Hepatic Steatosis and Steatohepatitis in Human Immunodeficiency Virus/Hepatitis C Virus–Coinfected Patients Juan Macı´as, 1 Juan Berenguer, 2 Miguel A. Japon, 3,4 Jos e A. Giron-Gonz alez, 5 Antonio Rivero, 6 Luis F. Lopez-Cort es, 2,4,7 Ana Moreno, 8 Manuel Marquez, 9 Jos e A. Iribarren, 10 Enrique Ortega, 11 Pilar Miralles, 3 Nicol as Merchante, 1 and Juan A. Pineda 1 Hepatic steatosis (HS) is frequent in human immunodeficiency virus (HIV)- and hepatitis C virus (HCV)-coinfected patients. Antiretroviral therapy (ART) and metabolic alterations could induce HS. However, a protective effect of ART has been reported in a paired biopsy study. Thus, our aim was to examine the changes and predictors of HS progression among HIV/HCV-coinfected patients with sequential biopsies. We also evaluated the rates of stea- tohepatitis and factors associated thereof. HIV-infected patients with detectable serum HCV RNA, who underwent two biopsies, separated at least by 1 year, were included in this retrospective study. HS progression was defined as increase in one or more HS grades. The median (interquartile range) time between biopsies was 3.3 (2.0-5.2) years. Among 146 individuals, HS at baseline was observed in 86 (60%) patients and in 113 (77%) in the fol- low-up biopsy (P < 0.001). Progression of HS was observed in 60 (40%) patients. HS regressed in 11 (8%) patients. Factors associated with HS progression were changes in fast- ing plasma glucose (FPG) between biopsies (per 10 mg/dL increase; odds ratio [OR] [95% confidence interval; CI] 5 1.4 [1.04-1.8]; P 5 0.024) and cumulative use of dideoxynu- cleoside analogs (per year; OR [95% CI] 5 1.5 [1.2-1.8]; P 5 0.001). Persistent steatohe- patitis or progression to steatohepatitis between biopsies was observed in 27 (18%) patients. Persistence of or progression to steatohepatitis was associated with progression 1 fibrosis stages between biopsies (OR [95% CI] 5 2.4 [1.01-5.7]; P 5 0.047). Conclu- sions: HS progresses frequently and regression is rarely observed in HIV/HCV-coinfected patients, including in those on ART. Cumulative exposure to dideoxynucleoside analogs and increases in FPG are related with HS progression. Stetatohepatitis is frequently observed in these patients and is linked to fibrosis progression. (HEPATOLOGY 2012;00:000- 000) H epatic steatosis (HS) is a common condition in hepatitis C virus (HCV)-infected patients with human immunodeficiency virus (HIV) coinfection. Previous cross-sectional studies have reported on frequencies of HS between 30% and 70%. 1-12 Besides its prevalence, the main clinical Abbreviations: ART, antiretroviral therapy; BMI, body mass index; CDC, Centers for Disease Control and Prevention; CI, confidence interval; DM, diabetes mellitus; ETR, end-of-treatment response; FPG, fasting plasma glucose; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HS, hepatic steatosis; IQR, interquartile range; IR, insulin resistance; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score; OR, odds ratio; SVR, sustained virological response; TGs, triglycerides. From the 1 Infectious Diseases and Microbiology Unit, Hospital Universitario de Valme, Seville, Spain; 2 Infectious Diseases Unit, Hospital General Universitario Gregorio Mara ~ non, Madrid, Spain; 3 Pathology Department, Hospital Universitario Virgen del Rocı´o, Seville, Spain; 4 Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocı´o/CSIC/Universidad de Sevilla, Seville, Spain; 5 Infectious Diseases Unit, Hospital Universitario Puerta del Mar, Cadiz, Spain; 6 Infectious Diseases Unit, Instituto Maimonides de Investigacion Biomedica de Cordoba, Hospital Universitario Reina Sofı´a, Cordoba, Spain; 7 Infectious Diseases Department, Hospital Universitario Virgen del Rocı´o, Seville, Spain; 8 Infectious Diseases Unit, Hospital Universitario Ramon y Cajal, Madrid, Spain; 9 Infectious Diseases Unit, Hospital Universitario Virgen de la Victoria, Malaga, Spain; 10 Infectious Diseases Unit, Hospital Donostia, San Sebastian, Spain; and 11 Infectious Diseases Unit, Hospital General Universitario de Valencia, Valencia, Spain. Received October 18, 2011; accepted April 10, 2012. This study was partly supported by a grant from Consejerı´a de Salud, Junta de Andalucı´a (exp. 0022/2007), from Fundacion para la Investigacion y la Prevencion del SIDA en Espa ~ na (FIPSE, exp. 36789/08), and from the ISCIII-RETIC RD06/006. J.B. and J.A.G.-G. are investigators from the Intensification of Research Activity Program of the Spanish National Health Service (reference I3SNS). J.A.P. and M.A.J. are recipients of intensification grants from the Fundacion Progreso y Salud of the Consejerı´a de Salud de la Junta de Andalucı´a (references AI-0021 and AI-0003, respectively). 1
Transcript
Hepatic Steatosis and Steatohepatitis in HumanImmunodeficiency Virus/Hepatitis C
Virus–Coinfected PatientsJuan Macıas,1 Juan Berenguer,2 Miguel A. Jap�on,3,4 Jos�e A. Gir�on-Gonz�alez,5 Antonio Rivero,6
Luis F. L�opez-Cort�es,2,4,7 Ana Moreno,8 Manuel M�arquez,9 Jos�e A. Iribarren,10 Enrique Ortega,11
Pilar Miralles,3 Nicol�as Merchante,1 and Juan A. Pineda1
Hepatic steatosis (HS) is frequent in human immunodeficiency virus (HIV)- and hepatitisC virus (HCV)-coinfected patients. Antiretroviral therapy (ART) and metabolic alterationscould induce HS. However, a protective effect of ART has been reported in a paired biopsystudy. Thus, our aim was to examine the changes and predictors of HS progression amongHIV/HCV-coinfected patients with sequential biopsies. We also evaluated the rates of stea-tohepatitis and factors associated thereof. HIV-infected patients with detectable serumHCV RNA, who underwent two biopsies, separated at least by 1 year, were included in thisretrospective study. HS progression was defined as increase in one or more HS grades. Themedian (interquartile range) time between biopsies was 3.3 (2.0-5.2) years. Among 146individuals, HS at baseline was observed in 86 (60%) patients and in 113 (77%) in the fol-low-up biopsy (P < 0.001). Progression of HS was observed in 60 (40%) patients. HSregressed in 11 (8%) patients. Factors associated with HS progression were changes in fast-ing plasma glucose (FPG) between biopsies (per 10 mg/dL increase; odds ratio [OR] [95%confidence interval; CI] 5 1.4 [1.04-1.8]; P 5 0.024) and cumulative use of dideoxynu-cleoside analogs (per year; OR [95% CI] 5 1.5 [1.2-1.8]; P 5 0.001). Persistent steatohe-patitis or progression to steatohepatitis between biopsies was observed in 27 (18%)patients. Persistence of or progression to steatohepatitis was associated with progression�1 fibrosis stages between biopsies (OR [95% CI] 5 2.4 [1.01-5.7]; P 5 0.047). Conclu-sions: HS progresses frequently and regression is rarely observed in HIV/HCV-coinfectedpatients, including in those on ART. Cumulative exposure to dideoxynucleoside analogsand increases in FPG are related with HS progression. Stetatohepatitis is frequentlyobserved in these patients and is linked to fibrosis progression. (HEPATOLOGY 2012;00:000-
000)
Hepatic steatosis (HS) is a common conditionin hepatitis C virus (HCV)-infected patientswith human immunodeficiency virus (HIV)
coinfection. Previous cross-sectional studies havereported on frequencies of HS between 30% and70%.1-12 Besides its prevalence, the main clinical
Abbreviations: ART, antiretroviral therapy; BMI, body mass index; CDC, Centers for Disease Control and Prevention; CI, confidence interval; DM, diabetesmellitus; ETR, end-of-treatment response; FPG, fasting plasma glucose; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HS, hepatic steatosis; IQR,interquartile range; IR, insulin resistance; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score; OR, odds ratio; SVR, sustained virologicalresponse; TGs, triglycerides.From the 1Infectious Diseases and Microbiology Unit, Hospital Universitario de Valme, Seville, Spain; 2Infectious Diseases Unit, Hospital General Universitario
Gregorio Mara~n�on, Madrid, Spain; 3Pathology Department, Hospital Universitario Virgen del Rocıo, Seville, Spain; 4Instituto de Biomedicina de Sevilla, HospitalUniversitario Virgen del Rocıo/CSIC/Universidad de Sevilla, Seville, Spain; 5Infectious Diseases Unit, Hospital Universitario Puerta del Mar, Cadiz, Spain; 6InfectiousDiseases Unit, Instituto Maim�onides de Investigaci�on Biom�edica de C�ordoba, Hospital Universitario Reina Sofıa, Cordoba, Spain; 7Infectious Diseases Department,Hospital Universitario Virgen del Rocıo, Seville, Spain; 8Infectious Diseases Unit, Hospital Universitario Ram�on y Cajal, Madrid, Spain; 9Infectious Diseases Unit,Hospital Universitario Virgen de la Victoria, Malaga, Spain; 10Infectious Diseases Unit, Hospital Donostia, San Sebastian, Spain; and 11Infectious DiseasesUnit, Hospital General Universitario de Valencia, Valencia, Spain.Received October 18, 2011; accepted April 10, 2012.This study was partly supported by a grant from Consejerıa de Salud, Junta de Andalucıa (exp. 0022/2007), from Fundaci�on para la Investigaci�on y la
Prevenci�on del SIDA en Espa~na (FIPSE, exp. 36789/08), and from the ISCIII-RETIC RD06/006. J.B. and J.A.G.-G. are investigators from the Intensification ofResearch Activity Program of the Spanish National Health Service (reference I3SNS). J.A.P. and M.A.J. are recipients of intensification grants from the Fundaci�onProgreso y Salud of the Consejerıa de Salud de la Junta de Andalucıa (references AI-0021 and AI-0003, respectively).
1
implication of HS is that it has been associated withliver fibrosis progression in several studies.2-7 Particu-larly, among patients without HIV infection with non-alcoholic fatty liver disease (NAFLD), those with steato-hepatitis are at increased risk of fibrosis progression.13
Thus, a better understanding of modifiable risk factorsthat may contribute to HS development is critical. Inthis sense, previous studies have implicated several meta-bolic factors, such as obesity, hyperglycemia, hyperlipid-emia, or lipodystrophy, in the development of HS.2-7 Inaddition, the use of certain reverse-transcriptase inhibi-tors, such as stavudine, didanosine, or efavirenz, hasbeen associated with HS in some studies.4,6,8,14 How-ever, other studies failed to find this association.1-3,5,7
Thus, the relationship between HS and antiretroviraltherapy (ART) remains to be elucidated.The main limitation to the current knowledge of
risk factors for HS is that the available informationderives mainly from cross-sectional studies. Moreover,only one of these reports, to our knowledge, evaluatedthe frequency of steatohepatitis in HIV/HCV-coin-fected patients.5 A recent longitudinal analysis of HIV/HCV-coinfected patients, who had undergone at leasttwo liver biopsies, examined the rates of steatosis pro-gression.15 The prevalence of HS at baseline was lowerthan that found in previous studies.1-11,14 At the fol-low-up biopsy, HS did not progress in the majority ofpatients. Among progressors, ART was associated witha lower risk of HS progression. The reasons for thesefindings are unclear. The racial background of thestudy cohort, overwhelmingly composed of HCV ge-notype 1–infected African Americans, may partlyexplain these striking results. Thus, there is a need foradditional studies assessing the rates of HS progressionand the risk factors for progression, including the roleof antiretroviral drugs, in HIV/HCV-coinfected sub-jects, as it has been claimed by some experts.16 Fur-thermore, there are no data on the changes in steato-hepatitis over time in HIV/HCV coinfection.In this study, we aimed at evaluating the changes in
HS between liver biopsies and the predictors of HSprogression among HIV/HCV-coinfected patients withsequential liver biopsies. We also assessed the rates ofsteatohepatitis and factors associated with the persist-ence and progression thereof in these patients.
Patients and Methods
Design and Study Population. This was a retro-spective study carried out in paired liver biopsies per-formed in HIV/HCV-coinfected patients who attendednine Spanish hospitals from January 1989 to January2008. An analysis of liver fibrosis progression in thesesequential biopsies has been previously reported on.17
HIV-infected patients were included in the presentstudy if they met the following: (1) active HCV infec-tion, as determined by detectable serum HCV RNA;(2) underwent two liver biopsies, separated by at least1 year; (3) liver biopsies have been performed as partof the assessment of HCV infection to establish theprognosis and/or to indicate treatment; and (4) no evi-dence of vascular, tumoral, biliary, or autimmune liverdisease. Individuals with cirrhosis detected at the firstliver biopsy were included for the present analysis. Bi-opsy samples with length lower than 15 mm or frag-mented specimens were deemed as inadequate, and thecorresponding patient was excluded.Antiviral Therapy. Patients received ART according
to the availability of drugs and the recommendationsof international guidelines and panels of experts inforce during the study period. Therapy against HCVinfection was prescribed according to the caring physi-cian criteria, based on consensus recommendations ineffect along the study period, usually guided by HCVgenotype and liver fibrosis stage. End-of-treatmentresponse (ETR) was defined as undetectable serumHCV RNA at the planned date of treatment cessation.Sustained virological response (SVR) was defined asundetectable serum HCV RNA 24 weeks after the endof treatment.Liver Histology. Liver steatosis and liver fibrosis
were scored blindly by a central pathologist (M.A.J.).HS classification was based on the proportion of hepa-tocytes containing fat droplets using Brunt’s criteria18
and was classified as follows: 0, absent steatosis; 1, lessthan 33% (i.e., mild HS); 2, 33%-66% (i.e., moderateHS); and 3, more than 66% (i.e., severe HS). Lobularinflammation was scored as follows: 0 ¼ no foci; 1 =<2 foci (excludes 2 foci �200 field); 2 ¼ 2-4(includes 2 and 4 foci �200 field) 3 ¼ >4 foci(excludes 4 foci �200 field). Cytologic ballooning was
Address reprint requests to: Juan A. Pineda, M.D., Ph.D., Infectious Diseases and Microbiology Unit, Hospital Universitario de Valme, Avenida de Bellavistas/n, 41014 Seville, Spain. E-mail: [email protected]; fax: þ34-955015757.CopyrightVC 2012 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.25791Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article.
2 MACIAS ET AL. HEPATOLOGY, Month 2012
classified as follows: 0, none; 1, few balloon cells; and2, many cells/prominent ballooning. The NAFLD ac-tivity score (NAS) was calculated as the unweightedsum of steatosis, lobular inflammation, and hepatocel-lular ballooning scores.19 Scheuer’s score20 was appliedto stage fibrosis as follows: 0, absent fibrosis; 1, portalfibrotic expansion; 2, extension of fibrosis to thelobule, but with few septa; 3, bridging fibrosis withnumerous septa, with architectural distortion withoutcirrhosis; and 4, cirrhosis. The length of biopsies wasrecorded to assess their adequacy. The median (inter-quartile range; IQR) length of the initial biopsy was17 (15-25) mm, whereas the respective figure of thesecond biopsy was 18 (16-25) mm.Statistical Analysis. The primary outcome variable
of the study was the progression in one or more gradesin Brunt’s score. The associations of the followingbaseline factors with HS progression were analyzed:gender, age, body mass index (BMI), self-reporteddaily alcohol intake, diagnosis of diabetes mellitus(DM) following the American Diabetes Association cri-teria, fasting plasma glucose (FPG), cholesterol and tri-glycerides (TGs), HCV genotype, and Centers for Dis-ease Control and Prevention (CDC) stage C. Inaddition, the following variables between biopsies andtheir relationship with HS progression were assessed:BMI, self-reported daily alcohol intake, FPG, cholesteroland TGs, response to treatment against HCV, CD4 cellcounts at liver biopsies, plasma HIV RNA viral load atliver biopsies, exposure to ART, and changes in fibrosisstage. Cumulative exposure to individual antiretroviraldrugs was calculated as the period in years receivingeach antiretroviral drug between biopsies. Blood testswere drawn within 1 month before the liver biopsies.The secondary outcome variable was persistence of
steatohepatitis between biopsies or progression to stea-tohepatitis in the final biopsy. Steatohepatitis wasdefined as NAS score �5.19 The associations betweenthe above-stated baseline and follow-up variables withpersistence of or progression to steatohepatitis wereassessed.Continuous variables are expressed as median
(IQR), and categorical variables are presented as num-bers (percentage). Mann-Whitney’s U test was appliedfor comparisons of continuous variables betweengroups. Comparisons between categorical variableswere made by the chi-square test or Fisher’s exact test,when appropriate. For comparisons between relatedgroups, Wilcoxon’s signed-rank test for continuous var-iables and McNemar’s test for the categorical oneswere used. Odds ratios (ORs) (95% confidence inter-vals; CIs) of variables potentially related with the out-
come variables were calculated by univariate logisticregression. Variables associated with the outcome varia-bles with a P value �0.2 in univariate analyses wereentered in multivariate logistic regression models. Mul-tivariate models were adjusted by the difference insample length between the first and second biopsies,regardless of its association with the outcome variables.Associations with a P value <0.05 after the multivari-ate analysis were considered significant. Statistical anal-ysis was carried out using the SPSS 19 statistical soft-ware package (SPSS, Inc., Chicago, IL).Ethical Aspects. The study was designed and con-
ducted following the Helsinki declaration. The EthicsCommittee of the Hospital Universitario de Valme(Seville, Spain) approved the study.
Results
Baseline Characteristics. One hundred and forty-six patients were included in the study. The character-istics of these patients at the initial biopsy are summar-ized in Table 1. All patients were Caucasians of Euro-pean ancestry. DM was diagnosed in 9 (6.5%)individuals. One hundred and twenty-five (86%)patients showed a BMI between 18.5 and 24.99kg/m2, 6 (4%) individuals showed a BMI lower than18.5 kg/m2, 11 (7.5%) had a BMI equal to or greaterthan 25 kg/m2 and lower than 30 kg/m2, and 4(2.7%) showed a BMI greater than 30 kg/m2. Themajority of patients received ART at baseline (Table1). The distribution of individual antiretroviral drugsprescribed during the follow-up and the cumulative ex-posure to them is listed in Table 2.Rate of Steatosis Progression. The median (IQR)
time between biopsies was 3.3 (2.0-5.2) years. HS atbaseline was observed in 87 (60%) patients. Mostpatients with HS at the initial biopsy presented grade1 HS (Fig. 1; refer to Supporting Table 1 for associa-tions with baseline HS). In the second biopsy, HS wasdetected in 113 (77%) patients, 49 (34%) of whombore grade 2 HS. The frequency of HS grades at thefirst and second biopsy is detailed in Fig. 1. The prev-alence of moderate and severe HS was higher in thefollow-up biopsies, compared with the baseline biop-sies (Fig. 1). Progression of at least one grade of HSwas observed in 60 (40%) patients at the second liverbiopsy, and 8 (5%) patients progressed two or moregrades of HS. Progression to grade 2 or 3 HS wasobserved in 34 (23%) patients. Only 11 (8%) patientsexperienced regression of one or more HS grades (referto Supporting Table 2 for detailed changes in HSbetween biopsies).
HEPATOLOGY, Vol. 000, No. 000, 2012 MACIAS ET AL. 3
Changes in Different Factors Between Liver Biop-sies. Median CD4 cell counts were 495 (338-660)cells/lL at the initial biopsy and 540 (427-700) cells/lL at the follow-up biopsy (P ¼ 0.021). At baseline,88 (60%) patients showed undetectable plasma HIV
RNA and 107 (73%) reached plasma HIV viremiabelow the detection level at the second biopsy (P ¼0.018). Twenty-nine (20%) patients at the first biopsyand 28 (19%) subjects at the second biopsy were notunder ART (P ¼ 0.882).The stage of liver fibrosis at the initial and follow-
up biopsies were as follows: stage 0, 29 (20%) ver-sus18 (12%); stage 1, 49 (34%) versus 39 (27%); stage2, 27 (19%) versus 42 (29%); stage 3, 30 (21%) ver-sus 24 (16%); and stage 4, 11 (7.5%) versus 23 (16%)(P ¼ 0.019). Among 69 patients who received therapyagainst HCV, 4 (5.8%) achieved SVR and 26 (38%)ETR.Factors Associated With Progression of Liver Stea-
tosis. Table 3 compares those with and without steato-sis progression. CD4 cell counts, plasma HIV RNA,and use of ART were not related with HS progression.Cumulative exposure to dideoxynucleoside analogs (i.e.,
Table 1. Baseline Characteristics of the StudyPatients (n 5 146)
Characteristics Value
Age, years* 37.8 (33-41)
Male gender, n (%) 107 (73)
Injecting drug users, n (%) 125 (86)
DM, n (%) 9 (6.5)
Alcohol intake >50 g/day,§ n (%) 31 (25)
BMI,* kg/m2 22.5 (21.9-23.4)
FPG,* mg/dL 90 (80-94)
TGs,*,k mg/dL 118 (98-180)
Cholesterol,*,k mg/dL 172 (150-192)
CDC stage C, n (%) 35 (24)
Nadir CD4 cell counts, cells/lL* 215 (100-340)
CD4 cell counts,* cells/mcL 495 (338-660)
Undetectable HIV viremia, n (%) 88 (60)
Antiretroviral therapy, n (%) 117 (80)
Estimated age at HCV infection, years*,† 20.4 (18-26)
ase; GGT, gamma-glutamyl transferase; BMI, body mass index; CDC, Centers for
Disease Control and Prevention; DM, diabetes mellitus; FPG, fasting plasma
glucose; TGs, triglycerides.
*Median (IQR).
†Available in 122 patients.
‡Available in 135 patients (10 patients with nontypable genotype and 1 not
available).
§Available in 127 patients.kAvailable in 132 patients.
Table 2. Antiretroviral Therapy During theFollow-up (n 5 146)
Drugs N (%) Median (IQR) Time, Years
Zidovudine 65 (45) 1 (0.8-2.0)
Abacavir 28 (19) 1.8 (0.9-2.6)
Tenofovir 37 (25) 2.7 (1.8-3.3)
Lamivudine 117 (80) 2.3 (1.3-3.4)
Dideoxynucleosides 89 (61) 1.7 (0.9-4.5)
Saquinavir 26 (18) 1.5 (1.1-2.8)
Indinavir 25 (17) 1.3 (1.1-2.2)
Nelfinavir 24 (16) 1.7 (1.3-2.4)
Lopinavir/r 10 (6.8) 2.2 (1.7-3.0)
Nevirapine 53 (36) 1 (0.5-3.0)
Efavirenz 51 (35) 1.3 (1-3)
Fig. 1. Frequency of grades of hepatic steatosis at the initial andfollow-up biopsies (n ¼ 146).
4 MACIAS ET AL. HEPATOLOGY, Month 2012
didanosine, stavudine, or zalcitabine) and efavirenz wasassociated with HS progression (Table 3; Fig. 2). Pro-gression of fibrosis one or more stages was not associ-ated with HS progression (Table 3). Increases in medianFPG were significantly higher among patients with HSprogression (Table 3). Changes in BMI, TGs, and cho-lesterol were not associated with HS progression (Table3).After the multivariate analysis, cumulative exposure
to dideoxynucleoside analogs and increases in FPGwere independently related with progression of HS
(Table 3). An analysis excluding patients with baselinecirrhosis yielded similar results (data not shown).Steatohepatitis Persistence or Progression. The
median (IQR) NAS score was 3 (3-4) for the first bi-opsy and 4 (3-4) for the follow-up biopsy (P ¼ 0.002;refer to Supporting Table 3 for associations with base-line steatohepatitis). The NAS score increased in 65(45%) and decreased in 35 (24%) individuals betweenthe initial and final biopsy. Steatohepatitis was detectedin 24 (16%) patients in the first biopsy and in 27(18%) subjects in the final biopsy (P ¼ 0.602).
Table 3. Factors Associated With Progression of One or More Grades of Steatosis Between Liver Biopsies
Characteristic
Steatosis Progression
(N ¼ 60)
No Steatosis
Progression (N ¼ 86)
Unadjusted
OR (95% CI)
P
Univariate
Adjusted OR
(95% CI)
P
Multivariate
Baseline age,* years 38.1 (33.9-40.6) 37.5 (32.7-41.0) 1.0 (0.9-1.1) 0.977
Male gender, n (%) 43 (72) 56 (65) 1.4 (0.7-2.8) 0.405
Alcohol intake >50 g/day during the follow-up 15 (28) 20 (25) 1.2 (0.5-2.5) 0.703
Treatment against HCV, n (%) 26 (43) 43 (50) 0.8 (0.4-1.5) 0.427
ETR or SVR,# n (%) 12 (46) 18 (42) 0.9 (0.4-2.1) 0.727
Time between biopsies,* years 3.4 (2.03-5.4) 2.9 (1.8-4.7) 1.04 (0.9-1.2) 0.511
Length difference between last and first biopsy, mm 0 (0-2) 0 (0-3) 0.96 (0.9-1.06) 0.539 0.96 (0.9-1.09) 0.548
BMI, body mass index; CDC, Centers for Disease Control and Prevention; DM, diabetes mellitus; FPG, fasting plasma glucose; TGs, triglycerides; ETR: End of
‡Available in 135 patients.kUndetectable viremia at baseline and follow-up biopsies.
§Cumulative time on each drug for patients exposed to them.
¶Per year of exposure.#Among 69 patients who received anti-HCV therapy.
HEPATOLOGY, Vol. 000, No. 000, 2012 MACIAS ET AL. 5
Steatohepatitis persisted in 9 (38%) of 24 patients.Among 122 individuals without steatohepatitis initially,18 (15%) showed progression (refer to Supporting Ta-ble 4 for detailed changes in NAS scores betweenbiopsies).Persistence of or progression to steatohepatitis was
related with fibrosis progression (Table 4). There was anonsignificantly longer exposure to dideoxynucleosideanalogs and to ART among patients with persistent orprogressive steatohepatitis (Table 4). Eleven (14%)patients with ART for <4 years and 10 (25%) subjectswith ART for �4 years showed steatohepatitis persist-ence or progression (P ¼ 0.119). After the multivariateanalysis, the only variable independently associatedwith persistent or progressive steatohepatitis was liverfibrosis progression (Table 4). An analysis excludingpatients with baseline cirrhosis yielded similar results(data not shown).
Discussion
HS severity progresses with time frequently in HIV/HCV-coinfected patients, both in those who receive ARTand in those who do not. HS regression is rarely observedin this setting. Cumulative exposure to dideoxynucleosideanalogs and increases in FPG are associated with HS pro-gression. In addition, steatohepatitis is frequentlyobserved in HIV/HCV-coinfected patients, and NASscore increases over time in these individuals. Steatohepa-titis tends to be associated with more-prolonged expo-sures to ART and dideoxynucleoside analogs. Impor-tantly, persistence of or progression to steatohepatitis islinked to fibrosis progression in HIV/HCV coinfection.The results of the herein reported study are in con-
trast with the study by Woreta et al. that assessed HS
progression in paired liver biopsies from HIV/HCV-coinfected patients.15 In that study, fewer patients pre-sented HS at baseline and HS did not progress inapproximately 90% of patients in the follow-up biop-sies.15 On the contrary, in our study, 60% of patientsshowed some degree of HS in the initial biopsy,increases of 1 stage in HS was observed in 40% ofpatients, and progression to moderate or severe HSwas observed in 23% of individuals. The reasons forsuch conflicting data are unclear. The participants inthe study by Woreta et al. were overwhelmingly HCVgenotype 1–infected African Americans,15 whereaspatients in the present study were Caucasians withinfection by more-diverse HCV genotypes. This maypartly explain the lower prevalence and progression ofHS in the study by Woreta et al., given that individu-als with African ancestry might have a lower propen-sity to develop NAFLD.21 However, a recent meta-analysis did not find a significantly different prevalenceof HS among HIV/HCV-coinfected African Ameri-cans.12 The high prevalence of HCV genotype 3 maypartially account for the higher rates of HS in ourstudy, given the association between this genotype andHS.2-4,11 Nevertheless, HCV genotype 3 was not asso-ciated with HS progression in our study.The role of ART in the development of HS is con-
troversial. We found that HS progression between liverbiopsies was associated with cumulative exposure todideoxynucleoside analogs. This finding is in agree-ment with previous cross-sectional studies.4,6,14
Dideoxynucleoside analogs, susc as didanosine, stavu-dine, and zalcitabine, are potent inhibitors of mito-chondrial DNA (mtDNA) polymerase-gamma, theenzyme responsible for mtDNA replication. mtDNAdepletion impairs respiratory chain activity and thusinhibits mitochondrial b-oxidation, finally causingabnormal deposition of fatty acids in hepatocytes.22
However, most reported cross-sectional studies failed tofind an association with ART or individual antiretroviraldrugs.1-3,5,7 One possible explanation might be differentexposures to dideoxynucleoside analogs across studies.Thus, the use of these drugs was highly prevalent in stud-ies finding an association between them and HS.4,6,14
We found an association between progression of HSand cumulative exposure to efavirenz in the univariateanalysis. Similar to our findings on dideoxynucleosides,the larger the time on efavirenz, the higher the fre-quency of patients with HS progression. There aresome data that support the mitochondrial toxicity ofefavirenz. In vitro, efavirenz induces bioenergetic stressin hepatic cells by inhibiting mitochondrial functionthrough an acute mechanism that is independent of
Fig. 2. Frequency of patients with progression of hepatic steatosisby the length of exposure to dideoxynucleosides, lamivudine, efavirenz,and nevirapine.
6 MACIAS ET AL. HEPATOLOGY, Month 2012
mtDNA replication.8 This leads to the accumulationof lipids in the cytoplasm through a mechanism medi-ated by the activation of adenosine monophospha-te&activated protein kinase.8 In vivo, efavirenz is asso-ciated with lipoatrophy,23 a mitochondrial toxicityinitially described among recipients of dideoxynucleo-sides. In the present study, the lack of an independentstatistical association between efavirenz and HS pro-gression might have been the result of the overwhelm-ing effect of dideoxynucleosides and the relativelysmall sample size of the efavirenz treatment group.Importantly, efavirenz is currently recommended as afirst-option drug to combine in initial ART regimens.
Thus, the risk of HS progression among patientsexposed to efavirenz needs further evaluation.Cumulative ART exposure was associated with a
lower risk of HS progression in a previous study.15 Inaddition, higher CD4 cell counts were also protectiveof HS progression.15 In our study, we found thatmarkers of response to ART, such as CD4 cell countsand undetectable HIV viremia, improved between liverbiopsies, confirming that most patients were receivingeffective ART. In spite of this fact, HS increased in fre-quency and severity in the follow-up biopsy, and thisobservation was not related to CD4 cell counts orHIV viremia changes. Moreover, we found that
Table 4. Factors Associated With Persistence of or Progression to Steatohepatitis Between Liver Biopsies
BMI, body mass index; CDC, Centers for Disease Control and Prevention; DM, diabetes mellitus; FPG, fasting plasma glucose; TGs, triglycerides; ETR: End of
‡Undetectable viremia at baseline and follow-up biopsies.
§Cumulative time on each drug for patients exposed to them.kPer year of exposure.
¶Among 69 patients who received anti-HCV therapy.
HEPATOLOGY, Vol. 000, No. 000, 2012 MACIAS ET AL. 7
cumulative dideoxynucleoside analog exposure was apredictor of HS progression, and that time on efavir-enz between biopsies was associated, in the univariateanalysis, with HS progression. Both dideoxynucleosideanalogs and efavirenz display mitochondrial toxicity.On the contrary, a drug with a very low risk of mito-chondrial toxicity, such as lamivudine, showed a statis-tical trend to less HS progression. Conflicting resultsbetween the present study and a previous report15 aredifficult to explain on the sole basis of racial andHCV genotype influences. Our study data are consist-ent with many previous findings. Thus, ART is associ-ated with increasing insulin resistance (IR), a mecha-nism involved in the pathogenesis of HS. Drugstypically related with mitochondrial toxicity, such asdideoxynucleosides and efavirenz, were associated withHS progression, whereas drugs without this side effect(i.e., lamivudine and nevirapine) were not. Prospectivestudies with uniform and predefined criteria for se-quential evaluation of HS are warranted to betterdefine the relationship between ART and specific drugsor families and HS progression. However, biopsy-basedstudies with this design will not be easily feasible,given the invasive nature of the technique and thenecessity of repeated measures over short periods.Only sequential evaluations of HS with an accuratenoninvasive technique can allow these studies.Metabolic factors are associated with HS. Obesity is
a well-characterized risk factor for HS. However, itsrole in HIV/HCV-coinfected patients seems to beweaker. BMI has been related with HS in cross-sec-tional studies2-4,10 and in the only longitudinal studyreported so far,15 but the magnitude of this associationwas small in cross-sectional studies.12 In our study,BMI was not related with progression of HS. Onepossible reason for the lack of association in our studyis that very few patients were overweight or obese.Indeed, the majority of patients were within the limitsof normal BMI. IR plays a central role in metabolicsyndrome and it is another factor implicated in HS. Inthis regard, we found that FPG was associated withHS progression. Several previous cross-sectional studiesfound an association between fasting glucose andHS.4,6,11 However, hypertriglyceridemia, another com-ponent of metabolic syndrome, was not associatedwith HS progression in the present study. In thisregard, a meta-analysis carried out in cross-sectionalstudies on HIV/HCV-coinfected patients failed toshow a relationship of hypertriglyceridemia with HS.12
Persistent steatohepatitis or progression to steatohe-patitis was observed in 18% of patients. This is a highrate of steatohepatitis, which is within the range
observed in morbid obesity or DM.13 Our results arein agreement with a previous cross-sectional study onHIV/HCV coinfection that reported on a prevalenceof steatohepatitis similar to the frequencies observed inthe herein reported study.5 Notably, we found thatpersistent steatohepatitis or progression to steatohepati-tis was associated with fibrosis progression in sequen-tial biopsies. This result is in agreement with studiesin paired liver biopsies among HIV-uninfected patientswith NAFLD, where fibrosis progression was observedin the subset of individuals with steatohepatitis.24,25
Moreover, cohort studies on NAFLD show thatpatients with steatohepatitis progress to more-seriousliver disease and have higher liver-related mortalitythan those only with HS.26,27
HS is very frequent in HIV/HCV-coinfectedpatients.1-12 HS has been linked with fibrosis pro-gression in previous cross-sectional studies on HIV/HCV coinfection2-7 and in the only previous studyon paired biopsies.15 Thus, HS itself is relevant inthe setting of HIV and HCV dual infection. Becauseof this, steatohepatitis was a secondary endpoint inthe present study. We did not find a correlationbetween HS and fibrosis progression, in agreementwith some cross-sectional studies.1,9-14 Rather, weobserved that patients with features of steatohepatitiswere at an increased risk of fibrosis progression. Dif-ferent rates of underlying steatohepatitis in previousstudies may account for the contradictory results onthe relationship between HS and fibrosis in coin-fected patients.This study has a few limitations. First, HS may
change fast in response to the modification of somefactors, such as alcohol intake, overweight, or drugs.These changes could be missed by a paired biopsystudy, particularly if the time between biopsies is verylong. Importantly, under- or overdiagnosing the inci-dence of HS in liver biopsies resulting from changes insuch modifiable factors may be a relevant reason fordiscrepant results among studies or for lack of detec-tion of some associations. This limitation is inherentin every liver biopsy-based study. Additionally, the useof scores to classify HS precludes the detection ofsmaller changes in HS, within each HS category. Theonly theoretical solution to these drawbacks would bea prospective study with a frequent schedule of nonin-vasive assessments of HS using a reliable procedure.Second, alcohol intake was self-referred by patients.Probably because of this, we did not find any associa-tion between alcohol and HS. In this regard, previouscross-sectional studies also failed to find this relation-ship.1,4-6,9 In fact, history of alcohol abuse was
8 MACIAS ET AL. HEPATOLOGY, Month 2012
associated with HS progression in a study on sequen-tial liver biopsies, but present reporting was not.15
Third, some antiretroviral drugs used during the pe-riod of study have become obsolete, particularlydideoxynucleoside analogs and unboosted proteaseinhibitors. A number of newer antiretroviral drugs areavailable to combine that may have no mitochondrialtoxicity and a better metabolic profile and thus poten-tially lead to lesser HS. Thus, data on the HS associ-ated with those newer drugs are needed. Fourth,patients who accept to undergo repeated liver biopsiesare highly selected. Usually, these patients are thosemore compliant with follow-up. Clinicians may indi-cate a follow-up biopsy if liver disease progression issuspected. Thus, rates of HS and steatohepatitis mightbe overestimated by paired liver biopsy studies as thestudy herein reported on. However, the frequency ofHS in the present study is in agreement with previouscross-sectional studies.1-11 Moreover, rates of steatohe-patitis were similar to those from a previous cross-sec-tional report.5 Finally, this study’s results may not beapplicable to other groups of patients, because very fewoverweight or obese individuals were included and theracial background of the study subjects was onlyCaucasian.In summary, HS is frequently detected in HIV/
HCV-coinfected patients with and without ART andhigh rates of progression to severe HS are observed inthem. This is a major concern, given that among indi-viduals with HS, those with features of steatohepatitisare at increased risk of fibrosis progression. Steatohepa-titis was not influenced by the suppression of HIV rep-lication, and this may be one of the reasons why fibro-sis progresses fast in HIV/HCV-coinfected patientsdespite controlling HIV replication. In addition, HSmay contribute to poorer response to interferon-basedtherapy against HCV. Cumulative exposure to dideox-ynucleoside analogs and, perhaps, to efavirenz are fac-tors associated with HS progression. Because of this,antiretroviral drugs with less potential impact on mito-chondrial toxicity should be prioritized in HIV/HCV-coinfected patients. Finally, the natural history of HSand steatohepatitis in HIV/HCV coinfection needsfurther investigation, particularly in patients receivingthe newer antiretroviral drugs.
References
1. Gaslightwala I, Bini EJ. Impact of HIV infection on the prevalenceand severity of steatosis in patients with chronic HCV. J Hepatol 2006;44:1026-1032.
2. Cast�era L, Loko MA, Le Bail B, Coffie P, De Ledinghen V, TrimouletP, et al. Hepatic steatosis in HIV-infected patients in France: compari-
son with HCV-monoinfected patients matched for BMI and genotype.Aliment Pharmacol Ther 2007;26:1489-1498.
4. McGovern BH, Ditelberg JS, Taylor LE, Gandhi RT, ChristopoulosKA, Chapman S, et al. Hepatic steatosis is associated with fibrosis,nucleoside analogue use, and hepatitis C virus genotype 3 infections inHIV-seropositive patients. Clin Infect Dis 2006;43:365-372.
5. Sterling RK, Contos MJ, Smith PG, Stravitz RT, Luketic VA, FuchsM, et al. Steatohepatitis: risk factors and impact on disease severityin human immunodeficiency virus/hepatitis C virus coinfection.HEPATOLOGY 2008;47:1118-1127.
6. Sulkowski MS, Mehta SH, Torbenson M, Afdhal NH, Mirel L, MooreRD, et al. Hepatic steatosis and antiretroviral drug use among adultscoinfected with HIV and hepatitis C virus. AIDS 2005;19:585-592.
7. Marks KM, Petrovic LM, Talal H, Murray MP, Gulick RM, GlesbyMJ. Histological findings and clinical characteristics associated with he-patic steatosis in patients coinfected with HIV and hepatitis C virus. JInfect Dis 2005;192:1943-1949.
8. Blas-Garcıa A, Apostolova N, Ballesteros D, Monle�on D, Morales JM,Rocha M, et al. Inhibition of mitochondrial function by efavirenzincreases lipid content in hepatic cells. HEPATOLOGY 2010;52:115-125.
9. Monto A, Dove LM, Bostrom A, Kakar S, Tien PC, Wright TL. He-patic steatosis in HIV/hepatitis C coinfection: prevalence and signifi-cance compared with hepatitis C monoinfection. HEPATOLOGY 2005;42:310-316.
10. Neau D, Winnock M, Cast�era L, Bail BL, Loko MA, G�eraut L, et al.Prevalence of and factors associated with hepatic steatosis in patientscoinfected with hepatitis C virus and HIV. J Acquir Immune DeficSyndr 2007;45:168-173.
11. Rodrıguez-Torres M, Govindarajan S, Sol�a R, Clumeck N, Lissen E,Pessoa M, et al. Hepatic steatosis in HIV/HCV co-infected patients:correlates, efficacy, and outcomes of anti-HCV therapy: a paired liverbiopsy study. J Hepatol 2008;48:756-764.
12. Machado MV, Oliveira AG, Cortez-Pinto H. Hepatic steatosis inpatients coinfected with human immunodeficiency virus/hepatitis C vi-rus: a metaanalysis of the risk factors. HEPATOLOGY 2010;52:71-78.
13. Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiol-ogy and natural history of non-alcoholic fatty liver disease and non-alco-holic steatohepatitis in adults. Aliment Pharmacol Ther 2011;34:274-285.
14. Pascual-Pareja JF, Camino A, Larrauri J, L�opez-Di�eguez M, MontesML, Gonz�alez-Garcıa J, et al. Factors associated with hepatic steatosisin human immunodeficiency virus and hepatitis C virus coinfectedpatients. Med Clin 2009;132:208-213.
15. Woreta TA, Sutcliffe CG, Mehta SH, Brown TT, Higgins Y, ThomasDL, et al. Incidence and risk factors for steatosis progression in adultscoinfected with HIV and hepatitis C virus. Gastroenterology 2011;140:809-817.
16. McGovern BH. Hepatis steatosis in HIV/HCV-coinfected patients:time to reevaluate! Gastroenterology 2011;140:772-775.
17. Macıas J, Berenguer J, Jap�on MA, Gir�on JA, Rivero A, L�opez-Cort�esLF, et al. Fast fibrosis progression between repeated liver biopsies inpatients coinfected with human immunodeficiency virus/hepatitis Cvirus. HEPATOLOGY 2009;50:1056-1063.
18. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Ba-con BR. Non-alcoholic steatohepatitis: a proposal for grading and stag-ing the histological lesions. Am J Gastronterol 1999;94:2467-2474.
19. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cum-mings OW, et al. Design and validation of a histological scoring systemfor nonalcoholic fatty liver disease. HEPATOLOGY 2005;41:1313-1321.
20. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassess-ment. J Hepatol 1991;13:372-374.
21. Mohanty SR, Troy TN, Huo D, O’Brien BL, Jensen DM, Hart J,et al. Influence of ethnicity on histological differences in non-alcoholicfatty liver disease. J Hepatol 2009;50:797-804.
HEPATOLOGY, Vol. 000, No. 000, 2012 MACIAS ET AL. 9
22. Maagaard A, Kvale D. Mitochondrial toxicity in HIV-infected patientsboth off and on antiretroviral treatment: a continuum or distinctunderlying mechanisms? J Antimicrob Chemother 2009;64:901-909.
23. Haubrich RH, Riddler SA, DiRienzo AG, Komarow L, Powderly WG,Klingman K, et al. Metabolic outcomes in a randomized trial ofnucleoside, nonnucleoside, and protease inhibitor-sparing regimens forinitial HIV treatment. AIDS 2009;23:1109-1118.
24. Fassio E, Alvarez E, Domınguez N, Landeira G, Longo C. Natural his-tory of non alcoholic steatohepatitis: a longitudinal study of repeat liverbiopsies. HEPATOLOGY 2004;40:820-826.
25. Harrison SA, Torgerson S, Hayashi PH. The natural history of nonal-coholic fatty liver disease: a clinical histopathological study. Am J Gas-troenterol 2003;98:2042-2047.
26. Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCul-lough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical andpathological severity. Gastroenterology 1999;116:1413-1419.
27. Rafiq N, Bai C, Fang Y, Shrishord M, McCullough A, Gramlich T,Younossi ZM. Long-term follow-up of patients with nonalcoholic fattyliver. Clin Gastroenterol Hepatol 2009;7:234-238.
