Diagnostic accuracy of noninvasive markers of steatosis, NASH and liver fibrosis in HIV-

monoinfected individuals at-risk of non-alcoholic fatty liver disease (NAFLD): results from the

ECHAM study

Maud Lemoine1, Lambert Assoumou2, Stephane De Wit3, Pierre-Marie Girard4, Marc Antoine Valantin5,

Christine Katlama5, Coca Necsoi3, Pauline Campa4, Anja D. Huefner6, Julian Schulze zur Wiesch6,

Hayette Rougier4, Jean-Philippe Bastard7, 8, Hartmut Stocker9, Stefan Mauss10, Lawrence Serfaty11, Vlad

Ratziu12, Yves Menu13, Jerome Schlue14, Georg Behrens15, Pierre Bedossa16, Jacqueline Capeau8,

Patrick Ingiliz17*, Dominique Costagliola*2 on behalf of the ANRS-ECHAM group.

* equally contributed

1 Department of Surgery and Cancer, liver unit, St Mary’s hospital, Imperial College London, UK

2 INSERM, Sorbonne Université, Institut Pierre Louis d’Épidémiologie et de Santé Publique (IPLESP),

Paris, France

3 Service des Maladies Infectieuses , CHU Saint-Pierre, Université Libre de Bruxelles , Brussels ,

Belgium

4 Saint-Antoine Hospital, Department of Infectious disease and tropical medicine, University Paris 6,

France

5 Pitie-Salpetriere Hospital, Department of Infectious disease and tropical medicine, University Paris 6,

France

6 Infectious Disease Unit University Medical Center, Hamburg-Eppendorf Germany

7 AP-HP, Hôpital Tenon, UF Bio-marqueurs Inflammatoires et métaboliques, Service de Biochimie, F-

75020, Paris

8 Sorbonne Université, Faculté de Médecine, F-75012 Paris ; INSERM, CRSA, UMR_S 938, F-75012

Paris, France

9 Department of Infectious Diseases, Vivantes Auguste-Viktoria-Klinikum, Berlin, Germany

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10 Center for HIV and Hepatogastroenterology, Düsseldorf, Germany.

11 Saint-Antoine Hospital, Department of Hepatology, University Paris 6, France

12 Pitie-Salpetriere Hospital, Department of Hepatology University Paris 6, France

13 Saint-Antoine Hospital , Department of Radiology, University Paris 6, France

14 Department of Clinical Immunology and Rheumatology, Hannover Medical School, Hannover,

Germany

15 Institute of Pathology, Hannover Medical School, Hannover, Germany

16 Beaujon Hospital, Department of Histopathology, Clichy, France

17 Center for Infectiology (CIB), Berlin, Germany

Running Title: Noninvasive markers of NAFLD in PLHIV

Corresponding author:

Maud Lemoine

Imperial College London

Department of surgery and liver cancer, liver unit

St Mary’s hospital

South Wharf Street

London, UK

Email: m.lemoine@imperial.ac.uk

Tel: + 44 (0) 20 331 25 212

Word count: 3,749

Tables: 3

Figures: 3

Suppl. Tables: 2

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Suppl. Figure: 1

References: 55

Introduction

Nonalcoholic fatty liver disease (NAFLD) is characterised by hepatic fat accumulation in the absence of

excessive alcohol consumption and is mainly associated with alterations observed in the metabolic

syndrome as central obesity, high blood pressure and impaired glucose and lipid homeostasis (1).

Following the global burden of obesity and diabetes in the general population, NAFLD has become the

most common cause of chronic liver disease in Western countries (2, 3) and is expected to become the

most frequent indication for liver transplantation by 2030 (4, 5).

Over the last years, the prognosis and life expectancy of people living with HIV (PLHIV) have greatly

improved and the burden of metabolic disorders has also become a concern with up to 70% of PLHIV

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being overweight or obese according to a recent population-based study in the US (median age 50

years (IQR 37-59), 73% males) (6) and up to 30% in the Age-HIV cohort in the Netherlands (median

age 53 years (IQR 48-60), 89% males) (7). In 2010 8% of PLHIV in France had a BMI ≥30 kg/m2

according to the French hospital database on HIV (8) A meta-analysis including 65 studies (from 1998

to 2005) estimated the prevalence of metabolic syndrome in HIV-infected populations between 17% and

31% according to the definition criteria (9). As a result, PLHIV represent a population at-risk of NAFLD

with an estimated prevalence at 35% (95% confidence interval (CI) 29-42) according to a recent meta-

analysis (10). This suggests that the prevalence of NAFLD in HIV-monoinfected patients may be higher

(35%) than the general population (25%) (2). HIV individuals might be at particular high risk of NAFLD

due to the impact of HIV infection and of exposure to some antiretroviral (ARV) drugs on lipid and

glucose homeostasis, adipose tissue, gut dysbiosis and accelerated aging (11, 12).

In contrast to HIV-uninfected individuals, data on NAFLD in PLHIV are limited and no recommendations

for its diagnosis and management have been developed so far. As a result, its clinical management is

highly heterogeneous.

NAFLD encompasses two main pathological conditions: 1/ non-alcoholic fatty liver (NAFL) i.e liver

steatosis as defined by steatosis in more than 5% of hepatocytes, with or without mild lobular

inflammation and 2/ non-alcoholic steatohepatitis (NASH), a more severe entity, defined by liver

steatosis (>5%), lobular inflammation and hepatocytes ballooning (13). NAFL is considered as a benign

condition that can however evolve towards NASH and its complications, fibrosis, cirrhosis and

hepatocellular carcinoma (13).

In HIV-uninfected individuals, NAFLD is associated with an increased prevalence of metabolic

comorbidities, cardiovascular events and mortality (14). The severity of liver fibrosis is the strongest

predictor of disease-specific morbidity and overall mortality, regardless other histological features (15,

16). As a result, the European guidelines for the study of the liver (EASL) currently recommend

screening for NAFLD and liver fibrosis patients who are obese or have a metabolic syndrome, using

noninvasive markers as a first step, and then considering liver biopsy in case of suspected NASH or

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advanced liver fibrosis (13). However, liver biopsy is an invasive and expensive procedure, requiring a

high level of expertise for sample analysis (17). Consequently, a variety of non-invasive methods have

been developed for the diagnosis of steatosis, NASH and liver fibrosis. Whilst the diagnosis of NASH

still relies on liver histology, liver steatosis can be detected and quantified using biochemical markers

(e.g SteatotestTM) or different imaging techniques (e.g magnetic resonance imaging (MRI), or the

controlled attenuation parameter (CAP) using transient elastography method). For the diagnosis and

staging of liver fibrosis, non-invasive methods including serum markers based on routine (e.g FIB-4,

aspartate aminotransferase (AST) to platelet ratio (APRI) APRI, NAFLD-fibrosis score (NFS)) or more

sophisticated laboratory tests (e.g FibrotestTM (FibroSureTM in USA) or FibrometerTM) and imaging

techniques (e.g Fibroscan®) have been developed over the last years. However, these tests have been

poorly validated in HIV-monoinfected patients and the diagnosis modalities of NAFLD are highly

heterogeneous in this population (10).

We prospectively enrolled HIV-monoinfected individuals at risk of NAFLD in a large European study

(ECHAM), and assessed the accuracy of noninvasive methods widely validated in HIV-uninfected

individuals i.e hepatic MRI proton-density-fat-fraction (MRI-PDFF), Fibroscan®/CAPTM and various

biochemical tests for the diagnosis of steatosis, NASH and liver fibrosis using liver histology as the

reference.

Patients and methods

Study population

ECHAM (European Cohort on HIV, Aging and Metabolic liver disease) is a multicenter European cross-

sectional study (7 centers from Belgium, France and Germany). HIV-1 infected adult individuals over 40

years of age, receiving antiretroviral therapy (ART) for at least 5 years and with a plasma HIV viral load

<400 copies/mL and CD4 cell count >100/mm3), were invited to participate to the study if they met at

least one following criteria: 1) persistently elevated liver function tests defined by transaminases ≥1.5

upper limit of normal (ULN) and/or gamma-glutamyltransferase (GGT) level ≥2ULN, confirmed in two

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blood samples within at least a three-month interval, or 2) metabolic syndrome defined by the 2009

international criteria (1), or 3) clinical lipodystrophy reported by the patient as previously described (18).

Participants were not eligible in the study if they met one of the following criteria: positive hepatitis B Ag

or C virus antibody, coinfection with HIV-2, use of intravenous drugs within the last six months,

excessive alcohol intake (>30 g/day), genetic hemochromatosis, autoimmune hepatitis, primary or

secondary biliary cirrhosis or cholangitis, alpha1 antitrypsin deficiency, Wilson’s disease, secondary

causes of NAFLD i.e ongoing prolonged steroid therapy, current therapy with amiodarone, tamoxifen,

methotrexate, nifedipine, or hycanthone, history of cancer chemotherapy, short bowel syndrome,

polycystic ovarian syndrome, Weber-Christian disease, active opportunistic infection except for candida

oesophagitis, ongoing cancer, pregnancy, uncontrolled congestive heart failure. Participants were

enrolled in the study after providing written consent. The study (clinicaltrial NCT02093754) was

approved by the local ethic committees and conducted according to the Helsinki declaration.

Demographic and clinical data

Demographic and clinical data were collected at time of enrolment and recorded in standardized forms

by trained investigators.

Biochemical measurement and calculation of tests of steatosis, NASH and fibrosis

Blood samples were collected after a 12-hour overnight fast for determination of liver function tests

(AST, alanine aminotransferase (ALT), GGT, alkaline phosphatase (ALP), total and conjugated

bilirubin), glucose, cholesterol (total, LDL, HDL), triglycerides (TG) and insulin. Measurements of

circulating insulin were centralized and performed using a high specific immunoassay (Architect;

Abbott Laboratories, Rungis, France). Insulin resistance (IR) was assessed by using the

Homeostasis Model Assessment Method index (HOMA-IR) as follows: fasting insulin (mU/L) x

fasting plasma glucose (mmol/L)/22.5 (19). IR was defined by a HOMA-IR index ≥2.5. FibroMaxTM

(20), the association of three tests: SteatoTest™ (21), NashTest™ (22) and FibroTest™ (23), was

calculated by the manufacturer (Biopredictive, Paris, France). Pre-determined cut-offs of steatosis

(0.57) (21), Nash (0.75) (22) and fibrosis (0.48) were used as recommended. APRI, FIB-4 and NFS

scores of liver fibrosis were calculated as follows: APRI= AST (/ ULN) / platelet count (10 9/L) x 100

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(24), FIB-4= (age (years) x AST (IU/L)) / (platelets (109/L) x (ALT (IU/L))) (25), NFS= (-1.675 +

0.037 x age (year) + 0.094 x BMI (kg/m2) + 1.13 x IFG/diabetes (yes = 1, no = 0) + 0.99 x AST/ALT

ratio - 0.013 x platelet count (x109 /L) - 0.66 x albumin [g/dL]) (26). Pre-defined thresholds were

used to distinguish F0-1 and F2-4 (24) (25, 26).

Serum adipokines measurement

Measurement of serum adipokines was centralized in the department of biochemistry and hormonology

(JC, Tenon hospital, Paris, France): Leptin, and high sensitivity (hs) IL-6 were measured by using an

enzyme-linked immunosorbent assay (ELISA) (Quantikine R&D Systems, Oxford, UK). Serum

adiponectin, which detects total full-length mature adiponectin, was measured by ELISA (ALPCO

Salem, NH, USA). High sensitivity C-reactive protein (hsCRP) was measured by immunonephelometry

(IMMAGE, Beckman-Coulter).

Hepatic magnetic resonance imaging proton-density-fat-fraction (MRI-PDFF)

Within one month after inclusion all participants underwent a two-phase contrast hepatic MRI-PDFF with

calculation of the hepatic fat fraction (FF) using a dedicated software. All examinations were based on In

Phase (IP) and Out of Phase (OP) T1 imaging acquisitions (with 6-11 echoes and T2* correction in

order to avoid the confusing effect of potential iron deposition). FF calculation in IP and OP were

standardised within each study centre as excellent linearity and high precision of PDFF measurements

have been proven in a meta-analysis, even when performed on various platforms (different field

strengths, vendors, reconstruction methods)(27). Nevertheless, for quality control, randomly selected

MRI-PDFF images and reports were reviewed by a single highly experienced radiologist (YM) blinded to

clinical patient information. We used a predetermined MRI-PDFF cut-off of 10% to distinguish moderate

to severe liver steatosis (usually corresponding to ≥ 30% on liver biopsy) from none or mild liver

steatosis (<30% on liver biopsy) as previously published (28).

Transient elastography (Fibroscan®)

Liver stiffness measurement (LSM) was performed when fasting (29), using transient elastography by

Fibroscan® device (Probe M, FS402, Echosens, France) with CAPTM (controlled attenuation parameter)

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measures. LSM were performed by experienced operators according to the manufacturer ’s protocol

(30). The value of LSM was expressed in kilopascal (kPa) as the median of 10 successful acquisitions.

Unreliable measurement was defined as interquartile range (IQR)/LSM of > 30% when LSM is ≥7.1 kPa

(31). The CAP values expressed in dB/m were simultaneously measured for the assessment of liver

steatosis measurements, colocalized to the valid liver stiffness measurements.

To estimate fibrosis stages, we used the following cut-offs, previously validated in HIV-uninfected

patients with biopsy proven NAFLD: 7.1 kPa, 8.7 kPa and 10.3 kPa for fibrosis stages F2, F3 and F4,

respectively (32).

Liver biopsy

Liver biopsy was indicated in case of suspected clinically significant fibrosis (F2) based on elevated

Fibroscan® (≥7.1 kPa) and/or FibrotestTM (>0.48) and was performed within one-three months after

these determinations. Percutaneous liver biopsy was performed using the Menghini technique. Liver

samples were formalin-fixed and paraffin-embedded for histological analysis. Liver histology was

interpreted by two highly experienced liver pathologists (PB and JS). Both pathologists were blinded to

the clinical information, including the results of non-invasive tests. In case of discrepancies, slides were

reviewed by a third highly experienced hepatopathologist. NAFLD/NASH and fibrosis were diagnosed

using the NASH-CRN scoring system (33).

Statistical analysis

Variables were summarized as proportions for categorical variables, median and 25th-75th percentiles

(IQR) for continuous variables. Nonparametric ROC analysis was conducted to assess the performance

of MRI-PDFF, CAP and SteatoTest for the diagnosis of steatosis; Fibroscan®, FibrotestTM, APRI, FIB-4

and NFS for the diagnosis of fibrosis; and NashTest and ALT for the diagnosis of NASH. The

performance was considered acceptable for AUROC between 80-90% and excellent if >90%. Student t-

test was used to compare the AUROC between methods used for the diagnosis of steatosis and

between methods used for the diagnosis of fibrosis. The performances were also assessed in terms of

sensitivity, specificity, positive and negative predictive values, and the likelihood ratio for a positive test

result (LR+) and the likelihood ratio for a negative test result (LR–) (34). Optimal cut-off values for CAP

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and ALT, which were not available in the literature, were selected to maximize the sum of sensitivity and

specificity.

Finally, we estimated the proportion of subjects correctly classified. Chi-square and Fisher’s exact tests

were used to compare categorical variables between groups, and Mann-Whitney test to compare

continuous variables between groups. All reported P values are two-tailed, with significance level at

0.05. All analyses were done with IBM SPSS statistics version 24 for Windows.

Results

Study population

Between March 2014 and November 2015, 420 participants were enrolled in the ECHAM study and 402

had a full liver assessment including valid Fibroscan® and/or FibrotestTM values. Of them 140 (35%) had

a Fibroscan® ≥7.1 kPa (n=67) and/or a FibrotestTM >0.48 (n=99) and were therefore eligible for liver

biopsy; 91 (64%) participants refused the procedure and 49 had a liver biopsy. Supplemental Figure 1

summarizes the flow chart of the study population. Participants with liver biopsy were mainly male

(92%), with median age 54 years (IQR 53-65), and median body mass index (BMI) 26 kg/m 2 (24-30).

Most of them (90%) had metabolic syndrome at inclusion. All participants with liver biopsy had

controlled HIV-1 infection with plasma HIV-1 RNA <400copies/mL, and a median CD4 cells count

610/mm3 (IQR 538-783) (Table 1). As compared to patients who had liver biopsy participants who

refused the procedure had more hypertension (81% versus 63%, p=0.024), higher serum triglycerides

levels (median 2.23 mmol/l (1.5-3.5) versus 1.70 (1.2-2.3), p=0.010) but lower GGT level (median 50

IU/l (IQR: 31-78) versus 76 (47-120 IU/l), p=0.012), and Fibroscan® values (median 6.9 kPa (IQR 5.3-

8.9) versus 8.5 kPa (6.6-10.5) p=0.014). Other biochemical parameters including ALT and AST levels

as well as MRI-PDFF, CAP score and other non-invasive tests of fibrosis were not statistically different.

Histopathological findings

No complication was observed after liver biopsy. Among the 49 patients who had a liver biopsy, 37

(76%) had a diagnosis of NAFLD as defined by liver steatosis >5%. NASH was diagnosed in 23 patients

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(47%) with 15 (15/23, 65%) having a NAS >4; liver fibrosis was reported in 31 (63%) patients, with one

third having significant fibrosis ≥F2 and two being diagnosed as cirrhotic (4%).

Participants without NAFLD had either normal liver or aspecific lesions. No vascular liver disease, in

particular nodular regenerative hyperplasia, was reported. Histopathological findings are summarized in

Table 2. Interestingly, among the two participants with cirrhosis, one had a normal Fibroscan® value at

6.6 kPa (with an IQR<30%), the other had a value at 46.4 kPa.

Characteristics of patients according to the presence of NASH or significant liver fibrosis are

summarized in Supplemental Tables 1 and 2. As compared to participants without NASH, participants

with NASH had higher BMI (p=0.032), waist circumference (p=0.015) and waist-to-hip ratio (p=0.022) as

well as higher fasting glucose (p=0.01), LDL (p=0.012) and transaminases levels (p<0.0001), HOMA

index (p<0.0001) and hs-CRP levels (p=0.047); the degree of liver steatosis measured by MRI-PDFF or

CAP was more important in NASH patients as compared to those without NASH (p<0.0001 and p=0.02,

respectively). As compared to participants with no or mild fibrosis (F0-F1), participants with liver fibrosis

≥F2 had higher liver steatosis as measured by MRI-PDFF (p=0.003) or CAP score (p=0.016), higher

insulin resistance as reflected by HOMA-index (p=0.011), QUICKI (p=0.007) or leptin/adiponectin ratio

(p=0.035) with higher fasting glucose levels (p=0.03). Transaminases levels (p<0.001) as well as serum

ferritin (p=0.014). were also significantly higher in participants with liver fibrosis ≥F2 than participants

with fibrosis F0-F1. The duration of drug exposure did not differ between patients with and without

NASH or between patients with and without significant liver fibrosis (Suppl. tables 1 and 2). Moreover,

we did not find any association between past exposure to zidovudine (AZT), didanosine (ddI),

zalcitabine (ddC) or stavudine (d4T) – which can induce mitochondrial toxicity and NAFLD- and the

severity of the liver disease (steatosis, NASH or fibrosis).

Diagnostic accuracy of non-invasive markers of liver steatosis and NASH

For the diagnosis of moderate to severe liver steatosis (≥30%), MRI-PDFF had an excellent

performance with an AUROC curve at 0.98 (95%CI 0.96-1). The CAP technique performed also very

well with an AUROC at 0.87 for an optimal cut-off identified at 280 dB/m. SteatotestTM had much lower

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performance with an AUROC at 0.67 (0.51-0.85), p<0.001 compared to MRI-PDFF and p=0.06

compared to CAP.

For the diagnosis of NASH, the NashTestTM had poor performance with an AUROC at 0.64 (95%CI

0.48-0.80) and only 61% of patients correctly classified. Interestingly, ALT level, with a cut-off at 36 IU/L,

performed well for the identification of patients with NASH with an AUROC of 0.88 (95% CI: 0.77-0.99)

(Table 3, Figure 2).

Diagnostic accuracy of non-invasive markers of liver fibrosis

For the diagnosis of clinically significant fibrosis (≥F2), Fibroscan® and FibrotestTM had poor

performance with similar AUROC at 0.61 and only half of patients were correctly classified. Using

Fibroscan®, three were classified as false negative including one patient having histologically confirmed

cirrhosis despite a Fibroscan® value at 6.6 kPa.

To discriminate patients with F2-F4 fibrosis from patients with fibrosis F0-F1, only APRI performed

statistically better than Fibroscan® and FibrotestTM with AUROC at 0.86 (p<0.02). The performance of

FIB-4 was acceptable with an AUROC at 0.81, but did not statistically differ to that observed with

Fibroscan® (p=0.08). NFS did not performed well, its AUROC was 0.71 (0.57-0.86) and similar to that

observed with Fibroscan® (p=0.37) (Table 3, Figure 3).

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Discussion

Using liver histology as the reference in a well-characterized population of HIV-monoinfected individuals

at risk of NAFLD, we found that the hepatic MRI-PDFF and CAPTM technique performed very well for the

diagnosis of liver steatosis. Whilst Fibroscan® and Fibrotest™ had poor performance for the diagnosis

of significant liver fibrosis (≥F2), APRI was identified as the best fibrosis marker and ALT (≥36 IU/l) as

the best NASH marker in this population.

Liver ultrasonography is frequently used in clinical routine practice for NAFLD screening. However, it

has limited performance for the detection of mild steatosis as defined by less than 30% of the

hepatocytes containing fatty deposits. In addition, liver ultrasonography is unable to accurately quantify

the hepatic fat content, and is highly operator-dependent (35). As a result, an increasing number of non-

invasive methods have been developed over the last years for the diagnosis and quantification of

hepatic fat content. MRI-PDFF has the advantage to quantify liver fat over the whole liver. Its excellent

performance for the diagnosis of liver steatosis (AUROC between 0.90 and 1) has been previously

reported in non-HIV individuals (36-38) but never in PLHIV.

The CAPTM technique, based on the properties of ultrasonic signals, which has been developed to

quantify ultrasound attenuation during measurement of liver stiffness using Fibroscan®, is a simple and

rapid procedure. Good correlation between CAP scores and histological grade of liver steatosis has

been previously reported in non-HIV patients with or without NAFLD (39, 40) but its optimal cut-off value

remains highly controversial. A meta-analysis including 11 studies, and a total of 2,076 subjects with

various chronic liver diseases, reported excellent accuracy of CAPTM for the detection of liver steatosis

but found cut-off values ranging from 214 to 289 dB/m (41). Using MRI-PDFF as a gold standard, a

recent study found an AUROC at 0.80 (95%CI: 0.70-0.90) of CAPTM for the diagnosis of FF>5%, with a

best cut-off at 288 dB/m, slightly higher than the cut-off we have identified (280 dB/m) for the diagnosis

of moderate to severe steatosis (FF>10%) (42). In PLHIV, this technique has been poorly assessed.

Only one study reported performance of CAPTM (using a relatively low cut-off: 240 dB/m) in PLHIV with

or without HCV coinfection. Its performance was good but decreased in patients with high BMI (43).

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Since many HIV centres have now access to Fibroscan® devices, CAPTM represents an accurate

alternative technique for the rapid identification of PLHIV with NAFLD.

For the diagnosis of liver fibrosis and cirrhosis, Fibroscan® is considered as a reliable method and is

now largely used for fibrosis screening in various liver diseases outside HIV infection. Very good

performance of Fibroscan® has been reported in NAFLD patients (32, 44, 45) with different thresholds

identified. However, using a cut-off at 7.2 kPa for the diagnosis of liver fibrosis F2 in HIV-uninfected

individuals with NAFLD, Petta et al. reported a sensitivity of Fibroscan® of only 60% and a high number

of false positive subjects (30%), especially in those with high BMI (46). More recently, the same group

suggested that Fibroscan® results should be interpreted according to the CAP scores to avoid

overestimation of the Fibroscan® values (47). Indeed, liver stiffness increases with the degree of

hepatic steatosis (48). In our study LSM and CAP values or liver steatosis grade on liver biopsy were

not correlated (r=0.12, p=0.462).

In HIV-monoinfected individuals, only two studies assessing the performance of Fibroscan® have been

published so far (49, 50). Both studies are from North America. Benmassaoud et al. included 17 patients

and reported an AUROC at 0.91 (95% CI 0.81–1.00) (50). Morse et al. analysed 59 patients with

persistently elevated transaminases who had liver biopsy (49). For the detection of mild fibrosis (≥F1)

Fibroscan® performed very well (AUROC 0.93) whilst APRI and FIB-4 had poor performance (AUROC

below 0.65). However, in this study the authors used cut-offs who have been validated for fibrosis F2

according to the Metavir system whilst they used the Ishak scoring system (49). In contrast to this study,

we found that that APRI and FIB-4 had good performance. This is in line with the study from Shah et al.

(51) who found good performance of FIB-4 for the detection of fibrosis in HIV negative individuals with

NAFLD (AUROC: 0.80). A recent meta-analysis on non-invasive tests for the diagnosis of liver fibrosis in

NAFLD (64 studies, a total of 13,046 patients with NAFLD) also reported acceptable performance of

FIB-4 and APRI (AUROC at 0.73 and 0.76, respectively) (52). Thus, these simple biochemical tests are

now proposed for fibrosis screening in patients with NAFLD (52).

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FibroTestTM had disappointing performance (AUROC: 0.61 (95%CI 0.44-0.78)) in our study although

good performance has been previously reported for the diagnosis of fibrosis F2 in HIV/HCV coinfected

patients (53) as well as non-HIV-infected patients with NAFLD (54). In NAFLD patients, NFS, a non-

patented and inexpensive test, may have similar performance than FibroTestTM (54). In our study NFS

had only poor performance (AUROC: 0.71).

Finally in our study the NashTestTM, originally developed in HIV-uninfected individuals with NAFLD (22),

had poor accuracy to identify patients with NASH with a sensitivity of 17% and an AUROC curve at 0.64

(95% CI: 0.48-0.80) (13) and ALT level performed much better to identify patients with NASH meaning

that in the absence of validated test of NASH, the identification of HIV-patients with NASH can be based

on the level of ALT ≥ 36 IU/l as a first step.

Of note, we do not believe that chronic inflammatory state observed in our HIV population may have

influenced the poor performance of some biochemical tests of Nash or fibrosis i.e NashTest, Fibrotest

and NFS since none of the inflammatory markers measured in our study (hsCRP, serum ferritin, IL-6,

leptin and adiponectin) were associated with the levels of NashTest or Fibrotest. Only higher levels of

IL-6 were observed in patients with significant fibrosis using NFS (2.9±1.65 versus 2.02±1.37,

P=0.021).

We acknowledge that our study has several limitations: first, the number of liver biopsies was limited

due to high refusal rate to liver biopsy (64%) as observed in the few histology-based studies including

HIV-monoinfected individuals (13-66 liver biopsies per study) indicating the difficulty of performing liver

histology in this population. As a result, we were unable to assess the performance of the tests for the

diagnosis of extensive fibrosis (F3) or cirrhosis. We cannot exclude higher performance of Fibroscan®

and biochemical tests for the diagnosis of F3-F4 fibrosis as reported in non-HIV patients with NAFLD

(52). Secondly, in our study the median length of liver samples was below 25 mm, the recommended

minimum size to evaluate accurately liver fibrosis. As a result, this might have contributed to the

discordance between histological analysis and noninvasive markers for the measurement of liver

fibrosis. Third, we analyzed a selected population at risk of NAFLD and fibrosis, therefore our results are

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not representative of the whole HIV-monoinfected population. Fourth, we did not use the XL probe

developed for obese subjects. However, a systematic review and meta-analysis, showed that the use of

the XL probe does not significantly increase the overall performance of fibrosis detection in patients with

NAFLD as compared to the use of the standard M probe (52). Moreover, only 11 (22%) of participants

had a BMI≥30 kg/m2. Finally, Fibroscan® measures the hepatic stiffness; whether liver stiffness

correlates with the amount of fibrosis as measured by collagen proportionate area (CPA) has been

poorly documented especially in PLHIV (55).

In conclusion, our study indicates that MRI-PDFF is an excellent technique to diagnose and quantify

liver steatosis in HIV-monoinfected patients at risk of NAFLD and CAP technique is another accurate

alternative to liver biopsy for the identification of PLHIV with NAFLD. Fibroscan® which is increasingly

used in PLHIV should be interpreted with cautious in HIV-monoinfected individuals with suspected

NAFLD. ALT and APRI should be considered as simple non-invasive markers for the detection of NASH

and liver fibrosis respectively, in this population.

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Acknowledgments

The authors would like to thank the patients, their families, the clinical research team. We also thank

Juliette Saillard, Soizic Lemestre, Daniela Behrendt and Doris Behrens for coordinating the study. We

are also grateful to Soraya Fellahi, Laëtitia Béraud and Ginette Marlin (Tenon Hospital Inflammatory and

metabolic biomarker laboratory) for her excellent technical expertise for the assessment of serum

adipokines. We are also grateful to Prof. Valerie Paradis for the third reading of liver histology.

Funding

The study was funded by the HIV-ERANET European program (01KI1205A) and French Research

Agency on HIV and viral hepatitis (ANRS)

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Figure legends

Figure 1: AUROC of non-invasive tests of liver steatosis

Figure 2: AUROCs of NashTest and ALT level for the diagnosis of NASH

Figure 3: AUROC of non-invasive tests of significant liver fibrosis (F2-F4).

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