Effects on mortality of a nutritional intervention for malnourished HIV-infected adults referred for antiretroviral therapy: A randomised controlled trial

Background: Malnourished HIV-infected African adults are at high risk of early mortality after starting antiretroviral therapy (ART). We hypothesized that short-course, high-dose vitamin and mineral supplementation in lipid nutritional supplements would decrease mortality. Methods: The study was an individually-randomised phase III trial conducted in ART clinics in Mwanza, Tanzania, and Lusaka, Zambia. Participants were 1,815 ART-naïve non-pregnant adults with body mass index (BMI) <18.5kg/m2 who were referred for ART based on CD4 count <350 cells/μL or WHO stage 3 or 4 disease. The intervention was a lipid-based nutritional supplement either without (LNS) or with additional vitamins and minerals (LNS-VM), beginning prior to ART initiation; supplement amounts were 30g/day (150kcal) from recruitment until 2weeks after starting ART and 250g/day (1,400kcal) from weeks 2 to 6 after starting ART. The primary outcome was mortality between recruitment and 12weeks of ART. Secondary outcomes were serious adverse events (SAEs) and abnormal electrolytes throughout, and BMI and CD4 count at 12weeks ART. Results: Follow-up for the primary outcome was 91%. Median adherence was 66%. There were 181 deaths in the LNS group (83.7/100 person-years) and 184 (82.6/100 person-years) in the LNS-VM group (rate ratio (RR), 0.99; 95% CI, 0.80-1.21; P = 0.89). The intervention did not affect SAEs or BMI, but decreased the incidence of low serum phosphate (RR, 0.73; 95% CI, 0.55-0.97; P = 0.03) and increased the incidence of high serum potassium (RR, 1.60; 95% CI, 1.19-2.15; P = 0.002) and phosphate (RR, 1.23; 95% CI, 1.10-1.37; P <0.001). Mean CD4 count at 12weeks post-ART was 25 cells/μL (95% CI, 4-46) higher in the LNS-VM compared to the LNS arm (P = 0.02). Conclusions: High-dose vitamin and mineral supplementation in LNS, compared to LNS alone, did not decrease mortality or clinical SAEs in malnourished African adults initiating ART, but improved CD4 count. The higher frequency of elevated serum potassium and phosphate levels suggests high-level electrolyte supplementation for all patients is inadvisable but the addition of micronutrient supplements to ART may provide clinical benefits in these patients. The NUSTART study was a blinded phase III individually-randomised controlled trial comparing in a two-stage protocol vitamin and mineral supplements in a lipid-based nutritional supplement (LNS-VM) with control LNS administered from recruitment at referral for ART until 6 weeks after starting ART. The primary outcome was mortality between recruitment and 12 weeks post-ART initiation. Secondary outcomes presented here are other serious adverse events (SAEs) and BMI, and CD4 count at 12 weeks. The trial was registered on the Pan-African Clinical Trials Register as PACTR201106000300631 (May 31, 2011). The study was conducted from August 2011 to December 2013 at two sites: the National Institute for Medical Research, Mwanza, Tanzania, and the University Teaching Hospital, Lusaka, Zambia. In Mwanza, patients were screened at six peripheral clinics and recruitment was conducted at a research clinic located at the Sekou Toure Regional Hospital. In Lusaka, patients were recruited from six peripheral clinics which referred to the University Teaching Hospital. At both sites, this resulted in patients with a wide range of socioeconomic and nutritional backgrounds. Previous work by our group at both sites confirmed that micronutrient deficiencies were prevalent and that multiple micronutrient supplementation could provide benefits [19-21]. HIV prevalence among adults in the Mwanza region is about 6% [22] and in Lusaka about 20% [23]. In both countries, at the time of the trial, ART was provided free for those with either CD4 lymphocyte count <350 cells/μL or WHO Stage 3 or 4 disease. About a third of patients starting ART in both countries have a BMI <18.5 kg/m2 [3,4]. Inclusion criteria were at least 18 years old, ART-naive (except for standard regimens to prevent maternal-to-child HIV transmission), BMI <18.5 kg/m2, requiring ART as determined by CD4 count <350 cells/μL or stage 3 or 4 disease, willing to undertake intensive ART follow-up in the study clinic, and providing written (or thumbprint if unable to write) informed consent. In the presence of oedema, patients with BMI <20 kg/m2 were considered; BMI was re-measured after loss of oedema, and the patient considered eligible if BMI was <18.5 kg/m2 and ART had not yet been initiated. Exclusion criteria were participation in a potentially conflicting research protocol or self-reported pregnancy. The LNS, made for the trial by Nutriset, Malaunay, France, contained about 60% calories as fat and 10% calories as protein and came in ready-to-eat packets. Within each treatment arm, the intervention products contained the same daily amounts of vitamins and minerals in both treatment stages (Table 1). Control LNS contained vehicle and flavourings similar to vitamin and mineral-enriched LNS (LNS-VM); it contained micronutrients intrinsic to the bulk ingredients but had no added vitamins or minerals. In the first stage, from recruitment to 2 weeks after starting ART, the products were given with minimal calories, i.e., 30 g/day, approximately 150 kcal/day. From 2 to 6 weeks after initiating ART patients were given 250 g/day, in two 125 g sachets, comprising approximately 1,400 kcal/day. In pilot work at the Mwanza site, the LNS products were found acceptable to a similar group of HIV patients [24]. Products were further evaluated by study staff at both African sites to confirm that the intervention and control preparations were acceptable and indistinguishable. Nutritional composition of trial supplements – amounts per day a aWhere nutrient contents are provided for both LNS and LNS-VM, these are values from analysis by the manufacturer, accounting for inter-batch variability; where values for only LNS-VM are given, these where not assessed in the prepared foods but refer to amounts added, that is, they do not include those intrinsic to the LNS. ART, Antiretroviral therapy; LNS, Lipid-based nutritional supplement; LNS-VM, LNS with added vitamins and minerals. The evidence base for micronutrient supplements for adults on ART is extremely limited, therefore, we based levels in the intervention supplement on previous work by the collaborators [8,9,17,25,26], established supplements used for severely malnourished children [7], and recent estimates of requirements for moderately malnourished children [27]. The fundamental basis for the formulation was three times the Recommended Nutrient Intake (RNI) for British women [28], but no iron during the first phase and only the RNI for iron during the second phase. Stability, safety, and micronutrient levels of the supplements were monitored regularly by the manufacturer, including in sachets kept for 18 months at the study sites, and found to be adequate. Randomisation was conducted by the Data Safety and Monitoring Board (DSMB) statistician using computer-generated blocks of 16 and stratified by site. An allocation code (letters A to H) indicating the contents of the supplement packets were known only to Nutriset and the DSMB statistician. A randomisation code linking the allocation code to study ID numbers was held by the DSMB statistician and site-based study pharmacists who did not know the package contents, had no direct contact with patients, and were instructed not to disclose packaging details to the clinical teams. Packages of LNS-VM and LNS, in both small and large dose formats, were delivered by the producer in lots designated by allocation code. Packets were then labelled with the study ID numbers by the clinic pharmacists at the time packets were dispensed. Eligible participants were recruited to sequential IDs (within sites) by clinic nurses with no access to either code. Participants were asked, on exit from the study, if they could guess their treatment. Only 616/1,256 (49%) patients said they could guess, and of these, 83% in both treatment groups guessed they were on the high vitamins and minerals supplement. At each visit, patients were provided with sufficient sachets of LNS or LNS-VM to last until their next scheduled visit. Adherence to both intervention supplements was monitored by asking participants to return empty packages at their next visit. Overall adherence was calculated as total empty packages returned divided by total packages expected to be consumed. When patients died or stopped attending study visits, data on returned packages was missing. In these cases, it was assumed that all of the packages given at their last study visit were consumed. A sensitivity analysis was carried out assuming that none of these packages were consumed. The primary outcome was mortality within the study period which was estimated to include about 4 weeks pre-ART and 12 weeks of ART. Calculations of sample size were based on two studies from urban Africa: one study in Cape Town [29], which separated the high mortality pre-ART phase from mortality after starting ART, and one from Lusaka, with a patient population similar to that expected in the proposed study [3]. We assumed a mortality rate in the control group of 25/100 person-years, based on post-ART mortality of Zambian patients starting ART with a BMI of 17 to 18.49 kg/m2. We calculated that 1,150 patients per treatment arm, with 5% loss to follow-up, would provide >90% power to detect a 50% reduction in mortality, similar to the mortality difference between Zambian patients with plasma phosphate above and below the median [8], and less than the mortality reduction achieved through multiple micronutrient supplementation of Thai patients with low CD4 counts before ART was locally available [25]. It became apparent, by June 2013, that the trial would be unable to recruit all planned participants within the funding available; however, mortality was higher than expected (see Results). The steering group asked the DSMB to consider futility, in addition to a planned interim efficacy analysis, at this point. The DSMB recommended that recruitment could cease. Therefore, recruitment stopped in July 2013 with a total of 1,815 participants which was estimated, considering actual mortality, as sufficient to detect, with 90% power, a reduction in mortality of 30%. Adults attending free HIV testing services at both sites, eligible for ART and fulfilling the study inclusion criteria, were deemed eligible for the trial. Before initiating ART as part of routine care, patients were screened, commenced on treatment for opportunistic infections, and counselled regarding lifelong treatment adherence. During this pre-ART period, the first stage study interventions were introduced. Medical care was provided primarily by local health services, although study staff treated and referred as necessary during the patients’ study visits. National treatment guidelines for ART regimen differed in the two countries: most Zambians were prescribed Efavirenz/Tenofovir/Emtricitabine, whereas a wider range of regimens were prescribed to Tanzanians. Patients were seen weekly from recruitment until the ART initiation visit, then at 2, 4, 6, 8, and 12 weeks after starting ART. Patients who were ill could come for unscheduled visits at any time. Patients were actively followed-up if they missed scheduled visits; patients and relatives were phoned, their ART clinics were contacted, and, in Mwanza, they were visited at home. We were thus able to ascertain the primary outcome, mortality, on a greater number of patients than those attending the week 12 visit for secondary outcomes. The primary outcome was death between recruitment and 12 weeks after starting ART, based either on reports from medical facilities or from relatives. In order to capture serious illnesses which did not result in death, a second outcome was hospitalisation, defined as at least overnight admission to a hospital, including inpatient facilities attached to clinics. Diagnoses and duration of hospitalisation were recorded. SAEs comprised a combination of events that resulted in death, hospitalisation, or permanent disability, or were life-threatening, as well as a drug overdose or cancer. Low serum phosphate and low or high serum potassium levels of US National Institutes of Health Division of AIDS (DAIDS [30]) grade 3 and 4 were considered severe laboratory adverse events. DAIDS does not set high ranges for phosphate but, because we were also interested in potential excesses from supplementation, we classified any above-normal limits as adverse events [31]. Analysis of magnesium levels was also planned, however for technical reasons, results were only available for few patients and did not show much of clinical interest or association with other data, and were therefore omitted. Alanine aminotransferase (ALT) at baseline and 12 weeks after starting ART was investigated in a non-random subset of patients recruited later in the trial following a report of raised ALT associated with high dose micronutrients in a trial of HIV patients in Tanzania [32]. Weight was measured at all visits and height at recruitment if the patient was able to stand; data from the screening visit were used if the patient was unable to stand. Measurements were taken in triplicate and the median used in analyses. Data for the final visit were used for all who attended up to 14 days before or after the official follow-up end date of 12 weeks since starting ART. Venous blood samples were taken at all scheduled visits. CD4 count (baseline and week 12 only) was measured by local central clinical services. In Lusaka, serum phosphate was measured spectrochemically on a Pointe 180 analyser (Bactlabs East Africa, Nairobi, Kenya). Sample results were accepted only from runs for which the external quality control (QC) sample from the same supplier was within expected range. Inter-assay coefficient of variation (CV) for this external QC was 7%. In Mwanza, serum phosphate was measured in an external laboratory (Bugando Medical Centre) using a Roche COBAS Integra 400 analyser. Serum potassium was measured by optical emission using Perkin Elmer Optima 7000 ICP (Perkin Elmer, South Africa). An external QC (Seronorm, Alere, Cheshire, UK) was run each day and values were within expected limits at both sites. CVs for potassium were 5% in Mwanza and 6% in Lusaka. Both sites experienced periods when the Optima machines were out of order so samples were sent to Bugando laboratory in Mwanza and the Centre for Infectious Diseases Research in Zambia (CIDRZ) laboratory in Lusaka. The Pointe 180 was used at both sites to measure ALT. Values for the external QCs (Bactlabs East Africa, Nairobi, Kenya) were very slightly under the expected ranges at both sites; inter-assay CVs were 14% in Lusaka and 10% in Mwanza. Data were double entered into OpenClinica data management system in Lusaka and into CSPro 4.1 and stored in MySQL databases in Mwanza. Analyses were conducted in STATA version 13.1. Primary statistical analysis was by intention-to-treat, and per protocol analyses were also conducted to account for adherence to treatments. The primary analysis compared mortality rates between treatment groups by Cox regression. Robust standard errors Cox regression was performed to determine effects of the intervention on hospitalisation rate and occurrence of clinical and laboratory SAEs accounting for repeat events. Proportional hazards assumptions were examined using cumulative hazard plots and Schoenfeld residuals [33]. Mean BMI and CD4 at 12 weeks were compared between groups using t-tests and linear regression, adjusting for baseline values. Pre-planned analyses determined whether treatment effects were modified by country, sex, initial BMI (< or ≥17 kg/m2), initial CD4 count (< or ≥100 cells/μL), or TB treatment pre-ART using Cox regression with an interaction term between treatment arm and potential effect modifier. Principal components analysis, used to describe the socio-economic status of the population [34], was conducted separately for each country since there were notable differences between sites; variables offered into the principal components analysis were housing characteristics, sanitation, water source, and ownership of electrical goods, animals, and modes of transport. Ethical approval was obtained from the research ethics committee of the London School of Hygiene and Tropical Medicine, the University of Zambia Biomedical Research Ethics Committee, and the Medical Research Coordinating Committee of National Institute for Medical Research, Tanzania. All participants provided written or thumbprint informed consent. Medical care of patients was according to national guidelines and provided through the local health services. Patients with low levels of serum electrolytes according to DAIDS criteria were provided appropriate electrolyte therapy.