46 research outputs found
Automated RIBA Hepatitis C Virus (HCV) Strip Immunoblot Assay for Reproducible HCV Diagnosis
A comparison between the CHIRON RIBA hepatitis C virus (HCV) processor and manual systems was performed by using 88 specimens repeatedly reactive by the second-generation HCV enzyme-linked immunosorbent assay (ELISA) (HCV 2.0 ELISA) and 111 random specimens from volunteer donors. For the second-generation RIBA HCV strip immunoblot assay (SIA) (RIBA HCV 2.0 SIA), test results correlated strongly between the manual and the automated runs (kappa value, 0.937). For the RIBA HCV 3.0 SIA, the correlation of the test results was also high (kappa value, 0.899). Among the specimens with positive results by RIBA HCV 2.0 and 3.0 SIAs, there was a very strong concordance of the test results between the manual and the automated runs with regard to the reactive bands. Nine samples had discordant results between the manual and the automated runs; this was probably attributable to increased variability in antigen scores close to the cutoff values for both tests. Run-to-run and within-run testing by the CHIRON RIBA HCV Processor System showed a very low rate of conflicting values. In conclusion, the CHIRON RIBA HCV Processor System is capable of performing RIBA HCV 2.0 and 3.0 SIAs accurately with minimal operator involvement. In addition, the CHIRON RIBA HCV Processor System shows excellent reproducibility, with the potential for operator-to-operator and site-to-site variability being greatly reduced. Our data indicate that this novel methodology may be very useful for supplemental anti-HCV testing of specimens repeatedly reactive by ELISA in routine clinical assessments and epidemiologic evaluations
Evaluation of a novel serotyping system for hepatitis C virus: strong correlation with standard genotyping methodologies.
Direct sequencing and analysis of viral genomes are definitive methods for identifying various hepatitis C virus (HCV) genotypes. However, HCV genome sequencing methods are cumbersome and unsuitable for analyzing large numbers of clinical samples. We have developed a convenient, reliable, and reproducible RIBA strip immunoblot assay system for determining HCV serotype. Briefly, the assay consists of an immunoblot strip on which there are five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS-4) and core regions of the genomes of HCV types 1,2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS-4 serotype-specific HCV peptide band in relation to the intensity of the human immunoglobulin G internal control bands on each strip. HCV core peptide reactivity is used only in the absence of NS-4 reactivity. We used this assay to successfully serotype a high percentage of sera from well-documented HCV-infected patients. Our serotyping results correlated 99% with the findings from the standard restriction fragment length polymorphism genotyping methods. Less than 5% of the serum samples were untypeable. For a selected group of alpha interferon-treated patients we observed that the nonresponders (76.2%) and a majority of the responders who relapsed (72.2%) had type 2 HCV infection. A small population (n= 8) of complete responders was split 3:4:1 as type 1, type 2, and type 3, respectively. Our data indicate that this new serotyping assay has the potential to be a highly specific and reliable method for typing of HCV infection in patients
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Automated RIBA HCV strip immunoblot assay: a novel tool for the diagnosis of hepatitis C virus infection in hemodialysis patients
Hemodialysis (HD) patients remain a high-risk group for hepatitis C virus (HCV) infection. Serological assays (enzyme-linked immunosorbent assays, ELISAs) are the only tests currently approved by the Food and Drug Administration in the United States for the diagnosis of HCV. The RIBA HCV Strip Immunoblot Assay (SIA) is an established method for supplemental testing of repeat reactive hepatitis C ELISA patients on HD. However, the current manual procedure is labor intensive, requiring subjective band scoring and result interpretation. Recently, the automated CHIRON RIBA HCV Processor System has been designed to perform RIBA supplemental testing. The CHIRON RIBA HCV Processor System consists of a bench-top instrument that provides objective evaluation of the RIBA immunoblot strips, by measuring the light differentially reflected from the developed bands and white background, creating a density of reflectance. The CHIRON RIBA HCV Processor System assesses the intensity of each of the reactive bands in relation to the intensity of the internal control bands on each RIBA HCV strip. Comparison between processor and manual protocols was performed using a large (n = 200) cohort of ELISA 3.0 HCV negative and positive patients on maintenance HD. The test characteristics of RIBA HCV 3.0 SIA were identical with manual and automated runs. The relative intensity values of antigenic bands by the CHIRON RIBA HCV 3.0 Processor System between anti-HCV positive and negative patients were significantly different; only 15 of 784 (1.9%) antigenic bands had borderline reactivities. The correlation of test results between manual and automated runs was very high (kappa value 0.989). Among positive results by RIBA HCV 3.0 SIA, there was a strong concordance between manual and automated runs with regard to the pattern of reactivity (kappa value 0.943). The discordant results between manual and automated protocols were attributable to increased variability of antigen scores close to the cutoff value for both tests. In conclusion, the CHIRON RIBA HCV 3.0 Processor System is capable of performing RIBA HCV 3.0 SIA in the HD population accurately with minimal operator involvement. The test characteristics of RIBA HCV 3.0 SIA were identical by manual and automated runs. There was a strong correlation between the results of the manual and automated runs; the few discordant results between the two procedures were mostly due to increased variability of antigen scores close to the cutoff value for both tests. The Centers for Disease Control and Prevention in the USA have recently included chronic HD patients among those persons for whom routine HCV testing is recommended; HCV-infected patients on HD often have a high rate of indeterminate results by manual RIBA technology which is operator dependent for band scoring and result interpretation. The CHIRON RIBA HCV 3.0 Processor System may be very useful for supplemental anti-HCV testing of ELISA repeat reactive specimens in clinical practice within dialysis units
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Epidemiology of Helicobacter pylori in chronic haemodialysis patients using the new RIBA H. pylori SIA
There are few data concerning the epidemiology of H. pylori in patients on chronic haemodialysis (HD) treatment. These surveys concerned small populations and were made with ELISA technique. However, ELISA-based assays do not differentiate between strains of H. pylori that are associated with ulcers. Recent literature reports that formation of ulcers correlates strongly with the expression of cytotoxin-associated protein (CagA) and vacuolating cytotoxin (VacA) of H. pylori.
A novel serological test (RIBA H. pylori strip immunoblot assay (SIA)) has been recently introduced, it uses the H. pylori lysate (Lys) along with two additional purified recombinant antigens derived from CagA and VacA of H. pylori.
To study the epidemiology of H. pylori using RIBA H. pylori SIA among chronic HD patients and blood donors as a control group. In addition, the activity of H. pylori was analysed by immunoblot technique in a group of patients with documented ulcers and normal renal function.
The prevalence of antibody towards H. pylori among HD patients, blood donors, and patients with documented ulcers was 56% (127/228), 53% (84/158), and 100%, (21/21) respectively; the difference was significant (P=0.0001). The frequency of anti-H. pylori-positive individuals was significantly higher in patients with documented ulcers than HD patients and blood donors, 21/21 (100%) vs 211/386 (55%), P=0.0001. The frequency of antibody to H. pylori in the HD population was significantly associated with race (P= 0.005); no relationship between anti-H. pylori antibody and numerous demographic, biochemical, and clinical features of patients was seen. The frequency of antibodies against virulent strains of H. pylori in HD patients and blood donors with H. pylori was 60% (76/127) and 61% (51/84) respectively; it was 86% (18/21) among individuals with documented ulcers. No significant difference among these three groups occurred.
The frequency of antibody towards H. pylori by RIBA H. pylori SIA was high both in HD patients and blood donors; patients with documented ulcers and normal renal function had significantly higher frequency of anti-H. pylori antibody. The anti-H. pylori antibody rate among HD patients was strongly associated with race. The prevalence of antibody against virulent strains of H. pylori did not change among HD patients and control groups. Studies in large cohorts of HD patients with documented peptic ulcer disease are in progress