Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA

Halfon, Philippe; Khiri, Hacène; Gérolami, V.; Bourlière, Marc; Feryn, Jean Marc; Reynier, Pascal; Gauthier, A.; Cartouzou, G

doi:10.1016/s0168-8278(96)80116-4

Cited by 126 publications

(87 citation statements)

References 26 publications

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“…It is well recognized that storage temperature can affect HCV-RNA stability and the recommended storage temperature for HCV-RNA is -70 o C (Halfon et al, 1996). The samples stored at 25 o C maintain their HCV-RNA titer during 14 days and at 5 o C were stable for at least 3 months, the stability of HCV RNA in plasma at -20 o C for RT-PCR reactivity at least 2.5 years at concentration equal to or higher that 100 IU/ml (Jose et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Epidemiology of Hepatitis C Virus Genotypes in Northeastern Thai Blood Samples

Barusrux

Sengthong²,

Urwijitaroon³

2014

Asian Pacific Journal of Cancer Prevention

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Background: Hepatitis C virus (HCV) infection is an important cause of liver cancer in Thailand. The highest prevalence of anti-HCV positive among Thai blood donors is found in the northeastern region. The present analysis of the genotype distribution among anti-HCV positive northeastern-Thai blood donors was conducted to provide a base for the epidemiological pattern of HCV infection in this region. Materials and Methods: A total of 112 HCV seropositive healthy blood donors were randomly selected and tested for the presence of HCV-RNA by RT-PCR. HCV-RNA positive samples were genotyped by direct sequencing at core region genomes and confirmed by phylogenetic analysis. Results: HCV viremia was found in 94.6% (106/112) of HCV seropositive blood donors. There were 3 major genotypes distributed among this population. HCV genotype 3a was the most prevalent (71.7%) followed by genotypes 1a (7.5%), 1b (7.5%), 6i (3.8%), 6f (2.8%) and 6n (1.9%). Conclusions: HCV genotype 3a in asymptomatic infections in northeastern Thailand is significantly higher than other previous reports. Subgenotype 6 prevalence is less than in neighboring countries and distribution patterns differ. The findings are relevant as predictors for using interferon therapy in this population.

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Section: Discussionmentioning

confidence: 99%

Epidemiology of Hepatitis C Virus Genotypes in Northeastern Thai Blood Samples

Barusrux

Sengthong²,

Urwijitaroon³

2014

Asian Pacific Journal of Cancer Prevention

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“…HCV RNA is not stable when stored at ambient or 4 o C for longer than a few days. 28 Multiple freeze-thaw cycles can decrease the integrity of the specimens, resulting in artefactual modifications of viral load. 21,29 All the specimens used for this study were exposed to a single freeze-thaw cycle before qualitative and quantitative evaluations.…”

Section: Discussionmentioning

confidence: 99%

Natural fluctuations of hepatitis C viral load in a homogeneous patient population: A prospective study

et al. 2000

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Hepatitis C virus (HCV) infection is the main cause of parenteral non-A, non-B hepatitis. 1-5 HCV consists of a heterogeneous mix of isolates defined by genotype, each of which is further classified into subtypes. A number of factors have been identified as important in predicting the outcome of disease progression. These include age at infection, viral type/subtype, viral load, quasispecies, and mode of infection. [6][7][8][9][10] Clinical heterogeneity in disease progression may reflect either viral heterogeneity or variations in host response. However, the fluctuations in viral load during the natural history of hepatitis C infection are poorly understood. The purpose of the present study was to determine if viral load remains at a steady state or is in dynamic flux during the course of an HCV infection. To avoid heterogeneity of risk factors and confounding variables in viral type/subtype, we studied a unique cohort of individuals all infected by anti-D/ blood product from a single source of HCV 1b. The sole source of the infectious agent was identified as HCV 1b-contaminated anti-D immunoglobulin. The contaminating HCV 1b was derived from a single donor. [11][12][13] The patients were of similar ethnogeographic background and had an absence of other risk factors for liver disease.Serum HCV RNA can be quantitated by a range of different technologies (reverse transcription-polymerase chain reaction [RT-PCR], RT-PCR coupled to different signal amplification systems such as colorimetric assays, and the branched chain technology). [14][15][16][17][18][19][20] The level of sensitivity achieved by the more recent versions of these technologies has improved qualitative assessment of HCV serum status. Clinically relevant sensitivities of 100 viral genomes per mL of serum are now readily achievable.The work reported here used the Roche Monitor system for amplification of the HCV. The high degree of reproducible quantification of viral load and a uniform linear range of amplification between the different versions of the Roche HCV Monitor assays for HCV of genotype 1b provide a means in which quantifications over several generations of assays are suitable for longitudinal studies. In addition, amplification of HCV genotype 1b by the Monitor system does not suffer from the reported difficulties associated with other genotypes. 21

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“…However, it has been difficult to predict the exact likelihood of a response in a given patient. This is mainly a result of the following problems: 1) most studies were retrospective, and quality of frozen sera depends on many parameters (temperature, time between sampling and storing, number of freezethaw cycles) 24,25 ; 2) there is no standardization of quantitative assays; and 3) the sensitivity and reproducibility of available assays relative to standards (e.g., samples serving to infect chimpanzees) is poorly evaluated. 26 Several studies have evaluated methods for the quantitation of HCV RNA, 27 such as quantitative RT-PCR 7,28,29 and, more recently, bDNA, 8,9,30 the semiquantitative Amplicor assay, 31 and the NASBA method.…”

Section: Discussionmentioning

confidence: 99%

Comparative Evaluation of Hepatitis C Virus Rna Quantitation by Branched Dna, Nasba, and Monitor Assays

et al. 1999

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Since its discovery in 1989, 1 it has been demonstrated that hepatitis C virus (HCV) is a major cause of chronic hepatitis throughout the world. The routine virological diagnosis is based on serological tests detecting antibodies to HCV. However, in the absence of an efficient culture system for HCV, or viral antigen assays, direct detection of HCV is based on nucleic acid amplification and hybridization methods. HCV RNA was principally detected with in-house nested polymerase chain reaction (PCR) methods, but new standardized assays such as Amplicor (Roche Diagnostics Systems, Basel, Switzerland) and nucleic acid sequence-based amplification (NASBA) (Organon Teknika, Boxtel, the Netherlands) have been developed to detect HCV RNA on a routine basis 2,3 or for research purposes. 4,5 Several studies have shown a relation between pretreatment viral load and the response to interferon (IFN) therapy, 6-10 creating a need for standardized HCV-RNA quantification assays. Currently, the most frequently used kit is the branched DNA (bDNA) assay (Quantiplex HCV RNA, Chiron Diagnostics, Emeryville, CA), a well-standardized signalamplification method. Other assays are based on HCV-RNA amplification (Amplicor HCV Monitor, Roche Diagnostics Systems, and NASBA HCV). The aim of this study was to compare the NASBA, bDNA, and Monitor HCV assays in terms of reproducibility, sensitivity, and linearity in various clinical settings and using standardized panels. Two qualitative methods (in-house nested PCR and Amplicor) were used as standards for sensitivity. Specificity was not a focus of this study, because specificity studies were performed by the manufacturers during the test registration procedure, and because we routinely obtain very good specificity in our lab. Moreover, negative samples were included in each series of tests (anti-HCV-and PCR-negative blood donors). MATERIALS AND METHODS Pelicheck HCV-RNA EUROHEP PanelThis panel was used to evaluate the sensitivity and linearity of each assay. It contains two dilution panels of the EUROHEP genotype 1 and 3 plasma standards. The plasma standards have been characterized in two collaborative EUROHEP studies. 11,12 The EUROHEP HCV-RNA genotype 1b and 3a plasma standard dilutions were prepared from a pool of plasma units negative for hepatitis B surface antigen, anti-hepatitis B core antibodies (Ab), anti-HCV Ab, anti-human immunodeficiency virus-1 or -2 Ab, anti-human T-lymphotropic virus type 1 Ab, syphilis, HCV RNA, hepatitis B virus DNA, and human immunodeficiency virus RNA. The undiluted plasma samples are not available. According to the EUROHEP group, the undiluted plasma corresponding to the genotype 1 panel should contain 38 Ϯ 8 ϫ 10 5 genome equivalents per milliliter (eq/mL) in the bDNA 2.0 assay, and 2.8 ϫ 10 5 copies per milliliter (cps/mL) in the HCV RNA Monitor assay. The potency of the

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Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA

Cited by 126 publications

References 26 publications

Epidemiology of Hepatitis C Virus Genotypes in Northeastern Thai Blood Samples

Epidemiology of Hepatitis C Virus Genotypes in Northeastern Thai Blood Samples

Natural fluctuations of hepatitis C viral load in a homogeneous patient population: A prospective study

Comparative Evaluation of Hepatitis C Virus Rna Quantitation by Branched Dna, Nasba, and Monitor Assays

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