Incidence and Risk Factors of Combined-Antiretroviral Therapy-Induced Hepatotoxicity among HIV Patients at the Bali District Hospital, Cameroon

Introduction The incidence of hepatotoxicity is life-threatening and can result to an end-stage liver disease in long-term patients on combined antiretroviral therapy (cART). Our study sought to evaluate the incidence and predictors of cART-induced hepatotoxicity (CIH) among long term users on cART in a rural District hospital. Methods This was a hospital-based cross-sectional study in the Bali District Hospital. Spectrophotometric method was use for the quantitative measurement of alanine-aminotransferase (ALT) and aspartate-aminotransferase (AST) levels. Patients with elevations of both ALT and AST were considered CIH. The Chi (χ2) square test, ANOVA and Kaplan Meier log-ranked/ survival analyses were used to analyse the data. Results Of the 350 participants enrolled [156 (44.6%) males and 194 (55.4%) females], aged 43.87 ± 0.79 years (range 20 – 84 years) included in this analysis, 26 (4.4%) experienced moderate CIH. We observed 57 (16.3%), 62 (17.7%) and 238 (68%) elevated levels ALT + AST, ALT and AST respectively. Two independent predictive factors of CIH were, the male sex and alcoholism during the study period. Conclusion The prevalence of CIH in HIV-infected patients in Bali was lower than that observed in previous studies. The duration of therapy had no influence on the frequency of CIH. Alcoholism and smoking showed significant differences in the development of CIH.


Introduction
The human immune deficiency virus/ acquired immunodeficiency syndrome (HIV/AIDS) is a devastating infection that remains a public health problem in Sub-Saharan Africa, is caused by either of two lentiviruses: HIV-1 or HIV-2 [1,2].Of the 36.7 million persons living with HIV (PLWHIV), with an estimated 2.1 million new infections, representing a rate of 0.3 new infections per 1,000 uninfected people worldwide, 19 million (51.8%) are in Sub-Saharan Africa [3,4].In Cameroon, the incidence of adult HIV has fallen consistently from 7.7% in 1999 to 4.3% in 2013 but has remained high among female sex workers, with an estimated prevalence of 3.6% (range; 1.5 -6.3%) within the ten Regions [5]. Highly Active Anti-Retroviral Therapy (HAART) is a combination of antiretroviral drugs for the management of HIV/AIDS [6,7].The first-line HAART recommended for use in Cameroon by the Ministry of Public Health (MOH) and the World Health Organization (WHO) areTenofovir /Lamivudine/ Efavirenz(TDF+3TC+EFV) [8,9].
Although HAART and combination antiretroviral therapy (cART) constitute the most significant interventions that have changed the landscape of HIV-related morbidity and mortality, there are also challenges of adverse drug reactions leading to dose modifications, changes or treatment discontinuations [10][11][12].
Highly Active Anti-Retroviral Therapy (HAART)-associated hepatoxicity/ cART-induced hepatotoxicity (CIH), arbitrarily defined as AST or ALT > 3 X upper limit of normal (ULN) in the presence of symptoms, or serum AST or ALT > 5 X ULN in the absence of symptoms, usually resolves without modification of therapy [13][14][15][16][17][18]. Hepatotoxicity, which is the most cited reason for the withdrawal of approved drugs, is damage caused by exposure to a drug or non-pharmacological agents [19],and is consequently associated with HAART/ cART [15,20] especially nevirapine-based HAART [21] or efavirenz-based drug-induced liver injury [22]. In isolated instances, serious and life-threatening conditions may arise.
Theclinical presentation of HAART/ cART-induced hepatotoxicity can range from mild asymptomatic increases in serum transaminases to overt liver failure [15,23,24]. Retrospective studies indicate that, the incidence of cART-related severe hepatotoxicity is approximately 10%, and life-threatening events occur at a rate of 2.6 per 100 person years [25]. The incidence of druginduced hepatotoxicity in the general African population is estimated to fall between 1/100,000 and 20/100,000 [26]. Hepatotoxicity due to ART may be related to agents from a number of classes including nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors [27]. The severity of hepatotoxicity may range from transient elevations in transaminase levels to hepatic failure and death, via a variety of mechanisms such as direct cell stress and disturbances in lipid/ sugar metabolism and steatosis, as seen with PI [27]. Co-infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) has consistently been associated with increased risk of ART-related hepatotoxicity [25]. Other risk factors associated with ART-related liver injury include pre-existing advanced fibrosis, pre-treatment of elevated ALT or AST, alcohol abuse, old age, female gender, first exposure to ART, significant increase in CD4 + cell count after ART initiation, concomitant tuberculosis medications and cocaine use [28].
While all antiretroviral drugs have some risks of hepatotoxicity, some are more implicated than others. The non-nucleoside reverse transcriptase inhibitors (NNRTI) typically cause either hypersensitivity reactions or direct drug toxicity and therefore have two peaks of onset: within days to weeks or several months after initiation [25].Nevirapine (NVP) is the NNRTI most associated with hepatotoxicity [21], though hypersensitivity reactions resulting in liver failure have been reported with the newer NNRTI etravirine.Efavirenz and stavudine can also cause hepatotoxicity but does so less frequently than NVP or etravirine [22].
The main objective of this study was to determine the incidence of hepatotoxicity and to assess the possible risk factors for developing cART-induced hepatotoxicity among PLWHIV at the Bali District Hospital.

Material and Methods
A retrospective study of HIV -infected patients was conducted to examine the incidence and predictors of cART-induced hepatotoxicity (CIH).

Study Area
The study was conducted at the state-owned Bali District Hospital in the North West Region of

Study Design and Population
A retrospective cross-sectional study was designed to examine the incidence and predictors of CIH.
Study participants were Persons living with HIV, who were on cART/ HAART. Participants aged ≥ 18 years and willingness to have HIV status confirmed from clinical records or by a point-of-care test were included in the study. Pregnant women, those younger than18 years, those on unprescribed medications and severely sick persons were excluded from the study.Participants with CD4 + > 500 cells/µL and viral load < 500 copies/mL were excluded from this analysis.

Data collection tool and Data Collection
The instruments used for the collection of data were, a well-organised laboratory form and patients' files. Data collected for analysis was defined as; socio-demographic information (age, gender and marital/educational status) and transaminase (ALT and AST) concentrations. The transaminase concentrations were obtained by spectrophotometric measurements.

Specimen Collection and analysis
3 mL of blood specimen was collected from each participant by venipuncture into 5 mL vacutainer dry test tubes and allowed to clot at room temperature. The specimens were latter centrifuged for five minutes at 2500 rpm, and the sera collected were assayed for ALT and AST according to manufacturers' instructions (Quimica, Italy).

Laboratory analysis:All the ALT and AST measurements wereperformed at the Bali District
Hospital Laboratory.
Hepatotoxicity was defined based on biochemical measurements as an elevation in serum ALT and/ or AST from the normal following the International Consensus Criteria and also on previous studies [15,19,23].The severity of the liver injury was indicated by category (graded) based on various enzyme levels (Grade 1/ Mild, Grade 2/ Moderate, Grade 3/ Severe, and Grade 4/ Acute Liver Failure) [6, 16].

Statistical Analysis
All data collected was entered into epi info 7. Univariate and multivariate Cox proportional hazards regressions, were performed to assess the predictors of cART-induced hepatotoxicity. The variables included in the multivariate model were those with either a theoretical importance or ones with a p<0.05 in theunivariable models.

Ethical considerations
The study was approved by the Regional Delegate of Public Health for the North West Region and the Higher Institute of Health Sciences of Bamenda University of Science and Technology (BUST) and was conducted in accordance with the Helsinki declaration [35]. An administrative clearance was obtained from the Director of the Bali District Hospital.All participants signed informed consent forms and all records were strictly confidential. were included for analysis in this study. A majority 154 (44%) of the study participants were in the age group 40 -< 60 years old and most of them 148 (42.3%) had at least the secondary education (Table 1).

Socio-Demographic Characteristics
Thirty-four males (29.6%, N = 57) and 23 females (40.4%, N = 57) among cases had cART-induced hepatotoxicity. Of the 350 study participants, 34 (9.7%%) were malnourished as defined by body mass index (BMI) of < 18.5 Kg/m 2 , 77 (22.0%) were overweight, 14 (4.0%) were obese and 64.3% were eutrophic (normal). Most of the participants 223 (63.7%) were married and just one-fifth (20.0%) of them were farmers (Table 1). There was no significant difference as regards age, BMI, cART regimen and duration of therapy between cases and controls.  Table 2). Table 2 shows the prevalence of elevated liver enzymes in the five cART treatment groups. Patients on TDF/3TC, had the highest incidence19 (30.6%) and 94 (39.5%) of elevated ALT and AST respectively, followed by TDF/3TC/EFV with incidence of 15 (24.2%) and 51 (21.4%) of elevated ALT and AST respectively. However, the overall prevalence of hepatotoxicity in the five treatment groups for the two liver enzymes was 57 (16.3%).  Table 2).

Clinical and Biochemical Spectrum of Participants
Results of the study indicated that there were five different cARTregimens ( Figure 1; Table 1)

Factors associated with cART-induced hepatotoxicity
Age was categorized into three groups and there was no difference betweencases and controls between these age groups. The nutritional status (as assessed by BMI) of study participants seems to  (Table 1).
Univariate analysis was also done using Cox Proportional hazard regression analysis for the variables in Table 3. Sex and alcohol consumption were observed to be predictors of cART-induced hepatotoxicity. In the multivariate model, the predictors of developing cART-induced hepatotoxicity include the male sex (Hazards Ratio (HR) = 1.6, 95% C.I = 0.9 -2.8), the cART regimen

General Characteristics
Hepatotoxicity is one of the most common adverse drug reactions associated with HAART/cART in Persons Living withHIV/AIDS. This increases the mortality rate in PLWHIV, rather than the HIV infection itself.
As observed in this study, age difference is not a determinant factor for ALT or AST elevations.This is supported by studies reported in Africa [14,36] and Zürich-Switzerland [37]which reported that age is not a risk factor for the development of hepatotoxicity in patients on cART/HAART.However, studies carried out elsewhere in Africa reported that age was significantly associated with drug-induced hepatotoxicity [15,31]. These differences could be due to the fact that more than 60% of our study participants were above 40 years of age.
These differences could be as a result of the fact that this study, weenrolled only HIV patients.

Hepatotoxicity
In the present study, the overall cART-induced hepatotoxicity among HIV-infected patients was 16.3%, which waslower compared to the 42.4 -54% reported in Cameroon [29,38],20.1 -32% in Ethiopia [23,32,39], 25% reported in Warsaw-Poland [12]and 19.7/100 person years in South Africa [28]. Our findings were higher compared to the 13.6% reported in Fako-Cameroon [30], 11.5% in Ethiopia [33] and 7.8% in Tanzania [36]. However, our study is consistent with the 16% reported in Zürich-Switzerland [37]and Taiwan-China [40]and the 15 -16.7%reported in Ethiopia [15,31]. This could be explained by the fact that the present study did not include tuberculosis and tuberculosis/ HIV co-infected patients, neither did it include hepatitis B and C viral infections as well as co-infections.
The finding of this study indicated that ALT and AST related hepatotoxicity were 17.7% and 68% respectively. This was in line with the findings of a study reported in Ghana [14] and lower when compared to findings reported in Yaoundé-Cameroon [38].
In this study, it was observed that the sex (male) and alcohol consumption were the independent predictors of cART-induced hepatotoxicity. The WHO clinical stage 2 of HIV, AZT/3TC/EFV regimen and cigarette smoking were significantly associated with hepatotoxicity (Table 3). Our finding of male being a predictor was in line with that of a study reported in Ethiopia [32] and different from females as reported in Ethiopia [23,31]. Alcoholism as reported in this study was in line with alcoholism reported in London [16], Zürich [37], as well as concomitant administration of other drugs [15,17,28].

Strengths and limitations of the study
Strengths of the study: The data used was collected by experienced scientists, using laboratory forms and patients' records.
Limitations of the study: A limitation to the study was that it was a cross-sectional study collecting data on the dependent and independent variables at the same time. It equally did not consider the concomitant administration of other therapeutic agents; anti-malaria drugs, herbs, as well as smoking and alcohol intake.

Conclusion
In conclusion, cART-induced hepatotoxicity is incident amongst HIV-infected patients seeking health care in the Bali District Hospital, regardless of the cART treatment group. Allof these hepatotoxic events were not severe and had no clinical significance. The male sex and alcoholism were associated with a higher rate of liver injury. Prospective studies focusing on the effects of cART/ HAART on hepatitis in HIV-infected patients are needed to confirm our findings.Combined antiretroviral therapy is frequent and a major concern amongst Cameroonian HIV patients and regular monitoring of liver enzymes during early therapy is recommended for proper identification and management of cART-induced hepatotoxicity.

Declarations Ethical approval and consent to participate
Ethical clearance was obtained from the North West Regional Delegation of Health and the Higher institute of Health Sciences of Bamenda University of Science and Technology (BUST)and was conducted in accordance with the Helsinki declaration [35]. An administrative clearance was obtained from the Director of the Bali District Hospital. Participation in the study was voluntary, and all participants signed informed consent.