Omana V. Nainan

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.

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.

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.

An outbreak of hepatitis a associated with green onions

BACKGROUND In November 2003, a large hepatitis A outbreak was identified among patrons of a single Pennsylvania restaurant. We investigated the cause of the outbreak and factors that contributed to its unprecedented size. METHODS Demographic and clinical outcome data were collected from patients with laboratory confirmation of hepatitis A, and restaurant workers were tested for hepatitis A. A case-control study was conducted among patrons who dined at the restaurant between October 3 and October 6, 2003. Sequence analysis was performed on a 315-nucleotide region of viral RNA extracted from serum specimens. RESULTS Of 601 patients identified, 3 died; at least 124 were hospitalized. Of 425 patients who recalled a single dining date at the restaurant, 356 (84 percent) had dined there between October 3 and October 6. Among 240 patients in the case-control study, 218 had eaten mild salsa (91 percent), as compared with 45 of 130 controls (35 percent) (odds ratio, 19.6; 95 percent confidence interval, 11.0 to 34.9) for whom data were available. A total of 98 percent of patients and 58 percent of controls reported having eaten a menu item containing green onions (odds ratio, 33.3; 95 percent confidence interval, 12.8 to 86.2). All restaurant workers were tested, but none were identified who could have been the source of the outbreak. Sequences of hepatitis A virus from all 170 patients who were tested were identical. Mild salsa, which contained green onions grown in Mexico, was prepared in large batches at the restaurant and provided to all patrons. CONCLUSIONS Green onions that were apparently contaminated before arrival at the restaurant caused this unusually large foodborne outbreak of hepatitis A. The inclusion of contaminated green onions in large batches that were served to all customers contributed to the size of the outbreak.

Risk Factors for Perinatal Transmission of Hepatitis C Virus (HCV) and the Natural History of HCV Infection Acquired in Infancy

BACKGROUND The goal of the present study was to assess risk factors for perinatal hepatitis C virus (HCV) transmission and the natural history of infection among HCV-infected infants. METHODS In a cohort study, 244 infants born to HCV-positive mothers were followed from birth until age > or =12 months. Maternal serum was collected at enrollment and delivery; infant serum was collected at birth and at 8 well-child visits. Testing included detection of antibody to HCV, detection of HCV RNA (qualitative and quantitative), and genotyping. HCV-infected infants were followed annually until age 5 years. RESULTS Overall, 9 of 190 (4.7% [95% confidence interval (CI), 2.3%-9.1%]) infants born to mothers who were HCV RNA positive at delivery became infected, compared with 0 of 54 infants born to HCV RNA-negative mothers (P=.10). Among HCV RNA-positive mothers, the rate of transmission was 3.8% (95% CI, 1.7%-8.1%) from the 182 who were human immunodeficiency virus (HIV) negative, compared with 25.0% (95% CI, 4.5%-64.4%) from the 8 who were HIV positive (P<.05). Three infected infants resolved their infection (i.e., became HCV RNA negative). In multivariate analysis restricted to HCV RNA-positive mothers, membrane rupture > or =6 h (odds ratio [OR], 9.3 [95% CI, 1.5-179.7]) and internal fetal monitoring (OR, 6.7 [95% CI, 1.1-35.9]) were associated with transmission of HCV to infants. CONCLUSION If duration of membrane rupture and internal fetal monitoring are confirmed to be associated with transmission, interventions may be possible to decrease the risk of transmission.

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

Diagnosis of Hepatitis A Virus Infection: a Molecular Approach

SUMMARY Current serologic tests provide the foundation for diagnosis of hepatitis A and hepatitis A virus (HAV) infection. Recent advances in methods to identify and characterize nucleic acid markers of viral infections have provided the foundation for the field of molecular epidemiology and increased our knowledge of the molecular biology and epidemiology of HAV. Although HAV is primarily shed in feces, there is a strong viremic phase during infection which has allowed easy access to virus isolates and the use of molecular markers to determine their genetic relatedness. Molecular epidemiologic studies have provided new information on the types and extent of HAV infection and transmission in the United States. In addition, these new diagnostic methods have provided tools for the rapid detection of food-borne HAV transmission and identification of the potential source of the food contamination.

An Outbreak of Hepatitis A Associated with Green Onions

Forty-three cases of serologically confirmed hepatitis A occurred among individuals who ate at restaurant A in Ohio in 1998. Serum samples from all restaurant A employees who worked during the exposure period were negative for IgM antibodies to hepatitis A virus (HAV). A matched case-control study determined that foods containing green onions, which were eaten by 38 (95%) of 40 case patients compared with 30 (50%) of 60 control subjects, were associated with illness (matched odds ratio, 12.7; 95% confidence interval, 2.6-60.8). Genetic sequences of viral isolates from 14 case patients were identical to each other and to those of viral isolates from 3 patients with cases of hepatitis A acquired in Mexico. Although the implicated green onions, which could have come from one of 2 Mexican farms or from a Californian farm, were widely distributed, no additional green onion-associated cases were detected. More sensitive methods are needed to detect foodborne hepatitis A. A better understanding of how HAV might contaminate raw produce would aid in developing prevention strategies.

Duration of Viremia in Hepatitis A Virus Infection

The duration of viremia and time course for development of IgM antibodies were determined prospectively in natural and experimental hepatitis A virus (HAV) infection. Serial serum samples from HAV-infected men (n=13) and experimentally infected chimpanzees (n=5) were examined by nested reverse-transcriptase polymerase chain reaction analysis to detect HAV RNA and by ELISA to detect IgM antibodies to HAV. Among infected humans, HAV RNA was detected an average of 17 days before the alanine aminotransferase peak, and viremia persisted for an average of 79 days after the liver enzyme peak. The average duration of viremia was 95 days (range, 36-391 days). Results were similar in chimpanzees. In addition, HAV RNA was detected in serum of humans and chimpanzees several days before IgM antibodies to HAV were detected. These results indicate that adults with HAV infection are viremic for as long as 30 days before the onset of symptoms and that the duration of viremia may be longer than previously described.

Molecular Confirmation of Hepatitis A Virus from Well Water: Epidemiology and Public Health Implications

An outbreak of hepatitis A in a rural river-island community was found to be associated with consumption of contaminated well water. Specimens from case-patients, the implicated well, and a cesspool suspected to be the source of contamination were all positive for hepatitis A virus (HAV) RNA by immunocapture reverse-transcription polymerase chain reaction. All isolates were identical over about 400 bases from two capsid-encoding regions of the genome, identifying the chain of transmission. Other wells up to 60 m from the cesspool also contained HAV RNA. In addition, HAV RNA was detected in the contamination source well 6 months after the initial contamination, when fecal coliform bacteria were no longer present. These findings demonstrate the utility of viral detection techniques to evaluate contaminated ground water.