Frequency distribution of HCV resistance-associated variants in infected patients treated with direct-acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) is a global public health problem. Second-generation direct-acting antivirals targeting non-structural regions on the viral genome are the cornerstone for treatment of chronic infection. However, resistance-associated variants (RAVs) have been reported to be associated with therapeutic failure. The aim of this study was to assess the frequency of variants, including RAVs, in the NS3, NS5A and NS5B regions at baseline in Brazilian patients with chronic hepatitis C with HCV genotypes 1a, 1b and 3a. METHODS: Serum samples from 13 patients were used to obtain viral RNA. Massively parallel sequencing was performed using genotype-specific amplicons and a panel of Ampliseq technology for all genotypes. RESULTS: Several non-synonymous substitutions were detected at baseline for 11 responders and pre-/post-treatment for two non-responders. HCV genotype 3a was found to have significantly more non-synonymous substitutions than HCV genotype 1 in the NS3 and NS5A regions. Analyses were conducted using quantitative and qualitative inter- and intrapatient comparisons. Variants that confer resistance to the treatment used by the patients were found in both responders and non-responders. CONCLUSIONS: A wide frequency distribution of RAVs was found at baseline, and this did not interfere with the achievement of a sustained response. Evaluation of the presence of RAVs requires additional study in order to determine clinical relevance.

Hepatitis C Virus-Infected Responders and Relapsers to Treatment Show Similar Genetic Profiles of IL28B and IL10 Single Nucleotide Polymorphisms.

Genotype 1 of hepatitis C virus (HCV) is the most prevalent worldwide. Pegylated-interferon and ribavirin therapy is still used in the developing world but has less efficiency in this genotype. Single nucleotide polymorphisms (SNPs) rs12979860 and rs8099917 (IL28B) and rs1800896, rs1800871, and rs1800872 (IL10) are related to treatment outcome, but previous studies clustered nonresponse and relapse patients. The aim of this study is to analyze the frequency of those SNPs in HCV genotype 1 for response, nonresponse, or relapse. Patients were classified according to treatment outcome. Genomic DNA was extracted by blood samples and SNPs were defined by PCR and sequencing. Data analysis was performed with R project. The frequency of rs12979860 CC was similar among responders (0.48) and relapsers (0.46) and lower among nonresponders (0.18). The same trend was observed for rs8099917 TT. rs12979860 CC showed a protective effect for relapsers compared to nonresponders (OR = 0.25) as it occurs with responders (OR = 0.17). Haplotypes 12979860/C rs8099917/T were associated with protection against the nonresponder phenotype compared to responders (OR = 0.27) or relapsers (OR = 0.37). Frequency of rs12979860 and rs8099917 is different between relapsers and nonresponders, but similar between relapsers and responders.

Tacrolimus Increases the Effectiveness of Itraconazole and Fluconazole against Sporothrix spp.

Calcineurin inhibitors - such as the clinically used drug tacrolimus - are active against important fungal pathogens, particularly when combined with azoles. However, tacrolimus has not been tested against sporotrichosis, an endemic subcutaneous mycosis with worldwide distribution. Here, we evaluated the activity of tacrolimus and cyclosporine A in vitro - as monotherapy and in combination with itraconazole or fluconazole - against yeasts of Sporothrix brasiliensis and S. schenckii, the main sporotrichosis agents in Brazil. We also analyzed the effect of tacrolimus treatment on intracellular neutral lipid levels, which typically increase after azole treatment. Tacrolimus inhibited the growth of yeasts from S. brasiliensis and S. schenckii reference isolates, with minimum inhibitory concentration (MIC) values (required for ≥50% growth inhibition) of 1 and 2 mg/L, respectively. Importantly, the combination of tacrolimus and azoles exhibited high synergy toward reference Sporothrix isolates. Tacrolimus combined with itraconazole significantly increased neutral lipid accumulation in S. brasiliensis, but not in S. schenckii. Clinical isolates of S. brasiliensis and S. schenckii were more sensitive to tacrolimus as monotherapy than feline-borne isolates, however, synergy between tacrolimus and azoles was only observed for feline-borne isolates. Cyclosporine A was effective against S. brasiliensis and S. schenckii as monotherapy (MIC = 1 mg/L), but exhibited no synergy with itraconazole and fluconazole. We conclude that tacrolimus has promising antifungal activity against sporotrichosis agents, and also increases the activity of the current anti-sporotrichosis therapy (itraconazole and fluconazole) in combination assays against S. brasiliensis feline-borne isolates.

DNA repair genes PAXIP1 and TP53BP1 expression is associated with breast cancer prognosis.

Despite remarkable advances in diagnosis, prognosis and treatment, advanced or recurrent breast tumors have limited therapeutic approaches. Many treatment strategies try to explore the limitations of DNA damage response (DDR) in tumor cells to selectively eliminate them. BRCT (BRCA1 C-terminal) domains are present in a superfamily of proteins involved in cell cycle checkpoints and the DDR. Tandem BRCT domains (tBRCT) represent a distinct class of these domains. We investigated the expression profile of 7 tBRCT genes (BARD1, BRCA1, LIG4, ECT2, MDC1, PAXIP1/PTIP and TP53BP1) in breast cancer specimens and observed a high correlation between PAXIP1 and TP53BP1 gene expression in tumor samples. Tumors with worse prognosis (tumor grade 3 and triple negative) showed reduced expression of tBRCT genes, notably, PAXIP1 and TP53BP1. Survival analyses data indicated that tumor status of both genes may impact prognosis. PAXIP1 and 53BP1 protein levels followed gene expression results, i.e., are intrinsically correlated, and also reduced in more advanced tumors. Evaluation of both genes in triple negative breast tumor samples which were characterized for their BRCA1 status showed that PAXIP1 is overexpressed in BRCA1 mutant tumors. Taken together our findings indicate that PAXIP1 status correlates with breast cancer staging, in a manner similar to what has been characterized for TP53BP1.

Interleukin 28B-related polymorphisms: a pathway for understanding hepatitis C virus infection?

AIM: To analyze the role of rs12979860 and rs8099917 polymorphisms in hepatitis C virus (HCV) genotype 1 infection of Brazilians. METHODS: A total of 145 adult patients diagnosed with genotype 1 chronic hepatitis C (CHC) who had completed a 48-wk regimen of pegylated-interferon α-2a or -2b plus ribavirin combination therapy were recruited from six large urban healthcare centers and 199 healthy blood donors (controls) from a single site between January 2010 and January 2012. Data on the patients' response to treatment was collected. Polymerase chain reaction-restriction fragment length polymorphism genotyping of the interleukin (IL)28B gene fragment encompassing the single nucleotide polymorphisms (SNPs) rs12979860 (C/T) and rs8099917 (T/G) was carried out for 79 of the CHC patients and 199 of the controls. Bi-directional amplicon sequencing of the two SNPs was carried out for the remaining 66 CHC patients. RESULTS: SNP rs12979860 genotyping was successful in 99.5% of the controls and 97.2% of the CHC patients, whereas the SNP rs8099917 genotyping was successful in 95.5% of the controls and 100% of the CHC patients. The genotype and allele distributions for both rs12979860 and rs8099917 were significantly different between the control and CHC patient groups, with significantly higher genotype frequencies of CC and TT in the controls (P = 0.037 and 0.046, respectively) and of TT and GG in the CHC patients (P = 0.0009 and 0.0001, respectively). Analysis of the CHC patients who achieved sustained virological response (SVR) to treatment (n = 55) indicated that the rs12979860 C allele and CC genotype were predictors of SVR (P = 0.02). No significant correlation was found between rs8099917 genotypes and treatment response, but carriers of the T allele showed significantly higher rates of SVR (P = 0.02). Linkage disequilibrium analysis of the group that achieved SVR showed a significant association between rs12979860 and rs8099917 (P = 0.07). CONCLUSION: The higher allele frequency of rs12979860 C and rs8099917 T observed in non-HCV-infected individuals may indicate a potential protective role for these IL28B-related polymorphisms.

Dynamics of resistance mutations to NS3 protease inhibitors in a cohort of Brazilian patients chronically infected with hepatitis C virus (genotype 1) treated with pegylated interferon and ribavirin: a prospective longitudinal study.

UNLABELLED: About sixty thousand new cases of Hepatitis C virus (HCV) infection are recorded in Brazil each year. These cases are currently treated with pegylated interferon (PEG-IFN) and ribavirin (RBV) with an overall success rate of 50%. New compounds for anti-HCV therapy targeted to the HCV NS3 protease are being developed and some already form the components of licensed therapies. Mapping NS3 protease resistance mutations to protease inhibitors or anti-viral drug candidates is important to direct anti-HCV drug treatment. METHODS: Sequence analysis of the HCV NS3 protease was conducted in a group of 68 chronically infected patients harboring the HCV genotype 1. The patients were sampled before, during and after a course of PEG-IFN-RBV treatment. RESULTS: Resistance mutations to the protease inhibitors, Boceprevir and Telaprevir were identified in HCV isolated from three patients (4.4%); the viral sequences contained at least one of the following mutations: V36L, T54S and V55A. In one sustained virological responder, the T54S mutation appeared during the course of PEG-IFN and RBV therapy. In contrast, V36L and V55A mutations were identified in virus isolated from one relapsing patient before, during, and after treatment, whereas the T54S mutation was identified in virus isolated from one non-responding patient, before and during the treatment course. CONCLUSIONS: The incidence and persistence of protease resistance mutations occurring in HCV from chronically infected patients in Brazil should be considered when using protease inhibitors to treat HCV disease. In addition, patients treated with the current therapy (PEG-IFN and RBV) that are relapsing or are non-responders should be considered candidates for protease inhibitor therapy.

A single nucleotide polymorphism, rs129679860, in the IL28B locus is associated with the viral kinetics and a sustained virological response in a chronic, monoinfected hepatitis C virus genotype-1 Brazilian population treated with pegylated interferon-ribavirin.

Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.

A single nucleotide polymorphism, rs129679860, in the IL28B locus is associated with the viral kinetics and a sustained virological response in a chronic, monoinfected hepatitis C virus genotype-1 Brazilian population treated with pegylated interferon-ribavirin

Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.

Association of IL-10, IL-4, and IL-28B gene polymorphisms with spontaneous clearance of hepatitis C virus in a population from Rio de Janeiro.

BACKGROUND: Cytokines play an important role in the regulation of the immune response. In hepatitis C virus (HCV) infection, cytokine levels may influence the outcome of acute HCV infection. Polymorphisms in cytokine genes have been associated to different expression levels in response to infection. This study was carried out to investigate the association of several cytokine gene polymorphisms with disease outcome in HCV-infected patients. FINDINGS: Patients with chronic or spontaneously resolved HCV infection were included in a cross-sectional study. A comparative analysis was performed between the groups regarding frequency distribution of the following cytokines' gene polymorphisms: IL-10 (-1082 A/G; -819 T/C; -592 A/C), IL-4 (+33C/T), IFN-γ (+874 T/A), TNF-α (-238 G/A and -308 G/A) and IL-28B (rs12979860 C/T and rs8099917 T/G). RESULTS: Eighteen patients with spontaneous viral clearance and 161 with chronic HCV infection were included. In the comparative analysis, the GG genotype of the IL-10 polymorphism -1082A/G was more frequent in patients with spontaneous viral clearance when compared to patients with chronic HCV (41.2% vs 6.2%; p = 0.001). This association was also found for the CC genotype of the IL-4 polymorphism +33C/T (72.2% vs 36.7%; p = 0.017) and the CC and TT genotypes of the IL-28B polymorphisms rs 12979860 and rs 8099917 (88.9% vs 30.3%; p < 0.001 and 88.9% vs 49.6%; p = 0.002). The IL10 (A-1082 G) and IL-28B (Crs12979860T) gene polymorphisms showed odds ratios of 12.848 and 11.077, respectively, and thus may have a greater influence on HCV spontaneous viral clearance. The IFN-γ (+874 T/A), TNF-α (-238 G/A and -308 G/A) polymorphisms did not show significant association with spontaneous viral clearance or chronicity. CONCLUSION: The G allele for IL-10 (-1082 A/G), the C allele for IL-4 (+3 C/T) and the C and T alleles for IL-28B (rs12979860 and rs8099917, respectively) are associated with spontaneous viral clearance in hepatitis C infection.

Dexamethasone treatment improves morphological and hematological parameters in chronic experimental schistosomiasis.

Schistosomiasis, a chronic disease with considerable social impact, is an important health problem in many countries. To investigate the possible use of immunomodulators as coadjuvants in the treatment of chronic Schistosoma mansoni infection, we evaluated the effect of dexamethasone on histological, hematological, and biochemical parameters that reflect disease severity and morbidity. Animals treated from the first day or after 35 days of infection, were analyzed. In both groups, dexamethasone: (1) induced a decrease in the number of granulomas in hepatic tissue without affecting the alanine aminotransferase profile, (2) reduced splenomegaly and hepatomegaly associated with disease, and (3) improved hemoglobin concentration, hematocrit values and reduced the percentage of reticulocytes, preventing the development of anemia that occurs in the chronic phase of infection. These data suggest that treatment with dexamethasone results in a mild course of murine schistosomiasis and point to this drug as a promising agent to complement S. mansoni specific treatment.

Dexamethasone, a drug for attenuation of Schistosoma mansoni infection morbidity.

To investigate the possible use of immunomodulators as coadjuvants in the treatment of chronic schistosomiasis, the study described in the present report evaluated the effects of dexamethasone on several parameters which reflect disease severity and morbidity. Parasitological, immunological, and histological parameters were analyzed in animals treated from the first day of infection or after 35 days of infection. In both situations, dexamethasone had no effect on the parasite burden but altered the egg distribution in tissue, indicating that under the schedule used it did not interfere with the development of adult worms or oviposition. Treated mice showed a decrease in the number of eggs in hepatic tissue, reduced granuloma sizes, reduced levels of granuloma maturation, and reduced collagen contents. Dexamethasone-treated mice also had decreased gamma interferon, interleukin-12 (IL-12), and IL-4 levels in serum and increased IL-10 levels in serum. Taken together, these data suggested a decrease in the severity of murine schistosomiasis and point to dexamethasone as a convenient and promising coadjuvant agent in the therapy of this infection.