Non-invasive diagnosis of liver fibrosis in chronic hepatitis C

Abstract

Assessment of liver fibrosis in chronic hepatitis C virus (HCV) infection is considered a relevant part of patient care and key for decision making. Although liver biopsy has been considered the gold standard for staging liver fibrosis, it is an invasive technique and subject to sampling errors and significant intra- and inter-observer variability. Over the last decade, several noninvasive markers were proposed for liver fibrosis diagnosis in chronic HCV infection, with variable performance. Besides the clear advantage of being noninvasive, a more objective interpretation of test results may overcome the mentioned intra- and inter-observer variability of liver biopsy. In addition, these tests can theoretically offer a more accurate view of fibrogenic events occurring in the entire liver with the advantage of providing frequent fibrosis evaluation without additional risk. However, in general, these tests show low accuracy in discriminating between intermediate stages of fibrosis and may be influenced by several hepatic and extra-hepatic conditions. These methods are either serum markers (usually combined in a mathematical model) or imaging modalities that can be used separately or combined in algorithms to improve accuracy. In this review we will discuss the different noninvasive methods that are currently available for the evaluation of liver fibrosis in chronic hepatitis C, their advantages, limitations and application in clinical practice.

Key Words: Liver fibrosis, Liver cirrhosis, Hepatitis C, Diagnosis, Elasticity imaging techniques

Core tip: There is increased interest in non-invasive markers of fibrosis, especially in chronic hepatitis C virus infection. Although several methodologies have been proposed over the last few years, the limited availability and concerns regarding the true accuracy of these techniques has restricted their clinical application. In this review we will discuss the different noninvasive methods that are currently available for the evaluation of liver fibrosis in chronic hepatitis C, their advantages, limitations and application in clinical practice.

INTRODUCTION

Assessment of liver fibrosis in chronic hepatitis C virus (HCV) infection is considered a relevant part of patient care and key for decision making. Liver fibrosis stage is probably the most robust prognostic factor in several liver diseases; including hepatitis C. Higher stages of fibrosis have been shown to be associated with progression to decompensated cirrhosis, the need for liver transplantation and liver-related death in HCV infection[1,2]. In addition, the severity of fibrosis may be used as a selection criterion for antiviral therapy and can indicate the need for further evaluations, such as surveillance for hepatocellular carcinoma (HCC) and esophageal varices screening[3,4]. For many years, liver biopsy has been considered the gold standard for staging liver fibrosis. Histological evaluation also provides information on necroinflammatory activity and other features such as steatosis and iron overload. Several scoring systems have been developed, the most common being the METAVIR, Scheuer’s, the Batts-Ludwig, the International Association for the Study of the Liver (IASL) and the Ishak scoring systems[5-9]. However, besides its advantages, liver biopsy is an invasive technique with associated morbidity. Minor complications are relatively common and about one fourth of patients have pain in the right upper quadrant or right shoulder after liver biopsy[10]. Severe complications are infrequent, with significant bleeding rates varying from 0.05% to 5.3% and mortality of less than 0.15% in the largest series[10]. The performance of liver biopsy for fibrosis staging has also been questioned and concerns regarding the possibility of sampling errors and significant intra- and inter-observer variability were raised over the last few years. Since biopsy represents 1/50000 of the liver, the heterogeneity of liver fibrosis in HCV infection and the inadequacy of sample size can cause considerable bias in the assessment of hepatic histology[11-13]. A study which included 124 patients with chronic HCV infection who underwent simultaneous laparoscopy-guided biopsies of the right and left hepatic lobes showed that 33.1% of the subjects had a difference of at least one stage between the two lobes[11]. Similarly, a study on virtual liver biopsy indicated that a non-fragmented specimen of at least 25 mm in length would be necessary to correctly evaluate fibrosis with a semiquantitative score, a goal not always achievable in daily practice[14].

Over the last few years, several noninvasive markers were proposed for liver fibrosis diagnosis in chronic HCV infection. Table 1 summarizes the major advantages and limitations of these methods in relation to liver biopsy. Besides the clear advantage of being noninvasive, a more objective interpretation of test results may overcome the mentioned intra- and inter-observer variability of liver biopsy. In addition, these tests can theoretically offer a more accurate view of fibrogenic events occurring in the entire liver with the advantage of providing frequent fibrosis evaluation without additional risk. However, in general, these tests show low accuracy in discriminating between intermediate stages of fibrosis and may be influenced by several hepatic and extra-hepatic conditions. In this review we will discuss the different noninvasive methods that are currently available for the evaluation of liver fibrosis in chronic hepatitis C, their advantages, limitations and application in clinical practice.

Table 1 Major advantages and limitations of liver biopsy and noninvasive fibrosis markers.

	Liver biopsy	Noninvasive markers
Advantages
	Validated scoring systems	Absence of significant discomfort and risks
	Differential diagnosis and associated conditions	Allows frequent re-evaluation
	Simultaneous evaluation of necro-inflammation	Objective interpretation
		Patient acceptance
Limitations
	Invasive	Low accuracy to discriminate between intermediate stages of fibrosis
	High cost	Nonspecific for the liver (biomarkers)
	Sampling errors and intra- and inter-observer variability	Influence of several extra-hepatic factors

NONINVASIVE MARKERS OF LIVER FIBROSIS

General considerations of noninvasive fibrosis markers

Because fibrosis denotes morphological changes, liver biopsy became the natural gold standard for staging the disease. However, the mentioned limitations of biopsy make it very difficult to interpret the “real” performance of surrogate markers of fibrosis in studies. In the vast majority of studies, the diagnostic performance of noninvasive markers of liver fibrosis was evaluated by calculating the area under the receiver operating characteristic (ROC) curve. The ROC curve is the plot that depicts the trade-off between the sensitivity and 1-specificity across a series of cut-off points when the diagnostic test is a continuously variable[15]. Mehta et al[16] demonstrated that, when a range of accuracies of biopsy and a range of prevalence of fibrosis are taken into account, even in the “best” scenario an area under receiver operating characteristic curve > 0.90 cannot be achieved even for a perfect marker. The perceived limitation in diagnostic accuracy of noninvasive markers of liver fibrosis is probably the major reason why these tests have not been widely adopted in clinical practice.

Noninvasive markers of liver fibrosis can be divided into two groups: serum biomarkers and imaging techniques. These methodologies will be presented separately and a combined approach will also be discussed in this review.

Serum biomarkers

Indirect (or class II) markers of liver fibrosis: This group comprises, in general, routine tests usually combined with other laboratory or clinical parameters in a specific model. The most commonly studied indirect markers in HCV infection include aspartate aminotransferase (AST), alanine aminotransferase (ALT), platelet count, gamma-glutamyltransferase (GGT), bilirubin, haptoglobin, apolipoprotein A1, and alpha-2-macroglobulin. Models that combined indirect markers are usually devised from retrospective studies and, as a rule for noninvasive fibrosis markers, are limited in discriminating between intermediate stages of fibrosis. Table 2 depicts the most common models including indirect markers proposed for fibrosis estimation in hepatitis C.

Table 2 Selected models including indirect markers of liver fibrosis.

Score (original reference)	Variables	Performance in HCV patients1
		Significant fibrosis (≥F2)			Cirrhosis (≥F4)
		Median AUROC	Median sensitivity2	Median specificity2	Median AUROC	Median sensitivity2	Median specificity2
APRI[24]	AST and platelet count	0.77	81%	95%	0.84	77%	94%
FIB-43[41]	Age, AST, ALT and platelet count	0.74	64%	79%	0.87	90%	92%
Forns index[48]	Age, platelet count, cholesterol levels, and GGT	0.76	88%	94%	0.87	98%	91%
Fibro index[50]	Platelet count, AST, and gamma globulin	0.76	94%	97%	0.86	70%	91%

¹Based on reference number 49;

²Sensitivity values are presented for the lower cutoff and specificity for the upper cutoff (when multiple cutoffs are presented); ³Model originally proposed for HIV/HCV co-infected patients. HCV: Hepatitis C virus; AUROC: Area under the receiver operating characteristic curve; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; GGT: Gamma-glutamyltransferase; HIV: Human immunodeficiency virus.

The AST/ALT ratio has been used for several years as a noninvasive method for assessing the severity of chronic liver diseases, including chronic HCV infection[17-20]. Although some studies have found promising results, its performance as a noninvasive marker of fibrosis is generally low, especially in the diagnosis of less advanced stages of fibrosis[21,22]. In a recent study, we showed that the AST/ALT ratio had a low diagnostic accuracy in detecting significant fibrosis (AUROC of 0.661) as compared to other simple models, such as APRI (AUROC 0.793) and FIB-4 (AUROC 0.811)[23].

The AST-to-Platelet Ratio Index (APRI) is calculated as (AST/upper limit of normal range)/platelet count (10⁹/L) × 100. This index was originally proposed by Wai et al[24] in 2003 and became one of the most studied noninvasive fibrosis markers in chronic HCV infection. The APRI is based on the rationale that worsening of fibrosis and increasing portal pressure are associated with reduced production of thrombopoietin by hepatocytes, increased platelet sequestration within the spleen and reduced clearance of AST[25-27]. A meta-analysis exploring the performance of this model in HCV infection was published in 2011 and included 40 studies and a total of 8739 subjects[28]. This study showed only a moderate degree of accuracy for APRI in the detection of HCV-related fibrosis. The summary AUROC of the APRI for the diagnosis of significant fibrosis (≥F₂ according to METAVIR), severe fibrosis (≥F₃), and cirrhosis was 0.77, 0.80, and 0.83, respectively[28]. In this meta-analysis, the best cutoff for diagnosing significant fibrosis was 0.7, with a summary sensitivity and specificity of 77% and 72%, respectively. For the detection of cirrhosis, the optimal cutoff was 1.0, with a summary sensitivity and specificity of 76% and 72%, respectively. A threshold of 2.0 exhibited a specificity of 91% for diagnosing cirrhosis, but a low sensitivity of 46%[28]. A major advantage of APRI is that it was validated in special populations, such as HIV/HCV co-infection[28-37], in whom the overall performance seems to be lower than in HCV mono-infected individuals[28], and adjusted cutoffs may increase sensitivity and specificity, as we previously demonstrated[30]. APRI also proved to be a valuable tool in hemodialysis patients with chronic HCV infection. In a study which included 203 subjects, we demonstrated good performance of this model, especially in excluding significant fibrosis, with a negative predictive value (NPV) of 93% for a prevalence of significant fibrosis of 24%[38]. These results were further validated in a cohort of 279 hemodialysis patients[39] and Canbakan et al[40] showed that APRI was superior to FibroTest^® in this population.

The FIB-4 is also a noninvasive method for the evaluation of liver fibrosis, based on simple variables such as age, AST, ALT and platelet count. It was initially proposed by researchers in the APRICOT study (AIDS Pegasys Ribavirin International Coinfection Trial) to evaluate the presence of liver fibrosis in HIV/HCV coinfected patients[41]. It was subsequently validated in a large cohort of HCV mono-infected patients in whom values < 1.45 had a NPV of 94.7% to exclude severe fibrosis (F₃-F₄) with a sensitivity of 74.3%[42]. A FIB-4 value higher than 3.25 had a positive predictive value (PPV) of 82.1% with a specificity of 98.2%[42]. The authors also showed a similar performance between FIB-4 and FibroTest^®[42]. Several other studies reported that the FIB-4 index had a variable degree of accuracy in HCV-infected subjects[43-47]. We performed a comparison between FIB-4 and APRI and found similar AUROCs for both models (0.811 vs 0.793)[23]. However, the proportion of biopsies that could have been correctly avoided was substantially higher with FIB-4 than with APRI (63% vs 47%) suggesting that FIB-4 is probably a more useful tool for incorporation into daily practice[23].

The Forns index is based on age and three additional simple tests: platelet count, cholesterol levels, and GGT[48]. In the original study, the AUROC was 0.86 for the estimation group and 0.81 for the validation group in diagnosing significant fibrosis[48]. However, in a recent systematic review, the median AUROC from 22 studies was 0.76 for significant fibrosis and 0.87 for cirrhosis[49]. When evaluating those studies that performed direct comparisons, the Forns index and APRI showed a very similar performance for both significant fibrosis and cirrhosis[49].

The Fibroindex was originally proposed for diagnosis of HCV-related fibrosis and includes the following variables: platelet count, AST, and gamma globulin[50]. In the original study, this model exhibited a higher AUROC (0.83) for diagnosing significant fibrosis as compared to APRI and the Forns index[50]. Fibro index was also correlated significantly with variation in fibrosis stage when a subset of 30 patients who had undergone a liver biopsy twice was evaluated[50]. Nevertheless, in the mentioned systematic review, the median AUROC for significant fibrosis detection was 0.76 and for cirrhosis was 0.86[49]. Direct comparisons showed no superiority of Fibroindex over APRI for both significant fibrosis and cirrhosis detection[49].

Direct (or class I) markers of liver fibrosis: Multiple etiologies of liver disease, including chronic HCV infection, can lead to liver fibrosis through integrated signaling networks that regulate the deposition of extracellular matrix[51]. This sequence of events drives the activation of hepatic stellate cells into a myofibroblast-like phenotype that is contractile, proliferative and fibrogenic[51]. Collagen and other extracellular matrix (ECM) components are deposited as the liver generates a wound-healing response to encapsulate injury[51]. The direct (or class I) markers of liver fibrosis are usually fragments of the liver matrix components produced by hepatic stellate cells during the process of ECM remodeling, usually reflecting the deposition or removal of ECM[52]. The most studied direct markers are hyaluronic acid (HA), YKL-40, laminin, fibronectin, alpha-2-macroglobulin, procollagen type I carboxy terminal peptide (PICP), procollagen type III amino-terminal peptide (PIIINP), N-terminal propeptide of type II collagen, metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) and transforming growth factor-b1 (TGF-b1).

As stated for indirect tests, direct biomarkers are usually combined in composite scores to increase the diagnostic performance (Table 3). It is important to point out that these models are not liver-specific and have limitations, such as low accuracy for intermediate stages of fibrosis and limited availability (patented formulas or tests not routinely performed). Below, we discuss the most studied models combining direct and indirect biomarkers for the diagnosis of liver fibrosis in chronic hepatitis C.

Table 3 Most studied models combining direct and indirect biomarkers for the diagnosis of liver fibrosis in chronic hepatitis C.

Score (original reference)	Variables	Performance in HCV patients1
		Significant fibrosis (≥F2)			Cirrhosis (≥F4)
		Median AUROC	Median sensitivity2	Median specificity2	Median AUROC	Median sensitivity2	Median specificity2
FibroTest[53]	Age, sex, serum haptoglobin, α2-macroglobulin, apolipoprotein A1, GGT, and total bilirubin	0.79	92%	96%	0.86	85%	81%
ELF[56]	Age, TIMP-1, PIIINP and hyaluronic acid	0.81	85%	70%	0.88	-	-
Fibrometer[61]	Platelet count, prothrombin index, AST, α2-macroglobulin, hyaluronic acid, blood urea nitrogen and age	0.82	69%	81%	0.91	-	-
FIBROspect II[63]	TIMP-1, α2-macroglobulin and hyaluronic acid	0.86	80%	70%	-	-	-
Hepascore[66]	Age, sex, α2-macroglobulin, hyaluronic acid, GGT and total bilirubin	0.79	66%	79%	0.89	72%	86%

¹Based on reference number 49;

²Sensitivity values are presented for the lower cutoff and specificity for the upper cutoff (when multiple cutoffs are presented). ELF: Enhanced liver fibrosis; HCV: Hepatitis C virus; AUROC: Area under the receiver operating characteristic curve; GGT: Gamma-glutamyltransferase; TIMP: Tissue inhibitor of matrix metalloproteinases; PIIINP: Procollagen type III amino-terminal peptide; AST: Aspartate aminotransferase.

The FibroTest^® score was originally proposed in 2001 and is one of the most validated models for predicting liver fibrosis in HCV-infected patients[53]. It is computed by accessing a proprietary website and entering the patient’s age, sex, and serum haptoglobin, α2-macroglobulin, apolipoprotein A1, GGT, and total bilirubin. A meta-analysis published in 2007 showed a pooled adjusted AUROC of 0.83 for the FibroTest^® in HCV patients[54]. However, the systematic review published by Chou and Wasson[49] revealed that when only studies that performed direct comparisons were considered, the APRI was associated with only a slightly lower AUROC than the FibroTest^® for significant fibrosis (median difference between AUROCs, -0.03; range, -0.10 to 0.07), but there was no difference for cirrhosis (median difference between AUROCs, 0.0; range, -0.04 to 0.06). In addition, the FibroTest^® was not superior to FIB-4 and other models such as the Fibrometer^® and Hepascore^® for the diagnosis HCV-related fibrosis, as shown in the same review[49]. Beyond the absence of a clear superiority of the FibroTest^® over other free and readily available models, and given the variability of components of assays and analyzers, FibroTest^® assays can only be performed in validated laboratories. In addition, the existence of hemolysis or Gilbert syndrome can lead to false-positive results and must be taken into account[55].

The enhanced liver fibrosis (ELF) score provides a single value by an algorithm combining age as well as quantitative serum measurements of TIMP-1, PIIINP and HA[56]. Age was removed from the simplified ELF score[56]. It was originally proposed in a cohort study including 1021 subjects with various chronic liver diseases (496 with chronic hepatitis C). In contrast to the good performance observed for alcoholic liver disease (AUROC 0.944) and non-alcoholic fatty liver disease (AUROC 0.870), a relatively low accuracy was observed in chronic HCV infection (AUROC 0.773)[56]. ELF was further validated in HCV patients[57-59], but it was not superior to APRI for the detection of both significant fibrosis and cirrhosis in studies that performed a direct comparison between the two models[49,58]. A recent study has shown that the ELF score is significantly influenced by gender (higher values in men) and age (higher scores in older persons), and this should be taken into account when interpreting the results of this scoring system[60].

The Fibrometer^® is a patented and commercially available proprietary panel of tests that combines platelet count, prothrombin index, AST, α2-macroglobulin, HA, blood urea nitrogen and age[61]. In addition to the typical results of fibrosis stage corresponding to the METAVIR system, the Fibrometer^® may also indicate the amount of liver fibrosis as a percentage of fibrous tissue within the liver (area of fibrosis)[62]. In the systematic review published by Chou and Wasson, the Fibrometer^® exhibited a pooled AUROC of 0.82 for significant fibrosis and 0.91 for cirrhosis[49]. Direct comparisons showed that the Fibrometer^® performed better than APRI and the FibroTest^® in detecting both significant fibrosis and cirrhosis[43,49].

FIBROspect II^® is also a commercially available panel that includes TIMP-1, α2-macroglobulin and HA levels[63]. It was generated from a cohort of 696 HCV-infected patients and exhibited an AUROC of 0.831 for diagnosing significant fibrosis in the original study[63]. FIBROspect II^® was subsequently validated[64,65] and the pooled AUROC in the mentioned systematic review was 0.86 for significant fibrosis[49].

The Hepascore^® is a patented model that combines age, sex and four biomarkers (α2-macroglobulin, HA, GGT and total bilirubin)[66]. It was devised in a cohort of chronic HCV patients[66] and further validated in several studies[67-72]. An interesting advantage of Hepascore^® is that it can be totally automated using a single analyzer and only one serum sample[69]. However, data from comparative studies showed that this test was not superior to APRI and FibroTest^® in diagnosing significant fibrosis and only slightly better for the detection of cirrhosis[49].

Imaging techniques

Transient hepatic elastography (TE) by FibroScan^®: TE, as assessed by FibroScan^® (Echosens, Paris, France), is a simple non-invasive method to measure liver stiffness, based on unidimensional transient elastography, a technique that uses elastic waves and low frequency ultrasounds (50 Hz). The equipment is composed of a probe, an in-built ultrasound system and an electronic unit for data processing. Through a transductor, low amplitude vibrations produced by the probe are transmitted to liver tissue. Simultaneously, the ultrasound system generates pulses that track and determine the rapidity of propagation of elastic waves within the parenchyma. The velocity of propagation is directly related to elasticity: the harder the tissue, the faster the propagation of elastic waves. Hence, tests with high results generally indicate the presence of significant fibrosis in liver parenchyma. The final result is the median of all valid acquisitions, which is considered to be representative of the hepatic elasticity, expressed in kilopascals (kPa), within a range of 2.5-75.0 kPa. The test is simple, fast (usually performed in less than 5 min), and can be easily carried out in both inpatient and outpatient settings. Neither special preparation nor laboratory tests are necessary.

TE has been evaluated in several non-viral chronic liver diseases[73-75] in both adults and children[76], it was, initially projected and then validated in patients with chronic hepatitis C[77-81].

Several studies have shown a significant correlation between TE and fibrosis stage, as assessed by the METAVIR scoring system[78-80], as well as by computer-assisted morphometric image analysis[81]. TE by the FibroScan^® shows a similar performance for predicting significant fibrosis and higher accuracy to identify liver cirrhosis, as compared to other noninvasive tests. This has been shown by Castera et al[78] who evaluated and compared the performance of FibroScan^®, FibroTest^® (FT, Biopredictive, Paris, France) and the AST-to-platelet ratio index (APRI) in 183 HCV patients. AUROCs of FibroScan^®, FibroTest^® and APRI for the diagnosis of significant fibrosis (F≥ 2) were 0.83, 0.85 and 0.78, respectively. To predict the presence of cirrhosis (F₄), the AUROCs for each method were 0.95, 0.87 and 0.83, respectively. The diagnostic superiority of TE was further confirmed in a study of 298 HCV patients by comparing the performance of FibroScan^®, FT, APRI, Lok index, platelet count (PC), prothrombin index (PI) and AST/ALT ratio (AAR) for the early detection of cirrhosis. In this study, TE was the most accurate method for predicting cirrhosis (AUROCs: TE 0.96 vs FT 0.82, Lok and APRI 0.80, PC 0.79, PI 0.73, AAR 0.61, P < 0.0001)[82].

The most validated TE cutoff points are 7.1 kPa to identify patients with significant fibrosis and 12.5 kPa to recognize those with cirrhosis[78,82,83]. TE values ≥ 14.6 kPa exhibit a positive predictive value of 90% to predict liver cirrhosis, with a positive likelihood ratio of 35.5[82].

TE was also evaluated in special populations of HCV patients, such as those with HCV-HIV co-infection[84,85] and post-transplant hepatitis C[86-88], with similar accuracy to that observed in the general population of HCV patients.

Besides estimating fibrosis stage, TE has been used to identify cirrhotic patients at risk of developing portal hypertension and liver-related complications[82,85,89,90]. However, several cutoff values have been used and many of these studies have found weak correlation coefficients, particularly among patients with high HVPG (> 10 mmHg). Altogether, these data indicate that the relationship between TE and HVPG is insufficiently linear to be clinically useful.

In contrast to HCV, few studies with appropriate methodology have evaluated the accuracy of TE in patients with chronic hepatitis B virus (HBV) infection[83,91-94]. An algorithm has been proposed to adjust the interpretation of TE values according to ALT levels. Values < 6.0 kPa and < 7.5 kPa accurately predict the absence of advanced fibrosis or cirrhosis in patients with serum ALT levels inferior to the upper limit of normality (ULN) and in subjects with ALT activity between 1 and 5 times the ULN, respectively[93]. Similarly, TE values > 9.0 kPa and > 12.0 kPa predict advanced fibrosis or cirrhosis in subjects with normal ALT and in those with ALT 1-5 times the ULN, respectively. A recent study suggested that TE exhibits a similar diagnostic performance in HBV infection as compared to HCV patients[83].

Acoustic radiation force impulse imaging: Although the acoustic radiation force impulse imaging technique has been developed by two companies, Siemens and Philips, most clinical studies have used the Siemens S2000 conventional ultrasound equipment which uses short-duration acoustic pulses (push-pulses) emitted with a frequency around 2.6 MHz. The compression induced by the pulse in the evaluated tissue generates shear waves which propagate perpendicularly into the tissue. These shear waves are tracked by the pulse-echo ultrasound acquisitions and their velocity of propagation is measured inside a small region-of-interest (ROI) of 5 mm × 10 mm located up to 8 cm in depth. The stiffer the tissue, the faster the shear wave velocity, which means that the speed of the shear wave increases with the severity of liver fibrosis[95-97]. The results are expressed in meters per second (m/s), ranging from 0.5 to 4.4 m/s (± 20% accuracy over the range). Higher values are generally found in the left hepatic lobe, but higher accuracy is obtained with ARFI measurements in the right hepatic lobe[98-100]. Although not formally recommended by the manufacturer, good quality technical parameters (especially an interquartile ratio < 30%) yielded better correlations between elastometry measurements and liver fibrosis, as well as higher accuracies for predicting fibrosis stages than those with inadequate parameters[101,102]. Evaluating 106 subjects with HCV infection, Bota et al[101] observed discordance of at least two stages of fibrosis between ARFI results and histological assessment in 31.7% of patients; in multivariate analysis, female gender and IQR ≥ 30% were associated with discordance. Finally, as observed with TE, high aminotransferase levels (> 5 times the upper limit of normal) are associated with higher liver stiffness as assessed by ARFI, which should be taken into account in order to interpret results adequately[103]. It is intuitive to consider that the impact of higher aminotransferase levels reflects the influence of higher levels of necroinflammatory activity, which has indeed been demonstrated by Chen et al[104].

ARFI elastography is an easy, fast (usually performed within five minutes), and reproducible noninvasive method for liver fibrosis assessment, especially in cirrhotic patients. However, ARFI reproducibility was lower in women, in patients with high BMI (≥ 25 kg/m²), in the presence of ascites and in the absence of liver cirrhosis[105]. Being included into a conventional ultrasound machine and the possibility of being performed in patients with ascites are relevant advantages of ARFI elastography over TE by FibroScan^®. A standard procedure should include measuring in a supine position with the convex transducer (4C1) without specific breathing maneuvers (the patient is simply asked to stop breathing for a moment). Elastometry values increase after food intake, and measurements should be performed in the fasting state (or at least 3 h after the last meal)[106,107].

Although in a limited number of studies, the method has been evaluated in a variety of liver conditions, such as metabolic liver diseases[108-112], autoimmune liver diseases[113], hepatic tumors[114,115], and chronic hepatitis B[116,117]. However, like TE, ARFI has been most studied in patients with chronic HCV infection[118-128]. In a pooled meta-analysis, Friedrich-Rust et al[129] evaluated the original data of 518 patients from eight studies (73% with HCV). The optimal cut-offs for diagnosing significant fibrosis (F≥ 2), advanced fibrosis (F≥ 3) and cirrhosis were 1.34, 1.55, and 1.80 m/s, respectively, with diagnostic accuracies (represented by AUROCs) of 0.87, 0.91, and 0.93, respectively. In a large, international multicenter study, Sporea et al[119] retrospectively evaluated 914 HCV subjects. They observed a significant positive correlation between liver stiffness by ARFI and fibrosis stage (Spearman r = 0.654; P < 0.0001), and a good diagnostic performance for predicting fibrosis stage according to the METAVIR score. Although with significant overlapping of ARFI measurements for fibrosis F₀-F₂, advanced fibrosis (F≥ 3) and cirrhosis could be accurately excluded. Generally, the correlation with histological fibrosis was similar between TE and ARFI elastography. Nevertheless, TE was better than ARFI for predicting the presence of liver cirrhosis and fibrosis (F≥ 1)[119]. A recent meta-analysis which included 13 studies and 1163 patients with different hepatopathies demonstrated that for the detection of both significant fibrosis (F≥ 2) and cirrhosis, the diagnostic performance of ARFI and TE were comparable, but ARFI showed a higher rate of reliable measurements as compared to TE[130].

Combined approach

Algorithms combining different fibrosis tests have been proposed to improve the accuracy of noninvasive methods for the correct diagnosis of liver fibrosis in HCV infection. They use two serum-based models either simultaneously or in a sequential procedure[72,131,132]. They may also be based on agreement between a blood test and an imaging technique[133,134]. The Leroy algorithm was proposed in a study that evaluated six non-invasive scores in 180 HCV patients[72]. In this approach, the APRI and FibroTest^® were calculated simultaneously and concordant results below the lower cutoffs (FibroTest^® < 0.22 and APRI < 0.5) could rule out significant fibrosis with a NPV of 94.1%. Results above the upper cutoffs (FibroTest^® > 0.59 and APRI > 2) exhibited a PPV for significant fibrosis of 96.7% and for extensive fibrosis (F₃-F₄) of 92.2%[72]. However, only 32% of patients presented concordant results. Therefore, a significant proportion of subjects are expected to require a liver biopsy when using this system[72]. The sequential algorithm (SAFE for fibrosis evaluation) biopsy algorithm was proposed in a study that included 2035 HCV patients[131]. The SAFE biopsy for simultaneous detection of significant fibrosis and cirrhosis produced only 52 (2.6%) misclassified cases, with an overall accuracy of 97.4%. However, as stated for the Leroy algorithm, liver biopsy would be required in the majority of cases (64%)[131].

A different approach was proposed by Castéra et al[133] in an algorithm combining FibroTest^® and TE simultaneously. In this method (called the Castera algorithm or Bordeaux algorithm), the diagnosis of significant fibrosis is based in the finding of concordant results for both methods, and liver biopsy is recommended in discordant results or for those individuals in whom liver stiffness measurement was not possible[133]. For the diagnosis of significant fibrosis, the number of saved liver biopsies was significantly higher using Castera than the SAFE biopsy algorithm (71.9% vs 48.3%, respectively). However, accuracy of the SAFE biopsy algorithm was significantly higher than the Castera algorithm (97.0% vs 87.7%, respectively).

More recently, a combination of Fibrometer^® and FibroScan^® was proposed in a study including 1785 patients with chronic hepatitis C[134]. The so-called FM+FS classification includes 6 fibrosis classes (F_0/1; F_1/2; F_2±1; F_2/3; F_3±1 and F₄) and requires no liver biopsy[134]. Using this approach, the proportion of discordant results decreased as compared to the SAFE biopsy and Castera algorithm. Although this new classification of liver fibrosis appears to be more a formalization of uncertainty, it may also be an astute tactic for the practical application of noninvasive fibrosis markers, allowing a more precise interpretation of its results. However, the employment of two relatively expensive methods will increase costs and may limit application of the FM+FS classification.

Although algorithms including every previously studied noninvasive marker are obviously not available, based on the above data, a simple and plausible approach would be to simultaneously perform two tests (serum and imaging technique), and reserve liver biopsy for discordant results (Figure 1). We suggest using models based on simple blood tests, such as APRI or FIB-4, as there is no clear evidence for the use of more complex and expensive tests in this setting. Similar methodology was proposed in other review articles[135,136]. Using this system, it is probable that liver biopsy will still be necessary in a significant proportion of patients. However, the number of misclassifications is likely to be low.

Figure 1 Suggested algorithm for diagnosing liver fibrosis in chronic hepatitis C. Patients with concordant results in two techniques (serum and imaging) may be followed without liver biopsy and histological analysis can be reserved for those with discordant results. HCV: Hepatitis C virus.

COST-EFFECTIVENESS OF NONINVASIVE MARKERS OF FIBROSIS

The total cost of each strategy for diagnosing liver fibrosis depends on several factors such as the need for hospitalization or sedation in the case of liver biopsy, potential risks, the modality of noninvasive tests and finally, the diagnostic accuracy. Although the procedure protocol varies greatly, liver biopsy usually requires short-term hospitalization, the administration of sedatives and specialized nursing staff for post-biopsy care. In addition, ultrasonography is often used to mark or guide the biopsy. These particularities and the need for interpretation are responsible for the relatively high cost of liver biopsy, ranging from $1200 in the United Kingdom[137] to $2200 in the United States[138]. There are limited data regarding the cost-effectiveness of noninvasive strategies for liver fibrosis assessment, especially for indirect markers. However, recent studies evaluating the FibroScan^® and FibroTest^® showed a favorable cost-effectiveness profile for both noninvasive tests as compared to the traditional approach based on liver biopsy[139-142]. There is a need for studies evaluating the cost-effectiveness of different strategies of noninvasive liver fibrosis assessment, such as the combined approach and the use of indirect markers. This information would be of special interest in low-income countries where the costs of liver biopsy are expected to be lower, but with limited availability.

CONCLUSION

The accurate diagnosis of liver fibrosis is essential for decision-making in chronic hepatitis C. Even though, over the last decade, remarkable achievements have been made in the noninvasive diagnosis of fibrosis, this is an evolving field and there is still room for improvement. It is possible that, in the near future, the incorporation of other methodologies such as genetic, proteomic, and metabolomics profiles will allow the diagnosis of fibrosis at earlier stages, even permitting the identification of stellate cell activation in pre-fibrotic stages. In addition, extensive validation of currently available tools, including the investigation of their prognostic value may extend the applicability of noninvasive fibrosis markers in clinical practice.

Although noninvasive tests are now routinely used in several countries, they are still very limited in differentiating between early stages of fibrosis, and this fact, at least in part, may be related to liver biopsy limitations. Along with the improvement in current noninvasive markers, there is also a need for changes in the way we look at fibrosis in the near future.