Our evolving understanding of the immunology of the hepatitis B virus (HBV) has led to the development of safe and effective therapies for preexposure and postexposure virus-specific prophylaxis. Perinatal transmission of HBV from mothers with chronic infection to their at-risk neonates remains a significant route for the perpetuation of the HBV carrier state, with its concomitant health risks, worldwide. This section outlines the evidence supporting antenatal identification of HBV-carrier mothers and targeted HBV immunoprophylaxis in their newborn children. Widespread adoption of such approaches, combined with ongoing HBV vaccination protocols in high-risk populations, including medical personnel, will make significant inroads against the overall prevalence of HBV-related disease.

Biology and Serology

Although an exhaustive discussion of HBV biology is beyond the scope of this chapter, an understanding of the basic structures and serologic tests associated with the virus is essential to understanding the logistics of perinatal transmission and prevention.

HBV is a small (42-nm) DNA virus that contains partially double-stranded DNA within its core.¹ Using its own DNA polymerase for replication, the virus is able to reproduce within a host's infected hepatocytes, drawing from the cell's pool of nucleotide precursors.

Much attention has been paid to the use of HBV-specific markers in serum to distinguish active from previous infection and to determine the relative infectiousness of a particular individual. Not surprisingly, these concerns are directly applicable to determining relative risks for vertical (maternal–fetal) transmission of the virus.

Hepatitis B surface antigen (HBsAg) is the HBV serum marker that has come to be used most commonly in clinical situations and screening protocols. Discovered by Blumberg and co-workers in 1965,² it initially was not known to be a virus-associated marker. The antigen, first isolated in the serum of an Australian aborigine during a study of serum protein polymorphisms (hence its being labeled the “Australia antigen”),³ was found incidentally to cross-react with the serum of multiply transfused patients. Later found to be present in the serum of institutionalized patients, it was even believed to be possibly associated with Down syndrome.⁴ Subsequent work by Blumberg's group and others established a link between the newly identified antigen and acute hepatitis B, an association confirmed by electron microscopy identification of particles dense with the antigen in the serum of patients who were acutely ill with hepatitis.⁵ Those particles now are known to represent incomplete portions of the viral envelope, synthesized in great excess during the process of virus replication. In addition, intact viral particles bear the surface antigen on their outer envelope. The presence of HBsAg in serum indicates infectivity, although such presence alone cannot distinguish acute from chronic infection, an often confusing exercise that requires a more complete elaboration of HBV-related serologies.

Although HBsAg is the first antigen detectable in the course of HBV infections, predating even the appearance of symptoms in those patients who become clinically ill, it is the predictable appearance and disappearance of other HBV-associated antigens and antibodies over time that allows patterns compatible with either acute or chronic infection to be described. Currently, six distinct antigens and antibodies are assayable in clinical specimens through the use of commercially available test kits and machinery. The agar gel precipitation techniques first used by Blumberg's group to demonstrate HBsAg antigen–antibody complex formation has given way to fully automated, multisample readers that capitalize on advances in molecular biology to detect the presence or absence of the specific immunogens in question.

The complete hepatitis B viral particle, also known as the Dane particle, after Dane and co-workers who described it in 1970,⁶ consists of the viral core surrounded by its HBsAg-rich envelope (Fig. 1). If the envelope is removed by the use of detergents in vitro, a viral core antigen (HBcAg) can be identified. Unlike HBsAg, HBcAg does not circulate free in serum and is found in blood only as an integral component of the internal viral nucleocapsid. A third antigen, the e antigen (HBeAg), is serologically distinct from both HBsAg and HBcAg. HBeAg is associated primarily with the core antigen in the virus's internal structure, but unlike HBcAg, it can be found circulating in serum, frequently in complexes with immunoglobulin.⁷ All three serologically unique antigens stimulate the production of equally distinct antibodies (HBsAb, HBcAb, and HBeAb) in the course of nonchronic host infection.

Also located within the viral core are the viral DNA and DNA polymerase. The presence of HBeAg has been closely correlated with both the infectivity of a particular patient's serum and the microscopic detection in serum of the HBV virus itself^8,9 and an increased risk for chronic liver disease.^10,11 Seropositivity for HBeAg should be taken as a marker of active viral replication, the most infectious phase of the disease, either in acute or in chronic illness. Practically, however, HBsAg is used in screening protocols because of the high concentrations of this antigen produced in response to viral presence and replication. More vigorous HBV serologic testing is performed, along with liver function evaluation, in HBsAg-positive individuals, both symptomatic and symptom-free, to characterize the nature and extent of their disease. The appearance of HBsAb in the serum of patients occurs in the setting of resolution of acute infection; it is this antibody that appears to confer protective immunity. However, both HBcAb and HBeAb have been shown experimentally to be protective against reinfection.^12,13,14,15 Still, the currently available HBV vaccine's efficacy is conferred by stimulating production of HBsAb by exposure to HBsAg; vaccine-related immunity can be distinguished from natural immunity in most cases by the absence of HBcAb in the serum of successfully vaccinated patients.¹⁶

Epidemiology, Transmission, and Prevention

Infection with HBV has been accepted as a health concern of worldwide importance, because 5% to 10% of those infected become chronic HBV carriers,¹⁷ and 25% to 30% of those carriers die as a result of long-term sequelae of HBV-related disease.¹⁸ Workers in an endemic area in Asia (Taiwan) found more than 15% of subjects screened in a general program to be HBsAg-positive; deaths in 54% of the carriers were attributable to primary hepatocellular carcinoma (PHC) and cirrhosis compared with 1.5% of deaths among noncarriers.¹⁹ Since that initial linkage, the virus has been shown to be the cause of approximately 80% of all cases of PHC globally.²⁰

In areas endemic for HBV, up to 20% of the general population is chronically infected, with perinatal/neonatal and childhood infections existing as a primary route for expanding the reservoir of carriers (Table 1). This is especially significant because the risk of chronic HBV infection for a child infected in the newborn period in the absence of prophylactic therapy is 70% to 90%.^21,22

Table 1. Global Distribution of Hepatitis B Virus (HBV) Infection

In areas of low endemicity for HBV carriage, however, such as the United States, screening programs for the general population have been targeted to decrease household, transfusion, sexual, and perinatal transmission risks among contacts of HBsAg-positive individuals. Population subsets have been identified that are at increased risk for HBV acquisition, and HBV vaccination is recommended for individuals within those groups who are serologically negative for HBsAg and HBsAb. The efficacy of a serum-derived HBV vaccine was demonstrated initially on a large scale in a cohort of more than 1000 homosexual men in the United States; this trial showed an antibody (HBsAb) response in 96% of those vaccinated, with an overall protective efficacy of 88% against all HBsAg-positive events for vaccine compared with placebo.²³ More recently, a recombinant vaccine consisting of purified HBsAg particles derived from yeast cells was licensed in the United States,²⁴eliminating even the theoretical (but never proven) risk of transmitting other viral agents with a serum-based vaccine.²⁵ Controlled trials in homosexual men showed an equivalent prevalence of acquired immune deficiency syndrome (AIDS) in groups receiving either the placebo or the serum-derived HBV vaccine.²⁶

Estimates in the United States tabulate approximately 200,000 new primary cases of HBV infection per year, only 25% of which are associated with acute symptomatic infection.²⁷ The dose of initial viral infection appears to correlate negatively with the risk of development of persistent disease. Survivors of fulminant hepatitis rarely have chronic infection, whereas experimental infections with low doses of virions result in longer incubation periods, milder clinical disease, and persistent antigenemia.²⁸

Blood and blood products are the most thoroughly established sources of hepatitis B infection, although HBsAg has been demonstrated in a variety of body fluids. Of those, however, only serum, saliva, and semen have been associated consistently with transmission in experimental models.^29,30,31 Despite the presence of HBsAg in feces, past attempts to produce infection using feces of experimentally infected subjects were unsuccessful, suggesting blood from the gastrointestinal tract to be the uncommon infectious vector present in feces of carrier individuals.³²

Percutaneous transfer of the virus is the most obvious route of transmission in the medical setting, either through blood products or through needle-stick accidents. Contact of infectious material with broken skin or mucous membranes also can result in effective transmission. Recent surveys, however, show that approximately 50% of health care workers at risk for contracting HBV have not been vaccinated against the virus.³³

Compared with other transmissible viruses, such as the human immunodeficiency virus (HIV), HBV is a fairly stable virus and remains infectious on household surfaces that may then contact mucous membranes, such as toothbrushes, baby bottles, razors, and eating utensils.^34,35 Although transmission in households is more common through sexual contact than through fomite contact,^36,37 nonsexual household transmission has been established as a route for HBV infection.^38,39,40 In areas of the world with higher HBV carrier rates than the United States, nonparenteral transmission would be expected to constitute the major route of person-to-person HBV infection. Vertical transmission is a major source; investigators in Taiwan estimated that 40% to 50% of HBsAg carriers became infected in the perinatal period.^21,41

Children born to carrier mothers who escape the neonatal period without evidence of infection are still at risk for childhood acquisition of HBV. One of the early vaccine trials conducted in Senegal showed that among children seronegative at the beginning of a randomized HBV vaccination trial, almost 10% acquired HBV infection in the absence of vaccination by the end of a 12-month follow-up period.⁴²

Immunoprophylaxis regimens to prevent HBV transmission in the perinatal period, to be discussed later in the chapter, were a direct extension of the success of these therapies in high-risk adult populations. Postexposure immunization was first demonstrated through the use of immunoglobulin preparations with high titers of HBsAb, when administered within 4 hours of experimental infection with HBV.⁴³ Before the development of an effective HBV-specific vaccine, transient preexposure prophylaxis was demonstrated using hyperimmune globulin (HBIG),^44,45 although such use of HBIG is now of purely historical interest in terms of understanding the evolution of therapeutic standards. Currently, postexposure treatment consists of a single dose of HBIG administered as temporally as possible to the exposure. Immediate therapy is, of course, optimal for maximal protection, although 75% efficacy has been shown when HBIG is administered within 7 days of exposure.⁴⁶ Although it does not increase the efficacy of HBIG therapy, a series of HBV vaccination also should be initiated if the exposure was within a setting of ongoing risk, such as a health care or institutional setting. This regimen consists of injections at 0, 1, and 6 months and results in high antibody titers in more than 90% of those younger than age 60 years.^47,48,49 Administration of HBV vaccine simultaneously with HBIG does not diminish the immunologic response to the vaccine.^50,51

Clinical Disease and Pregnancy

Hepatitis B is distinguished from the other viral hepatitides by its long incubation period (1–6 months), by the presence of extrahepatic symptoms in up to 20% of patients (arthralgia, rash, and myalgia thought to be a result of antigen–antibody complex deposition),^52,53 and, eventually, by the detection of HBV-specific serum markers (Fig. 2). The appearance of HBsAg usually predates any clinical symptoms by 4 weeks, on average, and remains detectable for 1 to 6 weeks in most patients.⁵⁴ In the 90% to 95% of patients in whom chronic infection does not develop, HBsAg titers decrease as symptoms diminish. The appearance of HBsAb defines the absence of the carrier state; titers increase slowly during the clinical recovery period and may continue to increase up to 10 to 12 months after HBsAg is no longer detectable. In most patients with self-limited, acute hepatitis B, HBsAb is detectable only after HBsAg titers in serum disappear.^55,56 A “window” of time has been described in which a patient still with clinical hepatitis is negative for both HBsAg and HBsAb. During this time, HBV infection still can be diagnosed by the detection of HBcAb, which begins to appear 3 to 5 weeks after HBsAg does. HBcAb titers may decrease in the first 1 to 2 years after infection, although the antibody is still detectable years after acute disease in most patients.⁵⁵ The appearance of HBeAg parallels that of HBsAg; in self-limited infections, HBeAb is detectable soon after the time that HBeAg disappears.

The chronic HBV carrier state usually can be predicted by HBsAg seropositivity for 20 weeks or more (Fig. 3). A test for HBV–DNA polymerase activity is positive in 50% of persistently HBsAg-positive patients, indicating ongoing viral replication⁵⁷; Dane particles also can be identified in serum from these patients through electron microscopy.⁵⁸ HBcAb is detectable in the serum of carriers at levels higher than those seen in either acute or recovering self-limited infections, and e-antigen markers are variable. HBeAb develops, with the disappearance of HBeAg, in one half to three quarters of carriers,^59,60 and its presence is inversely related to the relative infectivity index of a patient's serum.⁶¹

Acute HBV infection during pregnancy is treated mainly by supportive measures, as in the nonpregnant state. An increase in fulminance and mortality rates with acute HBV infection during pregnancy has been demonstrated in some HBV-endemic areas,^62,63 although other investigators in Western countries have suggested that these adverse outcomes were related more to health care conditions and maternal malnutrition.^64,65 No teratogenic association has been established for maternal HBV infections,^64,66 even though evidence of HBV infection at birth in children of HBV-carrier women have suggested the possibility of transplacental leakage of HBV-infected blood from mother to fetus in utero.^67,68,69 Encouragement is necessary to maintain adequate nutrition during the early symptomatic phase, and liver-metabolized drugs, if not avoidable, need to be monitored carefully through blood levels. Phenothiazine may be used, if needed, to control nausea and vomiting. In addition, household and sexual contacts of patients should be offered passive immunization with HBIG after their HBsAg seronegativity is established.

Universal screening protocols for prenatal patients have been advocated by a number of groups and are discussed more fully in the next section. Routine screening with HBsAg testing detects both chronic carriers and asymptomatic, acutely infected patients. A positive HBsAg result in early pregnancy should be followed-up by tests for liver function, as well as HBeAg and HBeAb; HBcAb is not helpful in distinguishing acute from chronic disease, and HBsAb rarely is present if HBsAg is still circulating. Repeating the tests for HBsAg and liver function later in pregnancy, however, does help to make the diagnosis and guide the need for perinatal prophylaxis of the neonate. Although a recent multicenter study indicated that treatment of chronic HBV carriers with alfa-interferon was effective in achieving remission, both biochemically and histologically, in one third of patients,⁷⁰ such therapy cannot be recommended in pregnant HBV carriers. No information currently exists to guide the use of genetic prenatal diagnostic procedures in HBsAg-positive women and the potential risk of producing in utero infection, although studies have demonstrated that such infection is possible in the face of preterm labor or placental abruption.^67,68,69 HBIG, administered periprocedurally to HBsAg-positive women, may protect the fetus from infection in a manner comparable with the use of Rh-immune globulin in Rh-negative women, but again, such statements are purely speculative and await further investigation before they take the shape of recommendations.

Perinatal Hepatitis B Virus Transmission

Discussion of perinatal HBV infection focuses on the following three major areas: (1) the transmissibility of the virus from mother to fetus; (2) the sequelae of neonatal infection; and (3) the effectiveness of currently available modalities for prophylaxis.

Finally, the extensive variance in prevalence rates worldwide requires that the feasibility of prenatal screening programs to identify carrier mothers be addressed.

The potential for vertical transmission of HBV at birth is significant. Most infants born to carrier mothers are HBsAg-negative at birth experience seroconversion in the first 3 months after delivery, suggesting acquisition of the virus at birth.^71,72,73,74 Mothers positive for both HBsAg and HBeAg are at highest risk for transmitting the virus; 85% to 100% of their offspring become infected, with 70% to 90% becoming chronic carriers. Mothers who are HBsAg-positive but HBeAg-negative, presumably indicating lower levels of replicating virus, do have a lower risk of transmitting the virus, but up to 35% of their children still will become carriers in the absence of neonatal therapy.^75,76,77,78 In addition to the long-term risks of HBV-related sequelae in chronic carriers, such as cirrhosis and hepatocellular carcinoma, both fulminant fetal neonatal hepatitis^79,80,81 and childhood-onset hepatic carcinoma,⁸² have been described in children born to HBsAg-positive mothers.

Early attempts at interrupting the perinatal transmission cycle used HBIG alone, administered in the neonatal period. Globulin alone had a protective efficacy against the carrier state of 70% to 75%, although the protection was not permanent, and many children eventually became infected after the passively acquired antibody was cleared, undoubtedly through household contact.^83,84,85 With the advent of the hepatitis B vaccine, trials were established to test its efficacy when administered in the newborn period, both alone and in conjunction with HBIG. A combination of HBIG and vaccine in the newborn period conferred significantly greater protection against perinatally transmitted HBV than even the vaccine alone, increasing efficacy from a range of 75% to 85% up to 90% to 95%.^{42,77,83–92} The small but identifiable percentage of babies who become infected, despite even combined HBV therapy at birth, is believed to represent in utero infection.^93,94 HBV DNA has been identified in abortus tissue extracted from an HBsAg-positive mother,⁹⁵ and other reports show evidence of intrauterine infection in clinical situations, increasing risks for transplacental leakage, such as preterm labor associated with placental abruption.^68,69 Still, combination HBV-specific immunotherapy provides the best opportunity to prevent the chronic carrier state in the offspring of HBsAg-positive mothers. In the United States alone, approximately 16,500 births occur in HBsAg-positive women each year, approximately 4300 of whom are also HBeAg-positive.⁹⁶ Infants born to these women should receive HBIG (0.5 mL) intramuscularly (IM), ideally within 12 hours of birth. HBV vaccine should be administered concurrently at a different site (0.5 mL IM) or can be administered up to 7 days after birth if it is not immediately available.⁹⁶ The timing of HBIG appears to be more critical than that of vaccine in achieving maximal effectiveness of passive–active therapy. Subsequent vaccination is performed, also 0.5 mL IM, at ages 1 month and 6 months. Follow-up for these infants is crucial, because one recent study confirms the concern that in the United States, groups at highest risk for HBV infection are also least likely to be compliant with follow-up care.⁹⁷

Hepatitis B Screening in Pregnancy

The unique opportunity to provide almost complete protection against perinatally acquired HBV infection makes antenatal identification of HBV carriers critical so that combined neonatal prophylaxis can be administered in a timely fashion. In nonendemic areas such as the United States, screening protocols were organized initially to test pregnant women who are in the HBV risk groups, as defined by the United States Public Health Service (Table 2).⁹⁸ Such recommendations, however, were not without problems. Reports from a number of groups working in geographically diverse areas around the country found that using risk groups alone for prenatal HBV screening would miss 40% to 60% of all HBsAg-positive parturients.^{99,100,101,102,103,104,105} Overall, in these studies, the HBsAg-positive rate ranged from 0.3% to 1.5% (Table 3). Even if risk factors were to be used to identify these women, however, evidence from one survey shows that only 60% of obstetricians could name more than two HBV risk groups, and less than 30% knew the recommended treatment for infants born to carrier mothers.¹⁰⁶

Table 2. Public Health Service Risk Groups for Prenatal Hepatitis B Virus (HBV) Screening Protocols (1984)

Table 3. Hepatitis B Surface Antigen (HBsAg) Positivity in Different U.S. Populations and Relation to Centers for Disease Control Risk Criteria

Such findings have led to recommendations by the Public Health Service⁹⁶ and, most recently, by the American College of Obstetricians and Gynecologists¹⁰⁷ that HBsAg screening be performed as part of routine prenatal testing in all pregnant women. An elegant cost-analysis study by Arevalo and Washington¹⁰⁸ shows that such a program, taking into account both acute and long-term costs of neonatally acquired HBV disease, is cost-effective at a prenatal population prevalence for HBsAg of only 0.06%. In countries where HBsAg carriage is endemic, funding for medical screening programs tends to be limited. In these settings, especially as the cost for HBV vaccine begins to decrease, workers have advocated consideration of empiric vaccination for all newborns.^109,110 Such a policy also has been recommended for children of Southeast Asian immigrants to the United States to prevent both perinatal and household acquisition of HBV.¹¹¹

However, protocols establishing prenatal HBsAg screening policies do not address the problematic issue of deliveries in inner-city populations among women with minimal to no prenatal care. Maternal HBsAg status, in the absence of prenatal testing, then can only be known in the 1 to 2 days after delivery, and the newborn may miss out on maximally effective HBV prophylaxis. Investigators have recognized this problem because most hospital laboratories perform HBsAg testing at best on a daily basis.¹¹² This fact is particularly important because evidence suggests that HBIG administered as perinatal prophylaxis may have limited efficacy if it is not administered as soon as possible after birth.¹¹³ A recent study has shown the rate of HBsAg carriage to be significantly higher among such unregistered women than in a comparison group enrolled in an inner-city clinic (7.8% vs 0.8%) and that the increase was specifically related to substance abuse. Among unregistered women with positive urine drug screens, moreover, the HBsAg-positive rate was 15%, and a maternal urine drug screen was suggested as a rapid screening test to target neonates at highest risk for HBV infection for prophylaxis, before the 24 to 48 hours required awaiting maternal HBsAg status.¹¹⁴

Impact of Perinatal Hepatitis B Virus Vaccination Programs

The efficacy of perinatal HBV vaccination programs in preventing infection in children born to carrier mothers has led to the inclusion of the HBV vaccine series in the American Academy of Pediatrics' current recommendations for childhood vaccines for the general population.¹¹⁵ Still, investigators have demonstrated that even in high-risk groups for perinatal HBV transmission, appropriate neonatal surveillance is critical. Among 426 children born to HBsAG-positive mothers in one longitudinal series, only 68% were completely vaccinated with the full three-dose sequence. Among the children followed-up, it was shown that the third vaccine dose was least likely to be received (64%). Serologic evaluation of the children in this well-conceived surveillance program showed 4% to have acquired chronic carrier status, with an additional 10% having evidence of resolved natural infection, as demonstrated by positive tests for antiHBc. Not surprisingly, incompletely vaccinated children were more likely to be HBsAg-positive than those completing the series (12% vs 1%; relative risk 7.9 [confidence interval = 1.5–41.2]).¹¹⁶ These findings underscore the need for reinforcement of complete vaccination for all children enrolled in the HBV vaccine series, particularly those born to carrier or other high-risk mothers.

The beneficial impact of adequate childhood vaccination for HBV recently has been demonstrated dramatically in reports from Taiwan, where large-scale mass vaccination programs were begun in 1984. Researchers there have conclusively proven a link between HBV and hepatocellular carcinoma (HCC) by showing a significant decrease in the average annual incidence of childhood HCC since the institution of the program.^117,118 The decrease in the rate of childhood HCC was also paralleled by a decrease in the rate of HBsAg carriage among children born before the vaccination program was started, suggesting a herd immunity effect from the mass inoculation of children in the much more infectious younger birth cohorts, and resulting in a lower rate of horizontal HBV infection among the older unvaccinated children.¹¹⁷ These researchers had demonstrated previously that 5 years into the institution of the vaccination program in Taipei, the HBsAg carrier rate in children younger than age 5 years had decreased from 9.3% in 1984 to 2% in 1989.¹¹⁹ These results even further bolster the need to identify HBsAg carrier mothers and provide timely and complete HBV vaccination to their children.

Description and Diagnosis of the Virus

The term “non-A non-B hepatitis” (NANBH) traditionally was used to describe the clinical picture of posttransfusion hepatitis in the absence of positive serologic markers for either hepatitis A or hepatitis B viral infections. Molecular investigation of the serum of such affected patients led to the reporting in 1989 of the identification of a novel viral agent, named hepatitis C (hepatitis C virus [HCV] ), with a uniquely sequenced viral genome.¹²⁰ That pioneering work led to the development of HCV-specific antibody assays, from the initial first-generation enzyme-linked immunosorbent assay (ELISA), through recombinant immunosorbent assays (RIBA), to the currently available third-generation ELISA and chemiluminescent assays, with increased diagnostic sensitivity as a result of their detection of several, rather than a single, nonstructural viral protein antigens.^{121,122,123,124,125}

While antiHCV testing includes initial screening with an immunoassay, the false-positive rate of approximately 1%, especially for a low-risk patient, raises the possibility of an improper diagnosis being given without appropriate confirmatory testing. In contrast to serologic diagnosis of HBV, in which certified laboratories are required to perform supplemental confirmatory testing before issuing a positive HbsAg test result, no such automatic “reflex” testing has been mandated for HCV antibody screening. In a patient at high risk for HCV infection with a positive antiHCV test result, the chance of a false-positive ELISA is exceedingly low. However, recognizing that most laboratories report positive antiHCV results using screening assays alone¹²⁶ despite previous recommendations,¹²⁷ the CDC expanded its HCV testing algorithm to include an option for supplemental testing based on “signal-to-cutoff” (s/co) ratios of positive assay results.¹²⁶ Because pregnant women do not constitute a high-risk group requiring HCV screening by CDC guidelines unless other risk factors exist (Table 4), both the importance of understanding the limitations of even the most currently available screening assays and the ability to interpret test results for patients are critical for clinicians who might be ordering HCV screening tests.

Table 4. Risk Factors Warranting Hepatitis C Screening: CDC Guidelines¹²⁷

HCV itself has been characterized as an enveloped single-stranded RNA virus of the family Flaviviridae, with a genomic structure that includes core (nucleocapsid), and envelope proteins at the 5' end and five nonstructural proteins extending to the 3' end of the genome. The newer antibody tests combine detection of nonstructural proteins toward the 3' end of the genome with detection of c22–3, a viral core antigen located just distal to the 5' conserved end of the genome. The improved detection rates with the use of these newer antibody tests have led one authority to say that, as useful as the first-generation assay had been, it is now virtually obsolete for diagnostic purposes.¹²⁸

Diagnosis of HCV infection also has been assisted by the use of the polymerase chain reaction (PCR) to amplify and detect extremely small amounts of HCV-RNA in serum, with both qualitative and quantitative tests currently available commercially.¹²⁹ PCR is still a technically demanding procedure, however, with a need to maintain stringent testing conditions to assure both accuracy and precision. Heparinized samples interfere with the polymerase in the assay, and sample storage techniques can have great impact on detectable viral particles. Prolonged sample storage at room temperature, for example, reduces PCR signal detection, as do repeated freeze-thaw cycles, even when the sample is otherwise stored appropriately at −70°C.¹³⁰

At least six distinct HCV genotypes have been identified with broad geographic variation,^131,132 and this mutability may limit the reproducibility of results from laboratory to laboratory, and from study to study, if primer standardization is not verified. Genotype prevalence of HCV infection varies widely by geographic location; variants of genotype 1 are the most prevalent types seen among infected individuals in the United States and Japan, for example.^133,134 This genotypic variability has been found to have a significant impact on disease progression and response to therapy for infected individuals, with genotype 1 associated with poorer outcomes and responses overall.^{135,136,137,138} Despite this fact, HCV genotype has not been determined to be an independent risk factor for perinatal HCV transmission.¹³⁹ The 5' noncoding terminal region of the HCV genome appears to be strongly conserved, although viral isolates with nucleotide sequence variability have been identified within that region as well.^140,141

Epidemiology

Soon after HCV was identified, a number of studies used screening with the first-generation assays for antiHCV to describe seroprevalence rates for groups perceived to be both at high and low risks for HCV infection. Among presumably low-risk volunteer blood donors in the general population, rates of 0.5% to 1.4% for antiHCV seropositivity were described.^142,143 Higher risk groups, using HBV infection patterns as a model, were shown to have higher rates of HCV antibody positivity, also. These high-risk groups have included patients in sexually transmitted disease clinics (seroprevalence 1.5%–6.2%),^144,145 commercial sex workers (2%–10%),^146,147 people with hemophilia (64%–86%),^142,148 and patients with drug abuse histories (56%–86%)^{145,149,150,151}

Overall, the principal risk factors for HCV transmission are blood and blood products, transfusion, and use of intravenous drugs. At least 90% of reported cases of posttransfusion hepatitis can be traced to HCV, usually within 5 to 10 weeks of the transfusion.^152,153,154 Although mass screening of banked blood products for HCV antibodies has reduced the risk of transfusion-associated HCV substantially, up to 10% of donors later implicated in transfusion-related HCV may be seronegative carriers at the time of blood donation.^{155,156,157,158} Current estimates place the risk of transfusion-associated HCV infection at approximately 1 in 100,000 per unit transfused (range 1 in 28,000 to 1 in 288,000).¹⁵⁶ This rate is significantly lower than was reported from the period before the use of HCV-specific assays for screening donated blood, when only surrogate markers of NANBH, such as elevated liver-function tests, were used. Still, it is higher than current estimates of transmission risks by transfusion for other viral vectors, specifically HIV (1 in 500,000 per transfused unit) and HTLV-1 (1 in 641,000 per unit).^156,159,160 In contrast, the risk of HBV transmission is approximately 1 in 63,000 per unit, so that HBV and HCV together account for 88% of the aggregate risk of transfusion-related infection of 1 in 34,000.¹⁵⁶ As the risk of HCV infection resulting from blood transfusions has diminished, a direct result of mass screening of blood products, the proportion of HCV infections attributable to drug use has markedly increased, from 20% to 60%.¹²⁷

Sexual transmission of HCV has been implicated variably. A report before the availability of specific HCV tests suggested that up to 11% of NANBH cases, in the absence of other identifiable risk factors, could be related to heterosexual activity, particularly multiple sexual partners.¹⁶¹ Subsequently, reports using antiHCV assays have shown lower rates of HCV transmission from seropositive partners, approximately 0% to 4%.^144,162 Sexual promiscuity was identified as an independent risk factor for HCV seropositivity in one recent study,¹⁶³ however, and an interaction increasing the risk of concomitant transmission of HCV and HIV has been described. In these reports, men with evidence of infection with both HCV and HIV were five-times more likely to transmit both viruses to a female partner than would have been expected by chance.^164,165 This potential interaction of HIV and HCV to increase transmissibility of either or both agents also has become important in describing issues surrounding maternal–fetal HCV transmission.

Pathogenesis and Clinical Issues

Acute HCV infection occurs after an incubation period of 30 to 60 days. Asymptomatic infection occurs in 75% of patients; the remaining 25% present with the typical manifestations of other viral hepatitides. Fulminant hepatitis and hepatic failure attributable to HCV, as compared with that from other viral hepatitis agents, are uncommon.

Chronic liver disease occurs frequently after acute HCV infection; at least 50% of disease progresses to chronicity, regardless of the mode of acquisition or severity of initial infection.^166,167,168 Chronic hepatitis C infection has also been associated with an increased risk of developing a monoclonal gammopathy, particularly in conjunction with genotype 2a/2c infections,¹⁶⁹ theorized to possibly be related to the impact of chronic antigenic stimulation over time by a long-standing latent viral infection such as HCV.¹⁷⁰ Similarly, chronic HCV infection has also been associated with an increased risk of developing B-cell lymphomas and cryoglobulinemia.^171,172,173 HCV antibody has been detected in serum from patients with both cryptogenic cirrhosis and HCC, although a linkage between the latter and HCV is controversial and geographically may be quite variable.^{174,175,176,177,178} Evidence also exists that coexisting HIV and HCV infections may accelerate progression of hepatic injury.¹⁷⁹

Pregnancy and Effects on the Fetus and Neonate

Seroprevalence data describing HCV antibody status among pregnant women or women of reproductive age emerged quickly as HCV testing became more readily available. In Taiwan, for example, where HBV infection is endemic, the prevalence of antiHCV among a cohort of pregnant women was reported as 0.6%.¹⁶² An early general seroprevalence study in Spain used 241 “healthy” pregnant women as part of a control group and showed an antiHCV positive rate of 1.2%. However, additional information related to other risk factors among these women was not provided.¹⁴² Another group of Spanish investigators reported a 2.9% antiHCV-positive rate among pregnant women, 17% of whom had no identifiable risk factors.¹⁸⁰

A number of studies to date have looked specifically at HCV antibody seroprevalence in prenatal populations in the United States. One, designed as a vertical transmission study, reported a 4.5% positive rate in a county hospital in New York; 74% reported a parenteral source of exposure, and 17% reported no risk factors. Of these, 17% (14 of 23) were also HIV-positive.¹⁸¹ Investigators in Dallas studied 1013 obstetric patients and found 2.3% to be positive for antiHCV. Risk factors for infection were specifically studied, with history of intravenous drug use, previous sexually transmitted disease, substance-abusing partner, and more than three lifetime sexual partners being significantly associated with HCV antibody positivity.¹⁸² A study from Philadelphia detected antiHCV antibodies in 4.3% of pregnant women screened, which was significantly higher than infection rates for HIV (0.5%), HTLV-1 (0.8%), and HBV (0.8% HBsAg). The relative risk of other coexisting viral infections was significantly higher among antiHCV positive women than for those who were antibody-negative. Risk factor-targeted screening would have failed to detect half the antiHCV-positive women in the study.¹⁸³ A follow-up study from this group, using a larger cohort of more than 1400 women from a heterogenous socioeconomic sample, showed an HCV seropositive rate of 3.2%, with only 19% having HCV–RNA-positive test results in newborn cord blood samples. Women who were antibody-positive were also more likely to need to undergo cesarean section, suggesting an increased risk of other coexisting obstetric complications in that subpopulation.¹⁸⁴

Information regarding the maternal–fetal transmission of HCV has continued to steadily accumulate. Although reported rates of transmission have been variable, overall it is encouraging that most series show the risk to be generally less than 5%. However, the definition of which mothers are most infectious dramatically alters the relative risks of vertical transmission. Before the identification of HCV, newborns of women with NANBH were followed-up serially with surveillance of transaminase levels. A study from Sweden showed no increased risk to the women followed-up during their pregnancies, although 16% (2 of 12) of their children had persistent unexplained transaminase elevations through infancy.¹⁸⁵ This report was consistent with an earlier study demonstrating alanine aminotransferase (ALT) elevations in 6 of 12 infants born to women with NANBH, followed-up to 8 weeks after delivery.¹⁸⁶

Surveillance of mothers and their newborns using HCV-specific antibodies in published reports has, on the whole, demonstrated low rates of transmission from antiHCV-positive mothers to their offspring.¹⁶² The designs of these earlier studies, however, were mostly retrospective, with limited neonatal follow-up. Subsequent work has used HCV–RNA as a marker of neonatal infection rather than antibody seropositivity only, with rates of newborn RNA retrieval averaging 5% to 10% when the mother is also HCV–RNA-positive.^181,187,188

More recent studies from Asia and Europe have established what appears to be a correlation between maternal viral titer of HCV near delivery and the risk of neonatal infection. These studies have used newer technologies to determine quantitative, rather than qualitative, PCR results. One team in Japan showed significantly higher (2 logs) HCV–RNA titers in transmitting mothers than in those whose infants remained uninfected (106 vs 104 HCV-RNA copies/mL).¹⁸⁹ Using similar techniques, another group in China drew similar conclusions, also demonstrating that perinatal transmission occurred only in those women whose serum was HCV–RNA positive.¹⁹⁰ A group from the United States, however, failed to establish a similar viral burden correlation in their series, suggesting a possible role for geographic variability in genotype and virulence in explaining the variance in results.¹⁸⁸ More recent reports have continued to confirm clearly that maternal viremia (usually defined as HCV–RNA detected in maternal blood) is a key determinant for vertical HCV transmission, regardless of maternal HIV status. Still, the independent impact of quantitative maternal viremia (“viral load”) on vertical transmission remains less uniformly established across the studies than the presence of viremia at all,^{191,192,193,194,195,196,197,198,199} in direct contrast to previous experience with vertical HIV transmission.

Recent investigators also have confirmed earlier reports that women coinfected with HCV and HIV had significantly higher rates of perinatal infection than women infected with HCV alone. Earlier, smaller series suggested rates ranging from 15% to 87% in the face of maternal coinfection.^200,201,202 The more recent series have narrowed but have not lowered the risk range of 23% to 44%.^{188,191,202,203,204,205,206,207}

The interaction between maternal and fetal humoral and immunologic is thought to be a critical contributor to both the occurrence and persistence of perinatally acquired neonatal HCV infection. The fact that maternal and/or neonatal coinfection with HIV increases the risk of vertical HCV infection suggests that HIV-infected infants, who are known to have early deficits in cell-mediated and humoral immunity, may be less able to clear small amounts of perinatally presented HCV than HIV-uninfected infants.^206,207 Although no large-scale longitudinal follow-up studies exist into the natural history of perinatal HCV infection through childhood, at least one published case report has documented clearance of neonatally documented HCV–RNA by age 24 months in a child born to an HIV-negative, HCV–RNA-positive mother.²⁰⁸ Therefore, an intact neonatal immune system may allow for HCV clearance in early infancy as can occur in HIV-uninfected adults. More recently, Italian investigators have presented provocative data demonstrating that HCV infection of maternal peripheral blood mononuclear cells (PBMNC) was a stronger independent predictor of vertical HCV transmission than either quantitative HCV–RNA titer or viral genotype in a cohort of HIV-uninfected women.²⁰⁹ These researchers had previously shown the presence of an HCV–RNA strand in infected PBMNC to be a marker of active viral replication rather than one of uptake of cellular debris, as had been alternatively proposed,²¹⁰ and its association with perinatal infection further emphasizes the complex interplay between maternal and fetal factors in determining the risk of neonatal HCV infection.

Although the impact of maternal HCV infection on perinatal HCV transmission has been extensively studied, the potential impact of pregnancy on maternal infection and HCV-related illness is less well-documented. One large study of more than 15,000 HCV-infected pregnant women in northern Italy, however, demonstrated a downward trend (almost 50% lower) in transaminase levels as pregnancy progressed, with no concomitant change in the proportion of women with documented viremia. The authors hypothesized that a favorable immuno-mediated positive effect of pregnancy on liver cell necrosis might be one explanation for these findings, although liver biopsy samples were not a routine prospective component of their study.¹⁹⁹ A group in China similarly reported that HCV levels did not change significantly as pregnancy progressed but did show a significant decrease in those levels at 1 and 3 months postpartum.²¹¹

Optimal route of delivery, if one exists, remains an area of controversy in the context of maternal HCV infection. This debate, to some degree, parallels the one that evolved regarding maternal HIV infection. However, unlike with HCV infection, maternal HIV viral load was a clearly established independent predictor of vertical transmission^212,213 and had a direct bearing on the current guidelines regarding route of delivery and maternal HIV infection, specifically when maternal HIV viral load is durably suppressed to less than 1000 RNA copies/mL.^214,215 Route of delivery appears to be much less clearly associated with risk of HCV transmission, however.^{191,193,195,196,199,206} While the rate of cesarean section in HCV-infected mothers is higher than that for uninfected women, this is thought to be more related to the higher rates of obstetric complications and comorbidities in a subgroup of women whose primary risk factor for HCV infection is illicit drug use.^173,193,199 Current consensus opinions, therefore, recommend cesarean delivery in HCV-infected women only for usual obstetric indications.^216,217

The experience with antiretroviral treatment in decreasing both maternal viral load and the risk of neonatal HIV infections^{218,219,220,221} raises the question of potential comparable treatment options in the context of maternal HCV infection. Recent advances in combination therapy of HCV infection in nonpregnant adults have made sustained normalization of transaminase levels and clearance of HCV–RNA a reality, even in individuals with the less favorable HCV genotype 1.^{136,137,138,222} More recently, the modification of interferon alfa-2a via a branched-chain polyethylene glycol moiety has produced a compound, peginterferon alfa-2a, with prolonged absorption, slower clearance, and a longer half-life than standard interferon, with once-weekly dosing possible.^223,224 Randomized trials have shown peginterferon to be superior to standard interferon, either alone or combined with ribavirin, for the treatment of chronic hepatitis C infection in adults.^225,226,227 While the use of ribavirin is contraindicated during pregnancy,²²⁸ interferon has been used safely for the treatment of T-cell leukemias during pregnancy,^229,230,231 and its potential role as an antiHCV therapy for both maternal and fetal/neonatal benefits warrants further exploration.

Finally, questions surrounding the safety of breastfeeding frequently arise from HCV-infected women. Many of these women have other risk factors, such as ongoing substance addiction or coexisting HIV infection, which preclude breastfeeding in general and override concerns about transmission of HCV through breast milk. For those women who have no other obstetric or medical issues that prevent nursing, limited data have failed to demonstrate breast milk as an effective route for HCV transmission. Although most of these studies were not designed to specifically address this topic, the authors' evaluation of mother–infant pairs enrolled in some of the published vertical HCV transmission studies failed to document any neonatal HCV infection in breast-fed infants, even when the mother was HCV–RNA-positive.^203,204,232 One study from China that did look at breast milk specifically found a correlation between maternal HCV serum titer and detection of HCV by means of PCR in breast milk; however, no infants in this small series (n = 15) became infected with HCV.²³² More recent studies from Australia,²³³ United Arab Emirates,²³⁴ and Switzerland²³⁵ have studied an additional 100 pregnancies in HCV-infected women who breast-fed (up to two-thirds of whom were HCV–RNA-positive in sera), with no detectable impact of breastfeeding on the risk of neonatal infection. As a result of the currently available supporting data, consensus opinions do not view maternal HCV infection as a contraindication to breastfeeding except, perhaps, in cases in which a mother experiences cracked or bleeding nipples.^216,217

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