Lipid Profile Derangements among Human Immunodeficiency Virus Infected Adults Receiving First Line Anti-Retroviral Therapy in Tikur Anbesa Specialized Hospital, Addis Ababa, Ethiopia: Comparative Cross-Sectional Study

Introduction: Dyslipidemia is becoming one of the common problems in human immunodeficiency virus infected patients receiving antiretroviral therapy. Data on lipid profile derangements induced by antiretroviral treatment in Ethiopia is scarce. The aim of this study was to assess the prevalence and patterns of lipid profile abnormalities among patients taking first line antiretroviral therapy in Tikur Anbesa hospital, Addis Ababa, Ethiopia. Methods: comparative cross sectional study was conducted between August and December 2012 in Tikur Anbesa Specialized Hospital in Addis Ababa. The study population consisted of 70 HIV positive individuals who had been receiving first line ART regimen for at least 6 months (treatment group) and 71 individuals with diagnosed HIV infection and who were not yet receiving antiretroviral therapy. An interviewer administered structured questionnaire was used to collect information. Lipid profile was determined after overnight fasting and dyslipidemia was diagnosed according to the United State National Cholesterol Education Program III criteria. Data comparison used chi-square test, Student t-test and logistic regressions. Result: The prevalence of dyslipidemia was higher in antiretroviral treatment group (80%) as compared to antiretroviral treatment naïve groups (57.7%). Total cholesterol >200 mg/dL was 45.7% in Antiretroviral Therapy groups and 21.1% in Antiretroviral Therapy naïve groups. Similarly low density lipoprotein cholesterol > 130 mg/dL was 40% vs 29.6%, triglyceride >150 mg/dL; 40% vs 32.4%, and high density lipoprotein cholesterol <40; 22.9% vs 16.9% in Antiretroviral Therapy and Antiretroviral Therapy naïve groups respectively, showing more lipid alteration in ART group. Use of ART was also significantly associated with high total cholesterol (>200 mg/dL) (p<0.002), total cholesterol / high density lipoprotein cholesterol ratio >5(P<0.026), an established risk indicator of coronary artery disease and triglyceride / high density lipoprotein cholesterol ratio > 2.4(p<0.036). Conclusion: Higher prevalence of dyslipidemia was observed among Antiretroviral Therapy treated groups as compared to ART naïve groups. Therefore lipid profiles should be screened in Antiretroviral Therapy treated populations periodically to monitor any changes in lipid profile.


Introduction
The introduction of antiretroviral treatment (ART) in the mid-1990s led to a marked reduction in morbidity and mortality from human immunodeficiency virus (HIV) infection [1]. In addition to improving quality of life and reducing acquired immune deficiency syndrome (AIDS) related deaths [2,3], ART treatment has been recognized to prevent HIV transmission by reducing viral load [4]. However, over time ART has been associated with an increasing number of metabolic abnormalities such as the development of dyslipidemia, insulin resistance, and human immunodeficiency virus lipodystrophy syndrome (HIV-LS). These metabolic change are known to contribute for the development of cardiovascular disease (CVD) and diabetes mellitus (DM), representing a challenge in the treatment of HIV infection [5][6][7][8][9]. Moreover, lipodystrophic body changes can jeopardize the quality of life of these patients, leading to low adherence to ART and subsequent virologic and clinical failure [7].
During the last decade, an increasing frequency of dyslipidemia has been observed among ART treated HIV-positive patients [5]. The prevalence lays somewhere in between 20% and 80% including hypertriglyceridemia (40-80%) and high total cholesterol (10-50%), with at least one physical abnormality in approximately 50% of patients depending on the type of drug regimen used [6,10]. It is reported that as high as 82.3% of first line ART and 76.9% pre-ART patients had at least one lipid profile abnormality (dyslipidemia) [11].
The mechanisms responsible for lipid profile changes in HIV/AIDS infected patients are proven to be complex and to date are not fully understood but are probably multifactorial. It is suggested that various conditions and complex interactions involving the direct and indirect effects of antiretroviral medications and HIV infection itself have played a role in development of dyslipidemia [5,9,12].
Lipid profile alterations in pre ART patients are associated to the host's response to systemic inflammation and persistent viral infection mediated by various cytokines including tumor necrosis factor (TNF), interleukins, and the interferons secreted by active immune cells in the adipose tissue [6]. Increased production of these cytokines and inflammatory responses enhance β-adrenergic stimulation of adipose tissue and thus advance adipose tissue lipolysis which in turn results in a secondary elevation in hepatic fatty acid levels, providing a stimulus for triglyceride synthesis and secretion as very-low-density lipoprotein (VLDL) particles [5].
Following the initiation of ART, more pronounced atherogenic changes in the lipid profile has been increasingly observed [13]. Initially it was associated with exposure to protease inhibitors (PI) but subsequently exposure to nucleoside reverse transcriptase inhibitors(NRTIs) particularly stavudine (d4T) and zidovudine (AZT) were recognized as being central to the development of this syndrome, even though it has been less well studied [1,14]. HIV patients exposed to NRTIs demonstrate mitochondrial dysfunction, manifested by depletion of Mitochondrial Deoxy Ribonucleic Acid (mtDNA) and reduced mitochondrial Ribonucleic Acid (mtRNA) expression. This is due to the ability of NRTIs to inhibit DNA polymerase-, the enzyme responsible for replication of mtDNA. These molecular effects resulted in impaired β-oxidationconversion of fatty acids to triglycerides that accumulate in myocyte and hepatocyte cytosol, causing hyperlipidemia, inhibition of preadipocyte differentiation, increased adipocyte apoptosis, and abnormally functioning subcutaneous adipose tissue with reduced storage capacity for circulating lipids. Finally it resulted in increased circulating free fatty acids, reduced adiponectin secretion, and lipid accumulation in non-adipose tissues such as liver (hepatic steatosis) and hepatic triglyceride accumulation [1]. Mitochondrial toxicity are also believed to disrupt metabolic pathways through changes in sterolregulatory binding proteins leading to insulin resistance and dyslipidemia [5].
Emerging evidences also indicate an association between Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) containing regimens and dyslipidemia and other metabolic changes [9,15], even though the mechanisms they induced dyslipidemia is unclear and still unexplained [16].
Different ART classes and even individual agents within each drug class can have disparate effects on lipid profile alteration which may determine selection of ART regimens for initiations of treatment. However, most of the previous studies explored vigorously on the effect of old ART regimens like stavudine (d4T) in lipid metabolism, which are now almost excluded from the combination therapy. Still the effect of recently approved ART regimens like TenofovirDisoproxilFumarate (TDF) on lipid metabolism remains fully unexplained particularly in sub Saharan African where most of HIV patients live. In this region, where 8 to 71% of patients initiating ART die within the first year of treatment, apart from baseline CD4 count, viral load, and stage of the disease, dyslipidemia is thought to be one of the contributing variables to high AIDS-related mortality [17]. In addition, patients in developing nation initiating ART may experience different rates and types of lipid abnormalities than patients in developed countries because of differences in genetic background, dietary intake, and lifestyle factors [18]. A better understanding of the prevalence and patterns of lipid metabolic derangements at early stage in both HIV infected patients and those initiated ART could be important to identify potential interventions as well as additional clinical measurements that can be used to improve the care of HIV patients.
Therefore the aim of this study was to determine and compare the prevalence and pattern of dyslipidemia in resource poor setting, where data are scarce.

Study setting and period
This study was done at Tikur Anbesa Specialized Referral hospital. This is the largest referral hospital in the country, which is located at the center of the capital city. The ART clinic hosted approximately 5015 clients, of which 2414 were on 1st line ART and the rest 133 and 2468 were on 2nd line ART and pre-ART respectively.

Study design
A comparative cross sectional study design (comparison between ART naïve and ART treated groups) was used to assess the prevalence and paterns of lipid profile derangements among HIV patients receiving first line ART with respect to treatment naive groups at TikurAnbesa specialized Referral Hospital, Addis Ababa Ethiopia, from August to December 2012.

Study populations
All adult HIV positive patients (≥18 years of age) visited TikurAnbesa specialized Referral Hospital ART clinic from August to December 2012 were our source population for cases and controls.
Cases were defined as adult HIV positive (≥18 years old) who had been on first line ART treatment continuously for at least six months duration and controls were HIV positive adults who were not yet receiving ART prior to time of data collection.
Those who had started/changed first line ART treatment within less than six months' time and those on 2nd line ART treatment (for cases), with known diabetics and cardiovascular disease, those using lipid lowering drugs, Pregnant/ breast feeding womens, were excluded from the study.

First-line ART regimens
As defined by the WHO, regimens that included nucleoside reverse transcriptase inhibitors (NRTIs): 3TC, AZT, or d4T, TDF, and nonnucleoside reverse transcriptase inhibitors (NNRTIs): NVP or EFV and or do not include PIs.

Sample size determination
The sample size was determined by using double proportion formula and taking the prevalence of Low Density Lipoprotein-Cholesterol(LDL-c) from previous cameroon study with similar context and proximate study settings [19]. LDL-cholestrol >130 was found in 46.4% of ART treated and 21% of ART neive gruop. By taking significance level (α) = 0.05, 95% confidence level, 80% power with a case to control ratio of 1:1. Adding 10% contingency, the calculated sample size was 141(61 for treatment groups and 61 for the ART naieve groups).

Sampling method
By using convenient sampling method, all consecutive treatment and treatment naïve individuals fulfilling the inclusion criteria and attending TikurAnbesa specialized hospital ART clinic during the study period were included until the required sample size was achieved.

Data collection procedure
Clinical and demographic data collection: Information on sex, age, specific ART type in use , ART start date, duration of treatment, duration of HIV infection, BMI, relevant signs and symptoms, CD4 count, co-infection/opportunistic infection, other chronic diseases and medications if any were collected by trained nurses using structured questionnaires and patients medical record.
Blood Sample Collection, Transport and Processing: Following a standard and safety collection procedure, about 5 mL fasting venous blood was taken from both the patients and the control groups by clinical nurses and senior laboratory technologist with SST TM test tube. Sera were separated after centrifugation at 3700 rotation per minute for 10 minutes, stored at -20°C with nunck tubes and thawed just before analysis.
Dyslipidemia was defined according to the US National Cholesterol Education Program III guidelines. These include TC level >200 mg/dL, LDL-c level>130 mg/dL, TG level >150 mg/dL, and HDL-c level < 40 mg/dL.

Statistical data analysis
Data were entered using EPIInfo version 3.5 software and analyzed using SPSS software version 20.0. Differences in mean values and proportions of altered lipid profiles between the two groups were assessed using Student t-test and X 2 test. Bivariate logistic regression models with odds ratios (ORs) and their 95% confidence interval (CIs) were used to estimate the association of independent variables to altered lipid profiles in both groups. Internal Comparisons among groups of various variables was also performed. P values < 0.05 were considered statistically significant.

Ethical considerations
The study was approved by Ethics and Research Committee of the department of biochemistry (protocol no. 0016/DERC, 2012) and department of internal medicine, School of Medicine, Addis Ababa University. Then written informed consent was obtained from each study participants after stating and introducing the purpose and procedures of the study to them clearly in understandable local language. Confidentiality was assured for all the information gathered from the participants by restricting the persons who access the data and personal identifiers were not also included in data collection questionnaire.

Result General characteristics of the study population
Of 141 participants selected, 141 study subjects responded, 70 (50%) participants who were on first line ART cases and 71(50%) participants who were ART naïve controls) with over all response rates of 100 %. The sex distribution in each group was almost equivalent (P = 0.9) (

First line antiretroviral drugs used in ART groups
The first line ART used in our study participants was a combination of 2NRTIs and 1NNRTIs. Half, 50% of participants in the treatment group were using TDF containing NRTIs while more proportion of groups, 58.6% was on EFV based regimes. All the combinations were included 3TC in common ( Table 2).
A high triglyceride to HDL-C ratio (≥2.4), a strong indicator of the insulin resistance syndrome, was detected in 42.3% of ART naïve participants and 60% of ART participants (p=0.04). There was no significant difference between the two groups on HDL-C<40 and LDL-C >130 (Table 3).

Changes in Lipid profile in different ART Regimen
Nucleoside Reverse Transcriptase Inhibitors (NRTIs): There were no statistically significant differences among groups taking d4T, AZT and TDF regimens against raised TC, LDL-c, TG values and low HDL-c value (p>0.05). However, AZT and TDF groups were 5.8 and 6.7 times more likely to develop TG >150 as compared with d4T groups; 1.3 and 1.5 times for LDL-c >130, and 1.7 and 1.9 times more likely to develop TC>200 than d4T groups, respectively (Table 4).

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
Nearly 50% of participants taking NVP based NNRTIs combination have TC>200 and TG >150. Also the prevalence of LDL-c >130 and TC/HDL-C ratio was higher in these groups. However the differences between the two regimens was not statistically significant for all lipid profiles (Table 5).

Discussion
This comparative cross sectional study assessed the prevalence and characteristics of lipid profile derangement in HIV infected adults receiving first line ART in resource limited setting, where data are scarce.
The prevalence of dyslipidemia was 80% in ART treated group and 57.7% in ART naïve group. For ART groups, it was comparable with other findings from Uganda (81.6%) and southern Ethiopia (79.6%) [11,20].
The difference between the two group may be due to changes in lipid metabolism induced by medium to long term exposures to ART [1]. The direct effects of ART on lipid metabolism, endothelial and adipocyte cell function, and mitochondria have been suggested for altered lipid profiles in these subjects [21,22]. Both decreased TG clearance and increased Very-low-density lipoprotein (VLDL) overproduction found in these patients could be also the reason for the increased serum TG and then consequent elevated cholesterol level observed [13,23]. High prevalence of these lipid abnormalities in pre ART HIV infected people might be associated with high levels of oxidative stress and lipid per oxidation associated with HIV/AIDS [21]. It may be also associated to the host's response to infection mediated by various cytokines including tumor necrosis factor (TNF), interleukins, and the interferons that increase serum triglyceride levels and decrease HDL-cholesterol [6].
An association between dyslipidemia and myocardial infarction (MI) and cardiovascular disease (CVD) has been recognized. The association between high serum cholesterol levels, especially high LDL-C, and CVD is causal and independent of other risk factors while increasing clinical evidence suggested that elevated triglycerides may be an independent risk factor for CVD. Low HDL-C can also act synergistically with other lipid to increase CVD [24].
Generally the present study observed a high prevalence of dyslipidemia and noted differences in the type and extent of dyslipidemia between ART and ART naïve groups. Accordingly we found statistically significant differences in the prevalence of TC>200, TC/HDL-c ratio>5 and triglyceride /HDL-c ratio >2.4 between ART treated group and pre ART group (p<0.05). Even though it is not statistically significant, the prevalence and the chances to have LDL-c>130, TG> 150 and LDL-C <40 was still higher in ART participants.
Significant difference in TC>200 between the two groups was also described by different authors [11,14,[19][20][21] However, all these studies did not include patients taking TDF containing regimens.
TC/HDL-c ratio ≥ 5 is an established sensitive marker of cardiac risk. Participants in ART group were four times more likely to have TC/HDL ratio ≥ 5 than participants in ART naïve group (p= 0.006, OR=4.0, 95%CI (1.5-10.8)). This was also in line with other studies from Cameroon and Ethiopia [11,19]. According to US National Institutes of Health report [25], Einhorn et al. [26], McLaughlin et al. [27], a high TG / HDL-C ratio ≥2.4 is a strong indicator of the insulin resistance syndrome. In contrast to the current study other studies reported significance difference between the two groups for LDL-c ≥ 130 [11,14,19] and TG>150 [11].
Higher proportion participants taking TDF and AZT containing regimens was diagnosed with lipid profile derangements as compared to those taking d4T containing regimens, in spite of small number of participants. Pefura Yone et al. [19] also found higher prevalence of raised value of TC, LDL-c, TG, and TC/HDL-c ratio > 5 in participants taking AZT compared to those taking D4T, while the prevalence of HDL<40 was similar in both regimens. However Tadewos et al. [11] reported no differences in the prevalence of these lipid abnormalities in AZT and d4T based. In contrast to this previous studies reported that smaller increases in TC, LDL, and triglyceride levels among patients taking TDF than other NRTIs (d4T and AZT [5,18,[28][29][30]. On the other hand d4T was more involved in the occurrence of lipid derangements as compared with other NRTIs [7,20,31,32].
For NNRTIs based regimens, we did not find significant difference in all lipid profile alterations between patients taking NVP and EFV based regimens. This was in agreement with other studies reports by Pefura Yone et al. [19] Tadewos et al. [11] and Padma priyadarsini et al. [14]. Divergence to the current finding, it has been stated that EFV has a deleterious effect on lipids when compared to, NVP based regimens [5,18]. These variations may be due to patient characteristics such as gender and race/ethnicity, mitochonderial haplotype and drug metabolism polymorphism which affects variations in lipid profile between populations taking the same antiretroviral drug. Also variations in the study settings and treatment duration may contribute to these differences.

Conclusion
The prevalence of dyslipidemia is high in HIV positive populations receiving first line ART as compared to ART naive. There is major difference in atherogenic lipid profile changes between ART and pre ART groups. Considering that these altered lipid profiles can be an independent risk factors for coronary artery diseases and myocardial infarction, treatment with first-line ART may actually have potential risks for cardiovascular health of HIV positive people receiving ART. The major limitation of this study was the absence of HIV negative control groups.

Recommendation
The present study illustrated high occurrence of altered lipid profiles in HIV infected patients receiving first line ART compared to treatment naïve individuals. However we need another study with large sample size and prospective cohort in nature is to explain fully the causal relationship between each class of ART and dyslipidemia.
We recommend that lipid profile measurements at baseline, which are not currently part of routine care in our countries. It could become an important parameter to increase survival and improve treatment outcome. Therefore lipid profiles should be screened before and after start of antiretroviral therapy; then periodically through treatment follow-up to monitor any rising trends.