Reply:

Abstract

Our article1 reported that viral RNA could not be detected in the peripheral blood mononuclear cells (PBMCs) of aviremic seropositive blood donors, a conclusion supported by another recent article which also concluded that there was no evidence for persistent hepatitis C virus (HCV) infection in PBMCs after clearance of viremia in plasma.2 Decreasing or disappearing anti-HCV antibody titers in aviremic subjects also argue against continued HCV protein expression.3 Whether other reservoirs of HCV RNA, such as hepatocytes, exist in aviremic subjects remains unclear with some4-6 although not all7, 8 studies finding no HCV RNA in the liver of treated sustained viral responders. We clearly acknowledged1 that in chronically viremic subjects, PBMCs could be a site of HCV replication and that in vitro mitogen plus cytokine treatment might increase PBMC-associated HCV levels.8-10 With respect to the relative sensitivities of the HCV RNA assays used, we performed two different tests for HCV RNA in PBMCs (cell-associated transcription-mediated amplification [CA-TMA] and reverse transcription nested polymerase chain reaction [RT-nPCR]). We demonstrated a sensitivity of 2-50 HCV RNAs in 5 million PBMCs for the CA-TMA assay and 15-150 HCV RNAs in 5 million cells using RT-nPCR (adjusted from a sensitivity of 1-10 HCV RNAs for RT-nPCR, because only a fraction of total PBMC RNA and derived complementary DNA is sampled).1 Pham et al. report a sensitivity for their RT-nPCR-NAH (nucleic acid hybridization) assay of <10 viral genome equivalent (vge)/mL for serum (and in their letter, <5 vge/million PBMCs). We quantified the sensitivities of our assays using serial dilutions of plasma samples of known viral loads (prequantified using a commercial viral load assay) in the presence of total cellular RNA, as well as of total RNA from the PBMC of viremic subjects and showed the actual replicate assay results.1 Pham et al. used dilutions of a plasmid without interfering cellular nucleic acids for “semiquantitative estimation” in a “preliminary experiment” whose results where not shown.10 The fraction of total complementary DNA derived from PBMCs or 250 μL serum they used as input for their nPCR was not specified.10 With regard to the total number of cell equivalents analyzed in respective assays, we indeed analyzed only 0.16 million PBMCs by RT-nPCR but in parallel, and with consistent results, also used CA-TMA on 2.5 million PBMCs,1 slightly more than the 1 million to 2 million PBMCs Pham and Michalak reported in their letter. We agree with Pham and Michalak that difference in assay sensitivity is a likely explanation for the reported discrepancy but feel that the more relevant assay is that used for detecting HCV RNA in plasma. In our case, we used an extensively validated plasma HCV RNA assay used for blood-donation screening (Transcription mediated amplification, Procleix human immunodeficieny virus-1/HCV assay, Gen-probe, with a 95% detection sensitivity of 30 HCV RNA/mL).11-13 This TMA assay was used in duplicate assays (consuming a total of 1 mL plasma) to identify a total of 69 plasma aviremic blood donors, none of whom possessed detectable PBMC-associated HCV RNA.1 Pham et al. analyzed and found HCV RNA in the PBMCs of two aviremic subjects (so classified using their RT-nPCR-NAH).10 We suspect that further testing of the plasma from these two subjects using TMA assays might have detected a low-level viremia. Also, mostly consistent with our results, Pham et al. reported in 2008 detecting HCV RNA in only one of 10 PBMCs from aviremic subjects (although stimulation with interleukin-2 plus mitogen and testing of purified immune cell subsets lead to the detection of HCV RNA in all 10 PBMCs).14 Our selection criterion for the absence of plasma viremia (based on a highly sensitive, extensively validated assay used in duplicate tests to increase sensitivity) may therefore have been more stringent than that used by Pham et al. and account for the discrepant results seen in PBMCs. Ultimately, we agree with Pham and Michalak that the question of a residual HCV reservoir in the PBMCs of truly aviremic subjects will best be resolved using the demonstrably most sensitive detection assays for HCV RNA in both plasma and PBMC. The use of the same HCV RNA quantitation standards15 by different groups will help compare assay sensitivities. Eric Delwart Ph.D.*, * Blood Systems Research Institute, Department of Laboratory Medicine, University of California, San Francisco, San Francisco CA.