Effects on body composition and handgrip strength of a nutritional intervention for malnourished HIV-infected adults referred for antiretroviral therapy: A randomised controlled trial

Lipid-based nutrient supplements (LNS) may be beneficial for malnourished HIV-infected patients starting antiretroviral therapy (ART). We assessed the effect of adding vitamins and minerals to LNS on body composition and handgrip strength during ART initiation. ART-eligible HIV-infected patients with BMI <18·5 kg/m 2 were randomised to LNS or LNS with added high-dose vitamins and minerals (LNS-VM) from referral for ART to 6 weeks post-ART and followed up until 12 weeks. Body composition by bioelectrical impedance analysis (BIA), deuterium ( 2 H) diluted water (D 2 O) and air displacement plethysmography (ADP), and handgrip strength were determined at baseline and at 6 and 12 weeks post-ART, and effects of LNS-VM v. LNS at 6 and 12 weeks investigated. BIA data were available for 1461, D 2 O data for 479, ADP data for 498 and handgrip strength data for 1752 patients. Fat mass tended to be lower, and fat-free mass correspondingly higher, by BIA than by ADP or D 2 O. At 6 weeks post-ART, LNS-VM led to a higher regain of BIA-assessed fat mass (0·4 (95 % CI 0·05, 0·8) kg), but not fat-free mass, and a borderline significant increase in handgrip strength (0·72 (95 % CI -0·03, 1·5) kg). These effects were not sustained at 12 weeks. Similar effects as for BIA were seen using ADP or D 2 O but no differences reached statistical significance. In conclusion, LNS-VM led to a higher regain of fat mass at 6 weeks and to a borderline significant beneficial effect on handgrip strength. Further research is needed to determine appropriate timing and supplement composition to optimise nutritional interventions in malnourished HIV patients. The study was conducted from August 2011 to December 2013 at the National Institute for Medical Research (NIMR), Mwanza, Tanzania and the University Teaching Hospital (UTH), Lusaka, Zambia. In Mwanza HIV-infected patients were screened at six peripheral ART clinics and recruitment was conducted at a research clinic located at the Sekou-Toure Regional Hospital. In Lusaka patients were recruited from six peripheral ART clinics and were referred to UTH for enrolment. HIV diagnosis and treatment followed local national guidelines(,19,20). The trial was registered at the Pan African Clinical Trials Registry as PACTR201106000300631 (http://www.pactr.org). HIV-infected patients who were being referred for ART in Mwanza and Lusaka were included in the trial if they met the following criteria: age 18 years and above, ART-naive (except for standard short-course regimens to prevent maternal-to-child HIV transmission), undernourished (BMI <18·5 kg/m2), eligible for ART according to national criteria at the time (CD4 count <350 cells/μl or had WHO stage 3 or 4 disease), willing to undertake intensive ART follow-up in the study clinic, and provided written informed consent. Patients were not invited into the trial if they were participating in a similar study or were pregnant by self-report. The NUSTART study was a phase III randomised controlled trial comparing in a two-stage protocol LNS-VM (intervention) v. LNS (control) given from recruitment at referral for ART until 6 weeks after starting ART(,16). In the first stage, from recruitment to 2 weeks after starting ART, participants in the control or treatment intervention were given 30 g/d LNS or LNS-VM, about 150 kcal (630 kJ)/d, and in the second stage, from 2–6 weeks after initiating ART, participants were given 250 g/d LNS or LNS-VM, about 1400 kcal (5860 kJ)/d. The LNS was manufactured for the trial by Nutriset (Malaunay, France) and came in ready-to-eat packets. Due to high nutrient requirements in HIV patients, the amounts of added vitamins and minerals in LNS-VM were three times the Recommended Nutrient Intake (RNI) for British women(,21), but to avoid possible deleterious effects of Fe during severe infections(,22), Fe was not included in the first stage, and in the second stage we provided one RNI only. We included bulk minerals, i.e. K, Mg and P, in both stages to address deficiencies of these minerals, correct electrolyte imbalance and promote tissue repletion. Further details of the intervention are in Supplementary Table S1 and published(,16). The primary trial outcome was mortality between recruitment and 12 weeks post-ART initiation(,16). Secondary outcomes presented here include effects on handgrip strength, fat mass and fat-free mass at 6 and 12 weeks post-ART initiation and during the follow-up period. BIA results were used as our main measure of body composition, both because we had results for the greatest proportion of participants and because it is a relatively cheap and feasible technique even in fairly poorly resourced settings. BIA produces results predicted from impedance, age, sex, weight and height and we wished to compare these results with the more direct measurements given by ADP and D2O. Randomisation was conducted by the Data and Safety Monitoring Board (DSMB) statistician using computer-generated blocks of sixteen and stratified by country. Packages of LNS-VM and LNS, in both small- and large-dose formats, were delivered by the producer in lots designated by allocation code. Clinic pharmacists not involved in recruitment or provision of care to study participants labelled intervention packets with the study identity numbers at the time packets were dispensed. Participants were recruited by clinic nurses with no access to the code and assigned sequential identity numbers (within sites) after they were found to be eligible and had signed informed consent. Both participants and recruiting staff were not aware of the group of the dispensed supplements and intervention and control supplements packets were of equal size, colour, and similar taste. Adherence to study supplements was modest, with only 39 % of participants consuming at least 75 % of their expected number of sachets of supplement(,16). As we reported earlier, by the end of recruitment, we had recruited 1815 patients(,16). This number was sufficient to detect, at 5 % significance, 90 % power and 25 % attrition by 12 weeks due to death or loss to follow-up, differences of 0·18 of a standard deviation in secondary continuous outcomes measured at 6 and 12 weeks. The study was conducted according to principles laid down in the Declaration of Helsinki. Ethics committees of the London School of Hygiene & Tropical Medicine, the University of Zambia Biomedical Research Ethics Committee, and the Medical Research Coordinating Committee of NIMR, Tanzania provided ethics clearances. Patients were enrolled after providing written or thumbprint informed consent and medical care of patients was provided according to national guidelines. Data on demographic and socio-economic status were collected at patient enrolment. Handgrip strength and body composition data were collected at enrolment (before ART initiation), and at the 6th and 12th week post-ART initiation. The time of starting ART was determined by factors outside the investigators' control and was a median of 21 (interquartile range 15–30) d after referral for ART(,16). Patients who did not attend study visits were reminded by telephone or those in Mwanza were also traced to their residences and encouraged to come to clinics for follow-up measurements. Patients were asked to fast overnight and were invited for anthropometry, handgrip strength and body composition measurements in the morning. While barefoot and with minimal clothing, weight was determined to the nearest 0·1 kg using a digital scale and height (baseline only) was measured to the nearest 0·1 cm using a stadiometer fixed to the office wall. Anthropometric measurements were taken in triplicate and medians were used during analysis. We assessed body composition using BIA using Tanita instrumentation (Tanita BC418) as the primary method. In addition, we used D2O (Cortecnet) and ADP (BodPod Model 2007A; Life Measurement Instruments/COSMED) in subsamples, limited by logistic and financial constraints to Zambian participants, to supplement BIA findings. All the methods assessed fat mass (kg) and fat-free mass (kg) and, in addition, BIA also produced results on trunk and segmental fat and fat-free mass, all expressed in kg. For the D2O technique, patients were asked to provide a 4-ml saliva sample (pre-dose saliva sample), after which they were asked to take a previously prepared dose of 30 g D2O using a straw from a 50-ml, screw-capped, leak-proof bottle. Then they drank 100 ml of drinking water from the same bottle to ensure all D2O was consumed. We collected two post-dose saliva samples at 3 and 4 h. All samples were collected into tightly capped cryogenic tubes and kept away from direct sunlight. While waiting for post-dose sample collection, patients were asked to refrain from walking, eating or drinking. On the day of collection, samples were transported in cool boxes to research laboratories at UTH, where they were stored at −20°C pending transfer to the Zambian National Institute for Scientific and Industrial Research in Lusaka for analysis. Enrichment of D2O in saliva samples was determined by Fourier Transform Infrared Spectrophotometer (FTIR Model 8400s; Shimadzu). Post-dose enrichment used the mean of the 3- and 4-h samples except where the 4-h enrichment was appreciably higher than the 3-h, in which case only the 4-h sample was used. Using post-dose enrichment data, the dilution space and total body water (TBW) were calculated using conventional formulae(,23). Fat-free mass was calculated as TBW/0·723(,24) and fat mass was calculated as body weight minus fat-free mass. From BIA, D2O and ADP fat and fat-free mass, and height measurements, fat mass index (FMI) was computed as fat mass (kg)/(height (m2)), and fat-free mass index (FFMI) as fat-free mass (kg)/(height (m2))(,25). We used FMI and FFMI in data management and fat and fat-free mass in evaluation of the effect of intervention. Handgrip strength was determined to the nearest 0·1 kg using a digital dynamometer (Takei Scientific Instruments). Four measurements were taken, with the mean of the two maximum measurements (one in each hand) reported. Venous blood samples were taken for CD4 count (baseline and week 12 only)(,16). Data were double entered into OpenClinica databases in Lusaka and into MySQL databases in Mwanza. Analyses were conducted in STATA version 13. The D2O technique is susceptible to chance errors resulting from incomplete dosing, sample contamination, or unrecorded fluid intake during the equilibration period. Therefore, at baseline, we excluded those with implausibly low fatness (fat mass <0 kg). We further excluded those with implausibly high fatness, given that the entire population had BMI <18·5 kg/m2; this resulted in excluding those with FMI >6 kg/m2, or FMI >4 kg/m2 if FFMI <11 kg/m2. For 6-week samples, we excluded those with implausibly low fatness (fat mass<0 kg). We also excluded those with poor agreement between BIA and ADP (difference >±7 kg TBW). For 12-week samples, we excluded those with implausibly low fatness (fat mass <0 kg), and implausibly high fatness (FMI >8 kg/m2). We also excluded those with poor agreement between BIA and ADP (difference >±7 kg TBW). Based on this we excluded fifty-six (10·5 %) at baseline, twenty (8 %) at 6 weeks and twenty (9 %) at 12 weeks. Baseline characteristics were presented as means and standard deviations if continuous variables and percentages if categorical variables to assess comparability of treatment arms. Socio-economic status was derived using principal component analysis of a list of housing characteristics and durable assets(,16,26). Outcome measures (i.e. fat mass, fat-free mass and handgrip strength) at 6 and 12 weeks post-ART were compared using linear regression with final estimate found by adjusting for baseline values, sex, age, BMI, CD4 count and socio-economic status. In addition, since results at 6 and 12 weeks could be analysed only for patients who survived and attended 6- and 12-week visits, we also investigated treatment effects using the detailed longitudinal data collected on all patients over the course of the study. We used piece-wise mixed-effects quadratic regression models to allow inclusion of data from patients who died or were lost to follow-up up prior to 12 weeks. The additional flexibility of cubic models and cubic splines was assessed but fit was considered adequate with quadratic models(,27). The time axis was split at the date of starting ART, allowing two lines with differing slopes to be fitted per person while restricting these lines to join at the date of ART initiation. For presentation, the marginal predictions after starting ART were based on the median time, 21 d, spent prior to starting ART; predictions pre-ART are not graphed because of the complexity of showing different lengths of time before ART. To assess comparability of BIA against D2O and ADP methods, we used the Bland–Altman method(,28). Based on this approach, we analysed differences (bias) in fat and fat-free mass between BIA and the other methods and their standard deviations (error) and calculated the limits of agreement (bias ± 1·96 error) to determine if the degree to which these methods differed was within a clinically acceptable range. We further assessed the dependency of bias on the mean of fat and fat-free mass for BIA and D2O and BIA and ADP using linear regression. Among both sexes, the acceptable range of error for fat and fat-free mass is thought to be 2 to 4·5 kg for males and 1·5 to 3·6 kg for females(,12).