During the mid-1990s, the development of highly active antiretroviral therapy (HAART) resulted in a significant decrease in morbidity and mortality among HIV-infected children (1–3). Nevertheless, many chronically HIV-infected patients exposed to HAART suffer from various kinds of toxicity (4–7). In HIV-positive adults, liver disease is becoming one of the leading causes of morbidity and mortality (8–11). Liver affection in HIV infection stems from several nonexclusive processes including a direct cytopathic effect of the HIV on liver cells (12,13), medication-related hepatotoxicity (14), opportunistic infection such as tuberculosis, HIV-induced metabolic conditions such as steatosis and steatohepatitis (15), and eventual concomitant chronic hepatitis B viruses or hepatitis C virus (HCV).
Children are particularly exposed to cumulative drug-related hepatotoxicity because of a longer life expectancy and the need for long-term control of viral replication (2,16). Furthermore, young children tend to have higher viral loads (17,18) and unfavourable pharmacokinetics profiles for some HAART drugs because of hepatic metabolism or enzyme polymorphism (19,20). Early recognition of hepatic events can improve the safe and effective use of HAART and enhance the survival of HIV-infected patients. Recently, Soriano et al (9) proposed a strategy for diagnosis, prevention, and treatment of antiretroviral drug-related liver injury.
Nevertheless, to date, few studies exploring different parameters of liver function have been conducted in HIV-infected children. Only 2 studies published in 1999 and 2003 reported data from liver biopsy in these patients (21,22). Results in terms of Metavir scores were rather reassuring, but seem insufficient for formal conclusions. Furthermore, although liver biopsy remains the gold standard for liver assessment (23), its invasiveness, sampling errors, variability in interpretation, and expense do not make it an ideal routine follow-up examination (24,25). Recognition of hepatotoxicity in patients, therefore, routinely relies on the quantification of serum liver enzymes, but their poor correlation with histological features in liver disease (26,27) and the lack of a homogenous definition for hepatotoxicity (8) render serum liver enzymes insufficient for monitoring liver condition on a regular basis.
During the last decade, new noninvasive biological and radiological tools allowing repeated assessment of the liver have been developed. These tests, respectively, are analyse hepatic fibrosis (FibroTest [FT], Forns index, aspartate aminotransferase [AST] to platelet ratio index [APRI] and transient elastography), necroinflammatory activity (ActiTest [AT]), and liver steatosis (SteatoTest [ST]). FibroTest, AT, and ST (BioPredictive, Paris, France) are patented artificial intelligence algorithms comprising routinely available laboratory serum markers with excellent diagnosis and prognosis accuracy (28–33). Forns index identifies patients without significant liver fibrosis (Metavir stages F0–F1) with a high accuracy (34), whereas APRI detects patients with advanced fibrosis (>F2) and cirrhosis with a good sensibility (35–37). Transient elastography (TE) or Fibroscan (EchoSens, Paris, France) is a novel, rapid, and totally painless bedside method to assess liver elasticity on the basis of 1-dimensional transient elastography (38). Transient elastography measures are well correlated with advanced stages of fibrosis and cirrhosis in adults and children (39–41).
The aims of this study were to evaluate the feasibility of noninvasive hepatic procedures in HIV-infected children in routine follow-up and to determine the gross prevalence of liver affection in this population. We searched for the existence of correlations between the biological algorithms FT/AT/ST results and the degree of liver tissue impairment measured by TE (stiffness) and ultrasound (steatosis). We also conducted an analysis on the influence of HIV disease severity and exposure to HAART on hepatotoxicity.
MATERIALS AND METHODS
We conducted a cross-sectional study in HIV-1 chronically infected children followed at the University Hospital of Nice, Department of Paediatric Haematology. Inclusion criteria were materno-foetal transmission, age 8 to 18 years, life expectancy >6 months, Lansky score (42) >70%, and informed parental consent and child assent.
Patients' sex, age, weight, height, nadir CD4 T-cell count (lowest CD4 count in the patient's medical history), highest viral load, HIV staging according to the Centers for Disease Control and Prevention (CDC) classification (43), past and present exposure to HAART drugs, and exposure duration for each drug were recorded. Patients were considered to be in a prepubertal stage in the absence of breast development for girls and with a testicular volume below 4 mL for boys (44). The existence of lipoatrophy or lipohypertrophy was assessed by 2 practitioners for each child according to the European Paediatric Lipodystrophy Group definition (45). Peripheral blood T lymphocytes and subclass assays (CD4, CD8) were conducted by flow cytometry. Real-time plasma HIV RNA values were determined using the Cobas Ampliprep-Cobas TaqMan HIV Monitor assay (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Undetectable viral load was defined as polymerase chain reaction-RNA below 1.60 log10 or 40 copies per mm3.
Assessment of Hepatic Function
Standard Biological Measurements
All of the blood samples were collected after an overnight fasting. Total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), apolipoprotein A1, and total cholesterol and triglycerides measurements were evaluated according to the manufacturer's instructions on the same automate (Integra 800, Roche Diagnostic).
FT, AT, and ST
FibroTest and AT combine the quantitative results of 6 serum biochemical markers (α-2 macroglobulin, haptoglobin, GGT, total bilirubin, apolipoprotein A1, and ALT) in patented algorithms (29). SteatoTest includes the patient's weight and height and 9 serum biochemical markers (AST, total cholesterol, and triglycerides in addition to the previous 6 markers) (30). As recommended by Biopredictive, values <0.25 for these tests were considered normal.
Forns index combines age, platelet count, GGT, and cholesterol in the following formula: 7.811 − 3.131.ln(platelet count) + 0.781.ln(GGT) + 3.467.ln(age) − 0.014(cholesterol). As recommended by Forns et al (34), values <4.21 were considered normal.
APRI is the ratio of the quantitative AST result to the platelet count. As recommended by Wai et al (35), values >1 were considered normal.
Transient Elastography or Fibroscan
The TE technique is used to quantify hepatic fibrosis in a totally noninvasive and painless manner, with no contraindications for the patient. A mechanical pulse is generated at the skin surface, which is propagated through the liver. The velocity of the wave is measured by ultrasound. This velocity is directly correlated to the stiffness of the liver, which reflects the degree of fibrosis. All of the measurements were performed according to the manufacturer's instructions (Echosens, Paris, France) by the same trained operator (R.T.). Patients were placed in supine decubitus position with the right arm in abduction. The probe was placed between 2 ribs. The measurement area was located by A-mode images provided by the probe transducer. Ten measurements were performed for each patient. The median value was assumed to be representative of liver stiffness. A set of measurements was considered to be reliable if the success rate was at least 60% and the interquartile range was less than one third of the median liver stiffness value. Because the limiting factor for TE in children is the distance between 2 ribs, only children older than 8 years were included in the study. Indeed, at the time we conducted this study the adapted children probe was unavailable.
Because TE norms have not yet been established in healthy children devoid of liver disease, a control cohort of 19 healthy sex- and age-matched children was included. A TE was performed by the same operator in the same conditions as for the HIV-positive patients.
Patients underwent abdominal ultrasound to detect liver steatosis and fibrosis. Because ultrasound is considered to be operator dependent, the examination was performed twice by 2 separate radiologists (C.B., C.L.).
Results are expressed as mean and range values. For biochemical standard hepatic markers, data are also furnished as multiples of the upper limit norm for age and sex to preclude age and sex influence. To confirm immunovirological stability for each patient, the latest and ongoing values for CD4 T-cell counts and percentages and for HIV-1 polymerase chain reaction-RNA were compared using the Wilcoxon signed rank test for paired groups. Comparisons of qualitative parameters were done with the Fisher exact test. Correlations between quantitative values used regression model. Adjusted R2 values (adjusted R2) are furnished. Quantitative results between subgroups of patients were compared with the Mann-Whitney U test. All of the statistical analyses were performed using Statview software 5.0 for Windows (SAS Institute, Cary, NC). P values below 0.05 are considered to indicate statistical significance.
Twenty-six HIV-1 chronically infected children were included. There were 15 girls and 11 boys, with a median age of 12.9 years (8.3–17.3). Twenty-three patients were monoinfected, 3 were HIV–HCV coinfected. Five were HIV stage N of the CDC classification, 5 were stage A, 8 were stage B, and 7 suffered from AIDS definition illness (stage C). HIV CDC status could not be determined for 1 child because of the lack of past information (orphan).
At inclusion, mean weight was 44.1 kg (18.4–83.5) and mean height was 149.6 cm (116–175). The mean body mass index was 19.1 kg/m2 (13.7–29.9), which corresponds to the 44th percentile or a −0.15 SD for age and sex. Fifteen patients (58%) had lipodystrophy: 9 had peripheral lipoatrophy and 6 had central hypertrophy. No patient had hepatomegaly or jaundice at inclusion or in the past medical history.
Upon inclusion, mean nadir CD4 T-cell count was 345 cells/mm3 (10–870) and nadir CD4 percentage was 16.2% (2–36). The highest measured viral load in the patients' history was 5.69 log10 copies/mm3 on average (3.20–6.18). Mean CD4 T-cell count at inclusion was 699 cells/mm3 (164–1252) or 30.6% (9.4–45%), with a viral load of 3.79 log10 copies/mm3 (1.60–4.83). Eleven children had undetectable viral load. Patients' immunological and virological status was stable as no differences were found between CD4 T-cell counts, percentages, and viral loads when comparing data study values and those measured at the last assessment for each patient.
Each child had been exposed to an average of 6.5 antiretroviral therapy drugs since birth (2–12): 4.2 nucleoside reverse transcriptase inhibitors (NRTI) (2–7), 0.7 non-nucleoside reverse transcriptase inhibitor (NNRTI) (0–2), and 1.6 protease inhibitors (PI) (0–4). The drugs most frequently prescribed were lamivudine (92% of the children) for a mean duration of 59 months (4–113), stavudine (77%) during an average of 61 months (5–129), and zidovudine (69%) for an average of 54 months (1–118). The most prescribed PIs were the combination of ritonavir and lopinavir (65%) for a mean duration of 32 months (1–80) and nelfinavir (35%) for an average of 48 months (17–105). Efavirenz and nevirapine were prescribed in, respectively, 58% and 11.5% of the patients. At the time of enrollment, 11 patients received conventional HAART with 2 NRTIs and 1 PI, 6 received 2 NRTIs + 1 NNRTI, 2 patients were receiving dual NRTI association, 1 was receiving triple NRTI combination, and 2 were receiving 1 drug of each class (NRTI+NNRTI+PI). Four children were on planned treatment interruption.
Assessment of Hepatic Markers
All of the patients underwent blood testing, 23 had an abdominal ultrasound, and 21 had a Fibroscan. For the latter, the average measurement success rate was 96.7%. Five children did not have a Fibroscan and 3 did not have ultrasound because the devices or the operator was unavailable on the day they consulted. The 2 youngest children failed in having a TE measure because of technical difficulties because of their small corpulence. Twenty-six results were obtained for FT, AT, and ST. Two patients were excluded from FT and AT analyses because of aberrant scores. Global results for standard biological hepatic markers, noninvasive algorithm tests, and TE are reported in Table 1. Nine female patients (60%) had elevated AST and 4 (27%) had abnormal ALT. Fifteen patients (63%) had abnormal FT values and 8 (33%) had excessive AT scores. A distinction between results of the 23 HIV monoinfected and the 3 HIV–HVC co-infected patients is exposed in Table 2.
Four children (17%) had signs of mild liver steatosis on ultrasound. HIV-infected patients had significantly higher TE measures than matched healthy control children (P = 0.014, Table 3). Furthermore, loss of elasticity assessed by TE measures tended to increase with age in a linear manner in patients (adjusted R2 = 0.208, P < 0.03), but not in healthy controls (Fig. 1).
Comparisons of Test Results
For the 19 patients who underwent successful TE, results for TE, FT, and AT were compared. Patients with abnormal FT results also had significantly higher TE measures than patients with normal FT (P = 0.01) in spite of similar ages. Abnormal FT results were also associated with higher AT scores (P = 0.02, Table 4). The 4 patients with steatosis on ultrasound had normal ST results.
Risk Factors for Hepatotoxicity
We did not find any difference in terms of hepatic parameters regarding sex, pubertal stage, and existence or type of lipodystrophy. Significant differences were observed between asymptomatic patients (N stage of CDC classification) and all of the symptomatic children (stages A, B, C) (Table 5). N stage patients were on average older and had been exposed to more NRTI and PI drugs with longer exposure durations. They also had significantly higher FT results, Forns index, and TE measures (Table 5).
We analysed the relation between the exposure to HAART drugs and the observed hepatic abnormalities. There were statistical correlations between the total number of NRTI drugs since birth and Forns index (adjusted R2: 0.19, P = 0.03), and between the total number of NNRTI drugs and AT (adjusted R2: 0.24, P = 0.008). Considering individual HAART agents of each class for which at least 50% of all patients had been exposed, no drug demonstrated significantly associated hepatotoxicity.
A decade after the widespread use of HAART in the management of chronically HIV-infected children, there has been growing concern about their long-term toxicity. Surprisingly few paediatric data are available concerning liver toxicity. Liver biopsy, the gold standard for liver assessment, is an invasive procedure in paediatric practice. In the past, major indications for liver biopsy in HIV-1-monoinfected patients were persistent biological hepatic abnormalities, hepatomegaly, jaundice, and unexplained prolonged fever (21). The most common biopsy-derived diagnosis was opportunistic infections, mainly mycobacterium avium complex (46). As prevalence of opportunistic infections has dramatically decreased (47), there has been a reduction in liver biopsy indications. We did not conduct a liver biopsy in our patients because none had any of the recognized biopsy indications (21). Noninvasive procedures allowing harmless and repeated assessment of liver status and function were recently developed. We considered that these investigations could be useful tools in the management of HIV-1 chronically infected children in whom repeated liver biopsies could not be done.
In this cross-sectional study a high proportion of HIV-infected children showed biological signs of liver affection, with 12 patients presenting with elevated liver enzymes (AST, ALT, or both), 63% and 33% abnormal FT and AT results, respectively. Four patients (17%) had signs of mild steatosis on ultrasound in our cohort, whereas the prevalence of fatty liver in a school-age population is estimated to be around 2.6% to 5% (48,49). This high prevalence is concordant with that published by Gil et al (50), but would need to be confirmed in larger cohorts. Nevertheless, none of the ST results was abnormal for these 4 patients. This suggests either that ST is not sensitive enough for the detection of mild steatosis in children or that steatosis had been incorrectly identified on ultrasound. Although hepatic fibrosis has never been demonstrated in HIV-monoinfected children, we found biological (FT, Forns index) and radiological (TE) evidence of liver fibrosis in our patients. We did not find any ultrasound sign of fibrosis, either because it was absent or early or because of the poor sensitivity of ultrasound fibrosis detection (51). It is nonetheless possible that these perturbations reflect nonspecific injury rather than a true fibrotic process. Unfortunately, none of our patients had a liver biopsy to evaluate this possibility.
The only significant correlations that could be evidenced concerning the exposure to HAART drugs and the degree of hepatotoxicity were between the exposure duration to NRTI and Forns index, and between the exposure duration to NNRTI and AT results. Forns index and AT are recognized, respectively, as good indicators of mild liver fibrosis and of hepatic necroinflammatory injury (29,34). These correlations are, therefore, compatible with an infraclinical mechanism of continuous hepatic toxicity. The underlying mechanisms of HAART-derived liver toxicity are intricate and still poorly understood. Mechanisms proposed for NRTI-related toxicity include mitochondrial dysfunction caused by inhibition of mitochondrial DNA polymerase leading to steatosis and potential lactic acidosis (9,52,53). NNRTI-associated toxicity appears to be the result of hypersensitivity reactions (9,54,55). Indirect toxicity has also been reported through abnormalities of lipid and/or glycemic regulation, with lesions similar to those observed in nonalcoholic steatohepatitis (56). It is also possible that besides the direct effect of HAART on hepatocytes, variations in the intrahepatic immune system participate in this toxicity.
We recognise that there are several limits to this study. We acknowledge the fact that all of the algorithms we used have never been validated in children or in HIV-monoinfected patients. Nevertheless, we considered that even if the pathogenic processes leading to hepatic injury in HCV, HIV, or co-infected patients are multifactorial, their consequences are univocal at the cellular level, resulting in inflammation and/or necrosis.
Transient elastography has been used in various hepatic pathological conditions (38–41,57–59). Paediatric published data mainly imply children with primary liver disease (hepatitis B virus, HCV, biliary atresia) or with affections with well-recognized liver impact (cystic fibrosis, Wilson disease) (40). Because TE norms have been established in healthy adults (5.49 ± 1.49 kPa) (60) but not in children, age- and sex-matched controls were included. In our cohort, the average TE score was significantly higher in HIV-infected children than in controls. Furthermore, the liver's stiffness tended to increase with age in a linear manner in patients. It remains unclear whether this relation persists after the age of 18 years or if the curve flattens and the liver elasticity stabilises. This correlation between age and liver stiffness was found only among the HIV-infected children. We, therefore, hypothesised that HIV infection and/or continuous exposure to HAART were responsible for this relation. Compared with symptomatic patients, N-stage children had the longest exposure duration to NRTI and had higher FT, Forns index, and TE scores. This strongly suggests that HAART exposure is the leading cause of hepatic injury, assuming that viral replication is (at least partially) under control. We also found that children with abnormal values for biochemical algorithms, FT mainly, had increased liver stiffness compared to age-matched patients. Transient elastography and biochemical algorithms, therefore, seem to be potent complementary and concordant methods of noninvasive liver investigations.
Our results confirm that evaluation of hepatic disease with noninvasive procedures is feasible in HIV-1 chronically infected children. These patients are at risk for liver dysfunction as a consequence of their infection and, possibly, its treatment. This risk seems to increase with time. Biological and radiological signs of liver injury should, therefore, be monitored on a regular basis.
The authors thank all of the participating children and their families; Prof Marc Albertini, Prof Etienne Bérard for helpful advice on the research protocol; Dr Nicolas Sirvent for critical comments on the text; the radiologists who performed the abdominal ultrasounds: Dr Carole Leroux and Dr Corinne Boyer; Dr Frederic Berthier for advice on statistical analysis; and Marie Soumah, Christelle Falzon. Dr Céline Caruba for information on biochemical technologies.
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