Clinical Virology: Diagnosis and Virologic Monitoring

Phase

Lab characteristics

Acute HBV infection

– HBsAg positive <6 months, anti-HBc IgM positive

– HBeAg positive, HBV DNA positive

– Normal ALT or elevated ALT (symptomatic patients have usually >10× ULN)

Chronic “Immuntolerant” HBeAg positive HBV infection (high replicative HBsAg carrier)

– HBsAg positive >6 months, anti HBc positive

– HBeAg positive, anti-HBe negative, HBV DNA >2,000 IU/ml, usually >10⁶ IU/ml

– Normal ALT or ALT <2× ULN

– Histology: mild or no liver necroinflammation and no or slow progression of fibrosis

Chronic “immune reactive” HBeAg positive hepatitis B

– HBsAg positive >6 months, HBeAg positive

– HBeAg positive, anti-HBe negative, HBV DNA >2,000 IU/ml

– Elevation of ALT ≥2-fold ULN (persistent high or fluctuating levels)

– Histology: moderate or severe liver necroinflammation and more rapid progression of fibrosis

“Inactive” HBeAg negative HBsAg carrier (Low replicative HBsAg carrier)

– HBsAg positive >6 months, HBeAg negative, anti-HBe positive

– HBV DNA <20,000 IU/ml, ideally <2,000 and HBsAg <1000 IU/ml

– Normal ALT or <2-fold ULN (multiple measurements required)

– Histology: mild or no liver necroinflammation and no or mild fibrosis

Chronic HBeAg negative Hepatitis B

– HBsAg positive >6 months, anti-HBc positive

– HBeAg negative, anti-HBe positive, HBV DNA >2,000 IU/ml

– Elevation of ALT ≥2-fold ULN (persistent high or fluctuating levels)

– Histology: moderate or severe liver necroinflammation and more rapid progression of fibrosis

Hepatitis delta coinfection

– HBsAg positive, anti-HBc positive

– Anti-HDV positive

– If HDV-RNA positive: active infection

– Often HBeAg negative and low HBV DNA levels

Occult HBV infection

– HBsAg negative, anti-HBs negative

– Anti-HBc positive (confirmed)

– HBV DNA detectable

– Normal/elevated ALT serum activity

Anti-HBc only

– Anti-HBc positive (confirmed)

– HBsAg negative, anti-HBs negative or <10 IU/l

– Normal ALT serum activity

Clinical resolved HBV infection

– Anti-HBc positive and anti-HBs ≥10 IU/l

– HBsAg negative

– HBV DNA negative

– Normal ALT serum activity

Vaccinated

– HBsAg negative

– Anti-HBc negative

– Anti-HBs >10 IU/ml

ULN upper limit of normal, IU international units, ALT alanine aminotransferase

HBsAg is determined with great sensitivity and specificity using an enzyme immunoassay [10–12]. Quantitative testing is possible and becomes increasingly popular in recent years as a tool to further differentiate the various states of HBV infection [13, 14]. HBsAg reflects the transcriptional activity of the cccDNA and is a useful surrogate marker to predict the natural course of HBV-infection and for treatment guidance [15, 16]. Escape variations of HBsAg epitopes, which do not bind to the antibodies that are used for detection, may cause a false-negative test [17, 18]. Two assays for HBsAg quantification have become commercially available, the Architect HBsAg assay (Abbott Diagnostics, Abbott Park, IL, USA) and the Elecsys HBsAg II quant assay (Roche Diagnostics, Indianapolis, IN, USA). Results from both assays are strongly correlated (r > 0.96) [19, 20].

HBeAg is linked to the activity and the clinical course of HBV infection and may aid as a prognostic marker [21]. Therefore, regular testing for HBeAg/anti-HBe is useful in the evaluation of the clinical course of the disease and in monitoring the therapy to assess HBeAg to anti-HBe seroconversion. It should be noted that HBV variants with mutations in the precore or core promoter region exists [22]. In these cases, HBeAg is commonly not detected and hepatitis is the result. In this so-called HBeAg negative chronic hepatitis B , the patients are infected either with a variant virus, bearing nucleotide substitutions at position 1896 (G1896A) in the precore region of the preC/C gene, which prevents the HBe protein from being synthesized. The second group of polymorphisms is located within the core promoter region at positions 1762 (A1762T) and 1764 (G1764A), which down-regulate HBe protein production up to 70 %. In addition other mutations have also been described [23]

The presence of anti-HBc antibodies is associated with ongoing or resolved HBV infection and the detection of the IgM and IgG immunoglobulin classes can help to distinguish between acute or chronic HBV infection. Anti-HBc-IgM is found in high concentrations during acute hepatitis B and drops subsequently [24]. However, during acute exacerbation of chronic HBV infection, anti-HBc-IgM may increase [25]. Anti-HBc persists also in patients with resolved HBV infection and immunization, indicated by anti-HBs levels >10 IU/ml.

Detection of HBV DNA may help tremendously in the diagnosis of HBV infection and plays an important one for the prognosis of the disease [26, 27]. In case of unclear results for HBsAg and anti-HBc, detection of HBV DNA can confirm HBV infection. Furthermore HBV DNA is a major tool for the monitoring of HBV treatment. Both copies/ml and IU/ml are used as units in viremia. Five copies of HBV DNA correspond to about 1 IU. An international standard for HBV DNA concentrations has been defined by the WHO [28] and it is recommended to use IU/ml. The lower limit of detection is around 6–20 IU/ml with modern real-time PCR assays. To maintain comparability, the same test should be used in longitudinal assessment of a patient preferentially.

HBV Genotypes

There are eight know HBV genotypes (A–H) which have distinct geographical distributions. In Europe, genotypes A and D are most prevalent while in Asia genotypes B and C dominate. Genotypes E and F may have originated in aboriginal populations of Africa and the New World. In addition there are at least 24 subtypes. For example, genotype A has two subtypes: Aa (A1) in Africa and Asia and Ae (A2) in Europe and North America. There are also different subtypes for other genotypes with regional differences. Details see Chap. 3. The different genotypes have some clinical relevant differences. Genotype C has been associated with faster disease progression and HCC development [29]. Patients with genotypes A and B respond better to interferon alpha treatment compared to genotypes C and D [30]. However, HBV genotyping is not strongly recommended by current guidelines [2] because of the limited relevance. Although genotypes have been associated with response to IFN, the positive or negative predictive value is not high and other predictors (HBV DNA <10⁸ IU/ml, ALT >2–5× ULN, HBsAg kinetics) exist [2, 4].

Non-serologic/Molecular Assessments for HBV Infection

Although the diagnosis of HBV infection is a serologic diagnosis, further assessments are important to provide adequate care for the infected persons and prevent spreading of the disease. A thorough medical history should be taken, specifically asking for risk factors (e.g., blood transfusions, i.v. drug use) and family and partner history, which may facilitate further testing. Family members or partners of persons infected with HBV must be offered diagnostic testing for HBV infection and vaccinations.

A physical examination helps to identify persons with advanced liver disease. Laboratory testing for possible coinfections (HDV, HIV, HCV) is mandatory. Clinical chemistry laboratory tests for liver inflammation, blood count, as well as blood coagulation status help to assess liver function and the degree of liver injury. If advanced liver disease is suspected, additional tests (albumin, bilirubin and parameters for renal function) should be done to determine the synthesis output of the liver and detect possible complications. A liver ultrasound examination also aids in the assessment of the degree of liver injury and is indispensable in the diagnosis of hepatocellular carcinoma.

Liver biopsy is important for the diagnosis, staging and prognosis of HBV infection. Histological evaluation of a liver biopsy helps to clarify the following issues:

Assessment of the inflammatory activity (grading);

Assessment of extent of fibrosis (staging);

Etiologic statements (especially comorbidity).

Several scoring systems exists for the staging and grading of liver diseases (e.g., ISHAK, Desmet) [31, 32]. The histologic workup should be done by an experienced pathologist, especially in regard of potential concomitant liver diseases if the etiology is unclear and no firm diagnosis can be made by the assessment of seromarkers.

Presently, liver biopsy is still considered as gold standard with respect to fibrosis evaluation. However, if no doubt regarding the etiology exists, noninvasive tests like transient elastography can also be used to reliably assess the extent of fibrosis and even complications of liver cirrhosis (e.g., portal hypertension, hepatocellular carcinoma) [33–37]. However, influencing factors such as high ALT values need to considered [38].

Virologic Monitoring

Virologic Monitoring During Acute HBV Infection

Acute HBV i nfection should be monitored until anti-HBs seroconversion has occurred. Testing for HBsAg and HBV DNA as well as the liver enzymes help to monitor the clinical course.

Initially serum transaminase activity should be monitored closely until normalization to recognize a potential fulminant liver failure. Once serum transaminases have normalized, testing for HBsAg and anti-HBs every 3–6 months until HBsAg is negative and anti-HBs >10 IU/ml should be done. If HBsAg is negative and anti-HBs <10 IU/ml, follow-up every 12 months is required and HBV DNA should be added to the test regimen to test for occult hepatitis B.

Virologic Monitoring During Chronic HBV Infection

Follow-up for chronic HBV infection should be done initially every 3 months for the first year to assess the clinical activity of the disease over time. The definition of the phase of chronic hepatitis B (Table 10.1) can be difficult because ALT and HBV DNA can fluctuate [4] (see Fig. 10.1). Testing should include clinical chemistry (liver inflammation and synthesis parameters, blood count, blood coagulation parameters), HBsAg, HBeAg (if initially positive) and quantitative HBV DNA.

Fig. 10.1

Virological markers during the different phases of HBV infection

After the initial year, it is recommended to perform follow-up visits every 6 months. In case of cirrhosis, visits should include ultrasound evaluation for HCC [2, 4].

In dependence of the clinical course of the disease and the clinical activity follow-up visits can be adjusted. In patients with fluctuating ALT and HBV DNA follow-up visits every 3 months should be considered. In patients without cirrhosis showing normal results for ALT and a stable HBV DNA <2,000 IU/ml in longitudinal monitoring, follow-up visits may be scheduled every 12 months [2].

Virologic Monitoring During Antiviral Therapy

Before treatment initiation clinical chemistry laboratory tests, quantitative HBV DNA, quantitative HBsAg, HBeAg should be performed. HBV genotyping can be performed if interferon therapy may be an option but is not reimbursed in many countries (see above).

If patients are treated with interferon alpha, it is recommended to test HBV DNA and HBsAg after 12 weeks of treatment. HBsAg kinetics have a good negative predictive value. Different stopping rules for HBeAg positive and HBeAg negative patients exist [16].

During treatment with nucleoside or nucleotide analogues (NUC), measurement of HBV DNA is important to assess the treatment response and should be performed every 3–6 month. In addition, HBeAg, anti-HBe, and HBsAg should be monitored every 6 month preferentially. Twelve months after anti-HBe seroconversion, treatment with NUC may be stopped [4]. Follow-up visits should be scheduled as in patients without treatment; however, closer monitoring (i.e., every 4 weeks) should be strongly considered directly after treatment withdrawal to detect an early relapse. An increase in HBV viremia is a sign of therapy failure, which might be caused by resistance to the antiviral agents or low adherence [4, 39]. To detect all possible resistance mutations, sequencing of the polymerase gene is the preferred method of detection. However, before further resistance testing is done, it should be assessed whether the antiviral medications was taken on a regular basis. Furthermore, it should be noted, that recent discoveries have identified the immune system and not viral resistance as the cause for a slow treatment response [40].

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