丙型肝炎病毒抗原检测方法的建立

目的以特异性单克隆抗体为基础建立丙型肝炎病毒(HCV)抗原检测的酶联免疫吸附(ELISA)法,探索从血浆或血清中检测HCV抗原的可能性.方法利用我们制备的抗-HCV核心及NS3区单克隆抗体(McAbs),进行多种交叉组合模式的分析,确立实验室模式,并测定348份义务献血员血样,确定此方法的Cutofff值,并分析146份抗-HCV阳性及225份抗-HCV阴性血浆,阳性结果用套式PCR试剂盒确证.结果构建了以抗-HCV核心区单抗C39及NS3区单抗C7-6为包被抗体,以C39及NS3区C7-57为标记抗体的夹心ELISA检测模型,以C7为抗原,其检出灵敏度为5ng/ml,其Cutoff值为阴性对照均值±0.25.146份抗-HCV阳性样本中,11份为抗原反应阳性,225份抗-HCV阴性样本中,16份为抗原阳性,这些阳性样本经PCR检测后,23份为HCVRNA阳性.结论用单抗构建的HCV抗原检测方法分别从抗-HCV阳性及阴性样本中检测出HCV抗原反应阳性样本,并经PCR确证,表明直接从血浆样本中检测HCV抗原是可能的,这将对于HCV和基础研究及控制HCV的传播有重要意义.     

Prevalence of hepatitis B and hepatitis C virus coinfection in chronic liver disease

Coinfection with HBV and HCV may lead to serious consequences. The present study was done to find out the prevalence of coinfection in patients with chronic liver disease. From patients with hepatitis and chronic liver disease 1673 samples were received and analysed for HBsAg by ELISA. 1342 samples were analysed for anti HCV by third generation ELISA. 493 samples positive for HBsAg were also analysed for Anti HCV to see the prevalence of coinfection. 15(3.0%) were found positive for both HBsAg and anti HCV. Out of 15 patients with coinfection 4 (26.6%) had HCC. Prevalence of HCC in patients with coinfection was higher than either infection alone i.e. HBV-9.1% and HCV-16.5%.     

人类免疫缺陷病毒Ⅰ型和丙型肝炎病毒核酸联合检测试剂的评价

目的 了解人类免疫缺陷病毒Ⅰ型（HIV-1）和丙型肝炎病毒（HCV）核酸联合检测试剂检测的敏感性、特异性以及检测中国不同亚型样品的适应性。方法 用酶联免疫试剂对来自于不同地区的HIV感染者或可疑感染者献血员样品进行HIV抗体和HCV抗体检测。对HIV抗体阳性者进行抗体确证。用HIV-1和HCV核酸联合检测试剂对样品进行检测，对初次检测阳性者，用HIV RNA和HCV RNA鉴别试剂单独检测，区分是HIV RNA阳性或是HCV RNA阳性。结果 74份HIV和HCV抗体均阳性的样品，HIV／HCV RNA检测也为阳性，5份HIV和HCV抗体均阴性的样品，HIV／HCV RNA检测也为阴性；84份HIV抗体确证为阳性的样品，有82份检测为HIVRNA阳性，7份HIV抗体阴性的样品检测均为HIV RNA阴性，12份HIV抗体不确定的样品，有6份检测为HIV RNA阳性；81份HCV抗体阳性样品中有70份检测为HCV RNA阳性，22份HCV抗体阴性样品中有18份检测为HCV RNA阴性，4份检测为HCV RNA阳性。结论 该试剂的灵敏度较高，尤其在HCV抗体阴性样品中检出HCV RNA阳性者。因此，可用于献血员筛查，减少窗口期的传播。     

Relative prevalence of hepatitis B viral markers and hepatitis C virus antibodies (anti HCV) in Madurai, south India

(1) The positivity of HBsAg was 4% (75/1819) whereas anti HCV was present only in 0.75% (27/3574) of blood donors. (2) 5.3% (4/75) of hospital staff had HBsAg alone in their blood samples. One doctor, one Staff nurse, one Lascar and one Sanitary worker were positive for HBsAg. None of them were positive for anti HBcIgm and anti-HCV. (3) 29% (31/115) of suspected hepatitis cases were positive for any one of the viral markers or both. 21% (15/72) of males and 14% (6/43) of females were positive for HBsAg. Whereas only 4% (3/72) of males and 2% (1/43) of females were positive for anti HCW. Both HBsAg and anti HCV were found in 8% (6/72) of males only. The age group predominantly, i.e. in 29 out of 31 cases found positive for HBsAg and/or anti HCV, was above 15 years. Two remaining HBsAg positive cases belonged to the 5 to 14 years age group. 71% (84/115) of suspected hepatitis cases were negative for both HBsAg and anti HCV.     

Utility of HCV core antigen ELISA in the screening for hepatitis C virus infection in patients on hemodialysis

An enzyme immuno assay for hepatitis C core antigen was recently developed and its performance was compared with that of the hepatitis C virus (HCV) RNA in the screening of HCV infection in patients on hemodialysis. One hundred and eleven chronic renal failure patients undergoing haemodialysis between May 2003 and October 2004 were included in the study. All the patients were tested for anti HCV antibody, core antigen and RNA. Fifteen patients were anti HCV antibody positive, three patients were positive for HCV core antigen and RNA, three patients were positive for HCV RNA, while two patients were positive only for core antigen but negative for RNA. In anti HCV antibody positive patients, the core antigen was negative while the viral RNA continued to be present. Hence, relying solely on a single HCV core antigen assay may not be useful for a definite diagnosis of early HCV infection. The sensitivity and specificity of the assay were 60% and 83% respectively, while the positive predictive value was 14.3%, negative predictive value was 97.7% and the efficiency was 81.9%.     

聚合酶链反应杂交ELISA对肝炎患者血清HCV RNA的测定和分析

目的 应用丙型肝炎病毒（HCV）聚合酶链反应杂交酶联法（HCV－PCR－H ELISA）152份各型肝炎血清进行HCV RNA测定和分析。方法 利用标记生物素的寡核苷酸引物对患者血清进行PCR扩增，扩增产物与结合在微孔反应板的HCV特异探针快速杂交，通过抗生物素－酶联反应判定结果。结果 152份肝炎患者中，HCV RNA的检出率为57．9％（88／152），其中抗－HCV阳性组的检出率为81．8％（72／88）。抗－HCV与HCV RNA的总符合率为78．9％（120／152）。对5例丙型肝炎患者应用α干扰素治疗者追访显示，HCVRNA的消长与抗病毒药物疗效一致。结论 HCV－PCR－H ELISA方法简便、稳定、敏感、特异，为半定量指标，可应用于HCV感染的临床诊断和抗病毒药物疗效判定。     

Hepatitis B, C seroprevalence and delta viruses in HIV-1 Senegalese patients at HAART initiation (retrospective study)

The aim of this study was to determine hepatitis co-infection in a cohort of HIV infected patients at their inclusion in the Senegalese Initiative of ART Access. B, C, and D Hepatitis viruses serological markers were checked retrospectively on 363 stored plasma. For HBV, the Abbott laboratories equipment IMx was used to detect HBs Ag and anti Core Ab on negative HBs Ag samples. For HDV, anti Delta Ab was performed using the Abbott Murex Kit on all HBs Ag positive samples. For HCV, anti HCV Ab was detected by IMx as double screening test and confirmed by INNO-LIA(TM) HCV Core of Innogenetics laboratories. The statistical analysis was done with STATA V8. The study population was composed of 164 men and 199 women aged between 16 and 66 years. The immune and virological markers averages at their enrollment were 154 cell/mm(3) for TLCD4+ (n = 355 patients) and 4.9 log for viral load (n = 277 patients). HBs Ag was found in 61 patients or 16.8% and the prevalence of anti-HBc Ab was 83.2% (252/295). 2 patients or 3% on HBs Ag positive sample presents HBV/HDV co-infection Ab anti HCV was detects in 6 patients or 1.6% after confirmation and 2 patients had triple infection with HBV. These results showed that the prevalence of HBV and HCV in the population of persons living with HIV/AIDS in Senegal is similar to that found in the general population. Our data indicated that hepatitis pathology in the PLwHIV was essentially due to HBV. Further studies are needed to diagnose occult hepatitis in order to set up therapeutic strategies taking into account co-infections by hepatitis viruses in the ART programmes.     

1995至2000年北京地区散发性急性肝炎患者肝炎病毒抗体的检测与分析

目的 了解北京市地区散发性肝炎患者甲型、乙型、丙型和戊型肝炎病毒感染型别分布及重叠感染。方法 用EIA法检测1995斫2月至2000年12月北京地区散发性急性肝炎患者抗－HAVIgM、HBsAg／抗－HBcIgM、抗－HCVIgM/IgG和抗－HEVIgM/IgG。结果 214例散发性急性肝炎患者血清,抗甲、乙、内和戊型肝炎病毒IgM总阳性数155例,戊型肝炎76例。有9名患者检出2种肝炎病毒抗原或抗体阳性,其中在3名肝硬化患者和2名静脉吸毒者同时检测到HBV和HCV抗原或抗体,1例HBsAg阳性者检测到抗－HAVIgM,3例－HCVIgG阳性者中分别检测到2例抗－HBVIgM和1例抗－HEVIgM。肝炎病毒重叠感染的9名患者年龄在31岁至49岁之间。结论 北京地区散发性急性肝炎78％是由消化道传染的甲戊型肝炎病毒引起,戊型肝炎在四种肝炎中位居首位,其次为甲型肝炎、乙型肝炎和丙型肝炎。肝炎病毒重叠感染多见乙、丙型肝炎病毒合并感染或慢性乙、丙型肝炎患者合并甲型或戊型肝炎病毒感染。在我国对散发性病毒性肝炎的预防应引起高度重视。     

Co-infection with hepatitis C virus and human immunodeficiency virus among patients with sexually transmitted diseases in Pondicherry, South India

Hepatitis C virus (HCV), which is usually transmitted by blood and blood products is emerging as an important agent in the list of sexually transmitted diseases (STDs). The present study was undertaken to document the burden of HCV infection in individuals with STDs in this tertiary care hospital in South India. One hundred serum samples collected from individuals with STDs were tested for antibodies to HCV by a third generation ELISA. All the samples were also screened for HIV infection. Six out of 100 individuals were found to possess antibodies against HCV (95% confidence interval [CI=1.3-10.7%). Fourteen out of 100 samples were positive for HIV (95% CI=7-20.9%). The seroprevalence of HCV in HIV positive individuals was 21.4% (3/14) whereas the corresponding figure for HIV negative individuals was only 3.5% (3/86). The difference was found to be statistically significant (p<0.01).     

Dried blood spot samples: Optimization of commercial EIAs for hepatitis C antibody detection and stability under different storage conditions

This study was undertaken to optimize and compare the efficiency of two commercial EIAs for anti‐HCV detection (HCV Ab Radim, Pomezzia, Italy and ETI‐AB‐HCVK‐4 DiaSorin, Vercelli, Italy), in dried blood spot (DBS) samples. The long‐term stability of anti‐HCV on DBS samples stored at three environmental conditions was also evaluated at: 2–8°C, 20–25°C, and ?20°C. Paired DBS and serum samples were obtained from individuals with or without anti‐HCV. The type of elution buffer, sample and conjugate volume, sample incubation time and cut‐off values were evaluated. For both EIAs, a larger sample volume was used, and the cut‐off value determined by the manufacturer was employed for Radim EIA; however, ROC curve analysis was used for the DiaSorin EIA. The sensitivity and specificity of Radim EIA on DBS were 97.5% and 99.5%, respectively, and of DiaSorin EIA were 88.9% and 98.9%, respectively. Accurate results were obtained for a period of 117 days using DBS samples stored at all storage conditions, but storage at ?20°C resulted in the lowest variation among the absorbance values. Both EIAs demonstrated the same limit of detection (until dilution of 1:10⁴ with estimated viral load of 3.1 × 10^?1 UI/ml), but the Radim EIA was associated with the best performance because a low coefficient of variation was observed in the repetition and reproducibility studies. In conclusion, commercial EIAs can be optimized for anti‐HCV detection in DBS samples that are extremely stable at different conditions for more than 100 days. J. Med. Virol. 84:1600–1607, 2012. © 2012 Wiley Periodicals, Inc.     

广西HCV高危人群庚型肝炎病毒的新基因型核苷酸序列分析

目的 探讨广西HCv高危人群庚型肝炎病毒(HGv)的感染情况及其新基因型的核苷酸序列。方法 静脉药瘾者(IVDAs)85份、肝病患者(PLDs)80份和献血员(BDs)50份血清标本．用PCR法检测庚型肝炎病毒RNA，EIA法检测HBsAg、抗-HCV和抗-HIV；随机选出62份庚型肝炎病毒RNA阳性标本进行核苷酸序列分析，构建种系发生树作基因分型。结果 215份血清中HGv阳性者85份(39.53％)，HBsAg、抗-HGV和抗-HIV的阳性率分别为39.07％、42．79％和0；11份HGV RNA的测序结果证实其有3种基因型，其中5株为新基因型(亚洲型)，51份补测序，其中GBV—C型占3．23％，HGV型占30.65％，亚洲型占64．51％。结论 HGV的3种基因型中存在不同的基因亚型；广西IVDAs、PLDs和BDs中感染庚型肝炎病毒以亚洲型和HGV型为主。     

Prevalence of viral hepatitis B and C in riverside communities of the Tucuruí Dam,Pará, Brazil

Epidemiologically, the relevance of infection caused by hepatitis viruses is related mainly to their wide geographic distribution and the large number of infected individuals in all parts of the world. In this study, 668 residents from the islands around the Tucuruí Dam were selected. Blood samples were collected for investigation of serological markers (HBsAg, total anti‐HBc, anti‐HBS, and anti‐HCV) by enzyme immunoassays. HCV‐positive subjects were tested using RT‐PCR and RFLP for the identification of viral genotypes. Among the 668 subjects studied, 1.9% were HBsAg positive, 28% were total anti‐HBc positive, and 41.9% were anti‐HBs positive. The anti‐HBs marker alone (vaccine response) was detected in 25.7% of the volunteers. Anti‐HCV antibody was detected in 2.2% of the subjects and genotype 1 was the predominant genotype (70%). The results indicate an intermediate level of HBV and HCV endemicity in the region studied, as well as low HBV vaccination coverage. J. Med. Virol. 84:1907–1912, 2012. © 2012 Wiley Periodicals, Inc.     

Diagnosis of occult hepatitis C without the need for a liver biopsy

The diagnosis of occult hepatitis C virus (HCV) infection is based on the presence of HCV‐RNA in the liver. This study aimed to evaluate the use of combining non‐invasive assays to diagnose occult HCV. A total of 122 patients with occult HCV (HCV‐RNA in the liver without detectable anti‐HCV and serum HCV‐RNA) and 45 patients with cryptogenic chronic hepatitis (without HCV‐RNA in the liver and negative for anti‐HCV and serum HCV‐RNA) were included. HCV‐RNA was tested in peripheral blood mononuclear cells (PBMCs) and in 2 ml of ultracentrifuged serum. Anti‐core HCV was examined by a non‐commercial enzyme‐linked immunosorbent assay. All controls were negative for the three HCV markers studied. Among patients with occult HCV, 36% were anti‐core HCV positive, 57% had serum HCV‐RNA after ultracentrifugation, and 61% had HCV‐RNA in PBMCs. Combining the results of the assays, 91% of the patients were positive for at least one marker. Intrahepatic HCV‐RNA load was significantly higher in patients who were positive simultaneously for the three HCV markers than in patients who were negative for all markers (P = 0.006) and than in those with one or two HCV markers (P = 0.039). Replication of HCV in liver was detected more frequently in patients with three (93%, P = 0.002), two (82%, P = 0.001), and one HCV marker (73%, P = 0.011) than in those without markers (27%). In conclusion, testing for all these markers allows diagnosis of occult HCV without the need for a liver biopsy and these assays may help to elucidate the clinical significance of occult HCV infection. J. Med. Virol. 82:1554–1559, 2010. © 2010 Wiley‐Liss, Inc.     

Hepatitis C virus infection among pregnant women in Yaounde,Cameroon: prevalence,viremia, and genotypes

Central Africa is considered to be an area of high endemic hepatitis C infection. To determine the prevalence of anti-HCV antibodies, HCV RNA, and the genotype distribution in Cameroon, 1,494 pregnant women attending antenatal care units in Yaounde, Cameroon were screened for HCV infection. Anti-HCV antibodies were detected with a 3rd generation ELISA (Monolisa anti-HCV plus version 2, BioRad, Richmond, CA). All anti-HCV antibody-positive sera were then tested with another 3rd generation ELISA (AxSYM) HCV version 3, Abbott Laboratories, Abbott Park, IL) and subsequently for HCV RNA (Amplicor HCV, Roche Diagnostics, Basel, Switzerland). Genotype was determined by phylogenetic analysis of the NS5b gene. Seventy-three pregnant women were found to be anti-HCV antibody positive by the first ELISA, but only 28 were anti-HCV positive by both ELISA. The prevalence of anti-HCV antibodies was thus 1.9% (28/1,494) (95% CI: 1.3-2.7%). 21/28 (75%) of the positive samples by both ELISA were HCV RNA positive. The 45 samples that were HCV antibody negative by the second ELISA were also HCV RNA negative. The HCV subtypes identified were 1a (24%), 2f (38%) and 4f (38%). In contrast to previous studies, anti-HCV antibodies were rare among pregnant women in Cameroon. The percentage of HCV seropositive pregnant women who had circulating HCV RNA was similar to that observed in Europe. Several HCV genotypes were found in Cameroon.     

Molecular analysis of transmission of hepatitis C virus in a nurse who acquired acute hepatitis C after caring for a viremic patient with epistaxis

A 23‐year‐old nurse (HC‐IP) developed acute hepatitis C. Intrafamilial transmission of hepatitis C virus (HCV) was suspected initially because her parents were carriers of HCV of the same genotype (1b) as that of Patient HC‐IP. However, the HCV isolate from Patient HC‐IP and those from her parents shared identities of only 92.4–92.7% in the 1,087‐nucleotide (nt) sequence within the NS5B region. It was then suspected that she contracted HCV infection during medical practice. Sixteen patients with antibodies to HCV (anti‐HCV) were hospitalized 1–3 months before she became positive for anti‐HCV. Upon analysis of stored serum samples, 14 of the 16 patients were found to be positive for HCV RNA, and 9 of the 14 viremic patients had genotype 1b HCV. Although the shared identities between the HCV isolate from Patient HC‐IP and those from eight of the nine patients were merely 90.6–93.9% within the 1,087‐nt NS5B sequence, the HCV isolate from the remaining one patient (HC‐P12) was 99.7% identical to that from Patient HC‐IP. Upon analysis of the E1 and E2 junctional region including hypervariable region 1 (283 nt), there was a close relationship (99.3–100%) between clones obtained from Patients HC‐IP and HC‐P12. Although the nurse HC‐IP had a finger injury, she took care of Patient HC‐P12, a 70‐year‐old man with HCV‐related cirrhosis and recurrent epistaxis, occasionally without wearing protective gloves. This study indicates the occurrence of HCV transmission by exposure of nonintact skin to blood in health care settings. J. Med. Virol. 81:1363–1370, 2009. © 2009 Wiley‐Liss, Inc.     

Prevalence of Hepatitis C virus infection in pregnant women and mother-child transmission in Ouagadougou, Burkina Faso

The aim of our study was to estimate the prevalence of Hepatitis C virus (HCV) among pregnant women and the rate of mother-child transmission. Over one month (April 26 to May 25, 2002) blood samples of 200 pregnant women who gave birth at the maternity of the university hospital and Gounguin center medical of Ouagadougou were tested for anti-HVC antibodies (Ac HCV) and anti HIV antibodies (Ac HIV). Infants born to mother tested positive for Ac HCV and their mother were tested for HCV-RNA. The prevalence of HCV (positive Ac HCV and HCV-RNA) was 2% in pregnant women (4/200). One case of mother-child transmission was found. The virus transmitted was 2a (A/C) genotype. The mother had a high titre of HCV-ARN, was co-infected by HIV and had had history of blood transfusion, excision and tattoo of the gums.