Hello, my name is Minh Dien Nguyen, and I'm from the Division of Gastroenterology and Hepatology at Stanford University Medical Center. If you have any questions, please do not hesitate to click on the button, Ask the Forum, where common issues regarding liver diseases are discussed. The title of my module is Interpretation of Viral and Other Serologies in Acute and Chronic Hepatitis. During this talk, I will discuss the interpretation of serologies for acute and chronic hepatitis A, B, and C. The learning objectives of this session would be to learn how to use serologies to diagnose acute viral hepatitis A, B, with hepatitis D, and hepatitis C, and hepatitis E. We will also learn how to use serologies to diagnose chronic viral hepatitis B, D, and C. An overview of the serologic diagnosis of acute hepatitis. In hepatitis A, the serology of choice is hepatitis A IgM antibody, and this is quite specific. For hepatitis B, we will rely on the presence of hepatitis B surface antigen and or hepatitis B IgM anti-core antibody. And the IgM anti-core antibody could be the only positive serologic test for acute hepatitis B if the patient is in a window period after hepatitis B surface antigen may have resolved. Diagnosis for hepatitis C will rely on hepatitis C antibody, and this may take some time to occur. So in the very acute settings in the first few weeks or a few months, this may or may not be positive yet. For hepatitis D, we will rely on the hepatitis D antibody test as a screening test, and this would be something to consider in patients with chronic hepatitis B or acute hepatitis B and could be positive in the setting of super infection of hepatitis B or co-infection. Hepatitis E IgM antibody is quite specific for acute hepatitis E and can be ordered. An overview for chronic hepatitis serologic diagnosis. For chronic hepatitis, the main ones we need to be concerned of is hepatitis B, C, and D. So for hepatitis B, the screening test would be hepatitis B surface antigen, and we could confirm this with hepatitis B DNA. And this could be positive in the setting of an active patient in replicative phase. For hepatitis C, the screening test would be hepatitis C antibody, and confirmatory test would be hepatitis C RNA. For hepatitis D, screening would be hepatitis B surface antigen and hepatitis D antibody, and we could request hepatitis D RNA for confirmation. So first, let's go into more details for hepatitis A. So hepatitis A virus is a PCORNA virus, and the serologic test available to us would be hepatitis A antibody IgM or IgG. Hepatitis A RNA testing is available mainly for research purposes and is not currently available commercially. Transmission route is fecal-oral. Transmission period would be 15 to 50 days. Mortality is very low, 0.1, 0.2% or so, but this can be significantly higher if the acute hepatitis A occurs in the setting of another chronic liver disease, such as chronic hepatitis B, and this has been well demonstrated in a large population-based study from Shanghai. Hepatitis A does not cause chronic hepatitis, and we have a very effective vaccine for hepatitis A, and the vaccine would be given at time zero and six months later. For patients with symptomatic acute hepatitis A, the treatment is largely supportive. The typical acute illness lasts about one to three weeks, but in some patients, it can become a prolonged illness, very rarely fulminant, mostly self-limited. The severity can increase with age, and these are the cases that we can see prolonged disease, prolonged jaundice. Extra-hepatic manifestations are rare, but can include skin rash, atrazia, relapse. With a second cycle, cholestatic hepatitis with prolonged jaundice is alluded to earlier. This causes vasculitis, aplastic anemia, red cell aplasia. Again, any of these would be very rare. And as mentioned earlier, it can be much more severe in patients with chronic liver disease. The next slide gives us a review on the serologic events in hepatitis A infection. So the first event is a shedding of hepatitis A RNA virus in the stool, and then hepatitis A RNA appearing in the serum. These tend to resolve within the next four to six weeks or so. ALT elevation tend to lag behind the appearance of hepatitis A RNA in the stool and the blood by a few weeks. And this tend to resolve in the next six, eight weeks or so. Hepatitis A IgM would peak at about two months after infection, and generally would resolve a few months later. IgG will occur fairly early as well, by two to four weeks, and this will remain lifelong. Hepatitis A serologies for acute infection would be hepatitis A IgM antibody. This would be diagnostic for acute hepatitis A. Hepatitis A does not lead to chronic infection. As mentioned earlier, hepatitis A vaccine is very effective and could be given. Patient who had prior exposure and infection would have lifelong or generally lifelong levels of detectable hepatitis A IgG antibodies and would not require vaccination again. Vaccination should be considered in patients with chronic liver disease or people traveling to endemic areas. Now let's go to some cases. This is serology of a patient with acute hepatitis. So this patient has a positive anti-hepatitis A antibody total, a positive hepatitis A IgM antibody, a negative hepatitis B surface antigen, a negative hepatitis B anti-core IgM antibody, and a negative hepatitis C antibody. Which acute hepatitis is this? Because the anti-hepatitis A IgM is positive, and this is diagnostic and specific for acute hepatitis A, this rules in acute hepatitis A quite definitively for us. With that, we will move on to hepatitis B. So hepatitis B illness could be acute or chronic. Acute hepatitis B is a largely asymptomatic disease with possibly a viral prodromal symptoms. Patients can have a little bit of fever and malaise. And usually, the patient is not aware of this. So most acute hepatitis B is not really diagnosed at the time of infection. Severe infection can occur if there is superinfection with hepatitis D or in the setting of immunosuppression. Chronic hepatitis B is much more common, or in another word, the patient presents much more commonly with chronic hepatitis B. And this is often an asymptomatic disease until very end stage, the liver disease or advanced HCC. Extra-hepatic manifestations are also rare and can include nephritis to nephrotic syndrome, vasculitis-associated athralgia. Chronic dual infection with hepatitis D leads to higher rates of progression to cirrhosis and liver cancer. Some overview of the characteristics of hepatitis B. Hepatitis B is a DNA hepatovirus. And they have many serologies, hepatitis B surface antigen, E antigen, hepatitis B core antibody, surface antibody, and E antibody. HBV DNA PCR is readily available. And that transmissions can be via sexual, vertical, from mother to babies, or a parenteral route. Incubation period is longer than hepatitis A, 15 to 180 days. Mortality of acute infection is a little bit higher, but still overall very low, 0.5 to 1% with acute infection. The chronicity rate is about 5% if individuals are infected as adults, and 95% if individuals are infected as children or infants. We have effective preventive measures with hepatitis B immunoglobulins for post-exposure prophylaxis or vaccine. Treatment, we also have many different treatments from injection with interferon to several well-tolerated and effective anti-hepatitis B medications, entecovir, tenofovir. The serologic course of acute hepatitis B infection usually starts out with appearance of hepatitis B surface antigen, followed by hepatitis B core antibody, IgM, as well as, and then later on IgG. And the IgM core antibody tends to be gone by about 32 weeks or so. And there is a window period between the appearance of the surface antibody and the disappearance of the surface antigen that is called the window period, in which the only serologic test that would be positive would be the hepatitis B core IgM. And this is tend to be between 20 to 30 weeks or so. In chronic infection, the hepatitis B surface antigen will persist. And the same for hepatitis B core antibody IgM. The patient may or may not have hepatitis B E antigen. Initially, E antigen could be positive, but there could be mutations develop and the patients can develop infections or chronic infections with a mutant type of virus that, despite active viral replication, the E antigen would be negative. So next is a table that hopefully can summarize the interpretation of hepatitis B serologic markers. If the patient's surface antigen is negative and surface antibody core antibody IgM and IgG were negative, so this patient has never been infected before. On the other hand, if we go down to the last line here, surface antigen negative, everything is negative, but if the core antibody is positive, this means that the patient has been infected before and has cleared the virus, but has not developed protective antibody. That scenario would be the patient in line four here, surface antigen negative, core antibody positive, and surface antibody positive. So this person was exposed before, cleared the infection, and developed protective antibody. We also have a scenario, the one next to the bottom here, when the patient have no evidence of chronic infection, negative surface antigen, no evidence of prior infection, negative core antibody, but has a positive surface antibody serology. So this person developed immunity by immunization, not from prior exposure. Patients who have positive surface antigen, these patients are chronically infected patients. So in these patients, the surface antibody clearly would be negative, the core antibody would be positive in both cases, but in the patients with also a positive core IgM, that patient has acute infection as opposed to chronic infection. Surface B has different genotypes, and these are distributed geographically. So North America, Western Europe, Africa, the most common one would be genotype A. B and C are prevalent in Asia. D is common in Southern Europe, Africa, and India. E, West Africa. F, Central and South America and Alaska. G, occasionally also in the U.S., France and Germany, and H, Central America. In studies that involves genotype A, B, and C, or mostly A, B, and C, genotype B is associated with less active disease and slower progression and lower incidence of liver cancer than genotype C. Genotypes A and B also tend to respond better to interferon therapy than genotype C and D. Some more serologies and interpretations of tests for hepatitis B. Hepatitis B can have different variants. Wild types are people with hepatitis B E antigen, and they have E antigen if they have active viral replication. The pre-core mutations are the patients who have mutations that abolishes the E antigen production. So in these patients, they can have active viral replication, but the E antigen would be negative, and the E antibody would be positive. Patient with core promoter mutation will downregulate the E antigen production. So if it downregulates enough, then the E antigen test may be negative. But if it's just partially downregulate, then the E antigen may still be positive. So in this case, the patient would have potentially a mixed viral population of wild type and mutant virus. And there are various treatment-induced mutations depending on which medications the patient use. We don't see this much in the U.S. or in the current time because the first-line therapies for hepatitis B in the last several years have been either enteglavir or tenofovir, and mutations are very rare with these newer medications. Ten, 20 years ago, treatment-induced mutations were a lot more common with the older drugs, such as lamivudine. The screening test for hepatitis B would be hepatitis B surface antigen, and we should also do the surface antibody test and offer vaccine for patient if the patient is not yet immune. Hepatitis B DNA PCR is expensive and should be used as confirmatory test for patients who are surface antigen positive, but should not be used as a screening test. Except for HIV positive and immunosuppressed patient, the HIV DNA PCR should be done because these patients may be so immunosuppressed that all serologic testings can be negative in actively infected patients. Hepatitis B core IgM should only be ordered if acute hepatitis B is suspected. If we are working up a chronic hepatitis patient, then we do not need hepatitis B core IgM. Hepatitis B surface antigen as well as hepatitis B core IgG should be tested in chemotherapy patients because if it's positive, we should consider antiviral therapy, prophylaxis, for patients with surface antigen, and in the case of core antibody patients, depending on the types of chemotherapy or immunosuppressant the patients get. For example, in patients who would undergo bone marrow transplant, even the core positive surface antigen negative patients will have a high rate of reactivation and antibiotic prophylaxis should be considered as well. Now let's go on a case. So in this next case here, we have positive total hepatitis A antibody, negative hepatitis A IgM, positive hepatitis B surface antigen, positive hepatitis B core IgM, and negative hepatitis C antibody. So because the hepatitis B surface antigen as well as the core IgM are positive, we can conclude that this patient has hepatitis B infection and acute hepatitis B infection. The patient also show a positive hepatitis A total antibody but negative IgM. So this patient either has received vaccination for hepatitis A or had been exposed to hepatitis A in the past and developed immunity. Another case for chronic hepatitis, so here we have positive hepatitis B surface antigen, positive core antibody, positive E antigen, negative E antibody, and negative hepatitis C antibody. So this patient has chronic hepatitis B virus and the wild type virus because the E antigen is also positive. Now next we have an asymptomatic healthcare worker that is negative for surface antigen, positive for hepatitis B core antibody, and also positive for hepatitis B surface antibody. So this means that the patient does not have chronic hepatitis B infection because surface antigen is negative. This patient has been exposed to hepatitis B before because the total core antibody is positive and this person also has immunity for hepatitis B with a positive hepatitis B surface antibody. And this immunity is most likely due to prior exposure to hepatitis B because the core antibody was also positive. Okay, another case, also an asymptomatic healthcare worker. Surface antigen is negative, core total antibody is negative, and surface antibody is positive. So this is a case of someone with immunity for hepatitis B from vaccine because there is no evidence of prior infection or exposure because the hepatitis B core antibody is negative. That concludes our session on hepatitis B. Next would be hepatitis C. This is a common infection in the general U.S. population with a prevalence about 1.7% or so. Acute hepatitis C is largely asymptomatic with possibly a viral prodromal symptoms and is often unrecognized, so just like hepatitis B. Chronic hepatitis C is also asymptomatic like hepatitis B until end-stage disease or advanced liver cancer develops. Other hepatic manifestations are also rare and also can include nephritis, nephrotic syndrome, cryoglobulinemia, and cutaneous tartar and arthralgia. So hepatitis C, as opposed to hepatitis B, is an RNA-FLAV-V virus. Hepatitis B is a DNA virus. Serology is much simpler. We only have a hepatitis C antibody. But for virology, we have very good, reliable hepatitis C RNA and hepatitis C genotypes. The primary route of transmission is parenteral. Incubation period is similar to hepatitis B, 15 to 160 days. Mortality in the acute setting is essentially zero. It's low in hepatitis B, but it can occur within hepatitis C that is almost never happen. The chronicity rate is 85%, even when adults are exposed to it, as opposed to only a 5% chronicity in hepatitis B in adults. We do not have a post-exposure globulin that can protect the patients, and we do not have a preventive vaccine. Currently, hepatitis C includes several oral direct-acting antiviral therapies. Now let's review the serological course for hepatitis C infection. So the hepatitis C antibody occurs within months of the exposure and infection, and will tend to last for years indefinitely. The hepatitis C RNA will also occur fairly quickly, in a matter of weeks, and would peak a few months later. Hepatitis C RNA would also persist for life in patients with chronic infection until they get successful treatment. But about 15% of the patients can resolve the infections spontaneously. And in these patients, the HCV RNA will resolve and will become undetectable. And this could occur within months or a few years. So let's review the diagnostic tests that we can consider for hepatitis C and the different situations here. So in chronic hepatitis C, the ALT can be normal or elevated. Hepatitis C antibody and RNA would both be positive. Hepatitis C can be called a carrier if the ALT is completely normal, and preferably liver biopsy is also normal, when the antibody and RNA are positive. The patient who have recovered from hepatitis C infection would have normal ALT, negative hepatitis C RNA, but the HCV antibody will persist. And these patients would not require any antiviral therapy. We can also have false positive of hepatitis C antibody. In this case, the RNA would be negative. And the patient does not have any risk factors for hepatitis C. Hepatitis C have six genotypes, six main genotypes, though a seventh genotype was recently proposed as well. Genotype 1, 2, and 3 are distributed worldwide. In the US, genotype 1 is the most common. Genotype 4 are restricted mostly in Africa and the Middle East. In Western countries, genotype 4 often occur in immigrants from endemic areas or in injection drug users. Genotype 5 is mostly in South Africa and expats from South Africa. Genotype 6 mainly in Southeast Asia and South China. Genotype 1, hepatitis C genotype, is traditionally one of the most important determinants of treatment response for antiviral therapy with interferon in the past. And to a large extent, it still affects our choice of direct acting antiviral therapies today. Hepatitis C screening is fairly simple. It only involves a hepatitis C antibody test, and it is recommended for all patients born between 1945 and 1965, the so-called birth cohorts, because the prevalence in these groups are very high. HCV screening is also recommended for patients with specific exposure risk, tattoos, injection drug users, cocaine snorting, HCV positive individuals, dialysis patients, and blood product risk recipients prior to 1992. One important thing to note is that patients from endemic areas often contract hepatitis C via iatrogenic exposure, and they should also be considered for screening even in the absence of specific exposure risk as mentioned above. Most of these patients would not be able to recall any specific exposure because these are just routine medical dental care that they have received from their home countries. Some of the endemic areas for hepatitis C include Egypt, about 18 percent, in parts of Southeast Asia, 5, 6 percent. The average prevalence for Asia is about 2 percent. It's also very high in many parts of the Middle East and Africa. The evaluation of patients with a positive hepatitis C antibody should first include hepatitis C RNA and hepatitis C genotype if the RNA is positive. Many labs now order a reflex testing. If RNA is positive, they would automatically do a genotype test. So let's review some serology sets for an injection drug users with acute hepatitis. So hepatitis A total antibodies negative, hepatitis A IgM negative, hepatitis B surface antigen negative, hepatitis B core IgM negative, hepatitis C antibody negative. So everything is negative. And the patient have acute hepatitis and he has a very major risk of exposure to hepatitis C. So it's likely that the patient may have contracted acute hepatitis C and early on the antibody is still negative. So if we check an RNA, then that would be positive and would confirm acute hepatitis C. Another scenario is with a remote intravenous drug users, drug users who has not been using for some time and he has chronic hepatitis. So on the serology for acute A and chronic hepatitis B are negative, but hepatitis C antibody is positive. So this patient most likely have chronic hepatitis C. And however, when we do hepatitis C RNA testing is negative. So in this patient with a very high risk for hepatitis C exposure, the most likely explanation for this is that the patient was exposed and was infected with hepatitis C in the past, but he clear his virus either on his own or by prior treatment. And that's why the hepatitis C RNA is negative. The next hepatitis that we will talk about will be hepatitis E. So hepatitis E in some ways are similar to hepatitis A in that it's usually an acute self-limiting infection that would resolve within four to six weeks, asymptomatic or causing acute hepatitis. It can lead to acute liver failure, but very rare unless it's in pregnant woman or patients with underlying chronic liver disease, immunosuppressed person, or patients who have a solid organ transplant history could develop chronic hepatitis E. So it is different from hepatitis A in that it could cause chronic disease, but in a very special setting. It's an RNA heparivirus. The only serology we have is hepatitis E antibody, IgM or IgG is not FDA approved. And hepatitis E RNA and genotypes are largely available in research lab. Transmission route is fecal-oral in the developing countries. And in developed countries, it's usually via consumption of undercooked or uncooked meat. Incubation, 15, 60 days, just like hepatitis B and C. Chronicity is low overall for acute infection, about 1%, but the third trimester pregnant woman, it can reach 10 to 30%. Chronicity can develop, but in special situation in immunosuppressed patients. We do not have specific treatment, so only supportive, and we do not have vaccine to prevent this, so hygiene is the key. So the next map here shows the areas of endemic hepatitis E. So you can see that it can cover most of the world here. So the hepatitis E prevalence would be high and the highest in parts of Africa, in the Indian subcontinent, many parts of the Middle East, as well as East Asia. So there are also different genotypes in hepatitis E. Genotype 1, Asia and Africa, 2 in Mexico and Africa, 3 worldwide, and genotype 4 has been reported in Asia. And there are some correlations with the route of transmission here. The most common mother-born hepatitis E outbreak in developing countries usually have genotype 1 and 2, and the genotype 3 and 4 associated with consumption of uncooked meat. Hepatitis E is clinically not distinguishable from other types of acute viral hepatitis, so we would have to rely on serologic diagnosis of hepatitis E IgM. Hepatitis E RNA can be done for a stool or blood specimen, but these are not widely available only in specialized research labs. So viral serologies are very helpful in the diagnosis of both acute and chronic hepatitis, as we have learned from this session. In the case of hepatitis C, we don't have a diagnostic test to differentiate between acute or chronic infection, so this would have to be differentiated largely by history. I hope you have enjoyed this presentation, and I invite you to access additional content on liver learning on this topic or any other related topics at your leisure. Thank you.

Minh Dien Nguyen

Stanford University Medical Center

viral serologies

acute hepatitis

chronic hepatitis

serological tests

hepatitis diagnosis

Fundamentals