Quantification of genotype 4 hepatitis C virus RNA by the COBAS AmpliPrep/COBAS TaqMan hepatitis C virus test
Authors: Jeffrey J. Germer, Carl E. Bommersbach, Dana M. Schmidt, Jordan L. Bendel, Joseph D. C. Yao
Journal: Hepatology (2009)

We read with interest the recent correspondence from Chevaliez et al.1 regarding failure of the COBAS AmpliPrep/COBAS TaqMan HCV Test (CAP/CTM; Roche Molecular Systems, Inc., Branchburg, NJ) to detect hepatitis C virus (HCV) RNA in two clinical specimens containing high viral loads of HCV genotype (GT) 4. We also noted that several evaluations of CAP/CTM have shown good correlation with other commercially available HCV quantitative assays2-5, whereas other evaluations have reported substantial viral load discordance, particularly with HCV GT 4.6-9 Due to these conflicting reports, we examined correlation between viral load results of CAP/CTM and the Versant HCV RNA 3.0 assay (bDNA; Siemens Healthcare Diagnostics Inc., Tarrytown, NY) among 100 unique clinical specimens submitted to Mayo Clinic between July 2007 and April 2009 for routine HCV GT determination and found to contain HCV GT 4 by the TruGene HCV 5'NC genotyping kit (TruGene; Siemens Healthcare Diagnostics Inc.). These 100 clinical specimens contained HCV GT 4 with an undetermined subtype (n = 1), 4a (n = 69), 4c (n = 13), 4e (n = 1), and 4i (n = 16) as determined by TruGene and originated from clinical laboratories located in all nine U.S. geographic regions (New England, n = 34; Mid-Atlantic, n = 11; East North Central, n = 13; West North Central, n = 11; South Atlantic, n = 11; East South Central, n = 1; West South Central, n = 9; Mountain, n = 5; Pacific, n = 4) and Mexico (n = 1). All 100 specimens yielding quantifiable HCV RNA by bDNA were also quantifiable by CAP/CTM with generally good correlation of results (Fig. 1), albeit with CAP/CTM showing under-quantification (mean difference = 0.44 log10 IU/mL; range, −0.10 to 1.57 log10 IU/mL) consistent with previous reports.6-9 Deming regression (top) and Bland-Altman (bottom) plots of Versant HCV RNA 3.0 Assay (bDNA) versus COBAS AmpliPrep/COBAS TaqMan HCV Test (CAP/CTM) results from 100 unique clinical serum specimens containing HCV genotype 4. ▵, results for specimens containing HCV sequences with an A-to-T substitution at nt 165; ●, results for all other specimens. Review of all 100 HCV 5′ noncoding region sequences previously generated by TruGene revealed no sequences containing the G-to-A substitution at nucleotide (nt) 145 associated with failure of CAP/CTM by Chevaliez et al.1 However, 6 of 100 (6%) sequencescontained the A-to-T substitution at nt 165 previously associated with under-quantification by CAP/CTM.6 Of the six specimens with this substitution at nt 165, four (4a, n = 1; 4c, n = 3) yielded under-quantification of >1.0 log10 IU/mL by CAP/CTM, whereas two yielded under-quantification of 0.83 (GT 4a) and 0.94 (GT 4c) log10 IU/mL. Of note, another specimen yielding under-quantification of >1.0 log10 IU/mL by CAP/CTM contained HCV GT 4i without the A-to-T substitution at nt 165. Chevaliez et al. described two clinical specimens containing HCV GT 4 strains (4h and 4l) that were undetectable by CAP/CTM and contained dual nt substitutions (i.e., a G-to-A substitution at nt 145 and an A-to-T substitution at nt 165). Their search of GenBank revealed that only 0.4% of HCV GT 4 sequences contained a G-to-A substitution at nt 145, whereas 8.1% of sequences contained an A-to-T substitution at nt 165. Our search of GenBank yielded only three HCV GT 4 sequences containing substitutions at both nt positions. The absence of G-to-A substitutions at nt 145 among our HCV GT 4 strains also suggests that dual nt substitutions are very rare, and one would be unlikely to encounter failure of CAP/CTM to detect HCV GT 4 strains found in the United States. However, our findings do support the previous report of greater under-quantification of HCV RNA by CAP/CTM in the presence of the A-to-T substitution at nt 165 than in the absence of this nt substitution. Jeffrey J. Germer*, Carl E. Bommersbach*, Dana M. Schmidt*, Jordan L. Bendel*, Joseph D. C. Yao*, * Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.