Harold S. Margolis

The Prevalence of Hepatitis C Virus Infection in the United States, 1988 through 1994

BACKGROUND Because many persons with chronic hepatitis C virus (HCV) infection are asymptomatic, population-based serologic studies are needed to estimate the prevalence of the infection and to develop and evaluate prevention efforts. METHODS We performed tests for antibody to HCV (anti-HCV) on serum samples from 21,241 persons six years old or older who participated in the third National Health and Nutrition Examination Survey, conducted during 1988 through 1994. We determined the prevalence of HCV RNA by means of nucleic acid amplification and the genotype by means of sequencing. RESULTS The overall prevalence of anti-HCV was 1.8 percent, corresponding to an estimated 3.9 million persons nationwide (95 percent confidence interval, 3.1 million to 4.8 million) with HCV infection. Sixty-five percent of the persons with HCV infection were 30 to 49 years old. Seventy-four percent were positive for HCV RNA, indicating that an estimated 2.7 million persons in the United States (95 percent confidence interval, 2.4 million to 3.0 million) were chronically infected, of whom 73.7 percent were infected with genotype 1 (56.7 percent with genotype 1a, and 17.0 percent with genotype 1b). Among subjects 17 to 59 years of age, the strongest factors independently associated with HCV infection were illegal drug use and high-risk sexual behavior. Other factors independently associated with infection included poverty, having had 12 or fewer years of education, and having been divorced or separated. Neither sex nor racial-ethnic group was independently associated with HCV infection. CONCLUSIONS In the United States, about 2.7 million persons are chronically infected with HCV. People who use illegal drugs or engage in high-risk sexual behavior account for most persons with HCV infection.

The Natural History of Community-Acquired Hepatitis C in the United States

BACKGROUND Chronic liver disease develops in more than half of patients with post-transfusion hepatitis C, but little is known about the natural history of community-acquired hepatitis C. METHODS In 1985 and 1986 we identified adults with acute non-A, non-B hepatitis in four counties in the United States and followed them prospectively. We used three markers to detect hepatitis C virus (HCV) infection in stored samples of serum: antibody to HCV (anti-HCV) detected by second-generation serologic assays; HCV RNA detected by polymerase-chain-reaction assay; and antibody to HCV antigen (anti-HCVAg) detected by fluorescent-antibody-blocking assay. RESULTS Of 130 patients with non-A, non-B hepatitis, 106 (82 percent) had HCV infection, 93 were positive for anti-HCV, and 13 were positive only for HCV RNA or anti-HCVAg. Chronic hepatitis developed in 60 (62 percent) of 97 HCV-infected patients followed for 9 to 48 months, with no relation to the risk factors for infection. Ten of the 30 patients who had liver biopsies had chronic active hepatitis. In samples collected 42 to 48 months after the onset of hepatitis, HCV RNA was detected in 12 of 13 tested patients with chronic hepatitis and in all 15 tested patients with hepatitis that had resolved. Anti-HCV persisted in all but two of the initially positive patients, for a rate of antibody loss of 0.6 per 100 person-years. CONCLUSIONS Patients with community-acquired hepatitis C have a high rate of chronic hepatitis. HCV may be a major cause of chronic liver disease in the United States, and in most patients HCV infection seems to persist for at least several years, even in the absence of active liver disease.

Molecular Cloning and Disease Association of Hepatitis G Virus: A Transfusion-Transmissible Agent

An RNA virus, designated hepatitis G virus (HGV), was identified from the plasma of a patient with chronic hepatitis. Extension from an immunoreactive complementary DNA clone yielded the entire genome (9392 nucleotides) encoding a polyprotein of 2873 amino acids. The virus is closely related to GB virus C (GBV-C) and distantly related to hepatitis C virus, GBV-A, and GBV-B. HGV was associated with acute and chronic hepatitis. Persistent viremia was detected for up to 9 years in patients with hepatitis. The virus is transfusion-transmissible. It has a global distribution and is present within the volunteer blood donor population in the United States.

Dengue: a continuing global threat

Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ∼50 million dengue infections and ∼500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future.

Genetic relatedness of hepatitis A virus strains recovered from different geographical regions

A pairwise comparison of the nucleic acid sequence of 168 bases from 152 wild-type or unique cell culture-adapted strains of hepatitis A virus (HAV) revealed that HAV strains can be differentiated genetically into seven unique genotypes (I to VII). In general, the nucleotide sequence of viruses in different genotypes differs at 15 to 25% of positions within this segment of the genome. Viruses from four of the genotypes (I, II, III and VII) were recovered from cases of hepatitis A in humans, whereas viruses from the other three genotypes (IV, V and VI) were isolated only from simian species developing a hepatitis A-like illness during captivity. Among non-epidemiologically related human HAV strains, 81 were characterized as genotype I, and 19 as genotype III. Within each of these major genotypes, there were two distinct groups (subgenotypes), which differed in sequence at approximately 7.5% of base positions. Each genotype and subgenotype has a characteristic amino acid sequence in this region of the polyprotein, with the most divergent genotypes differing at 10 of 56 residues. Strains recovered from some geographical regions belonged to a common (endemic) genotype, whereas strains from other regions belonged to several, probably imported, genotypes. Thus, HAV strains recovered in North America were for the most part closely related at the nucleotide sequence level, whereas in other regions, such as Japan and Western Europe, HAV strains were derived from multiple genotypes or sub-genotypes. These data indicate that patterns of endemic transmission can be differentiated from situations in which infections are imported due to travel.

Acute non-A-E hepatitis in the United States and the role of hepatitis G virus infection

BACKGROUND Little is known about the relation of the newly discovered hepatitis G virus (HGV) to the cause and clinical course of acute and chronic viral hepatitis. METHODS We selected patients from a surveillance study of acute viral hepatitis in four U.S. counties who had acute disease during 1985 to 1986 or 1991 to 1995. Serum samples were tested for HGV RNA by the polymerase chain reaction. RESULTS HGV RNA was detected in 4 of 45 patients with a diagnosis of non-A-E hepatitis (9 percent), 23 of 116 patients with hepatitis C (20 percent), 25 of 100 patients with hepatitis A (25 percent), and 32 of 100 patients with hepatitis B (32 percent) (P<0.05 for the comparison of hepatitis B with hepatitis non-A-E or C). The clinical characteristics of the acute illness were similar for patients with HGV alone and those with hepatitis A, B, or C with or without HGV infection. During a follow-up period of one to nine years, chronic hepatitis did not develop in any of the patients with HGV alone, but 75 percent were persistently positive for HGV RNA, as were 87 percent of those with both hepatitis C and HGV infection. The rates of chronic hepatitis were similar in patients with hepatitis C alone (60 percent) and those with both hepatitis C and HGV infection (61 percent). CONCLUSIONS The evidence from this surveillance study does not implicate HGV as an etiologic agent of non-A-E hepatitis. Persistent infection with HGV was common, but it did not lead to chronic disease and did not affect the clinical course in patients with hepatitis A, B, or C.

A Multistate, Foodborne Outbreak of Hepatitis A

BACKGROUND We investigated a large, foodborne outbreak of hepatitis A that occurred in February and March 1997 in Michigan and then extended the investigation to determine whether it was related to sporadic cases reported in other states among persons who had consumed frozen strawberries, the food suspected of causing the outbreak. METHODS The cases of hepatitis A were serologically confirmed. Epidemiologic studies were conducted in the two states with sufficient numbers of cases, Michigan and Maine. Hepatitis A virus RNA detected in clinical specimens was sequenced to determine the relatedness of the virus from outbreak-related cases and other cases. RESULTS A total of 213 cases of hepatitis A were reported from 23 schools in Michigan and 29 cases from 13 schools in Maine, with the median rate of attack ranging from 0.2 to 14 percent. Hepatitis A was associated with the consumption of frozen strawberries in a case-control study (odds ratio for the disease, 8.3; 95 percent confidence interval, 2.1 to 33) and a cohort study (relative risk of infection, 7.5; 95 percent confidence interval, 1.1 to 53) in Michigan and in a case-control study in Maine (odds ratio for infection, 3.4; 95 percent confidence interval, 1.0 to 14). The genetic sequences of viruses from 126 patients in Michigan and Maine were identical to one another and to those from 5 patients in Wisconsin and 7 patients in Arizona, all of whom attended schools where frozen strawberries from the same processor had been served, and to those in 2 patients from Louisiana, both of whom had consumed commercially prepared products containing frozen strawberries from the same processor. CONCLUSIONS We describe a large outbreak of hepatitis A in Michigan that was associated with the consumption of frozen strawberries. We found apparently sporadic cases in other states that could be linked to the same source by viral genetic analysis.

Evaluation of diagnostic tests: dengue

Dengue is an arthropod-borne flavivirus that comprises four distinct serotypes (DEN-1, DEN-2, DEN-3 and DEN-4) that constitute an antigenic complex of the genus flavivirus, family Flaviviridae. Infection by one serotype induces life-long immunity against reinfection by the same serotype, but only transient and partial protection against infection with the other serotypes1,2. Dengue virus infections can result in a range of clinical manifestations from asymp tomatic infection to dengue fever (DF) and the severe disease dengue haemorrhagic fever/dengue shock syndrome (DHF/ DSS). Most dengue infections are asymptomatic or cause mild symptoms, which are characterized by undifferentiated fever with or without rash. Typical DF is characterized by high fever, severe headache, myalgia, arthralgia, retro-orbital pain and maculopapular rash. Some patients show petechiae, bruising or thrombocytopenia. The clinical presentation of acute dengue infection is non-specific but 5–10% of patients progress to severe DHF/DSS, which can result in death if it is not managed appropriately. Plasma extravasation is the main pathophysiological finding of DHF/ DSS, which differentiates it from DF. DHF/ DSS is characterized by high fever, bleeding, thrombocytopenia and haemoconcentration (an increase in the concentration of blood cells as a result of fluid loss). Approximately 3–4 days after the onset of fever, patients can present with petechiae, rash, epistaxis, and gingival and gastrointestinal bleeding. Pleural effusion and ascites are common. Some patients develop circulatory failure (DSS), presenting with a weak and fast pulse, narrowing of pulse pressure or hypotension, cold and moist skin and altered mental state. Although there are no specific antiviral treatments for dengue infection, patients usually recover when the need for fluid management is identified early and electrolytes are administered3. It has been proposed that the classification of dengue disease should be simplified as severe and non-severe dengue. This simplified classification would make patient management and surveillance easier4. There is a need for specific, inexpensive dengue diagnostic tests that can be used for clinical management, surveillance and outbreak investigations and would permit early intervention to treat patients and prevent or control epidemics. Progress is being made in primary prevention, with several candidate dengue vaccines in late phases of development as well as improved vector control measures. Additionally, new techniques for the early detection of severe disease such as the use of biomarkers have the potential to decrease morbidity and

Antibody Levels and Protection after Hepatitis B Vaccination: Results of a 15-Year Follow-up

Context Although administration of hepatitis B vaccine for infants is routine practice in many countries, we do not know whether the protection that this vaccine offers lasts beyond 10 years. Such information is essential to develop policies about booster vaccination. Contribution Of 841 Alaska Natives who received 3 doses of hepatitis B vaccination during 19811982 and were followed for 15 years, 84% had protective levels of antibody to hepatitis B surface antigen that indicated continued protection. The greatest decline in antibody levels occurred in people who received vaccine before 4 years of age. Definite asymptomatic breakthrough infections occurred in 16 participants. Cautions Only about half of the initial cohort of 1578 was available for testing at 15 years. The Editors Universal vaccination of infants with hepatitis B vaccine is included in the immunization programs of most countries and has been shown to be effective in reducing the rate of chronic hepatitis B virus (HBV) infection (1, 2). Protection has been demonstrated in persons and populations vaccinated for 5 to 10 years, and rates of asymptomatic breakthrough HBV infection have been extremely low (3-9). However, the duration of protection afforded by hepatitis B vaccination beyond 10 years and the possible need for booster doses of this vaccine are unknown. Alaska Natives have a high prevalence of chronic HBV infection, primarily acquired during early childhood (10). Between November 1981 and May 1982, Alaska Natives residing in 15 villages in southwest Alaska were enrolled in a cohort study to ascertain the immunogenicity and long-term effectiveness of hepatitis B vaccination (11-14). We report data on the persistence of antibodies to hepatitis B surface antigen (anti-HBs), incidence of HBV infection, and the genetic characteristics of the HBV isolates in persons with breakthrough infections 15 years after initial vaccination of this cohort. Methods Participants and Data Collection A total of 1578 Alaska Natives who were serologically negative for hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (anti-HBc) were vaccinated on a 0-, 1-, and 6-month schedule with 3 doses of plasma-derived hepatitis B vaccine (Heptavax, Merck & Co., Inc., West Point, Pennsylvania) beginning in 1981 (11). Persons younger than 20 years of age received the 10-g dose, and adults received the 20-g dose. Of the 1578 persons vaccinated, 1436 (91%) were tested for an anti-HBs response 6 months after the last vaccine dose. From 1982 to 1992, serum specimens were obtained annually and once again during 1996 from as many of the 1578 consenting participants as possible. The Institutional Review Boards of the Alaska Area Native Health Service, the Indian Health Service, the Centers for Disease Control and Prevention, and the Yukon-Kuskokwim Health Corporation and the Norton Sound Regional Alaska Native health boards approved this study. All participants 18 years of age and older and parents of children younger than 18 years of age had provided signed informed consent to participate in the study; children older than 7 years of age gave verbal assent. The number of HBsAg-positive persons in each village was obtained from a registry used to follow patients with chronic HBV infection (15). Serologic Testing All serum specimens were tested for HBsAg, anti-HBs, and anti-HBc by radioimmunoassay using commercial test kits (Abbott Laboratories, Abbott Park, Illinois). At the initial testing of the cohort for anti-HBs, results were reported in sample ratio units. However, subsequent anti-HBs results were reported in milli international units (mIU) per mL using a World Health Organization reference standard (12-14). To ensure comparability of results over time, all serum specimens from each participant with sufficient volume (99.9% of all specimens collected during the study) were retested to determine anti-HBs levels in mIU/mL. Detection of HBV DNA and Nucleic Acid Sequencing Hepatitis B virus DNA was extracted from serum specimens (50 L) of participants with serologic markers of HBV infection by using commercially available reagents (MasterPure Complete DNA and RNA Purification Kit, Epicentre Technologies, Madison, Wisconsin), as described previously (16, 17). The HBsAg genomic region was then amplified by dilution cloning polymerase chain reaction by using previously described primers and methods to identify circulating variants of HBsAg (16, 17). The polymerase chain reaction products were purified and the nucleic acid sequence of the amplified region were determined by using prism dye or dRhodamine terminator cycle reactions (Applied Biosystems, Foster City, California) and automated sequencing (ABI Model 373 or 377, Applied Biosystems) (18). Sequence data were further analyzed by Sequence Navigator (ABI) and GCG software (19). Definitions The initial anti-HBs level was measured 6 months after the third dose of vaccine and 1 year after the first dose of vaccine. Participants with an initial anti-HBs level of at least 10 mIU/mL were considered vaccine responders. An anti-HBs level of 2 mIU/mL or greater was considered a positive result on subsequent specimens. A booster response was defined as a 2-fold or greater increase in anti-HBs levels between serologic test results. A definite HBV infection in a participant was defined as 1) 2 or more consecutive serum specimens positive for anti-HBc more than 1 year after the initial vaccine dose, 2) a single positive anti-HBc result with a positive HBV DNA result, or 3) any HBsAg-positive test result. A possible HBV infection was defined as a single positive or 2 nonconsecutive positive anti-HBc results. Participants who developed anti-HBc were interviewed for history of icterus or other clinical signs or symptoms compatible with acute hepatitis, and village and hospital medical records were reviewed for evidence of an illness compatible with viral hepatitis. Statistical Analysis Among persons who inadvertently received additional doses of hepatitis B vaccine during the follow-up period, anti-HBs results after the additional vaccine dose or doses were excluded from the analyses. Results for anti-HBs among persons with definite HBV infections were excluded from analyses after anti-HBc appeared. The primary outcomes in this study were the cumulative number of persons with a definite HBV infection during all follow-up years and the anti-HBs levels at the 15-year follow-up. The definitions of age classes (0 to 4 years, 5 to 19 years, 20 to 49 years, 50 years) were similar to those in a previously published analysis of this cohort (14). Although these data have been presented previously (11-14), we have provided the proportion of persons initially responding to vaccination. Quantitative anti-HBs levels are presented as geometric mean concentrations (GMCs). In bivariate analyses, analysis of variance was used to test the 15-year anti-HBs concentrations (log-transformed). Incidence rates of definite HBV infection were compared by using the Fisher exact test. We analyzed factors associated with anti-HBs levels over the 15 years after the first vaccine dose by using a linear mixed model (PROC MIXED in SAS version 9.1, SAS Institute, Inc., Cary, North Carolina) (19). We chose a longitudinal mixed linear model because it makes inferences by using information from the entire cohort collected at all follow-up time points. Levels of anti-HBs were log-transformed before analysis, and concentrations of 0 mIU/mL were assigned a value of 1.0 mIU/mL. Factors considered in the model were time (entered as a continuous covariate; linear and quadratic term were considered), age class at initial vaccination, sex, the log of the initial anti-HBs level, presence of an HBsAg-infected person in the household at the start of the study, and the proportion of village residents with chronic HBV infection at the end of follow-up, along with interaction terms involving time. Significance of 1 factor alone, such as age or sex, is called a main effect and is not of primary interest for this presentation. Of primary interest are the interaction terms between time and other factors. A significant interaction of time with another variable, such as sex, indicates that the decline in anti-HBs level differed between males and females. We obtained parameter estimates by using restricted maximum likelihood. We used an unstructured covariance matrix to account for dependence of observations across time within individuals. Backward elimination of statistically nonsignificant terms yielded a final model of main effects and time interaction terms. If the time interaction term was statistically significant, the main effect term remained in the model regardless of statistical significance. We used the Wald chi-square statistic to test covariates. Contrast tests were 2-sided, and an level of 0.05 was required. We used residual plots to evaluate model fit. A secondary outcome was a boost in anti-HBs level at the 11- or 15-year follow-up among persons without additional doses of vaccine. We used the chi-square test or the CochranMantelHaenszel test to compare age and sex of persons with a booster response to those of persons without a booster response at both follow-up years. All P values were exact where appropriate and were 2-sided; results were considered statistically significant at the 0.05 level. We conducted analyses by using StatXact4 (Cytel Software Corp., Cambridge, Massachusetts) and SAS software, version 9.1 (SAS Institute, Inc.). Missing Data Throughout the entire study, anti-HBs determinations were observed for 68% of all potential observational time points (Appendix Table 1). The 15 remote rural Alaskan study villages are accessible only by airplane. During each year, study personnel flew into each village for 1 to 2 days and attempted to recruit all available participants. Persons not available or out of the village for the day were considered miss

Incidence and Risk Factors for Acute Hepatitis B in the United States, 1982–1998: Implications for Vaccination Programs

From 1982-1998, enhanced sentinel surveillance for acute hepatitis B was conducted in 4 counties in the United States to determine trends in disease incidence and risk factors for infection. During this period, the reported incidence of acute hepatitis B declined by 76.1% from 13.8 cases per 100,000 in 1987 to 3.3 cases per 100,000 in 1998. Cases associated with injection drug use (IDU) decreased by 90.6%, men who have sex with men (MSM) by 63.5%, and heterosexual activity by 50.7%. During 1994-1998, the most commonly reported risk factor for infection was high-risk heterosexual activity (39.8%) followed by MSM activity (14.6%) and IDU (13.8%). Over half of all patients (55.5%) reported treatment for a sexually transmitted disease (STD) or incarceration in a prison or jail prior to their illness, suggesting that more than half of the acute hepatitis B cases might have been prevented through routine hepatitis B immunization in STD clinics and correctional health care programs.