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The microsporidian species Enterocytozoon bieneusi is a major cause of chronic diarrhea and malabsorption in patients with AIDS. Genotyping was performed on seven E. bieneusi strains for the first time in Tunisia. All the strains were isolated from stool samples of humans with immunodeficiency virus (HIV) infection. Analysis of the ribosomal RNA gene internal transcribed spacer (rDNA ITS) allowed the identification of three distinct genotypes previously described in other studies. Genotypes D and B were characterized in four and two respectively. The Peruvian genotype (Peru 8) was detected in the last isolate. These results indicate a genetic diversity in E. bieneusi strains from HIV Tunisian patients and suggest the coexistence of both zoonotic and anthroponotic route of transmission.

The purpose of the study was to obtain more reliable epidemiological data concerning Entamoeba (E.) histolytica infection in Tunisian food handlers using established molecular tools able to differentiate E. histolytica from E. dispar. From 2002 to 2005, 4,266 fresh stools specimens received in the setting of the National program of food handlers’ control were analysed by optical microscopy. Twelve (2.8 ‰) were positive for the presence of four nuclei cysts identified as E. histolytica/E. dispar. Extraction of DNA from the 12 samples, followed by specific amplifications of E. histolytica and E. dispar SSU rDNA, showed that 11 samples (92%) were positive for E. dispar and negative for E. histolytica. Sequencing analysis of 8 PCR products permitted to verify the results obtained with conventional PCR. The remaining sample was negative by PCR amplifying E. histolytica DNA or E. dispar DNA specifically, although it did not show any inhibition. It probably contains protozoan cysts genetically distinct from these two species but morphological similar. Estimation of relative proportions between E. histolytica and E. dispar in cyst carriers showed that all explored individuals harboured the non pathogenic E. dispar strains. This result highlights the need of use in this population of complementary tests that allow specific diagnosis and obviate unnecessary chemotherapy.

The authors report the identification of Leishmania strains isolated from the Centre and the South of Tunisia. 266 strains were isolated between 1998 and 2006 from human (n = 221 strains) and dogs (n = 45 strains) hosts. The isoenzymatic identification exhibits the presence of in total five zymodemes belonging to three Leishmania complexes: Leishmania infantum, L. major and L. killicki. All strains isolated from human and canine visceral leishmaniasis belonged to L. infantum. zymodeme MON-1 was the only one isolated from canine visceral leishmaniasis. However, it is predominant in human visceral leishmaniasis beside zymodeme MON-24 which was detected in two provinces of the Centre (Monastir and Kairouan) and zymodeme MON-80 isolated for the first time in Kairouan province. Three complexes are responsible for human cutaneous leishmaniasis: L. major MON-25 is the parasite the most frequently found in its classic foci in the Centre and the South of the country. L. infantum MON-24 was isolated for the first time in a small locality of Sfax (southern Tunisia) showing the appearance of a new focus of L. infantum. L. killicki was isolated in its original focus of Tataouine and in two new foci of the central part of the country (Sidi Bouzid and Kairouan).

In order to identify the phlebotomine sandfly populations in Tunisian leishmaniosis foci, an entomological survey was carried out through three entomological seasons (2002-2003-2004) in 19 visceral and cutaneous leishmaniosis areas, located in six bioclimatic zones. Sandfly collections were based on light and sticky traps placed around human leishmaniosis cases. 8,722 phlebotomine sandflies belonging to 12 species were collected. The dominance of subgenus Larroussius species in northern foci, Phlebotomus papatasi in south-western foci and their co-dominance in the centre of the country is in accordance with the distribution of Leishmania infantum and L. major in Tunisia. The low density found in the historical zoonotic cutaneous leishmaniosis focus of Metlaoui in the south-west may indicate the high competence of the local populations. Studied phlebotomine settlements have showed a low specific diversity in most of the studied sites. In L. infantum areas, the dominant species were respectively: P. perfiliewi in the cutaneous leishmaniosis site of the humid bioclimatic stage, P. perniciosus in the cutaneous and visceral leishmaniosis foci of semi-arid and arid bioclimatic stages and P. longicuspis in the visceral leishmaniosis focus of saharan bioclimate. In the zoonotic cutaneous leishmaniosis foci, P. papatasi was a dominant species. In the well-known southeastern foci of cutaneous leishmaniosis due to L. killicki, P. sergenti was a dominant species with P. perniciosus. In the central emerging foci of L. killicki, P. perniciosus was a dominant species in some sites whereas it was very rare in others. In these sites, the subgenus Paraphlebotomus was always present with a higher abundance of P. alexandri than P. sergenti.

Twenty cutaneous leishmaniasis (CL) cases were notified from December 2001 to February 2002, in a small village in the district of Oueslatia (governorate of Kairouan, central Tunisia) which is an endemic focus of infantile visceral leishmaniasis due to Leishmania (L.) infantum and that had never been concerned previously by CL. The parasite typing of two isolates obtained from two children that have never left the region has identified L. killicki. This species had only been reported previously in a limited focus of Tunisian Southeast. In October 2002, an epidemiological survey with isoenzym characterization of the parasite led in a well-known focus of zoonotic cutaneous leishmaniasis of South-West Tunisia also revealed the presence of L. killicki. These results suggest the spread of this species and stress the need of further investigations for a better control of CL in Tunisia.

La prévalence de la toxoplasmose a été étudiée dans le nord de la Tunisie chez 857 individus d'origine rurale et 564 individus d'origine urbaine. Les anticorps anti-toxoplasmiques ont été recherchés par immunofluorescence indirecte (IFI) et par ELISA. La prévalence de la toxoplasmose dans la population générale était de 58,4 %. Elle a significativement varié avec l'âge, passant de 24,5 % à dix ans à 52, I % à 20 ans, pour atteindre un maximum autour de 70 % vers l'âge de 30 ans. A partir de cet âge, les séroconversions semblent rares à en juger par la faible proportion des sérums à titres élevés en IFI (14,2 % avant 30 ans vs 3,7 % après 30 ans). La prévalence était significativement plus élevée en milieu urbain (67 % vs 52,8 %). Dans ce milieu, c'est entre dix et vingt ans, que les séroconversions semblent les plus fréquentes, la majorité des femmes étant alors immunisées à l'âge de la procréation. En milieu rural, l'immunisation, moins intense entre dix et 20 ans, fait que le nombre de femmes réceptives à la toxoplasmose à l'âge légal du mariage (18 ans) est plus important. Ce sont donc les femmes jeunes de ce milieu qui sont les plus exposées au risque d'infection toxoplasmique comme en témoigne la proportion élevée de sérum fortement positifs dans cette population (9,2 % pour les 18-30 ans vs 1,9 % pour les 30- 48 ans).

Objective – The epidemiological variations of visceral leishmaniasis (VL) in Tunisia are studied along with the clinical specificity of the disease in adults.Patients and methods – Six cases of parasitologically confirmed adult VL are described.Results – All patients tested negative for HIV. Clinical manifestations were not always evocative. In fact, two patients were apyretic and splenomegaly was absent in three. Biological disorders were more frequently noted, especially increase of erythrocyte sedimentation rate, anemia, and hyper γ globulinemia.Conclusion – The epidemiological profile of VL in Tunisia is slowly becoming identical to the North Mediterranean model characterized by a high proportion of adult cases. This may be due to the multiplication of immunodepressing factors and the decrease of specific immunization following the less frequent human-parasite contact during childhood. [Copyright &y& Elsevier]

Background: Dogs are considered the main domestic animals that may be a reservoir for Leishmania infantum, the agent of zoonotic visceral leishmaniasis (ZVL) in several countries of the world. The dog may host other Leishmania species, but its epidemiological role in the maintenance and spreading of these parasites is not completely elucidated. Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major, affects thousands of people every year and is particularly diffused in many countries of North Africa and Middle East Asia. In ZCL endemic countries, few reports of L. major-positive dogs have been reported, probably because most human cases occur in poor rural areas where the social role of the dog and its medical management is not well considered. The aim of the present study is to better understand the possible involvement of domestic dogs in the epidemiology of ZCL., Methods: Our research focused on a well-established endemic focus of ZCL, in the area of Echrarda, Kairouan Governorate, central Tunisia. A total of 51 dogs with no or mild clinical signs of vector borne diseases were selected in small villages where human cases of ZCL are yearly present. All dogs were sampled for the Leishmania spp. diagnosis, by using the following procedures: blood sample for serology and buffy coat quantitative polymerase chain reaction (qPCR), popliteal fine needle aspiration, and cutaneous biopsy punch for lymph node and skin qPCR., Results: The results demonstrated a high percentage (21.6%) of dogs positive at least at one or more test; the most sensitive technique was the lymph node qPCR that detected 8/11 positive dogs. Nine, out of the eleven positive dogs, resulted as infected by Leishmania infantum; ITS1-PCR-sequencing allowed Leishmania major identification in the remaining two cases, both from the popliteal lymph node samples, which can suggest a possible visceral spread of a cutaneous Leishmania species in the dog. Interestingly, one of the two L. major-positive dogs was living in the same house where 6-year-old children showed cutaneous lesions referred to as ZCL., Conclusions: To our knowledge, this is the first report of L. major-positive dogs in Tunisia, the  epidemiological role of which remains under investigation., (© 2024. The Author(s).)

Blood flow restriction training (BFRT) employs partial vascular occlusion of exercising muscle and has been shown to increase muscle performance while using reduced workload and training time. Numerous studies have demonstrated that BFRT increases muscle hypertrophy, mitochondrial function, and beneficial vascular adaptations. However, changes in cardiovascular hemodynamics during the exercise protocol remain unknown, as most studies measured blood pressure before the onset and after the cessation of exercise. With reduced perfusion to the exercising muscle during BFRT, the resultant accumulation of metabolites within the ischemic muscle could potentially trigger a large reflex increase in blood pressure, termed the muscle metaboreflex. At low workloads, this pressor response occurs primarily via increases in cardiac output. However, when increases in cardiac output are limited (e.g., heart failure or during severe exercise), the reflex shifts to peripheral vasoconstriction as the primary mechanism to increase blood pressure, potentially increasing the risk of a cardiovascular event. Using our chronically instrumented conscious canine model, we utilized a 60% reduction in femoral blood pressure applied to the hindlimbs during steady-state treadmill exercise (3.2 km/h) to reproduce the ischemic environment observed during BFRT. We observed significant increases in heart rate (+19 ± 3 beats/min), stroke volume (+2.52 ± 1.2 mL), cardiac output (+1.21 ± 0.2 L/min), mean arterial pressure (+18.2 ± 2.4 mmHg), stroke work (+1.93 ± 0.2 L/mmHg), and nonischemic vascular conductance (+3.62 ± 1.7 mL/mmHg), indicating activation of the muscle metaboreflex. NEW & NOTEWORTHY Blood flow restriction training (BFRT) increases muscle mass, strength, and endurance. There has been minimal consideration of the reflex cardiovascular responses that could be elicited during BFRT sessions. We showed that during low-intensity exercise BFRT may trigger large reflex increases in blood pressure and sympathetic activity due to muscle metaboreflex activation. Thus, we urge caution when employing BFRT, especially in patients in whom exaggerated cardiovascular responses may occur that could cause sudden, adverse cardiovascular events.

Cutaneous leishmaniasis (CL) caused by infection with the parasite Leishmania exhibits a large spectrum of clinical manifestations ranging from single healing to severe chronic lesions with the manifestation of resistance or not to treatment. Depending on the specie and multiple environmental parameters, the evolution of lesions is determined by a complex interaction between parasite factors and the early immune responses triggered, including innate and adaptive mechanisms. Moreover, lesion resolution requires parasite control as well as modulation of the pathologic local inflammation responses and the initiation of wound healing responses. Here, we have summarized recent advances in understanding the in situ immune response to cutaneous leishmaniasis: i ) in North Africa caused by Leishmania (L.) major , L. tropica , and L. infantum , which caused in most cases localized autoresolutives forms, and ii ) in French Guiana resulting from L. guyanensis and L. braziliensis , two of the most prevalent strains that may induce potentially mucosal forms of the disease. This review will allow a better understanding of local immune parameters, including cellular and cytokines release in the lesion, that controls infection and/or protect against the pathogenesis in new world compared to old world CL., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Saidi, Blaizot, Prévot, Aoun, Demar, Cazenave, Bouratbine and Pied.)

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.

Background: Colorectal cancer (CRC) is a major public health problem worldwide and in Tunisia. It ranks among the main cancers in terms of incidence and cancer-related cause of death. Its pathogenesis is currently considered to be multifactorial involving genetic and environmental factors. Recent studies have suggested that the gene encoding the β1 subunit of the IL-12 receptor, an important pro-inflammatory cytokine of the anti-tumor response, could be involved in the susceptibility to inherited CRC. Hence, it would be interesting to study the role of single nucleotide polymorphisms (SNPs) within the IL-12RB1 gene (rs401502 and rs11575934) in CRC susceptibility. Aim: Our purpose was to assess whether genetic variants IL-12RB1 +1196G/C (rs401502) and IL-12RB1 +705A/G (rs11575934) within the IL-12RB1 gene are associated with the sporadic CRC risk. Methods: A total of 110 Tunisian patients with sporadic CRC and 141 healthy control subjects were included in this study. Genotyping was performed by high-resolution melting (HRM) analysis. All results were confirmed by direct DNA sequencing or PCR-RFLP methods. Later, the allele frequencies and genotype distribution were established and compared between the control group and CRC patients. Results: The obtained results showed that the two target SNPs were in Hardy-Weinberg equilibrium (HWE) in both patients and controls. Minor allele frequencies of rs401502 SNP were 16.4% in CRC cases and 23.8% in controls. Mutant allele of rs11575934 SNP was present with 21.4% in CRC patients and 29.8% in control group. An association study showed a significant association of two target polymorphisms with CRC, according to the dominant genetic model with OR = 0.577, 95% CI = [0.343 to 0.972], p = 0.038 and OR = 0.547, 95% CI = [0.328 to 0.911], p = 0.02, respectively. Conclusion: In this study, we found, for the first time, a potential protective effect of two SNPs in the IL-12RB1 gene, namely rs401502 and rs11575934, in sporadic colorectal cancer in Tunisians., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KH declared a shared affiliation with the authors SD, HBS, NS to the handling editor at the time of review., (Copyright © 2022 Jelassi, Dhouioui, Ben Salah, Saidi, Mzoughi, Ammi, Bouratbine, Aoun, Zidi and Chelbi.)

We report the study of sandfly Leishmania infection in an area of low incidence of visceral leishmaniasis in Tunisia. Sandflies were collected monthly using CDC light-traps set in houses and animal shelters during May-November 2016 and 2017. All males were identified at the species level. A sample of 878 females including all gravid specimens was subjected to kDNA qPCR for Leishmania detection and parasite load estimation. Leishmania species were determined by ITS1 PCR sequencing, and species identification of infected sandflies was performed by DNA barcoding. Phlebotomus perfiliewi and P. perniciosus were the dominant species during the two-year period. However, comparison of their relative abundances showed that P. perniciosus was more abundant during peaks of 2017 with longer activity duration. Real-time kDNA PCR did not detect Leishmania infection in 2016, although it identified four positive specimens (0.7%) in 2017. All four infected specimens were identified as P. perniciosus . ITS1 PCR sequencing allowed L. infantum identification in one kDNA qPCR-positive specimen. This was a P. perniciosus gravid female with a high parasite load caught during the long-lasting peak of 2017. This work highlights the usefulness of multi-seasonal studies of sandfly dynamics and kDNA qPCR in screening Leishmania infection and determining L. infantum vectors in hypo-endemic foci of human leishmaniasis.

The ventricular-vascular relationship assesses the efficacy of energy transferred from the left ventricle to the systemic circulation and is quantified as the ratio of effective arterial elastance to maximal left ventricular elastance. This relationship is maintained during exercise via reflex increases in cardiovascular performance raising both arterial and ventricular elastance in parallel. These changes are, in part, due to reflexes engendered by activation of metabosensitive skeletal muscle afferents-termed the muscle metaboreflex. However, in heart failure, ventricular-vascular uncoupling is apparent and muscle metaboreflex activation worsens this relationship through enhanced systemic vasoconstriction markedly increasing effective arterial elastance which is unaccompanied by substantial increases in ventricular function. This enhanced arterial vasoconstriction is, in part, due to significant reductions in cardiac performance induced by heart failure causing over-stimulation of the metaboreflex due to under perfusion of active skeletal muscle, but also as a result of reduced baroreflex buffering of the muscle metaboreflex-induced peripheral sympatho-activation. To what extent the arterial baroreflex modifies the metaboreflex-induced changes in effective arterial elastance is unknown. We investigated in chronically instrumented conscious canines if removal of baroreflex input via sino-aortic baroreceptor denervation (SAD) would significantly enhance effective arterial elastance in normal animals and whether this would be amplified after induction of heart failure. We observed that effective arterial elastance (E a ), was significantly increased during muscle metaboreflex activation after SAD (0.4 ± 0.1 mmHg/mL to 1.4 ± 0.3 mmHg/mL). In heart failure, metaboreflex activation caused exaggerated increases in E a and in this setting, SAD significantly increased the rise in E a elicited by muscle metaboreflex activation (1.3 ± 0.3 mmHg/mL to 2.3 ± 0.3 mmHg/mL). Thus, we conclude that the arterial baroreflex does buffer muscle metaboreflex induced increases in E a and this buffering likely has effects on the ventricular-vascular coupling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mannozzi, Kim, Sala-Mercado, Al-Hassan, Lessanework, Alvarez, Massoud, Bhatti, Aoun and O’Leary.)

Introduction: travellers to endemic areas must know malaria, its risk factors and prophylactic measures. This can help to avoid severe cases of malaria and to prevent transmission in countries that are malaria-free. The purpose of this study is to assess Tunisian travellers´ knowledge about malaria, its transmission and prevention and their adherence to prophylactic measures., Methods: we conducted a survey based on two anonymous questionnaires (pre- and post-trip) among adults travelling to endemic countries. The 1st questionnaire was followed by a medical interview focusing on level of risk and recommended prophylactic measures., Results: two hundred and eighty-nine travellers were recruited. They mainly moved within sub-Saharan Africa (99%) for professional reasons (84,4%). The average age of subjects was 42.3 years and sex ratio (male/female) was 3.1. Prior to departure, only 53.3% of subjects were aware of the risk of malaria, and only 28% gave correct answers about modes of transmission. Recommendations for chemoprophylaxis were only known by 62.3% of subjects and only 43.6% intended to use chemoprophylaxis (p < 0.01). Better adherence to protective measures, including chemoprophylaxis, was reported after the trip, with attitudes qualified as good or excellent by 64.2% on return against 23.7% before the interview (<0.001)., Conclusion: Tunisian travellers knowledge of malaria is insufficient. Strengthening information through specialized consultations (whose usefulness has been demonstrated) is required., Competing Interests: Les auteurs ne déclarent aucun conflit d´intérêts., (Copyright: Sonia Kechaou-Cherif et al.)

Comprehensive detection and differentiation of intestinal protists mostly rely on DNA-based methods. Here, we evaluated next-generation sequencing of eukaryotic nuclear ribosomal genes (metabarcoding) for the detection and differentiation of intestinal eukaryotic protists in the stool of healthy Tunisian individuals. Thirty-six faecal DNA samples previously evaluated by microscopy and ameboid species-specific PCRs were tested. The hypervariable regions V3-V4 and V3-V5 of the 18S rRNA gene were amplified using three universal eukaryotic primer sets and sequenced using Illumina®MiSeq sequencing. In addition, real-time PCR assays were used to detect Dientamoeba fragilis , Giardia duodenalis , and Cryptosporidium spp. The metabarcoding assay detected Blastocystis (subtypes 1, 2, and 3) and archamoebid species and subtypes ( Entamoeba dispar , Entamoeba hartmanni , Entamoeba coli RL1 and RL2, Endolimax nana , Iodamoeba bütschlii RL1) in 27 (75%) and 22 (61%) of the 36 stool samples, respectively. Meanwhile, the assay had limited sensitivity for flagellates as evidenced by the fact that no Giardia -specific reads were found in any of the five Giardia -positive samples included, and Dientamoeba -specific reads were observed only in 3/13 D. fragilis -positive samples. None of the samples were positive for Cryptosporidium by any of the methods. In conclusion, a large variety of intestinal eukaryotic protists were detected and differentiated at species and subtype level; however, limited sensitivity for common flagellates was observed., Competing Interests: The authors declare that they have no competing interests., (© 2022 The Authors.)

Visceral leishmaniasis (VL) is a severe life threatening parasitosis requiring early management of cases. It is an emerging disease in the Mediterranean region with a spread of endemic areas and an increase in case incidence. The patient profile has also evolved with more affected adults, presenting generally non-specific symptoms. Hence the interest of a systematic biological confirmation. The microscopic detection of Leishmania amastigotes in bone marrow aspirates (BMA) smears is the gold standard diagnostic technique. However, it requires invasive sampling. Serological tests searching for specific antibodies remain highly contributory, but their interpretation must always take into account the epidemiological context and the patient's clinical and biological features. Currently, the Western-Blot represents the most specific serological technique for diagnostic confirmation. VL diagnosis has greatly improved by the introduction of both rapid diagnostic tests (RDTs) and molecular biological techniques. RDTs using recombinant rk39 antigen are easy to perform and deliver results in less than 30 minutes. Real-time PCR (Polymerase Chain Reaction) is currently retained as the best technique for VL diagnosis. It is efficient on simple blood samples, allowing to avoid invasive BMA needed for microscopy. In addition, real time PCR estimates parasite load which is helpful for the post-treatment follow-up. In any case, the choice of techniques to be used should be strategic and adapted to the local epidemiology as well as to the means available.

Background and objectives: Human cytomegalovirus (HCMV) and genetic polymorphisms of the chemokine receptor 5 have been suggested as factors associated with the progression of colorectal cancer (CRC). The aim of the study was to evaluate the associations of both CCR5Δ32 genetic deletion and/or HCMV virus infection with CRC in Tunisia., Materials and Methods: The association between HCMV and CRC was validated by Nested PCR technology performed for HCMV and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. Experiments were carried out on 40 tumor and 35 peri-tumor tissues, 100 blood from CRC patients and on 140 blood samples from healthy subjects and finaly serum samples of 80 patients with CRC and 100 healthy individuals. A conventional PCR has been optimized for the detection of CCR5Δ32 in100 CRC patients and 100 healthy subjects., Results: Our results show that HCMV is significantly active in 93% of patients compared to 60% in controls ( p < 0.0001, OR = 8.85, 95% CI: 3.82 -20.50). Compared to the healthy controls, the titers of IgG and IgM antiCMV antibodies in CRC patients were significantly higher than in healthy subjects ( p value < 0,0001 for IgG and IgM). Statistical analysis revealed a lack of association between CCR5Δ32 mutation and colorectal cancer ( p = 0.788, OR = 1.265, 95% CI: 0.228-7.011)., Conclusion: our data confirmed that the HCMV infection was related to the development of CRC and that CRC cells may be infected more favorably by HCMV. Given the importance of the CCR5 in inflammation and therefore CRC progression, further studies still needed to evaluate CCR5 role as a potential candidate gene for CRC susceptibility under other polymorphisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors (IZ)., (Copyright © 2021 Chelbi, Jelassi, Belfkih, Ben Amor, Saidi, Ben Salah, Mzoughi, Ben Dhifallah, Boujelben, Ammi, Bouratbine, Zidi and Aoun.)

Background: HLA-G is a non-classical class I gene of the human Major Histocompatibility encoding molecules with immune-modulatory properties. Expression of HLA-G is being largely studied in pathological conditions, such as tumors, viral infections, inflammation, and autoimmune diseases, grafted tissues, among others. HLA-G +3142C/G (rs1063320: dbSNP database) polymorphism is located in 3' UTR of HAL-G and plays a key role in determining the magnitude of gene and protein expression. The detection of HLA-G +3142C/G polymorphism in the most published report is done through polymerase chain reaction followed by enzymatic digestion. Therefore, it is so interesting to develop a rapid and sensitive assay to genotype HLA-G +3142C/G polymorphism. High-resolution melt analysis (HRM) is a technology that is based on the analysis of the melting profile of PCR products through gradual temperature increase. The aim of this work is to apply high-resolution melt method for genotyping the HLA-G +3142C/G polymorphism., Methods: DNA from 118 individuals was extracted from whole blood with QIAamp ® DNA blood mini kit (Qiagen, Germany). Primer couple was designed using Primer 3 online tools so as to have only one SNP in the target sequence for high HRM efficiency. Positive Controls were identified using DNA sequencing and used as reference when assigning genotypes for trial samples., Results: We were able to recognize the three genotypes with similar accuracy than DNA sequencing using high resolution melting method. Hardy-Weinberg equilibrium test shows that our population is in equilibrium for the studied SNP. Genotypes frequencies of +3142C/G polymorphism in Tunisian general population are 0.475 for heterozygote G/C, 0.186 for homozygote G/G and 0.339 for homozygote C/C., Conclusion: HRM is a cost-effective method suitable for SNP genotyping., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Algeria ranks second after Afghanistan for the incidence of cutaneous leishmaniasis (CL) worldwide. Here, we report a 34-years retrospective analysis of CL in Algeria and focused on the most affected region, the M'Sila province. All 66 cutaneous isolates corresponded to Leishmania ( L .) major . Our study of the sandfly and rodent fauna further highlighted the high density of Phlebotomus papatasi and additional phlebotomine species of medical importance, not previously identified in M'Sila. Wild rodents belonging to nine species were trapped in M'Sila, and Psammomys obesus and Meriones shawi were found infected by L. major . In addition, Leishmania infantum was isolated from two visceral leishmaniasis cases, one dog and its proven vectors ( P. perniciosus , P. longicuspis , and P. perfiliewi ) inventoried during the survey. The high incidence of CL in the M'Sila province is likely a consequence of the increase in minimum temperatures recorded that constitutes suitable conditions for establishing a high endemicity and leads to an explosive rise in leishmaniases cases in this region. A thorough investigation of the underlying risk factors is urgently needed to detect new cases earlier. All these would improve the preparedness to fight the disease.

Background: Transcranial sonography is increasingly used to aid clinical diagnoses of movement disorders, for example, to identify an enlarged area of substantia nigra echogenicity in patients with Parkinson's disease., Objective: The current study investigated characteristics of the midbrain at the anatomical plane for quantification of substantia nigra echogenicity. METHODS: Area of substantia nigra echogenicity, cross-sectional area of the midbrain, and interpeduncular angle were quantified in two groups of adults aged 18-50 years: 47 healthy non-drug-using controls (control group) and 22 individuals with a history of methamphetamine use (methamphetamine group), a cohort with a high prevalence of enlarged substantia nigra echogenicity and thus risk of Parkinson's disease., Results: In the control group, cross-sectional area of the midbrain (4.47±0.44 cm2) and interpeduncular angle were unaffected by age, sex, or image acquisition side. In the methamphetamine group, cross-sectional midbrain area (4.72±0.60 cm2) and area of substantia nigra echogenicity were enlarged compared to the control group, and the enlargement was sex-dependent (larger in males than females). Whole midbrain area and interpeduncular angle were found to be weak predictors of area of substantia nigra echogenicity after accounting for group and sex., Conclusions: History of methamphetamine use is associated with an enlarged midbrain and area of substantia nigra echogenicity, and the abnormality is more pronounced in males than females. Thus, males may be more susceptible to methamphetamine-induced changes to the brainstem, and risk of Parkinson's disease, than females., Competing Interests: The authors have declared that no competing interests exist.

The period between the infective sandfly bites and appearance of cutaneous leishmaniasis (CL) lesions is still hypothetical and little studied. This work aimed at assessing the incubation time of zoonotic CL (ZCL) due to Leishmania major using a standardized methodology. The retrospective analysis used the epidemiological, clinical, and biological information available in the database recording all the CL cases diagnosed at the Parasitology Department of the Pasteur Institute of Tunis during 2015-2019. It allowed for the selection of 92 privileged observations 1) of confirmed CL cases with presentation suggestive of ZCL form 2) living in northern regions free of ZCL 3) with a single infective trip of less than a week to ZCL foci during transmission season and 4) with accurate dates of travel and onset of lesions. Incubation length computed in this population ranged from 1 to 21 weeks, with a median of 5 weeks (interquartile range: 3-8.5 weeks).

Although several studies have investigated genetic diversity of Leishmania infantum in North Africa, genome-wide analyses are lacking. Here, we conducted comparative analyses of nuclear and mitochondrial genomes of seven L . infantum isolates from Tunisia with the aim to gain insight into factors that drive genomic and phenotypic adaptation. Isolates were from cured ( n =4) and recurrent ( n =3) visceral leishmaniasis (VL) cases, originating from northern ( n =2) and central ( n =5) Tunisia, where respectively stable and emerging VL foci are observed. All isolates from relapsed patients were from Kairouan governorate (Centre); one showing resistance to the anti-leishmanial drug Meglumine antimoniate. Nuclear genome diversity of the isolates was analysed by comparison to the L. infantum JPCM5 reference genome. Kinetoplast maxi and minicircle sequences (1 and 59, respectively) were extracted from unmapped reads and identified by blast analysis against public data sets. The genome variation analysis grouped together isolates from the same geographical origins. Strains from the North were very different from the reference showing more than 34 587 specific single nucleotide variants, with one isolate representing a full genetic hybrid as judged by variant frequency. Composition of minicircle classes within isolates corroborated this geographical population structure. Read depth analysis revealed several significant gene copy number variations correlating with either geographical origin (amastin and Hsp33 genes) or relapse (CLN3 gene). However, no specific gene copy number variation was found in the drug-resistant isolate. In contrast, resistance was associated with a specific minicircle pattern suggesting Leishmania mitochondrial DNA as a potential novel source for biomarker discovery.

Background: Zoonotic visceral leishmaniasis (VL) is endemic in the Mediterranean basin. However, large-scale comparative analyses of the commercial kits for the serological diagnosis of this neglected disease are lacking. This study compared the performances of four enzyme-linked immunosorbent assays (ELISA) and two immunochromatographic tests (ICT) as screening tests for the serodiagnosis of human VL in the Mediterranean region., Methodology/principal Findings: Serum samples from 319 patients living in France, Tunisia or Morocco were tested using two ICT (IT LEISH and TruQuick LEISH IgG/IgM Meridian) and four ELISA reagents (NovaLisa Leishmania infantum IgG, Bordier Leishmania infantum, Ridascreen Leishmania IgG, and Vircell Leishmania). The population with proven VL (n = 181) included 65 immunocompromised patients. Significantly higher percentages of false-negative results were obtained with all assays in immunocompromised patients, compared with the immunocompetent population. In the whole population, sensitivity and specificity ranged from 80.7% to 93.9% and from 95.7% to 100%, respectively. The maximum accuracy was observed with the Bordier and Vircell ELISA kits (96.2%), and the lowest accuracy with Ridascreen reagent (88.7%). New thresholds of positivity are proposed for the Bordier, Vircell and NovaLisa ELISA kits to achieve 95% sensitivity with the highest possible specificity. Western blot (WB), used as a confirmation method, showed 100% sensitivity and identified 10.1% of asymptomatic carriers among the control population from the South of France., Conclusions/significance: This is the first study that compared commercially available kits for VL serodiagnosis in the endemic region of the Mediterranean basin. It provides specific information about the tests' performance to help clinicians and biologists to select the right assay for VL screening., Competing Interests: The authors have declared that no competing interests exist.

Human leishmaniasis is a public health problem worldwide for which the development of a vaccine remains a challenge. T cell-mediated immune responses are crucial for protection. Peptide vaccines based on the identification of immunodominant T cell epitopes able to induce T cell specific immune responses constitute a promising strategy. Here, we report the identification of human leukocyte antigen class-I (HLA-I) and -II (HLA-II)-restricted multi-epitope peptides from Leishmania proteins that we have previously described as vaccine candidates. Promastigote Surface Antigen (PSA), LmlRAB (L. major large RAB GTPase) and Histone (H2B) were screened, in silico, for T cell epitopes. 6 HLA-I and 5 HLA-II-restricted multi-epitope peptides, able to bind to the most frequent HLA molecules, were designed and used as pools to stimulate PBMCs from individuals with healed cutaneous leishmaniasis. IFN-γ, IL-10, TNF-α and granzyme B (GrB) production was evaluated by ELISA/CBA. The frequency of IFN-γ-producing T cells was quantified by ELISpot. T cells secreting cytokines and memory T cells were analyzed by flow cytometry. 16 of 25 peptide pools containing HLA-I, HLA-II or HLA-I and -II peptides were able to induce specific and significant IFN-γ levels. No IL-10 was detected. 6 peptide pools were selected among those inducing the highest IFN-γ levels for further characterization. 3/6 pools were able to induce a significant increase of the percentages of CD4+IFN-γ+, CD8+IFN-γ+ and CD4+GrB+ T cells. The same pools also induced a significant increase of the percentages of bifunctional IFN-γ+/TNF-α+CD4+ and/or central memory T cells. We identified highly promiscuous HLA-I and -II restricted epitope combinations from H2B, PSA and LmlRAB proteins that stimulate both CD4+ and CD8+ T cell responses in recovered individuals. These multi-epitope peptides could be used as potential components of a polytope vaccine for human leishmaniasis., Competing Interests: The authors have declared that no competing interests exist.

We report here a case of simultaneous cutaneous and visceral manifestations due to Leishmania L. infantum diagnosed in an immunocompetent adult. We describe a 74-year-old woman from Tunis, Tunisia, who presented a biologically confirmed visceral leishmaniasis infection concomitant with arm ulceration which appeared 2 years before. Leishmania DNA was detected by ITS PCR in both buffy coat and dermal scrapping of the arm lesion. Sequencing revealed that the 2 isolated strains corresponded to L. infantum and were 100% identical. The symptoms of visceral leishmaniasis responded to amphotericin B with rapid healing. However, the skin lesion did not improve although Leishmania PCR on dermal sample became negative. This location is probably secondarily to lymphatic or blood dissemination during the systemic visceral leishmaniasis infection. It would be favored by the inflammatory environment induced by the basal cell carcinoma subsequently diagnosed., Competing Interests: The authors declare no competing interests., (© Imène Ben Abda et al.)

Cutaneous leishmaniasis (CL) remains one of the world's most prevalent neglected diseases, particularly in developing countries. Identification of the involved Leishmania species is an important step in the diagnosis and case management process. In this study, we tested simple, rapid, and highly sensitive loop-mediated isothermal amplification (LAMP) assays for Leishmania DNA species-specific detection from cutaneous lesions. Two LAMP assays, targeting cysteine protease B (cpb) gene, were developed to detect and identify Leishmania major and Leishmania tropica species. Loop-mediated isothermal amplification specificity was examined using DNA samples from other Leishmania species and Trypanosoma species. No cross-reactions were detected. The developed LAMP assays exhibited sensitivity with a detection limit of 20 fg and 200 fg for L. major and L. tropica , respectively. Both tests were applied on clinical samples of CL suspected patients living in endemic Tunisian regions and compared with kinetoplast DNA quantitative PCR (qPCR), microscopic, and conventional cpb-based polymerase chain reaction (PCR) assays. Our LAMP tests were able to discriminate between L. major and L. tropica species and showed a sensitivity of 84% and a specificity of 100%. However, when compared with the performance of the diagnostic tests with latent class analysis (LCA), our LAMP assays show a sensitivity of 100%. These assays can be used as a first-line molecular test for early diagnosis and prompt management of CL cases in public health programs.

Background: Visceral leishmaniasis is a zoonotic disease of major public health concern in several countries in the world. The local population awareness would improve prevention, early detection and treatment of both human and animal leishmaniasis., Methods: The aim of this survey was to assess the knowledge about visceral leishmaniasis in a sample of dog owners visiting the National School of Veterinary Medicine of Sidi Thabet, Tunisia, through a structured questionnaire., Findings: Two hundred dog owners were interviewed, 87% were men and 47% had higher education level. Ninety four per cent were from neighbouring districts to Ariana, where the National School of Sidi Thabet is located. Out of 200 respondents, 79 confirmed knowing leishmaniasis. The correct answers concerning canine visceral leishmaniasis (CVL) (77%) were significantly higher than those concerning human visceral leishmaniasis (HVL) (23%). Correct answers concerning CVL were given in part by previously diseased dogs' owners. The respondent could not explain what is exactly leishmaniasis, but the majority of questioned persons know that human and dogs are the most important hosts. Forty-four out 79 (56%) of the persons think that mosquitoes or insects are the vectors of leishmaniasis and 63% (53/79) knows that it is a zoonotic disease but 72% (38/53) were not able to define how., Conclusion: Despite the frequent visits to veterinarians for vaccination or other medical issues and their long experience in dog breeding, the sample of dog owners had not enough knowledge and several misconceptions regarding leishmaniasis. Large education programmes should be implemented in Tunisia to improve the knowledge of the Tunisian population, especially dog owners, concerning leishmaniasis.

Toxoplasmosis is an important zoonosis caused by an obligate intracellular parasitic protozoan, Toxoplasma gondii. The disease is distributed worldwide and can affect all warm-blooded vertebrates, including humans. The present review aimed to collect, compile and summarize the data on the prevalence of T. gondii infection in humans and animals in the five North African countries (Morocco, Algeria, Tunisia, Libya and Egypt). Published data from national and international databases were used. Distribution patterns and risk factors for T. gondii infection are discussed, focusing on biotic and abiotic factors. This review is a comprehensive epidemiological analysis of T. gondii infection in North Africa and will therefore be a useful tool for researchers. It can also be used to propose or enhance appropriate national toxoplasmosis control programs., (© Rouatbi et al., published by EDP Sciences, 2019.)