Diagnosing hepatic injury in HIV infection can be a herculean task for clinicians as several factors may be involved. In this study, we sought to determine the effects of antiretroviral therapy (ART) and disease progression on hepatic enzymes in HIV patients.
A case-control study conducted from January to May 2014 at the Akwatia Government Hospital, Eastern region, Ghana, The study included 209 HIV patients on ART (designated HIV-ART) and 132 ART-naive HIV patients (designated HIV-Controls). Data gathered included demography, clinical history and results of blood tests for hepatic enzymes. We employed the Fisher’s, Chi-square, unpaired t-test and Pearson’s correlation in analysis, using GraphPad Prism and SPSS. A P value < 0.05 was considered significant.
Median CD4 lymphocyte count of HIV-ART participants (604.00 cells/mm3) was higher than that of HIV-Controls (491.50 cells/mm3; P = 0.0005). Mean values of ALP, ALT, AST and GGT did not differ between the two groups compared (P > 0.05). There was a significant positive correlation between hepatic enzymes (ALP, ALT, AST and GGT) for both groups (p < 0.01 each). Duration of ART correlated positively with ALT (p < 0.05). The effect size of disease progression on hepatic enzymes for both groups was small.
Antiretroviral therapy amongst this population has minimal effects on hepatic enzymes and does not suggest modifications in therapy. Hepatic injury may occur in HIV, even in the absence of ART and other traditional factors. Monitoring of hepatic enzymes is still important in HIV patients.
Citation: Osakunor DNM, Obirikorang C, Fianu V, Asare I, Dakorah M (2015) Hepatic Enzyme Alterations in HIV Patients on Antiretroviral Therapy: A Case-Control Study in a Hospital Setting in Ghana. PLoS ONE 10(8): e0134449. https://doi.org/10.1371/journal.pone.0134449
Editor: Francesca Ceccherini-Silberstein, University of Rome Tor Vergata, ITALY
Received: January 19, 2015; Accepted: July 10, 2015; Published: August 6, 2015
Copyright: © 2015 Osakunor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Antiretroviral Therapy (ART), a term that refers to the use of the combination of three or more antiretroviral agents, since its introduction, has dramatically altered the treatment and life expectancy of Human Immunodeficiency Virus (HIV) patients for the better [1, 2]. In spite of the benefits of ART, adverse effects , of which hepatotoxicity is a common finding can lead to discontinuation, switch and non-adherence to therapy .
Diagnosing hepatic injury in HIV infection can be a herculean task for clinicians, as several factors may be involved. That which includes HIV itself, hepatitis viruses (B and C), systemic opportunistic infections, malignancies and drug-induced hepatotoxicity .
Mechanisms of hepatotoxicity from ART may result from the interaction between anti-retroviral agents and other drugs metabolized in the liver. Dose-dependent toxicity, increased half lives (decreased clearance rate) of antiretroviral agents resulting from treatment with drugs like antibiotics and increase in the levels of anti-fungal agents to hepatotoxic levels by anti-retroviral agents themselves, are amongst other mechanisms .
Studies have shown that in patients commencing ART, 14–20% will experience elevations in hepatic enzymes . Upon initiation of therapy however, most studies have found that mild hepatotoxicity exists amongst patients co-infected with hepatitis B or C virus [7–9] and after commencement of treatment for tuberculosis . In Tanzania, efavirenz and rifampicin-based hepatotoxicity occurred in HIV patients with or without Tuberculosis (TB) co-infection but these were reported to be mild and did not require modifications in therapy .
Meanwhile, other researchers have reported an increased incidence of hepatic injury in ART-treated patients and have identified hepatotoxic events [4, 12, 13] including life-threatening episodes  in patients on ART. In Uganda, there is documented evidence of the absence of hepatotoxicity during ART .
These confirm that incidence rates of hepatotoxicity during ART may vary across different populations and with different drug combinations. Giving the different definitions used in different studies, the overall frequency of grade 3 or 4 liver toxicity induced by ART in HIV patients ranges from 1% to 18% [15, 16].
In Ghana, there was more than a 200-fold increase in the number of HIV patients receiving ART from 197 in 2003 to over 45,000 in 2010 . With the increasing access to ART, there is the need to assess and monitor a plethora of co-morbidities that often present at the initiation or with the ageing of ART-treated patients.
To the best of our knowledge, this study is the first of its kind in Ghana. Thus, data on the extent of ART-related liver disease and the role of hepatic enzymes in monitoring this event, is limited.
In this study, we hypothesize that ART or HIV infection itself has hepatotoxic effects on HIV patients receiving treatment in Ghana. The purpose of this study was to determine the effect of ART and disease progression on hepatic enzymes, in a hospital setting in Ghana.
Materials and Methods
This case-control study was conducted at the Akwatia Government Hospital in the Eastern region of Ghana, from January to May 2014. The site is a district hospital that serves inhabitants of Akwatia and its surrounding areas.
Three hundred and forty-one (341) participants were involved in the study, that which consisted of 209 patients on ART (designated HIV-ART) and 132 ART-naive patients (designated HIV-Controls).
Patients were included in the study if they met the following criteria; diagnosed and confirmed HIV-positive, age ≥ 18 years, followed up by the Akwatia Government Hospital with frequent visits, and on ART for at least three (3) months. Patients were excluded if they had active or chronic viral hepatitis, were pregnant, on concurrent hepatotoxic drugs or on treatment with liver supportive drugs.
The study was approved by the Committee on Human Research, Publication and Ethics (CHRPE) of the School of Medical Sciences (SMS), Kwame Nkrumah University of Science and Technology (KNUST) / the Komfo Anokye Teaching Hospital (KATH). Participation was voluntary and written informed consent was obtained from each participant.
Data Collection and laboratory analysis
Demographic characteristics and clinical history were gathered using findings from a structured questionnaire and concurrent review of patient records.
About five (5) ml venous blood sample was taken from each participant, and dispensed into serum separator (SST) (3 ml) and ethylenediaminetetraacetic acid (EDTA) tubes (2ml). Serum biochemical assays [Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline Phosphatase (ALP) and Gamma-glutamyltransferase (GGT)] were performed immediately on the BT-3000 Plus Chemistry Analyser (Diamond Diagnostics, USA). CD4 lymphocyte count estimation was done on the BD FACS Count System (Becton Dickenson and Company, USA).
Data management and analysis
Data was entered into a Microsoft Excel spread sheet and analysed using GraphPad Prism version 6.0 (GraphPad software, San Diego, California, USA) and Statistical Package for the Social Sciences (SPSS) version 20 (SPSS Inc. Chicago, USA).
Case definitions for the various hepatic enzymes were as follows; ALT > 40.0 U/L for male or > 31.0 U/L for female, AST > 37.0 U/L for male or > 31.0 U/L for female, GGT > 51.0 U/L for male or > 33.0 U/L for female and ALP > 117.0 U/L for adults . Disease progression as indicated by CD4 lymphocyte count was; Stage1 (≥ 500 cells/mm3), Stage 2 (200–499 cells/mm3) and Stage 3 (< 200 cells/mm3) as per recommendations of the Centres for Disease Control and Prevention (CDC) [19–21]. Patients on ART were placed in three groups, based on duration of therapy; Group 1 (< 2 years), Group 2 (2–4 years) and Group 3 (> 4 years).
Categorical variables were expressed as frequencies and proportions and compared using the Fisher’s exact test or Chi-square test. Continuous variables were expressed as means ± SEM and compared using the unpaired t-test. To establish the relationship between variables, we performed the Pearson’s correlation co-efficient test. The Cohen's guidelines for eta squared was used in assessing the effect size of disease progression as follows; small = 0.02, medium = 0.06 and large = 0.13 . A P value of < 0.05 was considered statistically significant.
Table 1 shows the demographic and clinical characteristics of the study population. Mean age was 40.85 ± 0.63 and majority were female (76.8%). Amongst the HIV-ART population, 45.5% had been on ART for less than 2 years. Median CD4 lymphocyte count of HIV-ART participants (604.00 cells/mm3) was higher than that of HIV-Controls (491.50 cells/mm3; P = 0.0005). About half the population (50.1%) had CD4 counts ≥ 500 cells/mm3 with a significant difference between proportions of the two groups (P < 0.0001).
There was no significant difference in mean ALP (P = 0.8750), ALT (P = 0.6511), AST (P = 0.6128) and GGT (P = 0.0543) between the two groups (HIV-ART and HIV-Controls). [Table 2]
In Table 3, there was no significant difference between proportion of individuals with elevated hepatic enzymes, when compared between HIV-ART and HIV-Controls (P > 0.05).
When this was compared within and across gender, all enzymes but the GGT, did not differ significantly between HIV-ART and HIV-Controls. Of both groups compared, there were significantly more males than females with elevated GGT (P < 0.0001 each). [Table 4]
From Tables 5 and 6, there was a significant positive correlation between all hepatic enzymes (ALP, ALT, AST and GGT) for both the HIV-ART and HIV-Control groups (P < 0.01 each). Conversely, each of the enzymes is likely to rise concurrently with the rise of another. Duration of ART positively correlated with ALT (P < 0.05) but not with ALP, AST and GGT.
Table 7 presents the relationship between disease progression, as indicated by the CD4 lymphocyte counts and ART-related hepatic injury. The effect size of disease progression on hepatic enzymes for the HIV-ART group was small, as indicated by eta squared for the hepatic enzymes. This was similar for the HIV-Control group.
In spite of the benefits of ART, it may come with adverse effects , hence the question of discontinuation or alteration of treatment may come to play . In this study, we sought to determine the effect of ART and disease progression on hepatic enzymes, in a hospital setting in Ghana.
The results of the present study suggest that in this population, ART has minimal effects on hepatotoxicity. This may be due to the absence of other traditional risk factors such as hepatitis C virus and / or hepatitis B virus (HBV / HCV) co-infection, older age, high alcohol intake and use of illicit drugs that may contribute to the development of hepatic injury in adults [23, 24].
Upon HIV infection, it is expected that CD4 lymphocyte counts will drop with ageing of HIV infection, and ART is expected to help increase these numbers. We observed that the median CD4 count of HIV-ART participants was higher than that of HIV-Controls. Findings from other researchers have shown that, after ART, the median CD4 cell counts increase with time . Adherence to therapy may also be a contributing factor to achieving the purpose of therapy in HIV patients , thus the finding in the present study. However, since we were unable to assess adherence to therapy, we are unable to ascertain this fact. Recently, Obirikorang et al.,  found a lower CD4 lymphocyte count amongst HIV patients on ART than in those patients who were not. They attributed this to the fact patients who were not on ART had values not so low as to initiate ART, hence causing a possible masked increase in CD4 lymphocyte count amongst patients on ART when compared to ART-naive individuals . Hence, the suggestion of an increase in CD4 lymphocyte count upon ART initiation is still valid.
Hepatic enzyme elevations are common in HIV-infected patients, especially those treated with ART. Despite such reports, HIV-infected patients may present several risk factors for biochemical abnormalities, and a precise aetiology is rarely defined clearly . Most studies conducted on hepatotoxicity have shown the existence of hepatic injury associated with ART, but in the presence of other co-infections such as HCV  and even tuberculosis . In a retrospective study assessing severe hepatic injury during ART, co-infected patients had a 3.99 greater chance of developing severely elevated transaminase levels compared with patients not co-infected with HCV. This risk increased considerably in co-infected patients with identified alcohol abuse . We observed that of all the hepatic enzymes measured (ALT, AST, ALP and GGT), there was no significant difference in mean values between the two groups compared (HIV-ART and HIV-Controls). Thus in the absence of other traditional risk factors that increase the chances or cause hepatotoxicity, injury by ART may be minimal. On the contrary, a study conducted in Cameroon found elevations in both serum AST and ALT in patients on ART .
Reports have shown that slight increases in GGT may be due to contribution from other sources of GGT such as the prostate, pancreas and kidneys [29, 30]. In the case of the current study, higher proportion of males with elevated levels of GGT observed could be due in part, to contributions from extra-hepatic sources such as the prostate.
The observed small effect size of disease progression on hepatic enzymes could indicate that with careful studies, there is indeed an effect of disease progression on hepatic enzymes in HIV but this may not be clinically obvious. Mokondjimobe et al.,  reported that isolated naive HIV infection was associated with higher levels of ALT and GGT before and after adjusting for age and sex. Other researchers have also suggested that in the absence of ART, mild hepatotoxicity is seen in HIV patients . This has been attributed to the fact that HIV infection results from chronic immune system activation and inflammatory cytokine release and studies have shown that ALT and GGT are now considered markers of inflammation with immune dysfunction in HIV  and oxidative stress . As disease progresses and chronic inflammation increases, there is a high likelihood of increases in these hepatic enzymes.
The present study is limited by a number of factors. Our inability to determine the HIV viral RNA in study participants could not enable the determination of the relationship between viral load, ART regimen and liver enzymes, and thus the role of HIV itself on changes in liver enzymes. Furthermore, information on the number of ART regimens used by participants was not available, hence the effects of the number of regimens on liver enzymes was not determined. As this is a case control study, we were unable to determine pre-treatment liver enzyme levels and subsequently follow up with post-treatment levels. The inclusion of higher number of participants, information on antiretroviral exposure, increased observational time, and the use of a more restrictive criterion with regards to antiretroviral exposure is warranted.
Antiretroviral therapy amongst this population has minimal effects on hepatic enzymes and does not suggest modifications in therapy. In the absence of traditional risk factors for hepatotoxicity like alcohol use, viral hepatitis and illicit drug use, hepatic injury may be caused by HIV infection itself. This may however be seen only upon careful studies. Frequent measurement of hepatic enzymes is still important for HIV patients on and off ART, with and without other traditional risk factors for hepatotoxicity.
The authors appreciate the contributions of patients, staff and management of the Akwatia Government Hospital.
Conceived and designed the experiments: DNMO CO VF. Performed the experiments: DNMO CO VF IA MD. Analyzed the data: DNMO CO VF. Contributed reagents/materials/analysis tools: DNMO CO VF IA MD. Wrote the paper: DNMO CO VF.
- 1. Palella FJJ, Delaney KM, Moorman AC, O LM, Fuhrer J, Satten GA. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Eng J Med. 1998;338:853–60.
- 2. Berns JS, Kasbekar N. Highly Active Antiretroviral Therapy and the Kidney: An Update on Antiretroviral Medications for Nephrologists. Clin J Am Soc Nephrol. 2006; 1:117–29. pmid:17699198
- 3. Kontorinis N, Dieterich D. Hepatotoxicity of antiretroviral therapy. AIDS Rev. 2003;5(1):36–43. pmid:12875106.
- 4. Teklay G, Legesse B, Legesse M. Adverse Effects and Regimen Switch among Patients on Antiretroviral Treatment in a Resource Limited Setting in Ethiopia. J Pharmacovigilance. 2013;1:115. pmid:1000115.
- 5. Kreisberg R. Clinical problem-solving. We blew it. The New England journal of medicine. 1995;332(14):945–9. pmid:7661934.
- 6. Rodriguez-Rosado R, Garcia-Samaniego J, Soriano V. Hepatotoxicity after introduction of highly active antiretroviral therapy. AIDS. 1998;12(10):1256. pmid:9677182.
- 7. Heil EL, Townsend ML, Shipp K, Clarke A, Johnson MD. Incidence of Severe Hepatotoxicity Related to Antiretroviral Therapy in HIV/HCV Coinfected Patients. AIDS research and treatment. 2010;2010:856542. pmid:21490905; PubMed Central PMCID: PMC3065809.
- 8. Kalyesubula R, Kagimu M, Opio KC, Kiguba R, Semitala CF, Schlech WF, et al. Hepatotoxicity from first line antiretroviral therapy: an experience from a resource limited setting. African health sciences. 2011;11(1):16–23. pmid:21572852; PubMed Central PMCID: PMC3092323.
- 9. Cooper CL, Parbhakar MA, Angel JB. Hepatotoxicity associated with antiretroviral therapy containing dual versus single protease inhibitors in individuals coinfected with hepatitis C virus and human immunodeficiency virus. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2002;34(9):1259–63. pmid:11941553.
- 10. Ungo JR, Jones D, Ashkin D, Hollender ES, Bernstein D, Albanese AP, et al. Antituberculosis drug-induced hepatotoxicity. The role of hepatitis C virus and the human immunodeficiency virus. American journal of respiratory and critical care medicine. 1998;157(6 Pt 1):1871–6. pmid:9620920.
- 11. Mugusi S, Ngaimisi E, Janabi M, Minzi O, Bakari M, Riedel KD, et al. Liver enzyme abnormalities and associated risk factors in HIV patients on efavirenz-based HAART with or without tuberculosis co-infection in Tanzania. PloS one. 2012;7(7):e40180. pmid:22808112; PubMed Central PMCID: PMC3394799.
- 12. Chu KM, Boulle AM, Ford N, Goemaere E, Asselman V, Van Cutsem G. Nevirapine-associated early hepatotoxicity: incidence, risk factors, and associated mortality in a primary care ART programme in South Africa. PloS one. 2010;5(2):e9183. Epub 2010/02/23. pmid:20174653; PubMed Central PMCID: PMCPMC2822855.
- 13. Lucien KFH, Clement ANJ, Fon NP, Weledji P, Ndikvu CP. The Effects of Antiretroviral Treatment on Liver Function Enzymes among HIV-Infected Out Patients Attending the Central Hospital of Yaoundé, Cameroon. Afr J Cln Exper Microbiol. 2010;11(3):174–8.
- 14. Spengler U, Lichterfeld M, Rockstroh JK. Antiretroviral drug toxicity-Therapy. Reuters Health. 2002.
- 15. van Leeuwen R, Katlama C, Murphy RL, Squires K, Gatell J, Horban A, et al. A randomized trial to study first-line combination therapy with or without a protease inhibitor in HIV-1-infected patients. AIDS. 2003;17(7):987–99. pmid:12700448.
- 16. van Leth F, Phanuphak P, Ruxrungtham K, Baraldi E, Miller S, Gazzard B, et al. Comparison of first-line antiretroviral therapy with regimens including nevirapine, efavirenz, or both drugs, plus stavudine and lamivudine: a randomised open-label trial, the 2NN Study. Lancet. 2004;363(9417):1253–63. pmid:15094269.
- 17. Obirikorang C, Selleh PK, Abledu JK, Fofie CO. Predictors of Adherence to Antiretroviral Therapy among HIV/AIDS Patients in the Upper West Region of Ghana. ISRN AIDS. 2013;2013:7.
- 18. Hann HW, Wan S, Myers RE, Hann RS, Xing J, Chen B, et al. Comprehensive analysis of common serum liver enzymes as prospective predictors of hepatocellular carcinoma in HBV patients. PloS one. 2012;7(10):e47687. pmid:23112834; PubMed Central PMCID: PMC3480412.
- 19. CDC. 1992 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Morb Mortal Wkly Rep. 1993;41(no. RR–17):: 961–2.
- 20. CDC. RevisedGuidelinesforPerformingCD4+T-CellDeter- minations in Persons Infected with Human Immunodeficiency Virus (HIV). Morb Mortal Wkly Rep. 1997;46(no. RR–2):1–29.
- 21. Mandy FF, Nicholson JK, McDougal JS, Cdc. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Centers for Disease Control and Prevention. MMWR Recommendations and reports: Morbidity and mortality weekly report Recommendations and reports / Centers for Disease Control. 2003;52(RR-2):1–13. pmid:12583540.
- 22. Cohen J. Statistical Power Analysis for the Behavioural Sciences. 2nd ed. Hillsdale, NJ.: Lawrence Erlbaum Associates; 1998.
- 23. Nunez M, Lana R, Mendoza JL, Martin-Carbonero L, Soriano V. Risk factors for severe hepatic injury after introduction of highly active antiretroviral therapy. J Acquir Immune Defic Syndr. 2001;27(5):426–31. pmid:11511818.
- 24. Nunez M, Soriano V. Hepatotoxicity of antiretrovirals: incidence, mechanisms and management. Drug safety: an international journal of medical toxicology and drug experience. 2005;28(1):53–66. pmid:15649105.
- 25. Smith CJ, Sabin CA, Youle MS, Kinloch-de Loes S, Lampe JWFC, Madge S, et al. Factors Influencing Increases in CD4 Cell Counts of HIV-Positive Persons Receiving Long-Term Highly Active Antiretroviral Therapy. The Journal of infectious diseases. 2004;190(10):1860–8. pmid:15499544
- 26. Obirikorang C, Selleh PK, Abledu JK, Fofie CO. Predictors of Adherence to Antiretroviral Therapy among HIV/AIDS Patients in the Upper West Region of Ghana. ISRN AIDS. 2013;2013:873939. pmid:24386593; PubMed Central PMCID: PMC3872409.
- 27. Obirikorang C, Osakunor DN, Ntaadu B, Adarkwa OK. Renal function in Ghanaian HIV-infected patients on highly active antiretroviral therapy: a case-control study. PloS one. 2014;9(6):e99469. pmid:24921259; PubMed Central PMCID: PMC4055675.
- 28. Pol S, Lebray P, Vallet-Pichard A. HIV infection and hepatic enzyme abnormalities: intricacies of the pathogenic mechanisms. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2004;38 Suppl 2:S65–72. pmid:14986277.
- 29. Kubota S, Amino N, Matsumoto Y, Ikeda N, Morita S, Kudo T, et al. Serial changes in liver function tests in patients with thyrotoxicosis induced by Graves' disease and painless thyroiditis. Thyroid: official journal of the American Thyroid Association. 2008;18(3):283–7. pmid:18001177.
- 30. Littner Y, Bearer CF. Detection of alcohol consumption during pregnancy—current and future biomarkers. Neuroscience and biobehavioral reviews. 2007;31(2):261–9. pmid:16919733.
- 31. Mokondjimobe E, Longo-Mbenza B, Mampouya-Arrouse P, Parra HJ, Diatewa M. Inflammatory status hepatic enzymes and serum creatinine in HIV-, HIV+ and HIV-TB co-infected adult Central Africans. International journal of general medicine. 2012;5:961–5. pmid:23204860; PubMed Central PMCID: PMC3508561.
- 32. Ayelagbe OG, Akerele OP, Onuegbu AJ, Oparinde DP. Drug hepatotoxicity in HIV patients on Highly Active Antiretroviral Therapy [HAART] in Southwest Nigeria. IOSR-JDMS. 2014;13(5):67–70.
- 33. Nixon DE, Landay AL. Biomarkers of immune dysfunction in HIV. Current opinion in HIV and AIDS. 2010;5(6):498–503. pmid:20978393; PubMed Central PMCID: PMC3032605.
- 34. Yamada J, Tomiyama H, Yambe M, Koji Y, Motobe K, Shiina K, et al. Elevated serum levels of alanine aminotransferase and gamma glutamyltransferase are markers of inflammation and oxidative stress independent of the metabolic syndrome. Atherosclerosis. 2006;189(1):198–205. pmid:16405892.