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
Authors: Stéphane Chevaliez, Magali Bouvier-Alias, Laurent Castéra, Jean-Michel Pawlotsky
Journal: Hepatology (2008)

Accurate hepatitis C virus (HCV) RNA quantification is essential for the current and future management of chronic hepatitis C therapy. Assays based on real-time polymerase chain reaction (PCR) are now widely used in clinical virology laboratories.1 Two real-time PCR platforms are commercially available: Cobas TaqMan, which can be used with automated sample preparation in the Cobas AmpliPrep system [Cobas AmpliPrep–Cobas TaqMan (CAP-CTM), Roche Molecular Systems, Pleasanton, CA], and m2000RT, which can be used with automated sample preparation in the m2000SP device (Abbott Molecular, Des Plaines, IL).1 We have recently reported in HEPATOLOGY the results of a thorough evaluation of the intrinsic characteristics and clinical performance of the CAP-CTM platform, the most widely used and only Food and Drug Administration–approved real-time PCR assay for HCV RNA detection and quantification.2 We found that the CAP-CTM assay substantially underestimated HCV RNA levels in approximately 15% of HCV genotype 2 clinical specimens and 30% of HCV genotype 4 clinical specimens, probably because of nucleotide polymorphisms at the hybridization sites for the PCR primers and/or TaqMan probe in the 5′ noncoding region of the genome.2 Here we report two patients chronically infected with HCV genotype 4 in whom HCV RNA was undetectable in CAP-CTM, whereas a high HCV RNA level was found with two other assays. The first patient was infected with HCV subtype 4h. HCV RNA was undetectable in CAP-CTM (<12 IU/mL, target not detected), but high HCV RNA levels were found with the Abbott m2000SP/m2000RT real-time PCR assay and a third-generation branched DNA–based signal amplification assay (Versant HCV RNA 3.0 assay, Siemens Medical Solutions Diagnostics, Tarrytown, NJ): 5.4 and 5.0 Log10 IU/mL, respectively. The second patient was infected with subtype 4l. HCV RNA was undetectable in CAP-CTM (<12 IU/mL, target not detected), whereas levels of 6.0 and 5.7 Log10 IU/mL were found with m2000SP/m2000RT and the branched DNA assay, respectively. Two nucleotide substitutions with respect to the reference sequence were common to the two clinical specimens with negative HCV RNA detection in CAP-CTM: A to T at position 165 (a substitution found in other genotype 4 samples with underestimated HCV RNA levels2) and G to A at position 145, which is close to the former one (Fig. 1). A GenBank search among the more than 2500 HCV 5′ noncoding sequences deposited showed an A at position 145 in 0.3% of cases and a T at position 165 in 1.4% of cases (all genotypes). An A at position 145 was found in 0.4% and a T at position 165 was found in 8.1% of HCV genotype 4 cases. Alignment of the nearly full-length HCV 5′ noncoding sequence from the two patients with undetectable HCV RNA in CAP-CTM with respect to a reference HCV genotype 4 strain (F7157). The subtype of the HCV strain is indicated in parentheses. The nucleotide positions at which polymorphisms were observed in strain sequences from patients with underestimated HCV RNA levels in our previous study2 are highlighted in gray. Two nucleotide substitutions were found in both patients with undetectable HCV RNA in CAP-CTM with respect to the reference sequence: G to A at position 145 and A to T at position 165 (squared). The A at position 107, present in the two patients with undetectable HCV RNA in CAP-CTM, was the most frequent nucleotide in patients without HCV RNA level underestimation in the previous study.2 Abbreviations: CAP-CTM, Cobas AmpliPrep–Cobas TaqMan; HCV, hepatitis C virus. We describe here two patients infected with highly replicating HCV genotype 4 in whom the CAP-CTM assay repeatedly failed to detect HCV RNA. This failure could be related to nucleotide polymorphisms at positions 145 and 165. Although the manufacturer of the CAP-CTM assay did not disclose the positions of the assay primers and probe, the localization of these substitutions is compatible with a mismatch at the TaqMan probe level. CAP-CTM underestimation of or failure to detect HCV RNA in patients infected with HCV genotype 4 has a major clinical impact. Indeed, the incidence and prevalence of HCV genotype 4 are increasing in the Western world, especially among intravenous drug users,3 and a recent study has shown that 15% of European patients coinfected with human immunodeficiency virus carry HCV genotype 4.4 Modeling suggests that HCV genotype 4 could play an important role in the future HCV burden in industrialized countries.5 Further studies are warranted to assess the frequency of undetected HCV RNA with the CAP-CTM assay. Improvement in this assay is now urgently needed in order to ensure accurate quantification of HCV RNA, regardless of the HCV genotype, for optimal use in clinical trials and practice. In the meantime, its use should not be recommended for HCV RNA monitoring in patients infected with HCV genotypes 2 and 4. The authors are grateful to Françoise Darthuy, Alexandre Soulier, Laetitia Barbotte, and Pascale Trimoulet for their help. Stéphane Chevaliez* , Magali Bouvier-Alias* , Laurent Castéra , Jean-Michel Pawlotsky* , * National Reference Center for Viral Hepatitis B, C, and Delta, Department of Virology, Hôpital Henri Mondor, Université Paris 12, Créteil, France, INSERM U955, Créteil, France, Department of Hepatology, Hôpital Saint-André and Hôpital du Haut-Lévêque, Bordeaux, France.