Microbiological and physicochemical quality enhancement of treated wastewater using raw and chemically modified clays from Sidi Bouzid region, Tunisia.

Environmental discharge of wastewater represents a source of chemical and biological pollutants. This study firstly evaluates the microbiological and physicochemical quality of treated wastewaters collected from two wastewater treatment plants (WWTPs) located in two different Tunisian cities namely Sidi Bouzid (SB) and Gafsa (G). Then, the capacity of three raw and acid/base-activated local clays to enhance the quality of wastewaters was assessed. The results indicate that the quantities of enteric bacteria (oscillating from 1.381 × 103 to 1.4 × 108 CFU/100 mL), fungi (between 1.331 × 103 and 1.781 × 104 CFU/100 mL), as well as SARS-CoV-2 (between 4.25 × 103 and 5.05 × 105 CFU/100 mL) and Hepatitis A virus RNA (form 4.25 × 103 to 7.4 × 104 CFU/100 mL) detected in effluent wastewaters were not in compliance with the Tunisian standards for both studied WWTPs. Likewise for other indicators such as electrical conductivity (ranging 4.9-5.4 mS/cm), suspended matter (145-160 g l-1), chemical oxygen demand (123-160 mg l-1), biological oxygen demand 5 (172-195 mg l-1), chloride, Total Kjeldahl nitrogen (TKN) and phosphorus contents (710, 58-66 and 9.47-10.83 mg l-1 respectively), the registered values do not agree with the set standards established for wastewater treatment. On the other hand, the pH values fitted (oscillating from 6.86 (at G) to 7.24 (at SB) with the Tunisian standards for both WWTPs. After treatment, wastewaters showed better values for the microbiological parameters, especially for the clays designed as AM and HJ1, which eliminated 100% of viruses. In addition, when acid-activated AM clays were applied, a marked improvement in the quality of physicochemical parameters was obtained, especially for suspended matter (2 and 4 g l-1 for SB and G, respectively), TKN (5.2 (SB) and 6.40 (G) mg/l), phosphorus (1.01 (SB) and 0.81 (G) mg/l). Our results open perspectives for the possibility of efficiently using these specific clays in the enhancement of the quality of treated wastewaters.

Evaluation of three viral concentration methods for detection and quantification of SARS-CoV-2 in wastewater.

Wastewater-based epidemiology (WBE) could be useful as an early warning system for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic spread. Viruses are highly diluted in wastewater. Therefore, a virus concentration step is needed for SARS-CoV-2 wastewater detection. We tested the efficiency of three wastewater viral concentration methods: ultrafiltration (UF), electronegative membrane filtration and aluminum hydroxide adsorption-elution. We spiked wastewater with inactivated SARS-CoV-2 and we collected 20 other wastewater samples from five sites in Tunisia. Samples were concentrated by the three methods and SARS-CoV-2 was quantified by reverse transcription digital PCR (RT-dPCR). The most efficient method was UF with a mean SARS-CoV-2 recovery of 54.03 ± 8.25. Moreover, this method provided significantly greater mean concentration and virus detection ability (95%) than the two other methods. The second-most efficient method used electronegative membrane filtration with a mean SARS-CoV-2 recovery of 25.59 ± 5.04% and the least efficient method was aluminum hydroxide adsorption-elution. This study suggests that the UF method provides rapid and straightforward recovery of SARS-CoV-2 in wastewater.

Molecular characterization of enterovirus detected in cerebrospinal fluid and wastewater samples in Monastir, Tunisia, 2014-2017.

BACKGROUND: Enteroviruses (EVs) are considered the main causative agents responsible for aseptic meningitis worldwide. This study was conducted in the Monastir region of Tunisia in order to know the prevalence of EV infections in children with meningitis symptoms. Detected EV types were compared to those identified in wastewater samples. METHODS: Two hundred CSF samples collected from hospitalized patients suspected of having aseptic meningitis for an EV infection between May 2014 and May 2017 and 80 wastewater samples collected in the same time-period were analyzed. EV detection and genotyping were performed using PCR methods followed by sequencing. Phylogenetic analyses in the 3'-VP1 region were also carried-out. RESULTS: EVs were detected in 12% (24/200) CSF and in 35% (28/80) wastewater samples. EV genotyping was reached in 50% (12/24) CSF-positive samples and in 64% (18/28) sewage. Most frequent types detected in CSF were CVB3, E-30 and E-9 (25% each). In wastewater samples, the same EVs were identified, but also other types non-detected in CSF samples, such as E-17,CVA9 and CVB1 from EV species B, and EV-A71 and CVA8 from EV-A, suggesting their likely lower pathogenicity. Phylogenetic analysis showed that within the same type, different strains circulate in Tunisia. For some of the EV types such as E-9, E-11 or CVB3, the same strains were detected in CSF and wastewater samples. CONCLUSIONS: Epidemiological studies are important for the surveillance of the EV infections and to better understand the emergence of certain types and variants.

Dynamics of norovirus genotype change and early characterization of variants in children with diarrhea in central Tunisia, 2001-2012.

Human noroviruses (HuNoVs), especially GII.4 strains, are a major cause of gastroenteritis epidemics in both children and adults. Stool samples were collected from 113 Tunisian children with acute gastroenteritis in 2001 and 2002 and were retrospectively tested for HuNoVs. Fifteen (13.2%) of the 113 samples were positive for HuNoVs, all of which were genogroup II strains, and the GII.4-2004/Hunter variant was predominant (67%). We reconstituted the temporal circulation of HuNoV strains in central Tunisia between 2003 and 2012 using HuNoV isolates reported in our previous studies. A comparative analysis showed a dynamic change in the molecular profile of the HuNoV strains over a 12-year period. We found that GII.4-2004/Hunter strains were circulating as early as June 2002 and that GIX.1[GII.P15] HuNoVs were already circulating four years before this genotype was first reported in Japan in 2006. Our data suggest that epidemic strains of HuNoV circulate for several years in the pediatric population before becoming predominant. This study suggests that children from low-income countries with poor sanitation may play a significant role in the molecular evolution of noroviruses and the global emergence of new epidemic strains.

Type B coxsackieviruses and central nervous system disorders: critical review of reported associations.

Type B coxsackieviruses (CV-B) frequently infect the central nervous system (CNS) causing neurological diseases notably meningitis and encephalitis. These infections occur principally among newborns and children. Epidemiological studies of patients with nervous system disorders demonstrate the presence of infectious virus, its components, or anti-CV-B antibodies. Some experimental studies conducted in vitro and in vivo support the potential association between CV-B and idiopathic neurodegenerative diseases such as amyotrophic lateral sclerosis and psychiatric illness such as schizophrenia. However, mechanisms explaining how CV-B infections may contribute to the genesis of CNS disorders remain unclear. The proposed mechanisms focus on the immune response following the viral infection as a contributor to pathogenesis. This review describes these epidemiological and experimental studies, the modes of transmission of CV-B with an emphasis on congenital transmission, the routes used by CV-B to reach the brain parenchyma, and plausible mechanisms by which CV-B may induce CNS diseases, with a focus on potential immunopathogenesis.

Host-Range Shift Between Emerging P[8]-4 Rotavirus and Common P[8] and P[4] Strains.

In Tunisia, we observed that rotavirus P[8]-3 and P[4] strains in young children with gastroenteritis associate with secretor histo-blood group phenotype. In contrast, the emerging P[8]-4 strain, representing 10% of cases, was exclusively found in nonsecretor patients. Unlike VP8* from P[8]-3 and P[4] strains, the P[8]-4 VP8* protein attached to glycans from saliva samples regardless of the donor's secretor status. Interestingly, a high frequency of FUT2 enzyme deficiency (nonsecretor phenotype) was observed in the population. This may allow cocirculation of P[8]-3 and P[8]-4 strains in secretor and nonsecretor children, respectively.

Immunopathology in the brain of mice following vertical transmission of Coxsackievirus B4.

Coxsackie B viruses (CV-B) are associated with several central nervous system (CNS) disorders. These viruses are predominantly transmitted by fecal-oral route but vertical transmission can also occur. This work attempted to study the immune response ensuing vertical transmission of CV-B to the brain, and its eventual implementation in the brain pathogenesis. To this end, pregnant Swiss albino mice were inoculated with CV-B4 E2 or with sterile medium for control animals. At different ages after birth, brains were collected and analyzed for virus infection, histopathological changes and immune response. Infectious particles were detected in offspring's brain which demonstrates vertical transmission of the virus. This infection is persistent since the long lasting detection of viral RNA in offspring's brain. Some pathological signs including meningitis, edema and accumulation of inflammatory cells within and surrounding the inflammatory areas were observed. Immunoflorescence staining unveiled the presence of T lymphocytes and microgliosis in the sites of lesion for a long period after birth. Multiplex cytokines measurement upon supernatants of in vitro mixed brain cells and extracted mononuclear cells from offspring's brain has demonstrated an elevated secretion of the pro-inflammatory cytokines TNFα, IL-6 and IFNα and the chemokines RANTES and MCP-1. Hence, vertical transmission of CV-B4 and its persistence within offspring's brain can lead to pathological features linked to increased and sustained immune response.

Coxsackievirus B4 infection and interneuronal spread in primary cultured neurons.

Coxsackie B viruses (CV-B) are usually transmitted via the fecal-oral route and the virus gains the central nervous system (CNS) via the bloodstream. Nevertheless, other routes of spread of the virus to the CNS cannot be excluded, including the neuronal route. Neuronal cells, as well as non-neuronal cells (fibroblasts), were isolated from mice and inoculated with CV-B4 in the absence and presence of neutralizing serum. In the absence of neutralizing serum, virus titers recorded in neuron cultures and rates of infected neurons were non-significantly different compared to those recorded in fibroblast cultures. Higher cell mortality was noted among neurons than fibroblasts. The addition of neutralizing serum to neurons did not reduce significantly virus titers or rates of infected cells and cell viability was not significantly augmented, while virus titers and rates of infected fibroblasts were significantly reduced and their viability was significantly enhanced as well. Our results demonstrate the ineffectiveness of neutralizing serum to prevent neurons infection with CV-B4 which suggests a trans-synaptic transmission of CV-B4 between neurons.

Sapovirus in Wastewater Treatment Plants in Tunisia: Prevalence, Removal, and Genetic Characterization.

Sapovirus (SaV), from the Caliciviridae family, is a genus of enteric viruses that cause acute gastroenteritis. SaV is shed at high concentrations with feces into wastewater, which is usually discharged into aquatic environments or reused for irrigation without efficient treatments. This study analyzed the incidence of human SaV in four wastewater treatment plants from Tunisia during a period of 13 months (December 2009 to December 2010). Detection and quantification were carried out using reverse transcription-quantitative PCR (RT-qPCR) methods, obtaining a prevalence of 39.9% (87/218). Sixty-one positive samples were detected in untreated water and 26 positive samples in processed water. The Dekhila plant presented the highest contamination levels, with a 63.0% prevalence. A dominance of genotype I.2 was observed on 15 of the 24 positive samples that were genetically characterized. By a Bayesian estimation algorithm, the SaV density in wastewater was estimated using left-censored data sets. The mean value of log SaV concentration in untreated wastewater ranged between 2.7 and 4.5 logs. A virus removal efficiency of 0.2 log was calculated for the Dekhila plant as the log ratio posterior distributions between untreated and treated wastewater. Multiple quantitative values obtained in this study must be available in quantitative microbial risk assessment in Tunisia as parameter values reflecting local conditions.IMPORTANCE Human sapovirus (SaV) is becoming more prevalent worldwide and organisms in this genus are recognized as emerging pathogens associated with human gastroenteritis. The present study describes novel findings on the prevalence, seasonality, and genotype distribution of SaV in Tunisia and Northern Africa. In addition, a statistical approximation using Bayesian estimation of the posterior predictive distribution ("left-censored" data) was employed to solve methodological problems related with the limit of quantification of the quantitative PCR (qPCR). This approach would be helpful for the future development of quantitative microbial risk assessment procedures for wastewater.

Evolutionary dynamics of HBV-D7 subgenotype in Tunisia.

Hepatitis B virus (HBV) is the main cause of diseases liver related infecting more than 200 milion persons worldwide. HBV infection shows high level of prevalence in South-East Europe and in Mediterranean basin. In Tunisia, a country with an intermediate level endemicity, HbsAg prevalence ranges from 2 to 5%. Most of the HBV isolates from Tunisia were classified as subgenotype D7 whose circulation is restricted to a specific area of North Africa including Maghreb region. In this paper, the phylogeny of HBV-D7 isolated from 38 Tunisian patients was investigated by analyzing the S gene region of HBV. A Bayesian coalescent-based framework was used to estimate the origin of the HBV-D7 in the country. The Tunisian D7 isolates were found to share a common ancestor whose origin was traced back to 1958. Population dynamics indicated that HBV-D7 epidemic in Tunisia grew exponentially from 1960s to 1990s. After that, the curve reached a plateau around the years 2000 likely due to the implementation of the infant vaccination program in 1996. Epidemiological data suggested that the exponential growth phase was likely sustained by intra-familial transmission events occurring during infancy. Further characterization of HBV-D7 isolates should be performed to evaluate, in the post-vaccination era, the emergence of new transmission routes, and to monitor the efficacy of the vaccination program. J. Med. Virol. 89:469-475, 2017. © 2016 Wiley Periodicals, Inc.

Molecular characterization and phylogenetic analysis of highly pathogenic Vibrio alginolyticus strains isolated during mortality outbreaks in cultured Ruditapes decussatus juvenile.

In the summer of 2008 and 2009, a series of mortalities in growing out seeds of R. decussatus juveniles were occurred in the eastern Tunisian littoral. Nine predominant bacterial strains were isolated from dead and moribund juveniles and characterized as Vibrio alginolyticus. These isolates were subjected to biochemical and molecular characterization. All the Vibrio strains were tested for their susceptibility against the most widely used antibiotic in aquaculture as well as, the assessment of the presence of erythromycin (emrB) and tetracycline (tetS) resistance genes among the tested bacteria. The degree of genetic relatedness between V. alginolyticus strains was evaluated on the basis of the Entero-Bacterial Repetitive Intergenic Consensus (ERIC) and the Random Amplification of Polymorphic DNA-PCR (RAPD-PCR) approaches. We also looked for siderophore activity and the ability to grow under iron limitation. Furthermore, the pathogenic potential of the tested isolates was evaluated using R. decussatus larva and juveniles as infection models. On antimicrobial susceptibility test, Vibrio strains exhibited total resistance to at least four antibiotics. The MICs data revealed that flumequine and oxolinic acid were the most effective antibiotics to control the studied bacteria. Results also showed that studied antibiotics resistance genes were widely disseminated in the genome of V. alginolyticus strains. Both ERIC and RAPD-PCR fingerprinting showed the presence of genetic variation among Vibrio isolates. However, RAPD typing exhibited a higher discriminative potential than ERIC-PCR. Besides, we reported here for the first time the co-production of catechol and hydroxamte by V. alginolyticus species. The challenge experiment showed that most of Vibrio isolates caused high mortality rates for both larva and juveniles at 48-h post-exposure to a bacterial concentration of 106 CFU/ml.

Extended-spectrum ß-lactamases and plasmid-mediated quinolone resistance in enterobacterial clinical isolates from neonates in Tunisia.

This study was conducted to investigate extended-spectrum-ß-lactamase (ESBL) producing Enterobacteriaceae isolates from the Center of Maternity and Neonatology of Monastir, Tunisia. Fourty-six strains out of 283 were found to produce ESBL: Klebsiella pneumoniae (n = 37), Escherichia coli (n = 6), Enterobacter cloacae (n = 2), and Citrobacter freundi (n = 1). Genotyping analysis, using ERIC2 and RAPD, showed that strains were clonally unrelated. PCR amplification followed by sequencing revealed that all strains produced CTX-M-15. This enzyme was co-produced with TEM and SHV determinants in 34 and 36 strains respectively. The blaCTXM-15 gene was bracked by ISEcp1 and/or IS26 in 42 out of the 46 ESBL positive strains. The quinolone resistance determinants were associated to the ESBL producing isolates: we identified the qnrB1 gene in six isolates and the aac(6')-Ib-cr gene in five isolates. This epidemiological study shows the widespread of CTX-M-15 and qnr determinants among enterobacterial isolates from neonates hospitalized at the center of Maternity and Neonatology of Monastir suggesting either mother portage or horizontal transmission.

Coxsackievirus B4 vertical transmission in a murine model.

BACKGROUND: Life-threatening infections with type B Coxsackieviruses (CV-B) are frequently encountered among newborns and are partly attributed to vertically-transmitted virus. Our current study investigates this alternative way of contamination by CV-B, using a mouse model. METHODS: Pregnant Swiss mice were intraperitoneally inoculated with CV-B4 E2 at gestational day 10(G) or 17G. Dams and offspring were monitored for mortality and morbidity, and sampled at different time-points to document the infection and explore eventual vertical transmission. RESULTS: Inoculation at day 10G induced an important rate of abortion and a decrease in the number of delivered pups per litter, whereas inoculation at day 17G was marked by preterm delivery and significant behavioral changes in dams. Only one case of spastic paralysis and one case of pancreatitis were recorded among surviving pups. Seroneutralization revealed anti-CV-B4 neutralizing antibodies in infected dams and their partial transfer to offspring. Viral genome detection by RT-PCR and viral progeny titration in several tissues (dams' uteri, amniotic sac, amniotic fluid, placenta, umbilical cord, pancreas and heart) attested and documented CV-B4 vertical transmission to the majority of analyzed offspring. Virus detection in fetuses suggests transplacental transmission, but perinatal transmission during delivery could be also suggested. Vertically transmitted CV-B might even persist since prolonged viral RNA detection was noticed in the pancreas and heart from offspring born to dams inoculated at day 17G. CONCLUSION: This model of CV-B4 vertical transmission in mice, in addition to allow a better understanding of CV-B infections in fetuses and newborns, constitutes a useful tool to investigate the pathogenesis of CV-B associated chronic diseases.

Detection and Molecular Characterization of Hepatitis A Virus from Tunisian Wastewater Treatment Plants with Different Secondary Treatments.

UNLABELLED: 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 × 10(3) genome copies [GC]/ml) and influents (2.7 × 10(3) 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.

Chemical composition and in vitro evaluation of antioxidant, antimicrobial, cytotoxicity and anti-acetylcholinesterase properties of Tunisian Origanum majorana L. essential oil.

This study investigated the chemical composition and evaluated the antioxidant, antimicrobial, cytotoxic and anti-acetylcholinesterase properties of Tunisian Origanum majorana essential oil. The findings showed that the oil exhibited high activity, particularly in terms of reducing power and ß-Carotene bleaching, inducing higher IC50 values than BHT. The oil showed an important antimicrobial activity against 25 bacterial and fungal strains. In fact, the IZ, MIC and MBC values recorded for the bacterial strains were in the range of 8 ± 0-18.33 ± 0.57 mm, 0.097-3.125 and 0.39-6.25 mg/mL, respectively. The IZ, MIC and MFC values of the fungal strains varied between 11±0-28 ± 0 mm, 0.058-0.468 mg/mL and 0.234-1.875 mg/mL, respectively. A low cytotoxic effect was observed against cancer (Hep-2 and HT29) and continuous cell lineage (Vero), with CC50 values ranging from 13.73 to 85.63 mg/mL. The oil was also evaluated for anti-acetylcholinesterase effects, which showed that it exhibited significant activity with IC50 values reaching 150.33 ± 2.02 µg/mL.

BK and JC virus infections in healthy patients compared to kidney transplant recipients in Tunisia.

The human polyomaviruses BKPyV and JCPyV are members of Polyomaviridae family and after primary infections they persist as latent infection especially in the kidneys. BKVPy reactivation is mainly related to a renal nephropathy and JCV reactivation can induce the progressive multifocal leukoencephalopathy. The aim of this study was to investigate and to compare the presence of BKPyV and JCPyV in urine and plasma samples from immunocompromised and immunocompetent groups. The viral detection and quantification was done by a real time PCR from 100 healthy individuals and from 72 kidney transplanted patients (KTx) enrolled in a prospective study. Polyomavirus DNA urinary shedding was identified in 19% of healthy person, BKPyV in 6%; JCPyV more frequent in 13%. No individuals in this group developed polyomavirus viremia. BKPyV and JCPyV viruria was seen in 36% and 28% of KTx respectively, and 11% had a concomitant BKPyV and JCPyV viruria. Only BKPy viremia was detected in 5.5% of the KTx. In healthy persons, JCPyV shedding was associated with older individuals. However, in KTx, BKPyV was associated with younger age and male gender. No significant association was found between the patient's origin and BKPyV or JCPyV infection. In conclusion and consisting with previous reports, BKPyV and JCPyV prevalence and urinary loads were significantly higher in immunosuppressed compared to non-immunosuppressed individuals. In Addition and by contrast to KTx, JCPyV was more frequent than BKPyV in healthy individuals. Furthermore, the shedding of both polyomaviruses was differently associated with the age and the sex according to each population.

High potential of adhesion to biotic and abiotic surfaces by opportunistic Staphylococcus aureus strains isolated from orthodontic appliances.

Orthodontic and other oral appliances act as reservoir of opportunistic pathogens that can easily become resistant to antibiotics and cause systemic infections. The aim of this study was to investigate the ability of Staphylococcus aureus strains isolated from healthy patients with orthodontic appliances, to adhere to biotic (HeLa cells) and abiotic surfaces (polystyrene and dental alloy). Adhesive ability to polystyrene was tested by crystal violet staining and quantitative biofilm production on dental alloy surfaces was evaluated by MTT reduction assay. In addition, the presence of icaA and icaD genes was achieved by polymerase chain reaction (PCR). Qualitative biofilm production revealed that 70.6% of strains were slime producers. The metabolic activity of S. aureus biofilms on dental alloy surfaces was high and did not differ between tested strains. Moreover, all the isolates were adhesive to HeLa cells and 94% of them harbor icaA and icaD genes. Considerable adhesion and internalization capacity to the epithelial HeLa cells and strong biofilm production abilities together, with a high genotypic expression of icaA/icaD genes are an important equipment of S. aureus to colonize orthodontic appliances and eventually to disseminate towards other body areas.

Naturally Occurring Surface Antigen Variants of Hepatitis B Virus in Tunisian Patients.

In Tunisia, the prevalence of naturally occurring surface (S) gene variants of hepatitis B virus (HBV) has not been determined. In the present study, the prevalence of these variants was examined in terms of the clinical and viral state in a series of 99 Tunisian patients with HBV infection. The S genes were amplified and directly sequenced. Genotype D was predominant (98%), 40.4% isolates belonged to subgenotypes D7 and 1 to subgenotype D2. The most common subtype was ayw2 (95.9%). In total, 60.6% of the studied strains harbored S mutations. Several novel mutation patterns were detected. Interestingly, the presence of S mutations was significantly correlated with the D7 subgenotype, low HBV DNA and advancing age (≥35 years), and tended to be higher in liver cirrhosis than in chronic infection. The global prevalence of the major hydrophilic region variants was 12.1%, with substitution S143L/T as the most frequent (4%). Only 33.9% of S substitutions produced amino acid changes in the polymerase gene. In conclusion, a high prevalence of naturally occurring HBsAg variants was observed among Tunisian HBV carriers. Natural viral variability in a geographical region and duration of infection are among the major factors associated with the occurrence of S mutations.

Cytokine and cellular responses to human herpesvirus-6B in patients with B-acute lymphoblastic leukemia.

Primary infection with human herpesvirus-6 (HHV-6), is followed by its lifelong persistence in the host. Most T-cell responses to HHV-6 have been characterized using peripheral blood from healthy adults; however, the role of HHV-6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV-6 infection in patients with B-acute lymphoblastic leukemia (B-ALL) was analyzed. HHV-6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti- and pro-inflammatory cytokines (IL-4, IL-1, IL-6, IL-8, IL-12p70, IL-17a, TNF-α and IFN-γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV-6 virus loads in blood. Characterization of T-cell responses to HHV-6 showed low specific T-cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN-γ-producing T cells were detected in 0.03%-0.23% and in 0%-0.2% of CD4+T cells, respectively. Strong production of IL-6 was detected in medium supernatants of challenged T-cells whatever the HHV-6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF-α and IFN-γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV-6 in blood was identified, suggesting that HHV-6 is not strongly associated with development of B-ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV-6B specific immune responses may be below the detection threshold of the assays used.

Clinical surveillance for human astrovirus in Monastir region, Tunisia.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.