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.     

Hepatitis C virus infection in French hemodialysis units: a multicenter study

The aims of the study were: (i) to evaluate the prevalence of hepatitis C virus (HCV) antibodies (third generation tests) and RNA (standardized ultrasensitive RT-PCR assay) in a large cohort of hemodialysis patients, and (ii) to correlate HCV markers with bioclinical features and alanine-aminotransferase (ALT) activity. Antibodies were assayed by two methods in 1,323 patients (60% men, median age 65 years, median hemodialysis duration 3 years) attending 25 French hemodialysis centers including 9 self-care units. RNA was assayed using the Cobas Amplicor 2.0 method in pooled samples from 10 anti-HCV(-/-) patients and on individual samples from the other patients. Of the 16.3% patients (range 0-44%) tested (+/+) for HCV antibodies (anti-HCV), 2.3% tested (+/-) and 81.4% tested (-/-). 70% of the anti-HCV(+/+) patients and 3% of the HCV(+/-) patients were RNA(+). Pooled analysis revealed that 5/1077 anti-HCV(-/-) patients (0.5%) were RNA(+); all 5 displayed subsequently an increase in ALT and became anti-HCV(+/+). Mean ALT was higher (multiple of normal) in anti-HCV(+/+) RNA(+) patients than in anti-HCV(+/+) RNA(-) patients (0.46 +/- 0.08 vs. 0.22 +/- 0.07, P < 0.0001) and similar in all the RNA(-) patients, whatever their HCV antibody status. Multivariate analysis demonstrated that HCV status was linked to hemodialysis duration, previous kidney transplantation and positive anti-HBc. To summarize, the determination of the RNA status of anti-HCV(+/-) patients may have clinical relevance if a policy of isolation is contemplated. Standardized ultrasensitive RT-PCR assay combined with a pooling strategy is a promising method for use in epidemiological studies.     

HCV antibodies in saliva and urine

Infection with hepatitis C virus (HCV) is usually established by detection of serum antibodies (anti-HCV). This study was conducted in order to evaluate whether saliva and urine may substitute serum for anti-HCV detection. Serum, saliva, and urine were obtained simultaneously from 141 patients with a variety of liver diseases and from 52 patients with autoimmune diseases (systemic lupus erythematosus n = 27 and rheumatoid arthritis n = 25). The cell free fraction of saliva and urine samples was tested for anti-HCV using a modification of a serum anti-HCV kit. Western blot analysis was used as a confirmation method. Of the patients with liver diseases, 73 were anti-HCV-seropositive. Salivary and urinary anti-HCV could be detected in 66 (90%) and 36 (49%) of the anti-HCV-serpositive patients, respectively. The presence of anti-HCV in saliva or urine was not related to the severity of liver disease. All the anti-HCV-seronegative liver patients were negative for salivary anti-HCV and 22 (32%) had urinary anti-HCV. The patients with autoimmune diseases were all anti-HCV-seronegative. None had detectable salivary anti-HCV while 33 (63%) were positive for urinary anti-HCV. Western Blot analysis confirmed the presence of anti-HCV in all serum and saliva samples tested but only in 2/12 urine samples. The results suggest that saliva, but not urine, may serve as a substitute for serum for the determination of anti-HCV positivity. J. Med. Virol. 55:24–27, 1998. © 1998 Wiley-Liss, Inc.     

Surrogate markers are not useful for identification of HCV carriers in chronic hemodialysis patients.

Several diagnostic hepatitis C assays have been developed for the detection of antibodies to different antigens of the virus. This virus is the major cause of non-A, non-B hepatitis. Seventy-nine patients undergoing chronic hemodialysis and/or hemofiltration were tested for the presence of anti-HCV antibodies (anti-C-100-3 antibodies and anti-core antibodies), anti-hepatitis B core antibodies (anti-HBc), and aminotransferases (ALT). Seven patients were positive by one or more of the anti-HCV enzyme linked immunoassays (EIAs), while HCV-RNA was detectable in only four patients. These four patients had at least one, but not necessarily the same, positive anti-HCV EIA. HCV-RNA was not detected in patients who had no antibodies as determined by all six anti-HCV EIAs. All patients with a marker for HCV infection had persistent normal levels of transaminases. Three patients had elevated ALT values without a marker for HCV infection and suffered from hepatitis B virus infection. Anti-HBc was detected in 27/72 patients without any marker and in four patients with a marker of HCV infection. However, HCV-RNA was detectable in only one of these four anti-HBc positive patients. It is concluded that surrogate markers (anti-HBc and serum transaminases) are not useful for identification of HCV carriers in chronic hemodialysis patients.     

Antibodies to hepatitis C virus in uremic patients on continuous ambulatory peritoneal dialysis.

The prevalence of antibody to hepatitis C virus (anti-HCV) among 101 uremic patients receiving continuous ambulatory peritoneal dialysis (CAPD) was evaluated using a synthetic peptide-based HCV antibodies enzyme immunoassay. Thirty (29.7%) were found anti-HCV positive. This is significantly higher than 500 unselected paid blood donors (4.2%, P less than 0.0001). Among CAPD patients, anti-HCV positivity was found more frequently in patients who had received frequent and longer duration of hemodialysis previously (40.4% vs. 20.4%, P less than 0.05). These findings suggest that hemodialysis patients have a higher risk of HCV infection. At present, CAPD may be a suitable way to reduce the incidence of HCV infection in uremic patients.     

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.     

No evidence of hepatitis B virus activity in patients with anti-HBc antibody positivity with or without anti-hepatitis C virus antibody positivity.

BACKGROUND: The serological pattern of anti-HBc antibody positivity without both, HBsAg and anti-HBs antibody positivity may be present in up to 4% of the population of Europe and the United States. OBJECTIVES: The aim of the present study was to determine the hepatitis B virus (HBV) activity by detection of serum HBV DNA in patients with anti-HBc antibody positivity only and with confirmed anti-hepatitis C virus (anti-HCV) antibody positivity or without anti-HCV antibody positivity. STUDY DESIGN: A total of 141 patients positive for anti-HBc antibodies only, were investigated on serum HBV DNA load. Patients were classified into two groups: patients with confirmed positive anti-HCV antibodies (group 1) and patients without anti-HCV antibodies (group 2). RESULTS: Demographic data of patient groups were similar. In 66 of 70 patients with anti-HBc antibodies and anti-HCV antibodies (group 1), serum HCV RNA was detected; the remaining 4 patients were HCV RNA negative but the presence of anti-HCV antibodies was confirmed by the line probe assay. In none of the patients, with anti-HBc antibodies and without anti-HCV antibodies (group 2), serum HCV RNA was detected. In none of the patients, serum HBV DNA was detected. CONCLUSION: In this study, serum HBV DNA could not be detected in patients with anti-HBc antibodies only. There seems to be no need for determination of serum HBV DNA in patients without clinical evidence of chronic liver disease. Nevertheless, it would be useful to test patients with progressive liver disease and those, which belong to high-risk groups such as hemophiliacs, intravenous drug abusers, patients on hemodialysis, and immunocompromised patients.     

1年以上血透患者HCV感染状况的调查

目的 研究1年以上的长期血液透析患者丙型肝炎（HCV）感染状况。方法 用ELISA法和RT－PCR法检测137例长期血透患者血清中的抗－HCV和HCVRNA,并且同时检测谷丙转氨酶（ALT）和谷草转氨酶（AST）,计算其变动率。结果 透析时间超过1年以上的137例患者中仅抗－HCV阳性8例,仅HCV－RNA阳性13例,抗－HCV与HCVRNA同时阳性者24例,感染率34．3％。且透析时间小于2年的,HCV感染率为15％,透析时间大于2年以上的其感染率增至37．6％。结论 血透患者中HCV的感染应引起重视,透析的年限越长,被HCV感染的机率就越大。酶学指证的变动率不能作为长期透析患者HCV感染的敏感指标。     

Seronegative Hepatitis C Virus Infection

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. The routine diagnostics identifying HCV infection include testing for specific anti-HCV antibodies by enzyme-linked immnunosorbent assay and viral genetic material in serum or plasma. However, a small proportion of patients persistently infected with HCV, in whom anti-HCV are undetectable, constitute a serious diagnostic and possibly epidemiologic problem, as they could facilitate pathogen spread in the population. This type of infection is termed seronegative or serosilent. Seronegative HCV infection is currently of great interest to both scientists and physicians. The review presents epidemiological data concerning the prevalence of seronegative HCV infection in HIV/HCV co-infected individuals, hemodialysis patients, and blood and organ donors. The possible mechanisms behind this atypical course of infection are discussed. Furthermore, the differences between seronegative and occult infections and prolonged seroconversion are explained.     

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.     

Detection of HCV RNA in saliva, urine, seminal fluid, and ascites.

Approximately half of the patients with type C hepatitis do not have a history of parenteral exposure. The route of nonparenteral infection remains unknown. To evaluate the possible role of body fluids, the existence of hepatitis C virus (HCV) RNA in saliva, urine, seminal fluid, and ascites was examined by "nested" polymerase chain reaction (PCR). Amplification of the HCV 5' noncoding sequences was carried out. The amplified product was confirmed by Southern blot hybridization and restriction endonuclease digestion. Among 34 patients with chronic liver disease who were positive for anti-HCV and serum HCV RNA, the prevalence of HCV RNA in body fluids was 100% (7/7) in ascites, 48% (15/31) in saliva, 24% (4/17) in seminal fluid, and 7% (2/29) in urine. The body fluids collected from 3 healthy subjects and 5 patients with chronic liver disease who were positive for anti-HCV but negative for serum HCV RNA were all negative for HCV RNA. Hence, the potential infectivity of body fluids in patients testing negative for serum HCV RNA can probably be discounted. Conversely, the presence of HCV RNA in saliva and seminal fluid of patients positive for serum HCV RNA suggests sexual and household contact as likely modes of nonparenteral transmission of type C hepatitis. Furthermore, the high prevalence of HCV RNA in ascites and saliva may have important implications in medical and dental practice.     

Occult hepatitis C virus infection during an outbreak in a hemodialysis unit in Thailand

Control of hepatitis C virus (HCV) in hemodialysis populations is a major public health priority, but the preferred methods to prevent and rapidly detect HCV outbreaks in these populations remains subject to debate. We enrolled 231 hemodialysis patients at three dialysis centers in Chiang Mai, Thailand. Patients were followed every 6 months for 3 years and tested for the presence of serum HCV antibody and HCV RNA at each visit. We additionally isolated and tested peripheral blood mononuclear cells (PBMCs) for HCV RNA collected at the 30-month follow-up visit. Fifty-one study participants negative for anti-HCV at the baseline enrollment visit seroconverted over the course of the 3-year follow-up period. Of 11 individuals who transiently lost detectable serum HCV viremia, we were able to detect HCV RNA from the PBMCs of two individuals. Our results suggest that occult HCV infection may be common among hemodialysis patients, and serum HCV RNA testing may be supplemented with PBMC testing to maximize diagnostic sensitivity and aid in outbreak containment. Further work on the diagnostic implications of HCV compartmentalization in hemodialysis and other settings is urgently needed.     

Hepatitis virus infection among hemodialysis patients

AIM, PATIENTS AND METHODS: The high prevalence of anti-hepatitis C virus antibodies (anti-HCV) in hemodialyzed (HD) patients has been recognized since the early 1990s. Over the last decade, a significant decrease of anti-HCV prevalence among HD patients has been observed in many west European countries. In order to evaluate whether this trend is also present in Dialysis Center of Dubrava University Hospital, Zagreb, we tested HD patients for anti-HCV and HCV RNA in serum. ELISA 3 (Sorin) was used as a screening anti-HCV test, and confirmatory testing relied on western blotting (BioRad). HCV RNA was tested by HCV RNA PCR (Roche) and AMPLICOR, HCV test, version 2.0 (Roche). RESULTS AND DISCUSSION: The low prevalence of HCV infection is a consequence of the screening of blood donors with increasingly sensitive anti-HCV tests, followed by the progressive reduction of blood transfusion due to the availability of erythropoietin and the reinforcement of universral hygienic precautions and strict infection control in our HD unit. A contributing factor was the prevention of nosocomial transmission by the separation of anti-HCV positive from anti-HCV negative patients. Thus, the low prevalence of HCV infection in our HD center contributes-to an improved prognosis in end stage renal disease patients by additionally reducing the risk of nosocomial HCV infection.     

A novel double antibody sandwich-lateral flow immunoassay for the rapid and simple detection of hepatitis C virus

The objective of this study was to screen for antigens of the hepatitis?C virus (HCV) to establish a new double antibody sandwich-lateral flow immunoassay (DAS-LFIA) method for testing the presence of anti-HCV antibodies in human serum or plasma. A series of different recombinant HCV proteins in Escherichia coli cells were constructed, expressed, purified and the new DAS-LFIA strip was developed. The sensitivity and specificity of new the DAS-LFIA strip were evaluated by detecting 23?HCV-positive sera, a set of quality control references for anti-HCV detection that contain known amounts of anti-HCV antibodies, and 8?HCV-negative sera. A total of 300?clinical serum samples was examined by both the new DAS-LFIA strip and enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS?11.5 software. The sensitivity and specificity of the new DAS-LFIA strip were 100%. The lowest test line of the HCV DAS-LFIA strips was 2?NCU/ml. Additionally, the concordance between the new DAS-LFIA strip and ELISA methods was 94.33%. In conclusion, our new testing method is rapid, simple, sensitive and specifically detects the presence of anti-HCV antibodies in human serum or plasma. Therefore, it may be used for monitoring HCV.     

Detection of hepatitis C virus RNA in the cell fraction of saliva before and after oral surgery

The presence of hepatitis C virus (HCV) RNA in serum, whole saliva, and saliva from the sub-maxillary glands was investigated before and after oral surgery- The presence of HCV RNA (positive and negative-strand RNA) was determined in serum and saliva by a nested polymerase chain reaction in 26 anti-HCV positive patients, of whom 11 were coinfected with human immunodeficiency virus-1. Oral surgery was carried out on five occasions on four of the patients. HCV RNA was detected in the sera of 23 of 26 (88%) patients, and in the saliva of 4 of the 23 (17%) of the viremic patients. In all four cases, HCV RNA was detected only in the cell fraction derived from centrifugation of whole saliva. Negativestranded HCV RNA was not detected. At one of five occasions of oral surgery, HCV RNA was detected in saliva sampled immediately after surgery, but not before or 24 hours after surgery. The results suggest that HCV is present in saliva in less than 25% of HCV viremic persons. The presence of the virus in saliva is restricted to the cell fraction. Thus, saliva may serve as a possible, but low, nonparenteral transmission route of HCV. Contamination of saliva by blood during and after oral surgery may result in an increased risk of viral exposure. Except for trauma caused by sharp instruments during surgery, this might contribute to the higher HCV seropositivity found among dentists. © 1995 Wiley-Liss, Inc.     

Modified enzyme immunoassay to detect hepatitis C virus antibodies in oral fluid

Samples of oral fluid collected from 18 patients seropositive for hepatitis C virus (HCV) and 49 seronegative patients were tested for the presence of HCV antibodies with two modified serum-screening kits. The following sensitivities and specificities were obtained: HCV 3.0 assay, 72% and 98%, respectively, and Monolisa anti-HCV assay, 100% and 100%, respectively. The modified Monolisa assay demonstrated a striking concordance between serum and saliva samples. The use of oral fluids offers a convenient and noninvasive method applicable to HCV epidemiological studies and screening of high-risk groups.     

Viral hepatitis in hemodialysis patients

Hemodialysis patients are one of the high-risk groups for viral hepatitis, especially hepatitis C virus(HCV) infection. Although using recombinant human erythropoietin to treat anemia and introducing HCV testing of donated blood have been expected to reduce the incidence of HCV infection, occasional transmission of HCV to hemodialysis patients still occurs. The epidemiological and phylogenetic analysis provides the evidence for nosocomial infection of HCV in hemodialysis units. On the other hand, the discrepancy between results of anti-HCV antibody and HCV RNA is observed in some hemodialysis patients, indicating that the isolation of patients positive for anti-HCV antibody is not effective for the prevention of transmission of HCV. The strict enforcement of universal precaution such as carefully changing gloves should be more important for the prevention of nosocomial infection.     

Intrafamilial transmission of hepatitis C virus in hemodialysis patients

The prevalence of hepatitis C virus (HCV) infection in chronic hemodialysis patients ranges from 20 to 50% and these patients may serve as a reservoir of infection for their household contacts. The aim of this study was to investigate the prevalence of anti-HCV in hemodialysis patients and their families, and to evaluate possible routes of infection. One hundred eighty-six family members of 84 hemodialysis patients and 529 healthy adults were enrolled. The family members consisted of 50 spouses, 96 children, 11 parents, 29 siblings, and other relatives living together with the patients. Serum samples were collected for testing anti-HCV. Exposure to risk factors was obtained by a questionnaire and an interview. The results showed that prevalence of anti-HCV in hemodialysis patients was 44%, whereas in family members it was 5.4%, not significantly different from that of age-matched healthy adults (standardized morbidity rate = 1.51, P = 0.390). The anti-HCV rate in family members tended to increase with age, and a spouse of an infected hemodialysis patient had a higher risk of HCV infection than other family members (15% vs. 2.6%, odds ratio 6.6, P = 0.058). Except for the age factor, no difference was found between seropositive and se-ronegative family members with respect to risk factors such as blood transfusion, surgery, frequent injections, dental procedures, or acupuncture. It was concluded that, although the anti-HCV positivity of hemodialysis patients is high, the risk of HCV infection for their family members is not higher than that of the general population. Among family members, spouses of seropositive hemodialysis patients have the highest risk of HCV infection. These data imply that long-term intimate contact between spouses plays a key role in the intrafamilial transmission of HCV. © 1995 Wiley-Liss, inc.