Egypt has a high prevalence of chronic hepatitis C virus (HCV), with about 91% of patients infected with HCV genotype 4 1,2. HCV is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma, and the most common indication for liver transplantation. Also, schistosomiasis represents another important public health problem in Egypt, leading to hepatic schistosomiasis and eventually portal hypertension 3–8. Kamal et al.9 concluded that patients with concomitant schistosomiasis and HCV, predominantly genotype 4, have more advanced liver disease, with higher HCV RNA levels and more severe alterations in liver histology. They show more frequent progression to cirrhosis, hepatocellular carcinoma and higher liver-related mortality rates during follow-up 9. In contrast, Shiha and Zalata 10 concluded that schistosomal hepatic infection does not lead to more severe or progressive disease in patients with chronic HCV.
The accurate diagnosis of HCV-related fibrosis is crucial for prognosis and treatment decisions. Liver biopsy is considered as the gold-standard method for the assessment of liver fibrosis. Liver biopsy currently has three major uses: (a) for diagnosis, (b) for the assessment of prognosis (disease staging) and/or (c) to assist in making therapeutic management decisions 11. However, it is an invasive procedure, with some complications such as pain (40%) and other rare complications such as bleeding (0.5%) 12,13. Unfortunately, there are pitfalls of liver biopsy, which include sampling error, observer variation and lack of a universal scoring system of fibrosis 14–17.
In chronic HCV infection, hepatic steatosis is known to be closely related, acting as a cofactor aggravating hepatic fibrosis 18. There are two discrete forms of steatosis. Metabolic steatosis can coexist with HCV infection, regardless of the genotype, in patients with risk factors such as obesity and insulin resistance. The second form is a result of a direct cytopathic effect of genotype 3 virus infection and is related to the viral load 19,20.
Transient elastography (TE) (FibroScan) is a simple, rapid, noninvasive method. It is an ultrasound-based technique, used for the assessment of tissue stiffness 21. TE appears to be a valuable noninvasive tool to manage patients with chronic viral hepatitis in clinical practice 22–27.
The aim of the study was to determine the possibility of using TE by Fibroscan as a new noninvasive tool for the diagnosis and the monitoring of hepatic fibrosis among chronic hepatitis C genotype 4 patients instead of the standard invasive method (liver biopsy) (we also aimed at evaluating the effects of activity, steatosis and the presence of schistosomiasis on TE measurements).
Patients and methods
This cross-sectional study recruited 519 chronic HCV-infected patients aged 18–60 years, who were selected in the Specialized Medical Hospital, Mansoura University (Egypt), from attendees of the hepatology outpatient clinics, in the period between January 2009 and December 2010. The technique of the study focused on excluding patients having at least one of the following: clinical or radiological evidence of cirrhosis, those with coinfection with hepatitis B or other viruses and severe comorbid conditions, those with other liver diseases and patients with BMI of at least 30 kg/m2. Patients were enrolled after written informed consent was obtained.
All the included patients were subjected to thorough history taking and complete clinical examination with a series of investigations that are routinely performed in the pre-enrolment investigation for interferon therapy, including complete blood counts, the prothrombin time and liver function tests (serum bilirubin, ALT, AST, alkaline phosphatase, serum albumin and the prothrombin time) using commercially available kits; hepatitis B markers (hepatitis B surface antigen, and total and IgM antibodies against hepatitis B core antigen) were tested using commercially available enzyme immunoassay kits; circulating anti-HCV antibodies were tested using the Murex enzyme immunoassay kit; HCV-RNA was tested by the PCR using the Cobas Amplicor test (Roche Diagnostics, Indianapolis, Indiana, USA); autoimmune markers, ANA, ASMA, LKM1, AMA and TSH, were tested by the immunofluorescence technique; indirect hemagglutination test (IHA) was tested for schistosomiasis. Abdominal ultrasound, ECG and funds examinations were also performed.
All the 519 patients recruited were subjected to liver biopsy and TE (Fibroscan) within the same week.
Liver stiffness measurement
Fibroscan (Echosens, Paris, France) was based on one-dimensional TE, a technique that uses both ultrasound (5 MHz) and low-frequency (50 Hz) elastic waves 26. Fibroscan was performed with an ultrasound transducer probe mounted on the axis of a vibrator. A vibration transmitted from the vibrator towards the tissue induces an elastic shear wave that propagates through the tissue. These propagations were followed by pulse-echo sonographic acquisitions, and their velocity, which was directly related to the tissue stiffness, was measured. The harder the tissue, the faster the shear wave propagates. The tip of the probe transducer was placed on the skin between the rib bones at the level of the right lobe of the liver. The best measurement site for LSM was the one generally used for liver biopsy. Once the measurement area had been located, the operator pressed the probe button to start an acquisition. The measurement depth was between 25 and 65 mm below the skin surface. At least 10 successful measurements were performed for each patient, with the ratio of the number of successful measurements over the total number of acquisitions more than 80%. Results were expressed in kilopascal (kPs). The median value was considered as representative of the liver elastic modulus 26,27. Patients with unreliable liver stiffness measurements including those with markedly increased ALT and those with morbid obesity were excluded.
Liver histology and quantification of liver fibrosis
The liver biopsy was obtained according to the consensus recommendations of the Asian Pacific Association for the Study of the Liver (APASL) 28, which included obtaining biopsy using a16-G biopsy needle, preferable core length longer than 15 mm or a core containing at least ten portal tracts (a repeat pass was performed if the biopsy length was <1 cm), and the biopsy was performed only by experts with a minimum training of 50 biopsies carried out under supervision. All biopsy specimens were fixed in formalin and paraffin embedded, and each specimen was analyzed by two experienced pathologists blinded to the clinical data, results of the Fibroscan and results of each other. Using a special stain (Masson trichrome), fibrosis and the necroinflammatory activity were staged according to the METAVIR Scoring System 29, with reporting about steatosis 30 and tissue iron overload. Patients with unsuitable biopsy specimens were excluded.
Data were coded, entered into a computer and processed using PASW statistics 18 (IBM, Armonk, New York, USA) and Medcalc 11.6.1 (MedCalc Software, Mariakerke, Belgium). Patients’ background characteristics and laboratory investigation data were expressed as mean and SD, whereas the median value of TE measured by Fibroscan was calculated for different METAVIR stages of fibrosis. The Kruskal–Wallis nonparametric analysis of variance and the Kendall-τ coefficients of correlation (τb) were used to evaluate the relationship between histological fibrosis scores (METAVIR), and TE was measured by Fibroscan. The relationship between IHA and liver stiffness, activity and liver stiffness, and steatosis and liver stiffness were evaluated using the Mann–Whitney statistics.
The diagnostic performance of TE was assessed by the receiver operating characteristic (ROC) curve. The ROC curve was a plot of sensitivity versus −1 - specificity for all possible cut-off values. The most commonly used index of accuracy was the area under the ROC curve, with values close to 1.0 indicating a high diagnostic accuracy. Cut-off points that yielded a higher total sensitivity and specificity were optimized for different fibrosis stages (F≥2, F≥3, and F=4). The sensitivity, the specificity, the positive predictive value, the negative predictive value, the positive likelihood ratio (+LR) and the negative likelihood ratio (−LR) were calculated for each category. Sensitivity represented the true-positive cases (cases that were positive and identified by TE as such); specificity represented the true-negative cases (cases that were negative and identified as negative by TE as such); positive predictive value represented the proportion of participants with positive test results who were diagnosed correctly by TE; negative predictive value represented the proportion of participants with a negative test result who were diagnosed correctly by TE; +LR was the probability of a person who had the disease testing positive divided by the probability of a person who did not had the disease testing positive; −LR was the probability of a person who had the disease testing negative divided by the probability of a person who did not have the disease testing negative.
Table 1 demonstrates the demographic and the laboratory features of the studied patients. IHA for schistosomiasis was positive in about 81.5% (424) of cases.
Table 2 shows patients’ distribution with regard to METAVIR fibrosis stages, activity and steatosis. The majority of the patients had early fibrosis stages F0, F1, F2 (427 patients; 82.3%); 74 (14.3%) patients had advanced fibrosis; and a minority of 18 patients (3.5%) had complete cirrhosis (F4). Necroinflammatory changes were mild, whereas steatosis was found only in 63 (12.3%) patients and was mild, not exceeding an S2 score.
Table 3 demonstrates different measurements of Fibroscan according to the METAVIR score, and the median value (95% confidence interval) of liver stiffness compared with the METAVIR fibrosis stage: F0, 5.40 kPa; F1, 5.80 kPa; F2, 7.80 kPa; F3, 16 kPa; and F4, 26.30 kPa. Figures 1 and 2 demonstrate the distribution of the Fibroscan results and the relation between liver stiffness by TE and histological grades by liver biopsy. There was a significant statistical difference between the Fibroscan values and different histological stages (P<0.0001). Using the Kendall τ statistical correlation, the elasticity measured by Fibroscan was positively correlated with the biopsy results (τb=0.47, P<0.0001).
The vertical axis was the logarithmic scale of the elasticity score measured in kpa. The bottom and the top of the quadrangle were the first and the third quartiles, respectively. The length of the quadrangle represented the interquartile range. The lines through the middle of the quadrangle represented the median. Error bars were the minimum and the maximum values.
Figure 3 shows the receiver operator characteristics (ROC) curve to determine the whole population according to three different fibrosis stages: F0 and F1 versus F2, F3 and F4 patients (F≥2); F0, F1 and F2 versus F3 and F4 patients (F≥3); and F0, F1, F2 and F3 patients versus F4 patients (F=4). The areas under the ROC curves (95% confidence interval) were 0.816 (0.780–0.849) for F≥2, 0.919 (0.892–0.941) for F≥3 and 0.966 (0.946–0.980) for F=4.
Figure 4 represents a case with F4 METAVIR score, and at least 10 successful measurements were performed for the patient by Fibroscan, with a ratio of the number of successful measurements over the total number of acquisitions more than 80%. Results are expressed in kilopascals (kPs). The median value was considered as a representative of the liver elastic modulus.
Table 4 shows liver stiffness cut-off values; the optimal liver stiffness cut-off value, the sensitivity, the specificity and LRs were obtained for all enrolled patients. Apparent cut-off values for F≥2 (8.6 kPa) and F≥3 (10.2) were both close, but with a greater sensitivity and specificity for F≥3 than for F≥2. The cut-off value for F=4 was 16.3, with sensitivity 100% and specificity 90.62%, but the sample was small, which means that it could systematically lead to overestimation of the sensitivity and the specificity of the test under study, as there were only 18 true-positive cases in group F4 (3.5%) (Table 4).
In conducting a bivariate analysis of the IHA for schistosomiasis and the Fibroscan score using the Mann–Whitney test, there was no significant difference (P=0.387) between the IHA status and the Fibroscan score.
There was a significant relationship (P=0.043) between the elasticity and the activity. This relationship disappeared in controlling for the fibrosis stage. There was also no correlation between Fibroscan measurements and the steatosis degree (P=0.37).
The information derived from the current study affirms that the new noninvasive tool – TE by Fibroscan – can be used as an alternative technique to the standard invasive method (liver biopsy) to diagnose and monitor liver fibrosis among patients with HCV infection.
However, due to the fact that the patients recruited in this study were receiving interferon therapy, a small portion of them was reported to have cirrhosis (F4). The study reported a cut-off level of 16.3 kpa for the detection of cirrhosis (F=4) with an area under the ROC of 0.966, and a very high sensitivity and specificity (sensitivity 100% and specificity 90.62%). Although, this small number of patients who were detected by Fibroscan as cirrhotics may not represent the whole cirrhotic population, yet TE may be considered as a sensitive and reliable method for the diagnosis of cirrhosis among Egyptians.
Different studies reported different cut-off values, sensitivity% and specificity%. For different fibrosis stages, Castera et al.31 reported a cut-off level of cirrhosis of 12.5 kpa, with a sensitivity and a specificity of 87 and 91%, respectively, in 46/183 patients (25.1%). However, Ziol et al.27 found a cut-off level of 14.6 kpa with 86% sensitivity and 96% specificity in 49/251 patients (19.5%). Foucher et al.32 reported a cut-off value of 17.6 kpa, with 77% sensitivity and 97% specificity in 95/354 (27%) patients. The fact that the present study is based on certain exclusion criteria, including the exclusion of all liver specimens less than 1.5 cm or those with less than 10 portal tracts from the analysis, strengthened the findings of the current study. Furthermore, adding these exclusion criteria to the fact that the previously mentioned studies had different genotypes of HCV with different demographic characteristics and different numbers of enrolled patients may explain the result variations.
The study added a very important dimension through the identification of patients with significant fibrosis (F≥2). The current study reported that the cut-off level of significant fibrosis (F≥2) assessed by the METAVIR scoring system was 8.6 kpa, with sensitivity 65.95% and specificity 84.43%. However, the cut-off level of extensive fibrosis (F≥3) was 10.2 kpa with sensitivity 83.70% and specificity 89.23%. This was in accordance with other studies 27–32 that reported cut-off levels of significant fibrosis (F≥2) of 7.1, 8.8 and 7.2 kpa, with sensitivities of 67, 56 and 64% and specificities of 89, 91 and 85%, respectively.
One of the drawbacks of TE in this study was the inability to distinguish fibrosis among less advanced stages of liver fibrosis (F0 vs. F1 and F1 vs. F2). This finding is in accordance with that of Nudo et al.33.
Whereas TE by Fibroscan is influenced by major changes in the biochemical profile, such as hepatitis exacerbation in patients with stable biochemical activity, liver stiffness values did not change significantly, confirming the reliability of the technique under such conditions 34. The current study revealed that the activity could not be measured by liver stiffness measurements.
The current study was unable to affirm the potential influence of schistosomiasis on liver Fibroscan, as there was no significant difference between the IHA state and Fibroscan results (P=0.387). No previous studies could be found to assess this possible relation.
The strength of the current study lies in the large number of patients (519) in whom liver fibrosis assessment by biopsy was indicated for clinical purposes. Meanwhile, other reported studies were on small numbers of patients 35,36.
TE by Fibroscan may be a new noninvasive tool to diagnose and monitor liver fibrosis among patients with HCV infection.
Conflicts of interest
There are no conflicts of interest.
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