Effect of atorvastatin on subclinical atherosclerosis in virally-suppressed HIV-infected patients with CMV seropositivity: a randomized double-blind placebo-controlled trial

Participants and interventions

This study uses randomized control trial in which subjects are assigned to a double blind randomized with placebo-controlled clinical trial study with atorvastatin 20 mg in virally-suppressed HIV-infected patients.

Study setting and sample size

The study is taking place in a single center, the HIV integrated clinic Cipto Mangunkusumo Hospital, Jakarta. All patients receive government free antiretroviral program according to Indonesian guidelines. The first-line regimen consists of two nucleoside reverse transciptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI), and the second-line regimen of two NRTIs and one protease inhibitor (PI). The majority of patients belong to urban and mainly low- to middle-income families living in Jakarta. Assuming that the effect size is 0.1, standard deviation is 0.12, and the use of 2 sample t-test, the sample size needed for 90% power and 5% significant level is 32 per arm. With an additional 20% drop out rate, each arm (placebo and control group) will need 40 participants.

Eligibility criteria and recruitment

Patients, men and women aged between 20 to 45 years old, using stable ART (no adverse drug reactions, no pregnancy or current illnesses, understand lifelong adherence, and evidence of successful treatment) for at least a year with positive CMV IgG and viral load of HIV RNA <50 copies/mL are recruited to the study. Viral load, anti-CMV antibody, and lipid panel tests are done before defining the eligibility criteria. Participants are also required to not use any statin therapy 6 weeks before enrollment. Patients with the following conditions are excluded from the study:

Interventions and participant timeline

All potential participants receive an explanation about the purpose and method of the study and are informed of their rights to stop or refuse to take part in the study. A special mobile number is available for potential participants to obtain additional information should they wish. All potential participants are contacted again to schedule an appointment if they agree to continue with all of the procedures. After screening is completed, they will be interviewed and examined at the clinic area, which includes CIMT, FMD, and neurocognitive evaluation. The oral examination is conducted at the dentistry clinic and the transient elastography examination will be conducted at the hepatology clinic. Figure 1 presents a CONSORT diagram of the anticipated number of participants needed to be screened in order to reach a target of 80 participants randomized to atorvastatin or placebo.

Figure 1. Study trial flow diagram.

CONSORT diagram of the anticipated number of participants that need to be screened in order to reach a target of 80 participants randomized to atorvastatin or placebo.

Participants receive either 48 weeks of 20 mg atorvastatin or placebo once daily. We use oral atorvastatin 20 mg capsule in generic form and a placebo capsule prepared by the hospital pharmacist, composed of starch which is similar to atorvastatin capsule in size, shape, and color. Packagings are identical in appearance for both atorvastatin and the placebo. CMV = cytomegalovirus; DAA = direct-acting antiviral; LDL = low density lipid; CIMT = carotid intima media thickness; FMD = flow mediated vasodilatation; CAP = controlled attenuation parameter.

Study outcomes

The primary aim of this study is to compare the effect of 48 weeks of 20 mg daily atorvastatin versus placebo on CIMT in virally suppressed HIV patients with CMV seropositivity. Other aims are to assess:

Assignment of interventions

Participants who meet the inclusion criteria are randomized into atorvastatin groups and placebo groups with a ratio of 1:1 (40 subjects per group). Randomization using STATA version 14.2 based on randomization of permutation blocks with block 4 sizes is carried out by statistical analysts. The administration of study drug is home-based participant self-administration. Participants are asked to swallow the whole capsule without chewing or breaking it. The participants get the medication supply every month along with the prescription of antiretroviral drugs.

Masking

The code of each participant is given to pharmacists who are involved in the study. The pharmacists keep the code in the sealed envelope. Researchers, doctors, and participants do not know the results of randomization. The drug and placebo capsules are administered to patients by a staff member who is privy to the treatment. Each participant should return the unused capsules every month. At the end of the study, the pharmacist and related staff members will count the Medication Possession Ratio (MPR). The study will be discontinued if:

Data collection and management

Subjects who meet eligibility criteria are enrolled and have a baseline visit (week 0) within 6 weeks following the screening visit. Venous blood sample are collected for fasting blood glucose, ALT, CMV DNA, hsCRP, sCD14, VCAM, ICAM, and β2M examinations.

Each patient has a CIMT and FMD evaluation performed by a trained cardiologist using B mode ultrasonography. CIMT is defined as a double-line pattern visualized by echo 2D on both walls of the common carotid artery (CCA) in a longitudinal view. Two parallel lines (leading edges of two anatomical boundaries) form it: the lumen-intima and media-adventitia interfaces. High-resolution B-mode ultrasound images of the right and left common carotid artery will be used to measure carotid intima-media thickness. The patient is scanned in the supine position using 7–12 MHz linear array transducer (Philips Affiniti 70C, Phillips, UK). CIMT is calculated using both manual and semi-automated (Automated; QLAB, Phillips, UK) methods. Manual CIMT measurements are recorded from the far wall at 1 cm, 1.5 cm, and 2 cm intervals proximal to the carotid bulb. Automated measurements are also recorded from the far wall, using the same image, from the identical 1 cm section proximal to the carotid bulb. The carotid bulb is defined as the point where the far wall deviated from the parallel plane of the distal CCA. Mean manual and automated cIMT measurements for the right and left CCA are calculated from three consecutive cardiac cycles.

FMD is assessed in the brachial artery by ultrasonography. The participant has to abstain from exercise (≥12 hr), caffeine (≥12 hr), smoking or smoke exposure (≥12 hr), vitamin supplementation (>72 hr), and any medication (≥4 hr half-lives of the drug, non-steroidal anti-inflammatory agents for 1 day and aspirins for 3 days). We ensure that the participant is under fasting conditions or has only consumed low-fat meals. The measurements are performed on the right arm when the patient is in the supine position, after 10 to 20 minutes of rest in the supine position^36,37 The brachial artery is scanned longitudinally just above the antecubital crease with a linear multifrequency 5- to 12-MHz transducer B-mode. The diameter of the brachial artery is measured on the interface between media and adventitia of the anterior and posterior wall as a baseline. Gain settings are optimized to identify the lumen and the vessel wall interfaces and could not be modified during the examination. Hyperemia is induced by inflation of a pneumatic cuff of 200-250 mm Hg for 5 minutes on the most proximal portion of the forearm. Arterial diameter measurement is repeated just before (30-60 seconds) deflation of the cuff, then just after (30 to 60 seconds) sudden deflation of the cuff. Tracings is recorded on image and file and are read by two investigators (MS, LR) who are unaware of the participant’s clinical data. The measurements of basal and posthyperemia diameter will be used for the analysis. Flow-mediated vasodilation is expressed as the relative increase in brachial artery diameter during hyperemia and defined as 100× [(posthyperemia diameter−basal diameter)/basal diameter].

Transient elastography with CAP will be done by trained hepatologist using Fibroscan (Echosens®) device. The criteria for successful examination are 10 shots and an interquartile range (IQR) for liver stiffness of less than 20% of the median value. The cut-off value for NAFLD diagnosis is CAP measurement of above 238 dB/m and/or fibrosis score higher than 7.1 kPa³⁸.

A trained neurologist performs the neurocognitive evaluation. The cognitive tests are performed in week 0, week 24, and week 48. This study uses tests that are sensitive to domain most affected in HIV but can be delivered in relatively short time. This study uses Trail Making Test (TMT) A and B to measure executive function, Symbol Digit Modalities Test (SDMT) for speed of information processing, and Brief Visuospatial Memory Test Revised for learning and memory. These tests have already been validated in Indonesia and can be administered by non-neuropsychologists who have been trained³⁹. To determine cognitive impairment, we use local normative data that match age and education level.

A trained dentist will assess periodontitis using CPI, which was modified by the World Health Organization (WHO). This assessment uses a 0.5 mm ball tip periodontal probe with black band markers at 3.5, 5.5, 8.5, and 11.5 mm, and involve 10 teeth (17, 16, 11, 26, 27, 37, 36, 31, 46, and 47). The score ranges from 0 for healthy periodontal to score 4 for periodontal pocket ≥6 mm^40,41. Eligibility screen, informed consent, and physical examinations will be done within a week before taking study drugs. All other examinations will be done just before taking study drugs (week 0) and at the end of this study (week 48), except for neurocognitive and blood collection, which are done in baseline, in week 24, and at the end of this study in week 48, as seen in Table 1.

At the end study, a total drug consumption adherence will be assessed using MPR. Meanwhile, each patient has monthly clinical follow-ups as standard of care in the clinic. Study follow-up visits include clinical and laboratory procedures as presented in Table 1. MPR is the sum of the days’ supply for all fills of a given drug in a particular time period, divided by the number of days in the time period⁴².

Data management are managed in the HIV Integrated Care Unit, Cipto Mangunkusumo Hospital. All research staff have overall responsibility for maintaining the study CRFs. Corrections to the paper CRF documents must be made by striking through the incorrect entry with a single line (taking care not to obliterate or render the original entry illegible) and entering the correct information adjacent to the incorrect entry. Corrections to paper CRFs must be initialed and dated by the person making the correction. The researchers are responsible for maintaining accurate, complete, and up-to-date records. These forms are to be completed on an on-going basis during the course of the study.

Statistical analysis

Statistical analyses will be performed with SPSS (version 21). Subjects will be analyzed in the group to which they are randomized, regardless of the treatment received. A T test will be used to compare the mean CIMT changes of virally supressed HIV patients with CMV seropositivity in the atorvastatin group versus the placebo group between the baseline and week 48 to analyze primary endpoints. Meanwhile, secondary endpoints will be analyzed using linear regression with interaction to assess the magnitude of change in the effect of atorvastatin on CIMT associated with the baseline CMV copy number, and a proportional odds logistic regression model will be used to estimate the odds ratios of CPI in treating an increased level of CIMT. Other continuous endpoints will be analyzed using a 2 sample T-test. Missing data will be resolved by comparing subject characteristics in the drop out group with the remaining participants. If there is no difference in characteristics it would assumed that the missing data is at random.

Monitoring

The study is to be monitored by Cipto Mangunkusumo Hospital Research Unit. Research records for all study subjects including CRFs, laboratory, and other investigation data/results are to be maintained by the investigator in secure storage for five years. These records are to be maintained in compliance with all designated IRBs requirements. It is the investigator’s responsibility to retain copies of these study records until notified in that they can be destroyed. Any severe adverse event or unintended effects of trial intervention will be reported and assessed by the ethics committee.

Patient and public involvement

Participants begin involvement in the trial when the eligibility criteria are fulfilled and screening examinations are surpassed. Clear information about the trial, including participant’s role, is explained by study team and is written on informed consent form. Each participant has his/her right to decline or withdraw from the trial. The participant also has the right to receive any information regarding test result done during the trial. One of the participants and experienced team member are given the role of patient recruitment.

Ethics

This trial has been approved by Health Research Ethics Commitee–Universitas Indonesia and Cipto Mangunkusumo Hospital (HREC-FMUI/CMH) and registered in ClinicalTrials.gov (identification number 19-03-0272). Any amendments or modification regarding the protocol during the trial will be consulted and notified to HREC-FMUI/CMH. All participants are required to give their consent before joining the study. The consent form is written in Indonesian and is composed of the following details: participant’s name, date of birth, investigator contact, and explanation of the study purpose that is easily understood by non-medical personnel, course and duration of the study (as well as the freedom to withdraw at any time and guarantee of confidentiality), details of invasive procedures involved (blood collection), an explanation of blood remnant storage, the type of intervention (atorvastatin vs placebo), and possible side effects. The details of the informed consent form are explained by on-site research staff. Blood collections are collected by professional health workers and processed by lab analysts. The study team receives training on Good Clinical Practices (GCP) to ensure that sensitive research activities will be handled appropriately. The study maintains records of adverse events, as well as copies of the consent forms. All records are placed in a locked filing cabinet at the clinic that is only accessible by the research team. All team members are responsible for data security and record keeping. The datasets that will be used for analysis do not contain any identifying information, specifically, no names of participants are included in the datasets. Identifiers for the participants will be disposed of no more than three years after study completion. To protect confidentiality, identifiers are only accessible by the PI or project coordinator and are kept separated from other records with participants’ responses.

Participants presenting complaints or adverse event related to the trial will have an extra visit to medical personnel.

Dissemination plan

At the end of study analysis, the trial results will submitted and published in peer-reviewed journals. Links of publication will be provided in the applicable trial register. Authorship for all publications will be based on the criteria defined by the International Committee of Medical Journal Editors.