Fabián Cortés-Mancera

Etiology and Viral Genotype in Patients with End-Stage Liver Diseases admitted to a Hepatology Unit in Colombia

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the principal risk factor associated to end-stage liver diseases in the world. A study was carried out on end-stage liver disease cases admitted to an important hepatology unit in Medellin, the second largest city in Colombia. From 131 patients recruited in this prospective study, 71% of cases were diagnosed as cirrhosis, 12.2% as HCC, and 16.8% as cirrhosis and HCC. Regarding the risk factors of these patients, alcohol consumption was the most frequent (37.4%), followed by viral etiology (17.6%). Blood and/or hepatic tissue samples from patients with serological markers for HCV or HBV infection were characterized; on the basis of the phylogenetic analysis of HCV 5′ UTR and HBV S gene, isolates belonged to HCV/1 and HBV/F3, respectively. These results confirm the presence of strains associated with poor clinical outcome, in patients with liver disease in Colombia; additionally, HBV basal core promoter double mutant was identified in HCC cases. Here we show the first study of cirrhosis and/or HCC in Colombian and HBV and HCV molecular characterization of these patients. Viral aetiology was not the main risk factor in this cohort but alcohol consumption.

Molecular characterization of hepatitis c virus in multi-transfused Colombian patients

BackgroundHepatitis C virus (HCV) infects 170 million persons worldwide and is a public health problem. Considering that HCV is principally transmitted by exposure to infected blood, multi-transfused patients constitute one of the most important risk groups in developing countries. To explore the dynamics of this infection in Colombia, we performed a study to determine the genotypes of HCV in a cohort of multi-transfused patients.ResultsThe serum samples from patients positive for anti-HCV were evaluated for HCV RNA by nested-PCR of the 5’untranslated region (5’UTR). Viral genotype was determined by RFLP and/or automated sequencing. HCV subtype 1b was found in eight cases (66.7%) and subtype 1a in two cases (16.7%); seven isolates of subtype 1b were obtained from patients who had received the first transfusion before 1986. Either genotypes 2b (8.3%) or 3a (8.3%) were found in the remaining positive specimens.ConclusionsThis is the first HCV genotyping study developed in multi-transfused patients in Colombia where HCV subtype 1b was the most prevalent. The mutation G235A in the 5’UTR of three isolates generated an additional restriction site and an RFLP pattern different from those previously described for genotype 1.

Occult Hepatitis B virus infection Among blood donors in Colombia

BackgroundHepatitis B virus (HBV) surface antigen (HBsAg) screening in blood banks reduced the risk of HBV transmission through transfusion. However, the detection of occult HBV infection among blood donors is imperative for improving blood safety. The aim of this study was to determine the frequency of occult hepatitis B virus infection among blood donors in Medellin, North West Colombia and to characterize the viral genotypes and mutations.MethodsSerum samples from blood donors with the serological profile HBsAg-/Anti-HBc+ were evaluated by nested or hemi-nested PCR for HBV genome ORF C, ORF S and ORF X. A pairwise analysis was carried out with deduced amino acids sequence of overlapping S/P region.ResultsA total of 302 serum samples HBsAg-/Anti-HBc+ from donors recruited in a blood bank in Medellin were evaluated by PCR for the HBV genome. Six samples (1.98%) were identified as occult HBV infection. The cases were confirmed by sequencing and viral load analysis. All HBV strains were genotype F, subgenotype F3. The amino acid substitutions sY100H, sV184A, and sK141N were detected in ORF S and rtL108P, rtR110G, rtL180M, rtR192C, rtT150S, and rtL187V in ORF P.ConclusionsThis is the first report and characterization of OBI cases in blood donors in Colombia. Six from 302 donors HBsAg-/Anti-HBc+ were identified. The mutations rtL108P, rtR110G, rtR192C, rtT150S and rtI187V were characterized for the first time in these samples. Further studies are necessary to explore if these mutations could potentially impair HBsAg production.

Hepatitis B and Hepatitis C Infection Biomarkers and TP53 Mutations in Hepatocellular Carcinomas from Colombia

Hepatocellular Carcinoma (HCC) is a leading cause of cancer-related death worldwide. Globally, the most important HCC risk factors are Hepatitis B Virus (HBV) and/or Hepatitis C Virus (HCV), chronic alcoholism, and dietary exposure to aflatoxins. We have described the epidemiological pattern of 202 HCC samples obtained from Colombian patients. Additionally we investigated HBV/HCV infections and TP53 mutations in 49 of these HCC cases. HBV biomarkers were detected in 58.1% of the cases; HBV genotypes F and D were characterized in three of the samples. The HCV biomarker was detected in 37% of the samples while HBV/HCV coinfection was found in 19.2%. Among TP53 mutations, 10.5% occur at the common aflatoxin mutation hotspot, codon 249. No data regarding chronic alcoholism was available from the cases. In conclusion, in this first study of HCC and biomarkers in a Colombian population, the main HCC risk factor was HBV infection.

Hepatitis E Virus Genotype 3 in Colombia: Survey in Patients with Clinical Diagnosis of Viral Hepatitis.

Background Hepatitis E virus is a major cause of outbreaks as well as sporadic hepatitis cases worldwide. The epidemiology of this enterically transmitted infection differs between developing and developed countries. The aims of this study were to describe HEV infection in Colombian patients and to characterize the genotype. Methods A prospective study was carried out on 40 patients aged over 15 with a clinical diagnosis of viral hepatitis, recruited from five primary health units in the city of Medellin, Colombia. Fecal samples obtained from the 40 consecutives cases were analyzed for HEV RNA using nested reverse transcription PCR for both ORF1 and ORF2-3. The amplicons were sequenced for phylogenetic analyses. Results Nine (22.5%) cases of HEV infection were identified in the study population. Three HEV strains obtained from patients were classified as genotype 3. No significant association was found between cases of Hepatitis E and the variables water drinking source, garbage collection system and contact with pigs. Conclusions This is the first prospective study of hepatitis E in Colombian patients. The circulation of the genotype 3 in this population is predictable considering the reports of the region and the identification of this genotype from pigs in the state of Antioquia, of which Medellin is the capital. Further studies are necessary to establish whether zoonotic transmission of HEV is important in Colombia.

Automatic Image Segmentation Method for In Vitro Wound Healing Assay Quantitative Analysis

In vitro wound healing assay is a useful method to assess cell proliferation and migration, in this context quan-tification of the process is a critical step. Objective: to develop an automatic segmentation method for wound healing image analysis. Methods: sixty sequential images of two liver tumor-derived cell lines were acquired and processed with a region growing-based segmentation algorithm performed in MATLAB. Results: after image processing HepG2 cells pre-sented a low migration rate respect to SkHep1. Although there were visual differences during evaluation of automatic meth-odology with a manual segmentation carried out under the supervision of an expert, the average differences determined as percentage of wound region (%WR) in image segmentation were just of 0.97% (n=44) for HepG2 and 0.32% (n=16) in SkHep1. Conclusion: this novel approach is a simple alterna-tive to quantify wound healing assay on sequential images.

Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia

Background Hepatitis B virus (HBV) occult infection (OBI) is a risk factor to be taken into account in transfusion, hemodialysis and organ transplantation. The aim of this study was to identify and characterize at the molecular level OBI cases in patients with end-stage liver disease. Methods Sixty-six liver samples were obtained from patients with diagnosis of end-stage liver disease submitted to liver transplantation in Medellin (North West, Colombia). Samples obtained from patients who were negative for the surface antigen of HBV (n = 50) were tested for viral DNA detection by nested PCR for ORFs S, C, and X and confirmed by Southern-Blot. OBI cases were analyzed by sequencing the viral genome to determine the genotype and mutations; additionally, viral genome integration events were examined by the Alu-PCR technique. Results In five cases out of 50 patients (10%) the criteria for OBI was confirmed. HBV genotype F (subgenotypes F1 and F3), genotype A and genotype D were characterized in liver samples. Three integration events in chromosomes 5q14.1, 16p13 and 20q12 affecting Receptor-type tyrosine-protein phosphatase T, Ras Protein Specific Guanine Nucleotide Releasing Factor 2, and the zinc finger 263 genes were identified in two OBI cases. Sequence analysis of the viral genome of the 5 OBI cases showed several punctual missense and nonsense mutations affecting ORFs S, P, Core and X. Conclusions This is the first characterization of OBI in patients with end-stage liver disease in Colombia. The OBI cases were identified in patients with HCV infection or cryptogenic cirrhosis. The integration events (5q14.1, 16p13 and 20q12) described in this study have not been previously reported. Further studies are required to validate the role of mutations and integration events in OBI pathogenesis.

Evaluation of in silico tools for RNA secondary structures determination, using the Hepatitis C Virus IRES sequence as a model

The Ribonucleic acid (RNA) is a key molecule in different cellular processes, including catalysis. These functions depend not only on nucleotide sequences but also of RNA secondary structure (2D) [1]; this is the case of the extensively characterized IRES structure (Internal Ribosome Entry Site) in Hepatitis C Virus (HCV) RNA [2]. Although experimental techniques are available for studying molecular conformation and folding, their high cost limits a more extended use, therefore bioinformatics programs are currently used for RNA 2D prediction. How dissimilar methods are used in most available in silico tools [3-7] we evaluated the accuracy of five programs for RNA secondary structure prediction, using a HCV IRES domain II as a model [8]. Among assessed tools, MATLAB v7.10.0TM did not generate the expected shape for HCV domain II, resulting in the lowest Sensitivity (S) and Predictive Positive (PPV) values, while RNAShape 2.1.6, RNAstructure v 5.4 and Assemble 1.0.4 predicted the same topology, failing in pairings C19/G62 and G24/A56. Finally, Assemble 2.0 predicted the most acceptable topology of domain II, with the highest S and PPV values. Conclusion: None of the five programs could totally predict the topology of the structural model used.

Interacción entre el virus de la inmunodeficiencia humana y el virus GB tipo-C durante el estado de co-infección

The human immunodeficiency virus (HIV) infection is one of the most important problems in public health. It is estimated that 3 3 million people are infected around the world. HIV and GBV-C share the same transmission route, being frequent the co-infection. Since both viruses replicate in CD4+ lymphocytes, recent studies have described an interaction. Decreasing of HIV viral load and higher CD4 counts have been observed in co-infected patients, leading a better clinical outcome. Nevertheless, some epidemiological studies have shown contradictory results. Additionally, in vitro models report inhibition of HIV by E1, E2, NS3 and NS5A GBV-C proteins, resulting in a decreasing of p24 antigen. This review summarizes the principal findings about co-infection and mechanisms that have been proposed for HIV-1 inhibition.

Accurate Region-of-Interest Recovery Improves the Measurement of the Cell Migration Rate in the In Vitro Wound Healing Assay:

The wound healing assay is widely used for the quantitative analysis of highly regulated cellular events. In this essay, a wound is voluntarily produced on a confluent cell monolayer, and then the rate of wound reduction (WR) is characterized by processing images of the same regions of interest (ROIs) recorded at different time intervals. In this method, sharp-image ROI recovery is indispensable to compensate for displacements of the cell cultures due either to the exploration of multiple sites of the same culture or to transfers from the microscope stage to a cell incubator. ROI recovery is usually done manually and, despite a low-magnification microscope objective is generally used (10x), repositioning imperfections constitute a major source of errors detrimental to the WR measurement accuracy. We address this ROI recovery issue by using pseudoperiodic patterns fixed onto the cell culture dishes, allowing the easy localization of ROIs and the accurate quantification of positioning errors. The method is applied to a tumor-derived cell line, and the WR rates are measured by means of two different image processing software. Sharp ROI recovery based on the proposed method is found to improve significantly the accuracy of the WR measurement and the positioning under the microscope.