Molecular analysis of adenovirus strains responsible for gastroenteritis in children, under five, in Tunisia.

Purpose of work: Enteric Adenovirus (EAdV) is recognized as one of the most commonly identified agents responsible for severe acute gastroenteritis (AGEs) in the stools of infants.We sought to determine the rate of human adenovirus (HAdV) infections, and the genotypic characterization of circulating strains of HAdV in children under 5 years of age with AGEs in university and regional hospitals, located in the Center-East of Tunisia, from January 2014 to December 2016. Methods: A classic PCR was performed on 582 stool samples taken within 5 days of the onset of symptoms. Chosen positive samples were sequenced, and some of the results were confirmed by the Next Generation Sequencing technique (NGS). Partial nucleotide sequences of the Hexon gene obtained in this study were compared with the NCBI GenBank database using BLAST. Multiple sequence alignment and phylogenetic analysis were conducted using MEGA6 software. The phylogenetic tree was generated using the maximum-likelihood method and bootstrap analysis was performed with 1000 replications. Results: Out of 582 samples, 52 (8.93 %) cases were positive for HAdV, with a male predominance (57.4 %). Phylogenetic analyses showed that Tunisian HAdV strains clustered into five HAdV lineages corresponding to serotypes F41 (14/28), C2 (9/28), C5 (3/28), E4 (1/28), and A18 (1/28). HAdV was more frequent in children aged up to 12 months, as compared to the other age groups. The HAdV activity was noted in almost all the months of the year with a peak in autumn, in 2014 and 2015, and in winter in 2016. Conclusion: This study showed that infections with HAdV species were frequent in children suffering from AGE with the predominance of HAdV F41 and C2. This result underlines the importance of regular monitoring of circulating genotypes, and it could be useful for future epidemiological research.

Role of hepatitis C virus core antigen assay in hepatitis C care in developing country.

BACKGROUND: The World Health Organization (WHO) aims to achieve global hepatitis C elimination by 2030, defined as diagnosis of 90% of infected individuals and treating 80% of them. Current guidelines for the screening and diagnosis of hepatitis C infection denote using a relatively cheap screen with anti-hepatitis C virus (HCV) antibody immunoassay, followed by the much costlier molecular test for HCV RNA levels using polymerase chain reaction (PCR) assay to confirm active HCV infection. Simplification of the HCV evaluation algorithm to reduce the number of required tests could considerably expand the provision of HCV treatment especially in a developing country. This study investigates the performance of hepatitis C Core Antigen (HCV Ag) test by comparing HCV Ag results versus the results obtained with HCV ribonucleic acid (RNA) PCR which is considered the gold standard for the diagnosis of HCV infection. RESULTS: Among the 109 anti-HCV positive sera, 96 were positive for both HCV Ag (> 3 fmol/L) and HCV RNA (> 15 IU/mL); 8 were negative for both tests, while the remaining 5 were positive for HCV RNA only. Considering the HCV RNA as gold standard; the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HCV Ag test were found to be 95.05%, 100%, 100%, and 61.54%, respectively. HCV genotype was performed for 59 patients. The most common HCV genotype was genotype 1 (72.9%). Genotype 2 (15.3%) and genotype 3 (11.9%) were detected in the others samples. A high level of correlation was seen between HCV RNA and HCV Ag (r = 0.958, p < 0.001). The correlation for the samples that were genotyped 1 was significant (r = 0.966, p < 0.001). CONCLUSION: In our study, it was found that there was strong correlation between HCV RNA levels and HCV Ag levels. So, it can be used for a one-step HCV antigen test to diagnose active HCV infection.

Molecular characterization of respiratory syncytial virus circulating in Tunisia between 2015 and 2018.

Introduction. Respiratory syncytial virus (RSV) is the most frequently identified viral agent in children with lower respiratory tract infection (LRTI). No data are available to date regarding RSV genotypes circulating in Tunisia.Aim. The aim of the present study was to investigate the genetic variability of the glycoprotein G gene in Tunisian RSV strains.Methodology. Nasopharyngeal aspirates were collected from infants hospitalized for LRTI in five Tunisian hospitals. All specimens were screened for RSV by a direct immunofluorescence assay (DIFA). To molecularly characterize Tunisian RSV strains, a phylogenetic analysis was conducted. Randomly selected positive samples were subjected to reverse transcription PCR amplifying the second hyper-variable region (HVR2) of the G gene.Results. Among a total of 1417 samples collected between 2015 and 2018, 394 (27.8 %) were positive for RSV by DIFA. Analysis of 61 randomly selected RSV strains revealed that group A RSV (78.7 %) predominated during the period of study as compared to group B RSV (21.3 %). The phylogenetic analysis showed that two genotypes of RSV-A were co-circulating: the ON1 genotype with a 72-nt duplication in HVR2 of the G gene was predominant (98.0 % of RSV-A strains), while one RSV-A strain clustered with the NA1 genotype (2.0 %). Concerning Tunisian group B RSV strains, all sequences contained a 60-nt insertion in HVR2 and a clustered BA10 genotype.Conclusion. These data suggest that RSV-A genotype ON1 and RSV-B genotype BA10, both with duplications in the G gene, were widely circulating in the Central coastal region of Tunisia between 2015 and 2018.

Unexpected predominance of rotavirus G9P[8] strain in Tunisian adult diarrheal patients.

Introduction. Group A Rotavirus (RVA) is known to be a major cause of acute gastroenteritis (AGE) in children but its role as a potential pathogen in immunocompetent adults is probably underestimated.Aim. To compare RVA infections in patients from different age groups.Methodology. Fecal samples were collected from patients aged from birth to 65 years, hospitalized or consulting for AGE between 2015 and 2017. All samples were screened by RT-PCR for the detection of VP6 gene specific of RVA. RVA-positive samples were VP7 and VP4 genotyped using multiplex semi-nested RT-PCR. Full-length VP7 gene of G9-positive strains were sequenced and submitted for phylogenetic analysis.Results. Of 1371 stool specimens collected from children (<5 years; n=454), older children (5 to <15 years; n=316) and adults (15-65 years; n=601), 165 (12.0 %) were RVA-positive. RVA detection rates were significantly higher in children and adults than in older children (15.8 % and 12.1 Vs 6.3 %, respectively; P<0.001). While RVA infections were mostly detected during the coldest months in children, they were observed all year-round in patients aged >5 years. Although G1P[8] remained the most prevalent combination (41.7 %) detected in children, G9P[8] strains widely predominated in adults (58.1 %), followed by G2P[4] (12.9 %). All characterized G9 strains clustered in the modern lineage III.Conclusion. RVA play an important role in AGE not only in children but also in adults. The findings of a wide G9 predominance in patients >5 years highlights the need for continuing surveillance in both pediatric and mature populations.

Molecular characterization of group A rotavirus among children aged under 5 years in Tunisia, 2015-2017.

The aim of the present study was to report the molecular characterization of human group A rotaviruses (RVAs) circulating in Tunisia. Stool specimens were collected from children under 5 years of age who had been hospitalized or were consulting for gastroenteritis in Tunisian hospitals between 2015 and 2017. All samples were screened by reverse-transcription polymerase chain reaction (RT-PCR) for the detection of the VP6 gene specific for RVA. RVA-positive samples were further analysed for G/P genotyping by semi-nested multiplex RT-PCR. Among 454 tested samples, 72 (15.8 %) were positive for RVA. G1P[8] was the most prevalent detected strain (41.7%), followed by G9P[8] (32.8%), G2P[4] (7.5%), G12P[8] (7.5%), G1P[6] (3.0%), G2P[8] (1.5%) and G3P[8] (1.5%), with mixed infections in 4.5 % of cases. In the absence of a national anti-rotavirus vaccination strategy, RVAs remain the primary aetiological agent for gastroenteritis in Tunisian children.

Phylogenetic analysis of partial VP7 gene of the emerging human group A rotavirus G12 strains circulating in Tunisia.

PURPOSE: Group A rotavirus (RVA) is the leading cause of severe gastroenteritis in children younger than 5 years. The most common human G-types are G1-4 and G9. G12 genotype is currently emerging worldwide, becoming the sixth most prevalent RVA G-genotype. In Tunisia, an emergence of G12 RVA strains was observed. To understand the evolution and origin of these Tunisian G12 strains, phylogenetic analyses were conducted. METHODOLOGY: A total of 1127 faecal samples were collected from Tunisian children under 5 years consulting for gastroenteritis between 2009 and 2014. Samples were screened by ELISA for the presence of RVA antigen. RVA-positive samples were used for the detection of G12 RVA strains by semi-nested RT-PCR. G12-positive specimens were subjected to VP4 genotyping reaction. PCR products of the G12-positive samples were sequenced and characterized by phylogenetic analysis of partial VP7 gene sequence. RESULTS: Globally, 270 (24 %) stool specimens were RVA-positive. Fourteen presented the G12 genotype (5.2 %) and were found to be in combination with either the P[6] (50.0 %) or the P[8] (50.0 %) genotype. Phylogenetic analysis revealed that all characterized Tunisian G12 strains clustered in the modern G12 lineage III and appear to form three different subclusters. CONCLUSION: Thus, the Tunisian G12 strains may have originated from not a single, but at least three distinct ancestral G12 strains. Detailed molecular characterization of the entire genome of these strains remains essential to help determine the extent of genetic variation and the relatedness of Tunisian G12 RVA strains to G12 strains described worldwide.

Distribution of rotavirus VP7 and VP4 genotypes circulating in Tunisia from 2009 to 2014: Emergence of the genotype G12.

Group A rotavirus (RVA) represents the most important aetiological agent of diarrhoea in children worldwide. From January 2009 to December 2014, a multi-centre study realized through 11 Tunisian cities was undertaken among children aged <5 years consulting or hospitalized for acute gastroenteritis. A total of 1127 faecal samples were collected. All samples were screened by ELISA for the presence of RVA antigen. RVA-positive samples were further analyzed by PAGE and used for G/P-genotyping by semi-nested multiplex RT-PCR. Globally, 270 specimens (24 %) were RVA-positive, with peaks observed annually between November and March. Nine different electropherotypes could be visualized by PAGE, six with a long profile (173 cases) and two with a short one (seven cases). Mixed profiles were detected in two cases. Among the 267 VP7 genotyped strains, the predominant G- genotype was G1 (39.6 %) followed by G3 (22.2 %), G4 (13 %), G9 (11.5 %), G2 (5.2 %) and G12 (5.2 %). Among the 260 VP4 genotyped strains, P[8] genotype was the predominant (74.5 %) followed by P[6] (10.4 %) and P[4] (5.5 %). A total of 257 strains (95.2 %) could be successfully G- and P-genotyped. G1P[8] was the most prevalent combination (34.4 %), followed by G3P[8] (16.3 %), G9P[8] (10.3 %), G4P[8] (8.9 %), G2P[4] (4 %), G12P[6] (2.6 %) and G12P[8] (1.9 %). Uncommon G/Pgenotype combinations, mixed infections and untypeable strains were also detected. This is the first report, in Tunisia, of multiple detection of an emerging human RVA strain, G12 genotype. This study highlighted the need for maintaining active surveillance of emerging strains in Northern Africa.

Feline origin of rotavirus strain, Tunisia, 2008.

In Tunisia in 2008, an unusual G6P[9] rotavirus, RVA/human-wt/TUN/17237/2008/G6P[9], rarely found in humans, was detected in a child. To determine the origin of this strain, we conducted phylogenetic analyses and found a unique genotype constellation resembling rotaviruses belonging to the feline BA222-like genotype constellation. The strain probably resulted from direct cat-to-human transmission.

Molecular characterization of the NSP4 gene of human group A rotavirus strains circulating in Tunisia from 2006 to 2008.

Non-structural protein 4 (NSP4), encoded by group A rotavirus (RVA) genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. Recently, a new classification system for RVAs was proposed and a total of 14 NSP4 genotypes (E1-E14) are currently described. The most common NSP4 genotypes in humans are Wa-like E1 and DS-1-like E2. This report represents the first investigation on the genetic diversity of RVA NSP4 genes in Tunisia from 2006 to 2008. In the present study, the NSP4-encoding genes of human RVA strains with different G/P-genotype combinations were analyzed. NSP4 genes of 261 RVA-positive fecal samples were analyzed using a semi-nested reverse transcriptase-polymerase chain reaction and in addition the NSP4 gene of 46 representative RVA strains were sequenced. Phylogenetic analysis of the Tunisian NSP4 nucleotide sequences revealed the presence of two NSP4 genotypes. Genotype E1 was found to be associated with G1P[8], G3P[6], G3P[8], G4P[6] and G4P[8], whereas genotype E2 was associated with G2P[4], G2P[6] and G6P[9] samples. These results support the hypothesis that P[8] carrying RVA strains usually possess the E1 genotype, whereas P[4] carrying RVA strains usually possess the E2 genotype. P[6] carrying strains were found with both E1 and E2. The unusual G6P[9] strains possessed a E2 genotype with a possible animal origin. These results underline the need for further investigations to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of RVA infections and vaccine development.

Rotavirus strain diversity in the centre coast of Tunisia from 2000 through 2003.

An epidemiological survey investigating rotavirus infection in children was undertaken in the coastal region of Tunisia from January 2000 through September 2003. A total of 309 fecal specimens were screened by enzyme-linked immunosorbent assay and latex agglutination assay for the presence of group A rotavirus antigen. The detection rate was 26.2%. Rotavirus outbreaks showed a temperature-dependant pattern (P = .026) but no significant association with rainfall. Rotavirus strains isolated were analyzed by RNA polyacrylamide gel electrophoresis and were characterized antigenically by monoclonal antibodies to the VP6 subgroup. Eight RNA electropherotypes were identified, with 3 long and 5 short different RNA profiles. Among VP6 typeable strains, all isolates with a long electrophoretic pattern carried the subgroup II specificity, whereas those with a short profile belonged to subgroup I. In total, 48 rotavirus-positive samples were analyzed for G and P typing by reverse-transcription polymerase chain reaction. A total of 8 different G and P combinations were found: G1P[8] (35.7%), G1P[6] (21.4%), G2P[4] (4.8%), G3P[4] (4.8%), G4P[6] (4.8%), G8P[8] (4.8%), G3P[8] (2.3%), and G4P[8] (2.3%). Mixed infections were detected in 19.1% of stool samples. The emergence in Tunisia of unconventional types, such as G8VP7 specificity, highlights the need for a continual survey of the uncommon strains in North Africa.

Rotavirus infection and intussusception in Tunisian children: implications for use of attenuated rotavirus vaccines.

BACKGROUND: A licensed rotavirus vaccine was withdrawn from use because of an increased risk of intussusception. The association of rotavirus vaccination with intussusception raised concerns about a potential link between natural rotavirus disease and intussusception. The objectives of the present study were to determine whether an epidemiological association with natural rotavirus infection existed. METHODS: From 1984 to 2003, all children younger than 5 years with intussusception were retrospectively identified by medical charts, and from 1995 to 2003, a prospective surveillance study of rotavirus infection in children younger than 5 years was independently conducted. Epidemiological characteristics of intussusception and rotavirus infection were then compared. RESULTS: A total of 533 cases of intussusception and 146 cases of rotavirus infection were identified. The incidence of intussusception for infants younger than 1 year was 62/100,000 child-years. The age distributions of intussusception and rotavirus gastroenteritis overlapped, and a masculine predominance was noted in both cases. No significant association was observed between the monthly distribution of intussusception and rotavirus infection. CONCLUSION: The present study has not convincingly shown that rotavirus diarrhea plays a major role in intussusception. However, data about age and sex distributions supported the biologic plausibility of such an association.

Emergence and characterization of human rotavirus g9 strains in Tunisia.

Among human rotaviruses, G9 has emerged as the fifth most important genotype circulating globally. Ongoing surveillance of rotavirus in Tunisia during the past 10 years identified the first G9 strains in 2004. These strains exhibited the P[8] VP4 genotype and had a long RNA electrophoretype. The G9 strains were characterized by phylogenetic analysis of the VP7 gene sequence and showed high identity with other human rotavirus G9 strains belonging to the rotavirus VP7 lineage group III.

Respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity.

The relationships between host factors, virus strain, viral load, and illness severity in respiratory syncytial virus (RSV)-induced bronchiolitis are poorly defined. These relationships were evaluated prospectively in 81 previously healthy infants hospitalized with RSV bronchiolitis. Disease severity was determined by the respiratory rate, the duration of hospitalization, and whether patients during their hospitalization required pediatric intensive care unit admission or mechanical ventilation. RSV typing into subgroup A and B was obtained by RT-PCR-hybridization assay. The nasopharyngeal RSV viral loads were measured by real-time quantitative RT-PCR. Disease severity correlated significantly with the presence of risk factor (estimated gestational age < 37 weeks and/or birth weight < 2,500 g) and with chronologic age