No abstract

No abstract

Chronic infection with the hepatitis C virus (HCV) isThe identification of the hepatitis C virus (HCV) in 1989 led to the recognition that hepatitis C is a major cause of end-stage liver disease, accounting for more than 20% of liver transplantations in the United States in 1995. 1 Recognition of the importance of HCV infection has been the result of increasingly reliable methods of detection. An enzyme-linked immunoassay (EIA-1) to detect antibody to HCV (anti-HCV) was introduced in 1990, followed by a more sensitive and specific second-generation serological test (EIA-2) by the middle of 1992. Simultaneously, a second-generation recombinant immunoblot assay (RIBA-2) was introduced as a confirmatory test for the EIA. These assays for antibody were supplemented by sensitive methods to detect HCV RNA in serum, most notably by reverse-transcription polymerase chain reaction (RT-PCR) methods. The HCV RNA can also be characterized according to genotype and serum concentration. Application of these assays to serum from patients undergoing liver transplantation has better defined the role of HCV as a cause of end-stage liver disease and the clinical challenges of hepatitis C both before and after transplantation.Various aspects of HCV infection have been evaluated in liver transplantation, including rate of recurrence, 2-4 transmission from infected donors, 5,6 and the accuracy of serological assays. 7 However, these studies have generally examined isolated features of infection among relatively small numbers of patients or at a single center. We performed systematic testing for HCV in a large, multicenter, prospective study to evaluate donor and recipient predictors of posttransplantation infection, serological changes with transplantation, and genotype and viral levels before and after transplantation.

High prevalence of hepatitis C (HCV) and hepatitis G (HGV) viruses has been reported among hemodialysis patients with substantial heterogeneity of HCV genotypes throughout the world. We studied HCV prevalence, clinical significance, genotype distribution, and HGV coinfection in hemodialysis patients from Syria. Ninety (75%) of 120 screened patients were HCV antibody positive. Forty-nine (87.5%) of 56 HCV antibody-positive patients had HCV RNA detected by the polymerase chain reaction. The HCV genotyping was possible in 37 of 49 patients (76%). The HCV genotype distribution was genotype 1a, seven (19%); genotype 1b, 10 (27%); genotype 4a, 11 (30%); unmatched sequences, nine (24%). Phylogenetic analysis of unmatched sequences indicated that they represent two distinct and novel subtypes of HCV genotype 4. Hepatitis G virus RNA was detected in 29 (59%) of the HCV RNA-positive patients. No differences were identified between patients infected with HCV alone and those coinfected with HGV. These data demonstrate that HCV infection is common in this population with a genotype distribution predominantly made up of types 1 and 4. Coinfection with HGV had no effect on the outcome of HCV infection.

(1) Histologic evidence of recurrent hepatitis C is seen in 90% of liver allografts; (2) Histologic hepatitis C recurs with similar frequency in genotype 1b and non-1b recipients; (3) Genotype 1b is associated with more severe histologic disease recurrence than non-1b genotypes; (4) Genotype 1b appears to be associated with a higher degree of posttransplant fibrosis and cirrhosis than non-1b genotypes.

It has been suggested that prolonged formalin fixation and block storage adversely affect hepatitis C virus (HCV) ribonucleic acid (RNA) detection in tissue by reverse transcriptase-polymerase chain reaction (RT-PCR). We attempted to determine whether short-term perfusion fixation (3-5 days) or prolonged formalin storage adversely affects the detection of HCV RNA in paraffin-embedded tissue in comparison with 24-h fixation. Also, we examined the effects of prolonged storage of paraffin blocks on the sensitivity for HCV detection. We performed RT-PCR in formalin-fixed explanted livers from 20 liver allograft recipients known to be HCV positive (10 with specimens stored for 2-4 years and 10 with specimens stored for > 4 years). We compared the results of perioperative needle liver biopsy specimens fixed overnight with liver sections fixed by perfusion for 3-5 days and bulk liver tissue stored in formalin for years (mean, 6.25 years; range, 2-11 years). HCV RNA was detected in 100%, 85%, and 0% of specimens fixed for 24 h, 3-4 days, and years, respectively. We conclude that HCV can be readily detected in tissue fixed by formalin overnight, sensitivity decreases slightly with intermediate-length fixation, and HCV is rendered undetectable by prolonged fixation. In addition, retention of formalin-fixed tissue in paraffin blocks does not affect the sensitivity of HCV detection.

Infection with hepatitis B virus (HBV) remains a difficult worldwide challenge to public health. The World Health Organization estimates that more than one-third of the world's population has been infected with HBV (25). Epidemiological trends suggest there are currently 400 million HBV chronic carriers worldwide, with over 1 million deaths annually due to HBV-associated liver disease (13,23). HBV is the leading cause of cirrhosis and hepatocellular carcinoma globally (25; Centers for Disease Control and Prevention hepatitis fact sheet [www.cdc.gov/hepatitis]). The development and utilization of molecular diagnostic assays for the detection and quantification of HBV genomes have provided insight into the natural history of HBV and the pathogenesis of HBV infection as well as facilitated the monitoring of viral response to treatment (15,17). In addition, a quantitative evaluation of HBV DNA concentrations can provide valuable information on the levels of viral replication and may be useful as a prognostic indicator of liver disease (4, 21). A number of commercial assays are currently available for the quantification of HBV DNA in patient serum or EDTA-plasma, including hybridization-, signal-, and target-amplification-based technologies (5,11,(16)(17)(18)22). Selection of the optimal assay is dependent on the intrinsic performance characteristics of the methodology as well as the necessity to make appropriate clinical decisions in the context of HBV-associated disease (4, 15, 21).The VERSANT HBV 3.0 Assay (referred to herein as VER-SANT 3.0) is a third-generation branched-DNA (bDNA) assay for the direct quantification of HBV DNA in human serum and plasma. After HBV genomic DNA is released from the virions, the viral DNA is captured by a set of specific, synthetic oligonucleotide capture probes fixed in a microtiter well. A set of target probes (or label extender probes) then hybridizes to both the captured viral DNA and unique preamplifier probes. The capture probes and the target probes bind to conserved DNA regions throughout the entire HBV genome. The amplifier probes subsequently hybridize to the preamplifier probes, forming a bDNA complex. Multiple copies of an alkaline phosphatase-labeled probe are then hybridized to this immobilized complex. Detection is achieved by incubating the alkaline phosphatase-bound complex with a chemiluminescent substrate. The intensity of light emission is directly related to the amount of HBV DNA present in each sample, and results are recorded as relative light units by the luminometer. A standard curve is defined by light emission from quantitative standards containing known concentrations of recombinant DNA. Concentrations of HBV DNA in specimens are determined from this standard curve. This third-generation sandwich nucleic acid hybridization procedure differs from earlier bDNA assays by using the unique preamplifier probes to increase the number of labeled probes that can bind to the target, thereby

The Abbott RealTime human immunodeficiency virus type 1 (HIV-1) assay (ART) and the Cobas AmpliPrep/ Cobas TaqMan HIV-1 test (CTM) are commercially available assays for quantification of HIV-1 RNA in plasma. We evaluated performance characteristics, workflow, throughput, reliability, and direct costs of these assays. Both assays yielded good correlation of quantitative results (r ‫؍‬ 0.95) among clinical specimens, with a mean difference of ؊0.34 log 10 copies/ml. Testing of healthy donor plasma specimens yielded "target not detected" results by ART, with "HIV-1 RNA detected, <40 copies/ml" results for 3.3% (3 of 90 samples) of these specimens by CTM. Both the m2000sp/m2000rt (ART) and docked CAP/CTM96 (CTM) instrument systems were capable of operating with continuous, uninterrupted workflow. When daily maintenance and cleaning were included, ART and CTM run durations (5 h 52 min and 6 h 4 min, respectively) and hands-on times (53 min and 46 min, respectively) were similar for a run batch size of 24. While ART was more flexible in terms of run batch size, CTM required fewer user interventions and consistently produced higher specimen throughput rates at 8, 16, and 24 h. Assay run failure rates were 6.3% (1 of 16 runs) and 4.2% (1 of 24 runs) for ART and CTM, respectively (P ‫؍‬ 1.000), with invalid specimen result rates of 1.0% (5 of 495 specimens) and 2.8% (11 of 399 specimens), respectively (P ‫؍‬ 0.073). Direct reagent and consumable costs for each assay were comparable (difference of <10%). In selecting an assay for implementation, laboratories should consider how various assay and instrument features might impact laboratory operation and patient care.