anti-HCV Positive Sera Research Articles

Role of hepatitis C virus core antigen assay in hepatitis C care in developing country

BackgroundThe World Health Organization (WHO) aims to achieve global hepatitis C elimination by 2030, defined as diagnosis of 90% of infected individuals and treating 80% of them. Current guidelines for the screening and diagnosis of hepatitis C infection denote using a relatively cheap screen with anti-hepatitis C virus (HCV) antibody immunoassay, followed by the much costlier molecular test for HCV RNA levels using polymerase chain reaction (PCR) assay to confirm active HCV infection. Simplification of the HCV evaluation algorithm to reduce the number of required tests could considerably expand the provision of HCV treatment especially in a developing country. This study investigates the performance of hepatitis C Core Antigen (HCV Ag) test by comparing HCV Ag results versus the results obtained with HCV ribonucleic acid (RNA) PCR which is considered the gold standard for the diagnosis of HCV infection.ResultsAmong the 109 anti-HCV positive sera, 96 were positive for both HCV Ag (> 3 fmol/L) and HCV RNA (> 15 IU/mL); 8 were negative for both tests, while the remaining 5 were positive for HCV RNA only. Considering the HCV RNA as gold standard; the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HCV Ag test were found to be 95.05%, 100%, 100%, and 61.54%, respectively. HCV genotype was performed for 59 patients. The most common HCV genotype was genotype 1 (72.9%). Genotype 2 (15.3%) and genotype 3 (11.9%) were detected in the others samples. A high level of correlation was seen between HCV RNA and HCV Ag (r = 0.958, p < 0.001). The correlation for the samples that were genotyped 1 was significant (r = 0.966, p < 0.001).ConclusionIn our study, it was found that there was strong correlation between HCV RNA levels and HCV Ag levels. So, it can be used for a one-step HCV antigen test to diagnose active HCV infection.

CORRELATION BETWEEN THE ANTIBODY RESPONSE TOWARD SPECIFIC HCV PROTEINS AND HCV VIRAL LOAD

Background: Hepatitis C virus (HCV) is an RNA virus causing acute or chronic infection and affecting more than 2% of population worldwide. The firstline tests for diagnosis of HCV infection are 3rd or 4th generation enzyme immunoassays - ELISA and CIA. They indicate the presence of antibodies against HCV in serum. These tests are characterized by high sensitivity and specificity, but they cannot distinguish past, acute or chronic infection, and sometimes produce false positive results. Confirmatory tests, such as recombinant immunoblot-line immune assay (LIA), and quantitative PCR, are used to validate the positive antibody response. The recombinant immunoblot assay can be used to determine the specificity of antibody to HCV. The aim of the present study is to determine the correlation between the anti-HCV response in confirmatory immunoblot assay and the HCV viral load, measured by PCR. Materials and methods: Twenty-nine anti-HCV positive sera were included in the study. Third generation ELISA assay was used for anti-HCV screening of the samples and for detection of anti-HCV antibodies against specific HCV proteins. Third generation line immunoassay INNO-LIA HCV Score, based on the principle of an enzyme immunoassay, was used as a confirmatory test. The HCV viral load was measured by quantitative PCR method – Abbott Real Time HCV (Abbott Molecular Inc., USA) with linear sensitivity range from 1.08 Log 10 IU/ml (12 [IU/ml]) to 8.00 Log 10 IU/ml (100 000 000 [IU/ml]). Results: HCV RNA was quantified in all studied samples. Ten of 29 serum samples (34%, Group I) were HCV RNA negative. The rest of the samples were HCV RNA positive as follows: 3 serums were with minimal viral load from &lt; 12 to 10 000 IU/ml (10%, Group II); 3 serum samples –between 10 000 and 100 000 IU/ml (10%, Group III); 10 serum samples – between 100 000 and 1 000 000 IU/ml (34%, Group IV) and in 3 serum samples HCV RNA concentration was over 1 000 000 IU/ml (10%, Group V). Conclusion: HCV screening strategies involving anti-HCV detection by ELISA combined with recombinant immunoblot assay can be the method of choice in laboratories with limited equipment and finances.

Resistance mutations of NS3 and NS5b in treatment-naïve patients infected with hepatitis C virus in Santa Catarina and Rio Grande do Sul states, Brazil.

Hepatitis C virus (HCV) infection is a worldwide health problem. Nowadays, direct-acting antiviral agents (DAAs) are the main treatment for HCV; however, the high level of virus variability leads to the development of resistance-associated variants (RAVs). Thus, assessing RAVs in infected patients is important for monitoring treatment efficacy. The aim of our study was to investigate the presence of naturally occurring resistance mutations in HCV NS3 and NS5 regions in treatment-naïve patients. Ninety-six anti-HCV positive serum samples from blood donors at the Center of Hematology and Hemotherapy of Santa Catarina State (HEMOSC) were collected retrospectively in 2013 and evaluated in this study. HCV 1a (37.9%), 1b (25.3%), and 3a (36.8%) subtypes were found. The frequency of patients with RAVs in our study was 6.9%. The HCV NS5b sequencing reveled 1 sample with L320F mutation and 4 samples with the C316N/R polymorphism. The analysis of the NS3 region revealed the D168A/G/T (3.45%), S122G (1.15%), and V55A (2.3%) mutations. All samples from genotype 3a (36.8%) presented the V170 I/V non-synonymous mutation. In conclusion, we have shown that mutations in NS3 and NS5b genes are present in Brazilian isolates from therapy-naïve HCV patients.

Evaluation of three commercialized rapid point-of-care tests for detection of anti-hepatitis C virus antibodies in Burkina Faso

This study aimed to evaluate the diagnostic performance of commercially rapid point-of-care (POC) tests used for HCV antibodies detection. This is a case-control study conducted in Ouagadougou between December 2014 and January 2015. Three POC for HCV antibodies detection (SD Bioline HCV test®, Anti-HCV dipstick® and First response® HCV card test) marketed in Burkina Faso were evaluated. Architect anti-HCV assay and ImmunoComb® II HCV were combined and used as a reference test. All three tests were evaluated with a panel of 62 anti-HCV positive sera and 62 anti-HCV negative sera. The tests performance was calculated using the software OpenEpi. The three rapid POC tests had a specificity of 100% (95% CI: 94.17-100). However, the sensitivities were 33.87% (95% CI: 23.34-46.28) for the SD Bioline HCV test®, 41.94% (95% CI: 30.48-54.33%) for Anti-HCV dipstick® and 45.16% (95%CI: 33.42-57.47%) for First response® HCV card test. The tests evaluated in this study had good specificity but poor sensitivity for the HCV antibodies detection in Burkina Faso. The surveillance of HCV rapid POC tests through the validation of their accuracy in the local context before their approval must be strengthened. Key words: Hepatitis C virus, point-of-care, diagnostic test.

Investigation of Viremia Rates and Its Correlation With Anti-HCV Levels in Anti-HCV Positive Serum Samples

Investigation of Viremia Rates and Its Correlation With Anti-HCV Levels in Anti-HCV Positive Serum Samples

Age and gender distribution of Hepatitis C virus prevalence and genotypes of individuals of physical examination in WuHan, Central China.

Approximately 170 million people in the world are infected with Hepatitis C virus (HCV). There are no published population based studies about the prevalence of HCV genotypes and the associations of genotype and Infection frequency with gender and age in WuHan. We aimed to investigate the distribution of HCV prevalence and genotypes among different gender and age patients with chronic HCV infection in WuHan from 2011 to 2015. A total of 2685 anti-HCV positive serum samples from individuals of physical examinationwere recruited from the Renmin Hospital of WuHan University, Hubei Province in China from January 2011 to December 2015. From these 2685 anti-HCV positive serum samples, 496 samples were with a positive PCR for HCV RNA. The number of HCV infection showed an increase with year, but the annual infection rate has remained similar (χ2 = 2.94, P = 0.568). 2685 cases were infected with HCV from 2011 to 2015 in WuHan city of China. Blood transfusion (18.14 %) was the main routs of transmission, followed by Surgery (8.94 %). The highest prevalence of HCV infection was at the age group 50–59 (25.85 % of 2685) and the lowest prevalence was 0–9 (0.93 % of 2685). HCV genotype 1 was the most prevalent (73.39 %), followed by genotypes 2 (17.14 %), 3 (5.25 %) and 6 (3.22 %). Genotype 4 and 5 was not detected in these patients. The most prevalent subtype was subtype 1b (71.98 %), followed by genotypes 2a (17.14 %). Five patients had mixed infection across the HCV subtypes. Among all genotypes, genotype 1 was highest in both male (73.27 %) and female (73.47 %) patients, followed by genotype 2. Genotype 1 (male: 29.84 % of 496, vs female: 43.55 % of 496, χ2 = 20.07, P < 0.0001), genotype 2 (male: 6.25 % of 496, vs female: 10.89 % of 496, χ2 = 6.81, P = 0.009), and 6 (male: 1.41 % of 496, vs female: 1.81 % of 496, χ2 = 0.626, P = 0.401) were more common in female patients than males, while no significant gender differences were observed for genotype 6. Among age group 50–59 years Genotype 1 was most common in male patients (29.05 % of 148) followed in 20–29 years (23.65 % of 148), genotype 2 in the age group 60–69 (12 cases of 31) and genotype 3 in the age group 50–59 (4 cases of 13) and genotype 6 was most frequent in the age group 30–39 (4 cases of 7). The frequency of HCV prevalence was significantly higher in female patients compared to males before ages 60, while the opposite result was observed after 60 years. The most common HCV genotype in WuHan was subtype 1b followed by 2a and more common among women than males patients. Further studies are needed to collect a large number of samples to estimate the different epidemiology of the HCV genotypes, because the sample size of non-genotype 1b and 2a is not large enough and other factors like disease history/monthly income/etc. are not included in our study.

Correlation between HCV genotype and anti-HCV antibody level in hepatitis C patients

Objective To investigate the relationship between anti-HCV antibody level and hepatitis C virus genotype in the patients. Methods Total of 603 anti-HCV positive serum samples were collected during 2013 to 2014 by retrospective research method. HCV RNA were detected in anti-HCV positive samples by repeat test and the genotype were detected in HCV RNA positive samples.The distribution of anti-HCV level in different hepatitis C genotype patients was analyzed and the body's response to viral antibodies and viral genotype correlation with anti-HCV concentration interquartile range was explored. Rates among genotype groups were compared using chi-square test. Results Totally 412 of 603(68.33%) samples were anti-HCV positive by double reagent screening. 174(42.3%) samples were detected as HCV RNA positive. The distributions of different anti-HCV level in different genotype patients were 1a(n=8)1/8, 1/8, 4/8, 2/8; 1b(n=112)25.9%(29/112), 17.0%(19/112), 25.9%(29/112), 31.3%(35/112); 2a(n=14)3/14, 4/14, 5/14, 2/14; 3a(n=11)3/11, 6/11, 2/11, 0/11; 3b(n=16)4/16, 11/16, 1/16, 0/16; 6a(n=8)2/8, 2/8, 1/8, 3/8 with anti-HCV concentration interquartile range respectively. The anti-HCV concentration distribution was different in patients with different HCV genotypes. The anti-HCV concentration distribution in patients of 1b, 2a and 6a genotypes were evently, while anti-HCV level was relatively high in 1a (13.65) and relatively low in 3b (8.77). There were differences in different genotypes of antibody concentrations (χ2=35.2, P<0.05). Conclusions There was correlation between anti-HCV level and HCV genotype. Because there were fewer cases in some genotypes, it was necessary to investigate more samples to corfirm the above results.(Chin J Lab Med, 2016, 39: 95-98) Key words: Hepacivirus; Genotype; Hepatitis C Antibodies; RNA, Viral

Genotyping of hepatitis B and C virus Russian isolates for reference serum panel construction.

Approximately 2% and 5% of the world human population is estimated to be infected with HCV and HBV, respectively. Reference panels of HCV and HBV serum samples with defined genotypes and serotypes is necessary for monitoring of the specificity and sensitivity of diagnostic test kits. The aim of this study was to determine genotypes/serotypes of HBV and HCV circulating in Russia in order to construct a panel of reference sera containing these HCV genotypes and HBV serotypes. A total of 343 HBsAg-positive and 207 anti-HCV positive serum samples were collected from patients with HBV and HCV infection from different cities between years 2002 and 2010 in St. Petersburg, Krasnodar, Nizhny Novgorod, Novosibirsk, Barnaul, Gorno-Altaisk, and Khabarovsk. HBV DNA was found in 76.4% of HBsAg positive samples by PCR for the S gene and HCV RNA was found in 71.5, 70.0, and 64.7% of anti-HCV positive samples in the 5'UTR, Core, and NS5B regions, respectively. The prevalence and proportion of HBV genotype/serotype associations were as follows: A/adw2, 2.1%; D/ayw2, 54.0%; D/ayw3, 43.1%; D/adw2, 0.7%. A new combination of genotype D and adw2 serotype was discovered. The distribution of HCV genotypes was the following: 43.6%, b; 3.8%, 2a; and 52.6%, 3a. Russian National reference panels of HBV and HCV lyophilized sera were developed to monitor specificity and sensitivity of approved kits and for the certification of newly developed assays.

A chimeric protein encompassing hepatitis C virus epitopes is able to elicit both humoral and cell‐mediated immune responses in mice

Hepatitis C virus (HCV) infection is a worldwide health problem. Vaccines against this pathogen are not available and advances in this field are limited because of the high genetic variability of the virus, inaccessibility of animal models, and incomplete definition of immunological correlates of protection. In the present work, a chimeric protein, Eq1, encompassing HCV amino acid regions from structural antigens, was generated. Eq1 was expressed in GC-366 bacterial cells. After cell disruption, Eq1 was purified from the insoluble fraction by sequential steps of differential solubilization and metal chelating affinity chromatography. Eq1 was specifically recognized by anti-HCV positive human sera. Moreover, immunization of BALB/c mice with different doses of Eq1 formulated either in Alum or Freund's incomplete adjuvant elicited both humoral- and cellular-specific immune responses. Doses of 20µg of Eq1 induced the strongest cell-mediated immune responses and only the formulation of this dose in Alum elicited a neutralizing antibody response against heterologous cell culture HCV. All these data together indicate that Eq1 is immunogenic in mice and might be an interesting component of vaccine candidates against HCV infection.

High Prevalence of Anti-HCV Antibodies in Two Metropolitan Emergency Departments in Germany: A Prospective Screening Analysis of 28,809 Patients

Background and AimsThe prevalence of hepatitis C virus (HCV) antibodies in Germany has been estimated to be in the range of 0.4–0.63%. Screening for HCV is recommended in patients with elevated ALT levels or significant risk factors for HCV transmission only. However, 15–30% of patients report no risk factors and ALT levels can be normal in up to 20–30% of patients with chronic HCV infection. The aim of this study was to assess the HCV seroprevalence in patients visiting two tertiary care emergency departments in Berlin and Frankfurt, respectively.MethodsBetween May 2008 and March 2010, a total of 28,809 consecutive patients were screened for the presence of anti-HCV antibodies. Anti-HCV positive sera were subsequently tested for HCV-RNA.ResultsThe overall HCV seroprevalence was 2.6% (95% CI: 2.4–2.8; 2.4% in Berlin and 3.5% in Frankfurt). HCV-RNA was detectable in 68% of anti-HCV positive cases. Thus, the prevalence of chronic HCV infection in the overall study population was 1.6% (95% CI 1.5–1.8). The most commonly reported risk factor was former/current injection drug use (IDU; 31.2%) and those with IDU as the main risk factor were significantly younger than patients without IDU (p<0.001) and the male-to-female ratio was 72% (121 vs. 46 patients; p<0.001). Finally, 18.8% of contacted HCV-RNA positive patients had not been diagnosed previously.ConclusionsThe HCV seroprevalence was more than four times higher compared to current estimates and almost one fifth of contacted HCV-RNA positive patients had not been diagnosed previously.

Evaluation of the immunogenicity of liposome encapsulated HVR1 and NS3 regions of genotype 3 HCV, either singly or in combination

BackgroundHepatitis C virus displays a high rate of mutation and exists as a quasispecies in infected patients. In the absence of an effective universal vaccine, genotype-specific vaccine development represents an alternative. We have attempted to develop a genotype 3 based, liposome encapsulated HCV vaccine with hypervariable region-1 (HVR1) and non-structural region-3 (NS3) components.ResultsHCV RNA extracted from serum samples of 49 chronically infected patients was PCR amplified to obtain HVR1 region. These amplified products were cloned to obtain 20 clones per sample in order to identify the quasispecies pattern. The HVR1 consensus sequence, along with three variants was reverse transcribed to obtain peptides. The peptides were checked for immunoreactivity individually, as a pool or as a single peptide tetramer interspersed with four glycine residues. Anti-HCV positivity varied from 42.6% (tetramer) to 92.2% (variant-4) when 115 anti-HCV positive sera representing genotypes 1, 3, 4 and 6 were screened. All the 95 anti-HCV negatives were scored negative by all antigens. Mice were immunized with different liposome encapsulated or Al(OH)3 adjuvanted formulations of HVR1 variants and recombinant NS3 protein, and monitored for anti-HVR1 and anti-NS3 antibody titres, IgG isotypes and antigen specific cytokine levels. A balanced Th1/Th2 isotyping response with high antibody titres was observed in most of the liposome encapsulated antigen groups. The effect of liposomes and aluminium hydroxide on the expression of immune response genes was studied using Taqman Low Density Array. Both Th1 (IFN-gamma, Il18) and Th2 (Il4) genes were up regulated in the liposome encapsulated HVR1 variant pool-NS3 combination group. In-vitro binding of the virus to anti-HVR1 antibodies was demonstrated.ConclusionThe optimum immunogen was identified to be combination of peptides of HVR1 consensus sequence and its variants along with pNS3 encapsulated in liposomes, which could generate both cellular and humoral immune responses in mice deserving further evaluation in a suitable cell culture system/non-human primate model.

Geographic distribution of hepatitis C virus genotype 6 subtypes in Thailand

The nucleotide sequence of hepatitis C virus (HCV) genotype 6 found mostly in south China and south-east Asia, displays profound genetic diversity. The aim of this study to determine the genetic variability of HCV genotype 6 (HCV-6) in Thailand and locate the subtype distribution of genotype 6 in various geographic areas. Four hundred nineteen anti-HCV positive serum samples were collected from patients residing in - the central part of the country. HCV RNA positive samples based on reverse transcriptase- polymerase chain reaction (RT-PCR) of the 5'UTR were amplified with primers specific for the core and NS5B regions. Nucleotide sequences of both regions were analyzed for the genotype by phylogenetic analysis. To determine geographic distribution of HCV-6 subtypes, a search of the international database on subtype distribution in the respective countries was conducted. Among 375 HCV RNA positive samples, 71 had HCV-6 based on phylogenetic analysis of partial core and NS5B regions. The subtype distribution in order of predominance was 6f (56%), 6n (22%), 6i (11%), 6j (10%), and 6e (1%). Among the 13 countries with different subtypes of HCV-6, most sequences have been reported from Vietnam. Subtype 6f was found exclusively in Thailand where five distinct HCV-6 subtypes are circulating. HCV-6, which is endemic in south China and south-east Asia, displays profound genetic diversity and may have evolved over a considerable period of time.

418 INFLUENCE OF HEPATITIS C VIRUS (HCV) INFECTION ON ATHEROSCLEROSIS RISK IN HIV-INFECTED ADULTS

According to a new standard operating procedure, all 7683 patients who were seen in our emergency wards between May and November 2008 and in whom a blood sample was taken as part of the routine medical work-up were tested for the presence of anti-HCV antibodies. Anti-HCV positive patients were further analysed for the presence of viremia by real-time PCR. A total of 194 patients (2.5%) were found to be anti-HCV positive. 44% were female. The average age of these patients was 55 years. So far, 110 anti-HCV positive sera were further screened for HCV-RNA and 60% of them were HCV-RNA positive and 66% had elevated AST levels. Only 35% of HCV-RNA positive patients had a history of previous antiviral therapy. No difference in anti-HCV prevalence has been observed whether the patients from the traumatology or internal emergency ward were analysed. In conclusion, in patients of the Berlin Charite emergency wards the antiHCV prevalence was 5 times higher as expected form previous general population studies in Germany. In view of the progress made in recent years in the treatment of patients with HCV infection and the favourable outcome when treated at early stages, these data can be taken as a strong hint for a more general HCV screening at least in great urban areas.

Antigenic structure of the hepatitis C virus envelope 2 protein

The antigenic structure of the envelope 2 (e2) protein of the hepatitis C virus (HCV) was characterized by the use of 70 synthetic peptides and 131 human sera from persons with antibodies to HCV. Among 34 overlapping peptides spanning the e2 protein of HCV, two major antigenic regions were located to residues 484-499 and residues 554-569. The sequence of the two major antigenic regions of the e2 protein are partly well conserved within the described types of HCV. Both regions contain two Cys residues in close proximity, and the region at residues 554-569 contains a putative N-glycosylation site, which are factors that previously have been suggested to affect the immune recognition of the e2 protein. Using substitution peptide analogues where each position within residues 484-499 and 554-569 were sequentially substituted by Ala or Gly, the most essential residues for antibody binding were found to be the Pro-498, Ala-499, Ala-566, Pro-567, and Pro-568. All of these, except for the Pro-498 and Ala-566, are conserved among different HCV strains. Also, according to previous studies, position 496 often shows variations, which could be explained by position 496 being contained within the antigenic region at residues 484-499. Interestingly, none of the Cys residues at positions 486, 494, 564 and 569 were found to be essential for antibody binding, indicating that these are not essential in maintaining the e2 antigenicity of the peptides. In a material of 114 confirmed anti-HCV positive sera, derived from patients during the acute or the chronic phase of HCV infection, the prevalence of antibodies to the two major linear antigenic regions of the e2 protein was found to be 55% among HCV RNA-positive sera, and 53% among HCV RNA-negative sera. In conclusion, we have identified and characterized two major linear antigenic regions outside the two hypervariable regions of the e2 protein. Since these regions are accessible to the B cells of the infected host, these two regions are likely to be surface exposed either on the precursor polyprotein or the native e2 protein. Also, we could confirm that antibodies to the e2 protein co-exist with HCV viraemia.

Genetic characterization of hepatitis C virus strains in Estonia: Fluctuations in the predominating subtype with time

During the last decade, there has been a dramatic increase in intravenous drug use in young adults in Estonia with an increased incidence of both hepatitis B and C as a consequence. Since genetic data are limited regarding hepatitis C virus (HCV) strains in Estonia, the aim of the study was to characterize HCV strains in different risk groups to determine their relatedness to strains from other geographical regions. Three hundred fifty-three anti-HCV positive sera collected during 1994-2004 from hospitalized patients, blood donors and health care workers were used as source of HCV RNA. Two hundred nine (59%) of the sera were positive for HCV RNA by PCR directed to the 5'-UTR region. For 174 strains the HCV subtype was determined by analyses of the NS5B and/or the 5'UTR-core regions. 1b (71%) was the most common subtype followed by 3a (24%), 2c (2%), 1a (1%), and 2a (1%). The 1b and 3a strains were similar to strains from other regions of the former USSR. Within genotype 1b there were several HCV lineages. However, for 3a there seemed to be two separate introductions into Estonia. There was a relative shift from subtype 1b to 3a in 1999-2000 with a further replacement of 3a with 1b in intravenous drug users in 2001 and onwards (P < 0.05). However, both subtypes were found to co-circulate in the community independent of risk factors. One patient was infected with the 2k/1b recombinant presumed to originate from St. Petersburg being the first isolate of this recombinant recovered outside Russia.

Detection of antibodies to hepatitis C virus: False-negative results in an automated chemiluminescent microparticle immunoassay (ARCHITECT ® Anti-HCV) compared to a microparticle enzyme immunoassay (AxSYM HCV Version 3.0)

Background: Following an accidental observation of reduced sensitivity for detection of antibodies to hepatitis C virus (HCV) with a novel commercially available automated chemiluminescent microparticle immunoassay (CMIA, ARCHITECT ® Anti-HCV) compared to a well-established microparticle enzyme immunoassay (MEIA, AxSYM HCV Version 3.0), we wanted to explore whether this could be explained by a variation in marginal sensitivity, to be expected between highly sensitive assays of different formats, or represented a reduced sensitivity of the CMIA to certain antibody profiles. Objectives: To evaluate the ability of the CMIA to detect low concentrations of anti-HCV antibodies as defined by various patterns in the recombinant immunoblot assay (RIBA) and already detected in the MEIA system. Study design: All patient sera tested for anti-HCV reactivity during a period of 3 years (27,978) were evaluated. A total of 90 sera had a sample/cut-off ratio (S/CO) between 1.0 and 1.5 in the MEIA test and were available for further testing. Of these, 19 had a probable/possible presence of anti-HCV antibodies based on presence of at least two bands of ≥1+ strength in the RIBA, or because the patient was known to be anti-HCV positive. These 19 sera were tested with the CMIA. In addition, 16 sera with strong reactivity to various antigen combinations in the RIBA were serially diluted until testing negative in both microparticle test systems. Results: Seven of the 19 sera (37%) were negative (S/CO < 1.0) in the CMIA. At least 3 (16%) of these 19 sera were very likely to be true anti-HCV positive sera (from infants with known anti-HCV positive mothers). HCV-RNA was not detected in any of the sera tested. Testing of sera after serial dilution indicates that the CMIA has a lower sensitivity to c22- and c33c-antibodies than the MEIA, possibly also to c100-3-antibodies. Conclusion: Our findings indicate that ARCHITECT ® Anti-HCV is less sensitive than AxSYM HCV Version 3.0 in detecting antibodies to c22 and c33c in patients who have cleared their HCV-infection and have naturally declining levels of antibodies.

Soroprevalência e genótipos do vírus da hepatite C em pacientes com lúpus eritematoso sistêmico (LES) em Goiânia, Brasil

A infecção pelo vírus da hepatite C (HCV) desperta grande interesse em reumatologia por apresentar várias manifestações extra-hepáticas. Diversos estudos mostram associação elevada da infecção pelo HCV com manifestações reumáticas como dores musculares, crioglobulinemia mista, síndrome reumatóide, síndrome de Sjögren, vasculite, glomerulonefrite, fenômeno de Raynaud, poliarterite nodosa, miopatia, auto-anticorpos e outras manifestações de doença difusa do tecido conjuntivo. Em estudos anteriores realizados em nosso meio verificou-se a prevalência de 0,9% em gestantes, 1,4% em doadores de sangue, 1,8% em paciente com hanseníase e 2,0% em trabalhadores da área da saúde. OBJETIVO: determinar a prevalência da infecção pelo HCV em pacientes com lúpus eritematoso sistêmico (LES) atendidos no Serviço de Reumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade Federal de Goiás (HC-FM-UFG). MÉTODOS: foram entrevistados 175 pacientes com LES e amostras sangüíneas coletadas. Inicialmente, pesquisou-se a presença de anticorpos para o HCV (anti-HCV). As amostras anti-HCV positivas foram submetidas à detecção do RNA viral, pela reação em cadeia da polimerase (PCR) com primers complementares à região 5' não codificante do HCV. As amostras positivas foram genotipadas por line probe assay. RESULTADOS: a prevalência encontrada foi de 2,3% (4/175), e a identificação dos genótipos nas amostras RNA-HCV positivas revelou a presença do tipo 1 do HCV em três pacientes (75%) e do tipo 3 em um paciente (25%). Das quatro pacientes infectadas, três apresentaram evolução clínica favorável e uma evoluiu para óbito em razão de insuficiência renal por nefrite lúpica. CONCLUSÕES: a prevalência de HCV em pacientes com LES revelou-se um pouco maior que a encontrada anteriomente em gestantes, doadores de sangue e em hansenianos, comparável à encontrada em trabalhadores da saúde.

Combination hepatitis C virus antigen and antibody immunoassay as a new tool for early diagnosis of infection

Reduction of the window period of hepatitis C virus (HCV) infection represents an important goal in the transfusional and diagnostic setting. A prototype assay designed to simultaneously detect circulating HCV antigen and anti-HCV, has been developed. Aim of this study was to evaluate the performance of this new assay in terms of specificity and sensitivity and to compare its efficacy with commercial assays. To evaluate the specificity of the assay, 400 samples from the general population and 100 'difficult' sera, negative for anti-HCV, were tested. To assess sensitivity, the new test was used on 76 PCR-positive and anti-HCV negative sera, seven natural or commercial seroconversion panels that included 17 RNA-positive and anti-HCV negative sera and 31 anti-HCV positive sera, 20 weak anti-HCV positive sera, 80 viraemic and anti-HCV-positive sera from patients infected with different subtypes and 10 sera from patients with HBV-HCV or HIV-HCV co-infections. Of 500 anti-HCV negative samples, 499 (99.8%) were negative with a cut-off index <0.5, while one sample was within the grey zone. Of the 93 HCV-RNA positive and anti-HCV negative sera from patients and panels, 85 (91.4%) resulted positive, and one had the cut-off index in the grey zone. The reduction in the diagnostic window period observed with the new test and HCV-RNA assays were equal, on average, to 24 and 34.4 days respectively. All anti-HCV positive sera were positive. The new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or HCV-RNA detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible because of costs, organization, emergency and/or logistic difficulties.

Immunoreactivity studies on synthetic peptides deriving from immunodominant region of hepatitis C virus NS5a gene

To identify the location of major immunodominant antigenic region and study the relationship between the gene heterogeneity and immunoreactivity via detecting antigenic reactivity of synthetic peptides deriving from immunodominant region in different genotypes of hepatitis C virus (HCV) NS5a gene. In total, 305 non-identical 30-mer long and overlapping by 15 aa peptides derived from HCV NS5a region from codon 2,182 to 2,343 among 45 unique published HCV sequences in GenBank corresponding to different genotype were designed and synthesized. The amino acid sequences of all peptides were compared with DNA Star software. The antigenic reactivity of those peptides was detected with indirect ELISA with both anti-HCV and anti-NS5 positive serum. The sequences showed highly conserved among HCV genotype in regions 2,272-2,301 and 2,302-2,331 as compared to regions 2,212-2,241 and 2,257-2,286. The peptides basing on amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331 showed stronger immunoreactivity than any other peptides. Eighteen peptides derived from this region showed a broad immunoreactivity, 3 of them could react with 96% of anti-HCV positive sera. Whereas the immunoreactivity of the peptides derived from the region showing highly variable among HCV genotype was found to react more strongly with homologous-genotype sera. The major linear antigenic region was located at amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331; the short synthetic peptide derived from NS5a region at position 2,212-2,241, 2,272-2,301 and 2,302-2,331 can be used for efficient detection of HCV antibody; some peptides showed genotype specific immuunoreactivity.

Matrix effects in clinical immunoassays and the effect of preheating and cooling analytical samples.

Immunological reactions are influenced by various factors including antigens, antibodies and other variables. We focused on two items: i) matrix effects, especially of detergents and ii) temperature effects: preheating sera, especially effects on rheumatoid factor (RF) measurement and false-positive reactions in ELISAs, and cold storage of sera, especially effects on complement. Among various additives, detergents affected the agglutination reaction for fecal hemoglobin and hepatitis B surface (HBs) antigen. Some of the detergents examined abolished these antigenicities, however, polyethylenglycols enhanced the reactions. Heat-inactivation of sera at 56 degrees C for 30 min was employed in serological testing. However, in RF measurement, 10 min of preheating was sufficient to abolish C1q (subcomponent of C1), which could participate in the agglutination reaction. In ELISA for antibodies, false-positive reactions were caused by preheating sera. By the analyses of assays for antibodies to hepatitis C virus (HCV) and cardiolipin, it was found that they were induced by immunoglobulin G (IgG) modified by preheating. Cold storage induced activation of complement (cold activation) in anti-HCV antibody positive sera. CH50 titers in the sera were lowered by one cycle of freezing at -20 degrees C and thawing, and the decrease was affected by the containers.