Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enz... more Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enzyme immunoassay (EIA) to detect viral antigen and by reverse transcription-PCR amplification followed by ethidium bromide staining (PCR-ETBr) or nucleic acid hybridization (PCR-NA) to detect viral genetic material. A gradation of sensitivity was observed in the detection of virus by the three methods. In persons who had detectable virus, serial stool samples were found to be positive by EIA for up to 24 days after the peak elevation of liver enzymes. Viral genetic material could be detected by PCR-ETBr for up to 34 days and by PCR-NA for up to 54 days after the peak elevation of liver enzymes. After intravenous inoculation of tamarins with stool suspensions categorized as highly reactive for HAV (positive by EIA, PCR-ETBr, and PCR-NA), moderately reactive (positive by PCR-ETBr and PCR-NA), or weakly reactive (positive by PCR-NA), only tamarins infected with highly reactive stool suspensions (EIA positive) developed HAV infection. We conclude that positivity of stool specimens for HAV by PCR-ETBr or PCR-NA indicates a lower potential for infectivity, compared to that of EIA-positive stools. Hepatitis A virus (HAV) is most commonly transmitted by the fecal-oral route. Several aspects of the pathogenesis of HAV infection, including the duration of HAV excretion in stool and the duration of infectivity, are not fully characterized. In addition, the relationship between detection of HAV genetic material in stool and infectivity has not been established. During an outbreak of hepatitis A in an institution for the developmentally disabled in August 1990, we collected serial stool specimens from patients with HAV infection. These stool specimens were evaluated for the presence of HAV and for infectivity in tamarins. The objectives of these studies were (i) to assess the duration of HAV excretion detected by enzyme immunoassay (EIA) and the PCR in adults and (ii) to evaluate the relationship between detection of HAV antigens and genetic material in stools and animal infectivity. MATERIALS AND METHODS Patient identification and specimen collection. In August and September 1990, an outbreak of hepatitis A occurred among residents at an institution for the developmentally disabled in Boulder County, Montana. Overall, 36 of 174 residents at the institution developed HAV infection, defined as positivity for immunoglobulin M antibody to HAV (IgM anti-HAV). The average age of the patients was 39 years (range, 28 to 59 years). In a retrospective cohort study conducted to identify risk factors for the acquisition of hepatitis A, frozen strawberries were implicated as the source of infection. The details of this outbreak investigation have been presented in a previous publication (10). At the outset of the outbreak investigation, 13 residents were identified with symptomatic acute hepatitis A, and immune globulin (IG) was administered to 143 other residents determined to be susceptible by serologic testing. These residents were then monitored by weekly serologic testing for liver enzyme elevations and hepatitis A markers, for up to 8 weeks. During the subsequent 2 weeks, an additional 23 (16%) of these 143 residents became positive for IgM anti-HAV (Abbott Laboratories, North Chicago, Ill.). Of these 23 residents, 7 (30%) had symptomatic HAV infection (jaundice), and 16 (70%) were identified by IgM anti-HAV seroconversion. IgM anti-HAV is found only associated with acute cases of HAV and would therefore not be present in the IG. Serum specimens were tested at the Hepatitis Branch, Centers for Disease Control and Prevention, for total and IgM anti-HAV by EIA (Abbott Laboratories). Virus detection. For patients with elevated liver enzymes, stool specimens were collected and evaluated for evidence of HAV. Stool suspensions were prepared at a 20% (wt/vol) concentration in phosphate-buffered saline (pH 7.4) and clarified by centrifugation at 7,000 g for 10 min at 4°C. Virus antigen was detected by EIA with rabbit anti-HAV as the capture antibody and hyperimmune chimpanzee anti-HAV serum as the detector antibody (16). The presence of viral nucleic acid was determined by immunocapture of the virus followed by reverse transcription-PCR amplification using primers targeted to the VP1 amino terminus (13). Amplified products were separated by agarose gel electrophoresis followed by detection with ethidium bromide staining (ETBr) or nucleic acid hybridization (NA) (12). Animal infectivity of virus antigen of RNA-positive stools. Six anti-HAV-negative tamarins, Saguinus mystax, were divided into three groups of two animals each. The animals were cared for and housed under Association for the Assessment and Accreditation of Laboratory Animal Care International, Inc.-approved animal husbandry conditions at the Animal Resources Branch, Scientific Resources Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, as outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. Each group was intravenously inoc-ulated with a different stool specimen from patient 1. Intravenous inoculations were used to ensure optimal infectivity. The stool specimens were categorized by the level of detectable viral antigen or RNA. Group 1 was inoculated with 1 ml of filtered human stool suspension that was positive for viral antigen, as determined by EIA, and contained viral nucleic acid as determined by a strong ETBr band. Group 2 was inoculated with 1 ml of stool suspension that did not contain sufficient viral antigen to be detected by EIA but did contain viral nucleic acid, as determined by PCR amplification, and resulted in a weak band by ETBr. The third group of tamarins were inoculated with 1 ml of stool suspension that did not contain sufficient viral antigen for EIA detection but was positive for viral nucleic acid, as determined by PCR amplification followed by NA (PCR-NA). Serum specimens were collected twice a week and tested for liver enzyme levels and total anti-HAV (HAVAB; Abbott laboratories). Stools, collected daily and pre
Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enz... more Fecal excretion of hepatitis A virus (HAV) in 18 patients with HAV infection was evaluated by enzyme immunoassay (EIA) to detect viral antigen and by reverse transcription-PCR amplification followed by ethidium bromide staining (PCR-ETBr) or nucleic acid hybridization (PCR-NA) to detect viral genetic material. A gradation of sensitivity was observed in the detection of virus by the three methods. In persons who had detectable virus, serial stool samples were found to be positive by EIA for up to 24 days after the peak elevation of liver enzymes. Viral genetic material could be detected by PCR-ETBr for up to 34 days and by PCR-NA for up to 54 days after the peak elevation of liver enzymes. After intravenous inoculation of tamarins with stool suspensions categorized as highly reactive for HAV (positive by EIA, PCR-ETBr, and PCR-NA), moderately reactive (positive by PCR-ETBr and PCR-NA), or weakly reactive (positive by PCR-NA), only tamarins infected with highly reactive stool suspensions (EIA positive) developed HAV infection. We conclude that positivity of stool specimens for HAV by PCR-ETBr or PCR-NA indicates a lower potential for infectivity, compared to that of EIA-positive stools. Hepatitis A virus (HAV) is most commonly transmitted by the fecal-oral route. Several aspects of the pathogenesis of HAV infection, including the duration of HAV excretion in stool and the duration of infectivity, are not fully characterized. In addition, the relationship between detection of HAV genetic material in stool and infectivity has not been established. During an outbreak of hepatitis A in an institution for the developmentally disabled in August 1990, we collected serial stool specimens from patients with HAV infection. These stool specimens were evaluated for the presence of HAV and for infectivity in tamarins. The objectives of these studies were (i) to assess the duration of HAV excretion detected by enzyme immunoassay (EIA) and the PCR in adults and (ii) to evaluate the relationship between detection of HAV antigens and genetic material in stools and animal infectivity. MATERIALS AND METHODS Patient identification and specimen collection. In August and September 1990, an outbreak of hepatitis A occurred among residents at an institution for the developmentally disabled in Boulder County, Montana. Overall, 36 of 174 residents at the institution developed HAV infection, defined as positivity for immunoglobulin M antibody to HAV (IgM anti-HAV). The average age of the patients was 39 years (range, 28 to 59 years). In a retrospective cohort study conducted to identify risk factors for the acquisition of hepatitis A, frozen strawberries were implicated as the source of infection. The details of this outbreak investigation have been presented in a previous publication (10). At the outset of the outbreak investigation, 13 residents were identified with symptomatic acute hepatitis A, and immune globulin (IG) was administered to 143 other residents determined to be susceptible by serologic testing. These residents were then monitored by weekly serologic testing for liver enzyme elevations and hepatitis A markers, for up to 8 weeks. During the subsequent 2 weeks, an additional 23 (16%) of these 143 residents became positive for IgM anti-HAV (Abbott Laboratories, North Chicago, Ill.). Of these 23 residents, 7 (30%) had symptomatic HAV infection (jaundice), and 16 (70%) were identified by IgM anti-HAV seroconversion. IgM anti-HAV is found only associated with acute cases of HAV and would therefore not be present in the IG. Serum specimens were tested at the Hepatitis Branch, Centers for Disease Control and Prevention, for total and IgM anti-HAV by EIA (Abbott Laboratories). Virus detection. For patients with elevated liver enzymes, stool specimens were collected and evaluated for evidence of HAV. Stool suspensions were prepared at a 20% (wt/vol) concentration in phosphate-buffered saline (pH 7.4) and clarified by centrifugation at 7,000 g for 10 min at 4°C. Virus antigen was detected by EIA with rabbit anti-HAV as the capture antibody and hyperimmune chimpanzee anti-HAV serum as the detector antibody (16). The presence of viral nucleic acid was determined by immunocapture of the virus followed by reverse transcription-PCR amplification using primers targeted to the VP1 amino terminus (13). Amplified products were separated by agarose gel electrophoresis followed by detection with ethidium bromide staining (ETBr) or nucleic acid hybridization (NA) (12). Animal infectivity of virus antigen of RNA-positive stools. Six anti-HAV-negative tamarins, Saguinus mystax, were divided into three groups of two animals each. The animals were cared for and housed under Association for the Assessment and Accreditation of Laboratory Animal Care International, Inc.-approved animal husbandry conditions at the Animal Resources Branch, Scientific Resources Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, as outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. Each group was intravenously inoc-ulated with a different stool specimen from patient 1. Intravenous inoculations were used to ensure optimal infectivity. The stool specimens were categorized by the level of detectable viral antigen or RNA. Group 1 was inoculated with 1 ml of filtered human stool suspension that was positive for viral antigen, as determined by EIA, and contained viral nucleic acid as determined by a strong ETBr band. Group 2 was inoculated with 1 ml of stool suspension that did not contain sufficient viral antigen to be detected by EIA but did contain viral nucleic acid, as determined by PCR amplification, and resulted in a weak band by ETBr. The third group of tamarins were inoculated with 1 ml of stool suspension that did not contain sufficient viral antigen for EIA detection but was positive for viral nucleic acid, as determined by PCR amplification followed by NA (PCR-NA). Serum specimens were collected twice a week and tested for liver enzyme levels and total anti-HAV (HAVAB; Abbott laboratories). Stools, collected daily and pre
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