In-utero coxsackievirus B4 infection of the mouse thymus.

Type B coxsackievirus (CV-B) infections are involved frequently in the triggering of several autoimmune diseases such as myocarditis, dilated cardiomyopathy, pericarditis, pancreatitis, type 1 diabetes, encephalitis, thyroiditis or Sjögren's syndrome. Serological and virological evidence suggests that maternal infections during pregnancy can play a role in the appearance of these diseases in offspring. The current study aims to explore the effect of an in-utero CV-B infection on the fetal thymus, the central site for programming immunological self-tolerance. In this perspective, female Swiss albino mice were inoculated intraperitoneally or orally with the diabetogenic CV-B4 E2 strain at gestational days 10 or 17. Offspring were killed at different post-inoculation times, and their thymuses were analysed for evidence of infection and alterations in thymic T cell subsets. In-utero CV-B infection of the thymus was demonstrated during the course of vertical transmission, as attested by viral RNA and infectious virus detection in most analysed samples. No histopathological changes were evident. Thymic T cells were not depleted, despite being positive for viral RNA. As evidenced by flow cytometry analysis, CV-B infection of the fetal thymus induced significant changes of thymic T cell populations, particularly with maternal inoculation at gestational day 10. Altogether, these findings suggest that CV-B infection of the fetal thymus may play an important role in the genesis of autoimmune diseases.

Impact and long-term protection of hepatitis B vaccination: 17 years after universal hepatitis B vaccination in Tunisia.

Hepatitis B virus (HBV) vaccination has been part of the Expanded Programme of Immunization (EPI) in Tunisia since 1995. The aim of this study was to evaluate, for the first time, the impact of mass vaccination in Tunisia 17 years after this programme was implemented, and in parallel, assess the long-term persistence of anti-HBs antibody in the vaccinated Tunisian population. A total of 1422 students were recruited (703 vaccinated, 719 non-vaccinated). HBV seromarkers were checked. None of the students from either group had positive HBsAg. The overall prevalence of anti-HBc was 0·8%. A Significantly higher prevalence of anti-HBc was noted in unvaccinated students than in vaccinated (1·4% vs. 0·3%, P = 0·02). The overall seroprotection rate (anti-HBs titre ⩾10 mIU/ml) was 68·9% in vaccinated subjects. Seroprotection rates and geometric mean titres decreased significantly with increasing age, reflecting waning anti-HBs titre over time. No significant difference was detected between seroprotection rates and gender or students' area of origin. Incomplete vaccination was the only factor associated with an anti-HBs titre <10 mIU/ml. This study demonstrates the excellent efficacy of the HBV vaccination programme in Tunisia 17 years after its launch. However, a significant decline of anti-HBs seroprotection has been observed in ⩾15-year-old adolescents which places them at risk of infection. Additional studies are needed in hyperendemic regions in Tunisia.

Hepatitis E virus genotypes 1 and 3 in wastewater samples in Tunisia.

Hepatitis E represents an important public-health concern throughout the world. It is one of the leading causes of hepatitis in North Africa, Asia and the Middle East. In Tunisia, the true burden of HEV infection is still unknown. The objectives of the present study were to assess the occurrence of hepatitis E virus in Tunisia through the monitoring of urban sewage and to characterize the strains identified using molecular assays. A total of 150 sewage samples (raw and treated) were collected from three wastewater treatment plants located in the regions of Monastir and Mahdia and analyzed by nested RT-PCR using a qualitative assay targeting the methyltransferase gene in ORF1. Of these, only three samples (2 %) were found to be positive for HEV, one belonging to genotype 1 and two to genotype 3. The results of the present study indicate a low level of virus excretion among the Tunisian population. Both genotypes 1 and 3 are circulating in this country, however, possibly causing sporadic infections. The presence of the zoonotic genotype 3, known to be transmitted to humans mainly by swine and demonstrated in Tunisia for the first time in this work, raises the question of possible reservoir species, since pork products are not consumed in this country, pigs are not bred, and wild boar is not endemic. Further studies will be needed to gather information on the occurrence and diversity of HEV strains circulating among humans and animals in Tunisia, and on possible animal reservoirs.

Detection and genotyping of group A rotaviruses isolated from sewage samples in Monastir, Tunisia between April 2007 and April 2010.

AIMS: To ascertain the viral load, the distribution of G and P types of group A rotaviruses (RV-A) in sewage samples and to compare strains in clinical, animal and environmental samples. METHODS AND RESULTS: During our study from April 2007 to April 2010, 518 samples of raw and treated sewage were collected from two biological sewage treatment plants (STPs) located in the Monastir region, Tunisia. RV-A was detected by real-time RT-PCR in 375 (72·4%) sewage samples. According to the quantification results of RV-A, it appears that the viral load in raw and treated sewage of the two STPs was quite similar (P = 0·735). The genotyping of RV-A strains detected in sewage samples showed a great diversity with 10 G types and 8 P types. Most of them were described as common in humans, but we also detected genotypes commonly found in animals. All the genotypes detected in two previous studies performed in our laboratory on clinical and bovine samples were also found in environmental samples. However, some genotypes commonly found in animal were only found in sewage samples. CONCLUSION: The comparison of environmental, clinical and animal data suggests that STPs may convey not only human sewage but also animal wastes, both of them contaminated with numerous RV-A strains which are not efficiently eliminated by the sewage treatment process and may spread to surface waters. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates the potential release of human and animal RV-A into water sources, representing a public health risk, by inducing gastroenteritis in population, but also by increasing the risk of zoonotic transmission and formation of reassortant viruses which could get a higher infectious potential. Our findings also suggest that monitoring of sewage may provide an additional tool to determine the epidemiology of RV-A circulating in a given community.

Age- and gender-specific effects on VDR gene polymorphisms and risk of the development of multiple sclerosis in Tunisians: a preliminary study.

The vitamin D receptor (VDR) polymorphisms have been reported to be associated with multiple sclerosis (MS); however, evidence remains conflicting. In this report, we investigated the association between two single nucleotide polymorphisms (SNPs) TaqI and ApaI of VDR gene and risk development of MS. TaqI and ApaI SNPs were detected by PCR-RFLP from the DNA of 60 Tunisian patients with MS and 114 healthy controls. Our results show a significant difference of the allelic frequency distribution between the case and control groups for TaqI SNP (P = 0.01), but genotype frequencies were not significantly different (P = 0.07 and 0.23). When adjusting frequency distribution of different alleles and genotypes by age, we found that the difference between the T allele frequencies of this SNP in the group of patients age [15-24] in comparison with the control group of the same age group was statistically significant (P = 0.026). Moreover, frequency of the T allele was significantly higher in male patients compared with controls of the same sex (P = 0.017). However, neither the genotype nor the allele frequency distribution was significantly different between the MS and control populations for the ApaI SNP. Our preliminary results indicate that VDR gene polymorphism could be associated with susceptibility to MS. The role of VDR gene polymorphism should be further studied in other large populations, and the distribution of other polymorphism, such as FokI and BsmI, should be also analysed to confirm another susceptibility polymorphisms gene for MS and to obtain more adequate strategies for treatment of MS.

Antibacterial and antibiofilm activities of Laurus nobilis L. essential oil against Staphylococcus aureus strains associated with oral infections.

BACKGROUND: Laurus nobilis L. is an aromatic herb with relevant medicinal properties due to its important chemical composition and its potential therapeutic effects. In this study, we investigate the chemical composition, the antibacterial and the antibiofilms activities of Tunisian L. nobilis L. essential oils against clinical Staphylococcus aureus strains. METHODS: The chemical composition of L. nobilis L. essential oils was analysed by Gas Chromatography-Mass Spectrometry (GC-MS). The antibacterial activity of L. nobilis L. essential oils was evaluated in vitro against oral S. aureus (n=21) strains using broth microdilution method. The antibiofilm activity was assessed via Crystal Violet staining and MTT assays. RESULTS: Our results revealed that GC-MS assay exhibited 1.8-Cineole, methyl eugenol and α-terpinyl acetate as the major compounds in the essential oils. Moreover, the essential oil from Sousse exhibited the best bactericidal activity (MICs values ranged from 3.91 to 15.62mgm-1). Furthermore, this oil showed a strong biofilm inhibition effect above 70%, from a low sub-inhibitory concentration (1/16×MIC). MTT assay revealed that both essential oils displayed an excellent antibiofilm activity with eradication percentages ranging from 79.6±2.27 to 95.2±0.56. CONCLUSION: Our finding demonstrated that L. nobilis L. essential oils are able to inhibit oral S. aureus strains with important antibiofilm efficacy. It could have a promising role in the prevention of oral diseases.

Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea.

AIMS: To investigate the incidence, viral load and genetic diversity of bovine rotaviruses strains in Tunisia. METHODS AND RESULTS: A total of 169 faecal specimens, collected from diarrhoeic calves from several farms located in the central eastern regions of Tunisia, between January 2006 and October 2010, were analysed by semi-nested multiplex RT-PCRs for P and G genotypes identification or were genotyped by DNA sequencing. Positive samples were tested by TaqMan real-time RT-PCR to quantify the viral load. Group A bovine rotaviruses were detected in 15·4% (26/169) of the total studied cases of diarrhoea. Overall, G10 was the predominant G type, detected in 12/26 samples (46·2%) and G6 accounted for 42·3% (11/26) while P[11] was the predominant P type, detected in 12/26 samples (46·2%). Two P[5] genotypes (7·7%) were found in the collection. Dual G or P combination and genotype G8 were not found. The most common VP7/VP4 combinations were G6P[11] (30·8%; n = 8) and G10P[11] (11·5%; n = 3). The combination G10P[14] was seen in one sample, and partial typing was assessed in 53·8% (n = 14) of the cases. The viral load determined by real-time RT-PCR showed an average of 1·68 × 10(9) genome copies/g of faeces. CONCLUSION: Knowledge of P and G types could help us understand the relatedness of animal rotaviruses to viruses causing disease in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the viral load and P types of bovine rotaviruses have been determined in Tunisia, and this study contributes to a better understanding of the epidemiology of such viruses circulating in Tunisia. Nevertheless, continuous surveillance is necessary to detect the emergence of new variants.

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T(m) = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples.

Detection and characterization of Shigella species isolated from food and human stool samples in Nabeul, Tunisia, by molecular methods and culture techniques.

AIMS: This study was designed to isolate Shigella spp. strains from food and stool samples by a combination of PCR and culture methods and characterize their serotypes, antibiotic resistance profiles, virulence genes and pulsed-field gel electrophoresis (PFGE) patterns to investigate possible clonal relationships amongst strains circulating. METHODS AND RESULTS: Six Shigella spp. strains were isolated from 280 food samples against 16 Shigella isolates from 236 stool samples of symptomatic patients and asymptomatic food handlers during the period from January 2007 to December 2009 in Public Health Regional Laboratory of Nabeul. The detection of ipaH, ipaBCD, ial, ShET-1 and ShET-2 was performed by a PCR technique with specific primers. CONCLUSIONS: The use of PCR technique improved the rate of detecting Shigella in stool samples from 6·7 to 14% and in food samples from 2·1 to 8·6%. Percentage of Shigella isolates and ipaH-specific PCR demonstrated a marked pattern of seasonality, increasing in summer and fall seasons for human and food isolates. Amongst the environmental strains, 50% of isolates were invasive. However, for the 16 clinical strains isolated, nine were found to be positive for both ial and ipaBCD gene and 11 were found to produce ShET-1 and/or ShET-2. XbaI PFGE analysis revealed the presence of a predominant clone amongst Shigella sonnei strains recovered from different sources circulating in Nabeul, Tunisia, throughout the years 2007-2009. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the existence of Shigella in food samples and dispersion of different virulence genes amongst these isolates, which appear to constitute an environmental source of epidemic spread. The clonal relationships amongst strains isolated from food elements and human stools indicate the incrimination of different kinds of foods as vehicle of transmission of Shigella, which are usually escaped from detection by traditional culture methods.

Persistent infection of human intestinal Caco-2 cell line by Coxsackieviruses B.

Coxsackieviruses B (CV-B, Picornaviridae family, genus Enterovirus) are characterized by their ability to cause cytopathic effects in tissue culture. These viruses are considered highly cytolytic, but can establish persistence/latency in susceptible cells, indicating that a regulatory mechanism may exist to shut off viral protein synthesis and replication under certain situations. The persistence of coxsackieviral B RNA is of great interest because of its implication in the pathogenesis of several chronic human diseases. However, a few studies have dealt with the persistence of these viruses at the intestinal level. The aim of this study is to test the capacity of the six CV-B serotypes to establish persistent infection in human intestinal Caco-2 cell line. Ten CV-B isolates, including CV-B3 prototype strain (Nancy) and a recombinant isolate (B3-B4), were tested. Six CV-B isolates were found to establish persistent infections in Caco-2 cell line. Persistent replication was proved by the detection of viral RNA from cell cultures, VP1 capsid protein detection by immunofluorescence (IF) staining, and the release of infectious particles up to two months and a half after infection without any obvious cytolysis. In addition, our results suggest that the establishment of a persistent infection is serotype-independent.

[Seroprevalence and risk factors of human herpes virus 8 infection in Central-East Tunisia]. / Séroprévalence et facteurs de risque de l'infection par le virus herpès humain 8 dans le Centre-Est tunisien.

OBJECTIVE: Epidemiology of human herpesvirus 8 (HHV8) is still unknown in Tunisia. We aimed to assess the prevalence of HHV8 infection in adults and children from Central-East Tunisia and in patients with high risk of parenteral or sexual infection. METHODS: We enrolled 553 subjects: 116 blood donors, 100 pregnant women, 100 children, 50 subjects with sexually transmitted infections with positive HIV serology and 50 other without HIV infection, 107 multitransfused patients and 30 kidney transplant patients. Antibodies against HHV8 were tested using a sensitive indirect immunofluorescence assay. RESULTS: The seroprevalence of HHV8 was found to be 13.8% in blood donors, 13% in pregnant women and 12% in children. In healthy adult population, no association was found between HHV8 seropositivity and sex, sociodemographic characteristics, parenteral risk factors or serological markers of hepatitis B. Rates of HHV8 infection were significantly higher in patients having high-risk sexual behavior with or without HIV infection (P<10(-4)), in polytransfused patients (P<10(-4)) and in patients with kidney transplantation (P=0.001). CONCLUSION: Our findings suggest that HHV8 infection is widespread in Central-East Tunisia such as in the Mediterranean area. HHV8 infection appears to be acquired early in life, probably through saliva. HHV8 transmission by blood transfusion, subject of controversy in literature, is well established in our study. Early screening of this infection should be considered in populations with high risk of Kaposi's sarcoma in our areas.

[Kikuchi-Fujimoto's disease: Report of 4 cases and literature review]. / Maladie de Kikuchi-Fujimoto : à propos de quatre cas et revue de littérature.

INTRODUCTION: Kikuchi-Fujimoto's disease or histiocytic necrotizing lymphadenitis is a benign disease predominantly occurring in young women which etiology remains unknown and revealed by cervical lymphadenitis and/or prolonged fever. OBSERVATIONS: This report describes a survey of four patients who developed Kikuchi's lymphadenitis occurring concomitantly with LES in one case and actinomycosis in another case. The definite diagnosis is usually made through histopathological examination of a lymph node biopsy. The evolution was favorable under corticosteroid therapy in two patients, antibiotics in the third and only antipyretic in the fourth. CONCLUSION: The authors bring report through these three observations, the diagnostic difficulties, the therapeutic means of the disease of Kikuchi, as well as its evolutionary aspects.

Anti-inflammatory and analgesic activities of Tunisian Citrullus colocynthis Schrad. immature fruit and seed organic extracts.

BACKGROUND AND OBJECTIVES: Inflammations and immune-related diseases including rheumatoid arthritis are widespread in the entire globe. The treatment of these illnesses is mainly based on the use of synthetic and biotechnological drugs, in recent years. Tunisian traditional medicine is a potential source of new remedies namely Citrullus colocynthis Schrad. (Cucurbitaceae): endemic in southern Tunisia and used in folk medicine to treat many inflammation disorders. Our goal was to assess the in vivo analgesic and anti-inflammatory activities of Tunisian Citrullus colocynthis immature fruit and seed organic extracts (petroleum ether, chloroform, ethyl acetate, acetone and finely methanol extract). MATERIAL AND METHODS: Yields of prepared organic extracts are gravimetrically determined. For the analgesic and anti-inflammatory activities, we have used respectively, the acetic acid writhing test in mice and the carrageenan-induced paw edema assay in rats. RESULTS AND DISCUSSION: All extracts displayed an important analgesic and anti-inflammatory activities at different doses without inducing any side effects. This study has demonstrated the analgesic and anti-inflammatory activities of Citrullus colocynthis immature fruit and seed extracts. Experiment results provide scientific insight into the ancient practice of utilizing Citrullus colocynthis Schrad. as analgesic and as anti-inflammatory agents.

First molecular detection of Aichi virus in sewage and shellfish samples in the Monastir region of Tunisia.

The aims of our investigations were (1) to look for Aichi virus in environmental samples and (2) to compare the Aichi virus strains in both clinical and environmental samples in order to evaluate the role of environmental contamination as a possible vehicle for viral transmission. Aichi virus was detected in 15 (6%) sewage samples and in 4 (6.6%) shellfish samples. Aichi virus was identified for the first time in water samples. Phylogenetic analysis revealed several clusters that occurred sequentially in time, suggesting some parallelism in the evolution of environmental and human strains. Aichi virus present in sewage reflects the viruses circulating in the community.

Detection and molecular characterization of enteric viruses in environmental samples in Monastir, Tunisia between January 2003 and April 2007.

AIMS: A prospective study was performed to characterize the main human enteric viruses able to persist in sewage samples and in shellfish tissues, and to establish the correlation between environmental strains and viral infantile diarrhoea observed in the same area during the same period. METHODS AND RESULTS: A total of 250 sewage (raw and treated) and 60 shellfish samples were collected between January 2003 and April 2007 in Monastir region, Tunisia. Group A rotavirus (RVA) was detected in 80 (32%) sewage samples, norovirus (NoV) in 11 (4·4%) and enteric adenovirus (AdV) in 1 (0·4%). Among 60 shellfish samples collected near sewage effluents, one was contaminated by NoV (1·6%). CONCLUSION: Our data represent the first documentation in Tunisia, combining gastroenteritis viruses circulating in the environment and in clinical isolates. We observed a correlation between environmental strains and those found in children suffering from gastroenteritis during the same period study. This suggests the existence of a relationship between water contamination and paediatric diarrhoea. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results address the potential health risks associated with transmission of human enteric viruses through water-related environmental routes. The research findings will aid in elucidating the molecular epidemiology and circulation of enteric viruses in Tunisia and in Africa, where data are rare.

Coxsackievirus B4 and type 1 diabetes pathogenesis: contribution of animal models.

The role of enteroviruses, in particular type B coxsackieviruses (CV-B), in type 1 diabetes (T1D) pathogenesis is supported by epidemiological, clinical and experimental observations.The investigation of T1D pathogenesis benefits from the contribution of animal models called spontaneously diabetic. Among these animals the non-obese diabetic (NOD) mouse and the bio-breeding diabetes-prone (BBDP) rat present a genetic susceptibility manifested by the expression of an autoimmune diabetes similar to the pathology observed in human beings. Other models whose genetic predisposition is less known are of considerable contribution as well. Numerous major observations relative to several aspects of T1D pathogenesis in the context of CV-B infections, such as susceptibility, diabetogenicity, pancreatotropism, mechanisms of beta cells destruction and others, have been deduced thanks to investigations with animal models. Despite their limits, these models are necessary in improving our knowledge of the role of enteroviruses, like CV-B4, in the pathogenesis of T1D, and the recent advances ensuing from their contribution may have important therapeutic and preventive spin-offs.

Detection of the human Parvovirus B19 in nonimmune hydrops fetalis using immunohistochemistry and nested-PCR in formalin-fixed and paraffin-embedded placenta and fetal tissues.

The aim of the present study was to determine whether there is an association between Parvovirus B19 infection and hydrops fetalis setting in fetus and neonate. Twenty-nine samples were analyzed by three methods. Each sample was histologically examined for viral nuclear inclusions in fetal organs and placenta, then immunohistochemical study using Parvovirus B19 antibody that recognized the VP2 protein of the Parvovirus B19 capsid was done in tissue embedded in paraffin (lungs, liver, thymus, kidneys, heart and placenta). Nested-PCR analysis was done after DNA extraction from paraffin blocks and using specific primers of the Parvovirus B19 VP1 gene. Apparent causes of hydrops were eliminated such as metabolic diseases, cardiac failure or malformation. The standard histological study objects viral inclusion in one case (lung tissue). However, the immunohistochemical study was negative in all cases. Nested-PCR demonstrates the presence of the viral DNA in five cases. Our study demonstrates that the implication of Parvovirus B19 in hydrops fetalis must be affirmed by the use of more than one method. Nested-PCR is the most sensitive method in our study and can be easily used for the detection of Parvovirus B19 in formalin-fixed paraffin-embedded tissues.

Immunophenotypic analysis of bone marrow B lymphocyte precursors (hématogones) by flow cytometry.

The aims of this flow cytometry study were to quantify B lymphoid precursors known as hématogones across age and clinical conditions and to study the immunophenotypic profile of these benign immature B cells. A total of 406 consecutive marrow specimens were analyzed for hématogones using 4-color flow cytometry during a 19 month period (60% males and 40% females). The age range was 3 months to 89 years. Hématogones were present in 80% of the specimens. Morphologic analysis of the smears from each patient showed small numbers of hématogones (<13% of total cellularity). The B cell population was defined by CD19 + CD45 bright positivity, coexpression of other B lineage markers: CD20, CD22, CD10, CD29, CD38 and CD58 in addition to HLA-DR and CD34. In our study we found a significant decline in hématogones with increasing age but a broad range was found at all ages. Marrow from some adults contained relatively high numbers. Diagnosis in these patients included cytopenias, infections, and neoplastic diseases. Distinction of hématogones is critical for disease management particularly after therapy of paediatric B acute lymphoblastic leukaemia to monitor for minimal residual disease.

[Idiosyncratic drug-induced thrombocytopenia]. / Les thrombopénies médicamenteuses idiosyncrasiques.

Idiosyncratic drug-induced thrombocytopenia is a relatively rare and potentially serious side-effect in its moderate and profound forms (platelet count less than 100 x 10(9)L). It may be of central or peripheral mechanism, with consumption or immunological destruction. The main incriminated molecules are heparins, quinidine, sulfonamides and gold salts. Hemorrhagic manifestations are the rule and their severity is related to the level of thrombocytopenia and the involved molecule. Heparin-induced thrombocytopenia is more often associated with thrombotic events. Diagnosis relies on medical history and clinical criteria, which also establish the level of imputability. The role of serological tests is not yet clear but seems to be particularly interesting in difficult diagnostic situations and in heparin-induced thrombocytopenia. The treatment is based on the discontinuation of the incriminated drug and on supportive measures that depend on the severity of the clinical features.