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- Hepatitis A: Old and New

The specific detection of hepatitis A infection was first accomplished in 1973 using immune electron microscopy of fecal extracts to visualize virus-like particles 78. Antibody in convalescent-phase serum samples from persons with experimentally or naturally acquired infection aggregated the virus and permitted its visualization by electron microscopy. When serum collected before infection or early in the disease was used, few or no virus particles were identified. This research technique was successfully used to investigate the period of infectivity but could not be employed for routine diagnosis of large numbers of clinical samples. The next step was the development of complement fixation 200 and immune adherence 172 tests for detection of serum antibody to HAV antigens in 1974. This required a source of HAV antigen, supplied by liver extracts from marmosets infected with a Costa Rican strain of HAV, CR326.

Despite being cumbersome, the immune adherence test quickly provided a wealth of important data about hepatitis A infection. With the original description of the test came demonstration of simultaneous infection with both HAV and HBV; evidence that HAV antibodies were acquired early in life in areas of high prevalence; association of low socioeconomic status with seropositivity in areas of low incidence; persistence of antibody for at least 7 years; an antigenically related or identical virus infection in chimpanzees, grivets, and rhesus monkeys not experimentally infected; and detection of various quantities of antibody in lots of immune serum globulin 172.

Both the complement fixation test and the immune adherence test were used to examine seroconversion following experimental infection with the MS-1 strain of hepatitis 137. The complement fixation test was not as specific or as sensitive as the immune adherence test. Using the immune adherence test, seroconversion was demonstrated in 20 of 20 infected persons. Antibody was detectable soon after the onset of clinical hepatitis but was present within the first week in only 45%; in 20% detectable seroconversion was delayed for at least 2 weeks after disease onset. Nevertheless, a test that could be used for diagnosis of acute hepatitis A infection was now available. Hemagglutination assays for HBV surface antigen and antibody to HBV core and surface proteins were developed in 1970. Consequently, by 1975, acute viral hepatitis could be ascribed to either HAV or HBV, permitting the recognition of viral non-A, non-B hepatitis (hepatitis C and hepatitis E).

In 1975, solid-phase RIAs developed for the detection of HAV antigen were modified to measure antibody 203. A comparison between immune adherence, immune electron microscopy, and RIA demonstrated that each test was able to detect seroconversion following inoculation with MS-1 virus 69. Antigen partially purified from stool was equivalent to marmoset liver-derived viral antigen in these assays. RIAs were also modified to use minimal quantities of viral antigen and to assay anti-HAV IgM antibody 38.

A competitive binding assay (HAVAB; Abbott Laboratories, North Chicago, Ill.) was developed by 1978 to improve sensitivity 37. In this assay, antibody in patient serum competes with radiolabeled antibody for HAV. The assay was also adapted to measure anti-HAV IgM, which was detected in acute-phase sera but not in convalescent-phase sera 37. However, the absorption of IgG from samples to measure IgM reactivity was difficult to perform, and the resultant assay lacked reliable specificity. In 1980, an alternative technique was developed (HAVAB-M; Abbott Laboratories) in which IgM antibody was directly selected and anti-HAV activity was then measured 63. With these improvements, anti-HAV IgM antibody was detected at the time of onset of symptoms in most patients. IgM titers decreased in the weeks after onset and then became undetectable. This assay could thus be used to diagnose acute hepatitis A at the time of clinical symptoms.

Diagnostic Accuracy

The sensitivity, specificity, and positive predictive value of quantitating IgM-specific anti-HAV was determined in a cluster of cases that occurred in 1979 using normal blood donors as the control population 234. The sensitivity of IgM anti-HAV measurement for acute hepatitis was 100%, the specificity was 99%, and the positive predictive value was 88%. Since its introduction and widespread use, diagnostic difficulties have been uncommon. Occasionally, the test is negative at the time of clinical presentation, but repeat testing 1 to 2 weeks later usually demonstrates positivity 112. One possible explanation for this observation is that dilution of serum before assay, in order to prevent false-negative results, could result in loss of reactivity in sera with low titers. In two episodes of mild acute infection in vaccinees, the appearance of IgM anti-HAV positivity was delayed until convalescence 125, an observation that has not been explained. False-positive Epstein-Barr virus serologies 79 are less of a diagnostic problem than is prolonged positivity in the absence of hepatitis 130, 220.

The length of time that the IgM anti-HAV test remains positive varies 130. In 37 patients followed until the disappearance of antibody, the majority, 32 of 37 (86%), were IgM anti-HAV negative by 7 months after onset, defined as jaundice in all but two anicteric cases, for which the onset of symptoms was used. Twenty-six of 37 (70%) were negative for IgM anti-HAV by 4 months. All cases demonstrated a decrease in titer (positive test value closer to the negative cut-off) before becoming negative in the assay. In contrast, IgM anti-HAV positivity was prolonged beyond 7 months in five individuals whose last positive test was recorded between 9 and 12 months after onset. Eventually, they each had negative IgM anti-HAV test results. In most patients (47 of 50), the biochemical evidence of hepatitis had resolved either prior to or by the time of disappearance of IgM anti-HAV. Of the remaining three patients, two eventually normalized biochemical hepatitis and the third was lost to follow-up. In a second study, two of six patients were IgM anti-HAV positive (low titer) 30 to 32 months after the onset of hepatitis A 220. A diagnostic dilemma may arise if a patient has unrecognized chronic hepatitis before contracting HAV. The persistence of IgM anti-HAV positivity for more than 12 months together with an unrelated and unidentified cause of hepatitis could potentially lead to an incorrect diagnosis of chronic hepatitis A.

Future Assays

In vaccine trials, the detection of antibody often requires a more sensitive assay because vaccine-induced antibody titers are generally lower than those induced by natural infection. Noninvasive tests, i.e., not requiring blood samples, are also useful for screening large populations. The development of a highly sensitive assay that is specific for IgG anti-HAV and can measure antibody in saliva after vaccination is promising 182. The assay was validated using paired saliva samples from travelers undergoing vaccination. In practice, antibody titers are not measured routinely after vaccination since the response rate to the vaccine and its effectiveness are so high (see below), and a highly sensitive assay therefore has little utility. In contrast, a cheap and robust assay for screening prior to vaccination would be extremely useful in many clinical scenarios (see below). Most assays have used HAV produced in tissue culture as a source of antigen. In the future, recombinant HAV antigen may provide a less costly alternative 143.

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