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Hepatitis C
Chapter 44
Hepatitis C Roger Y. Dodd
INTRODUCTION For many years, hepatitis C was the most common infectious complication of blood transfusion in the United States. Indeed, a number of studies in the 1970s showed that more than 10% of blood recipients had biochemical evidence of what was then termed non-A, non-B hepatitis (NANBH).1,2 Subsequently, almost all of these cases were shown to be due to infection with the hepatitis C virus (HCV). Fortunately, the implementation of increasingly sensitive tests for HCV infection has now reduced the incidence of post-transfusion hepatitis C infection to levels that are essentially undetectable by direct study; by early 2001, the risk was estimated at about 1 infection per 1.4 million component units.3
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The symptoms of acute hepatitis C do not differ significantly from those of other viral hepatitides, although they may be somewhat less severe than those of hepatitis B. The incubation period is 7 to 8 weeks, but a wide range has been noted. It has been estimated that about 25% of acute cases may be accompanied by symptoms that, in some cases, may be quite mild. When present, symptoms include fatigue, anorexia, abdominal pain, and weight loss. A proportion (perhaps 20% to 30%) of patients with acute hepatitis C may become jaundiced. Symptoms may last for up to 10 weeks. Alanine aminotransferase (ALT) levels are elevated during the acute phase; such elevations tend to be moderate (i.e., around 300 to 400 IU/L) but may be quite high (up to 2000 IU/L). Acute disease is rarely fatal, with mortality rates of 1% or less. Approximately 20% of acute infections resolve. The remaining 80% (whether symptomatic or not) become chronic. The natural history of chronic HCV infection is not completely understood. Such chronic infections may last for the lifetime of the patient, although there is growing evidence that 26% to 45% of these cases may eventually resolve, as shown by the disappearance of detectable HCV RNA in the circulation and in some instances by the eventual loss of detectable antibody.4 Many infected individuals may remain completely asymptomatic for several years (perhaps even their whole lives), but a good number have histologic evidence of liver disease, including hepatitis, fibrosis, and cirrhosis. In some cases, hepatocellular carcinoma develops. Alter and Seeff 4 have published a framework representing the outcome of HCV infection that is based on a large number of published studies. They conclude that 20% of infected individuals resolve the acute infection. Of the remaining 80% who become chronically infected, 30% demonstrate a
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stable chronic hepatitis with a favorable outcome. Another 30% have severe, progressive hepatitis, and the remaining 40% may have some variable progression. Prospective studies have shown, however, that chronic symptomatic hepatitis may take an average of 10 to 14 years (and, frequently, much longer) to develop, whereas cirrhosis may take an average of 21 years and hepatocellular carcinoma almost 30 years. Although lifethreatening outcomes may be uncommon or considerably delayed, it is still true that the consequences of HCV infection are the leading indicator for liver transplantation in the developed world. From the perspective of transfusion medicine, this pattern of disease has two important consequences. First, although the immediate consequences of post-transfusion hepatitis C may seem to be largely trivial, the long-term outcomes cannot be ignored. Second, the preponderance of asymptomatic, chronic infection leads to a relatively large population of individuals who are at risk of transmitting the infection via blood donation.
TREATMENT In 1997, a National Institutes of Health Consensus Development Conference gave recommendations for treatment of chronic hepatitis C, applicable to individuals with significant histologic findings on biopsy, presence of HCV antibody, elevated ALT, and detectable levels of HCV RNA.5 In brief, such patients should initially be treated with 3 million units of interferon alfa three times a week for 12 months. In the absence of any response (i.e., normalization of ALT and loss of detectable HCV RNA), this therapy should be discontinued after 3 months and the patient should be considered for combination therapy with interferon and ribavirin. Overall, about 50% of patients show temporary improvement with this regimen, but ultimately, only about 25% of all patients show definitive resolution of disease. A subsequent Consensus Development Conference in 2002 updated these recommendations, emphasizing the value of combination therapy. These treatment recommendations have been updated by Liang and colleagues6 on the basis of experience with combination therapies. In fact, a review of two studies revealed that a sustained response to interferon alone was observed in 29% of patients after treatment for either 24 or 48 weeks.6 However, among those receiving combination therapy (interferon plus ribavirin), 33% showed a sustained response at 24 weeks, and 41% showed such a response after 48 weeks of therapy. It was also noted that treatment for more than 24 weeks was unnecessary in patients with HCV subtypes
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EPIDEMIOLOGY Hepatitis C virus is globally distributed and, with a few notable exceptions, the seroprevalence rate is remarkably constant from one region to another, usually on the order of 1% to 2%.10 Although some countries or localities have startlingly high rates, these may be due to cultural factors, including the use of traditional medicine practices; in Egypt, for example, numerous infections were attributed to a program involving injections for control of schistosomiasis.11 In the United States, the overall seroprevalence rate has been estimated at 1.8%, and 65% of persons infected with HCV are age 30 to 49.12 Transmission of HCV is primarily confined to parenteral routes. In the United States, it is clear that many of the currently identified infections are a result of previous exposure via illegal injection of drugs. In addition, many infections are believed to have resulted from blood transfusions given before the availability of sensitive tests.13 Other epidemiologic associations are the use of clotting factor concentrates before the implementation of effective viral inactivation in 1987, exposure in a health care setting, household exposure, multiple sexual partners, and low socioeconomic level.14 There has been considerable speculation about the natural transmission routes of HCV, which have presumably been responsible for maintaining the baseline viral prevalence levels over centuries or even millennia. Some evidence exists for sexual and perinatal transmission, but in both cases, it seems likely that transmission may occur only during early acute infection. HCV infection does not show the strong association with male-to-male sex that has been seen for hepatitis B and human immunodeficiency viruses (HIVs). The incidence of new HCV infections has declined by 80% or more since 1989. Clearly, there has been a major reduction in transfusion transmission of the virus, and the majority of new infections (about 60%) are attributable to injection drug use. Even though incidence is declining, it is anticipated that the burden of HCV disease will continue to rise over the foreseeable future because of the large number of chronically infected individuals.10,15 Studies on the epidemiology of HCV infection among blood donors are of significant importance in the context of strategies for donor selection and questioning. ConryCantilena and associates16 studied a population of 481 blood donors who were found to be reactive in screening tests for anti-HCV. Among the 241 with positive strip immunoassay (SIA) results, 27% had a history of transfusion, 68% had used cocaine intranasally, 42% had a history of intravenous drug use, and 53% reported a history of sexual promiscuity; ear-piercing among men was also significantly associated with a positive test result. Many of these risk factors were confounded. Nonetheless, these observations prompted the
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institution of donor questions regarding intranasal cocaine use; a formal requirement for this question was subsequently eliminated, however. It is of interest to note that, in a study performed in blood donors who tested positive for HCV RNA but negative for HCV antibody, 29.2% reported recent intravenous drug use. A history of recent incarceration was independently associated with nucleic acid amplification test (NAT) positivity.17 The prevalence of HCV infection in current injection drug users is estimated to be 79%, although this rate does appear to be declining.10 Donor history questioning can certainly be improved, but it is unclear how to obtain more accurate information on previous illicit drug exposures, whether they be recent or many years ago. Fortunately, individuals with past histories do not contribute to window period risk.
HEPATITIS C
2 and 3, because of the greater responsiveness of these subtypes to treatment. A number of studies have indicated that pegylated interferon is more effective than the standard product; it is now the preferred version, particularly for treatment of recurrent disease.7,8 Detailed recommendations for diagnosis, management, and treatment of hepatitis C have recently been published by the American Association for the Study of Liver Diseases.9 Current therapeutic approaches result in a sustained response in 42% to 82% of patients with chronic disease, depending on the viral genotype.
VIROLOGY The hepatitis C virus was first recognized in 1989 as the principal etiologic agent of NANBH. A small RNA genome segment was isolated from a presumptive viral pellet prepared from the plasma of an experimentally infected chimpanzee. When the encoded peptide was expressed in bacteria through the use of a lambda phage vector, it was reactive with antibodies present in convalescent serum from a patient with NANBH.18 This RNA fragment, termed 5-1-1, was used as a basis for the eventual sequencing of the entire genome of the virus. The 5-1-1 sequence was also used to develop the capture reagent (the c100-3 peptide) incorporated into the initial version of an HCV antibody enzyme immunoassay (EIA). This peptide represents a portion of the NS3 region of the HCV genome.19 Subsequently, additional peptides have been expressed and incorporated into improved versions of the HCV antibody test. The virus itself is an enveloped, single-strand RNA virus now classified as a separate genus (Hepacivirus) within the Flaviviridae. It has a positive-strand genome of barely less than 10,000 bases. The functional organization of the genome has been well-defined.20 Recently, there has been notable success in establishing a laboratory culture system for the virus, in which a replicative form of the viral genome has been used to infect cells in vitro. Virus produced from such systems has been shown to be infectious for chimpanzees.21,22 These culture systems offer considerable promise for the further study of the virus. Hepatitis C virus is characterized by considerable genetic variability, expressed at three levels: genotype, subtype, and isolate. There are six major genotypes of HCV, designated by the Arabic numerals 1 to 6. The RNA sequence varies between genotypes by some 25% to 35%, and this level of differentiation has probably emerged over periods of 500 to 2000 years. Subtypes within a genotype, designated by lowercase letters (a, b, etc.), represent RNA sequence variation of 15% to 25% that evolved over about 300 years. Individual isolates within a subtype may vary by 5% to 10% in RNA sequence.23–27 Overall, these studies indicate that the frequency of major subtypes in the United States is as follows: 1a, 42.6%; 1b, 29.2%; 2a, 2.7%; 2b, 8.1%; 3a, 4.7%. Additionally, there are hypervariable regions in the genome, and many quasispecies of HCV are likely to develop in an individual patient over time. In general, any variation below the level of subtype is unlikely to be reliable enough to differentiate sources of infection. The distribution of HCV
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subtypes may change with geography. It has been suggested that the clinical outcome of HCV infection and the response to treatment may vary with subtype. Genotypes 2 and 3 are more responsive to treatment than the more common subtypes 1a and 1b.5,9,28,29 The sequence of events after infection with HCV has been well-characterized at the level of viral markers in the circulation. Antibodies may be detected by version 3.0 tests an average of 70 days after exposure, although this period may be quite variable.30,31 However, some 40 to 60 days prior to the appearance of such antibodies, HCV RNA may be detected in the plasma. The HCV RNA levels rise rapidly (over a few days) to 105 to 107 copies/mL. This level is generally maintained at least until significant levels of antibody are expressed. It is now apparent that a soluble viral core antigen is also present and can be detected once the RNA levels exceed about 50,000 copies/mL.32,33 Elevated levels of ALT are frequently observed a few days before antibodies are detectable, but always after the steep rise in RNA levels. Interestingly, there is growing evidence that there may be occasional low levels of viral RNA in the circulation during the early eclipse phase, after infection and prior to the steep rise to peak levels of RNA.5,29 The implementation of widespread NAT for HCV RNA has been shown to detect a meaningful number of RNApositive, anti-HCV–negative donations. Stramer and colleagues34 reviewed the results of the first 3 years of testing in the United States. Among almost 40 million donations tested by the Chiron/Gen-Probe or Roche assays in small pools of plasma samples (16 and 24 samples per pool, respectively), 170 RNA-positive, anti-HCV nonreactive samples were identified. This reflected an overall rate of 1 in every 230,000 donations. Although there was no significant difference in the rates from the two different HCV RNA tests, the rate amongst donations tested by the more sensitive test for anti-HCV was 1 in 270,000. Within the Red Cross system, the rate was 1:251,000 for the first 3 years of HCV NAT, increasing to 1:221,000 in the following 2 years. The increase was not statistically signficant. Overall, of 156 evaluable cases, 51 would have been excluded as donors as a result of other test results, primarily ALT elevations, which occurred among 46 of the 51.34 Thus, in retrospect, ALT testing probably did have a modest impact on blood safety, but at significant cost in terms of blood supply. These data, along with other information showing that donor ALT levels were of little or no demonstrable value for interdicting other supposed transfusion-transmitted hepatitides, led to its elimination for blood component testing. In Stramer’s study, RNA-positive donors were entered into follow-up investigations. It was found that 75 of 90 enrolled donors seroconverted, as detected in samples
Table 44–1
collected a median of 35 days post-donation. The majority of those who failed to seronconvert had only been followed for 12 to 58 days. In a more comprehensive study of 67 RNA-positive donors, 7 failed to complete follow up, 2 had abortive infections, and 3 did not seroconvert after periods of 1.5 to 3 years. It is of interest to note that the median time to observed follow-up is approximately 50% of the supposed window period, although 35 days is probably something of an overestimate because of the sampling intervals in the study.34 Once antibodies are detectable, RNA levels may decrease or become variable. In the evaluation of samples from blood donors, HCV RNA is detected in about 80% of samples that are reactive on a version 3.0 EIA and confirmed reactive on the version 3.0 SIA.35 The frequency of RNA detection declines with the strength of the antibody response (as defined by the number and intensity of bands on the SIA).36 It seems likely that the antibody-positive, RNA-negative samples actually represent resolved infections. In some cases, antibodies may eventually decline to undetectable levels. There is a vigorous humoral and cytotoxic immune response to HCV, but in most cases, it is clearly unable to eliminate the virus. The mechanisms underlying the ability of the virus to escape the immune response are not well understood. The infecting virus often generates a wide variety of quasispecies; further, the representation of these variant forms changes rapidly over time.20,37 Yet it appears that these changes may contribute only in part to viral persistence.
SEROLOGIC TESTS FOR HCV INFECTION Two licensed EIA tests for HCV antibodies are currently available in the United States. Although both use recombinant viral antigens as the solid-phase capture reagent, they differ in their physical format and in the number and nature of viral antigens used (Table 44–1). The test manufactured by Ortho Clinical Diagnostics uses a microplate solid phase and has been designated as a 3.0 version by the manufacturer. The peptides that are coated onto the microplate well are known as c22-3 from the C (core) region, c200 from the NS3NS4 regions, and an NS-5 peptide. The test manufactured by Abbott Laboratories uses a polystyrene bead solid phase and is designated as a version 2.0 test. The capture reagents for this test are c22-3, and c33 and c100-3 (both from the NS3/ NS4 region). Both test procedures use an antiglobulin conjugate to detect the analyte antibodies. The performance characteristics of the tests, representing the manufacturers’ claims (see Table 44–1), have been validated by extensive clinical trials, and the tests have been licensed as biologics by the U.S. Food and Drug Administration (FDA). Other tests on fully
Enzyme Immunoassay Tests for Anti-HCV: Components and Performance Characteristics
Version of Test (manufacturer)
Peptides
Sensitivity*
Specificity*
1.0 (Ortho Clinical Diagnostics) 2.0 (Abbott Laboratories) 3.0 (Ortho Clinical Diagnostics)
C100 HC34 (c22), HC31 (c33), c100 c22, c200‡, NS-5
81% 85.7%† 88.1%†
>99.4% 99.83% 99.95%
*
Based on manufacturers’ claims in product inserts. Based on a diagnosis of chronic non-A, non-B hepatitis—ALT elevated >6 months, HBsAg negative. ‡ Includes c33 and c100 sequences. †
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●
Positive: Two or more bands with an intensity equal to, or greater than, that of the weak positive control band, plus nonreactive SOD band.
●
●
Negative: No band with a greater intensity than the weak positive control. Indeterminate: Only one band reactive or any pattern in association with a reactive SOD band.
HEPATITIS C
automated platforms are available elsewhere in the world and are soon expected to be licensed in the United States. Most diagnostic reagents are defined as devices, but those that are used in the preparation of blood and blood products are required to meet the more stringent biologics requirements, because blood itself is defined as a biologic. The sensitivity of these tests was defined in trials using patients diagnosed with NANBH. Because this diagnosis is not specific, the significance of the sensitivity figures is unclear. Of more importance now, at least in the context of blood donor screening, is the ability of the test method to detect infection at the earliest possible time during the seroconversion period. Studies of seroconversion panels suggest that currently available tests detect antibodies, on average, 70 to 80 days after exposure to the source of infection and 40 to 60 days after the initial detection of HCV RNA in the plasma. In addition, it should be noted that earlier versions of the EIA tests clearly failed to detect some infected individuals.38 This is not surprising, because only a very limited number of viral epitopes were included in the capture reagent. Current tests for antiHCV are whole-antibody assays. No tests are yet available for specific detection of immunoglobulin M anti-HCV. Although these EIA tests have high sensitivity and specificity, there is a possibility that some reactive test results are nonspecific. When the EIA is used to screen blood donors, its actual positive predictive value is 70% to 80%.36 Consequently, it is recommended that all asymptomatic individuals who test as reactive on EIA repeatedly should be further tested with an additional, more specific procedure. Currently, only one such immunologic method is licensed and available in the United States. This is a strip immunoblot assay (RIBA 3.0) that is constructed by application of recombinant or synthetic viral peptides representing c22, c33, c100, 5-1-1, and NS5 regions to nitrocellulose strips in a fixed pattern. The c-100 and 5-1-1 peptides are present in the same band on the strip. The expression carrier protein for the recombinant viral antigens is also applied (superoxide dismutase, SOD), as are strong and weak positive controls. Patient or donor samples are added to the strips and, after washing, adherent antibodies are detected by an appropriate enzyme-conjugated antiglobulin and visualization reaction. The number and intensity of bands are scored, and the result is interpreted as follows:
Table 44–2 summarizes the results of testing a large number of volunteer blood donations with HCV EIA and SIAs. Unlike the situation with HIV, there does not seem to be a common pattern for the sequence of appearance of reactive bands in the blot during seroconversion with HCV. However, a number of studies have defined the relationship between particular bands, or band patterns, and the likelihood that a sample will also contain detectable HCV RNA. For example, Dow and colleagues35 reviewed data from 177 blood donor specimens that were reactive on EIA and tested positive on the version 3.0 SIA. Among 82 samples with four positive bands, 69 (84.1%) were RNA-positive. Of the 54 samples with three positive bands, 40 (74.1%) were RNA-positive, whereas only 14 (34.1%) of the 41 samples with two positive bands were RNApositive. Among the samples with indeterminate SIA results, the frequencies of RNA-positive results were 3 of 154 for c22, 1 of 220 for c33, 1 of 191 for c100, and 0 of 380 for NS5.35 Thus, a few of the indeterminate patterns may be associated with the presence of RNA and, therefore, of active HCV infection. Similar data were published by Dodd and Stramer.36
TESTS FOR HCV RNA Tests for HCV RNA serve an important role in diagnosis and patient management.9,39 A variety of procedures is available, all of which depend on nucleic acid amplification, with one exception. The reverse transcriptase polymerase chain reaction (RT-PCR) is perhaps the most familiar. Viral RNA is reverse-transcribed to DNA, and two primers are used to define a sequence for repetitive amplification using a temperature-resistant DNA polymerase and a temperature-cycling protocol. A variety of methods may be used to detect the resulting amplified sequence, including visualization in gels, hybridization with labeled probes, and detection of amplicons in real time. Both qualitative and quantitative procedures are available.5,40,41 In most cases, a conserved segment of the 5′ untranslated region of the genome is selected for amplification. PCR-based assays are available commercially (e.g., Roche Molecular Systems) as well as from independent reference laboratories; alternatively, they may be developed in-house with the use of standard technologies.
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Table 44–2 Results of Testing 19.2 Million Blood Donations with HCV 3.0 EIA and Version 3.0 SIA and Percentage of RNA-Positive Samples among Subgroups of SIA-Positive Subjects Number of Subjects with SIA Finding (% RNA+)
HCV EIA RR N = 30,680 SIA >2 bands 2 bands only
Positive (% RNA+)
Indeterminate
Negative
19,541
4898
6241
17,139 (82%)* 2402 (42%)†
NA NA
NA NA
*
Sample of 200 tested. 2347 tested. EIA, enzyme immunoassay; HCV, hepatitis C virus; NA, not applicable; RR, repeatedly reactive; SIA, Strip immunoblot assay. Data courtesy of Susan L. Stramer, Ph.D.(personal communication).
†
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Another technique, known as nucleic acid sequence– based amplification (NASBA; a proprietary technology from Organon-Technika),42 and the very similar transcriptionmediated amplification (TMA; a proprietary technology from Gen-Probe Inc.) can be performed without temperature cycling. Two enzymes are used to produce an RNA amplicon, which can be detected by a variety of methods, including the hybridization protection assay. It should be noted that Chiron owns patent rights to the HCV genomic sequence used for amplification. Sample collection, stability, and preparation for amplification are all important and must be properly controlled. HCV RNA is quite labile, and it is preferable, if not essential, to collect specimens in EDTA. Samples should be maintained at refrigerated temperatures and tested with minimal delay. A number of different methods may be used to prepare the RNA for testing, including conventional extraction from ultracentrifugal pellets, extraction on silica, and probecapture techniques. Finally, in a method known as the branched-chain DNA assay (B-DNA), a probe is labeled with a large branched DNA molecule that carries many copies of the detection label. This method does not amplify the target nucleic acid; rather, it provides a system in which numerous label molecules may be associated with a single target sequence. As might be expected, this technique is not as sensitive as amplification. However, the lack of sensitivity can be exploited to differentiate those patients with high levels of circulating RNA.43,44 Within the United States, almost all blood donations have been tested for HCV RNA since 1999.34 Two methods are in use, RT-PCR(Roche) and TMA (Chiron/Gen-Probe). To date, all such testing has been performed on small pools of plasma samples, with current pool sizes of 24 for the Roche procedure and 16 for the Chiron/Gen-Probe method. Both tests have been licensed for routine use by the FDA and both have an analytical sensitivity of 12 or fewer copies of RNA/ mL by probit analysis and a 95% detection level of 30 to 60 copies/mL. The overall sensitivity of the testing is, of course, proportionately reduced in pooled testing. A number of methods are available for the determination of genotype and subtype. Not all methods are able to discriminate every genotype or subtype, however. Available methods may be broadly separated into those that depend on immunologic differentiation and those that directly detect variation in nucleic acid sequences. In the former case, specific peptides derived from the NS4 region are used to probe for antibodies in the specimen. Nucleic acid–based techniques include sequencing amplicons from selected genomic areas, PCR using genomespecific primers, DNA-enzyme immunoassay (DEIA), restriction fragment length polymorphism (RFLP) analysis of amplicons, and differential hybridization of amplicons with specific probes. Two of these approaches are commercially available (GEN-ETI-K DEIA kit and INNO-LiPA HCVI and HCV II). The reader is referred elsewhere for further details.23,45 Relatively little information is available about the impact of HCV genotype on the sensitivity of diagnostic tests, and some data suggest little variation in the analytic sensitivity of NAT.46 However, a recent publication reviewing the performance characteristics of numerous blood screening assays for HCV antibodies suggests that most tests are not seriously impacted by genotype variations (El-Nageh, in press).
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DIAGNOSTIC ALGORITHM The Centers for Disease Control and Prevention (CDC) has published an algorithm for diagnostic testing for HCV among asymptomatic individuals (Fig. 44–1).14 This algorithm is somewhat different from that recommended for blood donor screening, in that it explicitly permits the use of NAT to confirm a repeatedly reactive EIA result. However, NAT-negative samples must be further evaluated by an SIA. This algorithm also provides useful guidance in the context of treatment, but a more comprehensive guide to treatment was published in 2004.9 Given that blood donors are now routinely evaluated by NAT for HCV RNA, it is hoped that these results may be incorporated into the notification and management of seropositive donors.37,45
IMPACT OF BLOOD DONOR SCREENING AND TESTING FOR HCV Results of Testing In 2001, the frequency of positive results among firsttime blood donations as defined by RIBA was 0.3%, which is about one fifth of the national prevalence rate of 1800 per 100,000. The incidence of new infections in the donor population is 1.9 new infections per 100,000 person-years; the corresponding national incidence figure is 13.4 per 100,000 person-years.47 Thus, the donor rate is about one fifth of the national rate. These differences are attributable, at least in part, to the procedures used to recruit safer populations for donation and to the measures used to question presenting donors about their medical and behavioral histories. However, it should be
EIA for anti-HCV Positive (repeatedly reactive)
Negative
Negative
Indeterminate
Positive
Additional laboratory evaluation
Stop
OR
RIBA™ for anti-HCV
Stop
Negative (nonreactive)
RT-PCR for HCV RNA Positive
Medical evaluation
(e.g., RT-PCR, ALT)
Negative RT-PCR and normal ALT
Positive RT-PCR or abnormal ALT ALT
Stop
Alanine aminotransferase
Anti-HCV Antibody to HCV EIA
Enzyme immunoassay
RIBA™
Recombinant immunoblot assay
RT-PCR
Reverse transcriptase polymerase chain reaction
Figure 44–1 Algorithm for hepatitis C virus (HCV) testing among asymptomatic individuals. (From Centers for Disease Control and Prevention: Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR Morb Mortal Wkly Rep 1998;487[RR-19]:1–39.)
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Risk of Post-transfusion HCV Infection It is clear from a number of published studies that donor screening and testing measures have had a profound impact on the incidence of post-transfusion hepatitis C. Indeed, prospective studies have not demonstrated any infections since the implementation of the so-called multi-antigen tests for antibodies to HCV. But there is also substantial evidence for the efficacy of a variety of screening approaches used even before this time. The essentially complete elimination of commercial donation of whole blood had a major effect on reducing the frequency of post-transfusion hepatitis. It is also believed that a further reduction was seen as a result of more stringent donor questioning to reduce the risk of transmission of HIV and acquired immunodeficiency syndrome (AIDS), although subsequent information implies that the major effect would have come from a reduction in the number of injection drug users. The first study to clearly demonstrate the impact of testing measures on hepatitis C infection was published by Nelson and colleagues.49 They evaluated samples from a large population of patients undergoing cardiac surgery, using the first-generation test for anti-HCV. These researchers found that the risk of infection was 0.45% per unit prior to the implementation of any testing. After implementation of testing for ALT and anti-HBc, the rate of infection dropped to 0.19%. Finally, once the version 1.0 EIA test was implemented, the rate dropped to 1 per 3300 units (0.03%), a reduction of 84%.49 A subsequent reevaluation using the more sensitive version 2.0 test on the blood recipients suggested that the actual risk was closer to 1:1700.50 Once the version 2.0 test was introduced for blood donor screening, however, the frequency of residual infection declined profoundly. As pointed out previously, cases of hepatitis C were no longer observed in prospective studies, and risk estimates then had to be developed on the basis of the length of the window period (as determined from post-transfusion infections) and the incidence of new infections within the donor population. In a landmark publication in 1996, Schreiber and colleagues estimated the residual risk of HCV infection at 1 per 103,000 donations, based on a window period of 82 days and an incidence rate of 4.84 per 100,000 personyears.51 Subsequent evaluations account for a 12- to 13-day reduction in the window period attendant on the implementation of the version 3.0 EIA and an overall decline in the incidence of HCV infection to 2.09 per 100,000 person-years. The latter figure translated to a residual risk of 1:276,000 repeat donations. The addition of HCV NAT was estimated to markedly reduce this risk to 1:1.935 million repeat donations. In the same paper, it was observed, on the basis of the results of NAT, that the incidence of HCV infections was 2.4 times higher among first-time donors.52 Allowing for a 23% frequency of first-time donors in the Red Cross system, this translated to an overall risk of 1:1.39 million donations.3 The relative stability of the detection rates for HCV RNA and the lack of change in prevalence
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rates for HCV antibody53 suggest that this risk is not changing significantly, at least through the end of 2003. It is interesting to speculate about the higher incidence of infections among first-time donors, but there are no definitive data. One possibility is that there are more testseekers among people presenting to donate for the first time, although this is not necessarily supported by published data on interviews of RNA NAT-positive donors, because both first-time and repeat donors were found to be positive and to acknowledge risk factors for infection.17 It is also possible that the process of giving blood has an educational impact and that individuals who learn about risk factors for bloodborne infections defer themselves from future donation. Finally, it should be noted that there are cases in which HCV has been transmitted by blood units that have been fully tested for HCV, even after the implementation of NAT.54 Such cases have usually been detected as a result of lookback and are too few to offer any quantitative estimate of residual risk. Window-period theory would strongly suggest that such cases would occur, and this is supported by the detection of HCV RNA in window-period samples, using ultrasensitive test methods. It is of interest to speculate on the minimal infectious dose of HCV. Busch has developed models based on the conservative assumption that it is as low as one genome equivalent per 20 mL of plasma. Using this assumption and back-calculating, the theoretical window period from the observed doubling rate of HCV plasma RNA during early infection has led to estimates that are largely compatible with currently accepted window period estimates.55
HEPATITIS C
noted that only a very few donors are actually deferred on the basis of their response to risk questions, in part because donors do not always provide complete answers48 but also perhaps because potential donors make a conscious decision to avoid giving blood so as not to have to answer the questions.
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A positive test result for HCV antibodies does not provide any information about the duration of infection, even if accompanied by a positive finding for HCV RNA. Consequently, if a blood donor is found to be HCV antibody positive, it is possible that prior donations from that individual were infectious for HCV. This could occur by two broad mechanisms. First, a previous donation could have been made in the infectious but seronegative window period. Second, the prior donations could have been collected at a time when a less sensitive test was in use or even before testing was initiated. Accordingly, a focused lookback program has been initiated to locate, notify, test, and, if appropriate, treat recipients of such potentially infectious prior donations. Studies in Canada and elsewhere suggest that up to 70% of such recipients may indeed have been infected.56,57 However, in the United States, the effort appears to have been less productive. A team from the CDC reported that an estimated 98,484 blood components were identified as potentially infectious.58 Of these, 85% had been transfused. This interim study found that lookback had been completed for 80% of the transfused products; 69% of the recipients had died. Of those living, 78% were successfully notified that they had received a potentially infectious blood component. It was estimated that, of recipients notified, 49.5% were tested for anti-HCV; of those, 18.9% were seropositive, but 32% of these individuals were already aware that they were seropositive. Thus, at the time of publication of the study, it was estimated just over 1000 individuals were newly notified of unexpected HCV infection; this estimate translated to a national figure of 1520, on the
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assumption that the lookback process was to be completed. The figure represents fewer than 1% of all individuals who may have been infected as a result of transfusion.58 It should be noted that this component of the lookback program was restricted only to donations that were identified as a result of testing with multi-antigen tests (i.e., versions 2.0 or 3.0). It is likely that, as lookback is extended to cover donors initially identified by the version 1.0 test, the proportional yield will be greater, but the efficiency of the process will certainly be affected by availability of required records. On the other hand, the essential completion of achievable lookback affecting recipients of blood donated prior to the implementation of testing means that almost all available cases now represent lookback from incident cases of HCV infection. The yield of this aspect of lookback is now extremely low, and such yield will be further reduced as the impact of NAT is manifested. The ethical imperatives for lookback continue, but the public health benefits of this process are becoming vanishingly small.
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In its way, the history of hepatitis C has been as remarkable as that of HIV and AIDS. In retrospect, hepatitis C was the blood-borne agent most commonly transmitted by transfusion in the United States, right up until the early 1990s. The legacy of this problem will be seen for many years, as the long-term health consequences of HCV infection become manifest. The almost complete elimination of the problem of transfusion-transmitted HCV is the result of many years of dedicated study of an intractable problem. There were no simple solutions; the virus was refractory to laboratory study, and there was a truly frustrating inability to identify any serologic markers of infection. Indeed, only at the very end of the 20th century was a naturally occurring, circulating viral antigen recognized and then only in the few weeks preceding seroconversion. As with HIV, the first laboratory approach to abrogating transfusion-transmitted HCV infection was the development of serologic tests. In the case of HCV, however, this development was achieved through the combination of years of study of the disease, development of animal models, and the painstaking application of new recombinant technology. It is fitting that Harvey Alter and Michael Houghton received the 2000 Lasker award for this work, but hundreds of others contributed over many years. Serologic and nucleic acid testing have essentially eliminated the risk of transfusion-transmitted HCV. Plasma derivatives are prepared from highly tested starting material and are further treated with advanced inactivation procedures. It is of interest to note, however, that whereas the early tests removed a substantial fraction of antibody-positive units, they failed to identify all infectious units, probably leading to the unexpected occurrence of HCV in recipients of some immunoglobulin products. Despite the success of testing for HCV, there continue to be barriers to other aspects of management of this virus. As with many viral diseases, treatment options for HCV are limited and incomplete. More baffling, however, are the difficulties inherent in understanding and manipulating the interactions between the immune system and HCV. Development of an effective vaccine seems to be a particularly elusive goal.
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REFERENCES 1. Aach RD, Szmuness W, Mosley JW, et al. Serum alanine aminotransferase of donors in relation to the risk of non-A, non-B hepatitis in recipients. The Transfusion-Transmitted Viruses Study. NEJM 1981;304:989–994. 2. Alter HJ, Purcell RH, Holland PV, et al. Donor transaminase and recipient hepatitis: Impact on blood transfusion services. JAMA 1981;246: 630–634. 3. Dodd RY. Current safety of the blood supply in the United States. Int J Hematol 2004;80:301–305. 4. Alter HJ, Seeff LB. Recovery, persistence, and sequelae in HCV infection: a perspective on long-term outcome. Semin Liver Dis 2001;20:17–35. 5. Management of Hepatitis C. NIH Consensus Statement 1997;15:1–41. 6. Liang TJ, Rehermann B, Seeff LB, Hoofnagle JH. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med 2000; 132:296–305. 7. Carrat F, Bani-Sadr F, Pol S, Rosenthal E, et al. Pegylated interferon alfa-2b vs standard interferon alfa-2b, plus ribavirin, for chronic hepatitis C in HIV-infected patients: A randomized controlled trial. JAMA 2004;292:2839–2848. 8. Jacobson IM, Gonzalez SA, Ahmed F, et al. A randomized trial of pegylated interferon alpha-2b plus ribavirin in the retreatment of chronic hepatitis C. Am J Gastroenterol 2005;100:2453–2462. 9. Strader DB, Wright T, Thomas DL, Seeff LB. Diagnosis, management and treatment of hepatitis C. Hepatology 2004;39:1147–1171. 10. Shepard CW, Finelli L, Alter MJ. Global epidemiology of hepatitis C virus infection. Lancet Infect Dis 2005;5:558–567. 11. Frank C, Mohamed MK, Strickland GT, et al. The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt. Lancet 2000;355:887–891. 12. Alter MJ, Kruszon-Moran D, Nainan OV, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. NEJM 1999;341:556–562. 13. Alter MJ. Hepatitis C virus infection in the United States. J Hepatol 1999;31:88–91. 14. CDC. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR Morb Mortal Wkly Rep 1998;47 (RR-19):1–39. 15. Alter MJ. Epidemiology of hepatitis C and lookback. Hematology 1999;1999:418–421. 16. Conry-Cantilena C, VanRaden M, Gibble J, et al. Routes of infection, viremia, and liver disease in blood donors found to have hepatitis C virus infection. NEJM 1996;334:1691–1696. 17. Orton SL, Stramer SL, Dodd RY, Alter MJ. Risk factors for HCV infection among blood donors confirmed to be positive for the presence of HCV RNA and not reactive for the presence of anti-HCV. Transfusion 2004;44:275–281. 18. Choo Q-L, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359–362. 19. Kuo G, Choo Q-L, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362–364. 20. Simmonds P. Genetic diversity and evolution of hepatitis C virus—15 years on. J Gen Virol 2004;85:3173–3188. 21. Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11:791–796. 22. Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 2005;102:9739–9740. 23. Lau JYN, Mizokami M, Kolberg JA, et al. Application of six hepatitis C virus genotyping systems to sera from chronic hepatitis C patients in the United States. J Infect Dis 1995;171:281–289. 24. Simmonds P, Holmes EC, Cha T-A, et al. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol 1993;74:2391–2399. 25. McOmish F, Yap PL, Dow BC, et al. Geographical distribution of hepatitis C virus genotypes in blood donors: an international collaborative survey. J Clin Microbiol 1994;32:884–892. 26. Simmonds P, Alberti A, Alter HJ, et al. A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatology 1994;19: 1321–1324. 27. Zein NN, Persing DH. Hepatitis C genotypes: Current trends and future implications. Mayo Clin Proc 1996;71:458–462. 28. Martin P. Hepatitis C genotypes: the key to pathogenicity. Ann Intern Med 1995;122:227–228. 29. Cooreman MP, Schoondermark-Van de Ven EME. Hepatitis C virus: Biological and clinical consequences of genetic heterogeneity. Scand J Gastroenterol 1996;31(Suppl 218):106–115.
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44. Halfon P, Khiri H, Gerolami V, et al. Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA. J Hepatol 1996;25:307–311. 45. Lau JYN, Davis GL, Prescott LE, et al. Distribution of hepatits C virus genotypes determined by line probe assay in patients with chronic hepatitis C seen at tertiary referral centers in the United States. Ann Intern Med 1996;124:868–876. 46. Forman MS, Valsamakis A: Increased sensitivity of the Roche COBAS AMPLICOR HCV test, version 2.0, using modified extraction techniques. J Mol Diagn 2004;6:225–230. 47. Dodd RY. Current estimates of transfusion safety worldwide. In Vyas GN, Williams AE (eds). Advances in Transfusion Safety. Basel, Karger, 2005, pp 3–10. 48. Williams AE, Thomson RA, Schreiber GB, et al. Estimates of infectious disease risk factors in US blood donors. JAMA 1997;277:967–972. 49. Donahue JG, Murioz A, Ness PM, et al. The declining risk of post-transfusion hepatitis C virus infection. NEJM 1992;327:369–373. 50. Nelson KE, Ahmed F, Ness P, Donahue JG. The incidence of post-transfusion hepatitis: Reply. NEJM 1993;328:1280–1281. 51. Schreiber GB, Busch MP, Kleinman SH, et al. The risk of transfusiontransmitted viral infections. NEJM 1996;336:1685–1690. 52. Dodd RY, Notari EP, Stramer SL. Current prevalence and incidence of infectious disease markers and estimated window-period risk in the American Red Cross blood donor population. Transfusion 2002;42:975–979. 53. Zou S, Notari EP, Stramer SL, et al. Patterns of age- and sex-specific prevalence of major blood-borne infections in United States blood donors, 1995 to 2002: American Red Cross blood donor study. Transfusion 2004;44:1640–1647. 54. Schüttler CG, Caspari G, Jursch CA, Willems WR, Schaefer S. Hepatitis C virus transmission by a blood donation negative in nucleic acid amplification tests for viral RNA. Lancet 2000;355:41–42. 55. Busch MP, Glynn SA, Stramer SL, et al. A new strategy for estimating risks of transfusion-transmitted viral infections based on rates of detection of recently infected donors. Transfusion 2005;45:254–264. 56. Long A, Spurll G, Demers H, Goldman M. Targeted hepatitis C lookback: Quebec, Canada. Transfusion 1999;39:194–200. 57. Christensen PB, Groenboek K, Krarup HB, Danish HVL. Transfusionacquired hepatitis C: the Danish lookback experience. Transfusion 1999;39:188–193. 58. Culver DH, Alter MJ, Mullan RJ, Margolis HS. Evaluation of the effectiveness of targeted lookback for HCV infection in the United States— interim results. Transfusion 2000;40:1176–1181.
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30. Busch MP, Korelitz JJ, Kleinman SH, et al. Declining value of alanine aminotransferase in screening of blood donors to prevent posttransfusion hepatitis B and C virus infection. Transfusion 1995;35:903–910. 31. Busch MP. HIV, HBV and HCV: New developments related to transfusion safety. Vox Sang 2000;78:253–256. 32. Couroucé AM, Le Marrec N, Bouchardeau F, et al. Efficacy of HCV core antigen detection during the preseroconversion period. Transfusion 2000;40:1198–1202. 33. Tanaka E, Ohue C, Aoyagi K, et al. Evaluation of a new enzyme immunoassay for hepatitis C virus (HCV) core antigen with clinical sensitivity approximating that of genomic amplification of HCV RNA. Hepatology 2000;32:388–393. 34. Stramer SL, Glynn SA, Kleinman SH, et al. Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid-amplification testing. NEJM 2004;351:760–768. 35. Dow BC, Buchanan I, Munro H, et al. Relevance of RIBA-3 supplementary test to HCV PCR positivity and genotypes for HCV confirmation of blood donors. J Med Virol 1996;49:132–136. 36. Dodd RY, Stramer SL. Indeterminate results in blood donor testing: What you don’t know can hurt you. Transfus Med Rev 2000;14:151–160. 37. Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 2000;288: 339–344. 38. Alter MJ, Margolis HS, Krawczynski K, et al. The natural history of communityacquired hepatitis C in the United States. NEJM 1992;327: 1899–1905. 39. Gretch DR, Dela Rosa C, Carithers RL Jr, et al. Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications. Ann Intern Med 1995;123:321–329. 40. Lunel F, Mariotti M, Cresta P, et al. Comparative study of conventional and novel strategies for the detection of hepatitis C virus RNA in serum: Amplicor, branched-DNA, NASBA and in-house PCR. J Virol Methods 1995;54:159–171. 41. Hawkins A, Davidson F, Simmonds P. Comparison of plasma virus loads among individuals infected with hepatitis C virus (HCV) genotypes 1, 2, and 3 by Quantiplex HCV RNA assay versions 1 and 2, Roche monitor assay, and an in-house limiting dilution method. J Clin Microbiol 1997;35:187–192. 42. Damen M, Sillekens P, Sjerps M, et al. Stability of hepatitis C virus RNA during specimen handling and storage prior to NASBA amplification. J Virol Methods 1998;72:175–184. 43. Sangiovanni A, Morales R, Spinzi GC, et al. Interferon alfa treatment of HCV RNA carriers with persistently normal transaminase levels: a pilot randomized controlled study. Hepatology 1998;27:853–856.
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Hepatitis C Roger Y. Dodd
INTRODUCTION For many years, hepatitis C was the most common infectious complication of blood transfusion in the United States. Indeed, a number of studies in the 1970s showed that more than 10% of blood recipients had biochemical evidence of what was then termed non-A, non-B hepatitis (NANBH).1,2 Subsequently, almost all of these cases were shown to be due to infection with the hepatitis C virus (HCV). Fortunately, the implementation of increasingly sensitive tests for HCV infection has now reduced the incidence of post-transfusion hepatitis C infection to levels that are essentially undetectable by direct study; by early 2001, the risk was estimated at about 1 infection per 1.4 million component units.3
THE DISEASE III 592
The symptoms of acute hepatitis C do not differ significantly from those of other viral hepatitides, although they may be somewhat less severe than those of hepatitis B. The incubation period is 7 to 8 weeks, but a wide range has been noted. It has been estimated that about 25% of acute cases may be accompanied by symptoms that, in some cases, may be quite mild. When present, symptoms include fatigue, anorexia, abdominal pain, and weight loss. A proportion (perhaps 20% to 30%) of patients with acute hepatitis C may become jaundiced. Symptoms may last for up to 10 weeks. Alanine aminotransferase (ALT) levels are elevated during the acute phase; such elevations tend to be moderate (i.e., around 300 to 400 IU/L) but may be quite high (up to 2000 IU/L). Acute disease is rarely fatal, with mortality rates of 1% or less. Approximately 20% of acute infections resolve. The remaining 80% (whether symptomatic or not) become chronic. The natural history of chronic HCV infection is not completely understood. Such chronic infections may last for the lifetime of the patient, although there is growing evidence that 26% to 45% of these cases may eventually resolve, as shown by the disappearance of detectable HCV RNA in the circulation and in some instances by the eventual loss of detectable antibody.4 Many infected individuals may remain completely asymptomatic for several years (perhaps even their whole lives), but a good number have histologic evidence of liver disease, including hepatitis, fibrosis, and cirrhosis. In some cases, hepatocellular carcinoma develops. Alter and Seeff 4 have published a framework representing the outcome of HCV infection that is based on a large number of published studies. They conclude that 20% of infected individuals resolve the acute infection. Of the remaining 80% who become chronically infected, 30% demonstrate a
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stable chronic hepatitis with a favorable outcome. Another 30% have severe, progressive hepatitis, and the remaining 40% may have some variable progression. Prospective studies have shown, however, that chronic symptomatic hepatitis may take an average of 10 to 14 years (and, frequently, much longer) to develop, whereas cirrhosis may take an average of 21 years and hepatocellular carcinoma almost 30 years. Although lifethreatening outcomes may be uncommon or considerably delayed, it is still true that the consequences of HCV infection are the leading indicator for liver transplantation in the developed world. From the perspective of transfusion medicine, this pattern of disease has two important consequences. First, although the immediate consequences of post-transfusion hepatitis C may seem to be largely trivial, the long-term outcomes cannot be ignored. Second, the preponderance of asymptomatic, chronic infection leads to a relatively large population of individuals who are at risk of transmitting the infection via blood donation.
TREATMENT In 1997, a National Institutes of Health Consensus Development Conference gave recommendations for treatment of chronic hepatitis C, applicable to individuals with significant histologic findings on biopsy, presence of HCV antibody, elevated ALT, and detectable levels of HCV RNA.5 In brief, such patients should initially be treated with 3 million units of interferon alfa three times a week for 12 months. In the absence of any response (i.e., normalization of ALT and loss of detectable HCV RNA), this therapy should be discontinued after 3 months and the patient should be considered for combination therapy with interferon and ribavirin. Overall, about 50% of patients show temporary improvement with this regimen, but ultimately, only about 25% of all patients show definitive resolution of disease. A subsequent Consensus Development Conference in 2002 updated these recommendations, emphasizing the value of combination therapy. These treatment recommendations have been updated by Liang and colleagues6 on the basis of experience with combination therapies. In fact, a review of two studies revealed that a sustained response to interferon alone was observed in 29% of patients after treatment for either 24 or 48 weeks.6 However, among those receiving combination therapy (interferon plus ribavirin), 33% showed a sustained response at 24 weeks, and 41% showed such a response after 48 weeks of therapy. It was also noted that treatment for more than 24 weeks was unnecessary in patients with HCV subtypes
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EPIDEMIOLOGY Hepatitis C virus is globally distributed and, with a few notable exceptions, the seroprevalence rate is remarkably constant from one region to another, usually on the order of 1% to 2%.10 Although some countries or localities have startlingly high rates, these may be due to cultural factors, including the use of traditional medicine practices; in Egypt, for example, numerous infections were attributed to a program involving injections for control of schistosomiasis.11 In the United States, the overall seroprevalence rate has been estimated at 1.8%, and 65% of persons infected with HCV are age 30 to 49.12 Transmission of HCV is primarily confined to parenteral routes. In the United States, it is clear that many of the currently identified infections are a result of previous exposure via illegal injection of drugs. In addition, many infections are believed to have resulted from blood transfusions given before the availability of sensitive tests.13 Other epidemiologic associations are the use of clotting factor concentrates before the implementation of effective viral inactivation in 1987, exposure in a health care setting, household exposure, multiple sexual partners, and low socioeconomic level.14 There has been considerable speculation about the natural transmission routes of HCV, which have presumably been responsible for maintaining the baseline viral prevalence levels over centuries or even millennia. Some evidence exists for sexual and perinatal transmission, but in both cases, it seems likely that transmission may occur only during early acute infection. HCV infection does not show the strong association with male-to-male sex that has been seen for hepatitis B and human immunodeficiency viruses (HIVs). The incidence of new HCV infections has declined by 80% or more since 1989. Clearly, there has been a major reduction in transfusion transmission of the virus, and the majority of new infections (about 60%) are attributable to injection drug use. Even though incidence is declining, it is anticipated that the burden of HCV disease will continue to rise over the foreseeable future because of the large number of chronically infected individuals.10,15 Studies on the epidemiology of HCV infection among blood donors are of significant importance in the context of strategies for donor selection and questioning. ConryCantilena and associates16 studied a population of 481 blood donors who were found to be reactive in screening tests for anti-HCV. Among the 241 with positive strip immunoassay (SIA) results, 27% had a history of transfusion, 68% had used cocaine intranasally, 42% had a history of intravenous drug use, and 53% reported a history of sexual promiscuity; ear-piercing among men was also significantly associated with a positive test result. Many of these risk factors were confounded. Nonetheless, these observations prompted the
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institution of donor questions regarding intranasal cocaine use; a formal requirement for this question was subsequently eliminated, however. It is of interest to note that, in a study performed in blood donors who tested positive for HCV RNA but negative for HCV antibody, 29.2% reported recent intravenous drug use. A history of recent incarceration was independently associated with nucleic acid amplification test (NAT) positivity.17 The prevalence of HCV infection in current injection drug users is estimated to be 79%, although this rate does appear to be declining.10 Donor history questioning can certainly be improved, but it is unclear how to obtain more accurate information on previous illicit drug exposures, whether they be recent or many years ago. Fortunately, individuals with past histories do not contribute to window period risk.
HEPATITIS C
2 and 3, because of the greater responsiveness of these subtypes to treatment. A number of studies have indicated that pegylated interferon is more effective than the standard product; it is now the preferred version, particularly for treatment of recurrent disease.7,8 Detailed recommendations for diagnosis, management, and treatment of hepatitis C have recently been published by the American Association for the Study of Liver Diseases.9 Current therapeutic approaches result in a sustained response in 42% to 82% of patients with chronic disease, depending on the viral genotype.
VIROLOGY The hepatitis C virus was first recognized in 1989 as the principal etiologic agent of NANBH. A small RNA genome segment was isolated from a presumptive viral pellet prepared from the plasma of an experimentally infected chimpanzee. When the encoded peptide was expressed in bacteria through the use of a lambda phage vector, it was reactive with antibodies present in convalescent serum from a patient with NANBH.18 This RNA fragment, termed 5-1-1, was used as a basis for the eventual sequencing of the entire genome of the virus. The 5-1-1 sequence was also used to develop the capture reagent (the c100-3 peptide) incorporated into the initial version of an HCV antibody enzyme immunoassay (EIA). This peptide represents a portion of the NS3 region of the HCV genome.19 Subsequently, additional peptides have been expressed and incorporated into improved versions of the HCV antibody test. The virus itself is an enveloped, single-strand RNA virus now classified as a separate genus (Hepacivirus) within the Flaviviridae. It has a positive-strand genome of barely less than 10,000 bases. The functional organization of the genome has been well-defined.20 Recently, there has been notable success in establishing a laboratory culture system for the virus, in which a replicative form of the viral genome has been used to infect cells in vitro. Virus produced from such systems has been shown to be infectious for chimpanzees.21,22 These culture systems offer considerable promise for the further study of the virus. Hepatitis C virus is characterized by considerable genetic variability, expressed at three levels: genotype, subtype, and isolate. There are six major genotypes of HCV, designated by the Arabic numerals 1 to 6. The RNA sequence varies between genotypes by some 25% to 35%, and this level of differentiation has probably emerged over periods of 500 to 2000 years. Subtypes within a genotype, designated by lowercase letters (a, b, etc.), represent RNA sequence variation of 15% to 25% that evolved over about 300 years. Individual isolates within a subtype may vary by 5% to 10% in RNA sequence.23–27 Overall, these studies indicate that the frequency of major subtypes in the United States is as follows: 1a, 42.6%; 1b, 29.2%; 2a, 2.7%; 2b, 8.1%; 3a, 4.7%. Additionally, there are hypervariable regions in the genome, and many quasispecies of HCV are likely to develop in an individual patient over time. In general, any variation below the level of subtype is unlikely to be reliable enough to differentiate sources of infection. The distribution of HCV
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subtypes may change with geography. It has been suggested that the clinical outcome of HCV infection and the response to treatment may vary with subtype. Genotypes 2 and 3 are more responsive to treatment than the more common subtypes 1a and 1b.5,9,28,29 The sequence of events after infection with HCV has been well-characterized at the level of viral markers in the circulation. Antibodies may be detected by version 3.0 tests an average of 70 days after exposure, although this period may be quite variable.30,31 However, some 40 to 60 days prior to the appearance of such antibodies, HCV RNA may be detected in the plasma. The HCV RNA levels rise rapidly (over a few days) to 105 to 107 copies/mL. This level is generally maintained at least until significant levels of antibody are expressed. It is now apparent that a soluble viral core antigen is also present and can be detected once the RNA levels exceed about 50,000 copies/mL.32,33 Elevated levels of ALT are frequently observed a few days before antibodies are detectable, but always after the steep rise in RNA levels. Interestingly, there is growing evidence that there may be occasional low levels of viral RNA in the circulation during the early eclipse phase, after infection and prior to the steep rise to peak levels of RNA.5,29 The implementation of widespread NAT for HCV RNA has been shown to detect a meaningful number of RNApositive, anti-HCV–negative donations. Stramer and colleagues34 reviewed the results of the first 3 years of testing in the United States. Among almost 40 million donations tested by the Chiron/Gen-Probe or Roche assays in small pools of plasma samples (16 and 24 samples per pool, respectively), 170 RNA-positive, anti-HCV nonreactive samples were identified. This reflected an overall rate of 1 in every 230,000 donations. Although there was no significant difference in the rates from the two different HCV RNA tests, the rate amongst donations tested by the more sensitive test for anti-HCV was 1 in 270,000. Within the Red Cross system, the rate was 1:251,000 for the first 3 years of HCV NAT, increasing to 1:221,000 in the following 2 years. The increase was not statistically signficant. Overall, of 156 evaluable cases, 51 would have been excluded as donors as a result of other test results, primarily ALT elevations, which occurred among 46 of the 51.34 Thus, in retrospect, ALT testing probably did have a modest impact on blood safety, but at significant cost in terms of blood supply. These data, along with other information showing that donor ALT levels were of little or no demonstrable value for interdicting other supposed transfusion-transmitted hepatitides, led to its elimination for blood component testing. In Stramer’s study, RNA-positive donors were entered into follow-up investigations. It was found that 75 of 90 enrolled donors seroconverted, as detected in samples
Table 44–1
collected a median of 35 days post-donation. The majority of those who failed to seronconvert had only been followed for 12 to 58 days. In a more comprehensive study of 67 RNA-positive donors, 7 failed to complete follow up, 2 had abortive infections, and 3 did not seroconvert after periods of 1.5 to 3 years. It is of interest to note that the median time to observed follow-up is approximately 50% of the supposed window period, although 35 days is probably something of an overestimate because of the sampling intervals in the study.34 Once antibodies are detectable, RNA levels may decrease or become variable. In the evaluation of samples from blood donors, HCV RNA is detected in about 80% of samples that are reactive on a version 3.0 EIA and confirmed reactive on the version 3.0 SIA.35 The frequency of RNA detection declines with the strength of the antibody response (as defined by the number and intensity of bands on the SIA).36 It seems likely that the antibody-positive, RNA-negative samples actually represent resolved infections. In some cases, antibodies may eventually decline to undetectable levels. There is a vigorous humoral and cytotoxic immune response to HCV, but in most cases, it is clearly unable to eliminate the virus. The mechanisms underlying the ability of the virus to escape the immune response are not well understood. The infecting virus often generates a wide variety of quasispecies; further, the representation of these variant forms changes rapidly over time.20,37 Yet it appears that these changes may contribute only in part to viral persistence.
SEROLOGIC TESTS FOR HCV INFECTION Two licensed EIA tests for HCV antibodies are currently available in the United States. Although both use recombinant viral antigens as the solid-phase capture reagent, they differ in their physical format and in the number and nature of viral antigens used (Table 44–1). The test manufactured by Ortho Clinical Diagnostics uses a microplate solid phase and has been designated as a 3.0 version by the manufacturer. The peptides that are coated onto the microplate well are known as c22-3 from the C (core) region, c200 from the NS3NS4 regions, and an NS-5 peptide. The test manufactured by Abbott Laboratories uses a polystyrene bead solid phase and is designated as a version 2.0 test. The capture reagents for this test are c22-3, and c33 and c100-3 (both from the NS3/ NS4 region). Both test procedures use an antiglobulin conjugate to detect the analyte antibodies. The performance characteristics of the tests, representing the manufacturers’ claims (see Table 44–1), have been validated by extensive clinical trials, and the tests have been licensed as biologics by the U.S. Food and Drug Administration (FDA). Other tests on fully
Enzyme Immunoassay Tests for Anti-HCV: Components and Performance Characteristics
Version of Test (manufacturer)
Peptides
Sensitivity*
Specificity*
1.0 (Ortho Clinical Diagnostics) 2.0 (Abbott Laboratories) 3.0 (Ortho Clinical Diagnostics)
C100 HC34 (c22), HC31 (c33), c100 c22, c200‡, NS-5
81% 85.7%† 88.1%†
>99.4% 99.83% 99.95%
*
Based on manufacturers’ claims in product inserts. Based on a diagnosis of chronic non-A, non-B hepatitis—ALT elevated >6 months, HBsAg negative. ‡ Includes c33 and c100 sequences. †
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●
Positive: Two or more bands with an intensity equal to, or greater than, that of the weak positive control band, plus nonreactive SOD band.
●
●
Negative: No band with a greater intensity than the weak positive control. Indeterminate: Only one band reactive or any pattern in association with a reactive SOD band.
HEPATITIS C
automated platforms are available elsewhere in the world and are soon expected to be licensed in the United States. Most diagnostic reagents are defined as devices, but those that are used in the preparation of blood and blood products are required to meet the more stringent biologics requirements, because blood itself is defined as a biologic. The sensitivity of these tests was defined in trials using patients diagnosed with NANBH. Because this diagnosis is not specific, the significance of the sensitivity figures is unclear. Of more importance now, at least in the context of blood donor screening, is the ability of the test method to detect infection at the earliest possible time during the seroconversion period. Studies of seroconversion panels suggest that currently available tests detect antibodies, on average, 70 to 80 days after exposure to the source of infection and 40 to 60 days after the initial detection of HCV RNA in the plasma. In addition, it should be noted that earlier versions of the EIA tests clearly failed to detect some infected individuals.38 This is not surprising, because only a very limited number of viral epitopes were included in the capture reagent. Current tests for antiHCV are whole-antibody assays. No tests are yet available for specific detection of immunoglobulin M anti-HCV. Although these EIA tests have high sensitivity and specificity, there is a possibility that some reactive test results are nonspecific. When the EIA is used to screen blood donors, its actual positive predictive value is 70% to 80%.36 Consequently, it is recommended that all asymptomatic individuals who test as reactive on EIA repeatedly should be further tested with an additional, more specific procedure. Currently, only one such immunologic method is licensed and available in the United States. This is a strip immunoblot assay (RIBA 3.0) that is constructed by application of recombinant or synthetic viral peptides representing c22, c33, c100, 5-1-1, and NS5 regions to nitrocellulose strips in a fixed pattern. The c-100 and 5-1-1 peptides are present in the same band on the strip. The expression carrier protein for the recombinant viral antigens is also applied (superoxide dismutase, SOD), as are strong and weak positive controls. Patient or donor samples are added to the strips and, after washing, adherent antibodies are detected by an appropriate enzyme-conjugated antiglobulin and visualization reaction. The number and intensity of bands are scored, and the result is interpreted as follows:
Table 44–2 summarizes the results of testing a large number of volunteer blood donations with HCV EIA and SIAs. Unlike the situation with HIV, there does not seem to be a common pattern for the sequence of appearance of reactive bands in the blot during seroconversion with HCV. However, a number of studies have defined the relationship between particular bands, or band patterns, and the likelihood that a sample will also contain detectable HCV RNA. For example, Dow and colleagues35 reviewed data from 177 blood donor specimens that were reactive on EIA and tested positive on the version 3.0 SIA. Among 82 samples with four positive bands, 69 (84.1%) were RNA-positive. Of the 54 samples with three positive bands, 40 (74.1%) were RNA-positive, whereas only 14 (34.1%) of the 41 samples with two positive bands were RNApositive. Among the samples with indeterminate SIA results, the frequencies of RNA-positive results were 3 of 154 for c22, 1 of 220 for c33, 1 of 191 for c100, and 0 of 380 for NS5.35 Thus, a few of the indeterminate patterns may be associated with the presence of RNA and, therefore, of active HCV infection. Similar data were published by Dodd and Stramer.36
TESTS FOR HCV RNA Tests for HCV RNA serve an important role in diagnosis and patient management.9,39 A variety of procedures is available, all of which depend on nucleic acid amplification, with one exception. The reverse transcriptase polymerase chain reaction (RT-PCR) is perhaps the most familiar. Viral RNA is reverse-transcribed to DNA, and two primers are used to define a sequence for repetitive amplification using a temperature-resistant DNA polymerase and a temperature-cycling protocol. A variety of methods may be used to detect the resulting amplified sequence, including visualization in gels, hybridization with labeled probes, and detection of amplicons in real time. Both qualitative and quantitative procedures are available.5,40,41 In most cases, a conserved segment of the 5′ untranslated region of the genome is selected for amplification. PCR-based assays are available commercially (e.g., Roche Molecular Systems) as well as from independent reference laboratories; alternatively, they may be developed in-house with the use of standard technologies.
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Table 44–2 Results of Testing 19.2 Million Blood Donations with HCV 3.0 EIA and Version 3.0 SIA and Percentage of RNA-Positive Samples among Subgroups of SIA-Positive Subjects Number of Subjects with SIA Finding (% RNA+)
HCV EIA RR N = 30,680 SIA >2 bands 2 bands only
Positive (% RNA+)
Indeterminate
Negative
19,541
4898
6241
17,139 (82%)* 2402 (42%)†
NA NA
NA NA
*
Sample of 200 tested. 2347 tested. EIA, enzyme immunoassay; HCV, hepatitis C virus; NA, not applicable; RR, repeatedly reactive; SIA, Strip immunoblot assay. Data courtesy of Susan L. Stramer, Ph.D.(personal communication).
†
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Another technique, known as nucleic acid sequence– based amplification (NASBA; a proprietary technology from Organon-Technika),42 and the very similar transcriptionmediated amplification (TMA; a proprietary technology from Gen-Probe Inc.) can be performed without temperature cycling. Two enzymes are used to produce an RNA amplicon, which can be detected by a variety of methods, including the hybridization protection assay. It should be noted that Chiron owns patent rights to the HCV genomic sequence used for amplification. Sample collection, stability, and preparation for amplification are all important and must be properly controlled. HCV RNA is quite labile, and it is preferable, if not essential, to collect specimens in EDTA. Samples should be maintained at refrigerated temperatures and tested with minimal delay. A number of different methods may be used to prepare the RNA for testing, including conventional extraction from ultracentrifugal pellets, extraction on silica, and probecapture techniques. Finally, in a method known as the branched-chain DNA assay (B-DNA), a probe is labeled with a large branched DNA molecule that carries many copies of the detection label. This method does not amplify the target nucleic acid; rather, it provides a system in which numerous label molecules may be associated with a single target sequence. As might be expected, this technique is not as sensitive as amplification. However, the lack of sensitivity can be exploited to differentiate those patients with high levels of circulating RNA.43,44 Within the United States, almost all blood donations have been tested for HCV RNA since 1999.34 Two methods are in use, RT-PCR(Roche) and TMA (Chiron/Gen-Probe). To date, all such testing has been performed on small pools of plasma samples, with current pool sizes of 24 for the Roche procedure and 16 for the Chiron/Gen-Probe method. Both tests have been licensed for routine use by the FDA and both have an analytical sensitivity of 12 or fewer copies of RNA/ mL by probit analysis and a 95% detection level of 30 to 60 copies/mL. The overall sensitivity of the testing is, of course, proportionately reduced in pooled testing. A number of methods are available for the determination of genotype and subtype. Not all methods are able to discriminate every genotype or subtype, however. Available methods may be broadly separated into those that depend on immunologic differentiation and those that directly detect variation in nucleic acid sequences. In the former case, specific peptides derived from the NS4 region are used to probe for antibodies in the specimen. Nucleic acid–based techniques include sequencing amplicons from selected genomic areas, PCR using genomespecific primers, DNA-enzyme immunoassay (DEIA), restriction fragment length polymorphism (RFLP) analysis of amplicons, and differential hybridization of amplicons with specific probes. Two of these approaches are commercially available (GEN-ETI-K DEIA kit and INNO-LiPA HCVI and HCV II). The reader is referred elsewhere for further details.23,45 Relatively little information is available about the impact of HCV genotype on the sensitivity of diagnostic tests, and some data suggest little variation in the analytic sensitivity of NAT.46 However, a recent publication reviewing the performance characteristics of numerous blood screening assays for HCV antibodies suggests that most tests are not seriously impacted by genotype variations (El-Nageh, in press).
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DIAGNOSTIC ALGORITHM The Centers for Disease Control and Prevention (CDC) has published an algorithm for diagnostic testing for HCV among asymptomatic individuals (Fig. 44–1).14 This algorithm is somewhat different from that recommended for blood donor screening, in that it explicitly permits the use of NAT to confirm a repeatedly reactive EIA result. However, NAT-negative samples must be further evaluated by an SIA. This algorithm also provides useful guidance in the context of treatment, but a more comprehensive guide to treatment was published in 2004.9 Given that blood donors are now routinely evaluated by NAT for HCV RNA, it is hoped that these results may be incorporated into the notification and management of seropositive donors.37,45
IMPACT OF BLOOD DONOR SCREENING AND TESTING FOR HCV Results of Testing In 2001, the frequency of positive results among firsttime blood donations as defined by RIBA was 0.3%, which is about one fifth of the national prevalence rate of 1800 per 100,000. The incidence of new infections in the donor population is 1.9 new infections per 100,000 person-years; the corresponding national incidence figure is 13.4 per 100,000 person-years.47 Thus, the donor rate is about one fifth of the national rate. These differences are attributable, at least in part, to the procedures used to recruit safer populations for donation and to the measures used to question presenting donors about their medical and behavioral histories. However, it should be
EIA for anti-HCV Positive (repeatedly reactive)
Negative
Negative
Indeterminate
Positive
Additional laboratory evaluation
Stop
OR
RIBA™ for anti-HCV
Stop
Negative (nonreactive)
RT-PCR for HCV RNA Positive
Medical evaluation
(e.g., RT-PCR, ALT)
Negative RT-PCR and normal ALT
Positive RT-PCR or abnormal ALT ALT
Stop
Alanine aminotransferase
Anti-HCV Antibody to HCV EIA
Enzyme immunoassay
RIBA™
Recombinant immunoblot assay
RT-PCR
Reverse transcriptase polymerase chain reaction
Figure 44–1 Algorithm for hepatitis C virus (HCV) testing among asymptomatic individuals. (From Centers for Disease Control and Prevention: Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR Morb Mortal Wkly Rep 1998;487[RR-19]:1–39.)
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Risk of Post-transfusion HCV Infection It is clear from a number of published studies that donor screening and testing measures have had a profound impact on the incidence of post-transfusion hepatitis C. Indeed, prospective studies have not demonstrated any infections since the implementation of the so-called multi-antigen tests for antibodies to HCV. But there is also substantial evidence for the efficacy of a variety of screening approaches used even before this time. The essentially complete elimination of commercial donation of whole blood had a major effect on reducing the frequency of post-transfusion hepatitis. It is also believed that a further reduction was seen as a result of more stringent donor questioning to reduce the risk of transmission of HIV and acquired immunodeficiency syndrome (AIDS), although subsequent information implies that the major effect would have come from a reduction in the number of injection drug users. The first study to clearly demonstrate the impact of testing measures on hepatitis C infection was published by Nelson and colleagues.49 They evaluated samples from a large population of patients undergoing cardiac surgery, using the first-generation test for anti-HCV. These researchers found that the risk of infection was 0.45% per unit prior to the implementation of any testing. After implementation of testing for ALT and anti-HBc, the rate of infection dropped to 0.19%. Finally, once the version 1.0 EIA test was implemented, the rate dropped to 1 per 3300 units (0.03%), a reduction of 84%.49 A subsequent reevaluation using the more sensitive version 2.0 test on the blood recipients suggested that the actual risk was closer to 1:1700.50 Once the version 2.0 test was introduced for blood donor screening, however, the frequency of residual infection declined profoundly. As pointed out previously, cases of hepatitis C were no longer observed in prospective studies, and risk estimates then had to be developed on the basis of the length of the window period (as determined from post-transfusion infections) and the incidence of new infections within the donor population. In a landmark publication in 1996, Schreiber and colleagues estimated the residual risk of HCV infection at 1 per 103,000 donations, based on a window period of 82 days and an incidence rate of 4.84 per 100,000 personyears.51 Subsequent evaluations account for a 12- to 13-day reduction in the window period attendant on the implementation of the version 3.0 EIA and an overall decline in the incidence of HCV infection to 2.09 per 100,000 person-years. The latter figure translated to a residual risk of 1:276,000 repeat donations. The addition of HCV NAT was estimated to markedly reduce this risk to 1:1.935 million repeat donations. In the same paper, it was observed, on the basis of the results of NAT, that the incidence of HCV infections was 2.4 times higher among first-time donors.52 Allowing for a 23% frequency of first-time donors in the Red Cross system, this translated to an overall risk of 1:1.39 million donations.3 The relative stability of the detection rates for HCV RNA and the lack of change in prevalence
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rates for HCV antibody53 suggest that this risk is not changing significantly, at least through the end of 2003. It is interesting to speculate about the higher incidence of infections among first-time donors, but there are no definitive data. One possibility is that there are more testseekers among people presenting to donate for the first time, although this is not necessarily supported by published data on interviews of RNA NAT-positive donors, because both first-time and repeat donors were found to be positive and to acknowledge risk factors for infection.17 It is also possible that the process of giving blood has an educational impact and that individuals who learn about risk factors for bloodborne infections defer themselves from future donation. Finally, it should be noted that there are cases in which HCV has been transmitted by blood units that have been fully tested for HCV, even after the implementation of NAT.54 Such cases have usually been detected as a result of lookback and are too few to offer any quantitative estimate of residual risk. Window-period theory would strongly suggest that such cases would occur, and this is supported by the detection of HCV RNA in window-period samples, using ultrasensitive test methods. It is of interest to speculate on the minimal infectious dose of HCV. Busch has developed models based on the conservative assumption that it is as low as one genome equivalent per 20 mL of plasma. Using this assumption and back-calculating, the theoretical window period from the observed doubling rate of HCV plasma RNA during early infection has led to estimates that are largely compatible with currently accepted window period estimates.55
HEPATITIS C
noted that only a very few donors are actually deferred on the basis of their response to risk questions, in part because donors do not always provide complete answers48 but also perhaps because potential donors make a conscious decision to avoid giving blood so as not to have to answer the questions.
LOOKBACK 44
A positive test result for HCV antibodies does not provide any information about the duration of infection, even if accompanied by a positive finding for HCV RNA. Consequently, if a blood donor is found to be HCV antibody positive, it is possible that prior donations from that individual were infectious for HCV. This could occur by two broad mechanisms. First, a previous donation could have been made in the infectious but seronegative window period. Second, the prior donations could have been collected at a time when a less sensitive test was in use or even before testing was initiated. Accordingly, a focused lookback program has been initiated to locate, notify, test, and, if appropriate, treat recipients of such potentially infectious prior donations. Studies in Canada and elsewhere suggest that up to 70% of such recipients may indeed have been infected.56,57 However, in the United States, the effort appears to have been less productive. A team from the CDC reported that an estimated 98,484 blood components were identified as potentially infectious.58 Of these, 85% had been transfused. This interim study found that lookback had been completed for 80% of the transfused products; 69% of the recipients had died. Of those living, 78% were successfully notified that they had received a potentially infectious blood component. It was estimated that, of recipients notified, 49.5% were tested for anti-HCV; of those, 18.9% were seropositive, but 32% of these individuals were already aware that they were seropositive. Thus, at the time of publication of the study, it was estimated just over 1000 individuals were newly notified of unexpected HCV infection; this estimate translated to a national figure of 1520, on the
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assumption that the lookback process was to be completed. The figure represents fewer than 1% of all individuals who may have been infected as a result of transfusion.58 It should be noted that this component of the lookback program was restricted only to donations that were identified as a result of testing with multi-antigen tests (i.e., versions 2.0 or 3.0). It is likely that, as lookback is extended to cover donors initially identified by the version 1.0 test, the proportional yield will be greater, but the efficiency of the process will certainly be affected by availability of required records. On the other hand, the essential completion of achievable lookback affecting recipients of blood donated prior to the implementation of testing means that almost all available cases now represent lookback from incident cases of HCV infection. The yield of this aspect of lookback is now extremely low, and such yield will be further reduced as the impact of NAT is manifested. The ethical imperatives for lookback continue, but the public health benefits of this process are becoming vanishingly small.
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In its way, the history of hepatitis C has been as remarkable as that of HIV and AIDS. In retrospect, hepatitis C was the blood-borne agent most commonly transmitted by transfusion in the United States, right up until the early 1990s. The legacy of this problem will be seen for many years, as the long-term health consequences of HCV infection become manifest. The almost complete elimination of the problem of transfusion-transmitted HCV is the result of many years of dedicated study of an intractable problem. There were no simple solutions; the virus was refractory to laboratory study, and there was a truly frustrating inability to identify any serologic markers of infection. Indeed, only at the very end of the 20th century was a naturally occurring, circulating viral antigen recognized and then only in the few weeks preceding seroconversion. As with HIV, the first laboratory approach to abrogating transfusion-transmitted HCV infection was the development of serologic tests. In the case of HCV, however, this development was achieved through the combination of years of study of the disease, development of animal models, and the painstaking application of new recombinant technology. It is fitting that Harvey Alter and Michael Houghton received the 2000 Lasker award for this work, but hundreds of others contributed over many years. Serologic and nucleic acid testing have essentially eliminated the risk of transfusion-transmitted HCV. Plasma derivatives are prepared from highly tested starting material and are further treated with advanced inactivation procedures. It is of interest to note, however, that whereas the early tests removed a substantial fraction of antibody-positive units, they failed to identify all infectious units, probably leading to the unexpected occurrence of HCV in recipients of some immunoglobulin products. Despite the success of testing for HCV, there continue to be barriers to other aspects of management of this virus. As with many viral diseases, treatment options for HCV are limited and incomplete. More baffling, however, are the difficulties inherent in understanding and manipulating the interactions between the immune system and HCV. Development of an effective vaccine seems to be a particularly elusive goal.
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30. Busch MP, Korelitz JJ, Kleinman SH, et al. Declining value of alanine aminotransferase in screening of blood donors to prevent posttransfusion hepatitis B and C virus infection. Transfusion 1995;35:903–910. 31. Busch MP. HIV, HBV and HCV: New developments related to transfusion safety. Vox Sang 2000;78:253–256. 32. Couroucé AM, Le Marrec N, Bouchardeau F, et al. Efficacy of HCV core antigen detection during the preseroconversion period. Transfusion 2000;40:1198–1202. 33. Tanaka E, Ohue C, Aoyagi K, et al. Evaluation of a new enzyme immunoassay for hepatitis C virus (HCV) core antigen with clinical sensitivity approximating that of genomic amplification of HCV RNA. Hepatology 2000;32:388–393. 34. Stramer SL, Glynn SA, Kleinman SH, et al. Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid-amplification testing. NEJM 2004;351:760–768. 35. Dow BC, Buchanan I, Munro H, et al. Relevance of RIBA-3 supplementary test to HCV PCR positivity and genotypes for HCV confirmation of blood donors. J Med Virol 1996;49:132–136. 36. Dodd RY, Stramer SL. Indeterminate results in blood donor testing: What you don’t know can hurt you. Transfus Med Rev 2000;14:151–160. 37. Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 2000;288: 339–344. 38. Alter MJ, Margolis HS, Krawczynski K, et al. The natural history of communityacquired hepatitis C in the United States. NEJM 1992;327: 1899–1905. 39. Gretch DR, Dela Rosa C, Carithers RL Jr, et al. Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications. Ann Intern Med 1995;123:321–329. 40. Lunel F, Mariotti M, Cresta P, et al. Comparative study of conventional and novel strategies for the detection of hepatitis C virus RNA in serum: Amplicor, branched-DNA, NASBA and in-house PCR. J Virol Methods 1995;54:159–171. 41. Hawkins A, Davidson F, Simmonds P. Comparison of plasma virus loads among individuals infected with hepatitis C virus (HCV) genotypes 1, 2, and 3 by Quantiplex HCV RNA assay versions 1 and 2, Roche monitor assay, and an in-house limiting dilution method. J Clin Microbiol 1997;35:187–192. 42. Damen M, Sillekens P, Sjerps M, et al. Stability of hepatitis C virus RNA during specimen handling and storage prior to NASBA amplification. J Virol Methods 1998;72:175–184. 43. Sangiovanni A, Morales R, Spinzi GC, et al. Interferon alfa treatment of HCV RNA carriers with persistently normal transaminase levels: a pilot randomized controlled study. Hepatology 1998;27:853–856.
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