Arch Virol (1997) 142: 459—464
Quantiﬁcation and genotyping of serum HCV-RNA in patients
with chronic hepatitis C undergoing interferon treatment
G. Gerken, P. Knolle, S. Jakobs, and K.-H. Meyer zum Bu¨ schenfelde
I. Medizinische Klinik und Poliklinik der Johannes Gutenberg Universita¨t,
Mainz, Federal Republic of Germany
Summary. Quantiﬁcation of serum HCV-RNA and HCV genotyping was
studied in 27 patients with chronic hepatitis C undergoing interferon treatment.
Pretreatment serum HCV-RNA levels were quantiﬁed using competitive RT-
PCR and compared to a quantitative RT-PCR assay based on co-ampliﬁcation
of HCV-RNA with a synthetic RNA standard. HCV genotyping was performed
using a line probe reversed hybridisation assay or direct solid-phase sequencing.
This study shows the feasibility of performing HCV-RNA quantiﬁcation. RT-
PCR based on co-ampliﬁcation HCV-RNA titer less than 6 ;10 genome
equivalents/ml serum did correlate with a complete sustained response to
interferon in chronic hepatitis C. HCV genotype 1b was predominantly
associated with a high non-responder rate. Future prospective trials will be
required to evaluate quantitative HCV-RNA levels and HCV genotyping as
response predicting parameters for interferon-treatment.
The development of polymerase chain reaction (PCR) has provided a sensitive
diagnostic tool for the detection of HCV-RNA in serum and tissue specimens.
Currently, RT-PCR is frequently used to identify low viremic HCV carriers or to
monitor antiviral treatment [1—4]. Nevertheless, the lack of standardized reac-
tion protocols and the considerable risk of false positive results have impeded
its routine application . Many attempts have been made to simplify and to
qualify HCV-RNA detection, for example by signal ampliﬁcation assays or
limiting dilution PCR [4—6].
One of the most promising approaches for determining HCV levels in patient
sera seems to be competitive PCR including varying amounts of the internal
standard [7, 8]. In its present form, however, this technique is very time-
consuming and expensive. In our own group we have established a new, less
complicated HCV-RNA quantitation method based upon co-ampliﬁcation of
the target-cDNA together with constant concentration of a competitor DNA
. Recently, the ﬁrst commercially available PCR-assay for sensitive quantita-