Significant hepatic histopathology in chronic hepatitis B patients with serum ALT less than twice ULN and high HBV-DNA levels in Indonesia
Abstract
OBJECTIVE: To study the prevalence of significant hepatic histopathology in chronic hepatitis B (CHB) patients with alanine aminotransferase (ALT) ≤ twice upper limit of normal (ULN) and its association with age, HBeAg status, hepatitis B virus (HBV)-DNA level and viral genotype.
METHODS: A prospective study was conducted over a 3-year period in treatment-naive CHB patients with ALT ≤ twice ULN. Patients with a history of acute flare hepatitis, use of alcohol and hepatotoxic drugs, hepatitis C, hepatitis D and human immunodeficiency virus (HIV) co-infection were excluded from the study. Hepatic histopathology was assessed according to the METAVIR scoring system.
RESULTS: A total of 145 patients were recruited, 81 (55.9%) of whom were male. The patients’ mean age was 41.50 ± 10.74 years (range 16–70 years). Significant hepatic inflammation was found in 59.3% of these patients, and significant hepatic fibrosis was found in 62.1%, the latter being associated with hepatitis B e antigen status, ALT levels and serum HBV-DNA, but not with their age group or viral genotype. Significant hepatic fibrosis was found in 24 of 35 CHB patients (68.6%) who were previously considered in an immunotolerance phase.
CONCLUSIONS The prevalence of significant hepatic histopathology in CHB patients with serum ALT levels ≤ twice ULN is high. Delayed antiviral treatment can be harmful.
INTRODUCTION
Serum alanine aminotransferase (ALT) level has been widely used in liver biopsies for initiating antiviral therapy.1 However, previous studies have shown that 50–90% chronic hepatitis B (CHB) patients had normal ALT levels.2,3 Another study by Lai et al. showed that among the CHB patients with normal ALT levels, 37% already had a significant hepatic histopathology, 18% had stage II fibrosis and 34% grade II or III inflammation.4
Current guidelines suggest that in CHB patients over 40 years with ALT level ≤ twice the upper limit of normal (ULN), a liver biopsy should be performed in those with positive hepatitis B e antigen (HBeAg) status and serum hepatitis B virus (HBV)-DNA level ≥ 105 copies/mL, or those with negative HBeAg and HBV-DNA level ≥ 104 copies/mL.1 However, this age recommendation was decided arbitrarily1 and there is no recommendation yet for CHB patients under 40 years of age with an ALT level ≤ twice ULN.
The prevalence of significant hepatic histopathology and its associated factors among CHB patients with ALT levels ≤ twice ULN have not yet been assessed in the Indonesian population, since the differences of genotype and sub-genotype distribution for evaluating the progression of liver disease among countries. Furthermore, there is no strong scientific evidence for the precise timing for performing a liver biopsy in these patients, especially younger patients. Therefore, our study aimed to find out the prevalence of significant hepatic histopathology, that is, a moderate to severe grade of necroinflammation and fibrosis, and its relevance to age, HBeAg status, HBV-DNA level and viral genotype in CHB patients with serum ALT levels ≤ twice ULN.
MATERIALS AND METHODS
Study design and subjects
This was a prospective study in the Cipto Mangunkusumo and Medistra Hospitals, Jakarta. Study participants were treatment-naive CHB patients who were admitted in our hospitals between 2007 and 2010. All had initial laboratory assessments consisting of liver biochemistry test, hepatitis B seromarkers and their HBV-DNA was measured. After the initial assessment, the patients were asked to visit their doctor regularly at 3-month intervals for serum ALT level monitoring for over 1 year. Participants included in this study were those who had a serum ALT level ≤twice ULN for at least two measurements within a 3-month period since the first laboratory test. Patients who experienced an episode of acute flare up of HBV within a year and an increased serum ALT level more than twice ULN were excluded from the final analysis. The cut-off values for ALT levels were 50 U/L for men and 35 U/L for women. Those who had a history of alcohol and hepatotoxic drug use, hepatitis C, hepatitis D, human immunodeficiency virus (HIV) co-infection, or histopathologically proven autoimmune hepatitis were also excluded. The patients were then divided into groups according to age (<40 vs≥40 years), HBeAg status (positive vs negative) and viral genotype. The informed consent of all patients and ethical approval were obtained before starting the study.
Liver biopsy
All patients underwent ultrasound-guided liver biopsy using a 16-gauge Menghini needle (Hepafix, B. Braun Melsungen AG, Melsungen, Germany) under local anesthesia. Hematoxylin–eosin staining was performed to assess the necroinflammatory grade and fibrosis staging was evaluated with Masson's trichrome staining. The tissue specimens were 15 mm in length including five portal tracts. The histopathology evaluation was performed by a senior pathologist who was blind to clinical and laboratory data. Fibrosis staging was based on the METAVIR scoring system (F0 = normal connective tissue, F1 = foci of perivenular and/or perisinusoidal fibrosis in zone 3, F2 = perivenular or pericellular fibrosis affecting zones 3 and 2, F3 = septal or bridging fibrosis and F4 = cirrhosis). Significant necroinflammatory grades were defined as A2–A3, whereas significant fibrosis was defined as F2–F4 according to the METAVIR scoring system.5,6 The presence of hepatic steatosis and autoimmune hepatitis in the histopathological specimens was also noted.
Laboratory procedures
Blood chemistry test was done using automated blood analyzer (Advia-Bayer, Fernwald, Germany). Hepatitis B serology markers, that is, hepatitis B surface antigen (HBsAg) HBeAg, and anti-HBc were assessed by an enzyme-linked immunosorbent assay using commercial kits. The quantitative serum HBV-DNA level assay was performed using the polymerase chain reaction technique (COBAS TaqMan System; Roche Diagnostics, Mannheim, Germany). The lower detection limit was 4700 copies/mL. The genotype analysis was performed using specific primers. All hepatitis B seromarkers and HBV-DNA were assessed on the first visit.
Statistical analysis
The characteristics of the participants are presented in frequency and percentage for the categorical data and mean ± standard deviation (or median and range). A bivariate analysis between significant hepatic histopathology and clinical factors was performed using the χ2 test. Mean comparison was tested using the Mann–Whitney U-test for skewed data. A P value <0.05 was considered significant. The statistical analysis was performed using SPSS 11.5 (SPSS Inc., Chicago, IL, USA).
RESULTS
A total of 145 patients, of whom 81 (55.9%) were men, were enrolled during the study period. The mean serum ALT level in male patients was 41.40 ± 19.47 (14–94) U/L, which in female patients was 31.74 ± 16.87 (12–70) U/L. The patients’ mean age was 41.50 ± 10.74 years (16–70 years, Table 1). There were 86 (59.3%) patients with necroinflammatory grades A2–A3 and 90 (62.1%) patients with fibrosis F2–F4 (Table 2).
| Characteristics | n | % | |
|---|---|---|---|
| Gender | |||
| Male | 81 | 55.9 | |
| Female | 64 | 44.1 | |
| Age (years, mean ± SD) | 41.50 ± 10.74 | ||
| Serum ALT levels | |||
| Normal | 103 | 71.0 | |
| 1–2 × ULN | 42 | 29.0 | |
| HBeAg status | |||
| Positive | 57 | 39.0 | |
| Negative | 88 | 61.0 | |
| Log10 serum HBV DNA level | |||
| (median [range]) | 5.85 (1.88–10.84) | ||
| Genotype (n = 109) | |||
| Genotype B | 86 | 78.9 | |
| Genotype C | 23 | 21.1 | |
| Hepatic steatosis | |||
| Yes | 48 | 33.1 | |
| No | 97 | 66.9 |
- HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; ULN, upper limit of normal; SD, standard deviation.
| Histopathology | n | % |
|---|---|---|
| Necroinflammation activity | ||
| A0 | 1 | 0.7 |
| A1 | 58 | 40.0 |
| A2 | 60 | 41.4 |
| A3 | 26 | 17.9 |
| Fibrosis stage | ||
| F0 | 4 | 2.8 |
| F1 | 51 | 35.2 |
| F2 | 55 | 37.9 |
| F3 | 30 | 20.7 |
| F4 | 5 | 3.4 |
Hepatic fibrosis stage F2–F4 was significantly associated with positive HBeAg status, ALT levels 1–2 × ULN, and higher serum HBV-DNA levels. There was no association between significant hepatic histopathology and age group, hepatic steatosis and viral genotype (Table 3). The necroinflammatory activity grade was not associated with any clinical factor.
| Variable | Fibrosis stage, n (%) | P value | Necroinflammatory grade, n (%) | P value | ||
|---|---|---|---|---|---|---|
| F0–F1 | F2–F4 | A0–A1 | A2–A3 | |||
| Age group | ||||||
| <40 years | 26 (47.3) | 42 (46.7) | 0.943 | 25 (42.4) | 43 (50.0) | 0.366 |
| ≥40 years | 29 (52.7) | 48 (53.3) | 34 (57.6) | 43 (50.0) | ||
| HBeAg status | ||||||
| Positive | 16 (29.1) | 41 (45.6) | 0.049 | 21 (35.6) | 36 (41.9) | 0.448 |
| Negative | 39 (70.9) | 49 (54.4) | 38 (64.4) | 50 (58.1) | ||
| Genotype | ||||||
| Genotype B | 31 (81.6) | 55 (77.5) | 0.616 | 36 (80.0) | 50 (78.1) | 0.813 |
| Genotype C | 7 (18.4) | 16 (22.5) | 9 (20.0) | 14 (21.9) | ||
| Hepatic steatosis | ||||||
| Yes | 20 (36.4) | 28 (31.1) | 0.514 | 19 (32.2) | 29 (33.7) | 0.849 |
| No | 35 (63.6) | 62 (68.9) | 40 (67.8) | 57 (66.3) | ||
| ALT level | ||||||
| Normal | 47 (85.5) | 56 (62.2) | 0.003 | 46 (78.0) | 57 (66.3) | 0.127 |
| 1–2 × ULN | 8 (14.5) | 34 (37.8) | 13 (22.0) | 29 (33.7) | ||
| HBV-DNA (mean log10 of copies/mL) | 5.2 ± 1.76 | 6.3 ± 1.94 | 0.002 † | 5.7 ± 2.08 | 6.0 ± 1.88 | 0.194† |
- χ2-test; †Mann–Whitney U test.
- ULN, upper limit of normal; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus.
- Bold text indicates HBeAg status as an independent factor for significant liver fibrosis.
We further analyzed a subgroup of patients presenting with high HBV-DNA levels, that is, more than 104 copies/mL for HBeAg-negative and more than 105 copies/mL for HBeAg-positive groups. There were 97 patients who fell in this category. Of these 97 patients, there were 35 (36.1%) patients under 40 years of age with positive HBeAg suggestive of the immunotolerance phase. Significant hepatic fibrosis was found in 24/35 (68.6%) (Table 4).
| HBeAg status | Age group | Fibrosis group (n[%]) | Total | |
|---|---|---|---|---|
| F0–F1 | F2–F4 | |||
| Negative | <40 years | 5 (33.3) | 10 (66.7) | 15 |
| ≥40 years | 12 (38.7) | 19 (61.3) | 31 | |
| Positive | <40 years | 11 (31.4) | 24 (68.6) | 35 |
| ≥40 years | 3 (18.7) | 13 (81.3) | 16 | |
- Bold text indicates the high prevalence of significant liver fibrosis in young chronic hepatitis B patients in immune tolerance phase.
DISCUSSION
Based on the current Asian Pacific Association for the Study of the Liver guidelines, serum ALT level is used as an indicator to initiate antiviral treatment for active chronic HBV infection. The serum ALT level reflects patients’ immune response against HBV infection and any increase of serum ALT level is believed to be associated with a certain degree of liver damage. All the laboratories in our country are still using the old cut-off value and since every laboratory all over the world has its own cut-off value, the normal cut-off value of ALT level might need to be reviewed.7–12
Our results showed that the proportion of significant hepatic histopathology was very high in the patients in this study, that is, 59.3% (95% CI 51.3–67.3%) with significant necroinflammation and 62.1% (95% CI 54.2–70.0%) with significant fibrosis. To our knowledge, this is the first study with such a large number of CHB patients with an ALT less than twice ULN and with significant hepatic histopathology, which is much higher than those in the previous studies where significant hepatic inflammation and fibrosis were between 30% (95% CI 21–39%) and 49.2% (95% CI 42.5–55.5%).4,13 Another study also reported a high prevalence of significant hepatic histology in normal or mildly elevated ALT levels which was seen only in genotype C patients.11 We know that genotype C is more relevant to progressive liver disease, however, our study showed the prevalence in both genotypes B and C, in which the distribution was predominant in genotype B. The possibility of over-diagnosis had been excluded by the adequacy of biopsy specimens (which include at least five portal tracts) and it was evaluated by a senior pathologist who was blinded to the clinical and laboratory data.
It is not yet clear what other factors caused the high prevalence of significant hepatic histopathology in our study participants. Alcohol consumption is not a problem since there were no alcohol drinkers among our patients. On the other hand, the use of herbal medicines and beverages are common among Indonesians; but unfortunately this factor was overlooked in the data collection in our study. Our results showed that 33.1% of the patients had some degree of hepatic steatosis; however, its presence did not differ between patients with or without significant hepatic histopathology. Our recent study showed that hepatic steatosis was found in 30% of CHB patients, which was related to metabolic factors such as obesity but not associated with liver disease progression (Lesmana, unpublished data).
We also found the prevalence of significant hepatic histopathology was quite high in patients under 40 years of age. Previous studies also showed that significant hepatic fibrosis was found at a younger age .8,11,14 This might be explained by the predominant vertical transmission of HBV infection in Asia where the infection is acquired prenatally.15
We had not found any significant difference in clinical factors studied between patients with a significant hepatic histopathology and normal or mild liver changes. However, significant hepatic fibrosis was presented predominantly in patients aged 40 years or more and in patients with negative HBeAg. These findings may reflect the longer course of hepatitis B infection in these patients; however, we could not rule out the possibility of CHB patients acquiring a recent infection by mutated HBV that showed negative HBeAg.
Most of our studied patients had been diagnosed with hepatitis B for many years previously and their ALT levels were regularly monitored for assessing the demand of antiviral treatment. Although the inclusion criteria of our study required only two measurements of ALT levels within 3 months, in fact, our patients had been monitored for 1 to 3 years before participating in this study. During this period, their ALT levels never increased more than twice ULN despite their high HBV-DNA levels.
Our study also found a significant number of CHB patients who already had significant hepatic fibrosis who were considered to be in the immunotolerance phase. Up to the time of writing there is no specific marker that can differentiate the immunotolerance phase from immunoactive phase with normal ALT at a younger age. We thus have to be very careful when dealing with younger patients, especially when there is a clear familial history of the disease.
