Leishmaniasis in Tunisia: History and New Insights into the Epidemiology of a Neglected Disease

In Tunisia, both cutaneous (CL) and visceral leishmaniases (VL) are historical diseases that have been described since the nineteenth century. Cutaneous form is more prevalent than the visceral one. It is caused by three taxa (Leishmania major, Leishmania infantum, and Leishmania killicki synonymous Leishmania tropica) and six zymodemes (MON-1, MON-8, MON-24, MON-25, MON-80, and MON-317). Among these dermotropic zymodemes, sand flies vectors and reservoir hosts were identified for only three ones. Transmission cycles of L. infantum MON-24 and MON-80 and L. killicki MON-317 are still unknown. Zoonotic CL is largely distributed and covers mainly the sub-arid and arid bioclimatic stages. Nevertheless, it has recently spread to the humid and sub-humid stages in northern Tunisia. Sporadic and chronic CL are less prevalent with limited geographical distribution. Visceral leishmaniasis (VL) is mainly infantile that affects children of <13 years. It is caused by the single taxon L. infantum. Transmission cycle of this parasite is zoonotic but not well elucidated. Three zymodemes are responsible for the genesis of VL (MON-1, MON-24 and MON-80). Only the transmission cycle of L. infantum MON-1 is identified. Geographically, VL is mainly distributed in the humid, sub-humid, and semi-arid bioclimatic stages of the country. Despite the large progress of knowledge in the ecoepidemiology of leishmaniases in Tunisia, many parameters of the transmission cycles of these taxa are still unknown and need further investigations to identify them

Culicoides spp. (Diptera: Ceratopogonidae) in Tunisia

Culicoides is a genus of biting midges in the family Ceratopogonidae. The female midges require blood meals for egg production. There are over 1000 species in the genus, which is divided into many subgenera. Several species are known to be vector of many diseases and parasites, which can affect animals. As vectors of viruses, Culicoides species are of the higher veterinary importance. More than 75 arboviruses, belonging to Bunyaviridae, Reoviridae and Rabdoviridae families, were isolated from different Culicoides species. In Mediterranean region, the principal vector of Bluetongue virus is represented by Culicoides imicola, and also other European Culicoides biting midges are implicated in virus transmission. Despite the virulence of these species and his colonisation in Tunisia, they are still considered as neglected area due to the rarity or the absence of programmes to control these biting midges. Thus, the available data on species composition, dominant species, breeding sites and host preferences are urgently needed to better understand these biting midges and to develop reliable tools to prevent the spread of other diseases that threaten human and animal life

Molecular and Epidemiology Data on Cystic Echinococcosis in Tunisia

Cystic echinococcosis (CE) or hydatidosis is a widespread zoonose in the world. In Tunisia, despite the deployed prevention program, CE remains a serious public health problem. With a human surgical annual incidence averaging 12.6/100000 inhabitants, Tunisia is one of the most endemic areas amongst the Mediterranean countries. Three Echinococcus species have been described: E. granulosus sensu stricto (G1 and G3 genotypes), E. canadensis, and E. equinus. CE, which commonly starts during childhood and described as a young adult disease, may be observed at any age. The liver and the lungs are the most commonly involved organs but the cyst can occur almost anywhere in the body. In production animals, the prevalence of CE is ranged from 16.42% to 40.42% in sheep, 8.56% in cattle, 6% in dromedaries, 2.9% in goats, and 8.48% in donkeys. The elevated number of stray and semi-stray dogs and their frequent contamination by E. granulosus infected viscera is the major cause of the CE spread. A high prevalence of E. granulosus infection has been reported in Tunisian dogs ranging from 3.75% to 27.1%, depending on the regions. Thus, the sanitary education concerning hydatidosis should be reinforced and efforts should be made to implement a targeted educational program

Morphological Keys for the Identification of Tunisian <em>Culicoides</em> Biting Midges (Diptera: Ceratopogonidae)

Culicoides biting midges are tiny blood-feeding insects of several diseases with veterinary and public health significance, including Bluetongue in ruminants, African horse sickness in equids and filarial diseases like Onchocercosis and Mansonellosis affecting various species such as humans. Their identification depends basically on the microscope examination of key morphological characters. Consequently, identification keys are important to any non experiment working with these biting midges. The Tunisian fauna of Culicoides biting midges consists of 35 species, whose morphological delineation may be troublesome for non-taxonomists. In response to this situation, and for the first time a key to the adult Culicoides species in Tunisia was prepared

Isolation and molecular characterization of recombinant Echinococcus granulosus P29 protein (recP29) and its assessment for the post-surgical serological follow-up of human cystic echinococcosis in young patients

We synthesized recombinant Echinococcus granulosus protoscolex recP29 antigen to be preliminarily assessed by ELISA and immunoblotting. RecP29-serology was carried out on 54 young patients with cystic echinococcosis (CE). Patients were classified into either cured (CCE) (n=40) or non-cured (NCCE) (n=14) CE patients. RecP29 ELISA showed a gradual decrease of antibody concentrations in all CCE cases that were initially (before treatment) seropositive to this antigen (25 out of 40) or that seroconverted following treatment. A complete seronegativity was reached within 3 years post-surgery in all of these cases. Conventional HCF ELISA yielded seronegativity in only 10% of initially recP29-seropositive CCE patients (P=0.086). Likewise, recP29 immunoblotting yielded seronegativity in 93% of 29 out of 40 initially recP29-immunoblot-positive CCE patients after 3 years follow-up, compared with 72% in the HCF immunoblotting (P=0.060). Eleven out of 14 NCCE patients were initially positive by recP29 ELISA, and 10 out of these maintained a marked anti-recP29 antibody reactivity until the endpoint of the follow-up period. All 14 NCCE cases were initially seropositive by recP29 immunoblotting, and 13 cases remained seropositive until the end of the study. Thus, recombinant P29 protein appears prognostically useful for monitoring those post-surgical CE cases with an initial seropositivity to this marke

Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications

Background: Leishmania (L.) killicki is responsible for the chronic cutaneous leishmaniasis. The taxonomic status of this parasite is still not well defined. It was suggested on one hand to include this taxon within L. tropica complex but also on the other hand to consider it as a distinct phylogenetic complex. The present work represents the more detailed study on the evolutionary history of L. killicki relative to L. tropica and the taxonomic implications. Methods: Thirty five L. killicki and 25 L. tropica strains isolated from humans and from several countries were characterized using the MultiLocus Enzyme Electrophoresis (MLEE) and the MultiLocus Sequence Typing (MLST) approaches. Results: The genetic and phylogenetic analyses strongly support that L. killicki belongs to L. tropica complex. The study suggests the emergence of L. killicki by a funder effect followed by an independent evolution from L. tropica, but does not validate the species status of this taxon. In this context, we suggest to call this taxon L. killicki (synonymous L. tropica) until further epidemiological and phylogenetic studies justify the L. killicki denomination. Conclusions: These findings provided taxonomic and phylogenetic informations on L. killicki and helped to better know the evolutionary history of this taxon

Effect of seasonal variation on the chemical composition and antioxidant and antifungal activities of Convolvulus althaeoides L. leaf extracts

The composition of polyphenols, chlorophylls and carotenoids of eight extracts of Convolvulus althaeoides L. leaves, harvested in two different seasons, winter and spring, and extracted by hot extraction method using four solvents (dichloromethane, chloroform, ethyl acetate and ethanol) with increasing polarity, were evaluated along with their antioxidant and antifungal activities. Qualitative and quantitative variations were observed in the composition based on two different high performance liquid chromatography systems, liquid chromatography-photodiode array detection coupled to either atmospheric pressure chemical ionization mass spectrometry or to electrospray ionization mass spectrometry, permitting the identification of 22 polyphenols, 11 chlorophyll derivatives and 10 carotenoid compounds. Polyphenolic compounds were predominant in extracts from leaves collected in winter, whereas pigments were predominant in the spring collections. Antioxidant activities of the extracts were determined by DPPH radical scavenging method, revealing a half inhibition concentration (IC50) ranging from 0.1369 ± 0.0272 mg g−1 to 0.432 ± 0.0018 mg g−1, with no correlation to seasonal fluctuation. Concerning antifungal assays, ethyl acetate and ethanol extracts have been shown to be the most active against dermatophytes (T. rubrum, T. menthagrophytes, M. canis), with inhibiting percentages reaching 100% with 50 mg mL−1. Moreover, ethyl acetate and ethanol extracts showed a maximum inhibition potential with minimum inhibitory/fungicidal concentrations ranging from 0.78 to 6.25 mg mL−1 on Candida spp. cultures. The winter collect of these extracts showed an inhibitory effect of 90% on Candida albicans germ tubes formation, at a concentration of 3.1 mg mL−1. In conclusion, seasonality seems to influence the quality and the quantity of natural substances from leaves of C. althaeoides L., which have major importance on the antioxidant and the antifungal effectiveness

A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.The dog tapeworm Echinococcus granulosus (E. granulosus) is a cosmopolitan parasite. The adult worms reside in the small intestine of their definitive hosts (dogs). Infective eggs are shed with the feces into the environment and are orally ingested by intermediate hosts where they develop into the metacestode (larval) stage, causing cystic echinococcosis (CE) in humans and livestock. Ten intraspecific genotypes of E. granulosus (G1 to G10) have been reported from different intermediate host species. Based on the recently established molecular phylogeny, E. granulosus is now considered a complex consisting of four species: E. granulosus sensu stricto (G1/G2/G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6–G10). Simple and highly discriminative molecular epidemiological approaches are needed to explore dynamics, life cycle patterns, and the pathogenicity of the members of this complex. We here introduce a one-step multiplex PCR (mPCR) protocol for the genotyping and discrimination of the different members of the E. granulosus complex, allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex, and (iii) genetic variants within the E. granulosus complex. The relatively complicated task of E. granulosus complex speciation and genotyping is clearly simplified by mPCR, and this technique therefore represents a useful tool for routine practice. (Author Summary)Fil: Boubaker, Ghalia. University of Berne; SuizaFil: Macchiaroli, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Prada, Laura Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Fernández, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Rosenzvit, Mara Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Ziadinov, Iskender. Universitat Zurich; SuizaFil: Deplazes, Peter. Universitat Zurich; SuizaFil: Saarma, Urmas. Universitat Zurich; SuizaFil: Babba, Hamouda. University of Monastir; TúnezFil: Gottstein, Bruno. University of Berne; SuizaFil: Spiliotis, Markus. University of Berne; Suiz

Cytokine Profiles in Toxoplasmic and Viral Uveitis

BackgroundUveitis is a major cause of visual impairment throughout the world. Analysis of cytokine profiles in aqueous humor specimens may provide insight into the physiopathological processes that underly retinal damage in this context MethodsUsing a multiplex assay, we determined the concentrations of 17 cytokines and chemokines in aqueous humor specimens obtained from patients with ocular toxoplasmosis or viral uveitis and compared these concentrations with those in specimens obtained from patients with noninfectious intermediate uveitis or cataract ResultsFive mediators (interleukin [IL]-8, monocyte chemoattractant protein-1, tumor necrosis factor-α, IL-4, and IL-10) were detected in >50% of patients in all groups. In contrast, IL-5 and IL-12 were specific for ocular toxoplasmosis, and granulocyte monocyte colony-stimulating factor and IL-1 were specific for viral uveitis; these mediators could present specific markers for diagnostic purposes. Interferon-γ, IL-6, and macrophage inflammatory protein-1β were common markers of ocular toxoplasmosis and viral uveitis. IL-17 was a common marker of ocular toxoplasmosis and intermediate uveitis ConclusionsWe found specific cytokine profiles for each type of uveitis, with large interindividual variations and no etiological or clinical correlations. Ocular cytokine mapping contributes to a better understanding of the physiopathology of specific forms of uveitis and provides guidance for new targeted treatmen

Association between genital tract infection and premature rupture of membranes: A retrospective case control study in Tunisia, North Africa

Premature Rupture of Membranes is responsible for most cases of neonatal death. In most of these cases, the causes of PROM havenot been established in Tunisia, although several risk factors have been described. Therefore, we set out to determine the presenceof an association between genital infections and PROM among Tunisian women. A case-control study was conducted among 251 womens to detect the presence of association between genital tract infection and Premature Rupture of Membranes.Cases had apremature membranes rupture and the controls had intact membranes or suffering from premature membrane rupture during thelatent phase of labour. Data were collected from the medical register including socio-demographic characteristics, obstetrics, andmedical history. Association between genital infections and premature rupture of membranes was estimated using the Odds Ratioand 95% CI. One risk factor was identified, including age. There is no association between the presence of Group B streptococcus (OR= 1.08; 95% CI 0.50-2.34), presence of Trichomonasvaginalis (OR= 2.45; 95% CI 0.15-39.83) and presence of Candidiasis (OR= 1.11; 95% CI 0.58-2.14) and premature rupture of membranes. Co-infection was not associated with premature rupture of membranes (OR= 0.43; 95% CI 0.45-6.07). There is no association between genital infections and PROM among pregnant Tunisian women. Keywords: Genital infections, premature rupture of membranes, risk factors, Monastir, TunisiaLa rupture prématurée des membranes est responsable de la plupart des cas de décès néonatal. Enn effet, les causes de la RPM n'ont pas été établies en Tunisie, bien que plusieurs facteurs de risque aient été décrits. Par conséquent, nous avons cherché à déterminer la présence d'une association entre les infections génitales et la RPM chez les femmes tunisiennes. Une étude castémoins a été menée auprès de 251 femmes pour détecter la présence d'une association entre l'infection des voies génitales et la rupture prématurée des membranes. Les cas avaient une rupture prématurée des membranes et les témoins avaient des membranes intactes ou souffraient d'une rupture prématurée de la membrane pendant la phase latente du travail. Les données ont été recueillies à partir du registre médical, y compris les caractéristiques sociodémographiques, l'obstétrique et les antécédents médicaux. L'association entre les infections génitales et la rupture prématurée des membranes a été estimée à l'aide du OR et de l'IC à 95%. Un facteur de risque a été identifié, incluant l'âge. Il n'y a pas d'association entre la présence de streptocoques du groupe B (OR = 1,08; IC à 95% 0,50-2,34), la présence de Trichomonasvaginalis (OR = 2,45; IC à 95% 0,15-39,83) et la présence de candidose (OR = 1,11; 95% CI 0,58-2,14) et rupture prématurée des membranes. La co-infection n'était pas associée aussi à la rupture prématurée des membranes (OR = 0,43; IC à 95% 0,45 à 6,07). Il n'y a pas d'association entre les infections génitales et la RPM chez les femmes tunisiennes. Mots-clés: Infections génitales, rupture prématurée des membranes, facteurs de risque, Monastir, Tunisi