Development and evaluation of molecular tools for detecting and differentiating intestinal amoebae in healthy individuals.

Amoebae are single-celled parasites frequently colonizing human gut. However, few molecular tools are available for accurate identification. Here, we evaluated a panel of polymerase chain reactions (PCRs) targeting Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba bütschlii. Thirty-six faecal samples (18 containing at least one amoeba species by microscopy and 18 microscopy negative for amoebae) were tested. Real-time PCRs were used for detection and differentiation of E. histolytica and E. dispar. Conventional PCR with Sanger sequencing were applied for detection and differentiation of E. coli, E. hartmanni, E. polecki, E. nana and I. bütschlii. All microscopy results were confirmed by DNA-based methods. However, more samples were positive for single and mixed amoebic species by DNA-based assays than by microscopy (22 vs 18 and 7 vs 1, respectively). DNA sequencing allowed identification of E. coli subtypes (ST1 and ST2), showed low intra-specific variation within E. hartmanni, identified two phylogenetically distinct groups within E. nana, and identified Iodamoeba at the ribosomal lineage level. Taking into account the high intra-genetic diversity within some of the species at the small subunit (SSU) rRNA gene level, amplification of SSU rRNA genes with subsequent sequencing represents a useful method for detecting, differentiating and subtyping intestinal amoebae.

High-resolution melting-curve (HRM) analysis for C. meleagridis identification in stool samples.

BACKGROUND: Cryptosporidiosis represents a major public health problem. This infection, caused by a protozoan parasite of the genus Cryptosporidium, has been reported worldwide as a frequent cause of diarrhoea. In the immunocompetent host, the typical watery diarrhea can be self-limiting. However, it is severe and chronic, in the immunocompromised host and may cause death. Cryptosporidium spp. are coccidians, which complete their life cycle in both humans and animals. The two species C. hominis and C. parvum are the major cause of human infection. Compared to studies on C. hominis and C. parvum, only a few studies have developed methods to identify C. meleagridis. AIM: To develop a new real time PCR-coupled High resolution melting assay allowing the detection for C. meleagridis, in addition of the other dominant species (C. hominis and C. parvum). METHODS: The polymorphic sequence on the dihydrofolate reductase gene (DHFR) of three species was sequenced to design primers pair and establish a sensitive real-time PCR coupled to a high-resolution melting-curve (HRM) analysis method, allowing the detection of Cryptosporidium sp. and discrimination between three prevalent species in Tunisia. We analyzed a collection of 42 archived human isolates of the three studied species. RESULTS: Real-time PCR coupled to HRM assay allowed detection of Cryptosporidium, using the new designed primers, and basing on melting profile, we can distinguish C. meleagridis species in addition to C. parvum and C. hominis. CONCLUSION: We developed a qPCR-HRM assay that allows Cryptosporidium genotyping. This method is sensitive and able to distinguish three Cryptosporidium species.

Polymorphism study of Cryptosporidium hominis gp60 subtypes circulating in Tunisia.

Cryptosporidium spp. are a major cause of gastrointestinal diseases in humans worldwide. While a single subtype of Cryptosporidium hominis has been shown to be responsible for several large outbreaks related to water contamination in developed countries, little is known about the epidemiology of C. hominis in developing countries. This study reports the first genetic characterization of C. hominis at the subtype level in several human populations in Tunisia using the gp60 gene. Eighteen isolates were identified as C. hominis by a restriction fragment length polymorphism (RFLP) analysis. The prevalence of this species in different human populations ranges from 1.53% to 13.04% with a high prevalence being reported in immunocompromised children (13.04%) followed by patients with malignent myeloma (5.5%) and HIV-infected patients (4.59%). The gp60 analysis on C. hominis isolates, performed in 14 cases, showed the presence of a single subtype family: "Ia". Different subtypes were identified within this family (A11G1R1, A12R3, A23G1R1, A26G1R1, A27G1R1, A28G1R1). The IaA26G1R1 subtype was the most dominant subtype described in this area (50%). Despite the high genetic diversity of Cryptosporidium spp, a low heterogeneity at the subtype level was observed within C. hominis circulating in Tunisia. This distribution is an indicator for intensive and stable anthroponotic cryptosporidiosis in this region. Besides, the presence of a unique genotype in 5 HIV-infected patients attending the same hospital ward suggests the possible occurrence of hospital-acquired infection and underlines the need to implement preventive measures to avoid nosocomial transmission.

Malaria outside a living in endemic areas: diagnosis and measures to take.

In spite of its elimination from Tunisia, malaria remains a public health concern due to the severity of the disease and risk of its reintroduction. The vulnerability of our country is related to the persistent anophelism and to the existence of a potential reservoir represented by imported cases. In the absence of a stay in an endemic area, the suspicion of malaria remains a rare event. However, in front of the possibility of other modes of contamination, this parasitosis must be evoked in any fever without obvious etiology. Especially as delayed diagnosis in non immune subjects may cause a severe illness and death. We propose to present the principal clinical and biological aspects of malaria and to specify the action to be taken when the infection is contracted outside a stay in endemic areas, in order to determine the origin of contamination.

Airport malaria: report of four cases in Tunisia.

Four cases of airport malaria were notified for the first time in Tunisia during the summer of 2013. All patients were neighbours living within 2 km of Tunis International Airport. They had no history of travel to malarious countries, of blood transfusion or of intravenous drug use. Although malaria transmission had ceased in Tunisia since 1980, autochthonous infection by local Anopheles mosquitoes was initially considered. However, this diagnostic hypothesis was ruled out due to negative entomological survey and the absence of additional cases.All cases were caused by Plasmodium falciparum. Clinical presentation was severe (important thrombocytopaenia and parasitaemia), because of relatively important delay in diagnosis (average of seven days). This indicates the need to consider malaria while examining airport employees or people living near international airports presenting with fever of unknown origin. It also stresses the need for effective spraying of aircrafts coming from malarious areas.

Evolution of the prevalence of intestinal parasitosis in the region of tunis from 1996 at 2012.

BACKGROUND: The prevalence of intestinal parasitosis is very different according to countries. Therefore, it is always interesting to update the data in Tunisia to better direct control measures. AIM: The objectives of this survey were to estimate the prevalence of intestinal parasitosis in the region of Tunis, to study their evolution and to establish various combinations of intestinal protozoa. METHODS: This is a retrospective study carried out over a period of 17 years from 1996 at 2012 and which involved 20033 individuals. Each subject had one or more stool examination which included a direct microscopic examination and a concentration by modified Ritchie technique. RESULTS: The prevalence of intestinal parasitosis was 12.55%. Entamoeba histolytica/dispar and Giardia intestinalis accounted respectively a prevalence of 0.51% and 1.48%. Hymenolepis nana was the most predominant helminth with a prevalence rate of 0.53%, followed by Enterobius vermicularis (0.21%). Two cases of Hookworms and seven cases of Strongyloides stercoralis were diagnosed. Polyparasitism concerned 16.59% of infected individuals. Significant combinations occured mainly for amoeba in particular Entamoeba histolytica/dispar and Entamoeba coli (r=0.232). CONCLUSION: Our study confirms the decrease of the prevalence of giardiasis and amebiasis, whereas helminthiases with direct transmission remain frequent.

Late relapse of imported Plasmodium ovale malaria: a case report.

We report the first case of an imported Plasmodium ovale relapse in a Tunisian man who developed malaria three years after leaving sub- Saharan Africa. A 29-year-old Tunisian man consulted in September 2011 because of a fever, myalgia, and headache that had begun eight days earlier and persisted despite treatment with oral antibiotics. On questioning, the patient stated that he had resided three years ago for six months in Ivory Coast, where he acquired malaria. He was treated with artemether-lumefantrine. The patient said he had no recent travel to any other malaria-endemic area and had not received a blood transfusion. A first microscopy of peripheral blood smears was negative for malaria parasites. The diagnosis was established 17 days after onset of symptoms. A repeat microscopic examination of blood smears confirmed the presence of Plasmodium ovale with a parasitemia lower than 0.1%. The patient was treated with artemether lumefantrine, followed by primaquine. This case emphasizes the possibility of relapse of some plasmodial species. It highlights the importance of repeating microscopic examination of blood when the diagnosis of malaria is suspected.

Contribution of Polymerase Chain Reaction for detection of malaria in Tunisia.

BACKGROUND: In Tunisia, detection of Plasmodium in asymptomatic individuals from endemic countries is a critical measure in national program of malaria eradication. The screening is based on microscopic examination of thick and thin blood smears. However, the performance of this diagnosis is closely related to the experience of biologist and the parasitaemia. AIM: The objective of this study was to evaluate the contribution of the PCR in the screening of malaria. METHODS: This prospective study involved 260 students from malaria endemic areas who were screened for malaria between september 2011 and june 2013. Each subject had a blood sample which was examined for malaria by microscopy and nested multiplex PCR. RESULTS: PCR detected the presence of Plasmodium in 13 blood samples (5%). While microscopy was positive only in nine cases (3.5%). The discordances involved five negative samples at microscopy and which were positive in PCR and a negative sample in PCR which was positive at microscopy. A mixed infection with Plasmodium falciparum and Plasmodium malariae was identified by PCR. For this case, microscopy diagnosed only Plasmodium falciparum specie. CONCLUSION: PCR is more efficient than microscopy in detecting low parasitaemia ; particularly observed in asymptomatic subjects. This technique allows to reduce asymptomatic carriage of Plasmodium and reduce the risk of a resumption of transmission of malaria in our country.

Heterogeneity of molecular resistance patterns in antimony-resistant field isolates of Leishmania species from the western Mediterranean area.

Antimonials remain the first-line treatment for the various manifestations of leishmaniasis in most areas where the disease is endemic, and increasing cases of therapeutic failure associated with parasite resistance have been reported. In this study, we assessed the molecular status of 47 clinical isolates of Leishmania causing visceral and cutaneous leishmaniasis from Algeria, Tunisia, and southern France. In total, we examined 14 genes that have been shown to exhibit significant variations in DNA amplification, mRNA levels, or protein expression with respect to resistance to antimonials. The gene status of each clinical isolate was assessed via qPCR and qRT-PCR. We then compared the molecular pattern against the phenotype determined via an in vitro sensitivity test of the clinical isolates against meglumine antimoniate, which is considered the reference technique. Our results demonstrate significant DNA amplification and/or RNA overexpression in 56% of the clinical isolates with the resistant phenotype. All clinical isolates that exhibited significant overexpression of at least 2 genes displayed a resistant phenotype. Among the 14 genes investigated, 10 genes displayed either significant amplification or overexpression in at least 1 clinical isolate; these genes are involved in several metabolic pathways. Moreover, various gene associations were observed depending on the clinical isolates, supporting the multifactorial nature of Leishmania resistance. Molecular resistance features were found in the 3 Leishmania species investigated (Leishmania infantum, Leishmania major, and Leishmania killicki). To our knowledge, this is the first report of the involvement of molecular resistance genes in field isolates of Leishmania major and Leishmania killicki with the resistance phenotype.

A new IgG immunoblot kit for diagnosis of toxoplasmosis in pregnant women.

The determination of the accurate immune status of pregnant women is crucial in order to prevent congenital toxoplasmosis. Equivocal results with conventional serological techniques are not uncommon when IgG titers are close to the cut-off value of the test, so that a confirmatory technique is needed. For this purpose, we developed a homemade immunoblot (IB) using soluble extract of Toxoplasma gondii tachyzoites and assessed it by testing 154 positive, 100 negative, and 123 equivocal sera obtained from pregnant women. In order to select the more valuable bands in terms of sensitivity and specificity, we used the Youden Index (YI). The highest YIs were those given by the 32, 36, 98, 21, and 33 bands. The simultaneous presence on the same blot of at least 3 bands showed a much higher YI (0.964) and was adapted as the positivity criterion. The analysis of results showed that our homemade IB correlated well with the commercial LDBIO Toxo II IgG® kit recently recommended as a confirmatory test (96.7% of concordance).

Contribution of specific anti-hydatid IgG subclasses in the diagnosis of echinococcosis primary infection and relapses.

BACKGROUND: Serological diagnosis of hydatid disease still faces problems of sensitivity, limiting its use to either diagnosis or post-surgical monitoring. The use of IgG subclasses seems to overcome these difficulties. The contribution of IgG subclasses was evaluated in the diagnosis of primary infested and hydatid cyst relapse patients. METHODS: A group of patients (n = 34) diagnosed for the first time with liver cystic echinococcosis (CE) and a group of patients with CE surgical recurrence were included. Enzyme-linked immunosorbent assay anti-hydatid antigens (HA) specific IgG1, 2, 3, and 4 subclasses were analyzed by ROC curves. RESULTS: ROC curve analyses demonstrated that IgG4 had the ability to discriminate between primary infested and relapsed groups whereas IgG2 was not discriminatory. The sensitivity of IgG4 was statistically higher in the relapsed cases group (97.1% versus 70.6%, p = 0.008). CONCLUSIONS: anti-HA specific IgG2 was the best marker of primary infestation whereas IgG4 was the best marker of relapse.

[Leishmania epidemiology, diagnosis, chemotherapy and vaccination approaches in the international network of Pasteur Institutes]. / Les leishmanioses vues au travers du réseau international des Instituts Pasteur.

Protozoan parasites of the genus Leishmania generate severe human diseases termed leishmaniases. Due to their frequency and the severity of certain clinical forms, these diseases represent a major public health problem and limit the economic growth in various developing countries. The presence of Pasteur Institutes in countries with endemic leishmaniasis has provided important incentives to develop a strong public health agenda in the Pasteur scientific community with respect to this important disease. A concerted effort is now coordinated through the recently created LeishRIIP platform (www.leishriip.org), which aims to identify synergies and complementary expertise between the eleven members of the international network of Pasteur Institutes working on various aspects of the disease including epidemiology, diagnosis, chemotherapy and vaccination.

ITS1 and cpb genetic polymorphisms in Algerian and Tunisian Leishmania infantum isolates from humans and dogs.

Leishmania (L.) infantum strains, isolated from varying hosts and clinical manifestations (cutaneous, visceral and canine leishmaniasis), were investigated in order to understand the genetic polymorphisms within this species in Algeria and Tunisia. Two DNA-based typing methods were tested in order to evaluate their effectiveness against Multilocus enzyme electrophoresis (MLEE), widely considered as the reference method for Leishmania parasite typing. On the other hand, MLEE is cumbersome, high-cost, time consuming and frequently does not detect intra-species genetic polymorphisms. In this work, we used two molecular target regions to discriminate L. infantum strains, Internal transcribed spacer 1 (ITS1) and the cysteine proteinase B (cpb). The ITS1 region offers good resolution for Leishmania discrimination but does not spotlight intra-species polymorphisms. In contrast, cpbE and cpbF PCR-Sequencing demonstrated a certain variability within CL and VL Algerian and Tunisian L. infantum isolates. Following phylogenetic analyses of 44 L. infantum isolates, two main groups were identified, a group with 39 bp deletion in the cpb sequence, composed of cutaneous, visceral and canine isolates from both countries with no significant clinical or geographic distribution; these samples were typed as MON-1, MON-24, and MON-80 zymodemes. A second group which presents a clear clusterization of Tunisian cutaneous strains belonging to the L. infantum MON-24. This group, with no deletion in the mature domain of the cpb gene sequence, should be further explored with a higher number of samples.

Study of ocular manifestations and humoral immune response in eyes of dogs with leishmaniasis.

BACKGROUND: Ocular manifestations in dogs with leishmaniasis are frequent and complications in affected tissues can lead to blindness. Immune processes play a very important role in the pathogenesis of ocular inflammation. Therefore, the immunology of ocular manifestations in dogs with leishmaniasis remains complex and poorly understood. OBJECTIVES: Estimation and characterisation of ocular and periocular manifestations in dogs naturally infected with Leishmania infantum and investigation of the production site of specific anti-Leishmania infantum IgG. METHODS: The present investigation used 53 confirmed dogs infected with Leishmania infantum, presenting ocular and periocular lesions, and 10 control non-infected dogs. Complete macroscopic ophthalmic examination of eyelids and globes was performed. Both total and anti-Leishmania infantum IgG antibodies were studied in sera and aqueous humour (AH) of all dogs by ELISA technique. A Goldmann-Witmer coefficient (C value) was calculated. RESULTS: The main ophthalmological findings were keratoconjunctivitis (71.7%; 38/53), hyperplasia of conjunctival lymphoid follicles (54.7%; 29/53), blepharitis (50.9%; 27/53) and uveitis (20.7%; 11/53). Ocular production of anti-Leishmania infantum IgG was detected in 73.6% (39/53) of infected dogs. There was no correlation between the antibody levels in AH and sera of the same dog. The mean anti-Leishmania infantum IgG in AH was higher in uveitis, followed by lesions affecting only the adnexa (p < 0.0001). The highest mean C values were observed for uveitis, conjunctivitis and keratitis. CONCLUSIONS: Our findings suggest that production of anti-Leishmania IgG in dogs infected with Leishmania infantum with ocular manifestations begin in situ and follows by a transfer of antibodies from the bloodstream to the AH.

Clinical and immunological spectra of human cutaneous leishmaniasis in North Africa and French Guiana.

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.

Canine Leishmania spp. infection in two distinct foci of visceral and cutaneous leishmaniasis in Tunisia.

Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) infantum and L. major, respectively, are endemic in Tunisia. The aim of the study was to assess canine Leishmania spp. infection prevalence as well as to identify the Leishmania species involved in two well-documented and geographically distinct VL and ZCL foci. One hundred seventy-six dogs were randomly recruited in the VL focus of Sbikha-Zaghouan (n = 100) and the ZCL focus of Echrarda-Nasrallah (n = 76). Physical examination and blood collection were systemically performed. Needle aspiration was done in case of lymph node (LN) enlargement. All sera were tested by ELISA. kDNA RT-PCR was performed on DNA extracts from (i) buffy coats of seropositive dogs and (ii) LN aspirates. Leishmania species identification was done by ITS1 PCR-sequencing. Thirty-three dogs (18.8%) were infected by Leishmania; 30 having anti-Leishmania antibodies and 3 were seronegative dogs with Leishmania DNA in LN aspirates. Prevalence of infection was significantly higher in VL foci than in ZCL foci (27% versus 7.9%, p = 0.002). Leishmania species was identified in 11 dogs and corresponded to L. infantum. Combination of serology and qPCR on LN aspirates seems to be the best option for canine leishmaniasis diagnosis. Infection is more frequent in VL foci and L. infantum is the only identified species.

Mediterranean visceral leishmaniasis : update on biological diagnosis.

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.

Diversity, Abundance and Leishmania infantum Infection Rate of Phlebotomine Sandflies in an Area with Low Incidence of Visceral Leishmaniasis in Northern Tunisia.

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.

Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia.

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.

[Knowledge of malaria and preventive attitudes of Tunisian travellers toward malaria endemic areas]. / Connaissances du paludisme et attitudes préventives des voyageurs tunisiens vers les zones d'endémie palustre.

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.