Comparison of Conventional PCR with Real-Time PCR and Branched DNA-Based Assays for Hepatitis C Virus RNA Quantification and Clinical Significance for Genotypes 1 to 5

Abstract

Present recommendations for the management of alpha interferon-based treatment in patients with chronic hepatitis C virus (HCV) infection are based on HCV RNA measurements before, during, and after antiviral therapy (6,22). Changes in HCV RNA serum concentrations during the early phase of interferon-based therapy have been analyzed based on complex models of viral kinetics and applied to the prediction of treatment outcomes (13,16,31). In different studies, a high predictive value for virologic nonresponse (98 to 100%) was observed for HCV genotype 1-and genotype 4-to 6-infected patients, with a decline in the HCV RNA serum concentration of less than 2 log steps between baseline and week 12 of (pegylated) alpha interferon-ribavirin combination therapy (3,5,8). Alternatively, an absolute HCV RNA concentration above 30,000 IU/ml may be used for decisions about early treatment discontinuation at week 12 (3). In addition, for genotype 1-and genotype 4-to 6-infected patients at week 24 of treatment, it is recommended that therapy be discontinued on the basis of detectable HCV RNA in serum by qualitative PCR-based assays (3,5,19). Recently, for patients infected with genotype 2 or 3, the HCV RNA concentration at baseline and viral decline at week 4 have been described as highly predictive for virologic response to pegylated alpha interferonribavirin combination therapy (4,18,30,32). Furthermore, for assessment of virologic response to currently developed direct antiviral drugs (i.e., protease and polymerase inhibitors), proper HCV RNA quantification for the different HCV genotypes is critical (1,14,25,26). WHO HCV international standard, 96/790 (2,7,11,17,23,24,27). However, continuing limitations are the lack of complete automation (22), the necessity for dilutions for quantification by standard PCR-based assays (6), the relatively low sensitivity of quantitative HCV RNA assays (31), and the need for different test systems for qualitative and quantitative HCV RNA measurements (31). Furthermore, standardization of results to IU are mainly based on HCV genotype 1 panels, and little is known about the variability of commercially available HCV RNA assays for quantification of different HCV genotypes.Real-time PCR methods for the quantification of HCV RNA have the advantage of linear amplification over a broad dynamic range, together with an integrated, automated detection system. With efficient HCV RNA extraction, they have the * Corresponding author. Mailing address:

Development of a Second Version of the Cobas AmpliPrep/Cobas TaqMan Hepatitis C Virus Quantitative Test with Improved Genotype Inclusivity

“…Among these, several report underestimation of HCV RNA levels in up to 30% of GT4 specimens compared to competitor assays (2,16,17,20,22). Recently, the hypothesis was established that these findings were linked to the occurrence of GT4 specimens harboring amino acid substitutions at positions 145 (G to A) and 165 (A to T) in the 5ЈUTR of the HCV genome (1,3,8,10).…”

“…The CAP/CTM assay is widely used in clinical research and practice, and it has been evaluated in a number of studies (2,12,14,16,17,20,22). Among these, several report underestimation of HCV RNA levels in up to 30% of GT4 specimens compared to competitor assays (2,16,17,20,22).…”

Diagnosis and Management of Hepatitis C Virus Infection

“…In most cases, this entails polymerase-based target amplification via PCR or isothermal methods, coupled with endpoint or real-time detection formats. Methods based on signal amplification, such as the VERSANT bDNA assays, 96 are being phased out. Because HCV is an RNA virus, PCR amplification has to be preceded by reverse transcription (i.e., RT-PCR).…”

Standardization of nucleic acid amplification tests in diagnostic molecular microbiology

“…Un problema comune alle metodiche di quantizzazione basate sulla reazione di PCR end-point che non è ancora stato superato dalle tecniche di quantizzazione in real-time, è rappresentato da una diversa efficienza di quantizzazione in rapporto al genotipo virale. Questo problema è oggetto di un importante dibattito soprattutto dopo l'introduzione di metodi di real-time PCR basati sulla tecnologia TaqMan, per virus ad alta variabilità genetica, come HIV e HCV (26). I dati più recenti di confronto tra quantizazzione mediante metodi non-PCR-based, rappresentati per lo più dalla tecnologia di branched-DNA e real-time PCR per HCV fanno emergere per alcuni assemblati TaqMan divergenze di quantizzazione superiori a 1 log, rispetto al bDNA, che sembrano essere genotipodipendenti (26).…”

“…Questo problema è oggetto di un importante dibattito soprattutto dopo l'introduzione di metodi di real-time PCR basati sulla tecnologia TaqMan, per virus ad alta variabilità genetica, come HIV e HCV (26). I dati più recenti di confronto tra quantizazzione mediante metodi non-PCR-based, rappresentati per lo più dalla tecnologia di branched-DNA e real-time PCR per HCV fanno emergere per alcuni assemblati TaqMan divergenze di quantizzazione superiori a 1 log, rispetto al bDNA, che sembrano essere genotipodipendenti (26). La tecnologia TaqMan, infatti, per la sua costruzione, risulta molto sensibile alla presenza di mis-match nella sequenza amplificata, che sono tanto più frequenti, tanto più elevata è la variabilità gentica virale.…”