We studied 288 consecutive chronic HBV carriers (192 males, mean age 48.4 years, range 20-78 year) and nine patients with acute hepatitis B followed-up at the Hepatology Unit of the University Hospital of Pisa, Regional Reference Center for Chronic Liver Disease and Hepatocellular Carcinoma. The study was approved by the Ethical Committee of the hospital and patients gave their written informed consent.

HBV carriers were classified, after a monthly follow-up of at least 12 mo, as inactive or active according to their virological profile. Inactive carriers had serum HBV DNA persistently < 10⁵ copies/mL (by COBAS Amplicor HBV Monitor, Roche, Basel, Switzerland) and IgM anti-HBc levels < 0.200 (by Core-M™ Axsym System, Abbott, Sligo, Ireland). Chronic hepatitis patients showed the presence of active viral replication (serum HBV-DNA levels persistently or intermittently ≥ 10⁵ copies/mL during the follow-up), IgM anti-HBc ≥ 0.200 and liver histology consistent with chronic hepatitis. Exclusion criteria: hepatitis D virus (HDV) or hepatitis C virus (HCV) coinfections, Child B or C cirrhosis.

We studied the correlation between LS and the stage of liver disease with single point FS measurements in 297 HBV carriers (288 with chronic infection: 208 untreated and 80 treated; nine with acute hepatitis B) and 50 blood donors as controls. Transient elastography was performed within 6 mo (median 3 mo, 75% of cases between 0 and 4.6 mo) from liver biopsy in 157 patients with biochemical and/or virological signs of liver disease and 21 inactive carriers. In 47 HBV carriers with inactive infection, 63 patients with cirrhosis (with previous histological diagnosis and actual ultrasonographic signs of cirrhosis) and nine patients with acute hepatitis, who did not undergo liver biopsies, FS was performed within 1 wk from US and Doppler examinations of the liver.

To study the correlation between LS and spontaneous or treatment-induced variations of biochemical activity, we enrolled 87 patients who underwent monthly blood controls and FS measurements at least every 6 mo. In case of ALT flares (ALT values ≥ 300 IU/L with increments of at least 2 SD above previous values), patients were monitored with blood and FS test every 2 wk for the first month and monthly thereafter until flare resolution. Transaminases and virological markers (HBV DNA and IgM anti-HBc) were tested on the same day of FS measurements. In treated patients, FS was monitored every 3 mo.

Transient elastography was measured by Fibroscan (EchoSens, Paris, France). All measures were performed by trained physicians on the right liver lobe through intercostal spaces in the patient lying on his back, with right arm in maximal abduction. The US guide was used to identify a target liver area, at least 6 cm thick, without major vascular structures. The procedure was considered valid if at least 10 validated measurements were performed, with a success rate (ratio between numbers of validated and total measurements) ≥ 60% and interquartile range (IQR) < 20%. LS was recorded in kPa as the median value of all measurements.

Liver biopsies were obtained using 16 G disposable needles (Hepafix B; Braun, Melsungen, Germany). Liver specimens (median 27 mm, range 11-50 mm) were stained with hematoxylin and eosin. Necro-inflammatory activity and liver fibrosis were scored according to Ishak[15]. Steatosis was graded semiquantitatively, as reported previously[4]. Patients in whom liver biopsy yielded specimens shorter than 15 mm and/or with less than 11 portal tracts were excluded from the analysis.

The included variables were sex, age, virological profile (HBeAg/anti-HBe status), liver disease co-factors [alcohol intake (≤ 60 or > 60 g/d), iron overload (present, in case of staining at histology and serum iron > 150 g/L and/or ferritin > 400 μg/L), hyperlipemia (cholesterol > 240 mg/dL and/or triglycerides > 250 mg/dL), diabetes (fasting plasma glucose > 140 mg/dL), overweight [body mass index (BMI) > 25 kg/m²]. The biochemical profiles were defined as: (1) persistently elevated ALT; (2) biochemical remission (persistently normal ALT for at least 12 mo, at monthly controls); (3) ALT flares (when ALT values increased ≥ 300 IU/L, with increments of at least 2 SD above previous values). Virological profiles included HBV-DNA and IgM anti-HBc levels. Liver biopsy features were: length; number of fragments; portal tracts number; necro-inflammation, fibrosis and steatosis scores. Cirrhosis at ultrasound (US cirrhosis) was defined when enlargement of left/caudate lobes, nodular liver boundaries, and micro-macronodular liver structure were present. We recorded in addition: the signs of portal hypertension (portal vein diameter > 12 mm; spleen volume > 45 cm²; esophagus or gastric varices); the transient elastography performance (values, rate of successful measurements and IQRs); the characteristics of therapy (schedule, dose, duration and response).

Data are expressed as mean ± SD. The logarithmic transformation was used for quantitative data when their distributions were not normal. The Pearson’s correlation coefficient was used to analyze the correlations between values of liver elastometry and fibrosis. Differences between subgroups were analysed using one-way ANOVA, Mann-Whitney rank sum test or Kruskal-Wallis test when appropriate. To identify factors independently correlated with LS, variables with statistical associations (P < 0.05) or trends (P < 0.10) at univariate analysis were included in multiple regression analyses. The diagnostic performance of transient elastography was evaluated by receiver operating characteristic (ROC) curve. By using the cut-off values with the highest sensitivity + specificity sum, we defined two different cut-off values of liver transient elastography to identify patients with significant fibrosis (Ishak score ≥ 3/6) or cirrhosis. Statistical analysis was performed by SPSS (version 10.0, SPSS Inc., Chicago, IL, USA) software package.

Overall 277 of 297 (93.3%) HBV carriers were suitable for the analysis: nine had acute hepatitis, 68 inactive infection, and the remaining 200 had chronic hepatitis. Six patients (2.1%) were excluded because their liver biopsies were < 1.5 cm and 14 (4.9%) because their elastographic measures failed (seven cases had BMI > 28). Eighty patients were under treatment [61 nucleos(t)ides, NA; 19 interferon, IFN]. Demographic and clinical characteristics of the 268 chronic carriers are reported in Table 1.

FS values were 4.6 ± 1.2 kPa in 50 blood donors, 12.3 ± 3.3 kPa in nine patients with acute hepatitis and 10.3 ± 8.8 kPa in 268 chronic HBV carriers (P < 0.001) (Table 2).

In 68 inactive carriers, the mean FS value was 5.0 ± 1.8 kPa. Seventeen of them had abnormal ALT and at histology showed steatohepatitis or steatosis. Their mean LS values were significantly higher as compared to HBV carriers with normal ALT and without dysmetabolic profile (6.9 ± 2.3 kPa vs 4.3 ± 1.0 kPa, P < 0.001) (Figure 1). As a result of chronic liver damage caused by factors other than HBV, these 17 inactive carriers were excluded from further analysis.

In the 171 untreated chronic HBV carriers, LS correlated significantly with fibrosis stage (r = 0.706, P < 0.001). At univariate analysis, in the 171 untreated HBV carriers, LS significantly correlated with age, sex, phase of infection (inactive vs active), BMI, ALT levels, biochemical remission and fibrosis stage, showing a correlation trend for HBV-DNA levels, alcohol intake and hyperlipemia (Table 3). At multivariate analysis, the phase of HBV infection (P < 0.001), ALT levels (P < 0.001), HBV-DNA levels (P = 0.042) and fibrosis stage (P < 0.001) were independently associated with LS (Table 3). In the separate analysis of the 83 untreated patients with chronic hepatitis, but without cirrhosis, the factors independently associated with LS were ALT levels (P = 0.001), fibrosis stage (S3-S4 vs S0-S2, P = 0.001) and necroinflammation score (≥ 10/18 vs < 10/18; P = 0.035) (Table 4).

In 80 treated patients, LS correlated with fibrosis stage (r = 0.453, P < 0.001), but the mean values were lower than untreated patients with a comparable stage of fibrosis (6.1 vs 6.4 kPa in S0-S2 patients; 8.5 vs 10.1 kPa in S3-S4 patients; 11.7 vs 15.7 kPa in S5-S6 patients; 17.2 vs 23.6 kPa in US cirrhosis patients) (Table 2), and the difference reached the statistical significance in patients with US cirrhosis only (P = 0.035). Fifty of them were under long-term NA treatment and in long-term biochemical remission, which was independently associated with FS values (P < 0.001, Table 5).

To identify the FS cut-offs for fibrosis ≥ S3 and cirrhosis, we analyzed untreated patients only. Area under ROC curve (AUROCs) for fibrosis ≥ S3 and cirrhosis were 0.966 and 0.973 (95% CI 0.942-0.989 and 0.952-0.994) (Figure 2) and their cut-off values were 7.5 and 11.8 kPa, respectively.

Fibrosis≥S3: The diagnostic performance of 7.5 kPa cut-off is reported in Table 6. Overall, 46 of 60 patients with elastography ≥ 7.5 kPa had fibrosis ≥ S3 (76.7% PPV) and 108 of 111 patients with FS < 7.5 kPa had S0-S2 fibrosis (97.3% NPV). Among the 14 patients with FS ≥ 7.5 kPa, but a fibrosis stage < S3, five patients had ALT levels > 300 UI/L at the time of FS measurement. None of the three patients with FS < 7.5 but fibrosis ≥ S3 had cirrhosis: one had S3 and two had S4 fibrosis at liver histology.

Cirrhosis: The diagnostic performance of 11.8 kPa cut-off is shown in Table 6. Thirty-two of 37 patients with elasticity ≥ 11.8 kPa had histological or US cirrhosis (86.5% PPV); 129 of 134 patients with FS values < 11.8 kPa did not have cirrhosis (96.3% NPV). All but one of the five non-cirrhotic (two with S3 and three with S4 fibrosis stage) patients with FS values ≥ 11.8 showed LS values ranging from 11.8 and 13.3 kPa; the remaining patient with 20 kPa FS value had S4 fibrosis and ALT levels > 300 UI/L at the time of FS measurement. Two of five cirrhotic patients with low FS (7.0 and 7.6 kPa respectively) were in prolonged spontaneous remission; the remaining three had elastometry values ranging between 8.9 kPa and 11.3 kPa.

In 87 patients, LS was monitored for a mean period of 19.9 ± 7.1 mo (range 6-36 mo): Seventy eight patients had chronic hepatitis (43 untreated and 35 treated) and nine had acute hepatitis. All patients underwent at least three FS measurements (mean 5.6, range 3-10).

Untreated patients: Thirty patients showed stable biochemical and virological profiles without disease progression: their LS did not change, showing minor fluctuations (12 mo/baseline FS mean ratio 1.00 ± 0.20; 24 mo/baseline FS mean ratio 0.99 ± 0.26). The remaining 13 patients experienced hepatitis flares. During flares, FS values increased 1.2 to 4.4-fold as compared to baseline values (mean variation 2.1 ± 1.0-fold), mean FS value during flares being 20.7 ± 12.3 kPa (range 8.6-42 kPa). LS variations paralleled the dynamic profiles of ALT: FS values reached the peak simultaneously with ALT in eight patients (61.5%), later, with 15-30 d of delay, in the remaining five (38.5%). Thereafter, FS values decreased with a latency of 30 d from the initial ALT decrease and returned to baseline values within 3 to 6 mo (Figure 3A). Patients with disease profiles characterised by ALT flares intervened by complete biochemical remission showed major variations of FS values during their hepatitis exacerbations, as compared to patients with persistent ALT elevations between flares (FS variation ranging from 1.4 to 4.4 in the former and from 1.2 to 1.6-fold in the letters, P = 0.019).

Acute hepatitis: In nine patients with acute hepatitis B, FS values at presentation ranged from 8.2 to 16.6 (mean 12.3 ± 3.3) kPa and reached a peak of 11.8 to 45.7 kPa (20.0 ± 11.6 kPa) at the time of ALT peak. They then declined progressively to 5.6 ± 1.1 (range 4.4-6.9) kPa, in association with the reduction of ALT levels (Figure 3B).

Treated patients: The FS monitoring started with treatment in 18 patients, when treatment was already ongoing in the remaining 13. Overall, FS values showed progressive declines during therapy, with a mean on-treatment/baseline ratio of 0.9 ± 0.4 at 6 mo and 0.7 ± 0.2 at 12 mo. In patients with persistent response to long-term nucleoside/nucleotide analogues treatment, FS values decreased progressively during their follow up, with mean yearly reduction (ratio between two consecutive FS values registered at 12 mo intervals) of 0.8 ± 0.2 at 24 mo, 0.8 ± 0.1 at 36 mo and 0.7 ± 0.1 at 48 mo from the beginning of therapy.

All responders showed decreased FS values during therapy (Figure 3C and D): 0.8 ± 0.2 at 6 mo and 0.6 ± 0.2 at 12 mo, as compared to baseline values, respectively. FS value declines were similar in responders to IFN as compared to responders to NA: 0.7 ± 0.2 vs 0.8 ± 0.2 6 mo/baseline ratio and 0.6 ± 0.1 vs 0.7 ± 0.2 12 mo/baseline ratio, respectively. Two non-responder HBeAg-positive patients showed an increase of 1.1 and 2.4 times the FS values between 3 and 6 mo, during hepatitis flares, followed by a progressive decline that reached baseline values after 12 mo.

The old measure of liver stiffness (LS) by hand palpation has had a new appraisal after the recent introduction of the objective measure of the speed of transmission of an elastic wave across the liver (transient elastography) registered by the new instrument Fibroscan^® (EchoSens, Paris, France). Originally, the new technique was proposed in clinical practice as a non-invasive, surrogate marker of fibrosis and many studies demonstrated good reproducibility and a high correlation between LS and liver fibrosis at histology. However, liver elasticity is influenced not only by fibrosis, but also by the presence and extent of liquid, lipid and inflammatory infiltrates within the liver. The evidence that Fibroscan (FS) is significantly influenced by major variations of liver inflammation (as we previously showed), in addition to variations of staging, prompted the new frontier of testing FS values in the management of patients with chronic hepatitis.

The course of liver disease in a significant proportion of chronic hepatitis B (CHB) patients is characterized by hepatitis exacerbations, intervened by prolonged remissions whose biochemical and virologic patterns can be mistaken with those of chronic inactive carriers. Thus, measuring LS might be useful to distinguish active from inactive HBV carriers. We addressed this question and present here the results of the cross-sectional and prospective studies of a large cohort of pedigreed hepatitis B virus (HBV) carriers (68 inactive carriers, 200 CHB and nine acute hepatitis B patients).

FS correlates with fibrosis in CHB patients and FS provides a reliable method to assess the overall status of liver disease in the carrier with chronic HBV infection. The mean FS values of HBV-inactive carriers were comparable to those of normal controls and significantly lower than those of CHB patients. Interestingly, in HBV inactive carriers with metabolic liver disease FS values were significantly higher than in HBV-inactive carriers without liver disease. All factors stemming for activity of liver disease, namely the phase of infection (active or inactive), HBV-DNA and ALT levels influenced LS at multivariate analysis. Accordingly, in untreated patients without cirrhosis, histological necrosis and inflammation and ALT were the only factors influencing FS in addition to fibrosis. Thus both necrosis and inflammation influence LS that qualifies as a very promising tool for the non-invasive diagnostic assessment of the liver in the HBV carrier. The best cut-off values for fibrosis and cirrhosis were significantly lower than in chronic hepatitis C (CHC) patients, studied in identical conditions (same center, instrument, operators and test timing), suggesting that FS is influenced also by the different histopathology features of CHB and CHC. This prospective study on patients with hepatitis B exacerbations confirmed 1.2 to 4.4-fold increases of FS values at the time of ALT flares. Similarly, LS paralleled ALT fluctuations in patients with acute hepatitis B. Finally, in treated patients followed up for 48 mo, LS declined yearly at about 0.2-folds, reaching values below the cirrhosis cut-off (11.8 kPa) in a proportion of patients who maintained evidence of cirrhosis. This observation may explain the worse diagnostic performance of FS in treated versus untreated patients. Altogether, these data indicate the non-linear correlation between the kinetics of LS and histological staging during antiviral treatment.

This study suggests that LS provides a useful non-invasive tool to monitor not only fibrosis, but overall liver disease in the chronic HBV carrier. In monitoring CHB patients, LS appears useful to highlight major changes of liver disease and to warrant a more appropriate timing for control liver biopsies.

HBV-inactive carriers mean chronic HBV infection without liver damage caused by HBV, characterized by low HBV-DNA serum levels, persistently normal ALT and undetectable levels of IgM anti-HBc, a marker of HBV-induced liver disease (below the cut-off for CHB). Biochemical remission means transient ALT normalization (spontaneous or induced by antiviral treatment) in patients with CHB.

In this study, authors perform a cross-sectional and longitudinal analysis of LS in HBV carriers, correlating this variable with stage of disease, liver inflammation and other factors that could influence FS measurements. They found a good diagnostic accuracy to detect cirrhosis and fibrosis higher than S3. The work is well performed and conclusions are correctly sustained.