Blood-borne non-A, non-B hepatitis: hepatitis C.

SEMINARS IN LIVER DISEASE-VOL.

1 1 , NO. 2, 1991

Blood-Borne Non-A, Non-B Hepatitis: Hepatitis C JOAN GENESCA, M.D., JUAN I. ESTEBAN, M.D., and HARVEY J. ALTER, M.D.

From the Liver Unit, Department of Internal Medicine, Hospital General Universitari Vall d'Hebron, Barcelona. Spain, and the Departnzenr of Transfusion Medicine, Notional Institutes of Health, Berhesda, Marylnnd. Reprint requests: Dr. Genesca, Liver Unit, Department of Internal Medicine, Hospital General Universitari Vall d'Hebron, 08035 Barcelona, Spain.

In this article we will present an overview of parenterally transmitted NANB hepatitis that will focus on its transfusion-associatcd form, incorporating recent data generated from the use of the new specific test for antiHCV antibodies. It is therefore an update of previous more extensive treatments of the s ~ b j e c t . " ~

EPIDEMIOLOGY Parenteral Transmission Transfusion-Associated Hepatitis: Cellular Products TAH remains the most commonly recognized and best characterized form of NANB hepatitis, and most of our knowledge of this disease has been obtained in this setting. As we will discuss later, although the incidence of TAH has been decreasing constantly and although the current incidence of TAH in the United States is not precisely known, the estimated frequency of hepatitis in transfusion recipients is 3 to 4%;13more than 90% of these cases arc attributable to NANB hepatitis. ''It is estimated that the recent introduction of anti-HCV testing of blood donors will further reduce the incidence of TAH by 50 to 70%. TAH is addressed in detail later in this article.

Transfusion-Associated Hepatitis: Plasma Products NANB hepatitis is a well-known complication of the treatment of patients with congenital or acquired coagulation disorders and has been reported after infusion of every type of replacement the rap^.'^-'^ The incidence of NANB hepatitis in multitransfused hemophiliacs varies depending on age, duration of exposure, the type of replacement therapy, and the donor source of plasma derivatives. The development of effective viral inactivation procedures for clotting factor concentrates has greatly diminished the incidence of NANB hepatitis in this risk group as discussed later. Studies carried out before the availability of anti-HCV testing and viral-inactivated coagulation components demonstrated annual NANB hepatitis attack rates of 2 to 6% in h e m ~ p h i l i a c s ~ and ' ~ ' ~a prospective incidence of NANB hepatitis as high as 65%. l 9 Overall, approximately 20 to 60% of hemophil-

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The term "non-A, non-B" (NANB) hepatitis was adopted 15 years ago to designate those cases of transfusion-associated hepatitis (TAH) that were serologically unrelated to any known hepatotropic virus.'.' An enormous amount of information has been accumulated over the years regarding the epidemiology and natural history of this infection, the recognition of other potential sources of infection, the preventive measures directed to diminish blood-borne transmission, and the treatment of the chronic sequelae of this disease. Despite this intensive investigation, and until very recently, the identification of the causative agent of NANB hepatitis had remained elusive. Indirect observations suggested the existence of more than a single agent, but the absence of a reliable serologic marker and the inability to isolate or characterize this agent or agents represented the most serious obstacle to our understanding of NANB hepatitis. Recently, the so-called epidemic form of NANB hepatitis has been defined epidemiologically and the re~ designation sponsible virus identified and ~ l o n e d ; ' .the hepatitis E virus has been adopted for this agent."n addition, in 1989 investigators from the Chiron Corporation reported the cloning of the major causative agent of the parenteral form of NANB hepatitis."' Using sophisticated molecular biologic approaches, the now termed "hepatitis C virus" (HCV) was identified as a positivesense, single-stranded, linear RNA virus with a genomic size of approximately 10 kb. The sequence of a major portion of the viral genome has been reported, and the virus has been related to members of the flavi- and pestivirus families. A more extensive review of this subject appears elsewhere in this issue of Seminars. A fusion protein (C 100-3) representing a dominant viral epitope of HCV was expressed in yeast and served as the basis for a specific assay for antibody to HCV (anti-HCV).XInitially as a radioimmunoassay, and subsequently as an enzyme-linked immunosorbent assay (ELISA), the preliminary evaluation of the anti-HCV test showed that HCV was a major cause of NANB hepatitis throughout the world.

SEMINARS IN LIVER DISEASE-VOLUME

iacs have elevated alanine aminotransferase (ALT) in the absence of active hepatitis B virus (HBV) infection.'"' A substantial proportion of these cases are thought to be chronic NANB hepatitis. Testing for anti-HCV in recipients of blood products has confirmed the suspected high rate of infection with the NANB agent. Worldwide studies have found anti-HCV in 50 to 90% of hemophilia patients.'5-" A 65% seroconversion rate to anti-HCV was also detected in the acute phase of NANB hepatitis in 83 prospectively followed Italian thalassemic patients." The different rates of seropositivity reported are accounted for by the inclusion of patients who have received a wide variety of blood products with different risks for NANB transmission. In fact, the prevalence of anti-HCV in hemophiliacs who have been heavily transfused with nontreated blood products is higher than 90%.3" Some studies have found a correlation between the presence of anti-HCV antibodies and the amount of clotting factor concentrate received, the existence of anti-human immunodeficiency virus (HIV) antibodies, serum HBV markers, and elevated ALT.'7.xL.3' Despite occasional reports in which NANB hepatitis followed the intravenous administration of immune globulin in agammaglobulinemic per~ons,".~?his blood product is considered safe and the NANB hepatitis cases were thought due to an aberration in the manufacturing process. Subsequent prospective evaluations of patients receiving intravenous immune globulin have failed to detect NANB hepatitis c a ~ e s . ~In' a study by Diirkop et al of 81 patients in whom NANB hepatitis developed after parenteral treatment with contaminated immune globulin, an anti-HCV seroconversion rate of 91 % was observed 6 to 12 months after e x p o s ~ r e . ~ "

Intravenous Drug Users Acute and chronic NANB hepatitis in intravenous drug addicts has been recognized since the 1 9 5 0 ~ . In '~ this population, 50% of acute hepatitis cases have been categorized as NANB he pa ti ti^.'^^" Moreover, some reports of sporadic NANB hepatitis4'-" have included cases occurring in drug addicts. Surveillance data from the Centers for Disease Control's (CDC) Sentinel Counties Study of Acute Hepatitis showed that in 1987 42% of patients classified as NANB hepatitis had a history of intravenous drug abuse.14 As expected, the prevalence of anti-HCV in drug addicts has been extremely high. Studies carried out in Europe and the United States have found anti-HCV in 70 to 92% of intravenous drug users without any significant ' ~ ~ " one study, pergeographic d i s t r i b ~ t i o n . ' ~ . ' ~ ~ Only formed in Germany, showed a relatively low prevalence of anti-HCV (48Y0).'~Studies have also demonstrated that 75% of cases of acute NANB hepatitis in drug addicts tested positive for anti-HCV.'5,47A follow-up study suggested that in drug users with acute hepatitis C , less severe chronic lesions developed than in nondrug u ~ e r s . ' ~

Hemodialysis and Renal Transplantation NANB hepatitis has become the most common cause of acute hepatitis in patients on chronic hemodi-

1 1, NUMBER 2, 1991

alysis. Prospective studies have identified almost 70% of acute hepatitis cases in hemodialysis patients as NANB hepatitis virus related," and annual attack rates for new acute NANB hepatitis have been estimated to be between 3 and 6 % , S O . S I Moreover, NANB hepatitis plays a significant role in the development of chronic hepatitis in dialysis units, since 15% of patients have elevated ALT in the absence of infection with any other hepatotropic viruS,sO.sl

Prevalence studies of anti-HCV antibodies in dialysis centers around the world have shown relatively low rates of HCV infection. Although a wide range of infected patients (1 to 33%) has been reported, the majority of studies have found prevalences of 10 to 200/0,25-?7.6.44h.~-S7Anti-HCV positivity has been correlated with an abnormal ALT, duration of dialysis, and presence of HBV markers. Although some studies have found a relationship between anti-HCV and antecedent . " ~ ~have ~ failed to find any of blood t r a n s f u ~ i o n , ~ ~others c o r r e l a t i ~ n . " ~ ~Indeed, ' - ~ ~ 9 to 18% of Japanese patients on chronic hemodialysis who had never received blood ~ ~ ' studhave been reported as anti-HCV p ~ s i t i v e . ~ 'More ies are needed to define the transmission mechanisms of HCV in hemodialysis units. Finally, in an outbreak of NANB hepatitis in a hemodialysis unit in the United States, 87% of the cases categorized as probable NANB hepatitis were found to be anti-HCV p ~ s i t i v e . ' ~ Organ transplant recipients are also at high risk of acquiring NANB hepatitis. In renal transplant recipients NANB hepatitis can be implicated in a substantial proportion of cases of acute and chronic he pa ti ti^.'"'^ A 6.5% incidence of acute NANB hepatitis after transplantation has been reported,'" and 7 1 % of cases of chronic liver disease in these patients have been attributed to NANB hepatitis." A recent study in 140 French kidney transplant recipients has shown a global prevalence of anti-HCV antibodies of 24.3%." Anti-HCV was more prevalent in patients with chronic active hepatitis (CAH) and without HBV infection (57.5%). Follow-up studies showed that 63% of the patients were anti-HCV positive prior to transplantation and that 30% of them lost the antibodies after the procedure. At the same time, 37% of the recipients acquired this marker after transplantation. A very similar picture has been reported in liver transplant recipients; patients previously anti-HCV positive can lose the antibodies after the procedure, whereas others acquire this marker after t r a n ~ p l a n t a t i o n . ~ ~

Nosocomial and Occupational Exposure NANB hepatitis has been documented in health care workers after accidental needlestick exposure to blood or ' even without overt percutaneous e x p o ~ u r e . ~Although examples of occupational-acquired NANB hepatitis are included in surveys of sporadic NANB hepatitis and in studies of hemodialysis unit^,^'."^^'".^ the real incidence of this form of transmission is largely unknown. Data from the CDC have shown that a history of medical or dental employment involving blood contact accounted for less than 5% of reported cases of acute NANB hepatitis." It is suspected that inapparent percutaneous transmission may be responsible for many of the episodes in which a definite identifiable exposure cannot be


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elicited. Very limited data from prevalence studies of anti-HCV in hospital and hemodialysis staff members have shown only four positive individuals in more than 300 tested,?5.45,~X.~.67 Outbreaks of nosocomial NANB hepatitis have been described in oncology and plasmapheresis unit^,^^.^' and in some reports recent hospitalization was the only risk factor identifiable in cases of sporadic NANB hepatitis. The frequency of NANB hepatitis after surgery without transfusion has been found to range between 0.2 and 2.1 %.70.7'In a recent report from Greece." 12 patients admitted to a hospital with acute icteric NANB hepatitis had been hospitalized in another hospital 2 to 3 months prior to the onset of the disease; 1 1 were tested for anti-HCV, and seven were found to be positive. Also, in an outbreak of NANB hepatitis in a plasmapheresis center in China attributed to inadequate disposal practice, 12 patients diagnosed as acute NANB hepatitis who could be tested for anti-HCV showed unequivocal seroconversion."

Nonparenteral Transmission Perinatal Transmission In studies performed prior to anti-HCV testing, transmission of NANB hepatitis from mother to offspring had been observed in infants born to mothers with acute NANB hepatitis during the third, but not the second, trimester of pregnancy.74Although the information available is rather limited, several studies carried out in more than 70 anti-HCV positive mothers from different areas of the world have failed to detect evidence of HCV ' - ~ ~one case from transmission to n e ~ b o r n s , " ~ . ~only Sweden seemed to be a possible perinatal transmission of HCV." By contrast, one study conducted in Italian anti-HIV positive mothers showed evident HCV perinatal transmission." In this study, almost 50% of newborns from anti-HIV and anti-HCV positive mothers had antiHCV seroconversion as evident from active production of anti-HCV antibodies between 6 and 12 months of age; approximately 50% of t h e ~ nwere also diagnosed as having acute NANB hepatitis. Interestingly, children with perinatal HCV infection had a very high rate of simultaneous HIV infection, and the H1V-related disease seemed to have a more rapid course in children with acute C hepatitis. Although HCV infection of newborns is uncommon, passive transfer of maternal antibody is frequent with anti-HCV generally falling to undetectable Allevels between 3 and 12 months after birth.75.77.7y though there are not enough data to assess the contribution of perinatal transmission to the maintenance of the reservoir of HCV carriers, this mechanism of infection is probably limited. Presumably, only when the mother has high levels of viremia,such as in acute hepatitis or in HIV coinfection, will this route of transmission play a significant role.

Sexual and Household Transmission Sexual transmission of NANB hepatitis probably occurs, although the role of this transmission mechanism

has not been fully defined. Secondary spread of NANB hepatitis to heterosexual partners of patients with parenterally transmitted NANB hepatitis has been documented rarely.") However, surveillance data from the CDC's Sentinel County Study of Acute Viral Hepatitis have shown that 10% of reported patients with NANB hepatitis had a history of heterosexual or household contact with a person who had hepatitis or had heterosexual activity with multiple partner^.^^.'^ A case-control study indicated that such personal contact or sexual activity was significantly associated with acquiring NANB hepatitis." Prevalence studies also from the CDC have found anti-HCV in 14% of persons with a history of household or sexual exposure to a contact with hepatitis and in 16% of heterosexuals with more than 2 partners in the preceding 6 month^.^' Other reports have also suggested the existence of heterosexual transmission; prevalence studies in patients attending clinics for sexually transmitted diseases have found anti-HCV in 5 to 23% of heterosexuals with no history of drug a b ~ s e . ~Another ' - ~ ~ study from Greece detected a seroprevalence of anti-HCV of 5% in 203 prostitutes who denied drug use or blood transfusion .'" Ideo and coworkersX found a seropositive rate of 8% in family members, including sexual contacts, of 42 anti-HCV positive patients. Annual attack rates of NANB hepatitis of less than , ~ ~ homosex3% have been reported in h o m o s e x ~ a l s and ual men account for a minimal proportion of sporadic NANB hepatitis c a ~ e s . ~ " na recent study of risk factors associated with NANB hepatitis, no homosexual men were identified among 74 patients with acute NANB hepatitis with no known source for infection." However, most of the studies carried out in Europe and the United States have shown a seroprcvalence of anti-HCV in homosexual men that. although low, is significantly higher than the prevalence observed in the normal blood donor population.?5.2h.15.h7.82.X4.Xh Percentages have ranged from 1.8 to 25%, but the majority of reports found anti-HCV in approximately 5% of homosexuals. Only two studies from Italy detected rates of infection higher than 20%.25.X4 Anti-HCV positivity was clearly associated with the presence of anti-HIV antibodies in all studies. Stevens78recently found that anti-HCV prevalence in homosexuals also correlates with the number of partners and the frequency of receptive anal intercourse, but that partners of anti-HCV positive homosexuals were not themselves anti-HCV positive. Despite this evidence for sexual transmission, other data suggest the relative rarity of this event. Esteban et all6 found that only 1 of 18 heterosexual partners of intravenous drug abusers and none of 19 heterosexual partners of patients with NANB TAH were anti-HCV positive. Everhart et alxy found no anti-HCV in 42 sexual partners of patients with TAH. Other reports have also found anti-HCV prevalences lower than 5% in sexual partners of persons belonging to different risk g r o ~ p s . * ~Furthermore, .~' whereas male homosexuals have prevalences exceeding 60% for most sexually transmitted disease markers. including HBV and HIV, as already noted, only a small percentage have antibody to HCV. The association of HCV transmission with HIV


HEPATITIS C-GENESCA,

SEMlNARS IN LIVER DISEASE-VOLUME

coinfection is of interest. In addition to the aforementioned studies in male homosexuals, Eyster et alY0demonstrated an association between HCV and HIV among heterosexual contacts of hemophiliacs; 7 of 130 (5%) of the female partners of hemophiliacs were anti-HCV positive when the index case was both anti-HCV and antiHIV positive compared to none of 30 female partners of hemophiliacs who were only anti-HCV positive. Although more studies will be needed to define the importance of sexual transmission, most reports suggest that this mechanism of HCV transmission infection is very inefficient. It is probable that sexual transmission relates to the titer of virus. Most HCV-infected individuals have low titers of virus as demonstrated by infectivity studies in chimpanzee. Occasional patients, however, have higher titers, which might be facilitated during the early stages of infection and during coinfection with HIV. The measurement of anti-HCV is not in itself sufficient to assess the potential for infectivity; there is need for an infectivity marker similar to hepatitis B e antigen (HBeAg) or for methods to quantitate HCV RNA by the polymerase chain reaction (PCR). Sporadic Hepatitis By definition, sporadic NANB hepatitis occurs as an individual episode unassociated with a specific recognized event, epidemic, or outbreak. The incidence of sporadic NANB hepatitis has been difficult to assess because of dissimilarities in the definition and, particularly, the inclusion of persons with percutaneous exposure in some studies. Surveys of patients presenting with acute hepatitis for medical care at large urban hospitals have shown that NANB hepatitis accounts for 6 to 46% of cases of viral hepatitis in urban area^.".'^.^'.^^."^ In the United States, NANB hepatitis accounts for 20 to 40% of acute viral he pa ti ti^.^^ Data from the CDC's Sentinel Counties Study of Acute Hepatitis have shown that in 1987 approximately 62% of patients with NANB hepatitis had a recognized risk factor for acquiring the hepati ti^.^^.^' A history of intravenous drug abuse was the predominant factor (42% of cases), followed by a history of heterosexual or household contact with a person who had hepatitis or heterosexual activity with multiple partners (lo%), history of recent blood transfusion (5%), and history of medical or dental employment (5%). Therefore approximately 40% of patients presenting with acute NANB hepatitis in the United States have no evident source of infection; the term sporadic NANB hepatitis should be restricted to these cases. Overall, 25 to 50% of patients from most epidemiologic studies of acute NANB hepatitis fail to recall any percutaneous exposure, suggesting that NANB hepatitis can be transmitted by other mechanisms. In an additional report by Alter et al8I in which 140 patients with acute NANB hepatitis were interviewed to identify unrecognized sources of NANB infection, 42% had no recognized risk factor. Aside from fewer years of education, none of the other known risk factors for hepatitis B transmission was found to differ significantly between NANB hepatitis patients and matched control subjects. In spite of this, unapparent or covert percutaneous

11, NUMBER 2, 1991

exposure through person-to-person contact might account for some of the NANB sporadic cases. This is supported by several observations: ( 1 ) the recognition, in some studies, of household exposure to a person with hepatitis as a risk-factor for acquiring acute NANB hepatitis, and the rather limited role of sexual transmission; (2) the increased incidence of anti-HCV reported in mentally handicapped children in institutions from Brussels," along with a clear correlation with HBV markers seroprevalence; and (3) the demonstration that a family history of liver disease was a variable independently associated with anti-HCV positivity in Spanish blood don o r ~Worldwide .~~ studies have also confirmed that HCV is responsible for the vast majority of sporadic NANB he pa ti ti^.^^.'^ Seroconversion rates have ranged from 30 to 80%, depending on the timing for anti-HCV testing after the acute episode. Presence of anti-HCV seems to correlate with a higher rate of chronicity. Additional environmental sources of NANB hepatitis transmission, demonstrated or suspected, include tattooing, ear piercing, and bites by h ~ m a n s . 'The ~ relationship of HCV to the arthropod-borne flaviviruses raises the possibility that HCV might also be vectortransmitted. However, there is no direct evidence for this and epidemiologic trends do not suggest such transmission. Transmission by fomites, saliva of an infected person, or following contact with primates has not been demon~trated.~~' In summary, although percutaneous routes are responsible for almost two thirds of NANB hepatitis cases, the remaining cases are transmitted by nonpercutaneous mechanisms or nonovert percutaneous routes. The extent to which each of them, particularly sexual, covert percutaneous, and vertical transmission, contribute to the global picture of acute and chronic NANB hepatitis will have to await further studies.

Hepatitis C Virus in Other Liver Diseases and Risk Groups Antibodies to HCV have been detected, at different prevalences, in almost every liver disease. In patients with hepatitis B surface antigen (HBsAg) positive chronic liver disease, anti-HCV has been reported in 7 to 18% of patients from southern Europe and the United States, 1 0 0 - I D 3 Inclusion of patients from other risk groups for HCV infection probably accounts for some of the differences in seroprevalence. Patients with chronic liver disease and inactive HBV replication (HBeAg and HBV DNA negative) have very high rates of HCV infection (17 to 40%),100-102 suggesting that HCV infection could be an important cofactor in the development of liver disease in such patients. Another interesting hypothesis might be that HCV could suppress HBV replication as does hepatitis delta virus (HDV). In HBeAg-positive chronic liver disease, the concomitant presence of antiHCV seems to indicate greater severity of the liver disease."' Very limited data from patients with chronic HDV infection have also shown an increased prevalence of anti-HCV. Patients with alcoholic liver disease also have a remarkably high rate of anti-HCV.67~100.104.105 Reported


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prevalences have ranged from 25 to 52%, and the presence of anti-HCV has been associated with clinical se~ ~ ~ ' survival,'0s ~~ and verity of the liver d i s e a ~ e , 'shorter the presence of features of CAH in the liver biopsy of alcoholic p a t i e n t ~ . ' ~ ~ ~ ' ~ " o m concern e has been raised regarding the specificity of these results, and the possibility that patients with more advanced liver disease, and therefore with higher levels of serum gamma globulin, might have higher rates of false positive results in the anti-HCV test. Additional studies with confirmatory tests should help to clarify this issue. Cryptogenic chronic liver disease, defined as chronic liver disease with no identifiable etiologic factor, is also associated with HCV infection. Antibodies to HCV have been reported in 68 to 80% of patients with cryptogenic chronic hepatitis and cirrhosis in Italy and Spain, l0I'.lO?. IOh Anti-HCV have also been detected in patients with a diagnosis of autoimmune hepatitis. Difficulties in defining autoimmune hepatitis are probably rcsponsible for the different anti-HCV rates reported. Patients with high titer antinuclear antibodies and a positive response to corticosteroids rarely have antiHCV.h7.'01.'02.'07 In contrast, patients in whom thc diagnosis of autoimmune hepatitis was based solely on the presence of autoantibodies have anti-HCV prevalences of 40 to 80%.2h~"'2~107~"'X Whether these results represent true HCV infection with associated autoimmune phenomena or nonspecific anti-HCV reactivities in patients with autoimmune disease is a currently unresolved issue.Io9Confirmatory tests should aid in the resolution of this dilemma. Antibodies to HCV are rarc in patients with primary biliary cirrhosis,""'.""~"" except for patients with antecedent blood transfusion. ""' Finally, seroprevalence studies in different areas of Africa have shown a very high prevalcnce of antiHCV. lo[). 1 I[',' ' 1 Problems with sample artifacts due to long storage and frequent handling of sera have made it difficult to assess the real impact of HCV infection in Africa.

CLINICAL ASPECTS Acute Disease The mean incubation period (from transfusion to the first elevated ALT value) of NANB TAH is intermediate between that for hepatitis A and hepatitis B, with a peak onset between 6 and 12 weeks and a range from 2 to 26 weeks." Cases that follow the administration of clotting factor concentrates, especially Factor VIII, often occur after an incubation interval of 4 weeks or less. ".lX" Compared with hepatitis B, the acute course of NANB hepatitis is more likely to be clinically mild, to have lower peak ALT levels (200 to 600 UIiliter), and to be less commonly icteric (25% of case^).^.^^^'^^"' Individual cases, however, may be severe and are clinically indistinguishable from severe cases of hepatitis A or B. Detection of TAH is dependent on the careful monitoring of patients because subclinical disease is so common. Sporadic NANB hepatitis, when reported, tends to be

more severe, since only ill patients seek medical attention. A clinical feature quite characteristic of NANB hepatitis is the episodic. fluctuating pattern of ALT; periods of elevated enzyme activity alternate with periods of normal or near normal ALT values. Alternately, NANB hepatitis patients may have persistently elevated ALT or an acute-elevation that resolves completely. As a rule, it is extremely difficult to establish convalescence accurately in patients with NANB hepatitis. Severe or fulminant hepatitis is an unusual outcome of NANB TAH,' however, the proportion of fulminant hepatitis cases attributable to NANB hepatitis has been reported to range from 25 to 44% in surveys from the United States and Europe. '".' l 4 Diagnosis in those cases, however, was based on serologic and historical exclusion, and the viral etiology was not established with confidence. The case-fatality rate for fulminant NANB hepatitis seems to be higher than that of hepatitis A or B. 1 1 3 . 1 1 Fulminant 3 cases of parenterally acquired NANB hepatitis have been reported in patients with underlying chronic liver disease, immunodeficiency, or malignancy, and in previous healthy sub.jects with sporadic hepatitis, lh.?3.SY.hU.Y1.'15 In a recent report from Japan, among patients with chronic B hepatitis in whom fulminant hepatic failure developed, 44% were found to have antiHCV. "" Extrahepatic manifestations of viral hepatitis, such as arthritis, rash, glomerulonephritis, and periarteritis nodosa, have been associated with hepatitis R ; rarc instances of prodromal arthritis have been described in patients with acute NANB hepatitis.'-"' In addition, transient agranulocytosis and aplastic anemia have both been and, indeed, most cases of hcpatitis-asdescribed, "-"' sociated aplastic anemia have been attributed by serologic exclusion to NANB hepatitis."" Aplastic anemia has also been reportcd to occur in 28% of patients undergoing liver transplantation for presumed fulminant NANB hepatitis;''' the aplasia occurred within I to 7 weeks after surgery, an event not noted following liver transplantation for other forms of liver disease. It is intriguing that anthropod-borne KNA viruses have also been associated with bone marrow failure and have been shown to infect hematopoietic stem cells."' The histologic features of acute NANB hepatitis are similar to that of classic viral hepatitis A and B except that parenchymal cells often show diffuse, irregular, eosinophilic granulation associated with many acidiophilic bodies and microvesicular steatosis. "' Sinusoidal cell activation is prominent, but lymphocytic infiltration is sparse within the lobule and the periportal areas. A distinctive bile duct lesion described as a piling up of the ductular epithelial cells into the lumen without disruption of the basement membrane has been seen in NANB hepatitis. Unfortunately, none of these lesions is found consistently enough to distinguish NANB hepatitis from the other viral hepatitis

Chronic Sequelae Persistent infection and chronic hepatitis are the hallmarks of HCV infection. Collected observations




from several studies (for review see Dienstag et al,'." Shih et al," Bradley and Maynard,lZJand Alter"') indicate that in approximately 50% of prospectively followed patients with NANB TAH, biochemical and histologic evidence of chronic hepatitis develops. Although in some studies the frequency of progression to chronicity in sporadic NANB hepatitis cases has been reported to be significantly lower (10% or less) than among TAH cases (40 to 60%),12hone large study of community-acquired NANB hepatitis (sporadic cases with no obvious source of exposure) has revealed a similar 50% chronicity rate."7 These chronicity rates calculated on the basis of sustained or intermittent ALT elevation fol- more than I year after the acute episode might well underestimate the real proportion of patients who become persistently infected after exposure to HCV. The generally asymptomatic nature of the illness and the characteristic fluctuation of ALT levels, often with prolonged intervals of normal ALT values (months to years), make the diagnosis of complete recovery (viral clearance) from HCV infection very difficult to ascertain. Although elevated ALT levels are likely to reflect ongoing viral replication as well as the degree of liver inflammatory activity, chimpanzee inoculation experiments have shown that persistent viremia may prevail in the absence of ALT e l e v a t i ~ n . "Furthermore. ~ biopsy proven chronic hepatitis has been documented both in patients with seemingly acute self-limited NANB TAH"' and in anti-HCV positive blood donors with persistently normal ALT levels.'"' The current anti-HCV assay may not be of help in identifying complete recovery. Although anti-HCV tends to persist in most patients who progress to chronic NANB hepatitis and to disappear in the majority of patients who recover clinically and biochemically. The documented occurrence of TAH in recipients of antiHCV-negative blood'".'72 and the finding of HCV RNA by PCR in the liver of patients with seronegative chronic NANB hepatitis'" suggest that neither normalization of ALT nor disappearance of anti-HCV necessarily imply complete recovery. Therefore it is possible that persistent infection (whether active or quiescent) occurs in the majority of HCV-infected individuals. How this virus regulates its lytic potential and avoids detection and elimination by the host's immune response is currently unknown. Chronic NANB hepatitis is not a benign disease. Histologic evaluation of cases with chronic NANB hepatitis in several studies of TAH"5 has shown that approximately 50% have CAH and 10% have cirrhosis when first biopsied. Severity of the liver damage in chronic NANB hepatitis has also been documented in the hemophiliac population. In one large study'3J biopsy specimens were obtained from 155 hemophilia patients. Although 64% had minimal changes, chronic persistent hepatitis (CPH) or some mild forms of CAH, 15% had established cirrhosis and an additional 7% had severe CAH that would likely progress to cirrhosis. Similar findings have been reported in other two recent studies studies among hemophiliacs.13"n of patients with sporadic chronic NANB he pa ti ti^,'^^."^ liver biopsies from 109 patients showed that 60% had CPH, 35% had CAH, and 5% severe CAH with signs of early cirrhosis. Long-term clinical and biochemical fol-

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low-up of patients with chronic NANB hepatitis has shown that in about 50% of the cases the disease is relatively benign and nonprogressive and may even remit spontaneously. Although rapidly progressive cases have been observed, with some TAH cases progressing to cirrhosis within 1 year of infection, in about 50% of the patients the disease tends to progress insidiously over time. Emerging as a consistent observation among investigators is the slow progression of such cases to cirrhosis. Overall, among patients with chronic NANB hepatitis followed for 5 to 10 years, cirrhosis has been shown to develop in 25%, irrespective of the source of infection (whether transfusion, clotting factor-associated or sporadic),125.1 X 1 7 X Some factors seem to influence the rate of progression from chronic hepatitis to cirrhosis and might explain disparities in the figures reported in different studies. Among them, age at exposure, duration of infection, and degree of liver damage at initial biopsy, have all been found to correlate with progression to cirrhosis. Mattsson et all" noted that of 92 patients with chronic NANB hepatitis 64% of patients older than 30 years had CAH compared with only 33% of those younger than 30. In the same study, follow-up biopsies after a mean of 5 years showed progression to cirrhosis in 27% of the posttransfusion cases whose mean age was 54 years, and in none of the sporadic cases whose mean age was less than 40. Similarly, Tito et all7' in their evaluation of 306 patients with chronic NANB hepatitis found that age and initial histologic grade were the only variables influencing progression to cirrhosis; after a mean follow-up of 8 years. cirrhosis developed in only 19% of patients younger than 50 years compared with 39% of those older than 50 years. Of those whose initial biopsy showed CPH, only 7% progressed to cirrhosis compared with 33% of those with moderate CAH and 47% of the cases with severe CAH. In a similar study, Hopf et a1'j7 found that of 62 cases with sporadic chronic NANB hepatitis, those with stable disease were younger (mean age, 33 years) and all had CPH, whereas those with progressive diseases (53% of whom ultimately progressed to cirrhosis) were older (mean age, 43 years) and most had CAH on initial biopsy. It is noteworthy that in this study, of the 34 patients with progressive disease, cirrhosis developed in only 7 (2 1 %) after 5 years of follow-up, but in 16 (70%) of the 23 followed for more than 5 years. The immune system of the host, associated alcoholism, and concomitant infection with the HBV may also influence prognosis. In one report"' in three patients with NANB TAH who were simultaneously infected with the HIV, symptomatic cirrhosis developed within 3 years of onset of hepatitis. Similarly, among anti-HCV positive intravenous drug users, liver damage is significantly more severe in those who are also infected with HIV."9 The relationship between HCV infection and excessive alcohol intake and infection by other hepatitis viruses has been discussed already. Although cirrhosis has developed in 25% of patients with chronic NANB hepatitis after 5 to 10 years of follow-up, NANB hepatitis-related cirrhosis appears very indolent compared with alcohol-related cirrhosis. Most of these patients are asymptomatic or only mildly symptomatic for years. In the study by Tito et al,"K of


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their 306 chronic NANB hepatitis cases (68% of whom had CAH on initial biopsy), clinical evidence of decompensated cirrhosis or portal hypertension developed in only 10% during a mean follow-up period of 8 years. The indolent nature of the disease is also exemplified by studies in Japan,''" which, in evaluating chronic liver disease related to blood transfusion, have shown that the interval from transfusion to the clinical presentation of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) may be extremely long; in this study. the mean interval to thc recognition of chronic liver disease was 14 years. to development of cirrhosis, 18 years, and to development of HCC, 23 years. Therefore, it is very likely that many patients with cirrhosis resulting from NANB TAH (many of whom are middle-aged individuals with serious underlying diseases) may not live long cnough for end-stage liver disease to develop. However, the fact that some such cases do eventuate in severe and sometimes terminal liver disease is wcll docutnented.Y.lI.l?.1?5 If there was any doubt concerning the significant morbidity and mortality of chronic NANB hepatitis, the availability of the new anti-HCV assay has shown that the blood-borne NANB agent is perhaps the leading cause of chronic liver disease in the Western world. As discussed earlier, anti-HCV testing has demonstrated that among patients from recognized risk groups for NANB hepatitis, the prevalence of chronic HCV infection is much higher than estimations based on elevated ALT levels and that anti-HCV is highly prevalent in patients with cryptogenic chronic hepatitis or cirrhosis.

Relationship to Hepatocarcinoma Although strong epidemiologic evidence implicates HBV in the pathogenesis of HCC,14'-'4'other factors such as alcohol or chronic NANB infection have also been implicated, particularly in developed countries. This has been supported by several case reports, in which patients with well-documented NANB TAH progressed from biopsy-proven chronic hepatitis to cirrhosis and, finally, HCC 7 to 18 years after the onset of hepatitis. I" '" Development of HCC has also been reported in a chimpanzee 7 years after inoculation with serum from a patient with chronic NANB hepatitis.IJX Suspicion that NANB infection was involved in a significant proportion of HCC cases was raised by Okuda et all4" who reported that among 113 nonalcoholic patients with HCC, 20% had no HBV markers and that an additional 49% had HBV markers typical of recovery from that infection; this suggested that up to 70% of these HCC cases might be NANB hepatitis related. Indeed, as it had been predicted, 70% of Japanese patients with HCC have been found to be anti-HCV p ~ s i t i v e . ' ~Similar " prevalences of anti-HCV (65 to 75%) among patients with HCC with or without concomitant alcoholic cirrhosis have been reported from Western Europe,'"-"' somewhat lower figures from the United States (34 to 54%) and Africa (30 to .54%).l5"n the report of HCC patients by Colombo et all" coexistence of anti-HCV and antiHBc was found nearly three times as often in patients with chronic hepatitis C with HCC compared with those

without HCC (54 versus 19%), suggesting a synergistic effect of HBV and HCV in the development of liver cancer. Concerns about the specificity of anti-HCV reactivity among patients with HCC have been diminished by confirmation of ELISA reactivity with a recombinant immunoblot assay (RIBA) and, in some cases. by detection of serum HCV RNA by PCR or HCV-specific antigen in the liver by i m m u n o f l u o r e ~ c e n c e . ~ Although prospective studies of the development of HCC among HCV-infected patients have not yet been conducted, the high frequency of HCV infection among patients with HBsAg-negative HCC suggests that this agent may play a role in the development of this cancer. Thc HCV genome does not appear to integrate into the host genome, but, even for hepatitis B, the exact molecular mechanism underlying the strong epidemiologic connection between HBV and HCC in the Orient has not been elucidated, even though random integration of HBV DNA into the host genome in the liver is a common event. An alternative explanation for the role of hepatitis viruses in HCC is that any condition that can result in cirrhosis, including alcoholism, hemochromatosis, and chronic HBV or HCV infection, can promote hepatocarcinogenesis. The common denominator in these conditions is that hepatocyte necrosis triggers a host response marked by chronic inflammation. fibrosis, and, importantly, hepatocyte regeneration. Presumable, this regenerative hyperplasia expands the population at risk for subsequent genetic changes (such as mutations and chromosomal rearrangements) that deregulate cell growth further and increase the chance of unifocal malignant transformation. If this common denominator theory is correct, then the coexistence of HCV and HBV, or either virus with alcohol, might increase the probability of tumor formation.

TRANSFUSION-ASSOCIATED NON-A, NON-B HEPATITIS Incidence Studies When the first studies on the incidence of TAH in the United States began in the early 1960s at the National Institutes of Health (NIH), hepatitis developed after transfusion in approximately 33% of open heart surgery patients, as assessed by ALT elevations that fulfilled criteria for TAH." Three facts were responsible for this high incidence of TAH: a large transfusion volume; a high (50%) portion of blood from commercial sources; and increased recognition of cases resulting from prospective monitoring of patients. Retrospective evaluation of stored sera revealed that, even prior to HBsAg screening of donors, only 25% of TAH was due to HBV and that 75% was caused by NANB agents. In 1970, the adoption of an all-volunteer donor system and the simultaneous utilization of first-generation tests for HBsAg resulted in a 85% reduction in TAH for an overall incidence of 10%. In 1973, the implementation of third-generation testing for HBsAg screening in blood donors reduced further the incidence of type B TAH to very low levels but did not appreciably change


HEPATITIS C-GENESCA.


the overall incidence that persisted at 9 to 10% through the early 1980s. In 198 1, pretransfusion donor ALT testing was instituted at the NIH, and when hepatitis incidence before and after ALT testing was analyzed, a decline in the incidence prior to the implementation of the surrogate test was found. No subsequent decline was observed in the 3 years after donor ALT testing was instituted, and therefore a beneficial impact of donor ALT testing was not documented. The incidence of TAH in open heart surgery patients at NIH from 1984 to 1986 ranged between 3.7 and 7.8% with an average incidence of 6.5%. The continuous decrease in the number of patients in the cardiac surgery program at NIH has made it difficult to assess the incidence of TAH during the past 4 years, and no large prospective studies of TAH have been conducted in the United States since 1980. The addition of donor screening for antibody to hepatitis B core antigen (anti-HBc) and for anti-HIV in conjunction with more rigid donor questioning and deferral between 1985 and 1987 has certainly had a further impact in reducing TAH. It is estimated that these combined measures reduced the incidence to approximately 3 to 4% during the period immediately prior to the implementation of mandatory anti-HCV testing. In the wake of these major changes in donor screening and the recent implementation of antiHCV testing, the current incidence of TAH in the United States needs to be assessed by a large multicenter prospective study. Experiences from other centers have shown a wide range of TAH incidence throughout the world. In large prospective studies conducted in the late 1970s in the United States in recipients of volunteer blood only, the incidence of TAH ranged between 7 and 17%. mostly between 7 and 12O/o ,' -. 70.1 I?.lS6-15X In thc large multicenter Transfusion Transmitted Virus Study (TTVS), overall NANB hepatitis incidence was 10.9% with a range of 5 . 4 to 15.3%, in the four participating centers."'Thc incidence o f hepatitis correlated with the source of donor blood, highest in a center using some commercial donors, next highest in a center using a high percentage of replacement donors selected by the patient, and lowest in a center using all Red Cross volunteer donor blood. Studies conducted outside the United States, primarily in open heart surgery patients, showed a wide range in TAH incidence, ranging from approximately 2 to 3% in Australia and the Netherlands to as high as 45% in Japan~ll.lS9.1h0Studies in Italy, Spain, Sweden, and Canada showed an incidence ranging between 9 and 21 %.'"I-I"' In all these studies, NANB hepatitis was the predominant form of viral hepatitis. Feinman and associate^'^^ in Canada demonstrated a 9% TAH incidence prior to 1985 and a subsequent decline to 2% following the introduction of multiple measures to interdict donors at high risk for HIV infection.'" The most recent data on the incidence of TAH comes from the Barcelona Posttransfusion Hepatitis Study, a contemporary prospective investigation of the incidence of TAH and the efficacy of different measures to decrease NANB TAH.I3' The baseline incidence of NANB hepatitis in Barcelona between August 1988 to July 1989 was 9.6%; this study immediately anticeded anti-HCV testing (see later).

I 1, NUMBER 2. 199 1

HCV Serology in Recipients with TAH When the new anti-HCV assay was being evaluated in 1988, the first step was to prove its specificity against a pedigreed panel of coded sera of proven infectivity in the chimpanzee.' In these initial studies, anti-HCV was detected in about 80% of cases of chronic NANB TAH from different countries. Extensive retrospective evaluation of the ELISA version of this antibody test in samples from recipients participating in prospective follow-up studies of TAH has shown seroconversion to anti-HCV in 50 to 100% of TAH cases throughout the world,?h.?7.131.132.lhX. I69 This wide variation in seroconversion rates in different studies among cases categorized clinically as NANB TAH may be due to a number of factors: ( 1 ) Misdiagnosis, which is more likely to occur in cases with mild and transient ALT elevations, may account for low anti-HCV seroconversion rates in some studies. Indeed, as the certainty of the diagnosis of NANB TAH increases, as in cases that become chronic, so does the proportion of cases that demonstrate antiHCV; (2) inadequate sampling of patients is another source of error. Seroconversion has been shown to occur in the majority of cases after a mean interval of 15 to 22 weeks p o s t - t r a n s f ~ s i o n " l but ~ in some cases may take a year or longer. Furthermore. in acute resolving cases, the antibody response may be very transient, lasting only a few weeks."' Hence, if thc patient was samplcd inadequately so that late samples were unavailable, or sampling intervals were too long, seroconversion could have been missed. In fact, in most prospective TAH studies in which sampling was adequate (frequent and prolonged), more than 85% of the cases of NANB TAH have proven to be due to HCV by anti-HCV seroconversjon; " I . I?' l 7 l I . l 7 ' and (3) insensitivity of the assay also lowers the rate of observed seroconversion. The determination of anti-HCV with currently licensed assays may not be sufficiently sensitive to establish the presence of HCV infection uniformly. It is almost certain that the frequency of HCV detection will increase as new epitopes arc cxplored and new assays are developed, as liver tissue is examined for HCV antigen and HCV RNA by immunofluorescence and in situ hybridization, respectively, and, cspecially, as PCR amplification to detect HCV RNA is applied to both serum and tissue. Detection of HCV in well-pedigreed NANB hepatitis cases should then well exceed 90% and possibly approach 100%. As already noted, the anti-HCV response in TAH parallels, at least to some degree, the course of the disease. Anti-HCV tends to persist indefinitely in chronic cases; in one study,"' 93% of the cases progressing to chronicity still had anti-HCV after a mean follow-up of 7 years. Conversely, among patients with acute resolving disease, the frequency of detection of anti-HCV seroconversion is considerably lower, about 50%,'" and among the latter anti-HCV tends to disappear with time. In one large outbreak of NANB hepatitis that occurred in Germany, after administration of contaminated immune globulin preparation in 1978 to 1979, anti-HCV seroconversion was demonstrated in 93% of the cases 6 to 12 months after treatment. However, when retested 10 years


154

later, 83% of those in whom chronic hepatitis developed still had anti-HCV, compared with only 15% of those who recovered.17' In this study, it was also shown that patients who recovered had significantly lower anti-HCV ELISA ratios at 12 months than those who progressed to chronicity, suggesting that the anti-HCV titer measured 12 months after infection may have prognostic value.

HCV Serology in Blood Donors With the current C-100-3-based ELISA antibody test, the seroprevalence of anti-HCV among volunteer blood donors does not show marked variations from country to country. Prevalences of 0.3 to 0.7% have been reported from Scandinavia, Germany, France, and ~ . ' ~ ~in Southern Europe the United K i n g d ~ m , ' ~whereas and Japan 0.9 to 1.2% of blood donors have been found to be anti-HCV reactive,1 % ) . 1". 174.17' In the United States, the overall prevalence of repeatedly reactive donors is about 0.6%. ' 6 7 . ' 7 h The overwhelnling evidence indicates that HCV is the major causative agent of TAH and that the presence of circulating antibodies against the C-100-3 recombinant peptide represents infectivity rather than immunity. These facts have prompted almost worldwide mandatory screening of blood donors for anti-HCV, although the specificity of the assay, a crucial issue for donor counseling, is still a matter of controversy. Indeed, retrospective evaluation of anti-HCV in stored samples from donor-recipient pairs from prospective studies of TAH have shown HCV transmission rates from anti-HCV-reactive I n a recent donors ranging from 17 to study, look-back investigation of infectivity in prospectively identified anti-HCV-reactive donors showed that 84% had been involved in HCV transmission."" In this study, a high ELISA ratio (sample optical density divided by cut-off value). an elevated ALT leveland the presence of abnormal liver histology in the donor were all found to correlate strongly with infectivity. Although, at present, there is no licensed confirmatory assay for the ELISA screening test, RIBA has been developed for confirmation of ELISA reactive samples. 17' In this assay, the sample is tested against a nitrocellulose strip in which two HCV-specific recombinant proteins, one produced in Eschrrichici coli (5- I- I ) and the other in yeast (C- 1003), and three control antigens (the superoxide dismutase used in the cloning procedure and two levels of human gamma globulin) have been precoated as separate bands. A sample is considered positive when it reacts with both HCV-specific antigens, indeterminate when it reacts with only one of them, and negative when it reacts with none. The RIBA test has been found to be highly predictive of infectivity. In one study,Ih7hepatitis developed in 78% of recipients of ELISA-positive1RIBA-positive blood, compared with only one of 14 recipients of ELISA-positiveiRIBA-negative blood. In general, RIBA positivity correlates with the intensity of the ELISA res ~ l t ; " " . ' ~nearly ~ ~ ' ~ "90% of samples with ELISA ratios over 5 are RIBA positive, whereas 70% of samples with ELISA ratios below 2 are RIBA negative. Overall, only one third of ELlSA repeatedly reactive blood donors in

and the United States are confirmed by RIBA,1h7.'7h about the same proportion has been reported to have detectable serum HCV RNA by PCR.I8' The confirmatory power of the RIBA test, however, is limited due to its relative insensitivity and the high proportion of indeterminate results. Although most donors with indeterminate or negative RIBA results are not infectious,"* a proportion of such donors have been reported to harbor circulating HCV RNA as detected by PCR or to have been implicated in HCV t r a n ~ m i s s i o n . ~ ~ " ~ ~ n f o r t u n a t e l y , current PCR techniques that amplify a nonstructural portion of the HCV genome cannot be considered a gold standard for confirmation of infectivity: (1) sequence diversity in the nonstructural regions of different HCV isolates has been shown to account for false-negative PCR result^;'^" (2) current cDNAiPCR assays may not be as sensitive as PCR assays for DNA genomcs; and(3) the methods for sample collection, storage, and freeze-thawing can influence the number of viral gcnomes available for RNA analysis."' With respect to RIBA sensitivity, retrospective studies have found an implicated RIBA-positive donor in 60 to 75% of NANB hepatitis cases.'"' Therefore the current anti-HCV screening assay will probably detect about 70% of HCV infectious donors. Consequently, although this test will represent a major advance in blood donor screening, in its present configuration it will fail to detect a significant number of HCV carriers.

Measures to Decrease Blood-Borne Transmission Donor Deferral and Autologous Donation No other element in blood transfusion history has so focused attention on the risks of transfusion and has brought such radical changes in blood donation practice than has the advent of thc acquired immune deficiency syndrome (AIDS). The adoption first, in the early 1980s, of voluntary self-exclusion of persons in AIDS-risk groups and then, in 1985, of screening blood donors for antibodies to HIV. have made the overall blood supply considerably safer than it was in the pre-AIDS era. In a sense, antibodies to HIV have the potential to be another surrogate marker for NANB hepatitis. The threat of AlDS has increased donor screening procedures, established a more responsible donor base, and allowed for pre- and post-donation self-exclusion. It has also emphasized the need for limiting blood transfusion as much as possible, as curtailed the use of blood from high-risk persons, and has permitted the adoption of additional safety measures, such as surrogate testing. Lastly, it has expedited research in inactivation procedures that will render blood and components safe even in the absence of testing. AlDS has also placed increased emphasis on predeposit and intraoperative utilization of autologous blood. However, despite its evident advantages, autologous blood donation, is not employed widely.IX7Autologous donation in elective cardiac surgery is a safe praetice and effective in reducing homologous blood needs significantly and, consequently, the risk of transfusion-


HEPATITIS C - G E N E S C A , ESTEBAN, ALTER


acquired diseases.'xx Whenever possible, autologous blood should be recommended by physicians for elective surgery. Intraoperative autologous blood salvage is another method of reducing exposure to homologous blood, especially in patients with large volumes of blood loss. I X Y

Surrogate T e s t s Before the advent of a specific test for NANB hepatitis, and given the magnitude of chronic consequences of this disease and the absence of effective therapeutic measures, attention was focused on indirect means to prevent NANB TAH. Among several tests appraised for their usefulness as markers of potentially infectious donor blood, elevated ALT activity or anti-HBc, or both, in donor blood showed a significant correlation with the risk of NANB hepatitis in blood recipients.'"'-"" Data from the TTVS and from prospective studies at NIH predicted an efficacy of 29 to 30% in reducing NANB hepatitis by excluding donors with elevated ALT, at the loss of 1.6 to 3.1% of the donor population.""."' Subsequently, reanalysis of the same data in both studies showed a significant association between anti-HBc in thc donor and the development of NANB hepatitis in the recipient,1 ~ 2 . 1 9 2These two studies, along with another study from Germany,"' predicted a combined efficacy for anti-HBc testing of 2 8 8 . In the German study, in which donors were already excluded for elevated ALT, anti-HBc testing offered an additional 42% predicted efficacy. Overall analysis suggested that implementation of donor blood testing for ALT and anti-HBc could reduce the frequency of NANB TAH by 30 to 45% at a cost of donor units in the range of 1.5 to 5%. In 1987, the American Association of Blood Banks and the American Red Cross elected to require universal screening of all blood donors for ALT and anti-HBc. This decision was made despite several concerns that included the limited sensitivity and predictive value of these surrogate tests, the difficulties in standardization of both tests, the additional cost, and the loss of donors. Moreover, a prospective, randomized controlled trial of the efficacy of surrogate markers was never performed in the United States, and the implementation of these markers was also coincident with the introduction of measures to prevent posttransfusional AIDS. These facts, along with the absence of large prospective studies of TAH, and the recent implementation of anti-HCV screening in blood banks will make it impossible ever to know the real impact of surrogate testing on NANB TAH. Implementation of surrogate marker screening in Europe has been a matter of controversy for some of the reasons already outlined, but mainly because of the associated blood donor loss that would be higher than 15% in some European countries. One study from Spain showed that the exclusion of anti-HBc-positive blood would not have significantly reduced the incidence of only prospective, randomNANB TAH hepatitis.""he ized controlled study of the efficacy of anti-HBc and ALT testing to prevent TAH was performed in Spain and did not find any significant difference in the incidence of

I I . NUMBER 2, 1991

TAH between recipients of screened and unscreened blood.'"' However, results must be interpreted with caution because of the small study population. Independent analysis of each surrogate marker showed that ALT testing, but not anti-HBc testing, could be useful in reducing TAH. Randomly selected anti-HCV positive donors have reported prevalences of anti-HBc positivity and elevated ALT that range from 0 to 20% and 8 to 45%. respectively, 130-I67 However, when anti-HCV positive donors are implicated in NANB TAH cases, the prevalence increases to 13 to 33% for elevated ALT and 36 to 53% for anti-HBc positivity."'."' Screening for surrogate markers would thus exclude from 30 to 40% of antiHCV-positive donors implicated in TAH cases. Conversely, approximately 90 to 95% of blood donors with either elevated ALT or anti-HBc are not anti-HCV pos;tive, I67.l97,19X When a donor is anti-HBc positive and has an elevated ALT, the probability of being anti-HCV positive is higher and ranges from 10 to IOU%, with a mean value of approximately 50%. '67.1')7.1'1X The question of whether surrogate tests should bc retained after the introduction of anti-HCV screening is a difficult one. The answer would depend on the efficacy of the new test and could be obtained ultimately only by performing a prospective, randomized controlled study in anti-HCV tested population. However, the estimated currcnt low of TAH would necessitate a very large and prohibitively expansive randomized trial. and it is probable that such a study will never be performed. Nonetheless, anti-HBc screening should probably be retained because it is a surrogate marker not only for NANB, but also for HBV, HIV, and, in general, for sexual promiscuity and intravenous drug abuse. The ALT test, on the other hand, is a marker for acute hepatitis of any etiology and could detect the seronegative acute phase of HCV infection and, possibly, carriers of a NANB hepatitis agent other than HCV. Inactivation P r o c e d u r e s The complete prevention of NANB TAH and, in general, of any blood-borne viral infection may rely on viral inactivation rather than on serology. There are currently three major approaches to viral inactivation of blood products: heat, lipid solvents, and photochemical (nucleic acid) inactivation. Two studies in chimpanzees documented the efficacy of heat treatment of lyophilized clotting factor concentrates at 60°C for up to 30 hours.'"~""' However, when this concentrate was administered to humans, " retransmission of NANB hepatitis o c c ~ r r e d . ~ More cent formulations of dry-heated clotting factors have shown that heating in a dry state at 80°C for 72 hours seems to completely inactivate HCV.'s.'"2-2" No cases of NANB hepatitis and no anti-HCV seroconversions have been observed after the infusion of this product. Moreover, PCR analysis of several samples of this concentrate has failed to detect HCV sequence^.'^' Similarly, heating concentrates in solution at 60°C for 10 hours (pasteurization) has proved to be a very safe procedure, since no case of NANB hepatitis was reported among 26 hemo-


156

ESTEBAN, ALTER

philiacs treated with this type of Factor VIII."'h However, HBV infection was reported subsequently in two additional hemophiliacs."" Recently, the so-called second-generation Factor VIII concentrates, which add a purification step of anionic exchange chromatography to pasteurization have not been implicated in any case of HCV transmis~ion.'"~Also, a preparation that enlploys immunoaffinity chromatography with monoclonal antibodies to fractionate and purify Factor VIII from plasma followed by heating appears to be highly effective.'"'.'"' Lipid solvents or detergents have also had considerable success in inactivating NANB agents. Perhaps the most encouraging results have been obtained with the combination of tri-(n-butyl-)-phosphate (TNBP) with the detergent sodium cholate. Prince and coworkers"' showed that this method inactivated both HBV and NANB virus suspended in Factor VIII concentrate when administered to chimpanzees. Subsequent evaluation in recipients of this product has shown no NANB hepatitis cases or anti-HCV serocon~ersion.'~.'" Although chloroform inactivation of NANB virus is well documented,?" administration of a Factor VIlI concentrate treated by heating and chloroform produced NANB hepatitis in all recipients of this product."" Methods using photochemical inactivation procedures that inactivate nucleic acid rather than destroy the membrane might be very useful, for this methodology could be applicable to cellular blood elements. Two such methods are currently under study and include P-propriolactone (BPL) in combination with ultraviolet (UV) lightu5 and psoralen in combination with long-wave UV light.''" Both methods inactivate a broad spectrum of viruses, including single- and double-stranded RNA and DNA viruses. The combination of BPL and UV light has been shown to inactivate 10"o 10' chimpanzee infectious doses 50 (CID,,,) of the H-strain of NANB hepatitis; by incorporating Tween 80 as an additional inactivating step, the quantity of BPL can be reduced, thus improving the recovery of the clotting factor^."^ Similarly, the combination of psoralen and UV light was capable of inactivating lo4 to 10' CID jo of both HBV and NANB virus in a variety of media. including Factor VIII concentrate.'" This method seems to be innocuous to red cells and is currently being evaluated for its effect on the functional integrity of platelets. Anti-HCV Testing There is no doubt that the most important currently available single measure to decrease blood-borne transmission of NANB hepatitis is the implementation of donor screening for anti-HCV. Retrospective analysis of prospectively followed TAH cases and their respective implicated donors has suggested that 60 to 85% of NANB hepatitis cases could have been prevented had anti-HCV screening been in use at the time these studies were performed.'3','3','h7It is estimated that the recent introduction of anti-HCV testing will result in a further 50% decline in TAH incidence in countries that are utilizing the surrogate assays and probably higher in countries that are not.lh7The only study that has evaluated the efficacy of anti-HCV screening prospectively has been

performed recently in Spain by Esteban and associates.'" In two contemporary and consecutive studies, an 80% reduction in the incidence of post transfusion hepatitis (from 9.6% to 1.8%) after the introduction of antiHCV testing was demonstrated. In the United States, although there are no recent prospective data, the projected 50% minimal reduction related to anti-HCV screening would decrease the TAH rate to approximately I to 2%. a dramatic improvement from the rates observed in the 1960s and 1970s and a close approximation to the background frequency of hepatitis in nontransfused, hospitalized patients.

Is There Any Other NAN6 Agent Besides HCV? Prior to the discovery of HCV, considerable evidence existed for the presence of more than a single NANB hepatitis agent. This hypothesis was supported by the observation of two or more distinct bouts of NANB hepatitis in drug addicts and hcmodialysis pat i e n t s , ~ ~and . ? ~the ~ occurrence of both long- and shortincubation NANB hepatitis, the latter particularly observed in hemophiliacs receiving Factor VIII concentrates.L'.lxThe strongest evidence accrucd from crosschallenge studies in chimpanzees, which demonstrated that different inocula from different sources could induce two distinct episodes of acute NANB hepatitis in the same animal, and by studies that appeared to distinguish a chloroform-sensitive agent that induced cytoplasmic tubular changes in chimpanzee hepatocytes (tubuleforming agent) from a chloroform-resistant agcnt that did not induce tubular change^."".'?^'-"^ Despite this cvidence, reports of second and third attacks of acute NANB hepatitis have been documented in chimpanzees following challcnge with the same in~ c u l u m . ' ' ~Similarly, Brotman et alX%ave also demonstrated that immunity to this agcnt may be ovcrwhclmcd in chimpanzees by reinoculation with a high titer of the same strain of virus. Finally, spontaneous reactivation of liver disease has been observed in a chimpanzee after prolonged normalization of ALT.""otentially, these observations could invalidate the conclusions of the cross-challenge studies. Furthermore, it has been previously estimated that the vast majority of NANB TAH cases were due to a single agent, indistinguishable from the NIH Hutchinson strain (tubule-forming The identification of HCV has largely confirmed the suspicion that one major agent is responsible for NANB hepatitis. HCV is the chloroform-sensitive tubule-forming agent,'; and serologic studies have demonstrated that it is implicated in the majority of NANB hepatitis acquired through parenteral and nonparenteral mechanisms. The question of other NANB viruses still remains for those cases that do not show anti-HCV seroconversion. Evidence that anti-HCV negative cases of NANB hepatitis might nonetheless be HCV related stems from the observations that: (1) the anti-HCV response may be very transient in cases that recover rapidly; (2) anti-HCV-positive hepatitis can result from the transfusion of blood that tests anti-HCV negative; (3) new anti-HCV assays employing additional HCV epi-



SEMlNARS IN LIVER DISEASE-VOLUME

topes detect anti-HCV in some cases negative by currently licensed assays; (4) HCV-related antigen can be detected by fluorescence in the liver tissue of some NANB hepatitis cases that test anti-HCV negative; and ( 5 ) HCV RNA can be detected in the serum and liver of some anti-HCV negative NANB hepatitis cases. While a second NANB hepatitis agent cannot be excluded, it is probable that if such an agent exists it will account for a very small part of the NANB hepatitis spectrum. It is estimated that no more than 10% of NANB hepatitis will be unrelated to HCV, and it is possible that with tests of increased sensitivity, with the use of PCR, and with examination of the liver by immunofluorescence and and in situ hybridization that the probability of an additional agent may be reduced to near zero.

recombinant vaccine a realistic expectation for the near future. Finally, elucidation of the mechanisms involved in viral persistence and hepatocellular damage during chronic HCV infection and identification of the essential steps in viral replication may provide clues for the design of new antiviral compounds (such as proteinase, helicase, or polymerase inhibitors) capable of suppressing viral replication without disturbing normal cell physiology.

REFERENCES I.

2.

PROSPECTS FOR THE FUTURE 3.

The present review has chronicled the major reccnt advances in our knowledge of NANB hepatitis after the successful cloning of its causative agent and the subsequent development of specific serologic assays. However, although novel data are now accumulating rapidly, we are still at the beginning of our understanding of HCV infection. Much work remains to be done to elucidate the pathophysiology of this disease and to develop fully effective preventive and therapeutic measures. Although, as already mentioned, introduction of anti-HCV testing will dramatically reduce the incidence of TAH, it is obvious that this screening test, in its present configuration, will fail to detect a significant proportion (25 to 30%) of HCV carriers. To increase thc scnsitivity of the anti-HCV test, a second-generation antibody assay has been developed that includes additional recombinant peptides, both structural (c-22) and nonstructural (c-33-c). Similarly, a new KlBA test that incorporates two new HCV-specific bands, corrcsponding to the previously mentioned peptides, has been designed for confirmation of ELISA-reactive samples. Preliminary ~ t u d i e s ' ~ ' . ' with ~ ' these second generation tests suggest that they can shorten the "window" period considerably between infection and seroconversion and detect additional infectious donors nonreactive in the first generation assay. Despite this improvement, a proportion of HCV carriers are not detected even with these second-generation assays. Although further refinements in the anti-HCV test, with the addition of new immunogenic epitopes and development of IgM-capture assays, are to be expected, there is clearly the need to develop HCV-antigen assays or to find some practical way to apply gene amplification technology to routine donor and patient screening. Since it has become clear that NANB TAH cases account for only a small fraction of the total number of HCV-infected individuals, the only reliable approach to the prevention of a large proportion of HCV infections will be the development of a vaccine and the implementation of mass vaccination programs in childhood. Pending the elucidation of epitopes that induce neutralizing antibodies, the cloning of HCV makes development of a

11, NUMBER 2, 199 1

4.

5.

6.

7. 8.

9.

10

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14

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fusion non-A, non-B hepatitis in c h i m p a n ~ e e s Physicochem: ical evidence that the tubule-forming agent is a small enveloped virus. Gastroenterology 88:773-779. 1985 Eder G , Bianchi L, Gudat F: Transmission of non-A, non-B hepatitis to c h i m p a n ~ e e s :A second and third episode caused by the same inoculum. In: Zuckerman AJ (Ed): Viral Hepatitis and Liver Disease. New York. Alan R. Liss, 1988. pp 550-552. Brotlnan B, Prince A M , Huima ' I Non-A. non-B hepatitis: Is there more than a single blood-borne strain'? J Infect Dis 15 l:618-625, 1985. Bradley DW, Krawczynski KZ, Cook EH, et al: Recrudescence of non-A, non-B hepatitis in persistently infected chimpanzees. In: Hopkins R. Field S (Eds): Viral Hepatitis, 2nd International Workshop. Edinburgh. Nuclear Enterprises, 1982, pp 43-48. Schiniizu YK, Feinstone S M , Purcell R H , et al: Non-A, nonB hepatitis: Ultrastructural evidence for two agents in experimentally infected chimpanrees. Science 205: 197-200. 1979. Lee S , Francis B. Ball V, et al: A second generation ELISA ( ~ 2 0 0 1 ~ 2 for 2 ) the detection of antibody to hepatitis C virus. Proceedings of the Second International Symposium on HCV. Los Angeles. November 1990, p 109. Lee S. Francis B. Ball V, et al: Improved detection of antibodies to hepatitis C virus in cases of non-A. non-B hcpatitis using a second generation ( ~ 2 0 0 1 ~ 2ELISA. 2) Proceedings of the Second International Symposium on HCV. Los Angeles, November 1990, p 109.



OSHA stiffens bloodborne rules, decrees free hepatitis B vaccine.

Hepatitis C.

Hepatitis C.

Hepatitis C.

Hepatitis C.

[Treatment of hepatitis C].

Screening for hepatitis C.

Hepatitis C virus.

[The hepatitis C virus].

Hepatitis C (chronic).

Steatosis and hepatitis C.

Hepatitis C and neutropenia.

Hepatitis B and C.

Urticaria and hepatitis C.

Hepatitis C virus.

Hepatitis C genotype 1.

New hepatitis C therapies.

Hepatitis C virus replication.

Hepatitis C and pregnancy.

Hepatitis C screening.

Hepatitis C virus upstanding.

[Hepatitis C virus].

Occult hepatitis C virus infection in patients with autoimmune hepatitis.

Type 2 autoimmune hepatitis and hepatitis C virus infection.