Serological evidence for the circulation of flaviviruses in seabird populations of the western Indian Ocean.

Birds play a central role in the epidemiology of several flaviviruses of concern for public and veterinary health. Seabirds represent the most abundant and widespread avifauna in the western Indian Ocean and may play an important role as host reservoirs and spreaders of arthropod-borne pathogens such as flaviviruses. We report the results of a serological investigation based on blood samples collected from nine seabird species from seven islands in the Indian Ocean. Using a commercial competitive enzyme-linked immunosorbent assay directed against the prototypic West Nile flavivirus, antibodies against flaviviruses were detected in the serum of 47 of the 855 seabirds tested. They were detected in bird samples from three islands and from four bird species. Seroneutralization tests on adults and chicks suggested that great frigatebirds (Fregata minor) from Europa were infected by West Nile virus during their non-breeding period, and that Usutu virus probably circulated within bird colonies on Tromelin and on Juan de Nova. Real-time polymerase chain reactions performed on bird blood samples did not yield positive results precluding the genetic characterization of flavivirus using RNA sequencing. Our findings stress the need to further investigate flavivirus infections in arthropod vectors present in seabird colonies.

Investigation of a leptospirosis outbreak in triathlon participants, Réunion Island, 2013.

We report herein the investigation of a leptospirosis outbreak occurring in triathlon competitors on Réunion Island, Indian Ocean. All participants were contacted by phone or email and answered a questionnaire. Detection and molecular characterization of pathogenic Leptospira was conducted in inpatients and in rodents trapped at the vicinity of the event. Of the 160 athletes competing, 101 (63·1%) agreed to participate in the study. Leptospirosis was biologically confirmed for 9/10 suspected cases either by real-time PCR or serological tests (MAT or ELISA). The total attack rate, children's attack rate, swimmers' attack rate, and the attack rate in adult swimmers were respectively estimated at 8·1% [95% confidence interval (CI) 4·3-14·7], 0%, 12·7% (95% CI 6·8-22·4) and 23·1% (95% CI 12·6-33·8). Leptospirosis cases reported significantly more wounds [risk ratio (RR) 4·5, 95% CI 1·6-13], wore complete neoprene suits less often (RR 4·3, 95% CI 1·3-14·5) and were most frequently unlicensed (RR 6·6, 95% CI 2·9-14·8). The epidemiological investigation supported that some measures such as the use of neoprene suits proved efficient in protecting swimmers against infection. PCR detection in rats revealed high Leptospira infection rates. Partial sequencing of the 16S gene and serology on both human and animal samples strongly suggests that rats were the main contaminators and were likely at the origin of the infection in humans.

Expression of a public idiotype by human monoclonal IgM directed to myelin-associated glycoprotein and characterization of the variability subgroup of their heavy and light chains.

Most studies using rabbit or mouse antisera failed to detect CRI between human IgM directed to MAG. We show here that 9 of 10 such IgM express a public CRI as defined by a nonhuman primate antiserum. Shared idiotype is likely involved in (or close to) the combining site of those IgM since antiidiotypic serum inhibited the binding of IgM to MAG and reacted with IgM having different variable regions of light and heavy chains. Partial aminoterminal sequence of heavy and light chains showed that anti-MAG IgM use either lambda chains (one IgM) or kappa light chains (six IgM) of different variability subgroups (V kappa IV in three instances, V kappa I in two, and V kappa II in one), whereas heavy chains belong to the VHIII (six IgM) or to the VHII (1 IgM) subgroup. These features distinguish these IgM from other human monoclonal IgM with a defined antibody activity, such as rheumatoid factors or cold agglutinins.

Effects of tropism and virulence of Leishmania parasites on cytokine production by infected human monocytes.

The nature of early interactions between Leishmania and macrophages which determine the outcome of infection can be related directly to parasite biological properties. Here we compared the capacity of L. major (Lm) strains, reported to be high (LmHV) and low virulent and (LmLV) in the mouse model and L. infantum (Li) strains, dermotropic (LiD) and viscerotropic (LiV), to infect and modulate cytokine production in human peripheral blood derived monocytes. Monocytes were infected with metacyclic promastigotes for 24, 48 and 72 h. Parasite burden was significantly higher in Lm- than in Li-infected monocytes. LmHV and LiD induced a significantly higher parasite burden than LmLV and LiV respectively. Cytokine production was evaluated in monocytes infected for 24 h. Contrary to interleukin (IL)-12p70, monocyte chemotactic protein-1 and transforming growth factor-beta production was increased significantly in infected monocytes with no differences between strains. Lm isolates induced significantly higher quantities of tumour necrosis factor (TNF)-alpha than Li isolates. Low levels of IL-10 were induced by all Leishmania strains and, interestingly, co-stimulation with lipopolysaccharide (LPS) was accompanied by a dramatic increase in IL-10 production by infected monocytes. In conclusion, Lm isolates displaying different levels of virulence in mice exhibited significant differences in parasite burden but similar abilities to modulate cytokine production in human monocytes. Li strains showed weaker infectivity and TNF-alpha inducing-capacity compared with Lm strains. The dramatic increase of IL-10 production in infected monocytes co-stimulated by LPS may play a role in disease progression considering the presence of LPS during bacterial superinfections observed during human leishmaniasis.

Quantitative detection of bcr-abl transcripts in chronic myeloid leukemia.

The optimal management of malignant haematological disorders depend on the degree of tumor load reduction after therapy. Chronic myeloid leukemia constitutes a clinical model for molecular detection and therapy surveillance of malignant disease since this entity was the first leukemia shown to be associated with a specific bcr-abl fusion gene in the patient's leukemia cells. Molecular monitoring of bcr-abl transcript levels by real-time quantitative PCR is increasingly used to assess treatment response in patients with chronic myeloid leukemia (CML). This has become particularly relevant in the era of imatinib therapy when residual levels of leukaemia usually fall below the level of detection by bone marrow cytogenetic analysis. We monitored bcr-abl transcript levels by quantitative real time PCR in 50 tunisian patients treated with imatinib for chronic myeloid leukemia in chronic phase for a median of 29 months (3-60) after they started imatinib.

[Immuno-modulating effects of eukaryotic expressing vectors of IL-12 and GM-CSF associated to DNA-based vaccination against experimental cutaneous leishmaniasis in BALB/c mouse]. / Effets immuno-modulateurs des vecteurs d'expressions eucaryotes de l'il-12 et du GM-CSF en association avec la vaccination à base d'ADN contre la leishmaniose cutanée expérimentale chez la souris BALB/c.

Different works of DNA based vaccination against leishmaniasis highlight the complexity of the induced immune responses to fight against the disease. In this work, we exploited the capacity of IL-12 and GMC-SF to activate immune cell mediators and effectors to induce a Th1 response, more capable of clearing the parasite. To generate these immunomodulating activities, we associated eukaryotic expressing vectors of murine IL-12 and GMC-SF to several DNA based vaccine candidates encoding to several L. (L.) major antigens, in the BALB/c mouse. When mice were challenged with a high parasitic load in the hind footpad, no additional protective effect could be generated. However, when the challenge was carried out in the inner face of the ear with a small parasitic load, the association of plasmids encoding to IL-12 and GMC-SF to DNA based vaccination, the protective effects were increased.

Genetically engineered colorimetric single-chain antibody fusion protein for rapid diagnosis of rabies virus.

The most widely used test for rabies diagnostics is the fluorescent antibody test, which is recommended by both the World Health Organization and the World Organisation for Animal Health (OIE). This test may be used directly on a smear, and can also be used to confirm the presence of rabies antigen in cell culture or in brain tissue for diagnosis. The colorimetric enzymes are usually coupled to an antibody by chemical means using cross-linking reagents. However, such non-specific procedures lead to heterogeneous conjugates, sometimes with reduced activity and specificity. To bypass these problems, genetic engineering has provided a way to create chimeric bifunctional molecules in which the variable domains of an antibody are genetically linked to unrelated protein tracers. In this study, we describe the successful production of a bifunctional chimeric protein based on alkaline phosphatase-fused anti-rabies virus glycoprotein scFv antibody fragment. We also report the antigen binding properties and the alkaline phosphatase activity of the recombinant conjugate protein. We established its value as a novel in vitro tool for detecting the rabies virus in brain smear in a one-step procedure; it presents a similar sensitivity and specificity to that obtained using standard reagents.

Dogs L. infantum infection from an endemic region of the north of Tunisia: a prospective study.

A follow-up study of 917 dogs was undertaken between 1994 and 1995 in the focus of visceral leishmaniasis in northern Tunisia. It permitted to assess the demography of the dog population, the importance of canine leishmaniasis (CL) and the determinants of seropositivity and mortality of dogs. Canine population was stable through time with an input of 231 dogs and an output of 218 dogs per year. The prevalence of seropositivity was 18% and 22.3% in 1994 and 1995 respectively and 90% of dogs were asymptomatic. Among 525 negative dogs in 1994 and reassessed in 1995, 78 seroconverted revealing an annual cumulative incidence of 14.74%. On the other hand, 23.47% (27/115) of seropositive dogs became negative in 1995. Age, presence of symptoms and density of dogs were independently associated with CL seropositivity. These results demonstrate the difficulty of control strategies of visceral leishmaniasis targeting the dog population.

Cross-idiotypic antigens among monoclonal immunoglobulin M from patients with Waldenström's macroglobulinemia and polyneuropathy.

The monoclonal immunoglobulin (Ig)M from 5 to 16 patients with Waldenström's macroglobulinemia and a polyneuropathy shared cross-idiotypic antigenic determinants as demonstrated by hemagglutination and hemagglutination inhibition experiments as well as by precipitin reactions. This reactivity was located to the Fab (and not Fc) fragment of the protein. The IgM from 73 patients with macroglobulinemia but without neuropathy all gave negative reactions. In contrast, the monoclonal IgG from a patient with polyneuropathy also possessed similar idiotypic determinants. Since cross-idiotypic determinants are usually related to the combining site of a monoclonal Ig, this finding suggests that the monoclonal Ig of these patients may mediate the nerve injury via their antibody activity, which could be directed either to a nerve antigen or to some component involved in the pathogenesis of the neuropathy.

High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris.

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc- (His)6 tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein. We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested posttranslational modifications may contribute to the size difference. The NH2-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.

[Clinical spectrum of cobalamin deficiency in Tunisia]. / Le spectre clinique des déficits en cobalamines en Tunisie.

PURPOSE: the aim of this study was to determine the prevalence of cobalamin (vitamin B12) deficiency in different populations of patients with clinical manifestations associated or secondary to cobalamin or folates deficiency and to analyse the demographic, clinical, paraclinical investigations in cobalamin deficient patients in Tunisia. METHODS: it was a prospective (1999-2001) multicenter study of 604 patients divided into four groups. The first group is composed of 478 consecutive patients with anaemia and/or macrocytosis with megaloblastic haemopoiesis on bone marrow examination without myelodyslasic or malignancy signs. The second group is made up of 34 patients with unexplained neurological symptoms without the presence of anaemia. The third group was composed of 82 invidious with isolated psychiatric disorders and the 10 patients with Hashimoto thyroïditis constituted the last group. RESULTS: serum cobalamin level was low in 98 %, 23%, 14% of cases, respectively, in the first three groups. Only one case of patients with Hashimoto thyroiditis has serum cobalamin deficiency. Pernicious anaemia (Biermer's disease) was established by dual isotope schilling examination in 103 patients among a sample of 120 serum cobalamin deficient patients (86%). The median age at presentation was 45.5 years. Severe chronic atrophic gastritis was diagnosed in 97.5% of patients with Biermer's disease. Serum antibodies against intrinsic factor and gastric parietal cells were detected in (42.5%) and (60.6%) patients, respectively; (25.5%) patients had the both types of antibodies. 23.4% patients were positive for antithyroid antibodies. Anti-nuclear antibodies were detected in 3% patients. CONCLUSION: an interesting finding of our study was the high frequency of cobalamin deficiency in Tunisia, particularly in relative young patients. Our patients had classic features of florid cobalamin deficiency (severe haematological manifestations and neuro-psychiatric disorders). The main underlying causes of such deficiencies were Biermer's disease. Subtle clinical manifestations should be recognized and investigated even in young patients at risk.

[Prevalence of Pneumocystis jiroveci pneumonia in Tunisian primary immunodeficient patients]. / Prévalence de la pneumopathie à Pneumocystis Jiroveci au cours des déficits immunitaires héréditaires observés en Tunisie.

UNLABELLED: Pneumocystis Jiroveci pneumonia (PJP) is a rare opportunistic infection in immunodeficient patients in Tunisia, as well as in other Africain countries including those with a high prevalence of AIDS. In the literature, PJP has been reported in primary immunodeficiency diseases (PID) namely SCID T-B- or T-B+ or X-linked hyper-IgM syndrome. OBJECTIVE: To evaluate the prevalence of PJP in the different PID observed in Tunisia. PATIENTS AND METHODS: This retrospective study concerned 290 cases of PID confirmed by immunological investigation including the study of specific and/or non-pecific humoral and cellular immunity. The identification of P. Jiroveci in patients suspected of pneumocystosis was achieved by parasitological investigation in bronchoalveolar lavages. RESULTS: A PID associated to a parasitologically confirmed pneumocystic infection was found in 9 out of 290 patients (3%) among whom the majority (7 patients) had an HLA class II combined immunodeficiency. The latter is an autosomic recessive disease which has been reported mainly in North African families. Indeed, this population is characterized by a high rate of consanguinity. Interestingly, no PJP has been observed neither in SCID T-B- or T-B+ nor in X-linked hyper-IgM syndrome. DISCUSSION AND CONCLUSION: PJP seems to be particularly frequent in HLA class II deficiency patients, since 7 out of 22 patients with this deficiency had a PJP (31%). Due to this defect, antigen presenting cells are unable to present the antigen to T lymphocytes demonstrating the critical role of CD4+ T lymphocytes responses in the immune response to this pathogen.

A case control study to assess risk factors for hepatitis C among a general population in a highly endemic area of northwest Tunisia.

A matched case-control study was undertaken in 2004 in Béja, north-western Tunisia, to evaluate potential risk factors for hepatitis C infection. Cases were anti-HCV positive subjects screened in 1996 serosurvey. HCV seronegative controls (5 per case) were selected in the proximity of cases and matched for age and gender. A standardized questionnaire was used to collect demographic, socioeconomic, social behavior, medical and surgical history information. Matched odds ratios (OR) and adjusted OR (AOR) and their 95% CI were calculated in multivariate analysis using logistic regression. 57 HCV positive cases (mean age 61.63 +/- 14,84; 68.4 % female) and 285 HCV negative controls (mean age 60.95 +/- 14.66; 68.4 % female) were enrolled. Multivariate analysis revealed that intravenous drug injections (AOR=1.96; 95%CI[1.02-3.8] p=0.045), past history of invasive procedures (AOR=2.53; 95%CI[1.21-5.29] p=0.0014) and medical history of hypertension (AOR=2.31; 95%CI [1.17-4.56]p=0.015) were significantly associated to HCV infection. These results suggest that nosocomial transmission of HCV infection in north-west Tunisia is common.

Abnormal expression of vimentin intermediate filaments in human lymphoid cell lines with deletion or translocation of the distal end of chromosome 8.

The expression of vimentin, the major polypeptide of the intermediate filament (IFM) cytoskeleton of lymphoid cells, was studied in normal and malignant human lymphoid cell lines. Cells from 24 of 27 Burkitt's lymphoma cell lines (BLCL) were found to have an absent (16 lines) or decreased (8 lines) expression of vimentin IFM. In contrast, non-Burkitt's malignant lymphoid cell lines (5 lines) and lymphoblastoid cell lines (LCL) derived from normal B-cells (45 lines) exhibited a well-developed vimentin IFM network. However, low expression of vimentin was also found in 3 LCL derived from patients with the Langer-Giedion syndrome, which is characterized by a deletion of the distal end of chromosome 8. Treatment of vimentin-negative BLCL and Langer-Giedion LCL with azacytidine led to a transient reexpression of vimentin.

Characterization of intermediate filaments expressed by Ewing tumor cell lines.

The histogenesis of Ewing sarcoma is still controversial; we therefore studied the expression of intermediate filaments (IF) in cell lines derived from Ewing tumors since identification of IF in tumor cells is considered a reliable marker of tissue origin and differentiation. All nine lines studied expressed vimentin IF; in addition, a small number of Ewing cells from three lines expressed keratin filaments. After treatment with phorbol esters, a high percentage of cells from these three lines synthesize keratin IF identified by immunoblotting as keratin 8 and 18 polypeptides, which are expressed by single epithelia and epithelial cells in early embryonic development. Furthermore cells from a fourth line synthesize keratins after transplantation in nude mice. These data indicate that, under certain conditions, undifferentiated Ewing cells may acquire an IF phenotype related to that of epithelial cells.

[RT-PCR use for the diagnostic of chronic myeloid leukaemia]. / Contribution de la RT-PCR au diagnostic de la leucémie myéloïde chronique.

The molecular analysis of chromosomal abnormalities associated with hematological malignancies allowed the identification of genes involved in theses rearrangements as well as of some recurrent mechanisms. Polymerase chain reaction (PCR) tools are now available to detect these rearrangements, allowing a better follow-up of these diseases. Chronic myeloid leukemia is a myeloproliferative disorder characterized by a reciprocal translocation t(9;22)(q34;q11) which results in a bcr-abl fusion gene. Retro-transcription polymerase chain reaction (RT-PCR) is used to detect bcr-abl to establish diagnosis and to monitor patients. We report here the results of 30 patients samples tested in the hematology laboratory at Pasteur Institute, diagnosed as chronic myeloid leukemia and monitored with RT-PCR. Our results highlight the interest of molecular tools to diagnose and monitor patients mainly when cytogenetic techniques are irrelevant such as cases with complex chromosomal rearrangements or when patients achieve Philadelphia negativity after treatment.

First-line thalidomide-dexamethasone therapy in preparation for autologous stem cell transplantation in young patients (<61 years) with symptomatic multiple myeloma.

Thalidomide-dexamethasone therapy was given in patients (<61 years) with previously untreated symptomatic multiple myeloma. The aim of this study was to assess the efficacy and toxicity of this combination as first-line therapy, and to determine its effect on stem cell collection and engraftment. During first-line therapy, thalidomide and dexamethasone were administered for 75 days (200 mg/day) and 3 months, respectively. The monthly dose of dexamethasone was 20 mg/m2/day for 4 days, with cycles repeated on days 9 to 12 and 17 to 20 on the first and the third month of therapy. After first-line therapy, a collection of peripheral blood stem cells (PBSC) was performed. Between May 2003 and September 2004, 60 patients were included. On an intent-to-treat basis, the overall response (> or =partial response) rate was 74%, including 24% of patients who obtained a complete remission. Grade 3-4 toxicities consisted of infections (12%), deep-vein thrombosis (3%), constipation (5%), and neuropathy (5%). A total of 58 patients (96%) proceeded to PBSC mobilisation and yielded a median number of 8 x 10(6) CD34+ cells/kg. First-line thalidomide-dexamethasone therapy is effective and relatively well tolerated in young patients with symptomatic multiple myeloma. This combination does not affect PBSC mobilisation.

[Association study between diabetic retinopathy and aldose reductase gene polymorphism in Tunisians]. / Etude de l'association entre la rétinopathie diabétique et un polymorphisme du gène de l'aldose réductase dans la population tunisienne.

INTRODUCTION: Aldose reductase (ALR2), the enzyme of the polyol pathway, may play an important role in the pathogenesis of diabetic microvascular complications, namely diabetic retinopathy. The study aimed to determine whether the aldose reductase gene is involved in diabetic retinopathy in the Tunisian population. MATERIAL: and methods: A case-control study was conducted in 47 type 2 diabetic patients who have diabetic retinopathy and 28 diabetic patients without diabetic retinopathy in spite of diabetes lasting for more than 5 years and over 10 years in 13 cases. We investigated the association between the (CA)n polymorphism located at 2.1 kb upstream of the transcription start site of ALR2 and diabetic retinopathy. The distribution of genotypes and alleles was compared between cases and controls by chi2 test using Epi info software. RESULTS: Genotyping of the two groups did not demonstrate any association between the alleles of this marker and diabetic retinopathy in the Tunisian population studied. DISCUSSION: An association between one of the alleles (Z - 2) of this microsatellite and diabetic retinopathy was identified in Chinese and Japanese patients with type 2 diabetes. Discordant results were obtained for the different populations studied. The lack of an association between diabetic retinopathy and ALR2 alleles indicates that the ALR2 gene is not a genetic marker of predisposition to diabetic retinopathy for type 2 diabetic patients in the Tunisian population studied.

Antibodies to human myelin proteins and gangliosides in patients with acute neuroparalytic accidents induced by brain-derived rabies vaccine.

Antibody responses to myelin antigens were analysed in 15 patients who developed acute neuroparalytic accidents (ANPA) during post-exposure rabies vaccination using a rabies vaccine prepared on brain tissues and in 30 individuals who were uneventfully vaccinated. High titers (> or = 100) of IgG and IgM antibodies to GM1 or GD1a gangliosides were detected by enzyme linked immunosorbent-assay (ELISA) in plasmas from ANPA patients but not in controls. These data suggest that antibodies to GM1 and GD1a gangliosides may play a pathogenic role in the demyelinating and/or inflammatory processes characteristic of rabies vaccine-induced acute neurologic complications.

Association analysis of HLA-class II and class III gene polymorphisms in the susceptibility to mediterranean visceral leishmaniasis.

HLA-DRB1, -DQB1, TNFalpha, TNFbeta, HSP70-2 and HSP70-hom genetic polymorphisms were analyzed in 156 unrelated patients who developed mediterranean visceral leishmaniasis (MVL) due to Leishmania infantum, and 154 unrelated healthy controls, who have got asymptomatic infection with this parasite and were selected on the basis of a positive leishmanin skin test (LST). A significantly reduced frequency of HLA-DR2 was observed among MVL patients (16.1%), compared with controls (26.3%) (relative risk = 0.54; p = 0.04). HLA-DR2/DR13 as well as HLA-DQB1*0201/- genotype frequencies were significantly lower in patients vs controls (relapse rate = 0.17 and 0.46, respectively; p < 0.05). However, using Bonferroni correction, none of these associations remained significant. No association was found, between either the -308 base pair TNFalpha gene polymorphism or the NcoI polymorphism in the first intron of the TNFbeta gene and susceptibility to MVL. Analysis of PstI and NcoI polymorphisms in the coding region of HSP70-2 and HSP70-hom genes, respectively, revealed a significantly higher frequency of homozygotes for the HSP70-2/PstI negative allele, among patients (21.8%) vs controls (12.6%) (relapse rate = 1.94; p = 0.04). Again, this result was not significant after using Bonferroni correction. These results do not support association between susceptibility to MVL and the MHC class II and class III loci analyzed in this study.