Mohamed Fethi Diouani

Leishmania Major Infection Among Psammomys Obesus and Meriones Shawi: Reservoirs of Zoonotic Cutaneous Leishmaniasis in Sidi Bouzid (Central Tunisia)

A study was undertaken between November 2008 and March 2010, in the focus of cutaneous leishmaniasis of Central Tunisia, to evaluate the role of Psammomys obesus (n=472) and Meriones shawi (n=167) as reservoir hosts for Leishmania major infection. Prevalence of L. major infection was 7% versus 5% for culture (p=not signifiant [NS]), 19% versus 16% for direct examination of smears (p=NS), and 20% versus 33% (p=NS) for Indirect Fluorescent Antibody Test among P. obesus and M. shawi, respectively. The peak of this infection was in winter and autumn and increased steadily with age for the both species of rodents. The clinical examination showed that depilation, hyper-pigmentation, ignition, and severe edema of the higher edge of the ears were the most frequent signs observed in the study sample (all signs combined: 47% for P. obesus versus 43% for M. shawi; p=NS). However, the lesions were bilateral and seem to be more destructive among M. shawi compared with P. obesus. Asymptomatic infection was ~40% for both rodents. This study demonstrated that M. shawi plays an important role in the transmission and the emergence of Leishmania major cutaneous leishmaniasis in Tunisia.

Comparative evaluation of specific ELISA and RFFIT antibody assays in the assessment of dog immunity against rabies

Two techniques are currently used to evaluate the humoral immune responses to rabies vaccination: ELISA, which detects binding antibodies to viral antigens and the WHO reference rapid fluorescent focus inhibition test (RFFIT), which assays in vitro virus-neutralizing antibodies. In this study, we have comparatively evaluated antibody responses of dogs reared either in an experimental kennel or living in field conditions after vaccination with a cell culture-derived rabies vaccine. In experimental conditions, both ELISA and RFFIT techniques were well correlated. However, in field conditions, they yielded discrepant results particularly in evaluating the residual rabies immunity before vaccine administration and in identifying seroconverted dogs. After rabies vaccination in field conditions, while similar antibody titres and seroconversion rates were obtained using either technique, the discrimination of a given dog according to the seroconversion threshold depended on the assay. We concluded, that whereas in experimental conditions, ELISA and RFFIT were well correlated, in field conditions ELISA yielded upper estimates. Consequently, RFFIT, although a cumbersome test, should continue to be considered as the reference rabies antibody assay technique. A seroconversion threshold of 0.5 IU/ml should be cautiously considered and a higher threshold (1 IU/ml) could be more appropriate in the evaluation of rabies immunity in the field in order to marginalize the interfering factors.

Fabrication of Electrochemical Model Influenza A Virus Biosensor Based on the Measurements of Neuroaminidase Enzyme Activity

Neuroaminidase (NA) enzyme is a kind of glycoprotein that is found on the influenza A virus. During infection, NA is important for the release of influenza virions from the host cell surface together with viral aggregates. It may also be involved in targeting the virus to respiratory epithelial cells. In this study, a model electrochemical influenza A viral biosensor in which receptor-binding properties have been based on NA was developed for the first time. The biosensors working principle is based on monitoring the interactions between fetuin A and NA enzyme. The assay was monitored step by step by using electrochemical impedance spectroscopy.

Detection of ESAT-6 by a label free miniature immuno-electrochemical biosensor as a diagnostic tool for tuberculosis

Tuberculosis is a worldwide disease considered as a major health problem with high morbidity and mortality rates. Poor detection of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis remains a major obstacle to the global control of this disease. Here we report the development of a new test based on the detection of the major virulent factor of Mtb, namely the early secreted antigenic target 6-kDa protein or ESAT-6. A label free electrochemical immunosensor using an anti-ESAT-6 monoclonal antibody as a bio-receptor is described herein. Anti-ESAT-6 antibodies were first covalently immobilized on the surface of a gold screen-printed electrode functionalized via a self-assembled thiol monolayer. Interaction between the bio-receptor and ESAT-6 antigen was evaluated by square wave voltammetry method using [Fe(CN)6]3-/4- as redox probe. The detection limit of ESAT-6 antigen was 7ng/ml. The immunosensor has also been able to detect native ESAT-6 antigen secreted in cell culture filtrates of three pathogenic strains of Mtb (CDC1551, H37RV and H8N8). Overall, this work describes an immune-electrochemical biosensor, based on ESAT-6 antigen detection, as a useful diagnostic tool for tuberculosis.

Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia.

Accurate detection and complete shape extraction of sand-flies using Gaussian mixture model

This paper presents a method for the accurate detection of the positions of moving phlebotomenae (sand-flies), and the extraction of their complete shape. The proposed method is based on the background subtraction approach, with a statistical background model found using Gaussian mixture model. Furthermore, a method based on the maximization of interclass variance, is used to eliminate wings of the phlebotomenae and then detect accurately its position. Results are further used to study the behaviour of phlebotomenae, and subsequently, improve the traps to fight against many diseases transmitted by these insects, especially leishmaniasis. The experimental results show the efficiency of the proposed algorithm to accurately detect the position and extract the complete shape of moving phlebotomenaes even in case of very blurry ones.

A new approach based on the serological tests for the diagnosis of tuberculosis in cattle

The diagnosis of bovine tuberculosis (TB) is still a challenge for a better control of the disease. Here we report the use of a simple ELISA test combined with a multilayer neuronal network analyzing method in order to diagnose TB in cattle from the north part of Tunisia. A panel of Mycobacterium bovis (Mbv)-specific recombinant proteins along with crud extracts were used to coat the 96 wells plates, namely the recombinant 10 kDa culture filtrate antigen (CFP-10), the 6 kD Early Secretory Antigenic Target (ESAT-6), the recombinant Esat-6/CFP-10 heterodimer along with the crud BCG proteins and Tuberculin purified protein derivative (PPD). In the current article, a new approach is described to compare their characterization degree to select the most discriminative antigens. The classification of subjects into two groups: TB+ and TB-subjects was affected by an artificial multilayer neural network and the statistical study to estimate the diagnosis of bovine tuberculosis and construct an optimal partition of the results. This method was applied on the serological tests of a set of cattles. The results are encouraging.

Automatic identification and behavioral analysis of phlebotomine sand flies using trajectory features

The present paper reports an automated approach for the characterization and analysis of the behavioral of sand flies; the method used is based on Gaussian mixture model and Kalman filter for the detection and tracking of sand flies, and then the extraction of an optimized set of features from the trajectory of flight is performed for the classification process. So, we propose here two optimized sets of features; the first one is used to identify sand flies among other insects, and the second is employed for the characterization of the behavioral change in the sand flies in the presence of a repulsive odor. These features are tested on three different classifiers; artificial neural network, support vector machine and K-nearest neighbor (KNN), and the results show an important improvement in the classification accuracy and confirm the effectiveness of our approach; the accuracy rate of the proposed method reached 88.6% for the identification of sand flies and 93.4% for the detection of their behavior change. Instead of the excessive use of pesticides over wide areas, the presented investigation is a key pillar of the development of an ecological way for a statistical information gathering about sand flies in order to fight against disease carried by those insects especially leishmaniosis and pappataci fever.

Diagnosis of Human Echinococcosis via Exhaled Breath Analysis: A Promise for Rapid Diagnosis of Infectious Diseases Caused by Helminths

Background Human echinococcosis is a neglected infectious disease affecting more than 1 million people globally. Its diagnosis is expensive and difficult because of lack of adequate resources in low-resource locations, where most cases occur. Methods A group of volunteers diagnosed with the 2 main types of echinococcosis and corresponding control groups were recruited from hospitals in Tunisia (32 patients with cystic echinococcosis and 43 controls) and Poland (16 patients with alveolar echinococcosis and 8 controls). Breath samples were collected from all patients and analyzed by gas chromatography coupled to mass spectrometry, and a specifically developed electronic nose system. Results The chemical analysis revealed statistically different concentrations of 2 compounds in the breath of patients with cystic echinococcosis compared to controls, and statistically different concentrations of 7 compounds in the breath of patients with alveolar echinococcosis compared to controls. The discrimination accuracy achieved by the electronic nose system was 100% for cystic echinococcosis and 92.9% for alveolar echinococcosis, while the discrimination accuracy between these 2 patient groups was 92.1%. Conclusion Here we advocate a noninvasive, fast, easy-to-operate and nonexpensive diagnostic tool for the diagnosis of human echinococcosis disease through exhaled breath analysis, suitable for early diagnosis and population screening.

Ultrasensitive detection of hazardous reactive oxygen species using flexible organic transistors with polyphenol-embedded conjugated polymer sensing layers

Here we report that superoxide, one of the hazardous reactive oxygen species (ROS), can be quickly detected by flexible organic field-effect transistors (OFETs) with the polyphenol-embedded conjugated polymer micro-channels. Rutin, one of the abundant polyphenols found in a variety of plants, was employed as a sensing molecule and embedded in the poly(3-hexylthiophene) (P3HT) matrix. The rutin-embedded P3HT layers showed randomly distributed micro-domains, which became bigger as the rutin content increased. The best transistor performance was achieved at the rutin content of 10 wt%, while the OFETs exhibited proper and controllable transistor performances even in the phosphate buffer solutions. The sensing test revealed that the present OFET sensors could stably detect superoxide using very small amount (<10 μl) of samples at extremely low concentrations (500 pM), while they exhibited outstanding stability and durability upon repeated detection and storage-reuse tests. Finally, the present flexible OFET sensors could deliver confident sensing results for the detection of superoxide generated from the mouse RAW264.7 macrophages.