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1.  Detection and Molecular Characterization of Hepatitis A Virus from Tunisian Wastewater Treatment Plants with Different Secondary Treatments 
Applied and Environmental Microbiology  2016;82(13):3834-3845.
Hepatitis A virus (HAV) is the main causative agent of hepatitis infection associated with waterborne outbreaks worldwide. In Tunisia, there is no specific surveillance system for HAV and current secondary wastewater treatment processes are unable to remove viral particles, which present a potential public health problem. Qualitative and quantitative analysis of HAV in 271 raw and treated wastewater samples from five sewage treatment plants (STPs) during 13 months was performed. Moreover, the efficiency of three secondary wastewater treatment processes (conventional activated sludge, extended aeration, and oxidation ditch activated sludge) was evaluated. Data obtained demonstrated that HAV is endemic in Tunisia and circulates with high prevalence in both raw (66.9%) and treated (40.7%) wastewater. HAV circulates throughout the year in the coastal areas, with the highest rates found during summer and autumn, whereas in central Tunisia, high levels were shown in autumn and winter. Total virus removal was not achieved, since no difference in mean HAV loads was observed in effluents (6.0 × 103 genome copies [GC]/ml) and influents (2.7 × 103 GC/ml). The comparison of the HAV removal values of the three different wastewater treatment methods indicates that extended aeration and oxidation ditch activated sludge had better efficiency in removing viruses than conventional activated sludge did. Molecular characterization revealed that the vast majority of HAV strains belonged to subgenotype IA, with the cocirculation of subgenotype IB in wastewater treatment plants that collect tourism wastewater.
IMPORTANCE This report provides important data on the incidence, behavior, seasonality, and genotype distribution of HAV in the environment in Tunisia, as well as the risk of infection derived from its occurrence in effluents due to inadequate wastewater treatment. In addition, these findings seem to confirm that the prevalence of HAV depends on socioeconomic level, sanitary conditions in the communities, sewage facilities, the locality, and the climate. The wide dispersion of HAV in effluents proves the inefficacity of the current wastewater treatment processes used in Tunisia to remove virus; therefore, establishment of tertiary treatment processes or replacement of the medium-charge activated sludge (conventional activated sludge) by the low-charge version (oxidation ditch activated sludge) is absolutely needed. Rapid detection of the HAV genome in wastewater may provide a timely warning sign to health authorities to implement population protection measures.
PMCID: PMC4907194  PMID: 27107113
2.  Cosavirus, Salivirus and Bufavirus in Diarrheal Tunisian Infants 
PLoS ONE  2016;11(9):e0162255.
Three newly discovered viruses have been recently described in diarrheal patients: Cosavirus (CosV) and Salivirus (SalV), two picornaviruses, and Bufavirus (BuV), a parvovirus. The detection rate and the role of these viruses remain to be established in acute gastroenteritis (AGE) in diarrheal Tunisian infants. From October 2010 through March 2012, stool samples were collected from 203 children <5 years-old suffering from AGE and attending the Children’s Hospital in Monastir, Tunisia. All samples were screened for CosV, SalV and BuV as well as for norovirus (NoV) and group A rotavirus (RVA) by molecular biology. Positive samples for the three screened viruses were also tested for astrovirus, sapovirus, adenovirus, and Aichi virus, then genotyped when technically feasible. During the study period, 11 (5.4%) samples were positive for one of the three investigated viruses: 2 (1.0%) CosV-A10, 7 (3.5%) SalV-A1 and 2 (1.0%) BuV-1, whereas 71 (35.0%) children were infected with NoV and 50 (24.6%) with RVA. No mixed infections involving the three viruses were found, but multiple infections with up to 4 classic enteric viruses were found in all cases. Although these viruses are suspected to be responsible for AGE in children, our data showed that this association was uncertain since all infected children also presented infections with several enteric viruses, suggesting here potential water-borne transmission. Therefore, further studies with large cohorts of healthy and diarrheal children will be needed to evaluate their clinical role in AGE.
PMCID: PMC5025138  PMID: 27631733
3.  Clinical surveillance for human astrovirus in Monastir region, Tunisia 
BMC Public Health  2016;16:57.
Astroviruses (AstVs) are enteric viruses that can cause gastroenteritis in children. This study is part of monitoring the circulation of astroviruses in children hospitalized and/or outpatients for acute gastroenteritis at the primary care center of Ouerdanine or at the Pediatric Department of the University Hospital Fattouma-Bourguiba (Monastir, Tunisia). The aims of our study were to know the prevalence of human astrovirus in clinical samples of children, characterize the strains and evaluate the infectivity of isolated strains on cell culture.
Fifty stool samples were collected from children under five years old in the region of Monastir (Tunisia) from October 2010 to June 2011. All specimens were subjected to RT-PCR amplification followed by sequencing and phylogenetic analysis.
The study shows a low prevalence of astrovirus (4 %) in children. The two positive samples obtained were HAstV type 3. Samples that were RT-PCR positive were cultured in CaCO-2 cells and the presence of infectious viral particles was confirmed. The phylogenetic analysis shows that the different HAstV-3 strains isolated in Tunisia are grouped into two clusters. The first cluster includes strains obtained in 2004, which belong to lineage HAstV-3a, while strains isolated in 2010 belong to lineage HAstV-3c.
This study is part of monitoring the circulation of astroviruses in children younger than five years old from Monastir region, Tunisia. The results show low prevalence (4 %). All genotyped samples belonged to lineage HAstV-3c, which could be presently emerging. Two different lineages have been isolated in Tunisia: HAstV-3a in 2004 and HAstV-3c in 2010.
PMCID: PMC4722761  PMID: 26796330
Human astrovirus; Clinical surveillance; RT-PCR; Cell culture; Tunisia
4.  Enterovirus Migration Patterns between France and Tunisia 
PLoS ONE  2015;10(12):e0145674.
The enterovirus (EV) types echovirus (E-) 5, E-9, and E-18, and coxsackievirus (CV-) A9 are infrequently reported in human diseases and their epidemiologic features are poorly defined. Virus transmission patterns between countries have been estimated with phylogenetic data derived from the 1D/VP1 and 3CD gene sequences of a sample of 74 strains obtained in France (2000–2012) and Tunisia (2011–2013) and from the publicly available sequences. The EV types (E-5, E-9, and E-18) exhibited a lower worldwide genetic diversity (respective number of genogroups: 4, 5, and 3) in comparison to CV-A9 (n = 10). The phylogenetic trees estimated with both 1D/VP1 and 3CD sequence data showed variations in the number of co-circulating lineages over the last 20 years among the four EV types. Despite the low number of genogroups in E-18, the virus exhibited the highest number of recombinant 3CD lineages (n = 10) versus 4 (E-5) to 8 (E-9). The phylogenies provided evidence of multiple transportation events between France and Tunisia involving E-5, E-9, E-18, and CV-A9 strains. Virus spread events between France and 17 other countries in five continents had high probabilities of occurrence as those between Tunisia and two European countries other than France. All transportation events were supported by BF values > 10. Inferring the source of virus transmission from phylogenetic data may provide insights into the patterns of sporadic and epidemic diseases caused by EVs.
PMCID: PMC4692522  PMID: 26709514
5.  Rotavirus P[8] Infections in Persons with Secretor and Nonsecretor Phenotypes, Tunisia 
Emerging Infectious Diseases  2015;21(11):2055-2058.
To determine whether rotavirus infections are linked to secretor status, we studied samples from children in Tunisia with gastroenteritis. We phenotyped saliva for human blood group antigens and tested feces for rotavirus. Rotavirus was detected in 32/114 patients. Secretor genotyping showed that P[8] rotavirus infected secretors and nonsecretors, and infection correlated with presence of Lewis antigen.
PMCID: PMC4622234  PMID: 26488868
rotavirus; rotavirus P[8]; human blood group antigens; HBGA; nonsecretor; secretor; gastroenteritis; children; viruses; Tunisia; Lewis antigen
6.  An outbreak of West Nile Virus infection in the region of Monastir, Tunisia, 2003 
Pathogens and Global Health  2014;108(3):148-157.
A West Nile (WN) fever epidemic occurred in the region of Monastir, Tunisia, between August and October 2003.
Aim of the study
We attempt to describe the epidemiology, clinical presentation, and outcome of patients with confirmed West Nile virus (WNV) infection.
Three groups of specimens were prepared. One was made up of serum only (n  =  43), the other of cerebrospinal fluid (CSF) only (n  =  30), and the third group was made up of both (n  =  40). These specimens were obtained from 113 patients. A serological diagnosis and evidence of WNV genome by nested reverse-transcriptase polymerase chain reaction (nRT-PCR) and TaqMan reverse transcription-polymerase chain reaction (RT-PCR) were carried out.
Thirty-eight cases (33.6%) were serologically positive. Results of nRT-PCR showed a total of 10 positive cases of WNV (8.8%) detected in group 1 (n  =  1/43), group 2 (n  =  5/30), and group 3 (n  =  4/40) whereas the PCR TaqMan showed 18 positive samples (15.9%) found in group 1 (n  =  3/43), group 2 (n  =  9/30), and group 3 (n  =  6/40). All TaqMan PCR positive cases were nRT-PCR positive. In addition, four serologically probable cases were confirmed by TaqMan PCR. The attempts to isolate WNV by cell culture were unsuccessful. Considering the results of TaqMan assay and the serological diagnosis, WNV infection was confirmed in a total of 42 patients. The main clinical presentations were meningoencephalitis (40%), febrile disease (95%), and meningitis (36%). Eight patients (19%) died. The highest case-fatality rates occurred among patients aged ≧55 years. The phylogenetic analysis revealed that isolates of WNV were closely related to the Tunisian strain 1997 (PAH001) and the Israeli one (Is-98).
West Nile virus is a reemerging global pathogen that remains an important public health challenge in the next decade.
PMCID: PMC4083177  PMID: 24766339
West Nile virus; Outbreak; TaqMan RT-PCR; Epidemiology; Meningoencephalitis
7.  Migration pattern of hepatitis A virus genotype IA in North-Central Tunisia 
Virology Journal  2015;12:17.
Hepatitis A virus (HAV) epidemiology in Tunisia has changed from high to intermediate endemicity in the last decades. However, several outbreaks continue to occur. The last reported sequences from Tunisian HAV strains date back to 2006. In order to provide an updated overview of the strains currently circulating in Tunisia, a large-scale molecular analysis of samples from hepatitis A cases was performed, the first in Tunisia.
Biological samples were collected from patients with laboratory confirmed hepatitis A: 145 sera samples in Tunis, Monastir, Sousse and Kairouan from 2008 to 2013 and 45 stool samples in Mahdia in 2009. HAV isolates were characterised by nested RT-PCR (VP1/2A region) and sequencing. The sequences finally obtained from 81 samples showed 78 genotype IA and 3 genotype IB isolates.
A Tunisian genotype IA sequence dataset, including both the 78 newly obtained IA sequences and 51 sequences retrieved from GenBank, was used for phylogenetic investigation, including analysis of migration pattern among six towns. Virus gene flow from Sfax and Monastir was directed to all other towns; in contrast, the gene flows from Sousse, Tunis, Mahdia and Kairouan were directed to three, two, one and no towns, respectively.
Several different HAV strains co-circulate in Tunisia, but the predominant genotype still continues to be IA (78/81, 96% isolates). A complex gene flow (migration) of HAV genotype IA was observed, with Sfax and Monastir showing gene flows to all other investigated towns. This approach coupled to a wider sampling can prove useful to investigate the factors underlying the spread of HAV in Tunisia and, thus, to implement appropriate preventing measures.
PMCID: PMC4327963  PMID: 25886374
HAV; Sequencing; Phylogenetic analysis; Viral gene flow
8.  New Parvovirus in Child with Unexplained Diarrhea, Tunisia 
Emerging Infectious Diseases  2014;20(11):1911-1913.
A divergent parvovirus genome was the only eukaryotic viral sequence detected in feces of a Tunisian child with unexplained diarrhea. Tusavirus 1 shared 44% and 39% identity with the nonstructural protein 1 and viral protein 1, respectively, of the closest genome, Kilham rat parvovirus, indicating presence of a new human viral species in the Protoparvovirus genus.
PMCID: PMC4214302  PMID: 25340816
parvovirus; diarrhea; feces; child; viruses; Tunisia; Tusavirus
9.  Divergent gyroviruses in the feces of Tunisian children 
Virology  2013;446(0):346-348.
The Gyrovirus genus consists of the immunosuppressive Chicken Anemia Virus (CAV) prototype and since 2011 three other viral species found in sera/tissues of chickens, human feces, and on human skin. Here the genomes of two other gyrovirus species were characterized in diarrhea samples from Tunisian children whose main ORFs shared amino acid identity of 46–59% with those of the previously characterized gyroviruses and were provisionally named GyV5 and GyV6. All known gyroviruses grouped into two clades with distinct genomic features including replacement of the VP2 overlapping Apoptin gene with a distinct ORF of unknown function. Previous reports of gyrovirus DNA in human blood and on human skins warrant studies of possible human tropisms for these newly characterized gyroviruses.
PMCID: PMC3804065  PMID: 24074598
Gyrovirus; diarrhea; feces; children
10.  Coxsackievirus B detection in cases of myocarditis, myopericarditis, pericarditis and dilated cardiomyopathy in hospitalized patients 
Molecular Medicine Reports  2014;10(6):2811-2818.
Coxsackieviruses B (CV-B) are known as the most common viral cause of human heart infections. The aim of the present study was to assess the potential role of CV-B in the etiology of infectious heart disease in hospitalized patients. The present study is based on blood, pericardial fluid and heart biopsies from 102 patients and 100 control subjects. All of the samples were examined for the detection of specific enteroviral genome using the reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis. Immunohistochemical investigations for the detection of the enteroviral capsid protein, VP1, from the biopsies were performed. The samples were cultured on confluent KB monolayer cell line for possible virus isolation. The epidemiological data were also collected. CV-B was detected in 28 of the 102 patients. The sequence analysis demonstrated that 27 strains were identical to CV-B3 and only one strain was identical to CV-B1. Furthermore, VP1 in the heart biopsies was detected in enterovirus-positive cases, as revealed by RT-PCR. Pericarditis infection was more frequent than myocarditis (P<0.05) or myopericarditis (P=0.05). The epidemiological data demonstrate that CV-B heart infections occur mainly during autumn and winter, and young male adults are more susceptible than adolescents or adults (P<0.5). The present findings demonstrate a higher prevalence of viral heart infections, suggesting that CV-B may significantly contribute to heart infections.
PMCID: PMC4227425  PMID: 25241846
coxsackievirus B; human heart infections; molecular diagnosis; immunohistochemical investigations; epidemiology
11.  Type II and type IV topoisomerase mutations in clinical isolates of Morganella morganii harbouring the qnrD gene 
The aim of this study was to show the emergence of the qnrD gene among fluoroquinolone-resistant Morganella morganii isolate. The occurrence of mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC,parE) genes was also investigated in this strain.
95 clinical Enterobacteria were screened for harbouring the qnrD gene. The clinical isolate of M. morganii was recovered from urine from a patient hospitalized in the urology unit at Fattouma Bourguiba Hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method. Quinolone susceptibility was studied with microbroth dilution technique. The investigations of plasmid mediated quinolone resistance (PMQR) and topoisomerases mutations were performed by polymerase chain reaction and nucleotide sequencing.
This isolate showed high level of resistance to quinolones. The MIC with microbroth dilution technique was 512 μg/ml for norfloxacin, 256 μg/ml for ofloxacin and ciprofloxacin and 64μg/ml for levofloxacin.
This strain was found to harbour the quinolone resistance determinant qnrD. In addition, this strain harboured two new gyrB mutations (S463A, S464Y) and one parC mutation (S80I).
This is the first report in Tunisia of qnrD determinant and tow new gyrB muations in M. morganii. The nosocomial infection due to this proteeae invites further study of its epidemiologic evolution.
PMCID: PMC4236555  PMID: 25106550
Morganella morganii; Quinolone resistance; Topoisomerase mutation; qnrD
12.  Study of Coxsackie B viruses interactions with Coxsackie Adenovirus receptor and Decay-Accelerating Factor using Human CaCo-2 cell line 
Decay Accelerating Factor (DAF) and Coxsackievirus-Adenovirus Receptor (CAR) have been identified as cellular receptors for Coxsackie B viruses (CV-B). The aim of this study is to elucidate the different binding properties of CV-B serotypes and to find out if there are any amino acid changes that could be associated to the different phenotypes.
Twenty clinical CV-B isolates were tested on CaCo-2 cell line using anti-DAF (BRIC216) and anti-CAR (RmcB) antibodies. CV-B3 Nancy prototype strain and a recombinant strain (Rec, CV-B3/B4) were tested in parallel. The P1 genomic region of 12 CV-B isolates from different serotypes was sequenced and the Trans-Epithelial Electrical Resistance (TEER) along with the virus growth cycle was measured.
Infectivity assays revealed clear differences between CV-B isolates with regard to their interactions with DAF and CAR. All tested CV-B isolates showed an absolute requirement for CAR but varied in their binding to DAF. We also reported that for some isolates of CV-B, DAF attachment was not adapted. Genetic analysis of the P1 region detected multiple differences in the deduced amino acid sequences.
Within a given serotype, variations exist in the capacity of virus isolates to bind to specific receptors, and variants with different additional ligands may arise during infection in humans as well as in tissue culture.
PMCID: PMC4035751  PMID: 24885774
CV- B; CaCo-2 cell line; Receptors; Phenotypes; Variants; TEER
13.  Enteroviral Central Nervous System Infections in Children of the Region of Monastir, Tunisia: Diagnosis, Laboratory Findings of Cerebrospinal Fluid and Clinical Manifestations 
Human enteroviruses (HEV) are one of the major causes of central nervous system (CNS) infections in pediatrics. A prospective study was conducted to assess the epidemiological, clinical, and laboratory characteristics of enterovirus (EV) infections of the CNS in children under 15-years-old, suspected of having viral CNS infections and admitted to the Pediatric Department of Monastir University Hospital, Tunisia. Enteroviral RNA was detected by 5′ NCR nested RT-PCR assay in 33 % (20 out of 60) of cerebrospinal fluid specimens, whereas only six samples (10 %) were EV positive in cell culture. EV-positive patients were clustered according to their clinical manifestations, predominantly diagnosed as aseptic meningitis (65 %) and meningoencephalitis (20 %). Fever, headache, vomiting, and neck stiffness were the most pronounced symptoms. Pleocytosis with the predominance of lymphocytes was observed in 60 % of EV positive specimens. Although patients suffering from EV infections were encountered throughout the year, most occurred during spring and summer months. Using VP1-2A nested RT-PCR and sequence analysis, three of the 20 positive HEV were identified as Echovirus (E)-9. This is the first report of a cluster of aseptic meningitis cases caused by E-9 in Monastir.
PMCID: PMC3550789  PMID: 24293816
Enterovirus; Cerebrospinal fluid; Children; Molecular typing; Epidemiology
14.  Seroprevalence and Molecular Characterisation of Human Hepatitis A virus in Serum Samples of Tunisian Patients with Clinical Symptoms of Viral Hepatitis 
The aim of the present study was to investigate the seroprevalence of Hepatitis A virus antibodies in patients with clinical symptoms of viral hepatitis and molecular characterization of the detected isolates. The present study deals with the seroprevalence and the genetic diversity of HAV in 400 Tunisian patients presenting in dispensaries (160 patients) and in University Hospitals (240 patients) with hepatitis symptoms between 2006 and 2008. The patients with acute hepatitis were mainly from rural regions. However, the total number of patients was decreased over time. The collected samples were from patients with hepatitis symptoms occurring mainly during January–March (36.7, 26, and 35.5%) and September–December (39.4, 43.4, and 35.5%) during the three years of study, respectively. However, HAV infection was established for only 110 among 400 patients. The detected isolates were clustered within sub-genotype IA. The present study constituted another report of the continued surveillance of HAV infection in the region of Monastir and the molecular characterisation of the detected strains.
PMCID: PMC3550819  PMID: 23729999
Hepatitis A virus; Anti-HAV IgM; RT-PCR; Tunisia
15.  Persistent Infection of Thymic Epithelial Cells with Coxsackievirus B4 Results in Decreased Expression of Type 2 Insulin-Like Growth Factor 
Journal of Virology  2012;86(20):11151-11162.
It has been hypothesized that a disturbance of central self-tolerance to islet β cells may play a role in the enteroviral pathogenesis of type 1 diabetes. Whether enteroviruses can induce an impaired expression of β-cell self-antigens in thymic epithelial cells has been investigated in a murine thymic epithelial (MTE) cell line. This cell line was permissive to the diabetogenic group B4 coxsackievirus (CV-B4) strain CV-B4 E2 and spontaneously expressed type 2 insulin-like growth factor (Igf2), the dominant self-antigen of the insulin family. In this model, a persistent replication of CV-B4 E2 was obtained, as attested to by the prolonged detection of intracellular positive- and negative-strand viral RNA by reverse transcription-PCR (RT-PCR) and capsid protein VP1 by immunofluorescent staining and by the release of infectious particles in culture supernatants. The chronic stage of the infection was characterized by a low proportion of VP1-positive cells (1 to 2%), whereas many cells harbored enteroviral RNA, as displayed by RT-PCR without extraction applied directly to a few cells. Igf2 mRNA and IGF-2 protein were dramatically decreased in CV-B4 E2-infected MTE cell cultures compared with mock-infected cultures, whereas housekeeping and interleukin-6 (Il6) gene expression was maintained and Igf1 mRNA was decreased, but to a lower extent. Inoculation of CV-B3, CV-B4 JVB, or echovirus 1 resulted in a low level of IGF-2 in culture supernatants as well, whereas herpes simplex virus 1 stimulated the production of the protein. Thus, a persistent infection of a thymic epithelial cell line with enteroviruses like CV-B4 E2 can result in a disturbed production of IGF-2, a protein involved in central self-tolerance toward islet β cells.
PMCID: PMC3457166  PMID: 22855493
16.  Ribosomal Initiation Complex Assembly within the Wild-Strain of Coxsackievirus B3 and Live-Attenuated Sabin3-like IRESes during the Initiation of Translation 
Coxsackievirus B3 (CVB3) is an enterovirus of the family of Picornaviridae. The Group B coxsackieviruses include six serotypes (B1 to B6) that cause a variety of human diseases, including myocarditis, meningitis, and diabetes. Among the group B, the B3 strain is mostly studied for its cardiovirulence and its ability to cause acute and persistent infections. Translation initiation of CVB3 RNA has been shown to be mediated by a highly ordered structure of the 5′-untranslated region (5′UTR), which harbors an internal ribosome entry site (IRES). Translation initiation is a complex process in which initiator tRNA, 40S and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of the mRNA. We have previously addressed the question of whether the attenuating mutations of domain V of the poliovirus IRES were specific for a given genomic context or whether they could be transposed and extrapolated to a genomic related virus, i.e., CVB3 wild-type strain. In this context, we have described that Sabin3-like mutation (U473→C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we analyzed the efficiency of formation of ribosomal initiation complexes 48S and 80S through 10%–30% and 10%–50% sucrose gradients using rabbit reticulocyte lysates (RRLs) and stage-specific translation inhibitors: 5′-Guanylyl-imidodiphosphate (GMP-PNP) and Cycloheximide (CHX), respectively. We demonstrated that the interaction of 48S and 80S ribosomal complexes within the mutant CVB3 RNA was abolished compared with the wild-type RNA by ribosome assembly analysis. Taken together, it is possible that the mutant RNA was unable to interact with some trans-acting factors critical for enhanced IRES function.
PMCID: PMC3634407  PMID: 23439549
coxsackievirus B3; 5′UTR; IRES; translation initiation; ribosomal complex assembly
17.  Evaluation of the cytotoxic effect and antibacterial, antifungal, and antiviral activities of Hypericum triquetrifolium Turra essential oils from Tunisia 
A number of bio-active secondary metabolites have been identified and reported for several Hypericum species. Many studies have reported the potential use of the plant extracts against several pathogens. However, Hypericum triquetrifolium is one of the least studied species for its antimicrobial activity. The aim of the present study was to evaluate the cytotoxic effect of the essential oils of Hypericum triquetrifolium as well as their antimicrobial potential against coxsakievirus B3 and a range of bacterial and fungal strains.
The essential oils of Hypericum triquetrifolium harvested from five different Tunisian localities (Fondouk DJedid, Bou Arada, Bahra, Fernana and Dhrea Ben Jouder) were evaluated for their antimicrobial activities by micro-broth dilution methods against bacterial and fungal strains. In addition, the cytotoxic effect and the antiviral activity of these oils were carried out using Vero cell lines and coxsakievirus B3.
The results showed a good antibacterial activities against a wide range of bacterial strains, MIC values ranging between 0.39-12.50 mg/ml and MBC values between 1.56-25.0 mg/ml. In addition, the essential oils showed promising antifungal activity with MIC values ranging between 0.39 μg/mL and 12.50 μg/mL; MFC values ranged between 3.12 μg/mL and 25.00 μg/mL; a significant anticandidal activity was noted (MIC values comprised between 0.39 μg/mL and 12.50 μg/mL). Although their low cytotoxic effect (CC50 ranged between 0.58 mg/mL and 12.00 mg/mL), the essential oils did not show antiviral activity against coxsakievirus B3.
The essential oils obtained from Hypericum triquetrifolium can be used as antimicrobial agents and could be safe at non cytotoxic doses. As shown for the tested essential oils, comparative analysis need to be undertaken to better characterize also the antimicrobial activities of Hypericum triquetrifolium extracts with different solvents as well as their purified fractions and their pure secondary metabolites.
PMCID: PMC3637322  PMID: 23360506
Hypericum triquetrifolium; Coxsakievirus B3; Essential oils; Bacteria; Fungi
18.  Sudden unexpected death related to enterovirus myocarditis: histopathology, immunohistochemistry and molecular pathology diagnosis at post-mortem 
BMC Infectious Diseases  2012;12:212.
Viral myocarditis is a major cause of sudden unexpected death in children and young adults. Until recently, coxsackievirus B3 (CVB3) has been the most commonly implicated virus in myocarditis. At present, no standard diagnosis is generally accepted due to the insensitivity of traditional diagnostic tests. This has prompted health professionals to seek new diagnostic approaches, which resulted in the emergence of new molecular pathological tests and a more detailed immunohistochemical and histopathological analysis. When supplemented with immunohistochemistry and molecular pathology, conventional histopathology may provide important clues regarding myocarditis underlying etiology.
This study is based on post-mortem samples from sudden unexpected death victims and controls who were investigated prospectively. Immunohistochemical investigations for the detection of the enteroviral capsid protein VP1 and the characterization and quantification of myocardial inflammatory reactions as well as molecular pathological methods for enteroviral genome detection were performed.
Overall, 48 sudden unexpected death victims were enrolled. As for controls, 37 cases of unnatural traffic accident victims were studied. Enterovirus was detected in 6 sudden unexpected death cases (12.5 %). The control samples were completely enterovirus negative. Furthermore, the enteroviral capsid protein VP1 in the myocardium was detected in enterovirus-positive cases revealed by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Unlike control samples, immunohistochemical investigations showed a significant presence of T and B lymphocytes in sudden unexpected death victims.
Our findings demonstrate clearly a higher prevalence of viral myocarditis in cases of sudden unexpected death compared to control subjects, suggesting that coxsackie B enterovirus may contribute to myocarditis pathogenesis significantly.
PMCID: PMC3462138  PMID: 22966951
19.  Chemical composition of 8 eucalyptus species' essential oils and the evaluation of their antibacterial, antifungal and antiviral activities 
In 1957, Tunisia introduced 117 species of Eucalyptus; they have been used as fire wood, for the production of mine wood and to fight erosion. Actually, Eucalyptus essential oil is traditionally used to treat respiratory tract disorders such as pharyngitis, bronchitis, and sinusitis. A few investigations were reported on the biological activities of Eucalyptus oils worldwide. In Tunisia, our previous works conducted in 2010 and 2011 had been the first reports to study the antibacterial activities against reference strains. At that time it was not possible to evaluate their antimicrobial activities against clinical bacterial strains and other pathogens such as virus and fungi.
The essential oils of eight Eucalyptus species harvested from the Jbel Abderrahman, Korbous (North East Tunisia) and Souinet arboreta (North of Tunisia) were evaluated for their antimicrobial activities by disc diffusion and microbroth dilution methods against seven bacterial isolates: Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae and Streptococcus pyogenes. In addition, the bactericidal, fungicidal and the antiviral activities of the tested oils were carried out.
Twenty five components were identified by GC/FID and GC/MS. These components were used to correlate with the biological activities of the tested oils. The chemical principal component analysis identified three groups, each of them constituted a chemotype. According to the values of zone diameter and percentage of the inhibition (zdi, % I, respectively), four groups and subgroups of bacterial strains and three groups of fungal strains were characterized by their sensitivity levels to Eucalyptus oils. The cytotoxic effect and the antiviral activity varied significantly within Eucalyptus species oils.
E. odorata showed the strongest activity against S. aureus, H. influenzae, S. agalactiae, S. pyogenes, S. pneumoniae and against all the tested fungal strains. In addition, E. odorata oil showed the most cytotoxic effect. However, the best antiviral activity appeared with E. bicostata. Virus pretreatment with E. bicostata essential oil showed better antiviral activity (IC50 = 0.7 mg/ml, SI = 22.8) than cell-pretreatment (IC50 = 4.8 mg/ml, SI = 3.33). The essential oil of E. astringens showed antiviral activity only when incubated with virus prior to cell infection. This activity was dose-dependent and the antiviral activity diminished with the decreasing essential oil concentration.
PMCID: PMC3475086  PMID: 22742534
Eucalyptus sp.; Essential oil; Principal Components Analysis; Hierarchical Cluster Analysis; Antibacterial activity; Antifungal activity; Antiviral activity
20.  The molecular epidemiology of circulating rotaviruses: three-year surveillance in the region of Monastir, Tunisia 
BMC Infectious Diseases  2011;11:266.
Rotavirus infection is the most common cause of severe, dehydrating, gastroenteritis among children worldwide. In developing countries, approximately 1440 children die from rotavirus infections each day, with an estimated 527,000 annually. In infants, rotavirus is estimated to cause more than 2 million hospitalizations every year depending on the income level of the country.
The purpose of this study was to estimate the proportion of rotavirus gastroenteritis and identify the distribution of circulating G and P genotype rotavirus strains among children consulting several dispensaries in the region of Monastir (outpatients departments) or admitted to Monastir University Hospital (inpatients department) with acute gastroenteritis.
This study was undertaken during a 3-year period from April 2007 to April 2010 in Tunisian children under 13 suffering from acute gastroenteritis. Group A rotaviruses were detected in stools by ELISA and genotyped using multiplex reverse transcription PCRs with type-specific primers on the basis of their outer capsid proteins. Statistical analyses were performed with SPSS software, version 19.
Of the 435 stool samples from children with acute gastroenteritis, 27.6% were positive for rotavirus A. The predominant G type was G1 (37.5%), followed by G3 (25%), G2 (17.5%), G4 (12.5%), G9 (2.5%) and three mixed-G infections G3G4 (2.5%) were identified.
Only P[8] (80.8%), P[4] (16.7%) and P[9] (0.8%) genotypes were found. The predominant single G/P combination was G1P[8] (37.5%), followed by G3P[8] (25%), G2P[4] (16.7%), G4P[8] (12.5%), G9P[8] (1.7%) and one case of the unusual combination G9P[9] (0.8%). The G-mixed types G3G4 combined with P[8] (2.5%). Infants less than 3 months of age were most frequently affected. The prevalence of rotavirus infection peaked in the winter season, when temperatures were low, and decreased in summer.
Rotavirus gastroenteritis is a common disease associated with significant morbidity, mortality, and economic burden. Epidemiological knowledge of rotavirus is critical for the development of effective preventive measures, including vaccines.
These data will help to make informed decisions as to whether rotavirus vaccine should be considered for inclusion in Tunisia's National Immunisation Programme.
PMCID: PMC3193173  PMID: 21967503
21.  Characterization of outbreak hepatitis a isolates in five Tunisian childcare centers 
Brazilian Journal of Microbiology  2011;42(3):1204-1212.
In the present study, epidemiological survey and molecular characterization of hepatitis A virus during an outbreak in five Tunisian childcare centers in El-Mahres during October and November 2006 were carried out. Five well-water and five drinking water samples were included in the present study. Serological investigation and molecular characterization were carried out. All patients were IgM seropositive and the viral genome was detected in all clinical and well-water samples whereas it was not detected in drinking water from the five childcare centers. Sequence analysis showed that all Tunisian strains belong to sub-genotype IA. The genetic profile of the VP1/2A junction showed that the outbreak isolates underwent an amino acid substitution which was absent in virus’s strains detected previously in Tunisia. Further studies need to be conducted to evaluate the emergence of the virus’s strains in clinical and water samples and more epidemiological data need to be collected about the risk factors which may contribute to acute hepatitis.
PMCID: PMC3768787  PMID: 24031743
Hepatitis A Virus; VP1 gene; VP1/2A junction
22.  Bacteriological and physico-chemical assessment of wastewater in different region of Tunisia: impact on human health 
BMC Research Notes  2011;4:144.
In many parts of the world, health problems and diseases have often been caused by discharging untreated or inadequately treated wastewater. In this study, we aimed to control physico-chemical parameters in wastewater samples. Also, microbiological analyses were done to reveal Salmonella strains and each Escherichia coli (E.coli) pathotype.
Sixty wastewater samples were collected from fifteen different regions of Tunisia. All physico-chemical parameters (pH, residual free chlorine, total suspended solids, biological oxygen demand, and chemical oxygen demand) were evaluated.
For microbiological analyses, samples were filtered to concentrate bacteria. DNA was extracted by boiling and subjected to polymerase chain reaction (PCR) using different pairs of primers.
The mean pH values recorded for the sampling point were above the WHO pH tolerance limit. The total suspended solids (TSS) concentrations varied between 240 mg/L and 733 mg/L in entrance points and between 13 mg/L and 76 mg/L in exit points. In entrance points, the studied wastewater has an average COD concentration that varied between 795 mg/mL to 1420 mg/mL. Whereas, BOD concentration of the wastewater ranged between 270 mg/L to 610 mg/L. In exit points, COD concentration varied between 59 mg/L and 141 mg/L, whereas BOD concentration ranged from 15 mg/L to 87 mg/L.
The bacteriological control of wastewaters showed that, in entrance points, Escherichia coli (E.coli) was detected at the rate of 76.6%. Three E.coli pathotypes were found: ETEC (53.3%), EAEC (16.6%) and EIEC (6.6%).
Concerning the ETEC isolated strains, 8 of 16 (50%) have only the heat-labile toxin gene, 5 of 16 (31.2%) present only the heat-stable toxin gene and 3 of 16 (18.7%) of strains possess both heat-labile toxin gene and heat-stable toxin gene. In exist point, the same pathotypes were found but all detected ETEC strains present only the "est" gene.
Concerning Salmonella isolated strains; percentages of 66.6% and 20% were found in entrance and exit points respectively.
Wastewaters contain a large amount of pathogenic bacteria that present a real impact on human health. Assessment wastewater treatment stations have to consider in account enterobacterial pathogens as potential pathogens that should be correctly controlled.
PMCID: PMC3123216  PMID: 21600052
Enteropathogenic bacteria; Escherichia coli; pathotype; microbiological quality; reclaimed wastewater
23.  Aichi Virus IgG Seroprevalence in Tunisia Parallels Genomic Detection and Clinical Presentation in Children with Gastroenteritis ▿  
Aichi virus has been described as a novel causative agent of gastroenteritis in humans. In this study, we report the seroprevalence distribution of Aichi virus in Tunisia. A panel of 1,000 sera was screened by applying an enzyme-linked immunosorbent assay for immunoglobulin G specific for Aichi virus. A considerable prevalence (92%) of antibody to Aichi virus was found across all age groups. The specific anti-Aichi virus antibodies increased with age, from a high rate (68.8%) in children under 10 years old to about 100% in persons more than 60 years old. We found a statistically significant increase in levels of antibody to Aichi virus according to the age of patients. Immunoglobulin M antibodies were detected among five children. A high frequency of Aichi virus monoinfections in hospitalized children with severe gastroenteritis was previously observed in Tunisia. Aichi virus causes diarrhea with dehydration, fever, and vomiting. This work is the first to establish a correlation between the high seroprevalence of specific Aichi virus antibodies, clinical presentation, and a high frequency of isolation of Aichi virus by genomic characterization in stools of children suffering from gastroenteritis. Our data show the importance and emerging character of Aichi virus in the viral etiology of pediatric gastroenteritis.
PMCID: PMC2897256  PMID: 20484569
24.  Detection and Genomic Characterization of Aichi Viruses in Stool Samples from Children in Monastir, Tunisia▿  
Journal of Clinical Microbiology  2009;47(7):2275-2278.
Aichi virus has been associated with acute gastroenteritis in adults and children. Stool samples were collected from 788 Tunisian children suffering from diarrhea. Aichi virus was found in 4.1% of the cases. The high proportion of monoinfections and the high frequency of hospitalizations support the role of Aichi virus in pediatric gastroenteritis.
PMCID: PMC2708530  PMID: 19474269
25.  Molecular Epidemiology of Norovirus Gastroenteritis Investigated Using Samples Collected from Children in Tunisia during a Four-Year Period: Detection of the Norovirus Variant GGII.4 Hunter as Early as January 2003 ▿  
Journal of Clinical Microbiology  2008;47(2):421-429.
Human noroviruses (NoVs) cause epidemic and endemic acute gastroenteritis in children and adults. To study the prevalence and genetic diversity of NoV in children in Tunisia, a total of 788 fecal samples were collected during a 4-year period in the region of Monastir, from children 12 years of age or younger, hospitalized or presenting in dispensaries with symptoms of acute gastroenteritis. NoV was detected by reverse transcription-PCR and confirmed by sequence analysis. This is the first report that describes the molecular epidemiology of NoV in Tunisian children: NoVs were characterized as the causative agent in 128 (16.2%) of the samples. Fourteen samples contained a mixture of two NoVs, and 33 samples were coinfected with additional enteric viruses. Eight distinct NoV genotypes were detected (GGI.2, GGI.4, GGII.1, GGII.4, GGII.8, GGII.14, GGIIb/GGII.2, and GGIIb/GGII.3). GGII.4 was the most prevalent genotype, accounting for 83 (64.8%) cases. Interestingly the GGII.4 variant Hunter, described as spreading all over the world in 2004, was found in Tunisia as early as January 2003. The delay of 1 year between the isolation in Tunisia and the worldwide emergence is somewhat surprising, considering the importance of the contacts between North Africa and Europe particularly. Nevertheless, this illustrates the idea that sporadic gastroenteritis cases may be a reservoir for emerging epidemic NoV strains.
PMCID: PMC2643701  PMID: 19109464

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