Gradual Increasing Dyslipidemia in Male Patients With Human Immunodeciency Virus Primarily Treated With Tenofovir Plus Lamivudine Plus Efavirenz for Three Years

Introduction: Since the development of antiretroviral therapy (ART) with TDF plus 3TC plus EFV, this specic regimen has not been studied enough with long-term lipid and uric acid monitoring. Methods: A prospective follow-up cohort study was performed. Sixty-one treatment-naive male patients with human immunodeciency virus (HIV) were divided into three groups based on their baseline CD4+ cell count (26, 12, and 23 patients in the <200, 200 to 350, and >350 groups, respectively). The lipid and purine metabolism parameters of the patients over 144 weeks were analyzed. Result: Within 144 weeks, TG, LDL-c, TC and HDL-c gradually increased, especially the latter two (P=0.001, 0.000, respectively). Moreover, the percentages of hypercholesterolemia, hyper LDL cholesterolemia, hypertriglyceridemia and low HDL cholesterolemia also gradually increased, especially the latter (P=0.0007). The lower the baseline CD4+ cell counts were, the higher the TG levels were and the lower the TC, LDL-c and HDL-c levels were. But there was signicant difference of only baseline LDL-c levels between the three groups (P=0.0457). No signicant difference of the UA level and the percentages of hyperuricemia was found between the different follow-up time point groups or between the three groups (all P>0.05). The risk factors for dyslipidemia included age, anthropometric parameters and follow-up weeks, and for hyperuricemia was virus load. Conclusions: Gradual increasing dyslipidemia was found in male patients with human immunodeciency virus primarily treated with tenofovir plus lamivudine plus efavirenz for three years. There-fore lipid metabolism parameters should be closely monitored during long-term ART with the TDF plus 3TC plus EFV regimen. (HIV) natural logarithmic transformation before statistical analysis. Quantitative data were expressed as χ±SD, and categorical data were expressed as rates or percentages. One-way ANOVA was used to compare metabolism parameters from baseline to 144 weeks, and a paired t-test was used to compare metabolism parameters between baseline and some follow-up time points. The Kruskal-Wallis H (K) test for K independent samples was used to compare the percentage of dyslipidemia and hyperuricemia from baseline to 144 weeks. The Mann-Whitney test for two independent samples was used to compare the percentage of dyslipidemia and hyperuricemia between baseline and some follow-up time points. One-way ANOVA was used to compare metabolism parameters between the three different CD4+T cell count groups at the same time point. Two-way ANOVA was used to compare metabolism parameters between the three different CD4+T cell count groups from baseline to 144 weeks. A p value<0.05 was considered statistically signicant. results gradual increasing dyslipidemia was found in male patients with human immunodeciency virus primarily treated with tenofovir plus lamivudine plus efavirenz for three years. The risk factors for dyslipidemia included age, anthropometric parameters and follow-up weeks, and for hyperuricemia was virus load. There-fore lipid metabolism parameters closely monitored during long-term ART with TDF plus 3TC plus EFV regimen. AIDS: Acquired immunodeciency syndrome; ART: Antiretroviral therapy; BMI: body mass index; BW: body weight; EFV: Efavirenz; HDL-c: high-density lipoprotein cholesterol; HIV: Human immunodeciency virus; HIVRNA: HIV viral nucleic acid; 3TC: Lamivudine; LDL-c: low-density lipoprotein cholesterol; TC: Total cholesterol; TDF: tenofovir; TG: Triglycerides; ULN: normal value; UA: uric acid.


Introduction
In recent years, the numbers of patients with human immunode ciency virus (HIV) and acquired immune de ciency syndrome (AIDS) have sharply increased. By the end of 2019, approximately 38 million people worldwide lived with HIV, and 33 million people died of HIV-related diseases [1]. At the end of October 2019, a total of 1,045,000 patients in China were living with HIV [2,3]. At the end of September 2018, a total of 262,000 patients died of HIV-related diseases in China [2,3].
The most effective treatment for AIDS is antiretroviral therapy, which can prolong life expectancy and improve quality of life [4,5]. When a patient's CD4 + cell count reaches more than 350/mm 3 and the viral load reaches undetectable levels within the rst year of starting treatment, AIDS patients are predicted to have a normal life expectancy [4,5]. The cumulative survival rates of AIDS patients have increased markedly [6][7][8]. As of 2017, 20.9 million patients with HIV had received antiretroviral treatment worldwide. However, metabolic abnormalities, cardiovascular risk factors, and osteoporosis have become important factors affecting the prognosis and quality of life of AIDS patients [9][10][11][12].
HIV infection itself and antiretroviral therapy (ART) treatment drugs can cause dyslipidemia. As a rst-line ART program launched since the National Twelfth Five-Year Plan in China, the tenofovir (TDF) plus lamivudine (3TC) plus efavirenz (EFV) regimen has a weaker effect on lipid metabolism. Our previous study showed that newly diagnosed male AIDS patients had decreased total cholesterol (TC) levels, uric acid (UA) levels and high-density lipoprotein cholesterol (HDL-c) levels as well as increased triglyceride (TG) levels, especially patients with CD4 + counts < 200/µl. The dyslipidemia and decreased UA levels gradually returned to normal at 4 weeks after initial ART with the TDF plus 3TC plus EFV regimen. This speci c regimen has not been studied enough using long-term lipid and uric monitoring, which is the focus of the current study.

Study population
A prospective cohort study was conducted on sixty-one male patients with HIV who were treatment-naive with the TDF plus 3TC plus EFV regimen at the Public and Health Clinic Centre of Chengdu from October 1, 2012, to December 31, 2017.
The inclusion criteria were as follows: age from 18 to 65 years old; gender is not limited; HIV-1 antibody positive according to enzyme-linked immune-sorbent assay and con rmed by Western blotting; CD4 + T cell count 500 µL within 30 days before enrollment; voluntarily signed informed consent and agreed to undergo follow-up analyses; no plan to move away from current address during the trial; no history of antiretroviral therapy.
The following exclusion criteria were used in this study: patients with acute infections; patients with opportunistic infections or AIDS-related malignant tumors at the time of enrollment; patients with opportunistic infection occurring within 3 months before enrollment and still in unstable condition within 2 weeks before enrollment; patients with hemoglobin < 9g/dL, white blood cell count < 2000/µL, neutrophil count < 1000/µL, platelet count < 75000/µL, serum creatinine > 1.5-fold upper limit of the normal value (ULN), aspartate aminotransferase/alanine aminotransferase/alkaline phosphatase > 3-fold ULN, total bilirubin > 2-fold ULN, serum creatine phosphokinase > 2-fold ULN, or creatinine clearance rate < 60 mL/min; women who were pregnant or lactating; current drug users; patients with severe mental or neurological diseases; patients with a history of alcoholism; and patients with severe digestive tract ulcers.
The participants were divided into three groups according to their baseline CD4 T cell counts: there were 26, 12, and 23 patients in the < 200, from 200 to 350 and > 350 cells/µl groups, respectively.

Measurement of anthropometric parameters
The subjects fasted overnight for at least 12 hours. At 8:00 am the next day after emptying stool and urine, anthropometric parameters, including height, body weight (BW), body fat weight, lean body mass weight, body mass index (BMI) and body fat percentage, were measured by specially trained researchers using a body fat measuring instrument.

Detection of laboratory indicators
The subjects fasted overnight for at least 12 hours. At 8:00 am the next day, the venous blood of those patients was drawn to measure TC, LDL-c, HDL-c, TG, UA, HIV viral nucleic acid (HIVRNA), and T lymphocyte subsets.
TC, TG, HDL-c, LDL-c and UA levels were measured by the enzymatic method of an automatic biochemical analyzer purchased from Zhejiang Eastern European Biological Products Company. HIVRNA was detected by uorescent quantitative PCR; T lymphocyte subsets (including CD3 + count, CD4 + count, CD8 + count, CD3+%, CD4+%, CD8+%) were measured by ow cytometry using a Beckman ow cytometer.
Databases were established according to the needs of the research by two researchers simultaneously collecting and entering data. All of the data were checked by the researchers to assess data integrity, authenticity, and accuracy.

Patient and public involvement
Patients and the public were involved in the development of the research question or in the design of the study.
Patients received oral and written information about this study; however, they were not involved in the recruitment and implementation of the study. In addition, the burden of the intervention was assessed by the patients themselves. After signing an informed consent form by the participants, they were assessed for eligibility prior to data collection.

Statistical methods
The Statistical Package for the Social Sciences software version 17.0 (IBM Inc., Armonk, NY, USA) and GraphPad Prism 8 (GraphPad) software were used for statistical analysis. TC, LDL-c, HDL-c, TG and UA levels normally distributed, and statistical analysis was conducted directly. Nonnormally distributed HIVRNA levels were subjected to natural logarithmic transformation before statistical analysis. Quantitative data were expressed as χ±SD, and categorical data were expressed as rates or percentages. One-way ANOVA was used to compare metabolism parameters from baseline to 144 weeks, and a paired t-test was used to compare metabolism parameters between baseline and some follow-up time points. The Kruskal-Wallis H (K) test for K independent samples was used to compare the percentage of dyslipidemia and hyperuricemia from baseline to 144 weeks.
The Mann-Whitney test for two independent samples was used to compare the percentage of dyslipidemia and hyperuricemia between baseline and some follow-up time points. One-way ANOVA was used to compare metabolism parameters between the three different CD4+T cell count groups at the same time point. Two-way ANOVA was used to compare metabolism parameters between the three different CD4+T cell count groups from baseline to 144 weeks. A p value<0.05 was considered statistically signi cant.

Ethical consideration
The study was approved by the hospital ethics committee of the Public and Health Clinic Centre of Chengdu (PJ-K2012-012-01). All patients gave written informed consent.

Baseline conditions
Sixty-one treatment-naive male patients with HIV in the Public and Health Clinic Centre of Chengdu from October 1, 2012, to December 31, 2017, were divided into three groups according to their baseline CD4 + T cell count: there were 26, 12, and 23 patients in the < 200, 200 to 350, and > 350 cell/µl groups, respectively. There were 42 cases of infection through homosexual contact, 13 cases of infection through heterosexual contact and 5 cases involving both types of sexual contact. The general information, baseline immunity and virological indicators, and lipid metabolism parameters of 61 patients are shown in Table 1.  1C) ranged from 21.31% at 12 weeks to 100.00% at 72 weeks.
Long-term dynamic changes in anthropometric parameters after treatment with TDF + 3TC + EFV The body weight ( Fig. 2A) and lean body mass weight (Fig. 2B) of patients did not change signi cantly over the 144 weeks (all P > 0.05). The body mass index (Fig. 2C), body fat weight (Fig. 2D) and body fat percentage Effect of baseline CD4 + cell count on lipid and purine metabolic parameters after treatment with TDF + 3TC +

EFV
The lower the CD4 + cell count at baseline was, the higher the TG levels ( Fig. 6D) and the lower the TC (Fig. 6A), LDL-c (Fig. 6B), HDL-c (Fig. 6C) and UA levels ( Fig. 7) were; moreover, these changes were maintained throughout the follow-up period after ART treatment. However, there was no signi cant difference in the change from baseline to 96 weeks between the three different CD4 + cell count groups (all P > 0.05). The difference of the LDLc levels (Fig. 6B) at baseline was signi cant between the three different CD4 + cell count groups (P = 0.0457). TC levels (Fig. 6A) and HDL-c levels (Fig. 6C) all gradually increased along with prolonged ART regardless of the CD4 + cell count at baseline.

The risk factors of lipid and purine metabolic parameters
According to Spearman correlation analysis, nonalcoholic fatty liver disease, age, body weight, BMI, body fat weight, and body fat percentage were all positively correlated with TC, TG and LDL-c levels; lean body mass weight was positively correlated with LDL-c levels; and follow-up duration was positively correlated with TC and HDL-c levels. In contrast, nonalcoholic fatty liver disease, body weight, BMI, body fat weight, body fat percentage and lean body mass weight were all negatively correlated with HDL-c levels ( Table 2). Based on multiple stepwise regression analysis, the risk factors for TC levels included body weight, age, lean body mass weight and followup duration, for TG levels included body weight, lean body mass weight, BMI and age, for HDL-c levels included BMI, CD3 + CD4 + count and body weight, and for LDL-c levels included BMI and follow-up duration (Table 3).  In addition, nonalcoholic fatty liver disease, body weight, lean body mass weight, CD3 + CD4 + cell count and virus load were all positively correlated with UA levels, while age was negatively correlated with UA levels ( Table 2). Based on multiple stepwise regression analysis, only virus load was a risk factor for UA (Table 3).
In this prospective 3-year follow-up cohort study we assessed baseline CD4 + cell count, age, anthropometric parameters, and immunological and virological indicators impacting on lipid and purine metabolic parameters in male patients with HIV undergoing primary treatment, and found that the lower the baseline CD4 + cell count was, the higher the TG levels were and the lower the TC, LDL-c and HDL-c levels were. The risk factors for lipid and purine metabolic parameters included age, anthropometric parameters, and immunological and virological indicators. After comparisons between different time points over the 144 weeks and comparisons with baseline levels, no signi cant difference in UA levels was found. The lower the baseline CD4 + cell count was, the higher the UA levels were, and these changes were maintained throughout the follow-up period after ART treatment; however, there was no statistical signi cance in the change from baseline to 144 weeks between the three different CD4 + T cell count groups. That is, the TDF + 3TC + EFV regimen and CD4 + T cell count at baseline had no long-term dynamic effects on purine metabolism.This nding was inconsistent with the literature report that a high CD4 + cell count was a risk factor for hypertriglyceridemia, while a CD4 + cell count less than 200 copies/mm 3 increased the risk of hypercholesterolemia [26]. Regardless of whether the initial treatment regimen was based on D4T, the risk of hyperlipidemia in HIV/AIDS patients aged 50 and above was signi cantly higher than that in young HIV/AIDS patients aged under 40 years [31].
To our knowledge, this prospective follow-up cohort study is the rst to investigate this speci c regimen with TDF plus 3TC plus EFV using with long-term lipid and uric acid monitoring and the risk factors for lipid and uric acid.
The results showed that gradual increasing dyslipidemia was found in male patients with human immunode ciency virus primarily treated with tenofovir plus lamivudine plus efavirenz for three years. The risk factors for dyslipidemia included age, anthropometric parameters and follow-up weeks, and for hyperuricemia was virus load. There-fore lipid metabolism parameters should be closely monitored during long-term ART with the TDF plus 3TC plus EFV regimen.
In this cohort study the proportion of patients with abnormal lipid metabolism during the whole study period was less than that reported in the literature. The reasons may be related to the younger age of patients in this cohort, the duration of follow-up was not too long, and the TDF + 3TC + EFV regimen had little effect on lipid metabolism.
The present study had some limitations. The sample size was small, and this was a single-center cohort study that only involved the TDF + 3TC + EFV regimen and male patients, and the duration of follow-up was not too long. Further multicenter studies, more ART regimens, increase the number of female patients and large-sample randomized controlled trials and longer the duration of follow-up are necessary.

Conclusions
Gradual increasing dyslipidemia was found in male patients with human immunode ciency virus primarily treated with tenofovir plus lamivudine plus efavirenz for three years. The risk factors for dyslipidemia included age, anthropometric parameters and follow-up weeks, and for hyperuricemia was virus load. There-fore lipid metabolism parameters should be closely monitored during long-term ART with the TDF plus 3TC plus EFV regimen.

Declarations
Ethics approval and consent to participate The study was approved by the hospital ethics committee of the Public and Health Clinic Centre of Chengdu (PJ-K2012-012-01). All patients gave written informed consent.

Consent for publication
All of participants understand that the information will be published without their child or ward's/their relative's (circle as appropriate) name attached, but that full anonymity cannot be guaranteed. All of participants understand that the text and any pictures or videos published in the article will be freely available on the internet and may be seen by the general public. The pictures, videos and text may also appear on other websites or in print, may be translated into other languages or used for commercial purposes. All of participants have been offered the opportunity to read the manuscript.

Availability of data and materials
All data, models, or code generated or used during the study are available from the corresponding author by request: Dafeng Liu, E-mail: liudf312@126.com

Competing interests
The authors declare that they have no competing interests.  Long-term dynamic changes in anthropometric parameters over 144 weeks after initial ART with TDF plus 3TC plus EFV in treatment-naive patients with HIV (n=61      Long-term effect of baseline CD4+ T cell count on UA level within 96 weeks after initial ART with TDF plus 3TC plus EFV in treatment-naive patients with HIV (n=61;26, 12, 23 cases in<200, from 200 to 350, >350 groups, respectively). Abbreviations: ART, active antiretroviral therapy. TDF, Tenofovir. 3TC, Lamivudine. EFV, Efavirenz.
UA, uric acid. One-way ANOVA was used to compare UA between three groups at the same time point, and Twoway ANOVA was used to compare UA between three groups from baseline to 96 weeks. Comparison between different time points and different CD4+ T cell count groups, all P>0.05.