Mucosal human papillomavirus detection and TP53 immunohistochemical expression in non-melanoma skin cancer in Tunisian patients.

BACKGROUND: Several studies have reported the oncogenic role of human papillomavirus (HPV) in non-melanoma skin cancer (NMSC) carcinogenesis. Considering that HPV could affect tumor protein 53 (TP53) degradation via E6 oncoprotein, we evaluated the expression of TP53 according to HPV infection and E6 expression. METHODS: Biopsy specimens from 79 NMSCs (28 squamous cell carcinomas, 21 keratoacanthomas and 30 basal cell carcinomas) were enrolled. Nested PCR was used to detect mucosal HPV (mHPV) DNA. Genotyping was performed by reverse line hybridization. Expression of TP53 and E6 was evaluated by immunohistochemistry. RESULTS: mHPVs were detected in 34.2% (27/79) of NMSC, with 92.6% (25/27) of high-risk HPV (HR-HPV) types. HPV16-E6-positive expression was observed in all HPV16-positive samples. TP53 high expression was found in 51.4% (37/72) of specimens. In this group, 78.4% were HPV-negative (P = 0.014). TP53 expression was negative in 8/10 of HPV E6-positive specimens. Multivariate analysis showed that TP53 was associated with HPV infection independently of histopathologic type (P = 0.005). CONCLUSION: This study showed a high prevalence of mHPV in NMSC. Active infections assessed by E6 expression are associated with loss of p53 function, highlighting the involvement of mHPV in NMSC carcinogenesis.

Distribution of high-risk human papillomavirus in women with ASC-US or LSIL in Tunisian women: Place of HPV testing for women triage in Tunisia.

The present study aimed to evaluate the distribution of oncogenic HPVs in Tunisian women diagnosed with ASC-US or LSIL in order to highlight the importance of HPV testing in the management of women with minor cytological lesions. The study involved 213 cervical samples from women aged from 18 to 82 years and diagnosed with ASC-US or LSIL. HPV detection and genotyping was performed by nested PCR followed by reverse Line Blotting. HPV DNA was identified in 161 cases (76.3%). Oncogenic HPV genotypes were detected in 53.1% of cases. The most frequent high-risk genotypes in this study were HPV16 (28.8%) followed by: HPV51 (9.6%), HPV18, HPV31 HPV56 (7.1%) and HPV45 (5.1%). Thus, 24 % of studied women were not infected by HPV and about 47% of infections are without oncogenic HPV. These results highlight the value of HPV testing in the decision algorithm of management of minor abnormalities lesions.

The E6 gene polymorphism of Human papillomavirus 16 in relation to the risk of cervical cancer in Tunisian women.

Human papillomavirus type 16 (HPV-16) is the most prevalent HPV type worldwide and in Tunisia and the major carcinogenic HPV type found in cervical precancers and cancers. Previous studies have reported that genetic diversity of HPV16-E6 oncoprotein might be associated with cervical intraepithelial neoplasia progression. In this study we aimed to investigate the prevalence of HPV-16 E6 variants in precancerous lesions in Tunisian population to assess potential correlation with disease severity. Positive HPV cervical samples were obtained from the Laboratory of Anatomy Pathology of Pasteur Institute of Tunis. Cytological study was performed to identify cervical precancerous lesions. HPVs were typed using Reverse Line Hybridization. Only samples with HPV-16 single infection were selected for HP16-E6 genetic diversity investigation. HPV-16 E6 gene amplification was performed by PCR using specific primers and sequenced by Sanger Sequencing. The multiple alignment of generated sequences was performed using MEGAX software. Phylogenetic tree was constructed using Maximum Likehood method. The ternary complex of E6, E6AP and p53 core domain was used to perform in silico point mutations and thermodynamic calculations to assess stability and binding affinity. Genetic analysis of Tunisian E6-HPV16 sequences showed the presence of three lineages: European (A), African (C) and Asian American (D). Interestingly, the EUR variants were identified as the dominant lineage of HPV-16 and HPV-16 E6 350 G (L83V) was the most detected mutation in precancerous lesions. Modelling data showed that African variants induced the largest destabilizing effect on E6 structure and decreasing thereby in the affinity toward E6AP. Therefore, women infected with European variants are associated with low and high intraepithelial lesions. The findings give useful information for personalized decision algorithms of intra-epithelial cervical neoplasia in Tunisian women.

PHINDaccess Hackathons for COVID-19 and Host-Pathogen Interaction: Lessons Learned and Recommendations for Low- and Middle-Income Countries.

Hackathons are collaborative events that bring together diverse groups to solve predefined challenges. The COVID-19 pandemic caused by SARS-CoV-2 has emphasized the need for portable and reproducible genomics analysis pipelines to study the genetic susceptibility of the human host and investigate human-SARS-CoV-2 protein interactions. To build and strengthen institutional capacities in OMICS data analysis applied to host-pathogen interaction (HPI), the PHINDaccess project organized two hackathons in 2020 and 2021. These hackathons are aimed at developing bioinformatics pipelines related to the SARS-CoV-2 viral genome, its phylodynamic transmission, and the identification of human genome host variants, with a focus on addressing global health challenges, particularly in low- and middle-income countries (LMIC). This paper outlines the preparation, proceedings, and lessons learned from these hackathons, including the challenges faced by participants and our recommendations based on our experience for organizing hackathons in LMIC and beyond.

TLR9 and Glioma: Friends or Foes?

Toll-like receptor 9 (TLR9) is an intracellular innate immunity receptor that plays a vital role in chronic inflammation and in recognizing pathogenic and self-DNA in immune complexes. This activation of intracellular signaling leads to the transcription of either immune-related or malignancy genes through specific transcription factors. Thus, it has been hypothesized that TLR9 may cause glioma. This article reviews the roles of TLR9 in the pathogenesis of glioma and its related signaling molecules in either defending or promoting glioma. TLR9 mediates the invasion-induced hypoxia of brain cancer cells by the activation of matrix metalloproteinases (2, 9, and 13) in brain tissues. In contrast, the combination of the TLR9 agonist CpG ODN to radiotherapy boosts the role of T cells in antitumor effects. The TLR9 agonist CpG ODN 107 also enhances the radiosensitivity of human glioma U87 cells by blocking tumor angiogenesis. CpG enhances apoptosis in vitro and in vivo. Furthermore, it can enhance the antigen-presenting capacity of microglia, switch immune response toward CD8 T cells, and reduce the number of CD4CD25 Treg cells. CpG ODN shows promise as a potent immunotherapeutic drug against cancer, but specific cautions should be taken when activating TLR9, especially in the case of glioblastoma.

The Prevalence, Genotype Distribution and Risk Factors of Human Papillomavirus in Tunisia: A National-Based Study.

There are limited national population-based studies on HPV genotypes distribution in Tunisia, thus making difficult an assessment of the burden of vaccine-preventable cervical cancer. In this context, we conducted a national survey to determine the HPV prevalence and genotypes distribution and the risk factors for HPV infections in Tunisian women. This is a cross-sectional study performed between December 2012 and December 2014. A liquid-based Pap smear sample was obtained from all women and samples' DNAs were extracted. Only women with betaglobin-positive PCR were further analysed for HPV detection and typing by a nested-PCR of the L1 region followed by next-generation sequencing. A multiple logistic regression model was used for the analysis of associations between the variables. A total of 1517 women were enrolled in this study, and 1229 out of the 1517 cervical samples were positive for the betaglobin control PCR and tested for HPV. Overall HPV infection prevalence was measured to be 7.8% (96/1229), with significant differences between the grand regions, ranging from 2% in the North to 13.1% in Grand Tunis. High-risk HPV genotypes accounted for 5% of the infections. The most prevalent genotypes were HPV 31 (1%), 16 (0.9%), 59 (0.7%). HPV18 was detected only in four cases of the study population. Potential risk factors were living in Grand Tunis region (OR: 7.94 [2.74-22.99]), married status (OR: 2.74 [1.23-6.13]), smoking habit (OR: 2.73 [1.35-5.51]), occupation (OR: 1.81 [1.09-3.01]) and women with multiple sexual partners (OR: 1.91 [1.07-3.39]). These findings underscore the need to evaluate the cost effectiveness of HPV vaccine implementation, contribute to the evidence on the burden of HPV infections, the critical role of sexual behaviour and socioeconomic status, and call for increased support to the preventive program of cervical cancer in Tunisia.

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.

Distribution of human papillomavirus in precancerous and cancerous cervical neoplasia in Tunisian women.

BACKGROUND: High-risk human papillomavirus (HR-HPV) are responsible for cervical cancer (CC) which represents the second most prevalent gynecological cancer among Tunisian women. Preventive strategies against CC are based on prophylactic vaccines that have not yet been implemented into the national vaccination program of Tunisia. Therefore, the present study aimed to investigate the HPV genotypes distribution in cervical neoplasia in Tunisian women in order to predict the impact of using current HPV vaccines on cancer prevention in Tunisia. METHODS: A total of 200 formalin-fixed paraffin embedded biopsies were collected in our study. DNA was extracted using Qiagen Mini prep kit. DNA quality was controlled by Beta Globin PCR. Only positive samples for Beta Globin test were used. HPV detection was performed by a nested PCR using PYGMY and GP5+/6+ primers. Genotyping was performed by Reverse Line hybridization using 31 probes. RESULTS: The mean age of participants was 38.97 years and 75% were over 30 years. Cervical neoplasia distribution according to age showed that CINII/CINIII was observed among women over 30 years old. All samples were positive for Beta Globin PCR. Overall HPV prevalence in cervical lesions was 83% (166/200). HPV was present in 65% of CINI, 82% of CINII/CINIII and 85% of CC. HR-HPV was statistically significantly associated with cervical intraepithelial neoplasia (p < 10-3). HR-HPV distribution according to lesion grade and cervical cancer showed that HPV16 and HPV18 were present in all lesions. For CINII/CINIII, HPV 35 (37.5%) was the most detected type, followed by HPV18 (33.3%) HPV 45 (28.5%) and HPV 16 (18.9%). HPV 45(57.5%), HPV 18 (53.3%) were the most detected in CC. HPV58, 59, 68 were only detected in CC and associated with HPV45, 18 and HPV16. HPV39, 31, 33, 52, 56 and HPV70 was associated only with CINI. CONCLUSIONS: Our findings can give useful information for vaccine implementation by helping the health policymakers to choose the most appropriate vaccine type in Tunisia.

Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples.

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/µl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

New insights in the clinical implication of HOXA5 as prognostic biomarker in patients with colorectal cancer.

BACKGROUND: Homeobox A5 (HOXA5) is a member of the HOX protein family which is involved in several carcinogenesis pathways, and is dysregulated in many cancer types. However, its expression and function in human colorectal cancer (CRC) is still largely unknown. OBJECTIVE: This study aimed to evaluate HOXA5 expression in Tunisian patients with CRC in order to define new potential biomarker. METHODS: An immunohistochemical labeling using an HOXA5 antibody was performed on 85 formalin fixed paraffin embedded specimens from patients with CRC. Six normal colon mucosa cases were used as controls. RESULTS: HOXA5 expression showed a cytoplasmic staining in both tumor and stromal/endothelial cells. Loss or low HOXA5 expression was seen in tumor cells in 74/85 cases (87.06%) and in stromal/endothelial cells, in 77/85 (90.59%). In control group of normal colon mucosa HOXA5 was moderately expressed in all the cases. The abnormal expression, was significantly associated to lymph nodes metastasis in tumor cells (p= 0.043) and in stromal/endothelial cells (p= 0.024). CONCLUSION: HOXA5 immunostaining results suggest the valuable role of this protein in colorectal carcinogenesis. Moreover, the association of lymph node metastasis to HOXA5 abnormal expression underlies its crucial role in colorectal cancer dissemination and prognosis.

Prevalence, Genotype Distribution and Risk Factors for Cervical Human Papillomavirus Infection in the Grand Tunis Region, Tunisia.

Implementation of Human Papillomavirus (HPV) vaccination should be considered a key cervical cancer prevention strategy in Tunisia, where Pap smear screening is not efficient. This study aims to estimate the prevalence and to identify risk factors associated with HPV infection among women from Grand Tunis, Tunisia. We conducted a cross-sectional study, between December 2012 and May 2013. Eligible women for this study were those aged 18-65 years, sexually active, who sought medical attention at their primary health care centre or clinic in Grand Tunis, Tunisia and who gave written consent. A liquid-based Pap smear sample was obtained from all women using a cervical brush. Only women with betaglobin positive test were further analysed for HPV detection and typing. A nested-PCR of the L1 region was performed followed by reverse line blot hybridization to facilitate the specific detection of 31 HPV genotypes. Multiple logistic regression modeling was used for the analysis of associations between variables with some considered possible confounders after checking for interactions. A total of 391 women were enrolled in this study and 325 out of the 391 cervical samples were positive for the betaglobin test. Overall HPV prevalence was 13.2% [9.8%-17.5%], with the following most prevalent HPV genotypes: HPV6 (40%), HPV40 (14%), HPV16 (12%), HPV52 (9%), HPV31 and HPV59 (7%), followed by HPV68 (4%). Mean age of HPV positive women was 40.7±0.92 years. Independently associated risk factors of HPV infection were smoking (OR:2.8 [0.8-9.6]), low income (OR:9.6 [1.4-63.4), bad housing type (OR:2.5 [1-6.8]), partner with multiple sexual relationship (OR:4.5 [0.9-22.9]) and single women (widowed, divorced, separated, never married) (OR:6.9 [1.1-42.2]). This study provides the first national-based estimate of HPV prevalence in Tunisia. Our findings contribute to the evidence on the current burden of HPV infection, the critical role of sexual behaviour and socioeconomic status and call for increased support for the screening program in Tunisia to prevent cervical cancer. These results allow us to evaluate the cost-effectiveness of vaccine program implementation in Tunisia in future.

Expression of Toll-like receptor 9 increases with progression of cervical neoplasia in Tunisian women--a comparative analysis of condyloma, cervical intraepithelial neoplasia and invasive carcinoma.

Toll-like receptors (TLRs) are expressed in immune and tumor cells and recognize pathogen-associated molecular patterns. Cervical cancer (CC) is directly linked to a persistent infection with high risk human papillomaviruses (HR-HPVs) and could be associated with alteration of TLRs expression. TLR9 plays a key role in the recognition of DNA viruses and better understanding of this signaling pathway in CC could lead to the development of novel immunotherapeutic approaches. The present study was undertaken to determine the level of TLR9 expression in cervical neoplasias from Tunisian women with 53 formalin-fixed and paraffin-embedded specimens, including 22 samples of invasive cervical carcinoma (ICC), 18 of cervical intraepithelial neoplasia (CIN), 7 of condyloma and 6 normal cervical tissues as control cases. Quantification of TLR9 expression was based on scoring four degrees of extent and intensity of immunostaining in squamous epithelial cells. TLR9 expression gradually increased from CIN1 (80% weak intensity) to CIN2 (83.3% moderate), CIN3 (57.1% strong) and ICC (100% very strong). It was absent in normal cervical tissue and weak in 71.4% of condyloma. The mean scores of TLR9 expression were compared using the Kruskall-Wallis test and there was a statistical significance between normal tissue and condyloma as well as between condyloma, CINs and ICC. These results suggest that TLR9 may play a role in progression of cervical neoplasia in Tunisian patients and could represent a useful biomarker for malignant transformation of cervical squamous cells.