Gradually increased dyslipidemia in human immunodeciency virus infected male patients with tenofovir plus lamivudine plus efavirenz primary treatment: a 3-year follow-up study

Since the start of highly active antiretroviral therapy (HAART) with TDF plus 3TC plus EFV, the long-term dynamic characteristics of lipid and purine metabolism in patients infected with human immunodeciency virus (HIV) was unclear and worth studying. Methods A prospective follow-up cohort study was the way. Sixty-one treatment-naive HIV infected male patients were divided into three groups based on the baseline CD4 + T cell count (26, 12, 23cases in < 200, from 200 to 350, > 350 three groups, respectively). Lipid and purine metabolism parameters of those patients within 144 weeks were analyzed. Result TG, TC, LDL-c and HDL-c level all gradually increased within 144 weeks, but statistical signicances of TC level and HDL-c level were only found (F = 4.214, 5.518, P = 0.001, 0.000 ,respectively). Moreover the percentage of hypercholesterolemia, hyper LDL cholesterolemia and hypertriglyceridemia all gradually increased, low HDL cholesterolemia gradually decreased, but there was only obvious difference of the latter (χ 2 = 16.105, P = 0.0007).Furthermore the lower the baseline CD4 + T lymphocyte counts, the higher TG level, the lower TC level, LDL-c level and HDL-c level, but only signicant difference of LDL-c level between three groups at baseline was found (F = 3.256,P = 0.0457).Although UA level and the percentages of hyperuricemia gradually increased within 144 weeks, but there was no signicant difference between different follow-up time points groups and between three CD4 + T cell count groups (all P (cid:0) 0.05). These ndings provide a reference for clinicians to monitor lipid metabolism parameters closely during long-term HAART with TDF plus 3TC plus EFV identifying the long-term dynamic characteristics of lipid and purine metabolism and its inuencing factors after HAART with TDF + 3TC + EFV. To our knowledge, this prospective 3-year follow-up cohort study was the rst to report the long-term dynamic effects of TDF + 3TC + EFV regimen and baseline CD4 + T cell count on lipid and purine metabolic parameters in HIV-infected male patients. results showed that in patients treated with TDF + 3TC + EFV regimen for 3 years, TC level, LDL-c level and HDL-c level gradually increased, especially TC level and HDL-c level, TG level rst gradually decreased and then gradually increased, moreover the percentage of hypercholesterolemia, hyper LDL cholesterolemia and hypertriglyceridemia all gradually increased, while the percentage of low HDL cholesterolemia gradually decreased. It shows that in the early stage of HAART disordered lipid metabolism was improved, low HDL cholesterolemia. But along with prolonged HAART, the proportion of hyperlipidemia and hypertriglyceridemia gradually LDL cholesterolemia hypertriglyceridemia after weeks, 21.43%,


Introduction
In recent years, immune-de ciency virus (HIV) infected and acquired immune de ciency syndrome (AIDS) patients has sharply increased. By the end of 2019 all over the world about 38 million people lived with HIV and 33 million people died of HIV-related diseases[1], at the end of October 2019 nationwide in China a total of 958,000 patients [2,3] lived with HIV,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 life quality [4,5].When CD4 + T 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 obviously increased [6][7][8]. As of 2017, 20.9 million HIV infected patients have received antiretroviral treatment all over the world. But metabolic abnormalities, cardiovascular risk factors, and osteoporosis have become important factors affecting the prognosis and life quality of AIDS patients [9][10][11][12].
HIV virus infection itself and HAART treatment drugs can cause dyslipidemia. But as a rst-line highly active antiretroviral therapy (HAART) program launched since the National Twelfth Five-Year Plan in China, Tenofovir (TDF) plus lamivudine (3TC)plus efavirenz (EFV) regimen has less effect on lipid metabolism, there are few reports about it in the literature. Our previous study has shown that newly diagnosed AIDS male patients had decreased total cholesterol (TC) level, uric acid (UA) level [13,14], highdensity lipoprotein cholesterol (HDL-c) level and increased triglyceride (TG) level, especially those with CD4 + count < 200/u1, this dyslipidemia and decreased UA level gradually returned to normal after 4 weeks since initial HAART with TDF plus 3TC plus EFV regimen [13,14]. But the long-term dynamic characteristicso ipid and purinemetabolism in those patients was unclear and worth being identi edin this study.

Study Population
A prospective cohort study was conducted on sixty-one male patients with HIV infected and treatmentnaive in the Public and Health Clinic Centre of Chengdu from October 1, 2012 to December 31, 2017.Among them the rst 50 cases have been involved in the literature [13][14][15]. Study was approved by the hospital ethics committee of the Public and Health Clinic Centre of Chengdu. All patients gave written informed consent.
The inclusion criteria were as follows: Age from 18 to 65 years old; Gender is not limited; HIV-1 antibody positive by enzyme-linked immune-sorbent assay test, and con rm by Western blotting;CD4 + T cell count 500uL within 30 days before enrollment; Sign informed consent voluntarily and guarantee to receive follow-up; No plan to move away from current address during the trial; subjects have not received any antiretroviral therapy before the trial.
The following exclusion criteria were used in this study: patients with acute infections; with opportunistic infections or AIDS-related malignant tumors at the time of enrollment; or an opportunistic infection occurred within 3 months before enrollment, and the condition is still unstable within 2 weeks before enrollment; the following results were detected during screening: hemoglobin < 9 g/dL, white blood cell count < 2000/uL, neutrophilscount < 1000/uL, platelet count < 75000/uL, 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;creatinine clearance rate < 60 mL/min;women during pregnancy and lactation; current drug users; with severe mental and neurological diseases;with a history of alcoholism who cannot be terminated; severe digestive tract ulcers.
The participants were divided in three groups according to the baseline CD4 T cell counts (26,12,23 cases in < 200, from 200 to 350 and > 350 cells/ul three groups, respectively).

Detection of Laboratory indicators
The subjects were fasted overnight for at least 12 hours. At 8:00 am the next day venous blood of those patients was drawn for total cholesterol (TC), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-c), triglyceride (TG), uric acid (UA), HIV viral nucleic acid (HIVRNA), T lymphocyte subsets.
Databases were established according to the needs of the research by two researchers simultaneously collecting and entering. About 30% of the data was randomly selected by the researchers to ensure data integrity, authenticity, and accuracy.

Patient and Public Involvement
Patients and public was involved in the development of the research question or in the design of the study. Patients will receive oral and written information about this trial, however, they will not be involved in the recruitment and conduct of the study. Besides, the burden of the intervention will be assessed by patients themselves. After signing an informed consent by the participants, they will be assessed for eligibility and data collection will begin. Dissemination of the general results (no personal data) will be made on demand.

Statistical Method
The Statistical Package for the Social Sciences software version 17.0 (IBM Inc., Armonk, NY, the USA) and GraphPad Prism 8 (GraphPad) software were used for statistical analysis. TC level, LDL-c level, HDL-c level, TG level and UA level had a normal distribution, and statistical analysis was conducted directly. Non-normally distributed HIVRNA level was performed naturallogarithmic trans-formation before statistical analysis. The measurement data were expressed as x ± SD for metrological data, rate or percentage for enumeration data. ONE 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 point. Kruskal-Wallis H (K) test for K independent samples was used to compare the percentage of dyslipidemia and hyperuricemia from baseline to 144 weeks. Mann-Whitney test for two independent samples was used to compare the percentage of dyslipidemia and hyperuricemia between baseline and some follow-up time point. ONE ANOVA was used to compare metabolism parameters between three different CD4 + T cell count groups at same time point. TWO ANOVA was used to compare metabolism parameters between three different CD4 + T cell count groups from baseline to 144 weeks. A p value < 0.05 was considered statistically signi cant.

Baseline conditions
Sixty-one treatment-naive HIV infected male patients in the Public and Health Clinic Centre of Chengdu from October 1, 2012 to Decmenber 31, 2017, were divided into three groups according to the baseline CD4 + T cell count (26,12,23 Table 1. In 61 patients the average CD4 + T cell count (Fig. 1A) gradually increase from 319.80cell/ul at baseline to 464.85cell/ul at 96 weeks after HAART, and the average viral load (Fig. 1B) decreased rapidly from 49846.32 IU/ml at baseline to reaching undetectable levels by the high-precision detection method at 72 weeks, and the percentage of viral load reaching undetectable levels (Fig. 1C) was from 21.31% at 12 weeks to100.00% at 72 weeks.

Effect of baseline CD4 + T cell count on lipid and purinemetabolic parameters after treatment with TDF + 3TC + EFV
The lower the CD4 + T cell count at baseline, the higher the TG level (Fig. 5D), and the lower the TC level (Fig. 5A), LDL-c level (Fig. 5B), HDL-c level (Fig. 5C) and UA level (Fig. 6), and the changes were maintained throughout the follow-up period after HAART treatment. However, there was no statistical signi cance from baseline to 96 weeks between three different CD4 + T cell count groups ( all P > 0.05).
In this prospective cohort study we aimed at identifying the long-term dynamic characteristics of lipid and purine metabolism and its in uencing factors after HAART with TDF + 3TC + EFV. To our knowledge, this prospective 3-year follow-up cohort study was the rst to report the long-term dynamic effects of TDF + 3TC + EFV regimen and baseline CD4 + T cell count on lipid and purine metabolic parameters in HIVinfected primary treatment male patients. The results showed that in patients treated with TDF + 3TC + EFV regimen for 3 years, TC level, LDL-c level and HDL-c level gradually increased, especially TC level and HDL-c level, TG level rst gradually decreased and then gradually increased, moreover the percentage of hypercholesterolemia, hyper LDL cholesterolemia and hypertriglyceridemia all gradually increased, while the percentage of low HDL cholesterolemia gradually decreased. It shows that in the early stage of HAART treatment disordered lipid metabolism was improved, espectially low HDL cholesterolemia. But along with prolonged HAART, the proportion of hyperlipidemia and hypertriglyceridemia gradually increased, espectially hyper LDL cholesterolemia and hypertriglyceridemia after 48 weeks, hypercholesterolemia after 120 weeks, which were from 9.84%, 8.2%, 24.59% at baseline to 21.43%, 21.43%, 50.00% at 144 weeks, respectively.
This study was a cohort study with a follow-up time of 144 weeks. 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 metabolism. It is still necessary to expand the sample size, increase the number of female cases, extend the follow-up time and increase other HAART regimens for further discussion.
Previous study found that high CD4 + cell count was a risk factor for hypertriglyceridemia, while less than 200copies/mm 3 CD4 + cell count increased the risk of hypercholesterolemia [29]. Regardless of whether the initial treatment regimen or based on D4T or not, the risk of hyperlipidemia in HIV/AIDS patients aged 50 and above is signi cantly higher than that in young HIV/AIDS patients aged under 40 [33]. In this prospective 3-year follow-up cohort study the long-term effect of baseline CD4 + T lymphocyte count on lipid parameters was that the lower the baseline CD4 + T lymphocyte count, not only the higher TG level, but also the lower TC level, LDL-c level and HDL-c level.
Literature reported that of patients lived with HIV for mean duration 17.4 years, 35.6% had ASCVD, of those without ASCVD 53-86% had intermediate or moderate-to-high 10-year ASCVD risk scores, the cardiovascular risk factors including HIV, 31.9% low high-density lipoprotein cholesterol, 79.3% needed to receive statin therapy [34].
Comparison between different time points within 144 weeks and compared with baseline, no signi cant difference of UA level was found. Although the lower the CD4 + T cell count at baseline, the higher the UA level, and the changes were maintained throughout the follow-up period after HAART treatment, but there was also no statistical signi cance from baseline to 144 weeks between three different CD4 + T cell count groups. That is to say that TDF + 3TC + EFV regimen and CD4 + T cell count at baseline had no long-term dynamic effects on purine metabolism.
The present study had some limitations. The sample size was small, and this was a single-center, cohort study which only involved TDF + 3TC + EFV regimen. A further multicenter, more HAART regimens involved, large sample randomized controlled trial is needed.     Long-term effect of baseline CD4+ T cell count on lipid metabolism parameters within 96 weeks after initial highly active antiretroviral therapy with TDF plus 3TC plus EFV in treatment-naive HIV infected patients(n=61;26, 12, 23 cases in<200, from 200 to 350, >350 groups, respectively).ONE ANOVA was used to compare lipid metabolism parameters between three groups at same time point and TWO ANOVA was used to compare lipid metabolism parameters between three groups from baseline to 96 weeks. A. TC  Long-term effect of baseline CD4+ T cell count on UA level within 96 weeks after initial highly active antiretroviral therapy with TDF plus 3TC plus EFV in treatment-naive HIV infected patients(n=61;26, 12, 23 cases in<200, from 200 to 350, >350 groups, respectively).ONE ANOVA was used to compare UA between three groups at same time point and TWO ANOVA was used to compare UA between three groups from baseline to 96 weeks. Abbreviations: TDF, Tenofovir. 3TC, Lamivudine. EFV, Efavirenz. UA, uric acid. Comparison between different time points and different CD4+ T cell count groups, all P>0.05.