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Occult hepatits C virus persistence: identification and characteristics.

Hepatitis C virus (HCV) is a single-stranded RNA virus of the Flaviviridae family that replicates by making the so-called "negative" strand. The virus infects at least 180 million people worldwide, and up to 85% of the inflicted become carriers of the virus having chronic liver disease. The current standard antiviral treatment with pegylated interferon-alpha (IFN-alpha) and Ribavirin is effective in only about 50% of chronically infected patients. There is no vaccination against HCV; and with thousands of new infections each year, this virus will remain a significant social and economical burden for many years to come.

It has been presumed that resolution of hepatitis C, as evidenced by normalization of liver function tests and negativity of serum HCV RNA, determined by the current standard laboratory assays, reflects viral eradication. Recent evidence from our laboratory and others, however, indicates that by employing more sensitive nucleic acid detection tests, low levels of HCV RNA can be identified in sera, lymphoid cells, and hepatic tissue for years after apparent complete recovery from hepatitis C. (1-6) This review will focus on recent identification of occult HCV infection and characteristics of its persistence.

Natural course of HCV infection

In the majority of newly infected patients, hepatitis C is a clinically asymptomatic disease characterized by the appearance of HCV RNA in serum, which is detectable in one to three weeks after exposure to virus by current clinical laboratory assays. Elevation for up to eight times in serum alanine aminotransferase (ALT) level, which is the hallmark biochemical indicator of liver injury, may be seen two to eight weeks after infection. Symptoms of the acute hepatitis, which are observed in one-third of the patients and may include malaise, weakness, jaundice, and abdominal pain, tend to subside as the ALT levels decline. (7) Antibodies to HCV (anti-HCV) are detectable by commercially available enzyme-linked immunoassays in 50% to 70% of patients at the onset of symptoms, increasing to more than 90% after three months, and completely disappearing 10 to 20 years after resolution of hepatitis. (8) Spontaneous recovery and apparently complete clearance of HCV RNA occur in 15% to 50% of patients with acute disease.

In contrast to transient acute hepatitis, chronic hepatitis C infection is defined by the persistence of HCV RNA in serum for at least six months. HCV RNA and ALT levels may fluctuate considerably, especially during the transition from the acute to chronic phase of the disease. Most patients with chronic hepatitis have an HCV RNA load between 1[0.sup.4] to 1[0.sup.6] international units (IU)/mL or 1[0.sup.5]-1[0.sup.7] virus genome equivalents (vge) per mL of plasma. Two-thirds of them may have persistently elevated ALT of approximately four times the normal upper limit level. The most clinically important outcome of chronic hepatitis is progressive liver fibrosis leading to cirrhosis in 5% to 20% of the patients over a 20- to 25-year period. HCV-positive cirrhotic patients are at a higher risk of developing end-stage liver disease (30% over 10 years) and hepatocellular carcinoma (a risk of 1% to 2% per year). (9) In addition to these clinically and laboratory well-defined forms of HCV infection, recent studies revealed the existence of an asymptomatic HCV infection which continues for years after resolution of symptomatic hepatitis and is characterized by carriage of serum HCV RNA at levels normally not detectable by the assays currently used for laboratory diagnosis.

HCV RNA detection assays

Several tests have been developed for the detection and quantification of HCV RNA. Among them are the FDA-approved manual and semiautomatic qualitative assays, such as Amplicor and Cobas Amplicor HCV version 2 from Roche, which have limits of detection of 50 IU/mL or 50 vge/mL to 135 vge/mL, as well as the transcription-mediated amplification (TMA) assay, such as VERSANT HCV from Bayer, which has a sensitivity of 10 IU/mL or approximately 50 vge/mL. In addition, measurement of HCV RNA levels, which is important in predicting the likelihood of response to therapy, has been made possible by quantitative assays employing either TMA or signal amplification (branched DNA) techniques. Currently, VERSANT HCV RNA version 3.0 assay from Bayer, which has a dynamic range of 615-7.7 X 1[0.sup.6] IU/mL, is the only one with FDA approval.

Recently, research assays applying reverse transcription of viral RNA to cDNA followed by two sequential rounds of polymerase chain reaction (RT-PCR) with HCV gene-specific primers, and validating the amplified products by nucleic acid hybridization (NAH) with recombinant HCV DNA as a probe, were developed. (1) These RT-PCR/NAH assays detect HCV RNA positive (vegetative) and negative (replicative) strands with a sensitivity of [less than or equal to]10 vge/mL (<5 IU/mL) and ~100 vge/mL (~50 IU/mL), respectively. Due to their extreme sensitivity, rigorous protocols ensuring the authenticity of HCV RNA amplification have to be followed. In addition, a quantitative, real-time RT-PCR detecting HCV RNA at a sensitivity of ~100 vge/mL was also recently established. (1), (3) Application of these new detection methods has uncovered previously unknown facts about the natural history of HCV infection, including identification of occult HCV persistence. (1)

Lymphotropism of HCV

Although HCV is considered to be primarily hepatotropic, accumulated evidence clearly indicates that the virus also invades and replicates in the immune system. In fact, the presence of both HCV RNA positive and negative strands has been demonstrated in lymphoid cells in both in vivo and in vitro settings. For example, T cells, B cells, monocytes, and dendritic cells from patients with chronic hepatitis C have all been shown to carry HCV genomes. (2), (10-12) Most recently, by applying assays of superior sensitivity mentioned above, replication of HCV has been seen to persist in lymphoid cells for years after spontaneous recovery or a sustained virological response to IFN-alpha/Ribavirin therapy. (1-3) In addition, low levels of HCV RNA in lymphoid cells have been detected in a significant proportion of patients with persistently elevated liver enzyme levels of unknown etiology. (13), (14) Along the same line, susceptibility of T and B cell lines as well as primary human T cells and macrophages to HCV infection have also been documented. (15-18)

Occult HCV infection

In our studies, using the highly sensitive RT-PCR/NAH assays, conclusive evidence was obtained for the presence of replicating HCV in persons who apparently completely recovered from hepatitis C and whose sera were repeatedly negative for HCV RNA by standard assays. We found that more than 80% of these individuals were serum HCV RNA positive at levels usually not exceeding 1[0.sup.2] vge/mL and that 30% to 40% of them also carried HCV RNA in circulating lymphoid cells at levels between 10 and 1[0.sup.4] vge per 1[0.sup.7] cells (see Figure 1). (1), (19)

[FIGURE 1 OMITTED]

However, when the cells, including those apparently negative for HCV RNA, were ex vivo treated with mitogens known to activate different immune cell subsets, e.g., phytohemagglutinin (PHA) to stimulate T cells, pokeweed mitogen (PWM) to induce B and T cells, and lipopolysaccharide (LPS) to activate monocytes and B cells, HCV RNA was detected in all of the individuals. (1), (2), (19) Interestingly, in the majority of the cases, synergistic stimulation of T and B cells and monocytes with mitogen cocktails led to a more pronounced upregulation of HCV RNA expression than did single mitogen treatments, suggesting that all three cell subsets are reservoirs of the virus (see Figure 2). This propensity was also found when the same cell subsets purified from hepatitis C patients were analyzed. (2), (19) Overall, the ex vivo synergistic mitogen stimulation of peripheral lymphoid cells allows for a more precise detection of silent HCV infection than by testing either sera or naive (untreated) lymphoid cell samples alone. Our original identification of commonly occurring occult HCV infection has been corroborated by recent data from several other laboratories. (3), (4), (6) This collective evidence challenges the previous notion that resolution of hepatitis C reflects complete virus eradication. Further, the invariable detection of replicating HCV genomes in lymphoid cells from individuals with either occult or symptomatic infection attests to the existence of an extrahepatic compartment for HCV replication.

[FIGURE 2 OMITTED]

In addition to consistent identification of HCV RNA in ex vivo stimulated peripheral lymphoid cells, HCV RNA has also been detected in liver tissue of asymptomatic individuals with a sustained response to antiviral treatment. (3), (5) Although these patients generally exhibit histologically apparent improvement after IFN-alpha/Ribavirin therapy, including partial regression of fibrosis, liver biopsies from many of them show evidence of persistent minimal inflammation or even of active chronic hepatitis. (3), (5)

Implications of occult HCV infection

Although investigations on clinical relevance of occult HCV infection have just begun, the data available at this point could help explain sustained HCV-specific T-cell responses observed in individuals decades after recovery from hepatitis C. (8), (20) In this regard, the presence of low amounts of the replicating virus could provide continuous antigenic stimuli beneficial to immunocompetent individuals in maintaining an effective antiviral immune response and keeping the occult infection under control. On the other hand, virus persisting at very low levels may provide a means for reactivation of infection when the host's immune system becomes compromised due to a disease or therapy. Indeed, reactivation of HCV infection has been documented in patients receiving immunosuppressive treatment. (21), (22) Along the same line, HCV RNA was also detected in sera of anti-HCV positive patients weeks after acquiring HCV-negative kidney (23) or bone marrow (24) transplants and immunosuppressive therapy. With respect to commonly observed HCV reinfection of liver allografts in patients with end-stage diseases caused by chronic hepatitis C, migration of lymphoid cells, including those carrying not readily detectable quantities of replicating virus, may constitute a main mechanism by which the transplanted liver becomes infected.

In summary, occult HCV infection is a common, if not an invariable, consequence of resolution of hepatitis C when measured by highly sensitive assays established recently in some research laboratories. The availability of assays detecting HCV RNA with a comparable sensitivity in clinical laboratories would improve the precision with which HCV is detectable among patients and in the general population. Furthermore, the persistence of traces of replicating HCV in lymphoid cells, from which viral infection may potentially rebound under favorable conditions, suggests that future treatments aimed at HCV elimination should take into consideration that both the liver and the lymphatic system are the sites of virus propagation.

References

(1.) Pham TNQ, MacParland SA, Mulrooney PM, Cooksley H, Naoumov NV, Michalak TI. Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C. J Virol. 2004;78:5867-5874.

(2.) Pham TNQ, MacParland SA, Coffin CS, Lee SS, Bursey FR, Michalak TI. Mitogen induced upregulation of hepatitis C virus expression in human lymphoid cells. J Gen H:S Virol 2005;86:657-666.

(3.) Radkowski M, Gallegos-Orozco JF, Jablonska J, Colby TV, Walewska-Zielecka B, Kibicka J, Wilkinson J, Adair DM, Rakela J, Laskus T. Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C. Hepatology. 2005;41:106-114.

(4.) Lee WM, Polson JE, Carney DS, Sahin B, Gale M Jr. Re-emergence of hepatitis C virus after 8.5 years in a patient with hypogammaglobulinemia: evidence for an occult viral reservoir. J Infect Dis. 2005;192:1088-1092.

(5.) Carreno V, Castillo I, Rodriguez-Inigo E, Lopez-Alcorocho JM, Bartolome J, Quiroga JA, Pardo M. Hepatitis C virus persists and replicates in the liver of the majority of sustained responder patients to antiviral treatment. Hepatology. 2005; 42 (S4):284A.

(6.) Lopez-Alcorocho JM, Rodriguez-Inigo E, Pardo M, Castillo I, Quiroga JA, Carreno V. Persistence of HCV infection in apparently healthy anti-HCV positive patients with constantly serum HCV-RNA negative and normal ALT levels. Hepatology. 2005;42:433A.

(7.) Racanelli V, Rehermann B. Hepatitis C virus infection: when silence is deception. Trends Immunol. 2003;24:456-464.

(8.) Takaki A, Wiese M, Maertens G, Depla E, Seifert U, Liebetrau A, Miller JL, Manns MP, Rehermann B. Cellular immune response persist, humoral responses decrease two decades after recovery from a single source outbreak of hepatitis C. Nature Med. 2000;6:578-582.

(9.) Strader DB, Wright T, Thomas DL, Seeff LB. Diagnosis, management and treatment of chronic hepatitis C. Hepatology. 2004;39:1147-1171.

(10.) Goutagny N, Fatmi A, De L, Penin VF, Couzigou P, Inchauspe G, Bain C. Evidence of viral replication in circulating dendritic cells during hepatitis C virus infection. J Infect Dis. 2003;187:1951.

(11.) Laskus T, Radkowski M, Piasek A, Nowicki M, Horban A, Cianciara J, Rakela J. Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes. J Infect Dis. 2000;181:442-448.

(12.) Morsica G, Tambussi G, Sitia R, Novati A, Lazzarin L, Lopalco L, Mukenge S. Replication of hepatitis C virus in B lymphocytes (CD19+). Blood. 1999;94:1138-1139.

(13.) Castillo I, Pardo M, Bartolome J, Ortiz-Movilla N, Rodriguez-Inigo E, de Lucas S, Salas C, Jimenez-Heffernan JA, Perez-Mota A, Graus J, Lopez-Alcorocho JM, Carreno V. Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown. J Infect Dis. 2004; 189:7-14.

(14.) Castillo I, Rodriguez-Inigo E, Bartolome J, de Lucas S, Ortiz-Movilla N, Lopez-Alcorocho JM, Pardo M, Carreno V. Hepatitis C virus replicates in peripheral blood mononuclear cells in patients with occult hepatitis C virus infection. Gut. 2005;54:682-685.

(15.) Radkowski M, Bednarska A, Horban A, Stanczak J, Wilkinson J, Adair DM, Nowicki M, Rakela J, Laskus T. Infection of primary human macrophages with hepatitis C virus in vitro: induction of tumor necrosis factor-alpha and interleukin 8. J Gen Virol. 2004;85:47-59.

(16.) Sung VM, Shimodaira S, Doughty AL, Picchio GR, Can H, Yen TS, Lindsay KL, Levine KM, Lai MM. Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection. J Virol. 2003;77:2134-2146

(17.) Shimizu YK, Iwamoto A, Hijikata M, Purcell RH, Yoshikura H. Evidence for in vitro replication of hepatitis C virus genome in a human T cell line. Proc Natl Acad Sci USA 1992;89:5477-5481.

(18.) Michalak TI, MacParland SA, Pham TNQ. De novo infection and propagation of wild-type hepatitis C virus in normal human T cells in vitro. Hepatology. 2005;42:265A.

(19.) Pham TNQ, Mulrooney-Cousins PM, Mercer SE, MacParland SA, Inglot M, Simon K, Michalak TI. Invariable detection of HCV in mitogen-induced lymphoid cells during occult infection continuing after recovery from hepatitis C (in preparation).

(20.) Cramp M E, Carucc P, Chokshi S, Williams R, Naoumov NV. Hepatitis C virus (HCV) specific immune responses in anti-HCV positive patients without hepatitis C viremia. Gut. 1999;44:424-429.

(21.) Melisko M E, Fox R, Venook A. Reactivation of hepatitis C virus after chemotherapy for colon cancer. Clin Oncol. 2004;16:204-205.

(22.) Vento S, Cainelli F, Longhi MS. Reactivation of replication of hepatitis B and C viruses after immunosuppressive therapy: an unresolved issue. The Lancet Oncol. 2002;3:333-340.

(23.) Melon S, Galarraga MC, Villar M, Laures A, Boga JA, de Ona M, Gomez E. Hepatitis C virus reactivation in anti-hepatitis C virus positive renal transplant recipients. Transplant Proc. 2005;37:2083-2085.

(24.) Zekri ARN, Mohamed WS, Samra MA, Sherif GM, EI-Shehaby AMR, EI-Sayed MH. Risk factors for cytomegalovirus, hepatitis B, and C virus reactivation after bone marrow transplantation. Transplant Immunol. 2004;13:305-311.

By Tram N.Q. Pham, PhD, and Tomasz I. Michalak, MD, PhD

Tram N.Q. Pham, PhD, is a postdoctoral fellow, and Tomasz I. Michalak, MD, PhD, is the head of Molecular Virology and Hepatology Research at the Faculty of Medicine, Memorial University in St. John's, Newfoundland, Canada. Dr. Pham is supported by a fellowship from the National Canadian Research Training Program in Hepatitis C. Dr. Michalak is a Senior Canada Research Chair in Viral Hepatitis/Immunology supported by the Canada Research Chair Program and funds from the Canadian Institutes of Health Research and the Canada Foundation for Innovation. The research was supported by grants EOP-41538 and MOP-77544 from the Canadian Institutes of Health Research.
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Title Annotation:CLINICAL ISSUES
Author:Pham, Tram N.Q.; Michalak, Tomasz I.
Publication:Medical Laboratory Observer
Geographic Code:1USA
Date:Feb 1, 2006
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