SARS-CoV2 RT-PCR assays: In vitro comparison of 4 WHO approved protocols on clinical specimens and its implications for real laboratory practice through variant emergence.

INTRODUCTION: RT-PCR testing on nasopharyngeal swabs is a key component in the COVID-19 fighting, provided to use sensitive and specific SARS-CoV2 genome targets. In this study, we aimed to evaluate and to compare 4 widely used WHO approved RT-PCR protocols on real clinical specimens, to decrypt the reasons of the diverging results and to propose recommendations for the choice of the genome targets. METHODS: 260 nasopharyngeal samples were randomly selected among the samples tested between Week-16, 2020 and week-16 2021, in the Institut Pasteur de Tunis, Tunisia, one of the referent laboratories of COVID-19 in Tunisia. All samples were tested by Charité, Berlin protocol (singleplex envelop (E) and singleplex RNA-dependent RNA polymerase (RdRp)), Hong Kong Universiy, China protocol (singleplex nucleoprotein (N) and singleplex Open reading frame Orf1b), commercial test DAAN Gene® (using the CDC China protocol), (triplex N, Orf1ab with internal control) and Institut Pasteur Paris protocol (IPP) (triplex IP2(nsp9) and IP4 (nsp12) with internal control). For IPP, a selection from samples positive by IP2 but negative with IP4 was re-tested by exactly the same protocol but this time in singleplex. New results were described and analyzed. RESULTS: In vitro analysis showed discordant results in 29.2% of cases (76 out of 260). The most discordant protocol is DAAN Gene® due to the false positive late signals with N target. Discordant results between the two protocol's targets are more frequent when viral load are low (high Ct values). Our results demonstrated that the multiplexing has worsened the sensitivity of the IP4 target. CONCLUSION: We provide concise recommendations for the choice of the genome targets, the interpretation of the results and the alarm signals which makes suspect a gene mutation.

The value of West Nile virus RNA detection by real-time RT-PCR in urine samples from patients with neuroinvasive forms.

INTRODUCTION: Routine laboratory screening is based on the detection of WNV specific IgM and IgG in blood and cerebrospinal fluid. Confirmation is then classically applied by real time RT-PCR (rRT-PCR) in Cerebrospinal fluid (CSF), which often gives negative results due to too short virorachia and late sampling. rRT-PCR was applied-for the first time for routine diagnosis purpose-on urine samples. METHODS: During 2018 outbreak in Tunisia, 107 patients presented WNV neurologic symptoms and were positive for WNV serology. Of them, 95 patients were sampled for urine and 35 were sampled for CSF. Qualitative rRT-PCR was performed on both type of samples. RESULTS: WNV RNA was detected in 50.5% of urine samples (48/95) and in 2.8% of CSF samples (1/35). WNV RNA was detectable from day 1 to day 41 from symptom onset, however, positive urine rate was 53.1% during the first 10 days from symptom onset. The proportions of urine-positive and urine-negative samples, based on day of collection, showed no statistical difference (p > 0.005). Cycle threshold (Ct) values ranged from 12 to 39, with no correlation with the day of collection. The lowest Ct value was detected for urine sampled on day 5 after symptom onset. A statistically significant difference was found between age groups of confirmed and non confirmed cases (p < 0.001). DISCUSSION/CONCLUSION: Our study reported the use of rRT-PCR on urine samples as a confirmatory diagnostic tool for WNV "probable cases" during an outbreak. Our findings underlined the reliability and the rapidity of this confirmatory tool, even late, and showed its superiority on CSF investigation.

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.

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.

Association of CCR5Δ32 Deletion and Human Cytomegalovirus Infection With Colorectal Cancer in Tunisia.

Background and objectives: Human cytomegalovirus (HCMV) and genetic polymorphisms of the chemokine receptor 5 have been suggested as factors associated with the progression of colorectal cancer (CRC). The aim of the study was to evaluate the associations of both CCR5Δ32 genetic deletion and/or HCMV virus infection with CRC in Tunisia. MATERIALS AND METHODS: The association between HCMV and CRC was validated by Nested PCR technology performed for HCMV and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. Experiments were carried out on 40 tumor and 35 peri-tumor tissues, 100 blood from CRC patients and on 140 blood samples from healthy subjects and finaly serum samples of 80 patients with CRC and 100 healthy individuals. A conventional PCR has been optimized for the detection of CCR5Δ32 in100 CRC patients and 100 healthy subjects. RESULTS: Our results show that HCMV is significantly active in 93% of patients compared to 60% in controls (p < 0.0001, OR = 8.85, 95% CI: 3.82 -20.50). Compared to the healthy controls, the titers of IgG and IgM antiCMV antibodies in CRC patients were significantly higher than in healthy subjects (p value < 0,0001 for IgG and IgM). Statistical analysis revealed a lack of association between CCR5Δ32 mutation and colorectal cancer (p = 0.788, OR = 1.265, 95% CI: 0.228-7.011). CONCLUSION: our data confirmed that the HCMV infection was related to the development of CRC and that CRC cells may be infected more favorably by HCMV. Given the importance of the CCR5 in inflammation and therefore CRC progression, further studies still needed to evaluate CCR5 role as a potential candidate gene for CRC susceptibility under other polymorphisms.

Increased endothelin-1 levels of BAL fluid in patients with Behçet's disease.

OBJECTIVE AND BACKGROUND: Pulmonary aneurysms and thrombosis constitute a significant cause of morbidity and mortality in Behçet's disease (BD). Various factors have been studied to explore the pathogenesis of vascular involvement in BD. As endothelin (ET) is known for its potent vasoconstrictor and proinflammatory properties, we supposed that it is involved during the inflammatory process of BD pulmonary vasculitis. METHODS: To investigate the role of ET in BD, ET-1 concentrations were measured in bronchoalveolar lavage fluid (BALF) of 18 nonsmoking BD patients with pulmonary manifestations and 12 control subjects. Immunoreactivity of ET-1 was also evaluated in alveolar macrophages (AMs) cytoplasm. RESULTS: ET-1 levels in BD-BALF were significantly higher than those of controls. ET-1 levels were correlated with the number of alveolar macrophages, but not with BAL-CD4/CD8 ratio. ET-1-immunoreactivity was found mainly in AM of BD-BAL. CONCLUSIONS: Increased ET-1 production from AM is associated with pulmonary BD manifestations.

Associations of vitamin D receptor gene polymorphisms FokI and BsmI with susceptibility to rheumatoid arthritis and Behçet's disease in Tunisians.

OBJECTIVES: Reports of immunomodulating effects of vitamin D suggest a need for examining allele and genotype frequencies of the vitamin D nuclear receptor gene (VDR) in patients with autoimmune diseases. T-helper-1 (Th1) counts in peripheral blood are increased in both rheumatoid arthritis (RA) and Behçet's disease (BD). We studied VDR polymorphisms in patients with these two diseases in Tunisia. METHODS: In 108 patients with RA, 131 patients with BD, and 152 controls, we studied FokI and BsmI VDR polymorphisms, using the restriction fragment length polymorphism technique. RESULTS: The FokI polymorphism alleles and genotype were significantly more common in the RA group than in the controls (P=0.001 and P=0.005, respectively). The FokI F allele and F/F genotype were significantly associated with BD (P=0.0003 and P=0.002, respectively). Furthermore, in the group with BD, the FokI polymorphism was significantly associated with the presence of vascular manifestations (P=0.006). In patients with RA, the FokI polymorphism was significantly associated with female gender (P=0.003). No significant associations were found between the Bsm1 polymorphism and RA or BD. CONCLUSION: The VDR F allele is associated with RA and BD in Tunisians.

Association of vascular endothelial growth factor polymorphisms with asthma in Tunisian children.

BACKGROUND: Previous studies demonstrated that the vascular endothelial growth factor (VEGF) was being implicated in the airways inflammation and remodeling process in patients with asthma. AIMS: We explored the relationship of three polymorphisms in the VEGF gene with asthma in both case control and family studies. METHODS: We Genotyped a total of 210 children with asthma, 224 unrelated controls and 160 parents for the +936 C >T (rs3025039), -634 G > C (rs2010963) and -2549 -2567 del 18 of the VEGF promoter region. The Mutations were identified with polymerase chain reaction followed by restriction fragment length polymorphism (RFLP) analysis for the +936 C > T, and -634 G > C polymorphisms. RESULTS: Of the three polymorphisms studied, a borderline association with asthma was found for the G allele in the -634 G > C polymorphism (p = 0.059). No Statistically significant differences were observed for both +936 C > T, and -2549 -2567 del 18 polymorphisms between asthmatic patients and controls, considering either allelic or genotypic frequencies. The distribution of genotypes according to the severity status revealed a significant differences for the +936 C > T, and -2549 -2567 del 18 polymorphisms. In addition, association was found with the haplotypes inferred by the three polymorphisms and asthma susceptibility. CONCLUSION: We suggest that VEGF Gene polymorphisms can be implicated in asthma.