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To the Editor:

We are pleased to read that Germer et al. confirm our results1 showing that the Cobas AmpliPrep/Cobas TaqMan assay (CAP/CTM; Roche Molecular Systems, Pleasanton, CA) underestimates hepatitis C virus (HCV) RNA levels in a substantial number of patients infected with HCV genotype 4. We recently reported two cases where the simultaneous presence of a G-to-A substitution at position 145 (G145A) and an A-to-T substitution at position 165 (A165T) of the 5′ noncoding sequence of HCV genome abolished HCV RNA detection in the CAP/CTM assay, but not in two other assays, including another real-time polymerase chain reaction test (m2000SP/m2000RT Real-Time Assay, Abbott Molecular, Des Plaines, IL) and the third-generation branched DNA (bDNA)-based signal amplification assay (Versant HCV RNA 3.0; Siemens Medical Solutions Diagnostics, Tarrytown, NJ).2

In order to confirm the role of nucleotide substitutions at positions 145 and 165 in the failure of CAP/CTM to accurately quantify HCV RNA, we generated in vitro RNA transcripts from a plasmid into which the 5′ noncoding sequence of a wild-type genotype 4 strain originating from an HCV-infected patient was inserted. Substitutions at positions 145 and 165 were introduced by means of site-directed mutagenesis. The transcripts harboring one or both substitutions, as well as the wild-type transcript, were quantified without extraction with the three assays described above. The results are shown in Table 1. Each single substitution was responsible for an approximately 2-log underestimation of HCV RNA levels in the Cobas TaqMan assay relative to the two other assays. In the presence of both G145A and A165T substitutions, HCV RNA levels were high in m2000RT and bDNA, but undetectable with the Cobas TaqMan assay.

Table 1. Quantification of In Vitro RNA Transcripts Including a Wild-Type Genotype 4 5'-Noncoding Sequence and Mutated Sequences at Positions 145 and/or 165, with Cobas TaqMan, m2000RT, and the Third-Generation Branched DNA Assay
HCV 5′ Noncoding SequenceMeasured HCV RNA Levels (Log10 IU/mL)
Real-Time PCR AssaysbDNA assay (Versant 3.0)
Cobas TaqManm2000RT
  • All samples were tested in duplicate.

  • *

    Target not detected.

Wild-type (G145 and A165)5.73 ± 0.136.05 ± 0.106.43 ± 0.01
Single mutant (G145A and A165)4.02 ± 0.136.00 ± 0.116.69 ± 0.02
Single mutant (G145 and A165T)4.28 ± 0.355.88 ± 0.206.30 ± 0.02
Double mutant (G145A and A165T)<1.08*5.95 ± 0.256.60 ± 0.18

The frequency of substitutions at positions 145 and 165 in GenBank reported by Germer et al. and ourselves2 is not negligible. We agree that the presence of the double substitution at positions 145 and 165 is rare, at least in the sequences reported in GenBank. Given the fact that the incidence and prevalence of genotype 4 infection is increasing worldwide, especially in the injection drug user population, and that HCV RNA quantification is critical to make treatment decisions, accuracy is essential. The current version of the CAP/CTM assay, which underquantifies approximately 15% of HCV genotype 2 and 30% of HCV genotype 4 samples,1 is not appropriate for use in patients infected with these genotypes. An improved version of this assay is currently in development. It is expected to bring accurate quantification of all HCV genotypes. Careful evaluation of this new assay will be needed in expert centers.

References

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  • 1
    Chevaliez S, Bouvier-Alias M, Brillet R, Pawlotsky JM. Overestimation and underestimation of hepatitis C virus RNA levels in a widely used real-time polymerase chain reaction-based method. HEPATOLOGY 2007; 46: 2231.
  • 2
    Chevaliez S, Bouvier-Alias M, Castera L, Pawlotsky JM. The Cobas Ampliprep-Cobas TaqMan real-time polymerase chain reaction assay fails to detect hepatitis C virus RNA in highly viremic genotype 4 clinical samples. HEPATOLOGY 2009; 49: 13971398.

Stéphane Chevaliez*, Jenna Fix*, Alexandre Soulier*, Jean-Michel Pawlotsky*, * National Reference Center for Viral Hepatitis B, C, and Delta, Department of Virology, Hôpital Henri Mondor, Université Paris 12, INSERM U955, Créteil, France.