Publications by authors named "Karim Aoun"

60 Publications

Cutaneous Leishmaniasis in Algeria; Highlight on the Focus of M'Sila.

Microorganisms 2021 Apr 29;9(5). Epub 2021 Apr 29.

Laboratoire d'Eco-épidémiologie Parasitaire et Génétique des Populations, Route du Petit Staoueli, Institut Pasteur d'Algérie, Dely-Brahim 16047, Algeria.

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 (.) . Our study of the sandfly and rodent fauna further highlighted the high density of 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 and were found infected by . In addition, was isolated from two visceral leishmaniasis cases, one dog and its proven vectors (, , and ) 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.
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http://dx.doi.org/10.3390/microorganisms9050962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146893PMC
April 2021

Contribution of the Toxoplasma ICT IgG IgM test in determining the immune status of pregnant women against toxoplasmosis.

J Clin Lab Anal 2021 May 15;35(5):e23749. Epub 2021 Mar 15.

Department of Parasitology, Pasteur Institute of Tunis, Tunis, Tunisia.

Background: An immunochromatography technology (ICT) rapid diagnostic test, the Toxoplasma ICT IgG-IgM , was recently developed. Our aim was to study its contribution to establish accurately the Toxoplasma immune status in Tunisian pregnant women using Western blot (WB) Toxo II IgG as a reference technique.

Methods: Thirty-nine sera were selected for the study from among 2,615 which were already tested by IgG and IgM ELISA. They displayed equivocal IgG titres (4.4-9 IU/ml) in absence of IgM (19 sera) or IgM anti-Toxoplasma antibodies in absence of IgG (titre <4.4 IU/ml) (20 sera). All these sera were additionally tested by WB Toxo II IgG .

Results: Immunochromatography technology Sensitivity in the detection either of low IgG titres in absence of IgM or of specific anti-Toxoplasma IgM was 100%. Only one serum with equivocal IgG titre by ELISA and negative with Toxo II IgG test revealed positive in ICT. However, this serum showed a P30 band in WB analysis. On the other hand, three sera positive in ELISA IgM and negative in ELISA IgG revealed positive in ICT and negative in WB Toxo II IgG , the reference test.

Conclusion: Results confirm the high sensitivity of Toxoplasma ICT IgG-IgM in detecting both specific anti-Toxoplasma IgG and IgM, and highlight the usefulness of this rapid test as a first or second-line Toxoplasma serological test in pregnant women.
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http://dx.doi.org/10.1002/jcla.23749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128306PMC
May 2021

Use of Immunomagnetic Separation tool in Leishmania promastigotes capture.

Acta Trop 2021 Mar 29;215:105804. Epub 2020 Dec 29.

Laboratoire de recherche LR 16-IPT-06 "Parasitologie Médicale, Biotechnologies et Biomolécules"(1), Institut Pasteur de Tunis, Université Tunis El-Manar, 13 Place Pasteur, BP 74, 1002, Tunis, Tunisia; Laboratoire de Parasitologie-Mycologie, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002, Tunis, Tunisia. Electronic address:

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media. Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling. Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture. These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.
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http://dx.doi.org/10.1016/j.actatropica.2020.105804DOI Listing
March 2021

Nuclear and mitochondrial genome sequencing of North-African isolates from cured and relapsed visceral leishmaniasis patients reveals variations correlating with geography and phenotype.

Microb Genom 2020 10;6(10)

Laboratoire de recherche, LR 16IPT06, Parasitoses médicales, Biotechnologies et Biomolécules, Institut Pasteur de Tunis, Université Tunis El-Manar, 13 Place Pasteur, Tunis, Tunisie.

Although several studies have investigated genetic diversity of in North Africa, genome-wide analyses are lacking. Here, we conducted comparative analyses of nuclear and mitochondrial genomes of seven . isolates from Tunisia with the aim to gain insight into factors that drive genomic and phenotypic adaptation. Isolates were from cured (=4) and recurrent (=3) visceral leishmaniasis (VL) cases, originating from northern (=2) and central (=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 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 mitochondrial DNA as a potential novel source for biomarker discovery.
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http://dx.doi.org/10.1099/mgen.0.000444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660250PMC
October 2020

Assessment of Incubation Period of Cutaneous Leishmaniasis due to in Tunisia.

Am J Trop Med Hyg 2020 11;103(5):1934-1937

Department of Parasitology and Mycology, Pasteur Institute of Tunis, Tunis, Tunisia.

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 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).
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http://dx.doi.org/10.4269/ajtmh.20-0439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646806PMC
November 2020

Evaluation of six commercial kits for the serological diagnosis of Mediterranean visceral leishmaniasis.

PLoS Negl Trop Dis 2020 03 25;14(3):e0008139. Epub 2020 Mar 25.

Département de Parasitologie-Mycologie, Université de Montpellier, Centre Hospitalier Universitaire de Montpellier, Centre National de Référence des Leishmanioses, MIVEGEC, Montpellier, France.

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.
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http://dx.doi.org/10.1371/journal.pntd.0008139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135331PMC
March 2020

Design of multi-epitope peptides containing HLA class-I and class-II-restricted epitopes derived from immunogenic Leishmania proteins, and evaluation of CD4+ and CD8+ T cell responses induced in cured cutaneous leishmaniasis subjects.

PLoS Negl Trop Dis 2020 03 16;14(3):e0008093. Epub 2020 Mar 16.

Laboratoire de Parasitologie Médicale, Biotechnologie et Biomolécules, Institut Pasteur de Tunis, Tunis, Tunisie.

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.
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http://dx.doi.org/10.1371/journal.pntd.0008093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098648PMC
March 2020

Unexpected diagnosis of basal cell carcinoma in a patient presenting with a secondary location of parasites in the skin.

Pan Afr Med J 2019 3;34. Epub 2019 Sep 3.

Department of Parasitology, Pasteur Institute of Tunis, Tunisia.

We report here a case of simultaneous cutaneous and visceral manifestations due to 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. 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 and were 100% identical. The symptoms of visceral leishmaniasis responded to amphotericin B with rapid healing. However, the skin lesion did not improve although 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.
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http://dx.doi.org/10.11604/pamj.2019.34.8.18946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850740PMC
December 2019

Development and Assessment of and Specific Loop-Mediated Isothermal Amplification Assays for the Diagnosis of Cutaneous Leishmaniasis in Tunisia.

Am J Trop Med Hyg 2019 07;101(1):101-107

Laboratory of Medical Parasitology, Biotechnology and Biomolecules LR 11 IPT 06, Institut Pasteur de Tunis, Tunis, Tunisia.

Cutaneous leishmaniasis (CL) remains one of the world's most prevalent neglected diseases, particularly in developing countries. Identification of the involved 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 DNA species-specific detection from cutaneous lesions. Two LAMP assays, targeting cysteine protease B (cpb) gene, were developed to detect and identify and species. Loop-mediated isothermal amplification specificity was examined using DNA samples from other species and species. No cross-reactions were detected. The developed LAMP assays exhibited sensitivity with a detection limit of 20 fg and 200 fg for and , 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 and 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.
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http://dx.doi.org/10.4269/ajtmh.19-0097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609195PMC
July 2019

Knowledge and attitudes of Tunisian dog owners regarding leishmaniasis.

Parasite Epidemiol Control 2019 May 24;5:e00098. Epub 2019 Feb 24.

Laboratory of Parasitology, Univ. Manouba, Institution of Agricultural Research and Higher Education, National School of Veterinary Medicine of Sidi Thabet, 2020 Sidi Thabet, Tunisia.

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.
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http://dx.doi.org/10.1016/j.parepi.2019.e00098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411496PMC
May 2019

Toxoplasma gondii infection and toxoplasmosis in North Africa: a review.

Parasite 2019 15;26. Epub 2019 Feb 15.

Laboratoire de Parasitologie, Univ. Manouba, École Nationale de Médecine Vétérinaire de Sidi Thabet, 2020 Sidi Thabet, Tunisia.

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.
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http://dx.doi.org/10.1051/parasite/2019006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376878PMC
March 2019

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

Parasitology 2019 05 14;146(6):821-827. Epub 2019 Jan 14.

Laboratoire de recherche 'Parasitologie Médicale, Biotechnologies et Biomolécules', LR 16-IPT-06,Université Tunis El-Manar, Institut Pasteur de Tunis, 13 place Pasteur,B.P. 74 1002 Tunis Belvédère,Tunisia.

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.
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http://dx.doi.org/10.1017/S0031182018002196DOI Listing
May 2019

Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

mBio 2018 11 6;9(6). Epub 2018 Nov 6.

Unité de Parasitologiemoléculaire et Signalisation, Institut Pasteur, Paris, France

Protozoan parasites of the genus adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast growth. Together our data draw a complex picture of genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. Protozoan parasites of the genus cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the , , and complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.
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http://dx.doi.org/10.1128/mBio.01399-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222132PMC
November 2018

Congenital Toxoplasmosis in Tunisia: Prenatal and Neonatal Diagnosis and Postnatal Follow-up of 35 Cases.

Am J Trop Med Hyg 2018 06 12;98(6):1722-1726. Epub 2018 Apr 12.

Research Laboratory Medical Parasitology, Biotechnology and Biomolecule LR11IPT-06, Institute Pasteur of Tunis, Tunis, Tunisia.

Congenital toxoplasmosis (CT) results from transplacental passage of to the fetus during acute maternal infection. Our study aims to report clinical and biological patterns of 35 cases of CT diagnosed at the department of the Parasitology of the Pasteur Institute of Tunis and to access the performance of prenatal and early postnatal diagnosis techniques. Serological screening of maternal infection was performed by Immunoglobulin (Ig) M and IgG detection and IgG avidity determination. Prenatal diagnosis was based on both DNA detection in the amniotic fluid and monthly ultrasound examinations. polymerase chain reaction analysis on amniotic fluid, performed only in 15 cases, detected 's DNA in five cases (33.3%). Ultrasound examination did not reveal any morphological abnormalities. Thirty newborns had serological criteria of infection. Congenital toxoplasmosis diagnosis was confirmed in 23 cases (76.6%) by immunoblot. Among the 35 born-infants, five (14.3%) were symptomatic: three had chorioretinitis at the first clinical ocular examination, one had neurological symptoms (seizures) with positive parasite DNA in cerebral spinal fluid, and one had both ophthalmological and neurological damages- chorioretinitis and intracranial calcifications in the computed tomography scan. Thirty-four of 35 infected children were treated with pyrimethamine-sulfadiazine combination. Four (11.7%) of the treated infants showed abnormal hematological values because of the treatment side effect. Serological rebound was observed in seven infants. A screening program and a diagnostic algorithm in pregnant women should be implemented in Tunisia to improve the follow-up of seronegative ones and to prevent CT cases.
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http://dx.doi.org/10.4269/ajtmh.17-0580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086171PMC
June 2018

Genetic diversity of Cryptosporidium isolates from human populations in an urban area of Northern Tunisia.

Infect Genet Evol 2018 03 7;58:237-242. Epub 2018 Jan 7.

Laboratoire de Parasitologie-Mycologie, LR, 11-IPT-06, Institut Pasteur de Tunis, 13 place Pasteur, 1002, Tunis, Tunisia.

Cryptosporidium is an enteric parasite infecting a wide range of hosts. It has emerged as an important cause of chronic life-threatening diarrhea in humans worldwide. Several subtypes of Cryptosporidium sp. have been described to be responsible for several large outbreaks related to water contamination in developed countries. However, there is a lack of information in the genetic diversity of Cryptosporidium among human population especially in developing countries. The present study aimed to update and report the genetic diversity of human Cryptosporidium spp. at the subtype level in an urban area of Tunisia using the 18S rRNA and gp60 gene. Genotyping of 42 Cryptosporidium positive isolates from different human populations at the 18S rRNA locus has identified three Cryptosporidium species: C. hominis (n = 20), C. parvum (n = 19), C. meleagridis (n = 2) and a co-infection C. hominis/C. meleagridis (n = 1). The sub-genotyping of these isolates at the 60-kda glycoprotein (gp60) locus was possible in 40 cases. It showed the presence of three subtype families (IIa, IIb and IIc) within C. parvum, a single subtype family within C. hominis and C. meleagridis isolates (Ia and IIIb respectively). Several subtypes were implicated in different human populations with the dominance of IaA26G1R1, IIaA15G2R1, IIdA16G1R1, IIdA22G2R1 and IIIbA26G1R1 variant respectively for C. hominis, C. parvum and C. meleagridis. The distribution of Cryptosporidium isolates in urban area of Northern Tunisia was dominated by the anthroponotic transmission via C. hominis species and the IIc subtype of C. parvum. However, zoonotic transmission is still possible in this region via zoonotic subtypes of C. parvum (IIa and IId) and C. meleagridis (IIIb). Subtype diversity was higher in this area.
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http://dx.doi.org/10.1016/j.meegid.2018.01.004DOI Listing
March 2018

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

Microb Pathog 2018 Feb 3;115:332-337. Epub 2018 Jan 3.

LR 11-IPT-06 Laboratory of Medical Parasitology, Biotechnology and Biomolecules, Pasteur Institute of Tunis, University Tunis El Manar, Tunisia.

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.
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http://dx.doi.org/10.1016/j.micpath.2017.12.070DOI Listing
February 2018

Use of an Interferon Gamma Release Assay (IGRA) to test T-cell responsiveness to soluble Leishmania infantum antigen in whole blood of dogs from endemic areas.

Vet Parasitol 2017 Nov 1;246:88-92. Epub 2017 Sep 1.

Department of Clinical Parasitology, Laboratoire de recherche LR 11-IPT-06 "Parasitologie Médicale, Biotechnologies et Biomolécules", Institut Pasteur de Tunis, Université Tunis El-Manar, 13 Place Pasteur, BP 74, 1002, Tunis, Tunisia. Electronic address:

Interferon-Gamma (IFN-γ) Release Assays (IGRAs) are easy tests that allow rapid screening of primed memory T-cells immunity in response to antigen. The aim of this study was to use IGRA to assess IFN-γ release in response to Soluble Leishmania infantum antigen (SLA) in whole blood of dogs living in endemic area of visceral leishmaniasis and to interpret IGRA results according to clinical examination, specific anti-Leishmania humoral response and presence of L. infantum DNA in blood. The study was carried out on 56 dogs living in greater Tunis area. Physical examination, quantitative serology and PCR on blood were used to characterize dogs' status in relation to Leishmania infection and disease. IGRA consisted on testing by ELISA for IFN-γ-secretion in whole blood after a 20-h challenge with SLA. PBS and Phytohemagglutinin (PHA) stimulations were used as controls. Four groups of dogs were characterized: 31 were negative by both serology and PCR, two had doubtful serology, 10 presented no to mild clinical signs but low antibodies levels and 13 were affected by Canine Leishmaniasis (CanL). In seronegative dogs, IGRA was little contributory in 4 puppies (age <6months) and 5 old dogs (median age=72months, IQR: 45-84 months) that didn't respond to PHA stimulation, IGRA was negative in 19 and positive in three animals with lymph node enlargement. In dogs with doubtful serology, IGRA was positive in one dog and negative in the other. In infected dogs with no to mild clinical signs, one dog exhibited high level of IFN-γ in absence of antigenic stimulation and all the other were positive by IGRA. CanL dogs showed variable IGRA results. Negative IGRAs (n=4) were shown in animals with the highest parasitic burden whereas positive IGRAs (n=5) were shown in dogs with negative PCR or low parasitic load. The 4 remaining dogs either didn't respond to PHA (n=2) or showed non-specific secretion in PBS tube (n=2). The results of this study showed that IGRA is a useful new tool that can assess exposure to Leishmania in dogs with no to mild clinical signs in endemic area. Further comparative investigations using assays exploring cellular immunity are needed to determine its accuracy.
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http://dx.doi.org/10.1016/j.vetpar.2017.08.029DOI Listing
November 2017

Temporal dynamics and Leishmania infantum infection prevalence of Phlebotomus perniciosus (Diptera, Phlebotominae) in highly endemic areas of visceral leishmaniasis in Tunisia.

PLoS One 2017 21;12(9):e0184700. Epub 2017 Sep 21.

Department of Parasitology, Research Lab: LR 11-IPT-06, Pasteur Institute of Tunis, University Tunis El-Manar, Tunis, Tunisia.

Phlebotomus perniciosus is one of the major vectors of Leishmania infantum in the Mediterranean basin. The aim of this work was (i) to provide information about abundance and temporal dynamics of this Larroussius species in a hot spot area of visceral leishmaniasis in Tunisia, (ii) to detect L. infantum DNA in wild caught female sandflies and (iii) to measure Phlebotomus perniciosus infection rate throughout the active season. Sandflies were collected monthly during one year using CDC miniature light-traps in house and in animal shelters. Male specimens were identified at species level according to morphological characters. Female specimens were conserved individually for molecular study. Leishmania infection was tested by kinetoplast DNA real-time PCR and ITS-1 PCR-sequencing. Subsequent sandfly species identification of infected specimens was done by mitochondrial cytochrome b sequencing. In one year period, overall 4,441 specimens (2230 males and 2211 females) were collected. Sandfly activity started in end-April and ended in early-November. Mean sandfly density in house was significantly lower than in animal shelters (51 ± 50 versus 504 ± 460 sandflies /CDC night, p<0.05). However, a higher proportion of females was found in house (58.4% versus 49.2%, p<0.001). Based on species identification of male specimens, Phlebotomus perniciosus was the dominant species (56% of the whole male sandfly fauna, p<0.0001). It showed two peaks of density in the active season, a sharp one in early May and a higher long lasting one from end-July to end-September. DNA was extracted from 190 female specimens randomly sampled and corresponding to 96 specimens from house and 94 from animal shelters. Twenty four female sandfly were infected by Leishmania infantum. All infected specimens were recognized as Phlebotomus perniciosus. Leishmania infantum infection rate in female sandflies was 2.3 fold higher in house than in animal shelters (17.7% versus 7.4%, p<0.05). In house, estimated number of infected specimens was the highest at the end of the active season. Abundance, dynamics of density and Leishmania infantum infection prevalence of Phlebotomus perniciosus in Tunisian hot spot of visceral leishmaniasis highlight the major role of this Phlebotominae species in L. infantum transmission.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184700PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608219PMC
October 2017

Identification of Leishmania by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry Using a Free Web-Based Application and a Dedicated Mass-Spectral Library.

J Clin Microbiol 2017 10 19;55(10):2924-2933. Epub 2017 Jul 19.

Laboratoire de Parasitologie-Mycologie, CHU Timone, Université d'Aix-Marseille, Marseille, France

Human leishmaniases are widespread diseases with different clinical forms caused by about 20 species within the genus. species identification is relevant for therapeutic management and prognosis, especially for cutaneous and mucocutaneous forms. Several methods are available to identify species from culture, but they have not been standardized for the majority of the currently described species, with the exception of multilocus enzyme electrophoresis. Moreover, these techniques are expensive, time-consuming, and not available in all laboratories. Within the last decade, mass spectrometry (MS) has been adapted for the identification of microorganisms, including However, no commercial reference mass-spectral database is available. In this study, a reference mass-spectral library (MSL) for isolates, accessible through a free Web-based application (mass-spectral identification [MSI]), was constructed and tested. It includes mass-spectral data for 33 different species, including species that infect humans, animals, and phlebotomine vectors. Four laboratories on two continents evaluated the performance of MSI using 268 samples, 231 of which were strains. All strains, but one, were correctly identified at least to the complex level. A risk of species misidentification within the , , and complexes was observed, as previously reported for other techniques. The tested application was reliable, with identification results being comparable to those obtained with reference methods but with a more favorable cost-efficiency ratio. This free online identification system relies on a scalable database and can be implemented directly in users' computers.
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http://dx.doi.org/10.1128/JCM.00845-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625378PMC
October 2017

Leishmania major large RAB GTPase is highly immunogenic in individuals immune to cutaneous and visceral leishmaniasis.

Parasit Vectors 2017 Apr 17;10(1):185. Epub 2017 Apr 17.

Laboratory of Medical Parasitology, Biotechnology and Biomolecules, LR11-IPT-06, Institut Pasteur de Tunis, Tunis, Tunisia.

Background: We previously identified a Leishmania (L.) major large RAB GTPase (LmlRAB), a new atypical RAB GTPase protein. It is highly conserved in Leishmania species while displaying low level of homology with mammalian homologues. Leishmania small RAB GTPases proteins have been involved in regulation of exocytic and endocytic pathways whereas the role of large RAB GTPases proteins has not been characterized yet. We report here the immunogenicity of both recombinant rLmlRAB and rLmlRABC, in individuals with immunity against L. major or L. infantum.

Methods: PBMC were isolated from individuals cured of L. major (CCLm) or from healthy individuals. The latter were subdivided into high or low IFN-γ responders. Healthy high IFN-γ responders, considered as asymptomatics, were living in an endemic area for L. major (HHRLm) or L. infantum (HHRLi). Healthy low IFN-γ responders (HLR) were considered as naïve controls. Cells from all volunteers were stimulated with rLmlRAB or rLmlRABC. Cytokines were analysed by CBA and ELISA and phenotypes of IFN-γ-producing cells were analysed by flow cytometry.

Results: Both rLmlRAB and rLmlRABC induced high significant levels of IFN-γ in CCLm, HHRLm and HHRLi groups. Phenotype analysis of rLmlRAB and rLmlRABC-stimulated T cells in CCLm individuals showed a significant increase in the percentage of specific IFN-γ-producing CD4+ and CD8+ T cells. rLmlRAB induced significant granzyme B levels in CCLm and HHRLm. Low but significant granzyme B levels were detected in naïve group. IL-10 was detected in immune and naïve individuals.

Conclusion: We showed that rLmlRAB protein and its divergent carboxy-terminal part induced a predominant Th1 response in individuals immune to L. major or L. infantum. Our results suggest that rLmlRAB and rLmlRABC proteins are potential cross-species vaccine candidates against cutaneous and visceral leishmaniasis.
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http://dx.doi.org/10.1186/s13071-017-2127-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393016PMC
April 2017

Isolated cervical lymphadenopathy: Do not forget atypical leishmaniasis.

Presse Med 2017 Apr 21;46(4):452-454. Epub 2017 Feb 21.

La Rabta Hospital, Infectious Diseases Ward, 1007 Tunis, Tunisia; Université Tunis El Manar, faculté de médecine, Tunis, Tunisia.

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http://dx.doi.org/10.1016/j.lpm.2017.01.011DOI Listing
April 2017

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

Tunis Med 2016 Jun;94(6):167-171

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.
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June 2016

Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples.

Acta Trop 2017 Mar 22;167:71-72. Epub 2016 Dec 22.

Department of Clinical Parasitology, Laboratoire de Recherche LR 11-IPT-06, Parasitologie Médicale, Biotechnologies et Biomolécules, Institut Pasteur de Tunis, Université Tunis El-Manar, Tunis, Tunisia. Electronic address:

Implementation of simple diagnostic tests using non-invasive collection of biological specimens is of great importance in the diagnosis of pediatric visceral leishmaniasis caused by Leishmania infantum. Latex agglutination kit (KAtex) is widely used in the diagnosis mainly in L. donovani endemic areas. However its utilization in L. infantum endemic regions remains limited and its use on noninvasive biological specimen apart urine was not reported. In this study, KAtex kit was used to detect Leishmania-related antigen in urine and oral fluid of 35 L. infantum visceral leishmaniasis cases and 62 controls including non-infectious disease and infectious disease controls (34 and 28 respectively). Sensitivity and specificity of urine based KAtex were 51.4% and 98.3% respectively, whereas, sensitivity and specificity of oral-fluid based KAtex were 80% and 88.3% respectively. Although, sensitivity of oral-fluid KAtex was high, its specificity varied significantly according to the presence or the absence of an infectious disease (71.4% versus 97%, p=0.01).
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http://dx.doi.org/10.1016/j.actatropica.2016.12.026DOI Listing
March 2017

Prevalence and Subtype Identification of Blastocystis sp. in Healthy Individuals in the Tunis Area, Tunisia.

Am J Trop Med Hyg 2017 Jan 5;96(1):202-204. Epub 2016 Dec 5.

Research Laboratory of Medical Parasitology, Biotechnology and Biomolecules (PMBB), Institut Pasteur of Tunis, Tunis, Tunisia.

Blastocystis sp. is currently the most common eukaryotic parasite found in humans. Despite its potential public health impact, epidemiological data regarding its prevalence and molecular subtype (ST) distribution in Maghreb are rarely reported. Therefore, the aim of this study was to determine the prevalence of the parasite in a cohort of healthy food handler Tunisian individuals and to acquire the first molecular data regarding the distribution of Blastocystis sp. STs in this country. Therefore, 524 fecal samples were collected, and 68 of them (13%) were identified as positive for the parasite by direct-light microscopy of smears. Seventeen samples of 100 negative by microscopy were also shown to be positive by real-time quantitative polymerase chain reaction. Among all the positive samples, 61 Blastocystis isolates were subtyped using partial small subunit ribosomal RNA gene analysis. ST3 was the most abundant (51%) followed by ST1 (30%), ST2 (16%), and ST4 and ST7 (both 1.6%).
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http://dx.doi.org/10.4269/ajtmh.16-0506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5239694PMC
January 2017

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

Tunis Med 2015 Dec;93(12):766-70

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.
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December 2015

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

Tunis Med 2015 Nov;93(11):687-91

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.
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November 2015

[Hemophagocytosis and disseminated intravascular coagulation in visceral leishmaniasis in adults: three new cases].

Pan Afr Med J 2015 1;22:96. Epub 2015 Oct 1.

Service de Médecine Interne B, Hôpital Charles Nicolle, Faculté de Médecine de Tunis, 1006 Bab Saadoun, Université de Tunis El Manar, Tunis, Tunisie.

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http://dx.doi.org/10.11604/pamj.2015.22.96.5662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732622PMC
September 2016

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

Tunis Med 2015 Jun;93(6):347-9

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.
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June 2015

[High risk areas for echinococcosis-hydatidosis in Tunisia].

Tunis Med 2015 Jan;93(1):33-7

Aim: The study was conducted in order to identify high risk areas for hydatidosis in Tunisia witch would be eligible for a Hydatidosis control program initiation.

Methods: The most recent epidemiological investigation on surgical incidence of hydatidosis was used to classify governorates according to their incidence rate. A "global hydatidosis risk score" was calculated for each governorate, combining some parameters related to the hygiene conditions of the population, the literacy rate, the canine density and livestock census. Spearman correlation coefficient was used to compare scores and surgical incidences. Mapping analysis has been conducted. The surgical incidence rate of hydatidosis classifies each governorate regarding occurrence of human cases. The global hydatidosis risk score, by governorate, pointed out the most exposed areas to the disease.

Results: The mapping analysis showed a good agreement between the incidence rate of the disease and the global hydatidosis risk score and made it possible to identify the population of the center and the west of the country as a most exposed population for the diseases.

Conclusion: In order to have a chance for implementation, hydatidosis control program should target the three jointed governorates of Kasserine, Siliana and Kef, which have the highest incidence rates and the worst scores.
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January 2015

Airport malaria: report of four cases in Tunisia.

Malar J 2015 Jan 28;14:42. Epub 2015 Jan 28.

Laboratoire de Parasitologie-Mycologie, Tunis, 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.
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http://dx.doi.org/10.1186/s12936-015-0566-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318208PMC
January 2015
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