Development and evaluation of an easy to use real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus.

West Nile Virus (WNV) causes a serious public health concern in many countries around the world. Virus detection in pathological samples is a key component of WNV infection diagnostic, classically performed by real-time PCR. In outbreak situation, rapid detection of the virus, in peripheral laboratories or at point of care, is crucial to guide decision makers and for the establishment of adequate action plans to prevent virus dissemination. Here, we evaluate a Loop-mediated isothermal amplification (LAMP) tool for WNV detection. Amplifications were performed comparatively on extracted viral RNA and on crude samples using a classical thermal cycler and a portable device (pebble device). qRT-PCR was used as gold standard and two sets of urine samples (n = 62 and n = 74) were used to evaluate the retained amplification protocols and assess their sensitivity and specificity. RT-LAMP on RNA extracts and crude samples showed a sensitivity of 90 % and 87 %, respectively. The specificity was 100 % for extracts and 97 % for crude samples. Using the device, the RT-LAMP on extracted RNA was comparable to the gold standard results (100 % sensitivity and specificity) and it was a bit lower on crude samples (65 % sensitivity and 94 % specificity). These results show that RT-LAMP is an efficient technique to detect WNV. RT-LAMP provides a rapid, sensitive, high-throughput and portable tool for accurate WNV detection and has potentials to facilitate diagnostic and surveillance efforts both in the laboratory and in the field, especially in developing countries.

Whole genome analysis of hepatitis B virus before and during long-term therapy in chronic infected patients: Molecular characterization, impact on treatment and liver disease progression.

Hepatitis B virus (HBV) infection remains a serious public health concern worldwide despite the availability of an efficient vaccine and the major improvements in antiviral treatments. The aim of the present study is to analyze the mutational profile of the HBV whole genome in ETV non-responder chronic HBV patients, in order to investigate antiviral drug resistance, immune escape, and liver disease progression to Liver Cirrhosis (LC) or Hepatocellular Carcinoma (HCC). Blood samples were collected from five chronic hepatitis B patients. For each patient, two plasma samples were collected, before and during the treatment. Whole genome sequencing was performed using Sanger technology. Phylogenetic analysis comparing the studied sequences with reference ones was used for genotyping. The mutational profile was analyzed by comparison with the reference sequence M32138. Genotyping showed that the studied strains belong to subgenotypes D1, D7, and D8. The mutational analysis showed high genetic variability. In the RT region of the polymerase gene, 28 amino acid (aa) mutations were detected. The most significant mutations were the pattern rtL180M + rtS202G + rtM204V, which confer treatment resistance. In the S gene, 35 mutations were detected namely sP120T, sT126S, sG130R, sY134F, sS193L, sI195M, and sL216stop were previously described to lead to vaccine, immunotherapy, and/or diagnosis escape. In the C gene, 34 mutations were found. In particular, cG1764A, cC1766G/T, cT1768A, and cC1773T in the BCP; cG1896A and cG1899A in the precore region and cT12S, cE64D, cA80T, and cP130Q in the core region were associated with disease progression to LC and/or HCC. Other mutations were associated with viral replication increase including cT1753V, cG1764A/T, cC1766G/T, cT1768A, and cC1788G in the BCP as well as cG1896A and cG1899A in the precore region. In the X gene, 30 aa substitutions were detected, of which substitutions xT36D, xP46S, xA47T, xI88F, xA102V, xI127T, xK130M, xV131I, and xF132Y were previously described to lead to LC and/or HCC disease progression. In conclusion, our results show high genetic variability in the long-term treatment of chronic HBV patients causing several effects. This could contribute to guiding national efforts to optimize relevant HBV treatment management in order to achieve the global hepatitis elimination goal by 2030.

SARS-CoV-2 infection in pediatric population before and during the Delta (B.1.617.2) and Omicron (B.1.1.529) variants era.

BACKGROUND: COVID-19, the coronavirus disease that emerged in December 2019, caused drastic damage worldwide. At the beginning of the pandemic, available data suggested that the infection occurs more frequently in adults than in infants. In this review, we aim to provide an overview of SARS-CoV-2 infection in children before and after B.1.617.2 Delta and B.1.1.529 Omicron variants emergence in terms of prevalence, transmission dynamics, clinical manifestations, complications and risk factors. METHODS: Our method is based on the literature search on PubMed, Science Direct and Google Scholar. From January 2020 to July 2022, a total of 229 references, relevant for the purpose of this review, were considered. RESULTS: The incidence of SARS-CoV-2 infection in infants was underestimated. Up to the first half of May, most of the infected children presented asymptomatic or mild manifestations. The prevalence of COVID-19 varied from country to another: the highest was reported in the United States (22.5%). COVID-19 can progress and become more severe, especially with the presence of underlying health conditions. It can also progress into Kawasaki or Multisystem Inflammatory Syndrome (MIS) manifestations, as a consequence of exacerbating immune response. With the emergence of the B.1.617.2 Delta and B.1.1.529 Omicron variants, it seems that these variants affect a large proportion of the younger population with the appearance of clinical manifestations similar to those presented by adults with important hospitalization rates. CONCLUSION: The pediatric population constitutes a vulnerable group that requires particular attention, especially with the emergence of more virulent variants. The increase of symptomatic SARS-CoV-2 infection and hospitalization rate among children highlights the need to extend vaccination to the pediatric population.

Molecular Epidemiology of SARS-CoV-2 in Tunisia (North Africa) through Several Successive Waves of COVID-19.

Documenting the circulation dynamics of SARS-CoV-2 variants in different regions of the world is crucial for monitoring virus transmission worldwide and contributing to global efforts towards combating the pandemic. Tunisia has experienced several waves of COVID-19 with a significant number of infections and deaths. The present study provides genetic information on the different lineages of SARS-CoV-2 that circulated in Tunisia over 17 months. Lineages were assigned for 1359 samples using whole-genome sequencing, partial S gene sequencing and variant-specific real-time RT-PCR tests. Forty-eight different lineages of SARS-CoV-2 were identified, including variants of concern (VOCs), variants of interest (VOIs) and variants under monitoring (VUMs), particularly Alpha, Beta, Delta, A.27, Zeta and Eta. The first wave, limited to imported and import-related cases, was characterized by a small number of positive samples and lineages. During the second wave, a large number of lineages were detected; the third wave was marked by the predominance of the Alpha VOC, and the fourth wave was characterized by the predominance of the Delta VOC. This study adds new genomic data to the global context of COVID-19, particularly from the North African region, and highlights the importance of the timely molecular characterization of circulating strains.

Evidence of Circulation of Several HAV Genetic Variants and Emergence of Potential Antigenic Variants in an Endemo-Epidemic Country before Vaccine Introduction.

Similar to several other countries in the world, the epidemiology of hepatitis A virus changed from high to intermediate endemicity level in Tunisia, which led to the occurrence of outbreaks. This study aimed to determine the genetic and antigenic variability of HAV strains circulating in Tunisia during the last few years. Genotyping using complete VP1 gene and VP1-2A junction confirmed the predominance of genotype IA, with co-circulation of several genetic and antigenic variants. Phylogenetic analysis including Tunisian and strains from other regions of the world showed the presence of at least two IA-variants within IA subgenotype. Amino-acid analysis showed several mutations in or close to epitope regions in the VP1-region. This study provides a baseline on the genetic and antigenic variability of HAV circulating strains before the introduction of vaccination into the national immunization schedule.

Strong association of functional polymorphism in IL-12B with susceptibility to chronic hepatitis B in Tunisia.

BACKGROUND: The chronicity or clearance of hepatitis B virus (HBV) infection depends on viral and genetic variables. The immune response against HBV is thought to be responsible for viral persistence or clearance. Cytokines such as interleukin 1-2B (IL1-2B) involved in the T-helper 1 system are key mediators in the defence mechanisms against viral infection and play a role in the regulation of HBV clearance during infection. We aimed to examine whether the polymorphic variant TaqI polymorphism in the 3'-untranslated region (3'-UTR; rs3212227) suspected to modulate interleukin-levels of IL-12B has an influence on the risk of development of chronicity after HBV exposure. METHODS: Genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism method for 236 patients with chronic hepatitis B (CHB) and 240 controls from different cities of Tunisia recruited in the Pasteur Institute of Tunisia between January 2017 and December 2018. RESULTS: We found that the IL-12B polymorphism was associated with a significantly increased risk of CHB in patients (p = 1 × 10-3 ; χ 2 = 10.31 and odds ratio [OR] = 2.14; 95% confidence interval [CI] = 1.30-3.52) when AC/CC variant genotypes were compared with the wild-type AA homozygote. Statistical significance was found when CHB-males were compared with CHB-females (p = 2 × 10-7 ; χ 2 = 26.62 and p = 1 × 10-3 ; χ 2 = 10.36, for genotypic and allelic frequencies, respectively). Also, CHB-patients carrying C-allele less than 50-years were at an increased risk of developing chronic HBV infection than patients more than 50-years (p = 6.1 × 10-5 ; χ 2 = 16.07). CONCLUSIONS: These results suggest that the C-allele would affect susceptibility to chronicity after HBV exposure in Tunisian patients especially for males less than 50-years. Age and sex have an influence on this polymorphism in CHB Tunisian patients.

In silico comparative study of SARS-CoV-2 proteins and antigenic proteins in BCG, OPV, MMR and other vaccines: evidence of a possible putative protective effect.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) is a viral pandemic disease that may induce severe pneumonia in humans. In this paper, we investigated the putative implication of 12 vaccines, including BCG, OPV and MMR in the protection against COVID-19. Sequences of the main antigenic proteins in the investigated vaccines and SARS-CoV-2 proteins were compared to identify similar patterns. The immunogenic effect of identified segments was, then, assessed using a combination of structural and antigenicity prediction tools. RESULTS: A total of 14 highly similar segments were identified in the investigated vaccines. Structural and antigenicity prediction analysis showed that, among the identified patterns, three segments in Hepatitis B, Tetanus, and Measles proteins presented antigenic properties that can induce putative protective effect against COVID-19. CONCLUSIONS: Our results suggest a possible protective effect of HBV, Tetanus and Measles vaccines against COVID-19, which may explain the variation of the disease severity among regions.

Seroprevalence of hepatitis A virus infection in Central-West of Tunisia.

Hepatitis A infections still represent a major global health concern. During the past years, a transition pattern of the hepatitis A epidemiology was noted in many parts of the world. In Tunisia, there is not a recent survey on age-specific hepatitis A virus seroprevalence. This study aimed to investigate the seroprevalence of hepatitis A virus infection in Central-West Tunisia, representative of regions with lowest socioeconomic level in the country, before vaccine implementation. Sera obtained from the blood samples of subjects were screened for the detection of hepatitis A virus. The seroprevalence was evaluated by detection of total antibodies to hepatitis A virus using commercially available immunoassay kits. A total of 1379 subjects, aged 5-75 years (mean age: 29.0 ± 17.3 years) were studied. The global anti-hepatitis A virus seroplevalence was 84.7% (95% confidence interval: [82.6-86.5]). A higher hepatitis A virus seroprevalence was showed in subjects aged 10-14 years compared to those aged less than 10 years (50.0% vs. 31.0%). In subjects aged 20-29 years, a rapid increase in the hepatitis A virus prevalence was noted; it reached 97.0%. The seroprevalence of anti-hepatitis A virus differed by zone of residence (81.1% in rural area vs. 72.4% in urban area, p = .005) and increased significantly with lower level of education (p = .019). There was no statistical significant seroprevalence difference between male and female: 84.2% versus 85.2%, respectively. Our study confirm the transition pattern of the hepatitis A virus endemicity in Tunisia from high to intermediate and provide an evaluation of the hepatitis A virus epidemiological situation before vaccine implementation.

Hepatitis a virus infection in Central-West Tunisia: an age structured model of transmission and vaccination impact.

BACKGROUND: The epidemiological pattern of hepatitis A infection has shown dynamic changes in many parts of the world due to improved socio-economic conditions and the accumulation of seronegative subjects, which leads to possible outbreaks and increased morbidity rate. In Tunisia, the epidemiological status of hepatits A virus is currently unknown. However, over the past years higher numbers of symptomatic hepatitis A virus infection in school attendants and several outbreaks were reported to the Ministry of Health, especially from regions with the lowest socio-economic levels in the country. The aim of this study was to investigate the current seroprevalence of hepatitis A virus antibodies in central-west Tunisia and assess the impact of hepatitis A virus vaccination on hepatitis A epidemiology. METHODS: Serum samples from 1379 individuals, aged 5-75 years, were screened for hepatitis A virus antibodies. Adjusted seroprevalence, incidence and force of infection parameters were estimated by a linear age structured SEIR (Susceptible-Exposed-Infectious-Recovered) compartmental model. A vaccine model was then constructed to assess the impact on hepatitis A virus epidemiology of 3 scenarios of vaccination strategies: one dose at 12-months of age, one dose at 6-years and one dose at 12-months and another at 6-years of age during 6 years. RESULTS: A rapid increase in anti-hepatitis A virus seroprevalence was noted during infancy and adolescence: 47% of subjects under 10-years-old are infected; the prevalence increases to 77% at 15-years and reaches 97% in subjects aged 30-years. The force of infection is highest between 10 and 30-years of age and the incidence declines with increasing age. The vaccine model showed that the 3-scenarios lead to a significant reduction of the fraction of susceptibles. The two doses scenario gives the best results. Single-dose vaccination at 6-years of age provides more rapid decrease of disease burden in school-aged children, as compared to single-dose vaccination at 12-months, but keeps with a non-negligible fraction of susceptibles among children < 6-years. CONCLUSIONS: Our study confirms the epidemiological switch from high to intermediate endemicity of hepatitis A virus in Tunisia and provides models that may help undertake best decisions in terms of vaccinations strategies.

Interleukin IL-1B gene polymorphism in Tunisian patients with chronic hepatitis B infection: Association with replication levels.

Approaches based on association studies have proven useful in identifying genetic predictors for many diseases, including susceptibility to chronic hepatitis B. In this study we were interested by the IL-1B genetic variants that have been involved in the immune response and we analyzed their role in the susceptibility to develop chronic hepatitis B in the Tunisian population. IL-1B is a potent proinflammatory cytokine that plays an important role in inflammation of the liver. Polymorphic gene IL-1 (-511, +3954) was analyzed in a total of 476 individuals: 236 patients with chronic hepatitis B from different cities of Tunisia recruited in Pasteur Institute between January 2017 and December 2018 and 240 controls. Genomic DNA was obtained using the standard salting-out method and genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For -511C>T polymorphism a significant association was found between patients and controls when comparing the genotypic (P = 0.007; χ2 = 9.74 and odds ratio [OR] = 0.60; confidence interval [CI] = 0.41-0.89) and allelic (P = 0.001; χ2 = 10.60) frequencies. When the viral load was taken into account a highly significant difference was found (P = 9 × 10-4 ; χ2 = 10.89). For +3954C>T polymorphism a significant association was found between patients and controls when comparing genotypic (P = 0.0058; χ2 = 7.60 and OR = 1.67; CI = 1.14-2.46) and allelic (P = 0.0029; χ2 = 8.81) frequencies. T allele can be used as a strong marker for hepatitis B virus disease for both polymorphisms.

Genotype Distribution and Prevalence of Human Pegivirus among High-Risk Populations in Tunisia.

OBJECTIVE: A recently discovered non-A-E hepatitis virus has been designated as human Pegivirus (HPgV). HPgV is prevalent in high-risk groups such as patients with hepatitis C virus (HCV), and it is of interest for patients who are at risk for transmitted infections. The aim of this study was to evaluate the prevalence of HPgV as well as the genotype distribution among patients in the Tunisian population who are infected with HCV and also in multitransfused patients. METHODS: A total of 144 patients were screened using RTPCR/nested PCR of the 5'-untranslated region (UTR); 14 cases were sequenced and phylogenetically analyzed. RESULTS: Seven (14.9%) subjects from the multitransfused group and 7 (7.2%) patients infected with HCV, respectively, were found positive for HPgV RNA. Sequencing and phylogenetic analysis of the 14 cases revealed that genotype 2a was the main genotype circulating in Tunisian patients. Genotype 2b was found in the amplified samples of 2 HCV-infected patients. CONCLUSION: This study enriches the limited data on HPgV prevalence in Tunisia, and shows, for the first time, the molecular epidemiology of the circulating strains in this country.

[Immunohistochemical analysis of mismatch repair proteins in colorectal adenomas]. / Analyse immunohistochimique des protéins de réparation de I'ADN dans les adénomes colorectaux.

BACKGROUND: The deficiency of mismatch repair system is one of the main pathways in colorectal cancer. This system consists mainly of four proteins: MLH1, MSH2, MSH6 and PMS2. Colorectal cancer develops in the majority of cases from precancerous lesions called adenomas. Only few studies have reported on the deficiencies of these proteins in adenomas. AIM: In this study we used immunohistochemistry staining in colorectal adenomas to assay functional status of MLH1, MSH2, MSH6, and PMS2 proteins. METHODS: 102 adenomas from 93 patients were collected in our institution during six years (2007-2012). The immunohistochemical technique was performed with 4 antibodies: MLH1, MSH2, MSH6 and PMS2. The loss of expression was retained if adenomatous cells were not stained with positive internal control. Staining was considered as abnormal if nucleus of adenomatous cells showed low nuclear staining and / or heterogeneous one, while positive internal control had normal staining. RESULTS: Loss of expression of MSH2 and MSH6 in adenomatous cells was found in only 1 case which was a tubular adenoma 3mm high-grade dysplasia. Abnormal staining of the adenomatous cells was noted in 23 cases (22.5%) for MSH2 and in 8 cases (7.8%) for MSH6. No cases showed loss of expression of MLH1 and PMS2. Abnormal expression of MSH2 and MSH6 was not correlated with sex of patients, the location of the adenoma, its grade of dysplasia and its histological type. CONCLUSIONS: Loss of Mismatch repair proteins expression is a rare event in adenomas. However, the abnormal expression levels are higher in our study compared to those reported in the literature. This could reflect a higher rate of microsatellite instability in our patients. Multicenter and larger studies with molecular biology techniques are needed.