Dysregulated Serum MiRNA Profile and Promising Biomarkers in Dengue-infected Patients

Objectives: Pathological biomarkers and mechanisms of dengue infection are poorly understood. We investigated a new serum biomarker using miRNAs and performed further correlation analysis in dengue-infected patients.Methods: Expression levels of broad-spectrum miRNAs in serum samples from three patients with dengue virus type 1 (DENV-1) and three healthy volunteers were separately analyzed using miRNA PCR arrays. The expressions of the five selected miRNAs were verified by qRT-PCR in the sera of 40 DENV-1 patients and compared with those from 32 healthy controls. Receiver operating characteristic (ROC) curve and correlation analyses were performed to evaluate the potential of these miRNAs for the diagnosis of dengue infection.Results: MiRNA PCR arrays revealed that 41 miRNAs were upregulated, whereas 12 miRNAs were down-regulated in the sera of DENV-1 patients compared with those in healthy controls. Among these miRNAs, qRT-PCR validation showed that serum hsa-miR-21-5p, hsa-miR-590-5p, hsa-miR-188-5p, and hsa-miR-152-3p were upregulated, whereas hsa-miR-146a-5p was down-regulated in dengue-infected patients compared with healthy controls. ROC curves showed serum hsa-miR-21-5p and hsa-miR-146a-5p could distinguish dengue-infected patients with preferable sensitivity and specificity. Correlation analysis indicated that expression levels of serum hsa-miR-21-5p and hsa-miR-146a-5p were negative and positively correlated with the number of white blood cells and neutrophils, respectively. Functional analysis of target proteins of these miRNAs in silico indicated their involvement in inflammation and cell proliferation.Conclusion: Dengue-infected patients have a broad “fingerprint” profile with dysregulated serum miRNAs. Among these miRNAs, serum hsa-miR-21-5p, hsa-miR-146a-5p, hsa-miR-590-5p, hsa-miR-188-5p, and hsa-miR-152-3p were identified as promising serum indicators for dengue infection.     

Changes in Hepatitis C Virus Genotype Distribution in Chronic Hepatitis C Infection Patients

Purpose: Identification of hepatitis C virus (HCV) genotypes is very important in the selection of antiviral treatment, dose adjustment of antiviral agents, determining the treatment duration and following-up of treatment response. We aimed to determine the distribution pattern of HCV genotypes in chronic hepatitis C infection (CHC) patients. Materials and Methods: We have included 106 CHC patients who were positive in the anti-HCV and HCV-RNA tests performed in our hospital during the 16-month period. Anti-HCV assays were performed on device using a chemiluminescent microparticle immunoassay, while HCV-RNA tests and HCV genotyping assays were performed by real-time polymerase chain reaction. Results: Of the 106 cases; genotype 1b was detected in 67.0%, genotype 3 was detected in 16.0%, genotype 1a was detected in 14.2% and genotype 2 was detected in 2.8% patients. Genotypes 4, 5 and 6 were not detected in our study group. There were no statistically significant differences between the gender and age groups according to the HCV genotype distribution. The genotype 3 detection rate (16%) was the highest rate among the studies compared with the other studies in our country. Conclusions: Events that cause social changes such as war and immigration and intense commercial and touristic activities affect and alter the HCV genotype distribution in HCV-infected patients. For this reason, further multicentre studies are required reflecting all the regions in order to determine the genotype distribution in HCV-infected patients at regular intervals.     

肝脏内源性microRNA调控乙型肝炎病毒基因的表达与复制

目的探讨肝脏内源性microRNA对乙型肝炎病毒(HBV)复制与基因表达的影响。方法通过miRNA靶点分析软件寻找与HBV序列之间相关联的肝脏内源性microRNA,体外化学合成相应的microRNA分子,将合成寡核苷酸及对照与1.3倍HBV全基因组真核表达质粒pUC18-HBV1.3采用Lipofectamine2000共转染HepG2细胞,转染48h后收集细胞培养上清;通过ELISA检测HBsAg、HBeAg的表达水平;Western印迹检测HBcAg的表达水平;Trizol抽提转染细胞RNA,逆转录后用荧光定量PCR检测HBVmRNA的水平;提取细胞基因组DNA,Southern印迹检测HBV的复制中间体。经以上检测从HBV蛋白表达、转录和复制水平评价相应的microRNA作用效应。结果生物信息学方法提示miR-16和miR-122存在与HBV基因组作用的可能结合位点。经试验初步证实miR-16可下调HBV蛋白的表达及HBVDNA水平;miR-122可下调HBsAg、HBeAg的表达,上调HBVmRNA的水平。结论肝脏内源性microRNA可以调节HBV的复制与基因表达。     

丙型病毒性肝炎患者血清瘦素的检测

研究丙肝患者血清leptin水平的变化。采用RIA检测65例丙肝组患者,80名对照组血清leptin水平,以及各种生化指标,比较各组的leptin水平以及leptin与各生化指标的相关性。结果显示慢性丙肝患者血清leptin水平较对照组明显升高(P<0.01);血清leptin水平与谷丙转氨酶(ALT)、谷草转氨酶(AST)呈显著性正相关(P<0.05);而与血糖(Glu)、总胆固醇(TC)无明显相关性(P>0.05)。慢性丙肝患者血清leptin水平升高且与肝脏炎症病变严重程度有关,leptin可以作为一个判断肝脏炎症严重程度的指标。     

丙型肝炎病毒F蛋白对p21基因的调节作用

目的研究丙型肝炎病毒（HCV）F蛋白对细胞p21基因转录、表达的影响。方法PCR扩增HCV1b来源的F基因，克隆至pcDNA3．1真核表达载体。F基因质粒转染HepG2细胞，48h后抽提细胞总RNA和总蛋白，实时定量PCR及Westem blot检测p21基因表达变化。结果HCVF蛋白在HepG2细胞内瞬时表达，相对于空载体对照（目标基因表达量为1），HCVF基因质粒转染细胞的p21转录水平为3．2，蛋白表达水平为1．4。结论HCVF蛋白增加p21转录和表达，其生物学效应值得进一步研究。     

输血后肝炎病例中丙型肝炎的分析

７３例输血后肝炎经血清学检测，乙型肝炎病毒表面抗原和／或抗ＨＢｃ阳性２１例，抗丙型肝炎病毒抗体阳性６２例，显示输出后肝炎以丙型肝炎为主，在６２例输后血丙肝中，８２．２６％的患者输血或输血浆后６个月血清抗ＨＣＶ阳转；输血血组与输血浆组比较，输注量与输血后丙型肝炎的发生无关。提示血浆比全血更易传播丙型肝炎。部分随访病例说明丙型肝炎慢性化趋势较高。     

Distribution of Hepatitis C Virus Genotypes in Istanbul,Turkey

Purpose: The hepatitis C virus (HCV) has seven main genotypes and multiple subtypes. The distribution of HCV genotypes varies across geographical regions worldwide. Updated estimates of HCV genotype distributions have a critical importance for developing strategies to manage or eliminate HCV infection. The aim of this study was to determine the distribution of HCV genotypes in patients with HCV admitted to a university hospital in Istanbul, Turkey. Materials and Methods: A total of 412 HCV RNA positive patients with 46.6% of males and 53.4% of females between January 2013 and September 2016 were included in the study. Genotyping of HCV of the study population was performed by a commercial reverse hybridisation line probe-based assay. Results: Genotype 1 (82.5%) was dominant genotype, followed by genotype 3 (10.7%), genotype 2 (4.6%) and genotype 4 (2.2%). Among patients with genotype 1, subtype 1a, 1b and undetermined subtype were 6.3%, 38.8% and 37.4%, respectively. It was observed that genotype proportion was dependent on gender and age of the patients. Genotype 1 and genotype 2 were more prevalent in females, whereas genotypes 3 and 4 were more prevalent in males. Genotype 1 in the older patients and genotype 3 in the younger patients were more prevalent. Conclusion: The majority of patients with HCV infection had genotype 1 (82.5%), followed by genotype 3, 2 and 4. Monitoring the change in HCV genotype distribution is critical for the development of effective strategies for HCV elimination.     

HBV-LP与HBV-DNA联检的临床应用

目的：探讨血清乙型肝炎病毒大蛋白与HBV-DNA联检在乙型肝炎患者诊治中的意义。方法：对162例HBV感染者及47名健康对照组血清采用ELISA检测乙型肝炎病毒大蛋白及乙型肝炎病毒标志物;FQ-PCR定量检测HBV-DNA。结果：162例HBV感染者血清中,HBV-LP浓度与HBV-DNA拷贝数间具有良好的正相关性（rs=0.64,P〈0.001）,不同HBV-DNA拷贝数组别间HBV-LP浓度存在差异显著性（P〈0.01）;HBV-LP与HBV-DNA、HBeAg间均关联显著（P〈0.01）。HBV-LP与HBV-DNA、HBeAg间阳性率均存在差异显著性（P〈0.05）,HBV-LP阳性率为84.57%,较HBV-DNA、HBeAg均敏感。其中HBV-LP在HBV-DNA阳性组中阳性率为91.18%（93/102）,DNA阴性组中阳性率为73.33%,均较HBeAg高;在58例HBV-DNA和HBeAg共同阴性的HBV感染血清中,HBV-LP检出42例阳性。结论：血清HBV-LP浓度与HBV-DNA联检有利于提高HBV感染者体内病毒复制、疾病进程、疗效与预后判断的血清学诊断与监测水平。     

Discrepancy between Serological and Virological Analysis of Viral Hepatitis in Hemodialysis Patients

Background and Aim: Viral hepatitis is a health threat for hemodialysis (HD) patients and it may be transmitted during treatment. Some patients categorized to have viral hepatitis were found to be non-viremic. To clarify the discrepancy between the serological tests in HD patients, we conducted the study.Methods: A total of 1681 HD patients was included. Blood samples were analyzed for hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibody (anti-HCV). Detection of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA were performed in either HBsAg (+) or anti-HCV (+) samples. HBV DNA/HCV RNA was also measured in a subset of HBsAg (-) and anti-HCV (-) patients. Liver function tests were analyzed and compared with the serological and virological tests.Results: The serological tests showed that 230 patients (13.7%) were HBsAg (+) and 290 (17.3%) were anti-HCV (+). We were unable to detect HBV DNA in 97 of 230 (42.2%) HBsAg (+) patients, and HCV RNA could not be found in 76 of 290 (26.2%) anti-HCV (+) patients. In 167 HBsAg (-) patients, only one showed a trace amount of HBV DNA. None of 151 anti-HCV (-) patients showed detectable HCV RNA. The prevalence rate of viral hepatitis remains high in Taiwanese HD patients: 13.7% for HBV and 17.3% for HCV. However, virological analysis showed 42.2% non-viremic rate for HBsAg and 26.2% non-viremic rate for anti-HCV.Conclusions: The findings might challenge the presently suggested principles of bed and machine dedication and the diagnosis of viral hepatitis in HD patients.     

乙型肝炎患者血清中庚肝病毒核酸的检测

目的:了解乙型肝炎患者HGV感染情况,探讨HGV感染与HBV感染的关系.方法:应用巢氏PCR对1104例乙型肝炎患者进行了血清HGV-RNA检测,以251名正常人为对照.结果:共检出血清HGV-RNA阳性者34例,阳性率3.08%.乙型肝炎患者血清HGV-RNA阳性率(3.17%)与正常人群(2.79%)相比无显著差异(P＞0.05).慢性乙肝患者血清HGV-RNA阳性率(4.78%)显著高于急性乙肝患者(0.96%)(P＜0.05).结论:HGV感染可表现为病毒携带状态、亚临床型和不同临床类型,乙型肝炎患者并不比正常人群更易感染HGV,HGV与HBV重叠感染可能与病情发展和慢性化的形成有关.     

Hepatitis C virus antibody in hepatocellular carcinoma in Taiwan.

The prevalence of antibody to hepatitis C virus (anti-HCV) was investigated in patients with hepatocellular carcinoma (HCC), and correlated with the clinical features. Anti-HCV was detected in 129 histology or aspiration cytology proven HCC patients and 54 healthy controls. Anti-HCV was examined by the HCV EIA (Abbott Laboratories). All healthy controls were anti-HCV-negative. Nineteen of 81 (23.5%) hepatitis B surface antigen (HBsAg)-positive HCC patients were positive for anti-HCV. Anti-HCV was found among 60.4% (29/48) of HCC patients without detectable HB-sAg. Forty-eight of 129 (37.2%) HCC patients were positive for anti-HCV. There was a significant difference in the prevalence of anti-HCV between patients with HBsAg (23.5%) and those without HBsAg (60.4%, P = 0.0001). However, irrespective of the status of HBsAg, there was no statistical difference in sex, age, routine liver function tests, alpha-fetoprotein concentration, or associated cirrhosis between patients with anti-HCV and those without. The results imply that hepatitis C virus may play a role in the pathogenesis of HCC.     

Hepatitis C virus: Quantitation and distribution in liver

The optimal method for viral quantitation and the most appropriate site for determining viral load in patients with chronic hepatitis C virus (HCV) infection are unknown. We developed a method for measuring HCV RNA in the liver with the following features: 1) efficient extraction of RNA from tissue (89% of RNA recovered); 2) accurate amplification using branched DNA with strong concordance between a single sample tested on multiple occasions either in the same or in different runs; 3) good sensitivity (95%) and specificity (100%). HCV RNA was detected in as little as 2 mg of tissue, and viral load determined in a needle biopsy was representative of viral load in other parts of the liver. Within individual livers, 68% of the samples quantitated were within 1.5-fold of the geometric mean, and 95% were within 2.2-fold of the geometric mean. The mean ratio of virus in the liver and serum was 103, range 17.4–286. A delay of 30 minutes before freezing the liver tissue resulted in a reduction in the measured viral load in some, but not all instances. A sensitive, specific, and reproducible method for quantitating HCV RNA in the liver has been developed. Measurement of viral load at one site was representative of viral load at other sites. While hepatic HCV RNA levels are consistently greater than serum levels, the ratio of liver to serum viral load varies widely. The clinical use of measurement of viral load in the liver remains to be defined. J. Med. Virol. 51:217–224, 1997. © 1997 Wiley-Liss, Inc.     

Mannose-binding Lectin <Emphasis Type="Italic">MBL2</Emphasis> Gene Polymorphisms and Outcome of Hepatitis C Virus-infected Patients

INTRODUCTION: Mannose-binding lectin (MBL) is involved in host's response to several infections including hepatitis B but little is known about MBL and hepatitis C virus (HCV) infection. The present study attempts to investigate whether MBL2 genotype and serum MBL levels affect the course of HCV infection. RESULTS AND DISCUSSIONS: We investigated the variant alleles in MBL2 gene promoter and exon-1 regions in 80 Caucasian HCV-infected patients. Mutations in MBL2 were determined by polymerase chain reaction and restriction fragment length polymorphisms analysis. Serum MBL levels were measured by ELISA. Polymorphism homozygosity in exon-1 region was significantly related to lower serum MBL levels (p < 0.001), to liver inflammation (p = 0.034, OR = 11.7) and, in a lesser degree, to fibrosis. Polymorphisms in promoter sites -221nt and -550nt were not shown to be related with serum MBL levels or progress to liver inflammation and fibrosis. Serum MBL levels were adversely associated with progression to fibrosis (p = 0.037). Response to antiviral treatment was related to hepatitis C virus genotype (p < 0.001, OR = 10.9), but not to MBL2 genotype or serum MBL levels. CONCLUSION: These findings suggest that polymorphisms in MBL2 gene exon-1 region are related to low serum MBL levels and progression of HCV infection to liver inflammation and fibrosis.     

Anti-extractable Nuclear Antigens (ENA) Antibodies in Patients with Chronic Hepatitis C before and after Treatment with Interferon

A high prevalence of serological markers classically associated with autoimmune hepatitis or other autoimmune diseases has been reported in patients with chronic hepatitis C. However, the prevalence of antibodies to extractable nuclear antigens (anti-ENA) are rarely reported in such patients and the effect of treatment with interferon (IFN) on their prevalence is not known. In the present study, serum samples collected from 44 patients with chronic hepatitis C and 44 patients with non-hepatitis C virus (HCV) infected liver diseases were tested for anti-ENAs (U1 RNP, Sm, Ro/SS-A, La/SS-B and Scl-70) antibodies by enzyme-linked immunosorbent assay (ELISA). In 26 patients with chronic hepatitis C who received IFN treatment, serum samples were also collected just after completion of IFN treatment, and/or at 6–40 months after completion of the treatment, and tested for these antibodies. Sixteen (36%) of 44 sera from patients with chronic hepatitis C were positive for at least one of the above anti-ENA antibodies, whereas only 7 (16%) of 44 sera from patients with non-HCV infected liver diseases were positive for such antibodies (p=0.0290). There was no significant difference in the prevalence of each of anti-ENA antibody between men and women. Results of anti-ENA antibodies in most IFN-treated patients with chronic hepatitis C did not change after treatment. However, in some cases serum anti-U1 RNP, anti-La/SS-B and anti-Scl-70 became negative or converted to the gray zone after completion of IFN treatment regardless of HCV elimination. Our results showed that the overall prevalence of anti-ENA antibodies was significantly higher in patients with chronic hepatitis C than in those with non-HCV-infected liver diseases. However, the disappearance of anti-ENA antibodies after IFN treatment in patients with chronic hepatitis C may be due to the immunomodulating effects of IFN rather than HCV elimination.     

Hepatitis C virus infection in 2,744 hemodialysis patients followed regularly at nine centers in Hiroshima during November 1999 through February 2003

Patients on maintenance hemodialysis (HD) are at increased risk of infection with hepatitis C virus (HCV). A prospective follow-up study on HCV infection from November 1999 to February 2003 was conducted in nine hemodialysis (HD) units in Hiroshima. A total of 2,744 HD patients were surveyed regularly for HCV RNA in serum. The prevalence of HCV RNA decreased from 15.7% (262/1,664) on the first survey to 12.9% (242/1,882) in the last one (P<0.05). This decrease may be attributed to the inclusion of patients with a lower prevalence of HCV RNA compared to patients leaving dialysis centers (111/1,080 [10.3%] vs. 132/862 [15.3%], P<0.01). During the 40 months of this study, 16 de novo HCV infections were documented in the nine HD units corresponding to an incidence of 0.33% per year. These cases included eight new HCV infections, three re-infections, and five infections that presumably occured in the window period when tested during the first survey. Our study shows that the annual incidence of de novo HCV infection during HD was 0.33%, and emphasizes the need for frequent serum HCV RNA testing and for stringent disinfection procedures in order to prevent the transmission of HCV in these settings.     

慢性丙型肝炎患者血浆内毒素和血清MBL变化及临床意义

目的:探讨了慢性丙型肝炎患者血浆内毒素和血清甘露聚糖结合凝集素(MBL)水平的变化及意义.方法:应用鲎试验和酶联法对31例慢性丙型肝炎患者进行了血浆内毒素和血清MBL测定,并与35名正常健康人作比较.结果:慢性丙型肝炎患者血浆内毒素和血清MBL水平均非常显著地高于正常人水平(P<0.01),且内毒素水平与MBL呈正相关...     

近十年广东地区丙型肝炎感染途径的特点

目的了解最近十年广东地区丙型肝炎病毒（HCV）感染途径的特点。方法 采用回顾性调查与前瞻性研究相结合的方法,进行定群随访观察。结果 感染途径明确者占34.5%（38/110）,包括静脉吸毒15.5%（17/110）,输血及血制品14.5%（16/110）和血液透析4.5%（5/110）;感染途径不明确,存在可疑感染因素者占30%（33/110）,包括家庭内传播3.6%（4/110）,小手术及注射史12.7%（14/110）,皮肤损伤性美容10.9%（12/110）,职业暴露2.7%（3/110）;不明感染途径者占35.4%（39/110）。各组之间年龄大小差异无统计学意义（P〉0.05）,静脉吸毒均为男性,职业暴露均为女性,与其他组比较,P〈0.001。结论 在广东地区,近十年HCV感染的多种途径并存,小手术及注射、皮肤损伤性美容正在成为与静脉吸毒、输血及血制品同等重要的感染途径。不明感染途径者无论数量还是比例都排第一位,是今后预防的主要问题。     

Hepatic and Renal Safety Profile of Tenofovir in HIV-Infected Patients with Hepatitis C,Including Patients on Interferon Plus Ribavirin

Abstract

The liver safety of tenofovir (TDF) was investigated in 142 HIV+ patients exposed to the drug for longer than 12 months. No evidence of liver enzyme elevations were seen, even in 66 patients with chronic hepatitis C virus (HCV) co-infection. Given that TDF is an adenosine analogue, like didanosine, exposure to ribavirin might increase intracellular phosphorylated TDF metabolites, which could result in a higher risk of nephrotoxicity. Signs of tubular dysfunction in blood or urine were not recognized in 17 HCV-HIV co-infected patients exposed to TDF during interferon plus ribavirin therapy.