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.

[Prevalence of mutants in the determinant region of hepatitis B surface antigen among Chinese carriers after receiving only active postexposure immunoprophylaxis].

OBJECTIVE: To determine the frequency of hepatitis B surface antigen (HBsAg) mutations in the alpha determinant region among children who developed chronic hepatitis B virus (HBV) infection after receiving only active postexposure immunoprophylaxis. METHODS: HBsAg mutations were determined by PCR-directed nucleotide sequencing and sequence-specific solid-phase PCR analysis(SS-SPPCR) for 97 Chinese carrier cases after hepatitis B vaccination, for 88 children born aged women controls, and for 95 population based children controls. RESULTS: Prevalence of amino acid substitutions as detected by direct sequencing among carrier cases, women controls, and children controls were 30.9%, 10.2%, and 5.3%, respectively. The most frequent amino acid substitutions observed were at residues 145, 126, and 133. However, there was no difference in the prevalence of 145 and 126 amino acid mutants as detected by a sensitive SS-SPPCR method between carrier cases and controls. The prevalence of 145 Arg and 145 Ala mutants that were detected by SS-SPPCR was 39.2%, 33.0% and 32.6% among carrier cases, women controls, and children controls, respectively. The total odds ratio was 5.41 for mutants detected by direct sequencing. Odds ratio were 34.55 and 33.39 among adw2 subtype and genotype B subjects for mutants detected by direct sequencing, respectively. CONCLUSIONS: The results show that hepatitis B virus mutants in the determinant are fairly consistent observed but without immune selective pressures; HBV variant strains may pre-existent as minor quasispecies. The prevalence of mutants is related to HBV subtypes and genotypes.

[Characterization and distribution of hepatitis B virus genotypes and subtypes in 4 provinces of China].

OBJECTIVE: To determine the distribution and genetic relationship of hepatitis B virus (HBV) genotypes and subtypes. METHODS: HBV genotypes and subtypes were determined by PCR and DNA sequencing among 280 chronic HBV carriers in 25 counties of 4 provinces (Hunan, Guangxi, Henan and Hebei) in China. RESULTS: Genotype B, C and D were detected in these regions. Genotypes C and B were the majority genotypes of HBV with 190 cut of 280 (67.9%) genotype C, 82 (29.3%) genotype B, and 8 (2.9%) genotype D. Adr, adw2, ayr, ayw1, ayw2 and ayw3 subtypes were determined among these carriers. Adr and adw2 subtypes were the leading subtypes of HBV, taking up 64.3% and 31.4%, respectively. Adr subtype was completely encoded by genotype C while majority of adw2 subtype was encoded by genotype B. An average rate nucleotide substitutions of 2.94 was seen among 280 Chinese HBV sequences. The average rate of nucleotide substitutions of genotype B (adw2 subtype) was 5.63 (5.48), but only 1.6 (1.51) for genotype C (adr subtype). CONCLUSION: The results suggested that there were significant differences in geographic distribution of HBV genotypes and subtypes; genotype B, in which mostly consistent with adw2 subtype, was a higher variable than genotype C (adr subtype).

Genetic relatedness of hepatitis A virus isolates during a community-wide outbreak.

In 1993-94, a community-wide outbreak of hepatitis A occurred in Stanislaus County, California. Stool specimens collected from a sample of 33 case patients were used to evaluate the duration of hepatitis A virus (HAV) excretion and the genetic relatedness of HAV isolates. Twenty-four percent of the patients had a stool sample positive for HAV antigen by enzyme immunoassay, whereas 91% had at least one stool positive for HAV RNA by RT-PCR amplification. Children were found to excrete low levels of HAV RNA for up to 10 weeks after the onset of symptoms. Analysis of the HAV VP1 amino terminus and VP1/P2A regions showed that a limited number of HAV isolates circulated during the epidemic and the majority of the cases were infected with the same strain.

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.

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.

Chronic liver disease in central Harlem: the role of alcohol and viral hepatitis.

For reasons not yet determined, chronic liver disease (CLD) has been a leading cause of excess morbidity and mortality in central Harlem. We conducted a case series and case-control analysis of demographic, clinical, epidemiological, and alcohol-intake-related information from patients with CLD and age- and sex-matched hospitalized control patients. Patients' sera were tested for markers of viral hepatitis. The presumed etiology of CLD among case-patients was as follows: both alcohol abuse and hepatitis C virus (HCV) infection, 24 persons (46% of case-patients); alcohol abuse alone, 15 (29%); HCV infection alone, 6 (12%); both alcohol abuse and chronic hepatitis B virus (HBV) infection, 3 (6%); and 1 each (2%) from: 1) schistosomiasis, 2) sarcoidosis, 3) unknown causes, and 4) alcohol abuse, chronic HBV, and HCV combined. In the case-control analysis, patients who had both alcoholism and either HBV (odds ratio [OR]: 6.3; 95% CI: 0. 5-334) or HCV (OR: 2.9; 95% CI: 1.3-6.2) were at increased risk for CLD, whereas patients who had only one of these three factors were not at increased risk for CLD. Patients who tested positive for the hepatitis G virus (HGV) did not have a significantly increased risk of CLD, and neither severity of CLD nor mortality was greater among these patients. Most patients in central Harlem who had CLD had liver damage from a combination of alcohol abuse and chronic viral hepatitis. Alcohol and hepatitis viruses appear to be synergistically hepatotoxic; this synergy appears to explain both the high rate of CLD in central Harlem and the recent reductions in this rate. Persons at risk for chronic HBV and HCV infection should be counseled about their increased risk of CLD if they consume excessive alcohol. Morbidity and mortality from liver disease could be decreased further by a reduction in alcohol consumption among persons who have chronic HBV and HCV infection, avoidance of needle sharing, and hepatitis B vaccination.

A multistate, foodborne outbreak of hepatitis A. National Hepatitis A Investigation Team.

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.

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.

Hepatitis C and G co-infection: response to interferon therapy and quantitative changes in serum HGV-RNA.

Hepatitis G virus (HGV), a positive sense RNA virus, is distantly related to hepatitis C virus (HCV): its genetic organization and identity are consistent with the Flaviviridae family. Coinfection with HGV occurs in 10% to 20% of HCV-infected subjects. These similarities raise two theoretical questions. First, could HGV coinfection play any role in the response of HCV to antiviral therapy and second, would this coinfected population have changes in serum HGV-RNA induced by interferon. To address these questions, 98 patients with documented chronic HCV underwent interferon therapy (3 million units three times a week) for 6 months. Response to therapy was categorized using standard biochemical criteria. Changes in HGV-RNA levels were evaluated before, during, and after interferon therapy by a quantitative branched DNA amplification research-based assay. Eleven of 98 (11%) patients with HCV infection had detectable serum HGV-RNA. There was no difference between the groups (HGV+ vs. HGV-) when baseline alanine aminotransferase (ALT) values, HCV-RNA levels, HCV genotype, histological severity, or other demographic features were analyzed. Interferon response was similar in both groups and HGV was not associated with outcome following therapy. Antiviral therapy appeared to induce a reduction in HGV-RNA load in five of nine patients coinfected with HCV serially tested. In two patients, the fall in serum HGV-RNA correlated with biochemical response, independent of changes in HCV-RNA. These observations indicate that a larger study of an HGV population is required to more clearly define the relationship between HCV and HGV coinfection and their response to antiviral therapy.

Detection of hepatitis A virus RNA in oyster meat.

Detection of low concentrations of viruses in shellfish is possible with nucleic acid amplification by PCR. Hepatitis A virus (HAV) has been detected in oyster meat by reverse transcription-PCR (RT-PCR). We developed a method to identify HAV RNA by RT-PCR of total RNA extracted from oyster meat contaminated by adsorption, bioaccumulation, or injection. With dot blot hybridization detection of amplicons from the RT-PCR, rapid screening of a large number of samples is feasible. As few as 8 PFU of HAV/g of oyster meat can be detected.

Sequence-specific, single-primer amplification and detection of PCR products for identification of hepatitis viruses.

A simple system to detect polymerase chain reaction (PCR) amplification products was developed. This detection method has the sensitivity and the specificity of nested primer PCR amplification or Southern blot hybridization of PCR product. Digoxigenin-labeled PCR products were hybridized with a biotinylated probe in liquid phase and captured on to microtiter wells coated with antidigoxigenin followed by detection with streptavidin-peroxidase. The sensitivity of this assay for the detection of hepatitis A virus, hepatitis B virus, and hepatitis C virus is equal to that of existing nucleic acid detection systems.

Identification of amino acids located in the antibody binding sites of human hepatitis A virus.

Antigenic mutants of human hepatitis A virus (human-HAV) were isolated by their resistance to neutralizing monoclonal antibodies raised to human-HAV. The nucleotide sequence determined for the capsid regions of 12 mutants identified amino acid changes that clustered in three non-overlapping sites; one in VP3 and two in VP1. All mutants had a change at amino acid residue 70 in VP3, indicating its primary importance for antibody binding. Ten mutants had two amino acid changes occurring in the VP3 site as well as one in one of the two VP1 sites. These data suggest that both sites in VP1 interact with the single VP3 site to form the immunodominant epitope of HAV. The amino acid changes found in the antigenic mutants of human-HAV selected in this study were located in the same positions as changes found in strains of HAV isolated from Old World monkeys. These simian strains of HAV are not recognized by most monoclonal antibodies raised to human-HAV, suggesting that the observed amino acid changes are part of the antibody binding site.

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 outbreak of hepatitis A caused by the consumption of raw oysters.

BACKGROUND: In August 1988 we investigated a multistate outbreak of hepatitis A caused by Panama City, Florida, raw oysters. METHODS: Cases of hepatitis A (HA) with onset in July-August 1988 were identified among persons who ate seafoods harvested in the coastal waters of Panama City, Florida. We conducted a case-control study, using eating companions of case-patients, and calculated attack rate (AR) per 1000 dozen raw oysters served. Enzyme immunoassay (EIA) and a polymerase chain reaction (PCR) technique were performed on samples of raw shellfish obtained from Panama City coastal waters. RESULTS: Sixty-one case-patients were identified in five states: Alabama (23), Georgia (18), Florida (18), Tennessee (1), and Hawaii (1). We found an increased risk of HA for raw oyster eaters (odds ratio = 24.0; 95% confidence interval = 5.4-215.0; P less than .001). The AR of HA in seafood establishments was 1.9/1000 dozen raw oysters served. The EIA and PCR revealed HA virus antigen and nucleic acid in oysters from both unapproved and approved oyster beds, in confiscated illegally harvested oysters, and in scallops from an approved area. CONCLUSIONS: The monitoring of coastal waters and the enforcement of shellfish harvesting regulations were not adequate to protect raw oyster consumers. More emphasis should be placed on increasing public awareness of health hazards associated with eating raw shellfish.

Hepatitis A outbreak in a neonatal intensive care unit: risk factors for transmission and evidence of prolonged viral excretion among preterm infants.

An outbreak of hepatitis A virus (HAV) infection in a neonatal intensive care unit (NICU) provided the opportunity to examine the duration of HAV excretion in infants and the mechanisms by which HAV epidemics are propagated in NICUs. The outbreak affected 13 NICU infants (20%), 22 NICU nurses (24%), 8 other staff caring for NICU infants, and 4 household contacts; 2 seropositive infants (primary cases) received blood transfusions from a donor with HAV infection. Risk factors for infection among nurses were care for a primary infant-case (relative risk [RR], 3.2), drinking beverages in the unit (odds ratio [OR], infinity), and not wearing gloves when taping an intravenous line (OR, 13.7). Among infants, risk factors were care by a nurse who cared for a primary infant-case during the same shift (RR, 6.1). Serial stool samples from infant-cases were tested for HAV antigen (HAV-Ag) by enzyme immunoassay and HAV RNA by nucleic acid amplification using the polymerase chain reaction. Infant-cases excreted HAV-Ag (n = 2) and HAV RNA (n = 3) 4-5 months after they were identified as being infected. Breaks in infection control procedures and possibly prolonged HAV shedding in infants propagated the epidemic in a critical care setting.

Sequence analysis of a new hepatitis A virus naturally infecting cynomolgus macaques (Macaca fascicularis).

A new isolate of hepatitis A virus (HAV), CY-145, was isolated from stool specimens obtained from cynomolgus macaques naturally infected with this agent. Sequence analysis of the capsid region of the genome indicated that this virus differed from other sequenced HAV strains by about 20% at the nucleotide level and 7% at the amino acid level. Two amino acid residues (residues 70 of VP3 and 102 of VP1), previously identified as constituting an immunodominant site and conserved in all sequenced HAVs, were changed in the CY-145 virus. Sequence analysis of a second cynomolgus HAV isolate (CY-55), which came from a different geographical location, showed the same amino acid replacement at these two sites. In addition both isolates had an amino acid substitution at the VP3-VP1 cleavage site. These data suggest that the cynomolgus HAV differs genetically and antigenically from all other sequenced HAVs.

Epidemiologic patterns of wild-type hepatitis A virus determined by genetic variation.

Hepatitis A virus (HAV) isolates from different parts of the world are a single serotype. However, genetic analysis of the VP1 genome region of published HAV sequences suggested that distinct genotypes of HAV could be defined based upon the geographic source of the original isolates. To circumvent the process of cell culture adaptation or animal passage, a 247-bp segment within the VP1 genome region of wild-type HAV was amplified by reverse transcription followed by polymerase chain reaction amplification in the presence of negative- and positive-sense primers. From the sequences obtained from 22 epidemiologically distinct HAV isolates, three genetic groups of HAV could be delineated. Two of the groups differed by 10%, while the third group differed from other isolates by approximately 20%. These investigations indicate that HAV isolates from different parts of the world can be differentiated genetically, which will facilitate studies of epidemiologic transmission.

Immunoglobulin and hepatitis B surface antigen-specific circulating immune complexes in chronic hepatitis with hepatitis delta virus infection.

IgM, IgG, and HBsAg containing circulating immune complexes (CIC) were determined, by conglutinin (K) and C1q assays, for assessing the role of CIC in hepatitis delta virus (HDV) infection in 54 HBsAg-negative controls and 85 HBsAg-positive patients with chronic hepatitis. The prevalence of HDV markers (HDV antigen and anti-HD) was 24.70% (21/85). CIC were a common feature of HDV infection with 95.24% of patients having at least one abnormal test result. The prevalence of elevated IgM-K, IgG-K, IgM-C1q, and IgG-C1q CIC were 85.71, 85.71, 57.14, and 85.71%, respectively. The prevalence of IgM class CIC were statistically higher in patients with HDV infection than in those without (P = .001 for the K assay and P = .023 for the C1q assay). There was no difference in the prevalence of IgG class CIC. Patients with HDV infection also have significantly higher median levels of IgM K-CIC (P = .002), IgG K-CIC (P = .049), and IgG C1q-CIC (P = .008). In patients with HDV infection, there was positive correlation between IgM C1q-CIC and transaminase levels (r = .519, P = .016 for AST; r = .500, P = .021 for ALT). There was no difference in the prevalence of HBsAg containing CIC between patients with HDV infection (76.19%) and those without (74.60%). In conclusion, IgM class CIC are the major CIC and correlate with disease activity in HDV infection. CIC may play a role in the pathogenesis of HDV infection.

Identification of virus components in circulating immune complexes isolated during hepatitis A virus infection.

Circulating immune complexes were isolated by conglutinin affinity chromatography during the course of hepatitis A virus infection in a chimpanzee. Characterization of circulating immune complexes showed that most of the hepatitis A virus-specific antibody was IgM, that IgG was present and that C3d and fibronectin were also present. Hepatitis A virus capsid polypeptides were identified in the circulating immune complexes and polypeptides in the molecular weight range of 63 to 67 kDa having immunological determinants common to both C3d and hepatitis A virus. Hepatitis A virus-RNA was detected in these circulating immune complexes using the polymerase chain reaction for in vitro amplification of nucleic acid and suggests the circulating immune complexes contain intact virus.