Molecular analysis of mixed infection with hepatitis C virus and human immunodeficiency virus in a patient infected simultaneously

A case of simultaneous infection with HIV and HCV characterized by a rapidly progressive clinical course was studied retrospectively over 3.5 years. Molecular analysis indicated interference between HIV and HCV and between HCV sub-types 1a and 1b. An ineffective immune response was suggested by the persistence and sequence conservation of the HCV HVR1 variants isolated during the follow-up. © 1996 Wiley-Liss, Inc.     

多重RT-PCR方法检测甲型流感病毒

目的建立一种快速、敏感、特异的多重RT-PCR,同时检测甲型流感病毒中的3个分型:甲型H1N1流感病毒,季节性H1N1流感病毒,季节性H3N2流感病毒,并将此方法应用到实验室流感病毒核酸检测技术中。方法利用甲型流感病毒3个分型病毒的引物,在同一个RT-PCR反应体系中,对疑似流感咽拭子标本进行检测。结果多重RT-PCR对甲型流感病毒中分型病毒有较高的灵敏度和特异性,可直接从疑似流感标本中同时进行甲型流感病毒分型检测。结论此实验中采用的多重RT-PCR具有与常规RT-PCR一样的特异性和敏感度,而且比普通RT-PCR和病毒分离法更快速,也更简便。     

Case report: Clearance of hepatitis C virus after changing the HAART regimen in a patient infected with hepatitis C virus and the human immunodeficiency virus

The effect of highly active antiretroviral therapy (HAART) on hepatitis C virus (HCV) infection remains uncertain. This report describes the case of a man with hemophilia with HIV–HCV coinfection with persistent disappearance of HCV RNA after changing the HAART regimen. He had been treated with zidovudine, lamivudine, and indinavir for initial HAART and the HIV RNA level had been undetectable for more than 8 years. He had suffered from chronic active hepatitis. The HAART regimen was changed to emtricitabine/tenofovir, atazanavir, and ritonavir because the patient preferred a once daily regimen. The HCV RNA level fell immediately and thereafter became undetectable by quantitative and qualitative assay at 5 and 7 months after the change of the HAART regimen, respectively. In contrast to other reported cases, he experienced neither increase of CD4+ T cells count nor ALT flare‐ups before HCV RNA clearance. The HCV RNA disappearance in this case may be due to the direct effect of HAART against HCV rather than restoration of cellular immunity to HCV. J. Med. Virol. 81:979–982, 2009. © 2009 Wiley‐Liss, Inc.     

Intermittent detection of hepatitis C virus (HCV) in semen from men with human immunodeficiency virus type 1 (HIV-1) and HCV

HCV is usually transmitted via the blood, but HCV RNA has been detected recently in seminal fluid. This study was done to study HCV seminal shedding and factors that could influence the presence of HCV in the seminal fluid of men coinfected with HCV and HIV-1. HCV and HIV-1 genomes were assayed in multiple paired blood and semen samples obtained from 35 men enrolled in an assisted medical procreation protocol. HCV RNA was found intermittently in semen samples from 9 patients (25.7%). Samples from 9 men with HCV RNA in their semen and 26 men without were compared to further analyze these parameters. No correlation was found between HCV RNA in the seminal fluid and age, HCV virus load, the duration of HIV-1 infection, HIV treatment, the CD4(+) cell count, HIV-1 virus load or HIV-1 detection in the semen. The intermittent detection of HCV RNA in semen samples support the systematic search for HCV RNA in semen and the use of processed spermatozoa in assisted medical procreation of infertile HCV serodiscordant couples.     

ICSI for treatment of human immunodeficiency virus and hepatitis C virus-serodiscordant couples with infected male partner

BACKGROUND: Assisted reproductive technology with semen washing can offer a significant reduction in risk of sexual and vertical transmission of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in serodiscordant couples with infected male partner. METHODS: Among couples coming to our centre for reproductive problems from January 2001 to December 2003, we selected 43 couples with seropositive male and seronegative female: 25 couples with HIV-seropositive males, 10 couples with HIV/hepatitis C virus (HCV)-seropositive males and eight couples with HCV-seropositive males. Sperm samples were washed and used for ICSI. RESULTS: Seventy-eight cycles of ICSI were performed. The mean fertilization rate was 70.34 +/- 20.14% (mean +/- SD). A mean number of 3.55 +/- 1.11 (range: 1-5) embryos of good quality was transferred for each patient. We obtained 22 pregnancies (21 singletons and one twin), with a pregnancy rate per transfer of 28.2% and an implantation rate per transfer of 15.2%. The cumulative pregnancy rate was 51.2%. At follow-up, no seroconversion was detected in any patient. CONCLUSIONS: Our data suggest that sperm wash and ICSI could be useful for reducing the risk of HIV and/or HCV transmission in serodiscordant couples with infected male wishing to have a child, irrespective of their fertility status.     

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R ² = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.     

Automated multiplex assay system for simultaneous detection of hepatitis B virus DNA, hepatitis C virus RNA, and human immunodeficiency virus type 1 RNA

We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).     

Hepatitis C virus genetic variability in 52 human immunodeficiency virus-coinfected patients

The aim of this study was to examine whether hepatitis C virus (HCV) pretreatment quasispecies complexity was linked to virological response or other clinical and biological parameters, in human immunodeficiency virus (HIV)-coinfected patients undergoing anti-HCV treatment. In addition, HCV quasispecies composition is described longitudinally in these patients before, during, and after treatment. The 52 HIV-coinfected patients were included in a randomized therapeutic trial. At inclusion, they had CD4(+) counts of >250/micro l, HIV plasma load of <10,000 copies/ml, and chronic HCV infection with genotype 1 (n = 27), 2 (n = 2) or 3 (n = 23). These values were compared at baseline with 32 HCV-only-infected, interferon-naive patients who were infected with genotype 1, 2, or 3 (n = 16, 1, or 15, respectively). HCV complexity was studied by single-strand conformation polymorphism (SSCP) in E2 hypervariable region 1 (HVR1), and diversity was evaluated at inclusion in 20 coinfected patients by sequencing four major SSCP bands. The baseline number of SSCP bands was identical in HIV-infected and control patients. In HIV-infected patients, HCV complexity was not predictive of sustained virological response to anti-HCV treatment and was unrelated to epidemiological factors, immunological parameters linked to HIV infection (CD4(+) counts, T CD4(+) proliferative responses to HIV-1 p24), protease inhibitor treatment, HCV plasma load, or genotype. HCV diversity was lower in genotype 2- and 3-infected patients. Six months after completion of the anti-HCV treatment, in comparison with baseline, SSCP profiles were modified in 13 of the 21 nonresponding coinfected patients with analyzable samples. In conclusion, in HIV-infected patients, HCV variability had no significant influence on virological response to anti-HCV treatment.     

Influence of HCV genotype and co-infection with human immunodeficiency virus on CD4(+) and CD8(+) T-cell responses to hepatitis C virus

The role of T-cells in clearance of hepatitis C virus (HCV) during acute infection is critical. The relevance of the immunological response in the control of HCV replication is less clear in chronic HCV infection. HCV-specific T-cell responses were examined in 92 interferon-naive individuals with chronic hepatitis C. A panel of 441 overlapping peptides spanning all expressed HCV proteins was used to measure HCV-specific T-cell responses, using flow cytometry after stimulating peripheral blood mononuclear cells (PBMCs) with different pools of these peptides. Most patients showed responses to at least one HCV protein, with NS5B for CD8(+) responses and E2 for CD4(+) responses identified most frequently. Both the prevalence and breadth of CD4(+) and CD8(+) responses were lower in co-infected patients, independently of the HCV genotype.     

The role of liver biopsy in the management of chronic hepatitis C in patients infected with the human immunodeficiency virus

We evaluated the role of liver biopsy in the management of chronic hepatitis C in HIV-infected persons. Patients included had abnormal alanine transaminase (ALT) levels, detectable HCV RNA, and an interpretable liver biopsy. Demographic, epidemiologic, clinical, laboratory, histological, and therapeutic data were recorded in all patients. We also registered the clinical diagnosis of cirrhosis (previous to biopsy) and whether the biopsy result deferred the decision of initiating therapy. During the 33-month duration of the study, 112 patients were included. The degree of fibrosis in liver biopsies was none or mild (F0 or F1) in 47 patients (42%) and was significant or severe in 65 (58%). Seventeen patients (15%) had histological cirrhosis. By logistic regression analysis, only portal hypertension (odds ratio [95% confidence interval], 5.3 [1.05-25.9], P = 0.04) was independently associated with significant fibrosis. Overall, cirrhosis was predicted before biopsy in 29 patients (26%) by the caregiving physician, but only 8 of these were confirmed histologically. The clinical prediction of cirrhosis before biopsy had a sensitivity of 47%, a specificity of 78%, a positive predictive value of 28%, and a negative predictive value of 89%. Histological findings changed the decision to initiate HCV therapy in 19 patients (17%) because of little or no fibrosis. Liver biopsy is a useful tool in the management of HCV-HIV-coinfected persons. In addition to allowing grading and staging of the disease far better than any other method or combination of methods, it is important for making management decisions for patients coinfected with HCV and HIV.     

Semen characteristics in human immunodeficiency virus (HIV)- and hepatitis C (HCV)-seropositive males: predictors of the success of viral removal after sperm washing

BACKGROUND: Human immunodeficiency virus (HIV)/hepatitis C virus (HCV)-seropositive males can now father children safely, avoiding transmission risks to the mother and the children using sperm washing and nested PCR (nPCR) techniques. Nevertheless, we still lack enough data to determine the reasons why approximately 10% of the performed sperm washes remain positive, thus forcing the repetition of the treatment. Semen quality in infected males is also essential to these procedures. We aimed to determine the predictive value of the semen parameters, sperm washing procedure and the infection status for the post-wash viral positivity, as well as the correlation between the semen and the disease features. METHODS: Semen characteristics were evaluated in 136 samples provided from 125 males. We also included a control group of 125 males matched by age and length of sexual abstinence. At the time of semen retrieval, 70 of them were infected with HIV (45 also with HCV, 64.3%), and 55 of them with HCV alone. nPCR for viral detection was performed in each sample. RESULTS: Thirteen out of 136 (9.5%) of the samples were positive for one or more viral detections (HIV RNA, HIV DNA and HCV RNA, when needed). From a total of 240 nPCR viral analyses, 16 were positive (6.6%). None of the seminal parameters were adequate to predict post-wash results, nor was a positive result dependent on the volumes used in the semen wash. A positive correlation was found between post-wash progressive motility and CD4 blood levels, as well as a negative correlation between progressive motility and time of evolution of the disease in HIV-infected males. CONCLUSIONS: Semen analysis, according to the World Health Organization criteria, of HIV- and HCV-affected patients showed no differences from that of non-infected males. Moreover, low CD4 blood levels, and a long evolution of the disease do not negatively affect sperm motility.     

Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions

We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecular-weight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission.     

HIV/HCV共感染患者病毒特异性CTL细胞定量研究

目的探讨病毒特异性CTL对HIVHCV共感染患者病情进展的影响机制。方法观察对象为HIVHCV共感染患者、单纯HIV感染者、单纯HCV感染者。采用四聚体技术,运用流式细胞仪检测病毒特异性CTL。前瞻性比较HIV、HCV特异性CTL在三组中的异同,并对HIVHCV共感染患者的HIV、HCV特异性CTL进行相关性分析。结果HIVHCV共感染组与单纯HIV感染组比较HIV特异性CTL,差异无显著性(P=0.586)。HIVHCV共感染组中HCV特异性CTL的百分数及绝对计数(0.37±0.29,3.52±3.79)均高于单纯HCV感染组(0.15±0.05,0.86±0.33),差异有统计学意义(P=0.001,P=0.002)。HIVHCV共感染组中的HIV特异性CTL与HCV特异性CTL存在正性线性相关(P<0.001),方程成立。并且各系数均有统计学意义,方程似然比(r2)0.761。结论HCV特异性CTL可能是HIVHCV共感染组中肝脏功能损伤加重的原因之一;HIV与HCV在同一患者存在相互影响。     

Human immunodeficiency virus enhances hepatitis C virus replication by differential regulation of IFN and TGF family genes

HIV co‐infection significantly impacts the natural history of hepatitis C virus (HCV) by increasing plasma HCV viral load, accelerating liver disease progression, and reducing rates of HCV clearance. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the impact of HIV on cytokine expression is unknown. In this study, an HCV continuous infection cell culture system was modified to permit co‐infection with HIV to test the hypothesis that virus‐induced disregulation of immune‐response genes, particularly interferons and TGF‐β, may create a permissive environment for the initial establishment of HIV/HCV co‐infection in the host. CCR5‐expressing Huh‐7.5 hepatoma cells were transduced with human CD4 antigen to allow HIV infection in vitro. Co‐infection of CD4⁺ Huh‐7.5 cells with HIV and HCV or co‐culture of HIV‐infected CD4⁺ Huh‐7.5 cells and HCV‐infected Huh‐7.5 cells increased the level of HCV RNA compared to HCV mono‐infection. Quantitative gene expression analysis revealed HIV‐induced up regulation of most tested IFN family genes when compared to HCV or co‐infection. HCV infection induced up regulation of many TGF family genes that were subsequently down‐regulated in the presence of HIV or HIV/HCV. Interestingly, co‐infection resulted in down regulation of several IFN genes and significant up regulation of TGF‐β genes leading to an overall enhancement of HCV replication. These data suggest that HIV infection may influence HCV replication in vitro by increasing levels of HCV RNA, possibly through the differential regulation of endogenous IFN and TGF family genes. J. Med. Virol. 84:1344–1352, 2012. © 2012 Wiley Periodicals, Inc.