利用双扩增聚合酶链反应检测非甲非乙型肝炎患者血清中丙型肝炎病毒RNA

利用丙型肝炎病毒（ＨＣＶ）５’－端序列合成两对引物，建立了灵敏、特异的ＨＣＶＲＮＡ双扩增聚合酶链反应检测方法。用此方法及第二代Ａｂｂｏｔｔ酶联抗－ＨＣＶ检测试剂盒，检测了４４例非甲非乙型肝炎患者血清及１０名抗－ＨＣＶ阴性健康人。在４４例患者中，４１例（９３％）ＨＣＶＲＮＡ阳性，３６例（８２％）抗－ＨＣＶ阳性，３３例（７５％）ＨＣＶＲＮＡ、抗－ＨＣＶ全部阳性。３例ＨＣＶＲＮＡ阴性，但抗－ＨＣＶ阳性，另外，有８例抗－ＨＣＶ阴性，ＨＣＶＲＮＡ阳性。１０名健康人ＨＣＶＲＮＡ均为阴性。结果表明，大部分（９２％）抗－ＨＣＶ阳性患者带有ＨＣＶ，但为了检测所有病毒血症患者，抗－ＨＣＶ检测是不够的，利用双扩增ＰＣＲ方法检测ＨＣＶＲＮＡ对于抗－ＨＣＶ阴性患者的诊断是非常有用的。     

Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction.

The specificity of first-generation enzyme-linked immunosorbent assays (ELIAs) for antibody detection in individuals with hepatitis C virus (HCV) infection has been questioned in some pathological situations. We observed a surprisingly high prevalence of anti-HCV antibodies in alcoholic patients, and thus, false-positive reactions in anti-HCV tests were strongly suspected. The introduction of new epitopes, particularly a core protein, C22 (second-generation tests), seems to increase the sensitivity of anti-HCV detection. In order to study the specificity of the second-generation tests, 60 serum samples from alcoholic patients found to be positive by the first-generation anti-HCV ELISA (Ortho) were reexamined by a second-generation anti-HCV enzyme immunoassay (Abbott) and a recombinant immunoblot assay (RIBA II; Chiron). Fifteen serum samples gave contradictory results when they were tested by the two assays. We performed nested polymerase chain reactions (PCRs) to confirm that the discrepancies that we observed could be due to the presence of low levels of anti-HCV antibodies, which were detected by a more sensitive test, or to unspecific positive reactions. Nested PCR revealed the presence of HCV RNA sequences in all anti-HCV-positive sera or sera that were weakly positive by ELISA. Anti-HCV positive by RIBA II was always correlated with the presence of viral RNA in serum, but HCV RNA was detected in RIBA II-negative sera. These results indicate that the specificity of the second-generation tests is an important improvement but that an HCV infection can still persist without detectable antibodies. PCR remains the reference assay to clear up controversial serology results and to detect HCV infection in patients with no anti-HCV-detectable immune response.     

A cost-effective algorithm for the diagnosis of Hepatitis C virus infection and prediction of HCV viremia in Cameroon

Conventional tests for antibody to Hepatitis C virus (HCV) and HCV RNA require considerable time before results are available, remain very expensive and are not adapted to many sub-Saharan African countries where HCV is endemic. The aim of this study was to evaluate the accuracy of an algorithm consisting of two HCV rapid tests to diagnose and predict HCV viremia in patients in Cameroon. Three hundred and twenty nine plasma samples were screened by two HCV rapid tests (ImmunoComb II HCV, PBS Orgenics and Hexagon HCV, Human). Previous evaluation of these samples for HCV antibodies (anti-HCV) by conventional third generation ELISA, considered as a reference test, indicated that 168 were anti-HCV negative and 161 positive. Among the 161 anti-HCV positive plasma, 114 (71%) were HCV RNA-positive by RT-PCR assay. The ImmunoComb II HCV test provided the more sensitive detection of anti-HCV (sensitivity: 99.4% with a 95% CI = 96-100%). Surprisingly, the second HCV rapid test, Hexagon HCV, showed a high capacity to identify non-viremic subjects amongst anti-HCV positive cases (93.6% [95% CI: 82-99%]). These results suggest an algorithm using ImmunoComb II HCV as a first test to screen anti-HCV positive subjects, and Hexagon HCV as a second test to discriminate between viremic and non-viremic HCV seropositive subjects.     

丙型肝炎病毒核心抗原在血液透析患者中的检测和应用

目的 探讨丙型肝炎病毒核心抗原酶联免疫吸附法在血液透析患者HCV感染检测中的应用.方法 对本院250名血液透析患者,用第三代酶联免疫吸附试剂盒检测抗.HCV抗体、酶联免疫吸附法检测HCV核心总抗原,用巢式RT-PCR法对阳性标本进行确认.结果 250例血液透析患者中抗-HCV抗体阳性43例(阳性率17.2%),HCV-cAg阳性45例(阳性率18%),经配对x2检验,P=0.839,差异无统计学意义;43例抗-HCV抗体阳性中RT-PCR检测结果 为36例HCV-RNA阳性,其中32例HCV-cAg也呈阳性,另外4例HCV-cAg阴性;45例HCV-cAg阳性标本中HCV-RNA结果 全阳性;13例核心抗原阳性但抗体阴性的患者样本中也含有HCV-RNA,抗-HCV抗体的漏检率为23.2%(13/56);核心抗原阳性标本测出病毒载量是(49258±28682)拷贝/ml,高于抗-HCV抗体阳性组(23938±10780)拷贝/ml(P<0.05);4例核心抗原阴性但抗-HCV抗体及RT-PCR均阳性标本的病毒载量是(306±161)拷贝/ml,明显低于HCV核心抗原阳性组(P<0.001).结论 HCV核心总抗原酶联免疫吸附试验将有益于由于免疫抑制状态、窗口期更长的血液透析患者,与抗-HCV的检测结果 具有互补性;HCV-cAg能很好反映HCV RNA,相对于PCR来说,HCV-cAg检测既具有成本效益好又可减少人力资源,具有广阔的临床应用前景.     

Prevalence of anti-hepatitis C virus antibody among pregnant women and blood donors at Bowen University Teaching Hospital,Ogbomoso, Oyo State,Nigeria

Hepatitis C virus is one of the emerging infectious diseases that can be transmitted through blood-to-blood contact. This study was carried out to determine the prevalence of anti-HCV antibodies among potential blood donors and pregnant women attending Bowen University Teaching Hospital (BUTH), Ogbomoso, Oyo State. This hospital-based study was conducted from December 2014 to September 2015. The study group (N = 279) included potential blood donors and pregnant women. Data on socio-demographic characteristics and potential risk factors were collected using a structured questionnaire. The presence of anti-HCV antibodies in serum samples of the studied subjects were detected using third-generation Enzyme Linked Immunosorbent Assay (ELISA) (WKEA Med Supplies Corp, China). Chisquare test was utilized to assess the association between the socio-demographic variables and HCV status. Logistic regression was done to determine the strength of association between risk factors and HCV status. Statistical significance was set at P ? 0.05. Overall seroprevalence of hepatitis C virus infection was found to be 1.79% consisting 0.36% of pregnant women and 1.43% of blood donors. None of the socio-demographic characteristics and potential risk factors among the study groups were significantly associated with hepatitis C virus infection. This study found a seroprevalence of anti-HCV antibody to be 1.79%, thus, screening of pregnant women and blood donors for HCV infections with the use of ELISA is recommended because of its important role in detecting the presence of anti-HCV antibody with utmost specificity and sensitivity.     

Use of combined detection of hepatitis C virus core antigen and antibodies to reduce the serological window-phase

OBJECTIVES: In this study, we aimed at evaluating the performances of a combined assay for the detection of hepatitis C virus core antigen and antibodies and comparing this test with conventional third generation Elisa. MATERIAL AND METHODS: Two hundred forty-one samples were included in this study and tested by Monolisa HCV Ag-Ab ULTRA, Biorad and compared to Monolisa Anti-HCV Plus. A comparative study was performed on a HCV seroconversion panel (Monolisa anti-HCV Plus, Biorad; Innotest HCV Ab IV, Innogenetics and Murex anti-HCV, Abbott). False positive samples were detected with western blot assay (INNO-LIA HCV Ab III, Innogenetics). Two anti-HCV negative haemodialysis patients with rise in ALT have been tested for RNA detection (Amplicor v2.0, Roche). RESULTS: Results obtained with Biorad Ag-Ab were in agreement with third generation ELISA on HCV seroconversion panel. From anti-HCV negative patients, four samples were found low positive with HCV Ag-Ab. Two anti-HCV negative haemodialysis patients/HCV RNA positive were also negative with HCV Ag-Ab and 13 low positive samples with Biorad Ab were found negative with Ag-Ab. CONCLUSION: The HCV Ag-Ab assay has a high specificity and sensitivity comparatively to conventional ELISA; but in our study we don't prove the reduction of the "serologic window" for detection of anti-HCV antibodies.     

Prevalence of hepatitis C virus infections in dialysis patients and their contacts using a second generation enzymed-linked immunosorbent assay

The prevalence of antibodies to the hepatitis C virus (HCV) was determined in 498 hemodialysis patients from three german dialysis units, 121 staff members and 42 family members using an enzyme-linked immunosorbent assay (ELISA) of the second generation which detects antibodies to a structural (C22) and to non-structural (C33c, C100, 5-1-1) recombinant antigens to HCV. Using the second generation ELISA 115 patients (23.1 %) were anti-HCV positive versus 77 (15.5%) when sera were tested by an ELISA of the first generation containing only a non-structural antigen (C100). In 34 of these 40 discordant sera antibodies against at least one viral protein was found by a recombinant immunoblot assay. Of 5 sera containing antibodies to only one viral protein (C22) 3 were HCV RNA positive by polymerase chain reaction. Epidemiological evaluation of the patients revealed that the prevalence of anti-HCV was correlated to the duration of dialysis but not to the number of blood transfusions. Of 121 staff members 2 (1.6%) and 2 of 42 family members (4.7%) were positive indicating a low risk of the patients' contacts of acquiring HCV infection.Supported by the Kuratorium für Dialyse und Nierentransplantation e.V., Emil-von-Behring Passage, W-6078 Neu Isenburg, FRG     

Seroprevalence of HIV,HBV, HCV,and HTLV among Pregnant Women in Southwestern Nigeria

Sexually transmitted infections (STIs) are major public health challenge especially in developing countries. This study was designed to determine the prevalence of Hepatitis B virus (HBV), Hepatitis C Virus (HCV), Human immunodeficiency virus (HIV), and Human T-cell lymphotropic Virus type I (HTLV-I) among pregnant women attending antenatal clinic, in Ladoke Akintola University Teaching Hospital, Osogbo, and South-Western Nigeria. One hundred and eighty two randomly selected pregnant women were screened for HBsAg, anti-HCV, anti-HIV and HTLV-1 IgM antibodies using commercially available ELISA kit. Of the182 blood samples of pregnant women screened whose age ranged from 15–49 years, 13 (7.1%), 5 (2.7%), 9 (4.9%), and 44 (24.2%) were positive for HBsAg, anti-HCV, anti-HIV, and HTLV-1 IgM antibodies, respectively. The co-infection rate of 0.5% was obtained for HBV/HCV, HBV/HIV, HIV/HTLV-1, and HCV/HTLV-1 while 1.1% and 0% was recorded for HBV/HTLV-1 and HCV/HIV co-infections, respectively. Expected risk factors such as history of surgery, circumcision, tattooing and incision showed no significant association with any of the viral STIs (P > 0.05). This study shows that there is the need for a comprehensive screening of all pregnant women for HBsAg, anti-HCV, anti-HIV and HTLV-1 to prevent mother to child transmission of these viral infections and its attending consequences.     

聚合酶链反应鉴定丙型肝炎病毒母婴传播的状态及其…

采用多种方法,动态检测了１１例丙型肝炎病毒感的孕妇所生的婴儿血抗－ＨＣＶ和ＨＣＶＲＮＡ。发现用合成肽酶联免疫吸附试验检测婴儿抗－ＨＣＶ阳性率显著低于第二低重组抗原ＥＬＩＳＡ;用２ｎｄＥＬＩＳＡ检测,６例婴儿脐血和静脉血抗－ＨＣＶ阳性,５例持续１－５月阴转,１例阳性持续１３个月。     

Prevalence of antibodies to the hepatitis C virus in pregnant foreign residents in France]

Blood transfusion and intravenous drug use are the two main modes of transmission of the hepatitis C virus (HCV). Sexual intercourse seems to play a less significant part in transmission of the HCV and data are still lacking on vertical mother-to-offspring transmission. HCV seroprevalence was determined in 1,084 pregnant foreign residents of France living in a single city (Limoges). Antibodies to the HCV were detected in sera using both the first and second generation Abbott ELISA kits. Sera yielding reproducible positive results with either kit were retested with a blocking test (HCV EIA Neutralization, Abbott) and a second generation RIBA (Ortho) for confirmation. For screening, use of tests detecting both nonstructural and structural antibodies improved results noticeably (5 of 16 sera). Use of confirmation tests was found to be indispensable. Overall seroprevalence was 1.47%. However, results varied across geographic regions or origin, ranging from 0% for Europe, 1.9% for North Africa and 1.78% for South-East Asia, to 4.76% for black Africa. These findings demonstrated the potential for mother-to-offspring transmission among women from high prevalence areas. A prospective study in African an Asian women is being considered to evaluate ineffectiveness and transmission using serial serologic tests and viral genome detection by polymerase chain reaction studies (PCR).     

套式PCR检测HBV与HCV垂直传播的比较研究

用套式ＰＣＲ法检测了２７例ＨＢｓＡｇ阳性母亲的新生儿脐带血ＨＢＶ ＤＮＡ，阳性３例（１１．１％），同时检测了７例抗－ＨＣＶ阳性母亲的新生儿脐带血ＨＣＶ ＲＮＡ及抗－ＨＣＶ，结果ＨＣＶ ＲＮＡ无１例阳性，抗－ＨＣＶ全部阳性，因此认为婴儿体内抗－ＨＣＶ是一种由母体被动获得的抗体，不能作为ＨＣＶ感染的客观指标。     

Community prevalence of hepatitis C viraemia: A polymerase chain reaction study

In order to estimate the prevalence of HCV carriage in an inner city health district, we undertook a polymerase chain reaction (PCR) based survey of sera collected from 1,002 patients attending general practitioners for reasons unrelated to liver disease. The series comprised 305 sample selected sera patients sample from sera 995 patients previously screened by C100 antigen-based anti-HCV tests. Overall, 7 patients were positive for HCV RNA. Four cases had anti-C100 antibodies to HCV, 2 were strictly negative but had high-normal/borderline optical densities by ELISA assay, while one was completely anti-HCV negative. All but one had normal liver function tests. Only 3/7 PCR positive cases had any serum marker for hepatitis B (HBV) exposure (2 HBsAg positive, 1 IgM anti-HBc positive). The minimum point prevalence of HCV carriage in this community is 0.7%, approximating the HBsAg carriage in the same population (1%). HCV carriage in this inner city population is considerably higher than would be predicted by blood donor surveys. A positive anti-HCV antibody (anti-C100) test is poorly predictive (～10%) of HCV RNA carriage in a general practice based population in which measurement of “surrogate” (HBV related) HCV markers would have detected only 3/7 cases of presumed chronic HCV carriage. © 1994 Wiley-Liss, Inc.     

Prevalence of anti-HCV and HCV viremia in hemodialysis patients in Taiwan.

Hepatitis C viral infection in 125 hemodialysis patients from Taiwan was studied using a second-generation anti-HCV immunoassay (EIA II) (Abbott HCV 2.0 EIA) and the polymerase chain reaction (PCR) to detect the HCV RNA in the serum. A total of 59 patients (47.2%) were positive by EIA II. In comparison, the conventional C100-3 anti-HCV assay was positive in 40 (32.0%). HCV RNA was found in 47 patients (37.6%). Patients with elevated serum transaminase level had a higher positive rate of anti-HCV and HCV RNA. The dialysis time was longer for those patients positive for anti-HCV than for those who were negative. A total of 57 of the 59 EIA II-positive cases had a history of blood transfusion. The HBsAg status did not influence the anti-HCV positivity. Among the 59 EIA II-positive patients, 66.1% were also positive for HCV RNA, and of the 47 HCV RNA-positive cases 83.0% were positive for EIA II. It is concluded that the high prevalence of specific HCV infection and HCV viremia was present in these patients. Prevention of cross-contamination during dialysis and blood screening before transfusion are important for the control of HCV infection in these patients.     

Hepatitis C virus in sickle cell disease

PURPOSE: To determine the prevalence of hepatitis C virus antibodies (anti-HCV) in patients with sickle cell disease. PATIENTS AND METHODS: Between 1983 and 2001, 150 patients from the Howard University Hospital Center for Sickle Cell Disease were screened for HCV antibody (52% women, 48% men, mean age 34 years). Frozen serum samples from 56 adult sickle cell patients who had participated in previous surveys (1983-92) of HIV and HTLV-1 serology and who were tested in 1992 for anti-HCV antibody--when commercial ELISA test (Ortho) became available--were included in this paper. Of the 150 patients in the study, 132 had sickle cell anemia genotype (SS), 15 had sickle cell hemoglobin-C disease (SC) and three had sickle beta thalassemia. Clinical charts were reviewed for history of blood transfusion, IV drug abuse, homosexuality, tattooing, iron overload, and alcohol abuse. RESULTS: Antibodies to HCV were detected in 53 patients (35.3%). Of the 55 patients who had frozen serum samples tested in 1992, 32 (58%) were reactive for anti-HCV, while only 21 of the 95 patients (22%) tested after 1992 were positive for HCV antibodies (P<0.001). Thirty-nine of 77 patients (51%) who received more than 10 units of packed red blood cells were positive for HCV antibody, and only 14 of 61 patients (23%) who received less than 10 units of packed red blood cells transfusion were positive for HCV antibodies (P<0.001). None of the 12 patients who never received transfusion were positive for HCV antibody. In the 53 anti-HCV positive patients, the mean alanine amino-transferase (ALT) value was 98- and 81 U/L, respectively, for males and females. These values were normal for the HCV-antibody negative patients. The aspartate amino-transferase (AST) and the total bilirubin were also higher in the anti-HCV positive patients compared to patients in the anti-HCV negative group. Forty-four patients (57.1%) who were transfused more than 10 units developed iron overload defined by a serum ferritin level higher than 1,000 ng/ml. A total of 20 of the patients with iron overload underwent liver biopsies. Seven of these 20 patients (35%) were HCV positive. These patients often had more severe liver disease and higher degree of iron deposition. CONCLUSION: The prevalence of HCV antibody and iron overload is directly related to the number of blood transfusions in patients with sickle cell disease. The prevalence of HCV infection has decreased significantly, since blood donor screening for HCV became available. Chronic HCV infection and iron overload place sickle cell patients at risk for significant liver disease.     

Antibodies to a putative hepatitis C virus polyprotein in Japanese patients with chronic liver disease.

To investigate the frequency of exposure to hepatitis C virus (HCV) in chronic liver disease, sera from Japanese patients were tested with the original anti-HCV assay (Ortho) and an anti-HCV assay based on synthetic peptides corresponding to a variety of regions in the HCV genome. Thirty-one (67%) of 46 patients with chronic non-A,non-B hepatitis were anti-HCV-positive by the Ortho ELISA, 20 of whom were also positive by ELISA based on synthetic HCV peptides. Eight (53%) of the 15 patients negative by the Ortho ELISA tested positive for anti-HCV by ELISA based on HCV peptides. Serum HCV RNA was detected in all cases positive for antibody to the HCV peptide and in 14 (78%) of 18 cases without antibody. Thirty-seven hepatitis B virus carriers were without anti-HCV by the Ortho ELISA and were negative for serum HCV RNA, six (16%) of whom were positive by ELISA based on HCV peptides. Antibody responses were directed against each synthetic HCV peptide used, with a considerable difference in incidence, indicating possible expression of the corresponding region in the course of HCV propagation. These findings indicate that exposure to HCV may be more common than expected based on the results of the Ortho ELISA.     

Low prevalence of hepatitis C virus infection among dentists in Taiwan

To evaluate whether hepatitis C virus (HCV) infection is an occupational hazard in the dental environment, serum samples collected in 1990–1991 from 461 dentists were tested for the antibody to HCV (anti-HCV) with first- and second-generation HCV enzyme-linked immunoassays (EIAs). Five of the 363 (1.38%) serum samples were reactive by the first-generation (C100–3) HCV EIA. Of the same 363 samples and the other 98 samples, 3 (0.65%) were reactive by the second-generation EIA. Those samples positive by the first- and/or second-generation HCV EIA were analyzed further by cDNA/polymerase chain reaction (PCR) to detect the presence of HCV RNA. Only 1 of the 5 first-generation EIA reactive samples were PCR positive. These results are comparable to the anti-HCV prevalence of healthy blood donors (0.95% by C100–3 assay) and pregnant women (0.63% by recombinant immunoblot assay). We conclude that the prevalence of HCV infection among dentists in Taiwan is low, and there is no increased risk of HCV infection through the practice of dentistry, at least in our area. © 1993 Wiley-Liss, Inc.