Nonalcoholic fatty liver disease (NAFLD) has become, during the last decade, the most common form of chronic liver disease in children and adults. It is closely associated with obesity and threatens to become a serious public health problem in the United States and many other countries. NAFLD is estimated to affect close to 10% of the American population ages 2 to 19 years, and this figure increases to 30% 40% among obese children.
In addition to being a common condition in children, several lines of evidence suggest that NAFLD is a potentially serious condition. A recent long-term longitudinal study has demonstrated that similarly to adults, NAFLD in children is a disease with the potential to progress (1). Some children in the present study presented with cirrhosis at the time of diagnosis; others showed progressive liver disease resulting in significant liver-related morbidity. Moreover, the presence of NAFLD in children may be a key indicator of the metabolic status and a good predictor for the development of type 2 diabetes mellitus. Thus, establishing the diagnosis of NAFLD is of utmost importance and represents a major challenge because the disease is generally silent and the gold standard for diagnosis is an invasive liver biopsy, a procedure that is not suitable for screening purposes.
Other screening measures have been used, including monitoring liver transaminases, and recently, the American Academy of Pediatrics recommended that serum aminotransferases (alanine transaminase and aspartate transaminase [ALT and AST]) be performed in all overweight children starting at age 10 years if their body mass index (BMI) is ≥95th percentile or between the 85th and 94th percentile with risk factors. ALT and AST are to be checked in addition to fasting glucose and the lipid profile; however, it has become clear that in both adults and children, liver enzymes performed poorly for NAFLD diagnosis, with two-thirds of patients with NAFLD showing normal levels of serum ALT and AST (2).
Regarding the use of imaging for screening purposes, hepatic ultrasonography (US) is the most commonly used modality largely because it is relatively inexpensive, widely available, and user-friendly (3). Several studies in adults have demonstrated that this technique is greatly sensitive and specific for the detection of NAFLD. Moreover, hepatic US can provide a good estimate of the degree or extent of hepatic steatosis present based on a series of US characteristics including hepatorenal echo contrast, liver echogenicity, visualization of intrahepatic vessels, and visualization of liver parenchyma and the diaphragm (4–7); however, the diagnostic accuracy of hepatic US and the utility for quantification of the degree of hepatic steatosis in children remain unknown. Thus, we conducted the present study to evaluate the utility of hepatic US for quantifying hepatic steatosis in a large, well-characterized pediatric population with biopsy-proven NAFLD.
PATIENTS AND METHODS
A total of 208 consecutive patients diagnosed with NAFLD examined at Bambino Gesù Children's Hospital from January 2005 to January 2010 were included in the study. The study was approved by the ethics committee of the Bambino Gesù Children's Hospital and Research Institute. Informed consent was obtained from each patient or responsible guardian.
Inclusion criteria were liver biopsy consistent with the diagnosis of NAFLD (8,9). Exclusion criteria were the presence of hepatic virus infections, alcohol consumption (≥140 g/week), history of parenteral nutrition, and use of drugs known to induce steatosis (eg, valproate, amiodarone, prednisone) or to affect body weight and carbohydrate metabolism. Autoimmune liver disease, metabolic liver disease, Wilson disease, and α-1-antitrypsin-associated liver disease were ruled out using standard clinical, laboratory, and histological criteria.
The BMI and BMI z score were calculated (10,11). Metabolic syndrome was defined as the presence of ≥3 of the following 5 criteria (12): abdominal obesity (defined by waist circumference ≥90th percentile for age) (13); hypertriglyceridemia as triglycerides >95th percentile for age, sex, and race (14); low high-density lipoprotein cholesterol as concentrations <5th percentile for age and sex (14); elevated blood pressure (BP) as systolic or diastolic BP >95th percentile for age and sex (15); and impaired fasting glucose or known type 2 diabetes mellitus (16,17). The degrees of insulin resistance (IR) and sensitivity were determined by the homeostasis model assessment for IR using the formula IR = (insulin × glucose)/22.5, by the insulin sensitivity index (ISI) derived from oral glucose tolerance test using the formula ISI = [10,000/square root of (fasting glucose × fasting insulin) × (mean glucose × mean insulin during oral glucose tolerance test)], and by the quantitative insulin sensitivity check index using the formula insulin sensitivity = 1/(log of fasting insulin + log of fasting glucose) (18–20).
The clinical indication for biopsy was to assess either the presence of nonalcoholic steatohepatitis (NASH) and degree of fibrosis or other likely independent or competing liver diseases. Liver biopsy was performed in all of the children, after an overnight fast, using an automatic core biopsy 18 gauge needle (Biopince, Amedic, Sweden) under general anaesthesia and ultrasound guidance. A Sonoline Omnia Ultrasound machine (Siemens, Munich, Germany) with a 5-MHz probe (5.0 C 50, Siemens) with a biopsy adaptor was used. Two biopsy passes within different liver segments were performed for each subject. The length of liver specimen (in millimetres) was recorded. Only samples with a length ≥15 mm and including at least 5 to 6 complete portal tracts were considered adequate for the purpose of the study (9). Biopsies were evaluated by a single-liver pathologist. Biopsies were routinely processed (ie, formalin fixed, paraffin embedded). Sections of liver tissue, 5-μm thick, were stained with hematoxylin-eosin, Van Gieson, PAS-D, and Prussian blue stain. Immunohistochemical staining with antibodies to α-1-antitrypsin was used to exclude α-1-antitrypsin deficiency–associated liver disease. Liver biopsy features were graded according to the NAFLD activity score (NAS) system proposed by Kleiner et al (21). Briefly, grade of steatosis was scored as 0 ≤5%; 1 = 5% to 33%; 2 ≥ 33% to 66%; 3 = 66%; grade of lobular inflammation was scored as 0 = no foci; 1 ≤ 2 foci/200 × field; 2 = 2 to 4 foci/200 × field; 3 = 4 foci/200 × field; and grade of ballooning was scored as 0 = none; 1 = few ballooning cells; and 2 = many cells/prominent ballooning. The grade of steatosis (0–3), lobular inflammation (0–3), and ballooning (0–2) were then combined to determine the NAS (0–8) as proposed. Fibrosis was scored as 0 = none; 1 = periportal or perisinusoidal fibrosis; 2 = perisinusoidal and portal/periportal fibrosis; 3 = bridging fibrosis; and 4 = cirrhosis (10). The liver biopsy samples were then classified as either definitive NASH (unequivocally fulfills previously described criteria for steatohepatitis), borderline diagnosis (some but not all histologic features of steatohepatitis), or simple steatosis (isolated fat deposition in hepatocytes).
Conventional hepatic US was performed within 1 month of the liver biopsy by a single radiologist using an Acuson S2000 ultrasound system (Siemens) with linear and convex transducers (frequency bandwidth 4–14 MHz). The radiologist was blinded to the clinical, laboratory, and histological data of the patients. The ultrasonographic steatosis score (USS) was calculated as follows: absent (score 0) steatosis was defined as normal liver echotexture; mild (score 1) steatosis as slight and diffuse increase in fine parenchymal echoes with normal visualization of diaphragm and portal vein borders; moderate (score 2) steatosis as moderate and diffuse increase in fine echoes with slightly impaired visualization of portal vein borders and diaphragm; and severe (score 3) steatosis as fine echoes with poor or no visualization of portal vein borders, diaphragm, and posterior portion of the right lobe (22).
Descriptive statistics were computed for all of the factors. These included means, standard deviations, and percentiles for continuous variables and frequencies for categorical factors. Univariable analysis was done to compare subjects with moderate/advanced steatosis to those with mild steatosis. Student t tests and Wilcoxon rank sum tests were used to compare continuous variables, Pearson χ2 tests were used for categorical variables and Mantel-Haenszel χ2 tests were used for ordinal factors. Spearman correlation coefficients were used to evaluate correlations between USS and clinical factors of interest. In addition, a multivariable logistic regression analysis was performed to assess factors associated with the presence of moderate/advanced steatosis. USS was forced into the model, and an automated stepwise variable selection was performed on 1000 bootstrap samples; factors that appeared in ≥40% of replications were kept in the final model. A P < 0.05 was considered statistically significant. SAS version 9.2 software (SAS Institute, Cary, NC) and R version 2.10.1 software (R Foundation for Statistical Computing, Vienna, Austria) were used to perform all of the analyses.
The main demographic and clinical features of the subjects included in the analysis are presented in Table 1. Of the 208 participating patients, 132 (64%) were boys. Overall, the median age at the time of first visit was 10.8 years (3.25–14.1 years). Children who had higher USS were significantly older at the time of first visit than those who had lower USS. Eighty-eight percent of the patients were obese. BMI percentile and waist circumference tended to increase with higher USS when compared between groups with USS 0 to 1 and those with USS 2 to 3. This was statistically significant with P values of 0.046 (BMI percentile difference) and 0.014 (waist circumference difference). When comparing the same 2 groups, there was no significant difference in serum ALT or AST levels. Serum alkaline phosphatase and gamma-glutamyl transpeptidase (GGT) were higher in the group with USS 2 to 3 as compared to those with USS 0 to 1.
Comparison of US Determinations With Histological Features on Liver Biopsy
The histological findings and USS of the subjects included in the analysis are presented in Table 2. Fifty-six patients (27%) had severe steatosis on biopsy, 87 patients (42%) had moderate steatosis, 63 patients (30%) had mild steatosis, and only 2 patients (1%) had no or minimal steatosis (<5% steatosis). On ultrasonographic examination, 46 patients (22%) had severe steatosis, 77 (37%) had moderate steatosis, 73 (35%) had mild steatosis, and 12 (6%) had no evidence of steatosis. Examining other individual histological features showed that 56% of the patients had stage 1 fibrosis with only 12% having stage 2 or 3 fibrosis. Regarding inflammation grade, most of the patients (73%) had grade 1 inflammatory activity. There was no inflammation on biopsy in 11.5% of the patients. Fifty-three percent showed no ballooning (grade 0), 23% had grade 1, and 24% had grade 2. Sixty-six patients (32%) had a diagnosis of NASH based on NAS, and 56 patients (27%) had isolated hepatic steatosis.
When dividing the patients into 2 groups based on USS (1 group with USS 0–1 and 1 group with USS 2–3), we found that there was a statistically significant difference between the 2 groups regarding steatosis on liver biopsy. Ninety-two percent of the patients with USS 2 to 3 also had moderate to severe steatosis on biopsy and a majority of the patients with USS 0 to 1 had mild or no steatosis on biopsy (P < 0.001). When comparing the other histological features in the 2 groups of patients based on USS, we found that there was no significant difference between the 2 groups regarding extent of fibrosis and inflammation. Patients with USS of 2 to 3 had more ballooning on biopsy than those patients with USS of 0 to 1.
Utility of USS for Detection of Hepatic Steatosis
Table 3 outlines the correlations between USS and clinical and histological features evaluated in the study. There was excellent correlation between USS and steatosis on biopsy with a Spearman coefficient of 0.80 (confidence interval [CI; 0.71–0.88], P < 0.001; Fig. 1). Using a cutoff value of 2 for the USS, the area under the receiver operating characteristics curve for ultrasonographic detection of moderate to severe steatosis was 0.87 (Fig. 2). Sensitivity and specificity for diagnosing moderate to severe steatosis with USS ≥2 were 79.7% and 86.2%, respectively. With a cutoff value of 3 for the USS, specificity for diagnosing moderate to severe steatosis on biopsy increased to 100%. Using multivariable logistic regression, we found that for each 1-unit increase in USS, there was a 27-fold increase in the odds ratio for having moderate to severe steatosis on biopsy. Correlation between USS and ballooning was statistically significant, although positive correlation was only fair (ρ = 0.4, CI (0.28–0.53), P < 0.001). The USS did not correlate significantly with inflammation or fibrosis. There was significant correlation between the USS and NAS; however, this correlation was primarily caused by the steatosis component of the NAS. A significant association was found between USS and BMI (P < 0.006), waist circumference (P < 0.001), and measurements of IR and sensitivity. Interestingly, serum ALT and AST levels were not associated with histological grade of steatosis and had no correlation with USS (Fig. 3).
The principal findings of the present study relate to the utility of hepatic US for detection and quantification of hepatic steatosis in children with NAFLD. The results of the present study, which included one of the largest cohorts of children with biopsy-proven NAFLD reported to date, demonstrate that USS correlates closely with severity of steatosis on liver biopsy. These results suggest that ultrasonographic examination is an excellent screening test for NAFLD in children and a useful noninvasive modality for quantifying hepatic steatosis. Moreover, we found that serum aminotransferases had poor predictive value regarding the presence or severity of fatty liver disease; thus, serum ALT and AST are not helpful in dictating whether further workup should be done in obese children in whom fatty liver is suspected.
US has long been recognized as a useful screening tool for NAFLD and is the most frequently used radiological modality in fatty liver evaluation (3). Most of the available data on the use of US for the diagnosis of steatosis have been retrospective and focused on adult populations (5–7). A recent study by Chiloiro et al (4) investigated the relation among ultrasonographic diagnosis of fatty liver, adipose tissue distribution, and metabolic profile in 94 moderately obese children with no available liver biopsy. Fatty liver on US positively related to anthropometric measurements as well as IR and other metabolic abnormalities associated with obesity, whereas no statistically significant association was found between fatty liver on US and increased serum ALT and AST levels. Our present study extends these observations and demonstrates a strong positive correlation between USS and degree of steatosis on liver biopsy while confirming the lack of association of serum transaminases with both fatty liver determination by US and liver biopsy. These findings are of utmost importance because current screening guidelines for identifying and performing further work-up of children at risk of NAFLD are also solely based on serum transaminase levels. Our current data, in conjunction with the growing literature addressing the limitations of serum liver enzymes (23,24) in the evaluation of NAFLD, suggest hepatic US to be a more valid alternative for screening the at-risk pediatric population.
We found that hepatic US was unable to make the distinction between NAFLD and NASH, a finding that is similar to previous reports in adult population studies (5,7). One prospective adult study looking at the use of US in detecting hepatic steatosis found that none of the US findings could distinguish between steatosis and NASH, with no significant association found between US findings and stage of fibrosis or grade of inflammation. Furthermore, the lowest amount of steatosis that could be detected via US with the greatest correlation with histological findings was ≥20% (5). Our study adds further strength to the finding that US sensitivity and specificity in quantifying steatosis are improved when steatosis is moderate to severe. A USS of 2 in our study had a sensitivity of almost 80% in diagnosing moderate to severe steatosis. Similar to what has been reported in the adult population, determination of USS in children was not able to distinguish between steatosis and NASH and was not useful for quantitation of inflammatory activity and stage of fibrosis. These results further support the need for development of novel reliable biomarkers for risk stratification and monitoring response to therapeutic intervention in children with NAFLD.
The main strengths of our study are the inclusion of a large group of consecutively recruited children with liver biopsy–proven NAFLD with the full spectrum of disease in whom US was performed within 1 month of liver biopsy procedure. The limitations of our study include the fact that the patients were seen at a large referral tertiary care medical center, so it is possible that results may not be extrapolated to children with NAFLD from the community. In addition, US examinations were read once by a single radiologist; therefore intra- and interobserver variability could not be evaluated. Finally, in the present study we were unable to evaluate the potential causes of US failure in detecting steatosis because only a small group of patients (n = 12) had USS of 0. Further studies evaluating US as a tool for longitudinal follow-up are warranted.
In summary, our results indicate that hepatic US is a useful tool for quantifying steatosis in pediatric patients who have suspected NAFLD, with USS strongly correlating with grade of steatosis on liver biopsy. Ultrasonogrpahic examination of the liver should be an initial screening tool, even in the face of normal liver enzymes, because these are poor predictors of fatty liver disease.
1. Feldstein AE, Charatcharoenwitthaya P, Treeprasertsuk S, et al. The natural history of non-alcoholic fatty liver disease in children: a follow-up study for up to 20 years. Gut
2. Browning JD, Szczepaniak LS, Dobbins R, et al. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology (Baltimore, Md)
3. Mazhar SM, Shiehmorteza M, Sirlin CB. Noninvasive assessment of hepatic steatosis. Clin Gastroenterol Hepatol
4. Chiloiro M, Riezzo G, Chiarappa S, et al. Relationship among fatty liver, adipose tissue distribution and metabolic profile in moderately obese children: an ultrasonographic study. Curr Pharm Des
5. Dasarathy S, Dasarathy J, Khiyami A, et al. Validity of real time ultrasound in the diagnosis of hepatic steatosis: a prospective study. J Hepatol
6. Hamaguchi M, Kojima T, Itoh Y, et al. The severity of ultrasonographic findings in nonalcoholic fatty liver disease reflects the metabolic syndrome and visceral fat accumulation. Am J Gastroenterol
7. Saadeh S, Younossi ZM, Remer EM, et al. The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology
8. Nobili V, Manco M, Devito R, et al. Effect of vitamin E on aminotransferase levels and insulin resistance in children with non-alcoholic fatty liver disease. Aliment Pharmacol Ther
9. Manco M, Nobili V. Intensive treatment and dietary fats in adolescents with nonalcoholic fatty liver disease. J Pediatr Gastroenterol Nutr
10. Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, et al. CDC growth charts: United States. Adv Data
11. Cole TJ, Bellizzi MC, Flegal KM, et al. Establishing a standard definition for child overweight and obesity worldwide: international survey. BMJ (Clin Res Ed)
12. Boney CM, Verma A, Tucker R, et al. Metabolic syndrome in childhood: association with birth weight, maternal obesity, and gestational diabetes mellitus. Pediatrics
13. Fernandez JR, Redden DT, Pietrobelli A, et al. Waist circumference percentiles in nationally representative samples of African-American, European-American, and Mexican-American children and adolescents. J Pediatr
14. American Academy of Pediatrics. National Cholesterol Education Program: report of the expert panel on blood cholesterol levels in children and adolescents. Pediatrics
15. Report of the Second Task Force on Blood Pressure Control in Children—1987. Task Force on Blood Pressure Control in Children. National Heart, Lung, and Blood Institute, Bethesda, Maryland. Pediatrics
16. Genuth S. Lowering the criterion for impaired fasting glucose is in order. Diabetes Care
17. Genuth S, Alberti KG, Bennett P, et al. Follow-up report on the diagnosis of diabetes mellitus. Diabetes Care
18. Abdul-Ghani MA, Matsuda M, Balas B, et al. Muscle and liver insulin resistance indexes derived from the oral glucose tolerance test. Diabetes Care
19. Katz A, Nambi SS, Mather K, et al. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endocrinol Metab
20. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care
21. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology
22. Kim SH, Lee JM, Kim JH, et al. Appropriateness of a donor liver with respect to macrosteatosis: application of artificial neural networks to US images—initial experience. Radiology
23. Schwimmer JB, Dunn W, Norman GJ, et al. SAFETY study: alanine aminotransferase cutoff values are set too high for reliable detection of pediatric chronic liver disease. Gastroenterology
24. Franzese A, Vajro P, Argenziano A, et al. Liver involvement in obese children. Ultrasonography and liver enzyme levels at diagnosis and during follow-up in an Italian population. Dig Dis Sci