Study design and setting

The study was a double-blind, randomized controlled trial conducted at Mbarara Regional Referral Hospital’s (MRRH) HIV clinic in Uganda from December 2017 to August 2020. MRRH with 350 bed capacity is located in Mbarara City, Southwestern Uganda. Every day, the hospital receives 1,200-1,500 patients including 300 HIV-positive patients from different tribal and socio-demographic locations in Uganda. The hospital was chosen as a suitable site for this study because it provides care and services to HIV- positive patients and is equipped with trained staff, nurses, counselors, physicians, pharmacists, and lab technicians.

Recruitment of the study participants

The study involved HIV patients aged 18–66 years who had been on ART for at least one year. A Research assistant screened HIV-positive patients on ART at MRRH HIV Clinic, to identify those aged 18 years plus, with a CD4 count ≤ 350 cells/µl, living within 60 km from the clinic, having normal hematological, liver and renal function tests, and able to sign the informed consent. The study excluded pregnant women, those using other herbal or supplementary medicines, and those with pre-existing opportunistic infections. Research assistants obtained informed consent from all participants.

Randomization, blinding, and preparation of treatments for the study participants

Participants were first stratified according to baseline CD4 levels: 350–250, 249–150, and ≤ 149 before randomization to the three groups. Using computer-generated numbers supplied by an independent biostatistician, the block randomization method was used to assign eligible participants to one of the three study groups: ART alone as the control group (CG), ART with A. annua group (AG), or ART with A. annua + M. oleifera group (AMG) at a 1:1:1 ratio. The study involved two herbal treatments, A. annua and A. annua + M. oleifera, with participants and healthcare providers blinded to the specific treatment names. This was intended to avoid bias during the study. Both intervention herbal materials underwent quality tests before use by the participants.

Study procedures

Research assistants received opaque parcels already fixed with study codes. These parcels contained either 4 g of A. annua and 10 g of M. oleifera which were to be taken at least 8 h apart from each of the daily ARVs dosing. All participants were encouraged to take note and report all side effects.

Preparation of study materials

Leaf powders from Ugandan plants, A. annua and M. oleifera, were authenticated by a botanist and stored in a herbarium located at Mbarara University of Science and Technology (MUST). The study pharmacist prepared and packaged A. annua and M. oleifera leaf powders in 4 g and 10 g packets, respectively, adhering to good manufacturing practices.

Administration of treatments

Participants in the AG self-administered 4 g of A. annua leaf powder daily at 8 am, consumed in porridge or water, 8 h apart from their routine ARVs dosing, for 12 months. Participants in the AMG consumed 4 g of A. annua and 10 g of M. oleifera powders, mixed with porridge or water daily at 8 am, 8 h apart from their routine ARVs dosing, for 12 months. Participants in the control group received a placebo made by mixing cornstarch powder with food color, in addition to ART. Both the treatment herbs and the placebo were identically packaged and presented similarly.

Participants received one-month herbal treatments in parcels with study numbers, dosing instructions, and other safety information, including storage and safety details. Participants were reminded by an SMS to take their herbal medicine every morning between 7 and 8 am and were asked to respond with a message or call prompt. The study team collaborated with the HIV clinic to ensure that participants received regular HIV care, including ART, as prescribed. Participants were informed that herbal medicines are not replacements for ART in HIV treatment, and they were advised to continue taking prescribed ARVs as usual and correctly during the consenting process.

The study followed participants for 12 months and they were reviewed monthly by the study clinician.

Study measurements

Blood samples were collected post-enrollment but before initiation of herbal treatments, and six and twelve months after treatment initiation to measure CD4 count, viral load, complete blood count, and liver and renal enzymes. A blood sample was taken at 1- and 2-weeks post-treatment to evaluate ARV plasma levels.

Study outcomes

As our primary outcome, the study measured changes in absolute and relative CD4 counts after twelve months of follow-up, comparing intervention groups with control. Secondary outcomes included viral load changes, CBC, and antiretroviral plasma levels.

The study collected qualitative data on socio-demographics, herbal usage, depression, health [16], food insecurity [19], alcohol use [20], HIV stigma [21], and social support from study participants at enrolment.

Study sample size and statistical analysis

The mean CD4 count of HIV-positive patients aged 18+, taking ART, and maintaining a mean CD4 count of ≤ 350 cells/µl for over a year at MRRH was 160 ± 110. With that mean, a sample size of 282 participants—94 in each of the three arms—was required to detect a 30% rise in CD4 counts with 80% power at 0.05 alpha, assuming a 10% loss to follow-up.

All data were cross-checked for completeness before entry into STATA Version 12 for statistical analysis, using both intention-to-treat [22] and per-protocol methods. The chi-square test statistic was used to analyze categorical data while the Wilcoxon rank-sum (Mann-Whitney) test was used to compare means between the intervention groups and the control for both the primary and secondary outcomes. The per-protocol analysis analyzed participant follow-up time and compared mean changes in CD4 count over the same follow-up time of measurements. Linear regression analysis was used to determine the impact of demographic and anthropometric parameters on the primary outcome. In all analyses, statistical significance was considered at a p-value less than 0.05. The same analysis plan was used for secondary outcomes.

Ethical approval

The study was reviewed and approved by the MUST Research Ethics Committee (with reference number 27/05–17) and the Uganda National Council for Science and Technology (with reference number NCT03366922). The study adhered to the Helsinki Declaration’s ethical guidelines and kept all participant identities confidential, ensuring all informed consent was obtained from all participants. All treatments administered to participants have previously been documented to be safe when used for prophylaxis or as a supplement [14, 23].

Data safety and monitoring

The data and safety monitoring committee was constituted and included senior medical scientists to ensure the safety of participating individuals. Three safety checks were done at 1 month, at 50%, and 75% of recruitment.