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.

Analysis of MTNR1A Genetic Polymorphisms and Their Association with the Reproductive Performance Parameters in Two Mediterranean Sheep Breeds.

Sheep farming plays an important economic role, and it contributes to the livelihoods of many rural poor in several regions worldwide and particularly in Tunisia. Therefore, the steady improvement of ewes' reproductive performance is a pressing need. The MTNR1A gene has been identified as an important candidate gene that plays a key role in sheep reproduction and its sexual inactivity. It is involved in the control of photoperiod-induced seasonality mediated by melatonin secretion. The aim of this study was to identify SNPs in the MTNR1A gene in two Tunisian breeds, Barbarine (B) and Queue Fine de l'Ouest (QFO). DNA extracted from the blood of 77 adult ewes was sequenced. Selected ewes were exposed to adult fertile rams. A total of 26 SNPs were detected; 15 SNPs in the promoter region and 11 SNPs in the exon II were observed in both (B) and (QFO) breeds. The SNP rs602330706 in exon II is a novel SNP detected for the first time only in the (B) breed. The SNPs rs430181568 and rs40738822721 (SNP18 and SNP20 in our study, respectively) were totally linked in this study and can be considered a single marker. DTL was associated with SNP18 and SNP20 in (B) ewes (p < 0.05); however, no significant difference was detected between the three genotypes (G/G, G/A, and A/A) at these two SNPs. Fertility rate and litter size parameters were not affected by SNP18 and SNP20. There was an association between these two polymorphisms and (B) lambs' birth weights (p < 0.05). Furthermore, the ewes with the A/A genotype gave birth to lambs with a higher weight compared to the other two genotypes for this breed (p < 0.05). There was not an association between SNP 18 and SNP20 and (QFO) ewes' reproductive parameters. These results might be considered in future sheep selection programs for reproductive genetic improvement.

rs401502 and rs11575934 Polymorphisms of the IL-12 Receptor Beta 1 Gene are Protective Against Colorectal Carcinogenesis.

Background: Colorectal cancer (CRC) is a major public health problem worldwide and in Tunisia. It ranks among the main cancers in terms of incidence and cancer-related cause of death. Its pathogenesis is currently considered to be multifactorial involving genetic and environmental factors. Recent studies have suggested that the gene encoding the ß1 subunit of the IL-12 receptor, an important pro-inflammatory cytokine of the anti-tumor response, could be involved in the susceptibility to inherited CRC. Hence, it would be interesting to study the role of single nucleotide polymorphisms (SNPs) within the IL-12RB1 gene (rs401502 and rs11575934) in CRC susceptibility. Aim: Our purpose was to assess whether genetic variants IL-12RB1 +1196G/C (rs401502) and IL-12RB1 +705A/G (rs11575934) within the IL-12RB1 gene are associated with the sporadic CRC risk. Methods: A total of 110 Tunisian patients with sporadic CRC and 141 healthy control subjects were included in this study. Genotyping was performed by high-resolution melting (HRM) analysis. All results were confirmed by direct DNA sequencing or PCR-RFLP methods. Later, the allele frequencies and genotype distribution were established and compared between the control group and CRC patients. Results: The obtained results showed that the two target SNPs were in Hardy-Weinberg equilibrium (HWE) in both patients and controls. Minor allele frequencies of rs401502 SNP were 16.4% in CRC cases and 23.8% in controls. Mutant allele of rs11575934 SNP was present with 21.4% in CRC patients and 29.8% in control group. An association study showed a significant association of two target polymorphisms with CRC, according to the dominant genetic model with OR = 0.577, 95% CI = [0.343 to 0.972], p = 0.038 and OR = 0.547, 95% CI = [0.328 to 0.911], p = 0.02, respectively. Conclusion: In this study, we found, for the first time, a potential protective effect of two SNPs in the IL-12RB1 gene, namely rs401502 and rs11575934, in sporadic colorectal cancer in Tunisians.

Rapid high-resolution melting method to identify human leukocyte antigen-G (HLA-G) 3' untranslated region polymorphism +3142C/G (rs1063320).

BACKGROUND: HLA-G is a non-classical class I gene of the human Major Histocompatibility encoding molecules with immune-modulatory properties. Expression of HLA-G is being largely studied in pathological conditions, such as tumors, viral infections, inflammation, and autoimmune diseases, grafted tissues, among others. HLA-G +3142C/G (rs1063320: dbSNP database) polymorphism is located in 3' UTR of HAL-G and plays a key role in determining the magnitude of gene and protein expression. The detection of HLA-G +3142C/G polymorphism in the most published report is done through polymerase chain reaction followed by enzymatic digestion. Therefore, it is so interesting to develop a rapid and sensitive assay to genotype HLA-G +3142C/G polymorphism. High-resolution melt analysis (HRM) is a technology that is based on the analysis of the melting profile of PCR products through gradual temperature increase. The aim of this work is to apply high-resolution melt method for genotyping the HLA-G +3142C/G polymorphism. METHODS: DNA from 118 individuals was extracted from whole blood with QIAamp® DNA blood mini kit (Qiagen, Germany). Primer couple was designed using Primer 3 online tools so as to have only one SNP in the target sequence for high HRM efficiency. Positive Controls were identified using DNA sequencing and used as reference when assigning genotypes for trial samples. RESULTS: We were able to recognize the three genotypes with similar accuracy than DNA sequencing using high resolution melting method. Hardy-Weinberg equilibrium test shows that our population is in equilibrium for the studied SNP. Genotypes frequencies of +3142C/G polymorphism in Tunisian general population are 0.475 for heterozygote G/C, 0.186 for homozygote G/G and 0.339 for homozygote C/C. CONCLUSION: HRM is a cost-effective method suitable for SNP genotyping.

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.

High-resolution melting-curve (HRM) analysis for C. meleagridis identification in stool samples.

BACKGROUND: Cryptosporidiosis represents a major public health problem. This infection, caused by a protozoan parasite of the genus Cryptosporidium, has been reported worldwide as a frequent cause of diarrhoea. In the immunocompetent host, the typical watery diarrhea can be self-limiting. However, it is severe and chronic, in the immunocompromised host and may cause death. Cryptosporidium spp. are coccidians, which complete their life cycle in both humans and animals. The two species C. hominis and C. parvum are the major cause of human infection. Compared to studies on C. hominis and C. parvum, only a few studies have developed methods to identify C. meleagridis. AIM: To develop a new real time PCR-coupled High resolution melting assay allowing the detection for C. meleagridis, in addition of the other dominant species (C. hominis and C. parvum). METHODS: The polymorphic sequence on the dihydrofolate reductase gene (DHFR) of three species was sequenced to design primers pair and establish a sensitive real-time PCR coupled to a high-resolution melting-curve (HRM) analysis method, allowing the detection of Cryptosporidium sp. and discrimination between three prevalent species in Tunisia. We analyzed a collection of 42 archived human isolates of the three studied species. RESULTS: Real-time PCR coupled to HRM assay allowed detection of Cryptosporidium, using the new designed primers, and basing on melting profile, we can distinguish C. meleagridis species in addition to C. parvum and C. hominis. CONCLUSION: We developed a qPCR-HRM assay that allows Cryptosporidium genotyping. This method is sensitive and able to distinguish three Cryptosporidium species.

Polymorphism study of Cryptosporidium hominis gp60 subtypes circulating in Tunisia.

Cryptosporidium spp. are a major cause of gastrointestinal diseases in humans worldwide. While a single subtype of Cryptosporidium hominis has been shown to be responsible for several large outbreaks related to water contamination in developed countries, little is known about the epidemiology of C. hominis in developing countries. This study reports the first genetic characterization of C. hominis at the subtype level in several human populations in Tunisia using the gp60 gene. Eighteen isolates were identified as C. hominis by a restriction fragment length polymorphism (RFLP) analysis. The prevalence of this species in different human populations ranges from 1.53% to 13.04% with a high prevalence being reported in immunocompromised children (13.04%) followed by patients with malignent myeloma (5.5%) and HIV-infected patients (4.59%). The gp60 analysis on C. hominis isolates, performed in 14 cases, showed the presence of a single subtype family: "Ia". Different subtypes were identified within this family (A11G1R1, A12R3, A23G1R1, A26G1R1, A27G1R1, A28G1R1). The IaA26G1R1 subtype was the most dominant subtype described in this area (50%). Despite the high genetic diversity of Cryptosporidium spp, a low heterogeneity at the subtype level was observed within C. hominis circulating in Tunisia. This distribution is an indicator for intensive and stable anthroponotic cryptosporidiosis in this region. Besides, the presence of a unique genotype in 5 HIV-infected patients attending the same hospital ward suggests the possible occurrence of hospital-acquired infection and underlines the need to implement preventive measures to avoid nosocomial transmission.