Molecular characterisation of a coxsackievirus A24 that caused an outbreak of acute haemorrhagic conjunctivitis, Tunisia 2003.

This study reports the genetic characteristics of coxsackievirus A24 isolates from Tunisia, including a coxsackievirus A24 variant (CVA24v) that caused an outbreak of acute haemorrhagic conjunctivitis (AHC) between September and November 2003. The virus genome was detected by PCR from conjunctival swabs obtained from patients with AHC. Four virus isolates were obtained from PCR-positive samples and were serotyped by sequence analysis of the VP1 and VP4 genomic region and by seroneutralisation. Phylogenetic analysis of the VP1, VP4 and 3C genomic regions was performed. Other Tunisian CVA24 isolates from paralytic cases and healthy individuals were also amplified, sequenced and included in the phylogenetic analysis. The epidemic strain belonged to the CVA24 serotype. Phylogenetic analysis of the 3C region of the genome revealed a strong relationship between the Tunisian epidemic strain and strains that caused outbreaks in Korea (2002) and Guadeloupe and French Guiana (2003). Phylogenetic analysis of the VP1 and VP4 regions showed a clear distinction between serotype CVA24 isolates from conjunctivitis and non-conjunctivitis cases. This is the first study to report an outbreak of AHC caused by CVA24v in the North African region.

Enterovirus detection in stool specimen: relevance for poliovirus and enterovirus surveillance.

Detection of enterovirus genome by PCR in clinical samples is now extensively used for the diagnostic of enterovirus infections given its rapidity and high sensitivity. In contrast, its use in surveillance programs targeting specific enterovirus serotypes remains less frequent. The most sensitive protocols are those amplifying in the 5'untranslated region (5'UTR). However the possibility to use sequence analysis of the 5'UTR amplicons for serotype identification is not yet well established. In this report, stool samples from polio suspected cases and their healthy contacts were tested. The results of direct detection of enterovirus genome by PCR and serotype identification based on sequence analysis of the PCR products in the 5'UTR were compared to those of standard cell-culture-based protocols. Standard protocols detected enterovirus isolates in 7.4% of cases while 9.8% of samples were positive by PCR. Serotype identification based on sequence analysis of amplicons showed concordant results with serotypes determined on virus isolates by seroneutralisation or sequencing in the VP1 gene in 39% of cases only. These results confirm that the use of PCR amplification from stool samples improves the sensitivity of enterovirus detection but do not recommend the use of sequence analysis of the 5'UTR PCR product to determine enterovirus serotype.

Enteroviruses in Tunisia: virological surveillance over 12 years (1992-2003).

This report is an overview of enterovirus epidemiology in Tunisia during a 12-year period from 1992 to 2003. A total of 4700 clinical samples were collected as part of the national poliovirus surveillance programme and the routine diagnostic programme for aseptic meningitis. Enterovirus detection was performed by isolation on cell culture according to World Health Organization recommended protocols. Serotype identification was performed by seroneutralization of the cytopathic effect using pools of specific antisera and sequencing in the VP1 region of the genome. Poliovirus isolates were assessed for their wild or vaccine-related origin by standard World Health Organization recommended methods (PCR, probe hybridization and ELISA). The results confirm the interruption of wild poliovirus circulation since 1995. A total of 236 non-polio enterovirus (NPEV) strains were isolated; seroneutralization allowed typing of 93 % (219 out of 236) of them. The antisera used allowed the identification of the most common enterovirus serotypes. The remaining 17 isolates were sequenced; 16 of them belonged to enterovirus serotypes that were not targeted by the antisera pools used. A total of 29 different serotypes of NPEV were detected in the country during the study period. Echoviruses of serotypes 6, 11 and 30 were the most frequently isolated, almost every year; other serotypes had a cyclic occurrence and others were detected during a limited period with very few isolates. The NPEV isolation rate varied from year to year but was steadily under 10 %, suggesting a relatively low prevalence of these viruses in comparison to that in other developing countries. A seasonal variation was also noted; the high transmission period starts in March and peaks in September-November. This study is the first report of the epidemiology of NPEV in Tunisia. These viruses are associated with various diseases and epidemiological data may help to clarify their impact on human health.

[Role of enteroviruses in aseptic meningitis in Tunisia]. / Place des entérovirus dans les méningites aseptiques en Tunisie.

Despite the favourable clinical outcome in most cases, viral meningitis can cause a serious public health problem especially when several cases occur during outbreaks. The first part of this work is a retrospective study conducted in three hospitals in Tunisia and covering a period of three years. It showed an incidence of viral meningitis 2.4. The second part of the study is a prospective one, it included 94 cases of aseptic meningitis notified during a period of 12 months. Virus isolation in cell culture was performed on CSF and stool samples, using cell lines sensitive to enteroviruses. A PCR to detect enteroviruses was also used in parallel. This study represents a first approach to viral meningitis in Tunisia. It highlights the importance of a regular surveillance of the disease and the contribution of molecular methods to a more sensitive diagnostic. However, cell culture remained necessary for viral isolation and serotyping.

[Retrospective study of viral causes of central nervous system infections in Tunisia (2003-2009)]. / Étude rétrospective des étiologies virales des infections neuroméningées en Tunisie (2003-2009)

OBJECTIVES: To determine the role of enteroviruses, Herpesviridae, West Nile virus and Sandfly Toscana virus in central nervous system (CNS) infections in Tunisia. METHODOLOGY: 847 cerebrospinal fluid (CSF) samples, 427 serum samples and 23 stool samples were collected from 1071 patients hospitalized for CNS viral infections from January 2003 through December 2009. All CSF samples were first tested by PCR to detect enteroviruses and Herpesviridae. In specific epidemic contexts in patients negative for these viruses, arbovirus infection was tested by ELISA. RESULTS: Virological testing was positive in 17.5% of cases. West Nile virus and enteroviruses accounted for 58% of them, enteroviruses 23.5%, Herpesviridae 8.5%, and Toscana virus 10%. West Nile virus infection was observed only in 2003, during an outbreak in coastal regions. Toscana virus circulated regularly throughout the study period. Enteroviruses were responsible for grouped cases of aseptic meningitis in both 2003 and 2005. Arboviruses and enteroviruses were detected mainly in summer and autumn. Herpesviridae were associated with sporadic cases of meningoencephalitis. CONCLUSION: This report on viral causes of CNS infections in Tunisia shows that West Nile virus and enteroviruses appear to circulate mainly during epidemics, while the circulation of Toscana virus seems continuous. Negative virus findings may be due, at least in part, to late sampling, inappropriate sample collection and transportation to the virology lab, or failure to test for the right virus. It is essential to promote collaboration between clinicians and biologists to maximize the likelihood of diagnosis.

[Seroprevalences of hepatitis A and E infections in Tunisia]. / Séroprévalences des infections à hépatite A et E en Tunisie.

OBJECTIVE: Viral hepatitis A (HAV) and E (HEV) infections are still frequent in many regions of the world, particularly in developing countries where sanitary conditions and socioeconomic level are frequently low. In this work, we have studied seroprevalences of these two infections in Tunisian children, teenagers and young adults. MATERIAL AND METHODS: The studied population included 3357 individuals from different regions of Tunisia and distributed in three groups 1 (n=1145), 2 (n=707) and 3 (n=1505) with a mean of age of 6.94, 12.84 and 20.71 years, respectively. RESULTS: Rates of HAV infection prevalence of 84.0, 90.5 and 91.7% were found within groups 1, 2 and 3, respectively. These rates are lower than those previously found in the country; thus, primary infection with HAV in Tunisia is progressively shifting to older ages, which is probably due to the improvement of sanitary conditions. Lower anti-HAV prevalences were found in costal regions as compared to the rest of the country. This difference may be due to the higher socioeconomic level of the population living in costal regions. Antibodies against HEV were assessed in individuals of group 3. A seroprevalence of 4.3% was found which indicates that, despite the absence of epidemics, the virus is circulating among the Tunisian population as sporadic cases. CONCLUSION: The present work contributes to a better knowledge of HAV and HEV infections in Tunisia and highlights the need of the establishment of a national program for virological surveillance of hepatitis cases and of further studies to monitor changes in the epidemiology of these infections.

[Identification of adenoviruses serotypes implicated in haemorrhagic conjunctivitis in Tunisia]. / Identification des sérotypes d'adénovirus impliqués dans les conjonctivites hémorragiques en Tunisie.

Human adenoviruses (ADV) are distributed worldwide; they are associated with a variety of diseases. Some ADV can be implicated in large epidemics of conjunctivitis, gastroenteritis and respiratory infections. Classical diagnosis of ADV infections is based on virus isolation on cell culture and identification of the serotype by neutralization test or hemagglutination inhibition assay. However, these methods have a lack of rapidity that makes them impractical in clinical situations. With the advent of PCR, the diagnosis of ADV was improved. In this work, we have used molecular techniques for the identification of ADV serotypes implicated in conjunctivitis in Tunisia. A total of 199 conjunctival swabs received between October 2000 and May 2005 were investigated. Serotype identification was performed using a PCR followed by restriction enzyme analysis in the hexon gene. Typing by sequencing of the PCR product was used to confirm the serotype identification. Among the 199 tested clinical specimens, 24% were positive for ADV. Two different profiles were observed: one predominant corresponding to the majority of the detected ADV; this profile is in favour of two distinct serotypes, ADV37 or ADV8; the second profile was specific of ADV4 and was found in one case observed in 2005. Sequencing confirmed two serotypes: ADV8 with an endemoepidemically circulation in our country and ADV4 that appeared sporadic. The present work showed the importance of molecular techniques not only for ADV detection but also for identification of the circulating serotypes. These techniques are practical and interesting mainly for the rapid virological investigation during epidemics.