Serum samples were stored during clinical visits and were used for virological testing (fig 1). Serum HCV RNA level was 7 Meq genomes/ml in January 1997 (first infection) and 6 Meq genomes/ml in October 1997 (second infection) (Versant HCV RNA 3.0 Assay (bDNA); Bayer, Puteaux, France). Sequencing in the 5′ non-coding and the NS5B region of the HCV genome confirmed the results of genotyping. The viral sequence isolated in January 1997 was genotype 3a and that isolated in October 1997 was genotype 1a. To exclude the persistence of low levels of HCV RNA between the two infections, all RT-PCR negative serum samples were tested using two methods: the new RT-PCR assay (Cobas Amplicor HCV monitor assay) with a detection limit of 100 copies/ml, and the transcription mediated amplification assay (TMA, Versant HCV RNA qualitative assay, Gen-Probe; Bayer) with a sensitivity of 50 copies/ml. HCV RNA was not detectable in any of the samples tested.
To rule out the fact that the patient originally had a mixed infection with genotype 1a and 3, we developed an allele specific fluorescent amplification on a LightCycler (Roche Diagnostics) which enables fluorescent resonance energy transfer to be monitored during PCR. Briefly, 10 μl of a 10−6 Amplicor positive product were amplified using two sets of primers, located in the 5′UTR region, and specific for HCV genotypes 1 and 3, respectively. Fluorescence was generated by hybridisation of a 5′ TAMRA, 3′ FAM labelled TaqMan probe, common to all genotypes. Primers and probes were chosen with the assistance of the computer program Oligo 4.0 (National Bioscience, Plymouth, Minnesota, USA).
Quantitative values were obtained from the crossing point (CP) number at which the increase in the signal associated with exponential growth of PCR products begins to be detected using LightCycler analysis software, according to the manufacturer’s manual. This numerical value, which represent a number of PCR cycles, is proportional to the number of generated amplicons and reflects the specificity of the reaction.
To determine if we could detect a mixture of HCV genotype 1 and 3 infection (with genotype 1 minority), we pooled under the following conditions two serum samples genotyped as 1 and 3 and quantified at the same level (7 Meq genomes/ml), respectively: pool A, 1 part of genotype 1 added to 102 parts of genotype 3; pool B, 1 part of genotype 1 added to 103 parts of genotype 3; pool C, 1 part of genotype 1 added to 104 parts of genotype 3.
The patient samples before treatment (January 1997) and during the ALT peak (October 1997) were tested in the same run. CPs obtained with both sets of primers on pools A, B, and C indicated that we were able to specifically detect 1 copy of genotype 1 in 104 copies of genotype 3 (table 1). Before treatment (November 96 and January 97), amplification permitted detection of HCV genotype 3, and HCV genotype 1 was undetectable. In the serum sample corresponding to the new infection (October 1997), amplification permitted detection of HCV genotype 1, and HCV genotype 3 was undetectable.
Results of amplification using real time polymerase chain reaction (PCR) with specific probes for hepatitis C virus (HCV) genotype 1 or HCV genotype 3