Identification of soluble and membrane antigenic markers of acquired toxoplasmosis by immunoblot.

The overall performance of quantitative assays in the detection of anti-Toxoplasma IgG is satisfactory, but discrepancies between assays are not uncommon especially when IgG concentrations are close to the limit of detection of the tests. The purpose of our study was to identify soluble and membrane antigens extracted from Toxoplasma gondii tachyzoites by immunoblot to select the most relevant antigenic bands to be used for qualitative serodiagnosis of acquired toxoplasmosis. We selected five relevant bands (98, 36, 33, 32 and 21 kDa) with soluble antigens and four relevant bands (42, 35, 32 and 30 kDa) with membrane antigens which gave high sensitivity and/or specificity in immunodiagnosis. The association on the same blot of at least three of the five relevant bands in the soluble antigen immunoblot showed the highest sensitivity/specificity (97.4%/99.0%, respectively). Our results indicate that immunoblot using soluble tachyzoite extract with simultaneous detection of at least three of the five bands (98, 36, 33, 32 and 21 kDa) represents a valuable test for serodiagnosis of acquired toxoplasmosis and should be further evaluated as a confirmatory test for sera which give discrepant results in quantitative assays.

Molecular methods for strain typing of Candida albicans: a review.

Candida albicans is one of the most medically important fungi because of its high frequency as a commensal and pathogenic microorganism causing superficial as well as invasive infections. Strain typing and delineation of the species are essential for understanding its biology, epidemiology and population structure. A wide range of molecular techniques have been used for this purpose including non-DNA-based methods (multi-locus enzyme electrophoresis), conventional DNA-based methods (electrophoretic karyotyping, random amplified polymorphic DNA, amplified fragment length polymorphism, restriction enzyme analysis with and without hybridization, rep-PCR) and DNA-based methods called exact typing methods because they generate unambiguous and highly reproducible typing data (including microsatellite length polymorphism and multi-locus sequence typing). In this review, the main molecular methods used for C. albicans strain typing are summarized, and their advantages and limitations are discussed with regard to their discriminatory power, reproducibility, cost and ease of performance.

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.

[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 the immunoblotting for the detection of immunoglobulin G Toxoplasma antibodies in immunocompetent patients]. / Apport de l'immunoblot dans la détection des IgG anti-toxoplasmiques chez le sujet immunocompétent.

UNLABELLED: The serological tests commonly used for the diagnosis of toxoplasmosis raise the problem of the interpretation of the borderline immunoglobulin G (IgG) levels and discordant results between various tests. OBJECTIVE: The purpose of our study was to evaluate the contribution of the immunoblotting in the detection of specific IgG in acquired toxoplasmosis of immunocompetent patients especially when levels are equivocal or discordant in enzyme-linked immunosorbent assay (Elisa) and indirect fluorescent antigen test (IFAT). MATERIAL AND METHODS: [corrected] We tested three groups of sera. The first included 87 positive sera, the second 33 negative sera, and the last one 29 equivocal sera. RESULTS: Results obtained with the first and the second group of sera led us to identify the bands 30kDa and 32kDa as markers of the toxoplasmic infection. The simultaneous presence of both bands showed a sensitivity of 91.5%, a specificity of 96.9%, a VPP of 98.7%, a VPN of 74.4% and a Youden's index of 0.88. Our findings suggest that the presence of these two bands is a reliable criterion for the confirmation of the presence of anti-toxoplasmic IgG in the corresponding serum. The immunoblot allowed us to ascertain serological status of 27 (93.1%) patients from the third group in which results were discrepant or equivocal in Elisa and/or in IFAT. CONCLUSION: Immunoblot is a useful serological test for detection of very low or equivocal titers.

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.

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.

[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.

Molecular typing of Candida albicans isolates from patients and health care workers in a neonatal intensive care unit.

AIMS: The aim of this study was to investigate the genetic relatedness between Candida albicans isolates and to assess their nosocomial origin and the likeliness of cross-transmission between health care workers (HCWs) and hospitalized neonates in a neonatal intensive care unit (NICU). METHODS: We retrospectively analysed 82 isolates obtained from 40 neonates and seven isolates from onychomycosis of the fingers of five HCWs in a Tunisian NICU by using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis with CA1 and CA2 as primers. RESULTS: In RAPD analysis, the discriminatory power (DP) of CA1 and CA2 primers was 0·86 and 0·81, respectively. A higher DP was achieved by combining patterns generated by both primers (0·92), while PFGE karyotyping exhibited the lowest DP (0·62). The RAPD-CA1/CA2 analysis revealed that 65·8% of isolates obtained from neonates derived from a limited number (6) of groups of genetically identical strains, that five temporal clusterings occurred during the study period and that three HCWs' isolates and 11 isolates obtained from six neonates were identical. CONCLUSIONS: These findings argue for the nosocomial transmission of C. albicans in our NICU and for the transfer of strains from HCWs to patients. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of relatedness between Candida species obtained from neonates and health care workers by using molecular techniques with high discriminatory power is essential for setting up specific control measures in order to reduce the incidence of nosocomial candidiasis.

[Treatment of severe hemophagocytic syndrome associated with visceral leishmaniasis]. / Traitement du syndrome d'activation macrophagique sévère associé à une leishmaniose viscérale.

The association of hemophagocytic syndrome (HS) and visceral leishmaniasis is a frequent disorder during infancy in endemic areas such as Tunisia. The range of severity of HS secondary to visceral leishmaniasis includes both pure biological forms that resolve with antimicrobial therapy and life-threatening emergencies that require specific treatment. We describe 2 cases of severe HS secondary to visceral leishmaniasis. The diagnosis of HS was based on the HLH-2004 diagnostic criteria. Therapy involved pentavalent antimonial (Glucantime) in both cases. The combination of corticosteroids with immunoglobulins, used in the 1st case, but introduced late, led to an unfavorable course and death. In the 2nd case, the specific treatment of HS was based on immunochemotherapy including etoposide and corticosteroids. Progression was favorable with a follow-up of 24 months. Etoposide containing therapeutic regimens can be proposed in severe forms of HS associated with visceral leishmaniasis.

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.

Molecular typing of clinical Candida strains using random amplified polymorphic DNA and contour-clamped homogenous electric fields electrophoresis.

AIMS: This report describes an investigation into the genetic profiles of 38 Candida albicans and 19 Candida glabrata strains collected from a dental hospital of Monastir (Tunisia) and the Laboratory of Parasitology, Farhat Hached Hospital of Sousse (Tunisia), using two typing methods: random amplified polymorphic DNA (RAPD) and contour-clamped homogenous electric fields (CHEF). METHODS AND RESULTS: The two methods (RAPD and CHEF electrophoresis) were able to identify clonal-related isolates from different patients. RAPD method using two primers (CA1 and CA2) exhibited the highest discriminatory power by discriminating 22 genotypes for C. albicans with CA1 oligonucleotides and 19 genotypes with CA2 primer. For C. glabrata, 17 genotypes were obtained when both primers CA1 and CA2 were combined. The CHEF karyotyping of C. albicans has discriminated only 17 different karyotypes. CONCLUSION: The genotype of each isolate and genotypic difference among C. albicans and C. glabrata isolates were patient specific and not associated with the site of infection, geographic origin or date of isolation. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of relatedness between Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for interruption of transmission of this yeast.

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.

[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.