University of Medicine and Pharmacy [302946]

University of Medicine and Pharmacy

„Iuliu Hațieganu”

Cluj-Napoca

Faculty of Medicine

License THESIS

Chronic hepatitis B in children

past and present situation at children´s hospital pathology department

Coordinator:

Assistant Professor Dr. Dan GHEBAN

Graduate:

Anja RETHER

2019

University of Medicine and Pharmacy

„Iuliu Hațieganu”

Cluj-Napoca

Faculty of Medicine

License THESIS

Chronic hepatitis B in children

past and present situation at children´s hospital pathology department

Coordinator:

Assistant Professor Dr. Dan GHEBAN

Graduate:

Anja RETHER

2019

General Review

1.1 Introduction

With more than 240 million individuals being chronically infected with hepatitis B worldwide, HBV infections still represent a [anonimizat],. Since the introduction of HBV vaccines in routine immunisation schedule in Romania in the year 1996, the prevalence of HBV infections in the general population has decreased dramatically.

Nowadays, [anonimizat] a liver biopsy for the diagnosis of hepatitis B in children.

These aspects raised the question of the importance of a microscopical histopathologic investigation obtained by an invasive liver biopsy at the children´s [anonimizat]-Napoca.

1.2 Routes of transmission

The hepatitis B virion is a 42 [anonimizat], partially double stranded DNA . [anonimizat], semen and saliva whereas only low concentrations are found in stool and breast milk.

As summarized in Table 1, the routes of transmission vary according to geographic regions.

Table 1 – Routes of transmission according to geographic regions

With about 4.4% prevalence, Romania is facing a high burden of chronic viral hepatitis due to insufficient infection control in the hospital setting decades ago. Horizontal household transmission of the virus is possible due to long term survival of the virus in the environment up to 7 days. Vertical intrauterine transmission to the embryo does not represent a [anonimizat]-born. Mothers who are HBeAg positive have a 70-90% [anonimizat]-HBe antibodies decreases the risk of transmission.

1.3 Clinical course

The clinical course of hepatitis B [anonimizat]- limited disease and a chronic disease.

The acute phase of a viral hepatitis B infection is asymptomatic in 65% of the cases. The remaining 35% [anonimizat]-[anonimizat], vomiting or jaundice.

Less than 1% [anonimizat] a transplant is not available.

Chronic viral hepatitis is defined as the presence of HBsAg in blood for more than six months . As depicted in Fig.1, the rate of chronicity depends on the age at the time of infection.

Figure 1 -[anonimizat], most of them being asymptomatic, therefore the disease is discovered incidentally, during routine examination. If symptoms are present, they are nonspecific like pain in the right upper quadrant, pruritus, joint pain or anorexia. As the liver disease progresses, signs and symptoms of portal hypertension id est splenomegaly, caput medusae may be found and finally a decompensated liver cirrhosis may be recognized by the presence of jaundice, ascites or peripheral edema . Extrahepatic manifestations may be present in 1-10% of HBV infected individuals but are more frequent in the acute phase. These manifestations include serum-sickness like syndrome, membranous glomerulonephritis, polyarteritis nodosa and papular acrodermatitis of childhood. Even though most of the time the evolution of the disease is rather benign, the lifetime risk of hepatocellular carcinoma of a person infected during childhood is about 9-24% and the incidence of cirrhosis 2-3% per year. There is a wide variation in clinical course and outcome of chronic HBV depending on the influence of various factors such as the severity of liver injury at the time of HBV replication arrest, alcohol consumption, co-infection with other viruses etc. .

1.4 HBV diagnostic

Nowadays, serologic assays are the first step of investigations in order to identify hepatitis B infections .

As summarized in Table 2, by evaluating specific HBV antigens and antibodies, one can distinguish an acute infection from a chronic course. The following immunologic markers should be taken into consideration:

HBsAg – Hepatitis B surface antigen

HBsAg is the „serologic hallmark„ of acute and chronic viral hepatitis B infection. It is the first antigen to appear approximately 1-10 weeks after exposure, representing the transcriptional activity of covalently closed circular DNA (cccDNA). The presence of HBsAg for more than six months implies a chronic infection. In case of a recent vaccination it is possible for 14 days to detect HBsAg. Chemiluminescent microparticle immunoassay (CMIA) is the technology of choice for the detection of HBsAg.

Anti- HBc antibodies – Hepatitis B core antibody

Hepatitis B core antigen is located on the surface of the nucleocapsid and their presence indicates active viral replication. These particles are not secreted into the blood .

Anti-HB core antibodies appear after HBsAg. IgM is the first one to rise, and remains detectable for about two years, whereas in patients who have recovered from an acute infection, IgG anti- HBc antibodies persist.

HBeAg and anti HBe antibodies

Hepatitis B envelope antigen is a viral protein of hepatitis B which is secreted and accumulates in serum indicating an active viral replication, during which transmission is possible. Seroconversion from HBe antigens to anti-HBe antibodies is directly related to a decrease in serum HBV and a remission of liver involvement .

Serum HBV DNA assays

In order to verify the test results of the serological markers and to quantify the viral load, nucleic acid testing for HBV DNA can be done.

The higher the viral load, the higher the infectivity. Real time PCR techniques are currently the preferred method to asses viral replication.

Biochemical markers

For the assessment and monitoring of liver disease, several biochemical markers are analysed: aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (AP), prothrombin time (PT), serum albumin and blood counts. The aminotransferases are not very sensitive as not all chronical infected individuals present with elevated test results. On the contrary, liver cirrhosis may be present with absolutely normal AST and ALT values. If a liver cirrhosis has installed, the synthetic functions of the liver including albumin and coagulation factors is impaired, leading to a prolonged prothrombin time and low serum albumin.

Table 2 – Summary of serological markers of hepatitis infection

1.5 Indications for liver biopsy

Nowadays, non-invasive methods are increasingly replacing the need for an invasive liver biopsy. Nevertheless, the invasive procedure for obtaining a tissue sample and its microscopic examination is still an important diagnostic tool for the following indications:

Abnormal liver function tests not explained by other investigations

If the laboratory results show abnormal liver function tests but other serological or imagistic techniques cannot find the cause for this abnormality, direct visualisation of the parenchyma showing pathological changes may help in finding the etiology. For instance, granulomatous hepatitis is a disease diagnosed only by liver biopsy.

Staging of chronic liver disease.

As the specificity of serological methods is often limited and elastography is not quite available, liver biopsy still represents the gold standard for staging liver fibrosis.

Chronic hepatitis

Liver biopsy is a very important tool for the evaluation and staging of chronic hepatitis. It offers the possibility to determine the severity of the disease by quantifying the amount of inflammation, necrosis or irreversible structural alterations and therefore contributing to the diagnosis of the disease. In addition to that, it can show histological evidence of etiological factors leading to the hepatitis.

Cholestatic liver disease

Regarding chronic obstructive biliary disease, a liver biopsy can determine irreversible damage and therefore result in the need of a liver transplant.

For the diagnosis of primary biliary cirrhosis, histologic evidence of nonsuppurative cholangitis and destruction of interlobular bile ducts represents the „sine qua non„

Cirrhosis

A liver biopsy can be done in order to confirm the clinical diagnosis of liver cirrhosis and to determine the etiologic factors of the injury. Furthermore, it is a useful tool to diagnose dysplasia and potential hepatocarcinoma, therefore determining the prognosis.

Alcoholic liver disease

A liver biopsy is not necessarily required in order to diagnose an alcoholic liver disease, but it can be done in order to evidence continuous alcohol abuse.

Liver transplantation

Even though a liver biopsy is not necessarily required before a liver transplant, it may be used to screen for conditions affecting the transplantation such as steatosis.

Space occupying lesions

Fine needle aspiration largely replaced the use of a more traumatic liver biopsy for the histological examination of a space occupying lesion. Core biopsies can be done for immunohistochemical staining in order to determine the origin of a tumour .

Fever of unknown origin

As microbiological and immunologic diagnostic methods have improved over time, the use of a liver biopsy for this cause has diminished. Nevertheless, liver biopsy is a possibility to diagnose several mycobacterial disorders including toxoplasmosis or histoplasmosis.

Systemic inflammatory or metabolic disease

Most of the metabolic diseases affect the liver, therefore a liver biopsy is an efficient tool in order to recognise specific pathological changes in the liver parenchyma of a metabolic storage disease e.g Gaucher or Amyloidosis.

Liver injury caused by therapeutic drugs

Possible histological findings encountered after therapeutic drug use include steatosis, cholestasis, acute viral hepatitis and many more depending on the agent.

Unexplained hepatomegaly

The two most common results of histopathological findings done for hepatomegaly are steatosis and nonspecific congestion which are clinically irrelevant.

1.6 The role and technical considerations of liver biopsies

Recently, the trend to replace invasive diagnostic methods with non-invasive ones, has limited the need for a liver biopsy due to the small but ever-present risk of unwanted complications. Nevertheless, there are some disadvantages related to these non-invasive methods e.g. their inability to accurately discriminate between early stages of fibrosis (F0-F2). In addition to that, when performing a liver biopsy one can evaluate the presence of other liver involving conditions such as a fatty liver disease, which can possibly be complicated by inflammatory changes or fibrosis. Non-invasive method used to classify mode and extend of liver inflammation are yet to be developed. Therefore, a liver biopsy still represents the gold standard for staging liver fibrosis and evaluating the degree of necro-inflammation.

There are several technical considerations one has to take into account in order to obtain an appropriate hepatic specimen for the histopathological examination. The quality of a specimen obtained varies with its size and the number of portal fields collected. Therefore, each biopsy should be obtained with a 16- or 14-gauge needle taking a 20- 25 mm long sample containing at least 11 portal tracts. There are different routes to access liver parenchyma which can be chosen under different circumstances.

Percutaneous biopsy

Ultrasound guided percutaneous biopsy has been the standard procedure for almost 50 years . This technique is easy to perform and appropriate for diffuse liver disease. On the other hand patient acceptance is low and the rate for complications ranging between 0,75- 14% and a mortality of 0,001% – 0,003%. Minor complications represented by transient and moderate pain, anxiety or abdominal discomfort are common (5-20%), whereas severe complications resulting in hemoperitoneum, biliary peritonitis or pneumothorax are rarely encountered.

Transjugular biopsy

This method is preferred in case the patient presents with ascites or an increased bleeding risk. The disadvantages are high costs due to the necessity of an interventional radiologist and an in-patient setting. If the sample obtained with a biopsy forceps is too small, it may interfere with adequate fibrosis staging.

Laparoscopic biopsy

A laparoscopic biopsy is applied if a focal or subcapsular lesion is present. The rate of detection of cirrhosis is higher due to the direct surface visualisation of the liver and in the case of possible complications for e.g. bleeding, it can be stopped instantly. This procedure is expensive and not available in all centres .

After the acquisition of a hepatic specimen an immediate fixation is necessary in order to ensure a good quality of the section .

1.7 Anatomical pathology of chronic hepatitis B

Macroscopically

The macroscopic appearance of a liver chronically infected with a hepatitis B virus can be that of a normal beefy red liver without any visible pathological changes . If the fibrosis is more advanced, focal or more diffused areas with a gritty structure or lobulated appearance indicating fibrosis of the septa and local regeneration are present . A yellowish discoloration of the hepatic tissue during these early stages indicates steatosis.

In case this process continues to progress, liver cirrhosis will install which can be easily recognized with the naked eye, by its diffuse nodularity and fibrous scarring . Commonly, hepatic cirrhosis caused by chronic viral hepatitis is macronodular or mixed macro- and micronodular but rarely exclusively micronodular . These nodules varying in size also vary in colour, ranging from beefy red to yellow due to fatty changes up to dark green, owing to cholestasis .

Histopathological

Routine stains, used in most of the laboratories for the examination of liver tissue are of 2 types; haematoxylin and eosin stains along with an iron and a trichome stain.

Acute hepatitis B pattern

The availability of serological markers and PCR technique eliminated the need for a liver biopsy, in order to confirm the diagnosis of an acute hepatitis stage. Microscopical histopathological examination of a liver specimen is performed only if the laboratory results are inconclusive. In this case the microscopical findings include lymphocytic infiltration of the portal tract, lymphocytic inflammation and swelling of hepatocytes. Furthermore, apoptotic bodies or Kupffer cells may be present, whereas ground glass opacities are absent. If the hepatitis is severe enough, bridging or confluent necrosis can be observed.

Chronic hepatitis B pattern

In the majority of cases a liver biopsy in chronic hepatitis is performed with the purpose of grading the inflammation and staging the fibrosis, as it is possible to recognize a hepatitis injury pattern, but difficult to determine its etiology.

Portal tract changes

The predominant pattern of histopathological changes in chronic hepatitis of any etiology is the lymphocytic infiltration of portal tracts. The grade of portal tract inflammation in chronic hepatitis B is commonly mild to moderate. The type of lymphocytes most commonly encountered in these lymphocytic infiltrates are

CD4+ T- helper/inducer cells and plasma cells . In 10- 20% of the cases lymphoid aggregates may be encountered and the presence of iron laden macrophages indicates the phagocytosis of hepatocyte debris. The terms „piecemeal necrosis„ or „periportal hepatitis„ were replaced by the term interface activity, describing inflammation of the portal tracts involving adjacent periportal hepatocytes, the so called limiting plate. If the inflammation is limited to the portal tracts not expanding into the lobule, it is considered inactive . Interface activity is not specific for chronic hepatitis B and can be encountered in various other etiologies, including autoimmune hepatitis, drug induced hepatitis or other viral infections leading to hepatitis. This invasion of mononuclear cells into the portal tract can lead to their enlargement .

Comparing acute with chronic viral hepatitis, portal infiltrates are much denser in the chronic form . In about 10% of the cases, the so called Poulsen lesions are present, recognized by lymphocytosis in association with stratification and vacuolization in the portal bile duct. Inflammation may also encompass inflammation of the endothelial cells lining portal veins leading to endothelitis, resulting in venous thrombosis .

Lobular changes

Minimal to mild lobular inflammation is encountered in 80% of cases infected with chronic hepatitis B, while the residual cases presenting with moderate lobular inflammation. Marked lobular inflammation is consistently detected with a HBV flare or a superinfection with viral hepatitis D. Irrespective of the grade, lymphocytes are the predominant cell type of infiltrates, followed by Kupffer cells in case of moderate or marked lobular inflammation. A frequently encountered histopathological aspect are sand glass nuclei, comprising hepatitis B core antigen (HBcAG) inclusions inside the nuclei of hepatocytes. These are best visualized using immunohistochemical stains and their quantity is positively correlated to the viral replication rate. These sand glass nuclei are not specific for viral hepatitis B and may be encountered in other pathologies including viral hepatitis D. If the grade of inflammation is more advanced, it is possible that the hepatocyte cytoplasm will also stain using immunohistochemistry, depending on the antibodies used and the HBeAG status. Another typical aspect of chronic hepatitis B are ground glass inclusions within the cytoplasm of hepatocytes. These histopathological peculiarities consist of hepatitis B surface antigen (HBsAg) located within the smooth endoplasmic reticulum of hepatocytes. Recent studies , suggest that two possible pre-S large surface protein(LHBS) mutations (pre S1 and pre S2) are responsible for the retention of HBsAg inside the smooth endoplasmic reticulum, (sER) leading to a blockage of nuclear transport of a DNA repair system ,resulting in genomic instability and consequently an increased risk for the development of hepatocellular carcinoma. This accumulation of viral protein triggers an impaired release of other cellular substances, contributing to the ground glass effect. Immunostains for HBsAg are the preferred technique in order to visualize ground glass containing hepatocytes. These are of 2 major types of pattern; Type I gound glass hepatocytes and Type II ground glass hepatocytes. Other less frequently encountered patterns are membranous staining and granular cytoplasmic staining. Pseudoground glass effects can be the result of drug effects on hepatocytes, this is not to be confused with chronic hepatitis B. In case of an HBV flare, moderate to marked active lobular inflammation in association with zone 3 periportal focal necrosis, bridging necrosis or confluent panacinar necrosis is present. The cell types involved in lobular hepatitis are identical to those found in interface activity, but their role distinction in scarring and progression of liver injury in chronic viral hepatitis remains a subject of further research . Focal necrosis is a term used to describe cell death of a larger group of hepatocytes within a lobule, which can be recognized by the presence of acidophil bodies (Councilman bodies), representing apoptotic hepatocytes and PAS positive, diastase resistant macrophages indicating previous cell death., or just cellular debris in association with collagen and reticulin fibres . If multiple lobules are affected by this pathological changes, confluent necrosis is present. . In case this process progresses, bridging necrosis involving several central veins and portal tracts installs with high risk of further evolution towards liver tissue scarring and cirrhosis .

Granulomas and hepatitis B

In rare cases of approximately 1-2% of chronic hepatitis B, the patient presents with granulomas of activated macrophages, the so called epithelioid histocytes surrounded by lymphocytes and fibroblasts. There are several different etiologies for hepatic granulomas, including infectious (HBV, Epstein- Barr virus, Mycobacterium tuberculosis) and non-infectious diseases such as sarcoidosis or primary biliary cirrhosis. That is why acid- fast bacillus testing should be done in order to exclude a mycobacterial infection. In contrast to sarcoidosis, hepatic granulomas caused by chronic hepatitis B do not contain polarizing material observed under polarizing light microscopy.

Liver cell dysplasia

Chronic infections with hepatitis B virus lead to liver cell changes previously referred to as liver cell dysplasia, but this term is no longer accurate, as its pre-neoplastic role is not yet fully understood . Nevertheless, more recent studies suggest a possible relation between liver cell change and development of hepatocellular carcinoma (HCC). There are 2 types of changes that occur in the liver cell. The first one is small liver cell change (SCC) which indicates the presence of aggregates of hepatocytes with a small cytoplasmic volume, but otherwise normal nuclear and cytoplasmic cytology leading to an increased nuclear to cytoplasmic volume . A particular feature of this small cell change is a higher proliferation rate than normal and hepatocytes leading to chromosomal instability, favouring the emergence of uncontrolled cell growth and therefore the development of HCC. However, the correlation is not yet fully established, with several studies showing a broad variety of results.

The second type of cell change is large liver cell change (LCC), indicating normal or increased cytoplasmic volume and nuclear changes including pleomorphism, multinucleation and hyperchromasia. None of the above listed findings is specific for viral hepatitis B infection, but rather for any chronic liver disease associated with advanced fibrosis.

Hepatocyte oncocytosis

The presence of hepatocyte oncocytosis is not specific for chronic hepatitis B infections, but it represents a more frequent finding than in other liver pathologies. Oncocytes can be recognised microscopically by their ballooned eosinophilic cytoplasm, containing abundant mitochondria responsible for its granular aspect. This finding is more common in cirrhotic liver tissue, but it could also be present in non-cirrhotic liver. The prognostic value of this finding needs to be determined.

Immunohistochemical stains (and in situ hybridisation, EM)

Immunohistochemical staining, using specific anti-HBs and anti-HBc antibodies is not necessary in routine liver biopsies in order to determine the stage and grade of the disease. Nonetheless, it represents a helpful diagnostic tool, in case the laboratory testing is not accessible. The pattern of immunohistochemical stains is in loose correlation with the clinical category of disease. There are three successive phases of chronic viral hepatitis B infection :

Virus tolerance Virus clearance Integration phase

During the immunotolerant phase, only mild hepatic inflammation is present. Immunohistochemical stains show predominantly nuclear HBcAg and cytoplasmic HBeAg, the latter indicating high viral replication rate. HBsAg is present in the cytoplasm and membrane of some hepatocytes, resulting in a honeycomb pattern.

During the viral clearance phase, the immune system eliminates infected hepatocytes, seroconversion from HBeAg to anti-HBe antibodies can take place and the viral replication rate decreases. Immunohistochemical stains during this phase reveal HBcAg in nuclear, cytoplasmic and membranous location. The presence of cytoplasmic HBcAg correlates with hepatocyte injury and histopathology reveals more severe lesions up to confluent necrosis. Small amounts of HBsAg are present in hepatocyte cytoplasm and membrane, as well as HBeAg in hepatocyte nucleus and cytoplasm. If the immune mechanism of elimination is not complete, the viral integration in the host genome persists. During this last phase, viral replication is not present but HBsAg which is synthetized by hepatocytes containing HBV DNA can be detected. HBsAg accumulates in groups of hepatocytes.

Fibrosing cholestatic hepatitis B

Fibrosing cholestatic hepatitis B is a rare subtype of chronic hepatitis B, most commonly present in immunosuppressed patients. In these cases, lobular cholestasis associated with hepatocyte swelling or ballooning degeneration may be present. Additionally, trichrome stain reveals ductal proliferation in the portal tract, as well as portal and pericellular fibrosis. Liver inflammation is typically mild, but due to the immunosuppression the viral load is high leading to a rapid progression towards cirrhosis.

Fibrosis

If the damage during the second phase is extensive, fibrous scarring may install. The extend of fibrosis is assessed by advancing stages of liver pathology . Trichrome or Sirius red are the stains of choice for the evaluation of hepatic fibrosis. The scarring usually originates as an extension of the portal tract, but pericellular and perivenular fibrosis may also be present. The collapse and condensation of the reticulin meshwork close to the central vein is found in areas of confluent necrosis, connecting adjacent central veins or central veins to other portal tracts . Long standing scars result in the deposition of mature collagen type I and type III, followed by elastin fibres . The underlying mechanism of hepatic fibrosis is supposed to be the activation of stellate cells and fibroblasts, resulting in an excessive synthesis of extracellular matrix and other inflammatory and profibrotic cytokines. This imbalance between matrix production and degradation leads to progressive liver fibrosis. Other characteristics of hepatic fibrosis are loss of hepatocytes, destruction of hepatic microarchitecture and proliferation of myofibroblasts. In contrast to that, liver regeneration is recognized by the thickening of hepatocyte plates, consisting of 2-3 hepatocyte cell lines. As the liver injury progresses, this regenerative process diminishes and the proliferative fibrous scarring continues . The fibrotic changes are reversible when viral activity is eliminated .

Hepatic cirrhosis

Hepatic cirrhosis, defined as ‘’regenerative nodules of hepatocytes surrounded by fibrosis„ . It is the common end point of many hepatic pathologies. For many years it was thought to be an irreversible end point, but more recent studies suggest that proper antiviral treatment that inhibits progression of fibrosis can lead to a regression of hepatic cirrhosis.

1.8 Grading and staging of chronic viral hepatitis

One of the most common reasons to perform a liver biopsy in chronic hepatitis is to assess the severity of liver injury.

At present, there is no universally accepted scoring system for the histopathologic findings in chronic hepatitis B. One out of several possibilities is the verbal diagnosis in English terms, used by many pathologists including the following four elements:

1. The principal diagnosis: Chronic Hepatitis

2. The grade of inflammation: Mild – Moderate – Marked

3.The stage: Without fibrosis; mild portal/ periportal fibrosis;

bridging fibrosis; cirrhosis

4. The etiology: e.g. consistent with viral hepatitis B

The grade considers the degree of inflammation.

Mild inflammation is present when interface hepatitis is spotty and rare and lobular hepatitis is uncommon (< 5 acidophilic bodies /ballooned cells/ inflammatory aggregates / low power field (lpf)).

Moderate inflammation is defined as interface hepatitis with < 50% of periportal circumference affected and lobular activities shows 5-20 apoptotic bodies/ ballooned cell/ inflammatory cells/ lpf.

In marked chronic hepatitis inflammation, the interface hepatitis involves > 50% circumference of most portal tracts and lobular activity is caused by > 20 acidophilic bodies/ ballooned cells/inflammatory aggregates. The stage evaluates the degree of fibrosis, a relatively permanent microarchitectural injury. Masson trichrome is the stain of choice for recognizing fibrosis. Mild fibrosis involves only zone 1 of the acinus, moderate fibrosis expands into zone 2 and marked fibrosis pathologically connects under normal conditions separated architectural structures. Mostly for research purposes, in 1981 the first numerical scoring system, the Knodell score was introduced, enabling statistical analysis of liver biopsies . This was a rather complex system with the disadvantage of combining the grade of inflammation with the stage of fibrosis. Several other semi-quantitative scoring systems developed during the years.

The four most commonly used scoring systems for clinical trials are the Scheuer system, Batts and Ludwig system, Ishak system and Metavir system. They all have in common the scoring of the grade of inflammation (portal/interface/lobular) and the stage of fibrosis (no fibrosis/portal fibrosis/ bridging fibrosis/cirrhosis). Each of the scoring systems subdivides and scores the main categories differently, but until now, there is no scoring recognized as being superior; each has its own advantages and disadvantages. They are most useful in the setting of clinical trials.

1.9 Specific aspects of hepatitis B in children

A striking difference between HBV infections acquired perinatally versus during adult life is the rate of chronicity. While only 5% of hepatitis B acquired during adult life becomes chronic, the percentage for neonatally acquired untreated cases lies at 95%. Below 6 years of age at the time of infection, the risk of chronicity is 30%.

There are three different phases of perinatally acquired hepatitis B infection.

Immune tolerance phase

The immune tolerant phase is characterized by high levels of HBV replication recognized by high HBeAg and serum HBV DNA levels. Despite this active viral replication, the serological liver markers ALT is usually normal and liver biopsy during this phase evidences only minimal changes . The mechanism of this contradiction is believed to be immune tolerance to HBV with a supressed T- cell host response. The individual may stay in this phase for 10 up to 30 years with a low chance (15%) of spontaneous HBeAg clearance during the first 20 years of infection . During this period, the child is usually asymptomatic with normal growth.

Immune clearance phase

During the next phase, (2nd to 3rd decade) the rate of spontaneous HBeAg clearance increases to 10-20% annually . Seroconversion of HBeAg to anti- HBe antibodies is characterized by a sudden rise of ALT and serum HBV DNA, triggered by a T- cell mediated inflammatory response resulting in immune mediated lysis of infected hepatocytes . Alpha- feto protein and anti- HBc IgM may increase and the symptoms may be similar to the onset of acute hepatitis. The continuous injury of hepatocytes during this immune active phase may lead to hepatic fibrosis.

HBeAg and serum clearance of HBV DNA is not always achieved during this phase, resulting in recurrent exacerbations .

Late non replicative phase (Inactive carrier)

The last phase is characterized by the absence of HBV DNA replication and normalisation of ALT similar to the features of adult chronic HBV infection. Complete resolution is present in case of a loss of HBsAg and appearance of anti- HBs antibodies; this is taking place in only 2% of affected individuals, determining a favourable prognosis .

This spontaneous resolution is even rarer (0,6- 1% per year) in affected children.

Despite this rather benign course, one has to take into consideration that the overall exposure time of hepatocytes to the virus in children, is considerably longer compared to an infection acquired in adulthood. This fact results in 3-5% of children suffering from hepatic cirrhosis and respectively 0,01-0,03% from hepatocellular carcinoma, even before reaching adulthood, the overall lifelong risk estimated to be 9-24% for hepatocellular carcinoma and 2-3% per year for hepatic cirrhosis . The management of HBV infections remains an unresolved issue, as the current therapeutic options are limited. Current studies do not recommend antiviral treatment during the immune tolerant phase.

Given the uncertainties about the natural history of chronic viral hepatitis B infection, the complication of adequate guidelines for management and follow up in paediatric patients, becomes a very complex issue resulting in a lack of precise paediatric guidelines. Current treatment guidelines recommend caution when considering antiviral treatment, as the course of the disease is generally mild and an enhanced requirement for safety is indicated. The decision to start antiviral treatment in paediatric patients is based on the phase of infection, the serum aminotransferase level, serum HBV DNA, histopathological findings on liver biopsy and family history of HCC. It is not mandatory to perform a liver biopsy before initiating antiviral treatment, but it is recommended in order to define the grade of inflammation and the stage of fibrosis.

If a child meets the criteria for treatment, Entecavir (ETV), Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide (TAF), PegαInterferon are the drugs of choice.

Monitoring of chronically infected children should be performed by checking the aminotransferase and HBeAg/ anti- HBeAb generally every 6 months, with the exception of new cases or a finding of increased aminotransferases indicating a check-up every 3 months. In case of inactive carriers, these investigations should be performed annually. HCC surveillance should include alpha- fetoprotein and abdominal ultrasound examination every 6-12 months, in correlation with the stage of fibrosis.

1.10 Alternatives to liver biopsy

As a result of the development of non-invasive serological markers, together with continuous progress in modern imagistic techniques, the role of a liver biopsy in the management of chronic hepatitis B has lost some of its indications. The trend is to replace invasive techniques by non-invasive ones, in order to avoid complications of the procedure.

1.Liver function tests (LFT)

Liver function tests are the first line of investigations used in order to screen for liver pathology. A major disadvantage of these investigations is that the values may be normal, even in significant liver damage.

In addition to that, there is no direct correlation between the degree of liver injury and laboratory test abnormalities, as these are not specific for different types of liver pathologies. Most commonly used tests are alanine aminotransferase (ALT) and aspartate transaminase (AST) representing markers of hepatocellular injury, the latter being less sensitive and specific to liver injury than ALT. Alkaline liver phosphatase (ALP) and gama- glutamyltransferase (GGT), indicating cholestasis.

Serum bilirubin is increased in case of disturbed anion excretion and prothrombin time (PT) and serum albumin reflecting the synthetic capacity of the liver.

Since liver fibrosis, resulting from excessive accumulation of extracellular matrix (ECM) is the hallmark of all chronic liver pathologies; recent studies are trying to replace the gold standard of liver biopsies with molecular serum markers of liver fibrosis considering LFT, extracellular matrix synthesis, extracellular matrix degradation and fibrolytic processes. A recent study performed on 1302 chronically infected HBV individuals established a novel algorithm for the assessment of liver fibrosis yielding better results than previous algorithm (29. Nevertheless, these biomarkers have a lower sensitivity and specificity than liver biopsy and their clinical use is controversial.

2.Ultrasonography

Considering the fact that liver ultrasonography is a non-invasive and cost-efficient technique, it represents the first line of imaging investigations in hepatobiliary disease. One of its other applications is its use in ultrasound guided liver biopsy. In relation to chronic hepatitis, the use of ultrasonography is limited due to low accuracy. The discrimination of different stages of liver pathology is difficult, with the exception of liver cirrhosis where a nodular hepatic surface, ascites, splenomegaly and hypertrophy of the left and caudate lobe are indirect signs of liver cirrhosis. A more recently developed technique is liver elastography which is „ measuring the velocity of propagation of a shock wave within the tissue„ in order to assess liver fibrosis. The more advanced the stage of fibrosis, the stiffer the liver parenchyma becomes, favouring a quicker wave propagation. The disadvantage of this technique is that other liver conditions such steatosis, inflammation or edema act as a confounding factor, tampering the results.

3.Computer tomography (CT)

CT is not the first line of imaging techniques used in chronic hepatitis, but it can be used in order to evaluate mass lesions e.g. hepatocellular carcinoma being the most frequent tumour in chronic hepatitis. After injection of contrast material, HCC shows a brisk enhancement during atrial phase.

4.Magnetic resonance imaging (MRI)

MRI plays a progressively important role in the evaluation of chronic liver disease. Combining conventional and functional sequencing results in the possibility to evaluate liver fibrosis and cirrhosis. Functional sequencing includes diffiusion weighted imaging (DWI), measuring the water diffusion and microcapillary blood perfusion leading to the quantification of the apparent diffusion coefficient, which is low in case of liver cirrhosis. Another functional sequencing is represented by perfusion weighted sequencing. It calculates most parameters of hepatic perfusion and is modified in case of hepatic fibrosis.

Clinical studies have suggested that MR elastography is an accurate diagnostic tool for the diagnosis of hepatic fibrosis by measuring the parenchymal stiffness.

Specific Review

2.1 Introduction

Since the beginning of the year 1996, most hospitals in Romania introduced an obligatory vaccination program for all neonates. This Romanian vaccination plan provides the administration of the fist vaccination dose during the first 24 hours after birth and the consecutive 2nd and 3rd dose after one, respectively six months. This concept was successful in preventing 70-95% of vertical HBV transmissions. The aim of this study is to evaluate the impact of this vaccination program from the point of view of an anatomic pathology laboratory. The introduction of non-invasive fibro scan techniques which eliminates the necessity of a liver biopsy for the diagnosis and evaluation of progression of hepatitis cases significantly influenced the role of anatomic pathology nowadays.

2.2 Materials and methods

In order to analyse the following data, we created a computerized database using Microsoft Excel. The data we collected are extracted from the Pathology Department of Children’s Hospital Cluj-Napoca. This database contains all the bioptic results obtained by the laboratory from 01.01.1994 till present. Applying this database, we created a list including all hepatic slides collected from biopsy or bioptic puncture at the Children’s Clinical Hospital during the time frame of 01.01.1994 until 01.01.2019. Using this list of pathologic pediatric hepatic biopsies between the mentioned time frame, we built a new selective database, including only the pediatric chronic hepatitis cases (viral and non-viral). This selective database is organized in such a way that the establishment of clinical-ethiological-pathological correlations that will be presented as results of this work, is facilitated. This database is the largest of its kind in Romania to date and it promotes an important survey on pediatric viral hepatitis in this region. The hepatic biopsies were obtained in the Infant Surgery Clinic through laparotomy whereas the hepatic bioptic punctures were performed at the Clinic for Pediatrics III using a percutaneous approach with age adjusted puncture needles.

2.3 Results

After the elimination of white biopsies which indicate invalidity due to insufficient or different tissue specimen, during the period of 01.01.1994 –01.01.2019 the anatomic pathology laboratory at the Children`s Hospital Cluj- Napoca received 632 hepatic biopsies/ bioptic punctures. The entities observed in this material as well as their percentual prevalence are illustrated in the graphic below Fig.2. Figure 2- Prevalence of diverse pathological entities in the 632 cases of percutaneous hepatic biopsies/bioptic punctures examined during 1994-2019.

The 430 cases of chronic hepatitis can be attributed to children between the age of 6 months up to 18 years as it is illustrated in the following Table 3.

Table 3- Age repartition of the 430 cases of chronic hepatitis

There are three parameters which are correlated to the age of distribution:

Figure 3 visualizes the percentage of prevalence in relation to age in years during childhood, whereas Figure 4 illustrates the average grade of inflammation identified and Figure 5 demonstrates the average stage of fibrosis during histopathological examination in relation to age distribution.

Figure 3- Age distribution of 430 cases with chronic hepatitis in children

Figure 4- Age distribution of the average level of inflammatory activity of the 430 cases with chronic hepatitis in children

Figure 5- Age distribution of the average level of fibrosis stage of the 430 cases with chronic hepatitis in children

Since the previous illustrations included any etiology of chronic hepatitis in children the following Fig. 6 aims to distinguish the different etiologies based on serological-clinical and histopathological basis.

Figure 6- Etiology of the 430 cases of chronic hepatitis registered.

As demonstrated in Fig.6, the etiology of an HBV infection (322 cases) predominate, being followed by another 56 cases in which the HBV is associated with HCV, resulting in 378 HBV infected cases. The other etiologies (HCV alone, drug induced hepatitis, primary sclerosing cholangitis, autoimmune hepatitis and cryptogenic hepatitis) were rarities compared to the HBV infection.

Further on the case analysis will refer exclusively to the 378 cases of chronic HBV infection.

The morphopathological examination of the hepatic specimen Fig.7 reveals an accumulation of mononuclear inflammatory cells around the portal triad indicating portal inflammation extending into the liver parenchyma involving the limiting plate, representing interface activity along with focal necrosis of the hepatocytes. These findings are typical in a chronic form of hepatitis B.

Figure 7- Typical microscopic image for a chronic B hepatitis. HEx100

As Fig. 8 visualizes, the follicular aggregation of lymphocytes is a finding more common encountered in chronic viral hepatitis C infections together with multifocal necrosis.

Figure 8- Typical image of chronic B+C hepatitis. HE x40

The following Fig. 9 states the importance of sequential liver biopsies for the follow up of chronically infected hepatitis B patients, evidencing a progression of disease in its natural evolution versus a regression of inflammation after the initiation of treatment.

Figure 9- Multibiopsied case followed in evolution, HE stain (x100) showing moderate inflammation in 2012 (a.), severe inflammation in 2013 (b.) and reduced inflammation in 2014 (c.) after treatment

Year repartition of the chronic hepatitis cases with HBV is depicted in the following graphic Fig. 10.

Fig.10 – Year repartition of the 378 HBV ± HCV chronic hepatitis biopsy cases.

As has been pointed out, in the year 1996 there was a maximum prevalence because in that year the anti HBV vaccination was introduced obligatory to all the new born as well as the gratuity of the interferon treatment, that led to bioptic documentation of the cases that were about to undergo the treatment. Age repartition of the cases can be seen in the following graphic Fig.11.

Figure 11- Age repartition of the 378 HBV ± HCV chronic hepatitis biopsied cases.

It must be remarked that over 2/3 of the cases are clustered between the age of 4 and 14 years. This age repartition curve suggests that majority of the chronic hepatitis cases in children are not the result of birth infection, but the result of a following infection during the first childhood, probably in the family.

This supposition is strengthened by the drastic drop of prevalence after 2004 indicating the practical disappearance of this mechanism of inter-familial transmission after introducing the anti- HBV vaccination in 1996.

Sex repartition of the chronic HBV infected cases can be seen in the following graphic Fig. 12. One can observe a preponderance of infection in boys. Statistical analysis (t Student) of this repartition proved to be insignificant. (p > 0.05).

Figure 12- Sex repartition of the 378 HBV ± HCV chronic hepatitis biopsied cases.

Figure 13- Fibrosis stage in the moment of biopsy based on sex.

By analysing Fig. 13 one notices that male cases are not only more frequent, but they are also diagnosed in a more fibrosis advanced stage or even cirrhosis, while the female cases (more rare) are diagnosed in a earlier stage of fibrosis.

As shown in the consecutive graphics (Fig.14, Fig.15), chronic hepatitis in children frequently presents with a high grade of necroinflammatory activity, due to the immunotolerance that at this age overlaps the physiological immune immaturity, while the fibrotic stage was present as frequent as 17% of cases being discovered straight in a cirrhotic stage.

According to this supposition, the cases should present an increasing necroinflammatory activity grade as the age advances and the immune maturity installs. All this accounted, the batch statistical analysis, pondered or non-pondered, didn’t prove this development resulting in an unconfirmed hypothesis.

Figure 14- Necroinflammatory grading of the 378 HBV chronic hepatitis biopsied cases.

Fig. 15- Fibrosis staging of the 378 HBV chronic hepatitis biopsied cases.

Fig.16- Pondered analysis of the cases based on the age group and activity level.

For the rare cases with unknown aetiology of a chronically evolving hepatitis in which biopsy is still indicated, we should practice etiological immunostainings. These immunostainings are only available for research purposes and not for current use. We still had the opportunity to perform these immunostainings on a small number of cases for Hepatitis B virus antigens. In the following images we present our results (Fig. 17- 24).

Figure 17(A)- HBsAg x100 immunostaining. Focal-diffuse placed cytoplasmatic staining pattern for the infected hepatocytes.

Fig.17(B)- HBsAg x100 immunostaining. Focal-diffuse placed cytoplasmatic staining pattern for the infected hepatocytes.

Fig.18- Detail. HBsAg x400. Focal-diffuse placed cytoplasmatic staining pattern for the infected hepatocytes.

Fig. 19- HBsAg x100. Nodular placed cytoplasmatic colour pattern for the infected hepatocytes.

Fig.20- HBsAg x200. Nodular placed cytoplasmatic colour pattern for the infected hepatocytes.

Fig.21- HBsAg x400. Membranal immunostaining pattern for infected hepatocytes.

Fig.22(A)- Immunostaining for HBcAg. Pure intranuclear immunostaining pattern, characteristic to the HBV virus replication phase.

Fig.22(B)- Immunostaining for HBcAg. Pure intranuclear immunostaining pattern, characteristic to the HBV virus replication phase.

Fig. 23- HBcAg immunostaining. Intra-cytoplasmic immunostaining pattern, characteristic to the viral clearance phase following interpherone therapy.

Fig. 24- HBcAg immunostaining. Negative immunostaining pattern, characteristic for the viral integration phase.

This immunoassays are expensive but they permit not only the identification of the HBV virus in cases with negative serology and unknown etiology but also to include the cases in a Desmet evolutive class.

The replicating phase of the virus, described through:

Light/Moderate necro-inflamatory activity

Nuclear immunohistochemical positivity of hepatocytes for HBcAg

Hepatocyte cytoplasmatic positivity HBeAg

Hepatocyte membrane positivity for HBsAg

The clearance phase, triggered by the interferon therapy is characterised by:

Intense/Moderate necro-inflammatory activity

Membrane, cytoplasmatic, nuclear HBcAg localisation

Membrane and focal cytoplasmatic HBsAg localisation

The viral integrated phase is characterized by:

Absence or reduced necro-inflammatory activity

Negative HBcAg immunostaining

Focal, cytoplasmic HBsAg immunostaining

This supplemental information can give an image about success and prognosis of the interferon or antiviral treatment.

2.4 Discussion

Chronic hepatitis represents the main reason for which a liver biopsy is performed during childhood.

In time, this fact suffered a major drop because the protocols for diagnosis and treatment of chronic hepatitis have progressed.

Before 2008, the histopathologic examination including grading and staging diagnosis was considered the gold standard and mandatory for a interferon treatment free of charge. After 2008 the etiologic diagnosis became exclusively based on serological tests, the grading remains based on biochemical tests and staging was completely replaced with non-invasive fibroscan examination. In this context the hepatic biopsy was not performed any more, remaining in the histopathologist competence only diagnosis of chronic hepatitis of unknown etiology rare metabolic diseases or of liver tumoral pathology.

In our data base, the histopathologic diagnosis of chronic B or B+C hepatitis, was made mostly in children aged between 4 and 14 years old, but cases were identified in the entire spectrum (0-18 yrs.) of childhood.

Analysis of our data base remark that, except a 2nd incidence peak in the years 2002-2004 (significantly inferior to the 1996 one), after the introduction of the anti HBV vaccination, the chronic hepatitis incidence at children dropped steadily reaching in the last years of mandatory biopsy (2007-2008) a number of just 5 cases / year of biopsies. After 2008, biopsy not being mandatory any more, no other cases were diagnosed by histopathologic examination.

We have analyzed separately the vaccinated cases after 1996, which were tracked as having HBV chronic hepatitis. These cases were in a number of 18 and presented the following repartition of necroinflammation grade and fibrotic stage.

cases

Average grade = 1,8 (0-4)

11.7% grade 4 (unlike the 2% general)

Average stage = 2,29 (1-3)

0 cirrhosis cases (less than 12 years of evolution for this cases)

Figure 25- HBV chronic hepatitis at vaccinated cases

As Fig.25 proves that the existence of these cases shows that the vaccination did not have a 100% efficiency or did not cover the entire infantile population. Although in these cases they have presented a severity level similar to the non-vaccinated cases, you can remark that the maximum necro-inflammatory grade was more often met in these cases because of the sensitised immune system after vaccination. None of these cases was diagnosed in cirrhotic stage.

Figure 26- Evolution in the last 20 years of chronic HBV infection

Another aspect of our analysis was the fact that boys were more frequently affected by chronic hepatis than girls, having a more rapid fibrogenic evolution.

2.5 Conclusions

Between 01.01.1994 – 01.01.2019 in the Pathology Department of the Children’s Clinical Hospital Cluj-Napoca, 632 pathologic hepatic fragments were analysed.

Out of the 632 interpretable hepatic fragments examined, majority of them (55%) presented chronic hepatitis lesions, of different aetiologies.

The 430 cases of chronic hepatitis diagnosed at children with the age between 6 months and 18 years, majority of cases (2/3) being clustered between 4 and 14 years with a maximum incidence between 7 and 8 years.

The necro-inflammatory activity grade was increasing with age while the fibrosis stage was decreasing with age, particular to the childhood period, where with age, the immunocompetence necessary to the elimination/integration of the virus and fibrosis develops gradually.

The resulting fibrosis is diluted and lysate with the normal growth process of the liver.

Overall the male hepatitis was more frequent (62.5%) than the female cases (37.5%). Also the female cases were diagnosed in earlier fibrotic stage while the male (majority) was diagnosed in advanced fibrosis or even cirrhotic stage.

Age distribution of the biopsied cases shows the augmentation of the interest for biopsies after 1996, the year of the introduction of the cost-free treatment (conditioned by the histopathological examination) with interferon, followed by the steady decrease of case number while the vaccination made its effect on decreasing the incidence of HBV chronic hepatitis.

Almost half the cases (46%) were diagnosed in advanced fibrotic phases (stage 3 or cirrhosis), the grand majority of these cases being male.

The change of chronic hepatitis treatment protocol, removed the liver biopsy for chronic hepatis from current practice, remaining indicated only for metabolic, tumoral or unknown aetiologies.

There is still a place for histopathologist in the management of chronic HBV hepatitis by practicing immunohistochemical stains for HBV antigens generating a more reliable diagnosis and therapeutic indications.

Bibliography

Tang LSY, Covert E, Wilson E, Kottilil S. Chronic Hepatitis B Infection:A Review. JAMA. 2018, 319(17), S. 1802-13.

Dandri M, Petersen J. HBV virology. [book auth.] Berg T, Rockstroh J, Sarazzin C, Wedemeyer H Mauss S. Hepatology – A clinical textbook. 8th. ed. Hamburg : Medizin Fokus Verlag, 2017, p. 85.

Pitigoi D, Rafila A,Pistol A,Arama V,Molagic V, Streinu-Cercel A. Trends in hepatitis B incidence in Romania, 1989-2005. Euro Surveill. January, January 2008, Vol. 13, 2.

Theise N.D, Bodenheimer Jr.H.C, Ferrell L.D. Acute and chronic viral hepatitis. [book auth.] Portman B, Ferrell L. Burt A. MacSween`s Pathology of the Liver. Edinburgh : Elsevier Limited, 2012, pp. 370-384.

Della Corte C, Nobili V, Comparcola C, Cainelli F, Vento S. Management of chronic hepatitis B in children: An unresolved issue. JGH Open. 2014, Vol. 29, 5, pp. 912-19.

Worl Health Organisation. Regional Office for Europe. [Online] 26 7 2018. [Cited: 28 10 2018.] http://www.euro.who.int/en/countries/romania/news/news/2018/7/universal-access-to-testing-and-treatment-is-key-to-eliminate-viral-hepatitis.

J, Petersen. Hepatitis B. : diagnostic tests. [book auth.] Berg T, Rockstroh J, Sarrazin C, Wedemeyer H Mauss S. Hepatology- A clinical textbook. Hamburg : Medizin Fokus Verlag, 2017, pp. 149-159.

Vivekanandan P, Singh O. V. Molecular Methods in the Diagnosis and Management of Chronic Hepatitis B. Expert Rev Mol Diagn. 2010, Vol. 10, 7, pp. 921- 35.

Mauss S, Berg T, Rockstroh J, Sarrazin C, Wedemeyer H. Hepatology – A clinical textbook. 8th. Hamburg : Medizin Fokus Verlag, 2017. pp. 45-50. 978–3–941727–22–9.

J, Liang T. Hepatitis B: The Virus and Disease. Hepatology. May, 2009, Vol. 49, 5.

Geller S. A., Petrovic L. M. Biopsy interpretation of the liver. 2nd. Philadelphia : Lippincott William & Wilkins, 2009. pp. 4-14. 9780781774680.

Kim K. A., Jeong S. H. The diagnosis and treatment of primary biliary cirrhosis. Korean J Hepatol. Sep, 2011, Vol. 17, 3, pp. 173-9.

Grünlage F, Lammert F. Assesment of hepatic fibrosis and steatosis. [book auth.] Berg T, Rockstroh J, Sarrazin C, Wedemeyer H Mauss A. Hepatology – A clinical textbook. 8th ed. Hamburg : Medizin Fokus Verlag, 2017, pp. 429-31.

Bedossa P, Paradis V. Cellular and molecular techniques. [book auth.] Portmann B, Ferrell L Burt A. MacSweens pathology of the liver. Edinburgh : Churchill Livingstone Elsevier, 2012, pp. 79-86.

MS, Torbenson. Biopsy interpretation of the liver. Philadelphia : Wolter´s Kluwer Health, 2015. p. 1.

M.S, Torbenson. Biopsy interpretation of the liver. Philadelphia : Wolters Kluwer Health, 2015. pp. 63-67.

Wuang HC, Huang W, Lai MD, Su MJ. Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer Sci. Aug, 2006, Vol. 97, 8, pp. 683-8.

Hsieh YH, Chang YY, Su IJ, Yen CJ, Liu YR, Liu RJ,etc. Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis. J Pathol. Jul, 2015, Vol. 236, 3.

Drebber U, Kasper HU, Raterin J, Wedemeyer I, Schirmacher P, Dienes HP. Hepatic granulomas: Histological and molecular pathological approach to differential diagnosis. Liver Int. Jul, 2008, Vol. 28, 6.

Park NY, Roncalli M. Large liver cell dysplasia: A controversial entitiy. J hepatol. Nov, 2006, Vol. 45, 5, pp. 734-743.

Park YN. Update on precursor and early lesions of hepatocellular carcinomas. Arch Pathol Lab Med. June, 2011, Vol. 135, 6.

Desmet VJ. Liver tissue examination. J Hepatol. 2003, Vol. 39, 1, pp. 43-9.

Burt A, Portman B, Ferrell L. MacSween's Pathology. Edinburgh : Elsevier Limited, 2009. S. 429-435. 978-0-7020-3398-8.

Torbenson MS. Biopsy Interpretation of the Liver. Philadelphia : Wolter´s Kluwer Helath, 2015. p. 45.

Hansen T, Christensen E. Cirrhosis and liver fibrosis are potentially reversible. Ugeskr Laeger. Nov, 2015, Vol. 177, 48.

Torbenson MS. BIOPSY INTERPRETATION OF THE LIVER. Philadelphia : Wolters Kluwer, 2015. pp. 110-116.

Rosenthal P, Ling SC, Belle SH, Murray KF, Rodriguez-Baez N et al. Combination of entecavir/ peginterferon alfa-2a in children with HBeAg-positive immune tolerant chronic hepatitis B virus infection. Hepatology. October, 2018, Vol. 10.1002, 30312.

Lampertico P. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol. March, 2017, Vol. 67, 2, pp. 370-98.

Zhu, M.-Y., et al. A Novel Noninvasive Algorithm for the Assessment of Liver Fibrosis in Patients With Chronic Hepatitis B Virus Infection. J Viral Hepat. 2017, Vol. 24, 7.

Valls C, Andía E, Roca Y,Cos M, Figueras J. CT in hepatic cirrhosis and chronic hepatitis. Seminars in Ultrasound, CT and MRI. February, 2002, Vol. 23, 1.

Man S.C, Schnell C.N, Sas V, Buzoianu A.D, Gheban D. Autoimmune hepatitis with sclerosing cholangitis in a patient with thiopurine methyltransferase deficiency: case presentation. Rom J Morphol Embryol 2017, 58(1): 211–217