Occult Hepatitis C Virus Infection Revisited with Ultrasensitive Real ...

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JOURNAL OF CLINICAL MICROBIOLOGY, June 2008, p. 2106–2108 0095-1137/08/$08.00⫹0 doi:10.1128/JCM.00345-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 46, No. 6

Occult Hepatitis C Virus Infection Revisited with Ultrasensitive Real-Time PCR Assay䌤 Philippe Halfon,1* Marc Bourlie`re,2 Denis Ouzan,3 Damien Se`ne,4 David Saadoun,4 Hace`ne Khiri,1 Guillaume Pe´naranda,5 Agnes Martineau,1 Vale´rie Oule`s,2 and Patrice Cacoub4 Laboratoire Alphabio, Ho ˆpital Ambroise Pare´, Marseille, France1; De´partement d’He´pato-Gastroente´rologie, Ho ˆpital Saint-Joseph, Marseille, France2; De´partement d’He´pato-Gastroente´rologie, Institut Arnault Tzanck, Saint-Laurent du Var, France3; Universite´ Pierre et Marie Curie-Paris 6, CNRS, UMR 7087-AP-HP, Ho ˆpital Pitie´-Salpeˆtrie`re, Service de Me´decine Interne, Paris, France4; and De´partement Biostatistiques, CDL Pharma, Marseille, France5 Received 19 February 2008/Returned for modification 2 April 2008/Accepted 18 April 2008

Occult hepatitis C infection is regarded as a new entity that should be considered when diagnosing patients with a liver disease of unknown origin. Using an ultrasensitive real-time PCR assay, we demonstrated that occult hepatitis C virus (HCV) infection cannot be found in peripheral blood mononuclear cells of patients with cryptogenic liver diseases, HCV-associated systemic vasculitis, or connective tissue diseases. The significance of such occult infection must be elucidated. detected by the presence of HBV DNA using a sensitive realtime PCR (detection limit of 40 IU/106 cells) in the four groups. The group 1 patients (n ⫽ 22; mean age, 51.6 ⫾ 12.9 years; 12 men) presented with LSAR. LSAR were assessed every 3 to 6 months from the onset of their discovery. The patients were repeatedly negative for serum anti-HCV antibodies and HCVRNA and showed no signs of muscle, autoimmune, or genetic diseases. Testing for human immunodeficiency virus, hepatitis A, hepatitis B antigen, and hepatitis E was negative by enzyme immunoassay (Architect; Abbott, Laboratories, Rungis, France). The patients reported no use of toxic drugs or any other liver-toxic products. Table 1 shows the main demographic, clinical, and biological characteristics. Fifteen of the 22 group 1 patients showed high ␥-glutamyl transpeptidase levels, whereas only four patients were overweight. The group 2 patients (n ⫽ 21; mean age, 55.8 ⫾ 15.6 years; 15 men) had HCV-associated systemic vasculitis and were HBV negative (HBsAg and HBV DNA). Of the 21 patients, 13 were serum HCV RNA positive and none had been treated with pegylated interferon. The group 3 patients (n ⫽ 27; mean age, 52.0 ⫾ 16.8 years; 10 men) had systemic connective tissue disease: i.e., 7 had systemic lupus 7, 2 had Takayasu arteritis, 3 had nonviral polyarteritis nodosa, 2 had essential mixed cryoglobulinemia, 2 had primary antiphospholipid syndrome, 2 had autoimmune hemolytic anemia, 1 had Sjo ¨ gren’s syndrome, 1 had Still’s disease, 1 had Buerger’s disease, 1 had temporal arteritis, 1 had Wegener’s granulomatosis, 1 had polymyositis, 1 had limited scleroderma (CREST syndrome), 1 had rheumatoid arthritis, and 1 had Churg-Strauss vasculitis. Among the patients in group 3, 4/27 were serum anti-HCV antibody positive and HCV RNA positive. All patients were negative for HBV serum markers. The group 4 patients (control group; mean age, 62.0 ⫾ 15.4; no men) included 20 serum anti-HCV antibody-positive infected genotype 1 patients with elevated alanine aminotransferase levels (1.5 to 4 times upper limit of normal values) and

Occult hepatitis C virus (HCV) infection has been described in patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but have HCV RNA in the liver (2–5, 13–15, 17). Because of the invasive nature of liver biopsies, researchers have attempted to increase the sensitivity of diagnostic tests for occult HCV infection in peripheral blood mononuclear cells (PBMC) (4, 9, 11). One important question regarding the transmission of occult HCV infection was whether the virus could replicate in PBMC. Only a limited number of studies have shown the presence of HCV RNA in PBMC of patients with cryptogenetic liver disease. Moreover, data have suggested differential regulation of HCV RNA in the serum and PBMC compartments and pointed out the relevance of PBMC in the detection of HCV RNA (1). No data have been reported about these occult infections in patients with systemic connective tissue diseases, and no negative data on occult hepatitis have ever been reported. The aim of this study was to investigate occult HCV infection by using ultrasensitive real-time PCR assays to assess HCV-RNA in PBMC of patients with long-standing abnormal results (LSAR) of unknown origin of liver function tests, systemic vasculitis, or connective tissue disease. A total of 70 patients were included: 22 patients (group 1) with LSAR of liver-function tests, 21 patients (group 2) with HCV-associated systemic vasculitis, and 27 patients (group 3) with connective tissue disease. The control group included 20 patients (group 4) with positive serum HCV RNA. HCV replication was assessed in PBMC by a modified real-time PCR assay (detection limit of 15 IU/106 cells). The sensitivity of our method was validated by detection and dilution of quantified HCV-infected sera added to PBMC from patients without HCV infection. Occult hepatitis B virus (HBV) infection was

* Corresponding author. Mailing address: Laboratoire Alphabio, 23 rue de Friedland, Ho ˆpital Ambroise Pare´, 13006 Marseille, France. Phone: (33) 4 91 25 41 00. Fax: (33) 4 91 79 20 44. E-mail: philippe [email protected]. 䌤 Published ahead of print on 30 April 2008. 2106

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TABLE 1. Characteristics of the three groups of patients studied Result for: Parameter

Group 1 (LSAR; n ⫽ 22)

Group 2 (HCV-associated systemic vasculitis; n ⫽ 21)

Group 3 (connective tissue diseases; n ⫽ 27)

Age in yr (mean ⫾ SD) No. (%) male Aspartate transaminase (UNLa ⫾ SD) Alanine aminotransferase (UNL ⫾ SD) ␥-Glutamyl transpeptidase (mean IU/liter ⫾ SD) No. (%) with liver fibrosisb F0 F1 F2 F3 F4 Test not done No. (%) with inflammatory activityd A0 A1 A2 A3 Test not done

53 ⫾ 13 11 (50) 1.86 ⫾ 1.55 1.97 ⫾ 0.98 55 ⫾ 30

56 ⫾ 16 15 (71) 1.18 ⫾ 0.60 1.20 ⫾ 0.58 26 ⫾ 10

52 ⫾ 17 10 (37) 1.36 ⫾ 1.40 1.21 ⫾ 0.79 28 ⫾ 7

8 (36) 6 (27) 1 (5) — — 7 (32)

1 (5) 3 (14) — 2 (10) 3 (14) 12 (57)

2 (7) —c 1 (4) — 1 (4) 23 (85)

12 (54) 1 (5) 2 (9) — 7 (32)

1 (5) 5 (24) 3 (14) — 12 (57)

3 (11) — 1 (4) — 23 (85)

a

UNL, upper normal limit. Liver fibrosis scores run from none (F0) to high (F4). —, not detected. d Inflammatory activity scores run from none (A0) to high (A3). b c

positive serum HCV RNA assessed with standard real-time PCR (11). Seventy HCV-negative patients were included. HCV replication was assessed in PBMC by a modified real-time PCR assay (detection limit, 15 IU/106 cells). Serum samples were stored at ⫺80°C before analysis with the usual storage cautions (12). HCV RNA was detected using a modified COBAS TaqMan HCV assay with a detection limit of 15 IU/106 cells. Detection of HCV RNA in PBMC was performed on 1 million PBMC prepared with a BD CPT tube (BD Diagnostics). The RNA extraction step was performed using silica beads (NucliSens; Organon Teknika S.A., Fresnes, France), and the real-time PCR was done with the Cobas Taqman HCV assay according to the manufacturer’s instruction’s using the CTM 48 (Roche Diagnostics, Meylan, France). The sensitivity of our method was validated by detection of HCV-infected sera diluted in negative PBMC. HCV genotype 1 serum quantified at 1,500 IU/ml was diluted by a factor of 10, and 1 ml of diluted serum was added to 1 million PBMC prepared as described above. The mixture of PBMC and HCV genotype 1 sera was then quantified by the COBAS TaqMan assay (11). The detection limit was 15 IU/106 cells. In all 22 group 1 patients with LSAR, no HCV RNA was detected in PBMC. Among the 21 group 2 patients with HCVassociated systemic vasculitis, HCV-RNA was detected in PBMC of 10 patients among the 13 who were serum HCV RNA positive and in 0/8 of patients who were serum HCV RNA negative. Among the 27 group 3 patients with systemic connective tissue disease, HCV RNA in PBMC was detected in 4/27 patients who were serum HCV RNA-positive. All of the PBMC of the controls were positive. We are the first study to use an ultrasensitive real-time PCR assay to demonstrate that occult HCV infection cannot be found in the PBMCs of pa-

tients with cryptogenic liver diseases, HCV-associated systemic vasculitis, or connective tissue diseases. Researchers have long sought to establish whether HCV replicates outside the liver, because detection of HCV RNA in extrahepatic reservoirs has important implications for disease transmission and progression and treatment efficacy (2, 7, 15, 16). The concept of occult hepatitis has been reported by few teams, and no negative data have been reported up to now (4–6, 8, 9, 11). One of the limits of this study is the absence of liver sample analysis. Nevertheless, testing for HCV RNA in PBMC has been demonstrated to be reliable for identifying patients with an occult HCV infection when a liver biopsy is not available (10). In the studies performed by Castillo et al. and Pardo et al. in occult HCV infection, up to 70% of patients also present with HCV RNA in PBMC (5, 13). Similar results were found in the group 2 patients of our study: 10/13 (77%) patients were HCV RNA positive in PBMC. Further studies of longer duration are needed to clarify the role of “silent” HCV infection in patients with LSAR on liver function tests, yet we have some reservations about the existence of the so-called “occult” infection and we encourage publications of negative data on this topic. REFERENCES 1. Blackard, J. T., L. Smeaton, Y. Hiasa, N. Horiike, M. Onji, D. J. Jamieson, I. Rodriguez, K. H. Mayer, and R. T. Chung. 2005. Detection of hepatitis C virus (HCV) in serum and peripheral-blood mononuclear cells from HCVmonoinfected and HIV/HCV-coinfected persons. J. Infect. Dis. 192:258– 265. 2. Carreno, V. 2006. Occult hepatitis C virus infection: a new form of hepatitis C. World J. Gastroenterol. 12:6922–6925. 3. Carren ˜ o, V., J. Bartolome´, I. Castillo, and J. A. Quiroga. 2008. Occult hepatitis B virus and hepatitis C virus infections. Rev. Med. Virol. [Epub ahead of print.] doi:10.1002/rmv.569. 4. Carreno, V., M. Pardo, J. M. Lopez-Alcorocho, E. Rodriguez-Inigo, J. Bartolome, and I. Castillo. 2006. Detection of hepatitis C virus (HCV) RNA in the liver of healthy, anti-HCV antibody-positive, serum HCV

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