans strains isolated from neonates (NN) suffering from systemic candidosis and from nurses in order to determine the relatedness between NN and health workers' strains. Thirty-one C. albicans strains were isolated from 18 NN admitted to the NICU of the neonatology service of Farhat Hached Hospital of Sousse, Tunisia and suffering from systemic candidosis, together with five strains recovered from nurses suffering from C. albicans onychomycosis. Two additional strains were tested, one from an adult patient who developed a systemic candidosis and the second from an adult with inguinal intertrigo. All strains were karyotyped by pulsed-field gel electrophoresis (PFGE) with a CHEF-DR II system. Analysis of PFGE patterns yielded by the 38 strains tested led to the identification of three pulsotypes that were designated I, II and III, and consisted of six chromosomal bands with a size ranging from 700 to >2500 kbp. The most widespread was the pulsotype I, which was shared by 17 NN and the five nurses' strains. The identity between NN and nurses' strains is very suggestive of a nosocomial acquisition from health-workers.

Alternaria alternata infection causing rhinosinusitis and orbital involvement in an immunocompetent patient.

Invasive fungal rhinosinusitis is a rare infection that occurs primarily in immunocompromised patients. The fungal pathogen Alternaria alternata is rarely associated with rhinosinusitis. We report a case of A. alternata rhinosinusitis in an immunocompetent patient.

Demodicosis revealing an HIV infection.

We report an observation of facial and upper limb demodicosis, revealing a human immunodeficiency virus infection. After an initial improvement with metronidazole, worsening of skin lesions related to immune reconstitution inflammatory syndrome was observed, requiring the use of steroids.

[Contribution of leukoconcentration in the diagnosis of Kala-azar in Tunisia]. / Apport de la leucoconcentration dans le diagnostic du Kala-azar en Tunisie.

OBJECTIVE: The authors had for aim to determine the role of leukoconcentration in the diagnosis of visceral leishmaniosis in immunocompetent children. MATERIALS AND METHODS: A study was made on leukoconcentration in blood samples of 84 immunocompetent children presenting with visceral leishmaniosis, hospitalised in the paediatric units of Sousse and Kairouan (Tunisian center) between April 1996 and March 2005. RESULTS: The study group included 34 girls and 50 boys (sex-ratio = 1.47) aged six months to ten years. In this group, 47 patients (56%) presented with positive leukoconcentration. The number of leishmania detected ranged from 1 to 64 per slide; parasitism of PMN leucocytes was noted in nearly half of the cases. CONCLUSION: Parasitemia is frequent in the Mediterranean Kala-azar; therefore leukoconcentration on peripheral blood can be proposed as a first intention examination for the diagnosis of visceral leishmaniosis in immunocompetent children.

[Infantile cryptosporidiosis in Sousse area, Tunisia]. / La cryptosporidiose infantile dans la région de Sousse, Tunisie.

OBJECTIVES: The aim of this study was to evaluate infantile cryptosporidiosis prevalence in central Tunisia. POPULATION AND METHODS: Stool samples from 34,020 immunocompetent children were investigated for oocysts through a 12-year study. RESULTS: Cryptotosporidia were detected in 0.32% (108) of samples. Ninety three per cent of children were less than five years old. Incidence was much higher in summer and autumn. Leading clinical symptoms were diarrhea, fever and vomiting. Symptoms spontaneously resolved in all patients. In three cases, infection was asymptomatic. CONCLUSION: Data analysis indicate that cryptosporidiosis is not uncommon in diarrheic central Tunisian children.

[Invasive aspergillosis in the leukemic patient]. / Aspergillose invasive chez le leucémique.

Invasive pulmonary aspergillosis (IPA) remains a life threatening complication in immuno-compromised and especially in neutropenic patients. We report our experience in the diagnosis and therapeutic management of IPA in 8 patients with acute leukemia. All patients were neutropenic (PNN < 100/mm3, mean duration = 37 days) when IPA was diagnosed. Clinical signs included fever above 39 degrees and cough in all cases, chest pain in 4 cases, hemoptysis in 3 cases, rales in 5 cases. Chest x ray showed one lesion in 4 cases and multiple lesions in 4 cases. The diagnosis of IPA was established by bronchoalveolar lavage (BAL) in 5 cases, tissue biopsy in one case, positive sputum in one case and it was highly probable in one case. Thoracic computed tomographic (CT) scans were preformed after diagnosis confirmation of IPA and showed one or multiple lesions with air crescent signs. Serological tests were positive in 4 cases late in the course of IPA. All patients were treated with i.v. Amphotericin B. Outcome was favorable in 5 cases and three patients died by massive hemoptysis (in two cases) and systemic aspergillosis (in one case). Early diagnosis and appropriate treatment are essential to improve IPA prognosis.

[Double fungemia. Report of four Tunisian cases]. / Les fongémies mixtes. À propos de quatre cas tunisiens.

UNLABELLED: Fungemia is classically caused by a single species and the detection of more than one species in blood samples is uncommon. We report four cases of mixed fungemia (MF) diagnosed in the parasitology-mycology laboratory of Farhat-Hached hospital in Sousse, Tunisia. The MF episodes occurred in two neonates and two adults suffering from acute myeloid leukemia. Two fungal species were detected concomitantly within the same blood culture in all cases. Species combination was detected by the subculture of the blood culture on Candida ID(®) chromogenic medium in three cases and on Sabouraud agar in one case. Predisposing factors were: indwelling catheters (4/4), broad-spectrum antibiotics (3/4), neutropenia (2/4), exclusive parenteral nutrition (2/4) and Candida colonization (1/4). Patients presented febrile sepsis with no response to broad-spectrum antibiotic therapy in all cases. Outcome under antifungal treatment was favorable in two cases and the two other patients died. CONCLUSION: MF appears similar to the more common monomicrobial fungemia. The use of chromogenic media in routine can improve the detection of MF episodes allowing appropriate antifungal therapy.

Evaluation of Chitine synthase (CHS1) polymerase chain reaction assay in diagnosis of dermatophyte onychomycosis.

BACKGROUND: Onychomycosis is one of the most prevalent dermatophytic diseases. Mycological methods used in the conventional diagnosis may not be optimal. PCR was reported as a reliable alternative in the diagnosis of dermatophytosis. MATERIALS AND METHODS: A PCR method based on the amplification of the chitin synthase 1 gene was developed. The study included 119 strains of dermatophytes and non dermatophytic fungi, eight dermatophytic reference strains and 201 nail specimens from patients with dermatophytic onyxis. DNA extraction was carried out by using the QIAamp DNA extraction kit (Quiagen). RESULTS: PCR positivity was based on the production of a specific 432 bp fragment. None of the investigated non dermatophytic strains was positive. Sensitivity of PCR was higher as compared to mycological examination (90.5% vs. 81.1%). PCR was positive in 31 onyxis cases with positive direct examination but negative or contaminated culture. In contrast, PCR was negative in 10 cases where both direct examination and culture were found positive. CONCLUSION: PCR is an adequate tool for the diagnosis of dermatophytic onychomycosis. It is much adapted to cases where culture is negative or contaminated by overgrowing molds, which makes the identification of the causal agent problematic.

Twenty-nine cases of invasive aspergillosis in neutropenic patients.

INTRODUCTION: Invasive aspergillosis is a life-threatening infectious complication in hematological patients undergoing immunosuppressive chemotherapy. PATIENTS AND METHODS: We report 29 cases of invasive aspergillosis diagnosed in the Sousse Farhat Hached hospital Hematology unit, Tunisia, between 2002 and 2010. RESULTS: The most frequent disease (65.5%) was acute myeloid leukemia. All patients were severely neutropenic (<500/mm(3), mean duration=27 days). Pulmonary invasive aspergillosis was suggested in 28 (96.5%) cases. The most frequent respiratory signs were cough (64.3%), chest pain (53.6%), and hemoptysis (50%). The chest X-ray showed suggestive lesions in 60.7% of cases. CT scans revealed nodules with cavitation in 65% of cases, a halo sign in 20% of cases, and nodules in 15% of cases. Galactomannan antigenemia was positive in 88%, mycological examination positive in 51.6%, and seroconversion was noted in 35.7% of the cases. Invasive pulmonary aspergillosis was classified, according to EORTC/MSG criteria, as probable in 26 cases, possible in one case, and proven in one case. Aspergillus flavus was the dominant species in pulmonary invasive aspergillosis accounting for 73.7% of isolates. Extrapulmonary involvement was suggested in 39.3% of cases, the most frequent were sinusitis and brain abscess. Primary cutaneous aspergillosis was observed in one case. The overall mortality rate was 64.2%; the 12-week survival rate was 71.4%. CONCLUSION: Our results are correlated to published data. A. flavus was the most frequent species in our region.

Immunochromatographic rK39 strip test in the serodiagnosis of visceral leishmaniasis in Tunisia.

The performance of the rK39 strip test in the diagnosis of Tunisian visceral leishmaniasis (VL) was evaluated and compared with that of immunofluorescent antibody test (IFAT). A total of 929 sera, including 574 from VL patients, 54 from cutaneous leishmaniasis (CL) patients, 42 from patients with other protozoan diseases, 152 from patients with non-parasitic diseases and 107 from healthy controls, were used in the study. The sensitivity and specificity of the rK39 strip test were 87.1 and 94.4%, respectively. Sixteen CL sera showed positive results, suggesting that the rK39 strip test is not restricted to Leishmania donovani complex detection. IFAT was comparatively more sensitive (98.9%) but slightly less specific (90.7%). Despite cross-reactivity shown by CL sera, the rK39 strip test can be recommended for the routine diagnosis of VL in Tunisia, as VL and CL are distinct clinical entities.

Biosorptive uptake of methylene blue using Mediterranean green alga Enteromorpha spp.

Batch biosorption experiments were carried out for the removal of methylene blue, a basic dye, from aqueous solution using raw and dried Enteromorpha spp., Mediterranean green alga. A series of assays were undertaken to assess the effect of the system variables, i.e. contact time, solution pH and sorbent amount. The results had showed that sorption capacity was optimal using 6-10 solution pH range (i.e. maximum adsorption capacity of 274 mg/g). The minimum sorbent concentration experimentally found to be sufficient to reach the total removal of the dye molecules from the aqueous solution was 5 g/L. Besides, equilibrium data were fitted using five linearisable isotherm models. The related results showed that the experimental data were very well represented by the Langmuir model for the linear regression analysis and both the Langmuir and Redlich-Peterson isotherm models for the non-linear analysis. In both cases, such modelling behaviour confirms the monolayer coverage of methylene blue molecules onto energetically homogenous Enteromopha surface. In addition, an exhaustive comparative study was done to situate this marine biomass among other proposed sorbents.