Author s response to reviews

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1 Author s response to reviews Title: Daily consumption of ready-to-use peanut-based therapeutic food increased fat free mass, improved anemic status but has no impact on the zinc status of people living with HIV/AIDS: a randomized controlled trial Authors: Adama Diouf (adama.diouf@ucad.edu.sn) Abdou Badiane (badiane21@yahoo.fr) Noël Manga (noelmagloiremanga@yahoo.fr) Nicole Dossou (nicole.dossou@ucad.edu.sn) Papa Sow (papasalifsow@gmail.com) Salimata Wade (salimata.wade@ucad.edu.sn) Version: 1 Date: 05 Nov 2015 Author s response to reviews: 1. Major Comments Reviewer #1: This is a small study which basically shows that use of an energy-dense supplement leads to more rapid accumulation of lean body mass in wasted HIV infected adults. Most of these adults were not on ART at enrollment, I struggled to find data on how many did start ART during the study, I assume all patients did. So food+art were better at restoring normal body habitus than ART alone. If there was anyone that did not start ART this should be noted. In the section Results/clinical and nutritional characteristics, page 9, line 19 of the manuscript, we wrote Over 70% of the patients in each group were on ART without specifying at what moment. Indeed, 19/26 and 19/25 were ART+ in the RUTF and Control group, respectively, but we didn t specified the number of patients in each group. For more precision, we will include the following sentence page 9, line 19 at enrollment, 19/26 and 19/25 patients were ART+ in the RUTF and Control group, respectively. Reviewer #1: So food+art were better at restoring normal body habitus than ART alone. If there was anyone that did not start ART this should be noted.

2 We agree with the reviewer that food + ART was better at restoring lean body mass than ART alone. Indeed, at the end of the study, 14/20 patients in the RUTF group were still ART+ and 6 ART-. Change in fat free mass between ART+ and ART- patients in the RUTF group, as indicated in Table 1 (see attached file), show that % change was significantly higher in the ART+ (p = ) than in the ART- patients (p = 0.032) indicating that food+art were better at restoring fat free mass that ART alone. Because, we wanted to make the manuscript shorter, we have not included these data on the submitted paper. We propose to insert in the section Results/Effect of the supplement in body composition, (page 11, line 4-7) the following sentence In the supplemented group, fat free mass increased more significantly in the patients on ART (+11.7%, n = 14; p = ) than in those who did not received ART (+6.2%, n = 6; p = 0.032). In the section Conclusion, in page 15, lines 19-20, we will add the following sentence Within the supplemented group, food + treatment were better at restoring fat free mass and resolving anemia than treatment alone, because such conclusion concern both fat free and anemia (see response to the reviewers, page 5) Reviewer #1: There are a couple of major problems with the manuscript which suggest that it should be refocused. A key piece of new information is publication from Lee Miller content/early/2015/06/24/jn abstract shows that phytate actually does not affect zinc absorption. This is a major shift in thinking about zinc bioavailability and the developing world. This explains why all your efforts to reduce phytate had no effect. Zinc status is a continuum, so while a plasma zinc of > 66 is normal, there is probably little difference between someone who is 64 and 68 µg/dl. You happened to be working with a group of people with marginal zinc status, which increased, but not dramatically. But think of all that zinc was needed to create new lean tissue, and this diet was adequate. First we thanks the reviewer for the paper of Miller et al. (2015) and we agree with him that such information would have an important implication in zinc bioavailability in infants and young children in developing world in the future. However, this recent paper of Miller et al. (2015) concern infants and young children and does not challenge the results previously published by the same authors in 2007 (ref 23 of our manuscript: we apologize; there is a mistake in the reference, the first author name (Leland V Miller is missing), indicating that primary factors affecting zinc absorption in adults are the quantity of zinc and phytates in the diet. As phytates has been expected to exert a high influence on total zinc absorption, we effectively put our efforts on increasing the amount of zinc and decreasing the phytates content of the supplement consumed by the adult PLWH.

3 The results of our study show that plasma zinc concentration does not respond to the food supplement. This lack of response, although most of the patients were zinc-depleted at enrollment particularly the supplemented group that received additional zinc, was consistent with 2 published studies from our laboratory that used plasma zinc concentration to assess the impact of zinc fortification (ref 24 and 49 of the manuscript). Indeed, zinc regulation is a complex dynamic. Physiological requirements of zinc are defined as the amount of zinc to be absorbed to counterbalance the total loss of endogenous zinc (high during HIV-infection) and the amount of zinc retained in the newly formed tissues. It is more likely that the additional zinc provided to the RTUF group, was used to create the additional 2 kg fat free mass (+3.7 g/kg/day) rather than increasing the plasma circulating zinc. In the manuscript, we concluded that plasma zinc concentration may not be a sufficient sensitive indicator of zinc status. We agree with the reviewer that the diet was zinc adequate, therefore, we recommended in our conclusion that, supplementation of adult PLWH with 100 g RUTF in the early stage of infection could be beneficial. Reviewer #1: Iron is very sensitive to inflammation. But you actually did not measure any indicators of iron status. Hemoglobin came up nicely. Reviewer #2: The paper reads well, and I have only one minor comment. The supplemented group had a higher iron intake compared to the control group of several nutrients, including iron. Iron supplementation has been suspected to potentially have negative effect on HIV infections. See for example these two papers: Jacobus DP. Randomization to iron supplementation of patients with advanced human immunodeficiency virus disease--an inadvertent but controlled study with results important for patient care. J Infect Dis Apr;173(4): McDermid JM1, Hennig BJ, van der Sande M, Hill AV, Whittle HC, Jaye A, Prentice AM. Host iron redistribution as a risk factor for incident tuberculosis in HIV infection: an 11-year retrospective cohort study. BMC Infect Dis Jan 29;13:48. doi: / It would be good if the author could review these and similar studies and discuss whether a similar negative effect would be possible with this food supplement. We thanks the reviewer 2 for the papers (McDermid et al.,2013 and Jacobus DP, 1996). These references are now cited in the manuscript (page 13, line 3 and 6) and include in the section/references, number 38 and 39 (page 21, line 4-9). The results on iron status of the patients have not been included in the manuscript, because we wanted to keep the manuscript short. In fact, iron status of the patients was measured at enrollment (25 in the group RUTF and 23 in Control group), at discharge (25 in the group RUTF and 23 in Control group) and after 9 weeks home follow-up (20 in the group RUTF and 17 in Control group) using plasma ferritin

4 concentration which was adjusted to infection/inflammation status measured by AGP and CRP according to Thurnam et al. (Thurnham DI, McCabe LD, Haldar S, Wieringa FT, Northrop Clewes CA, McCabe GP. Adjusting plasma ferritin concentrations to remove the effects of subclinical inflammation in the assessment of iron deficiency: a meta-analysis. Am J Clin Nutr 2010, 92:546-55). Iron deficiency anemia was defined as Hb < 12 µg/dl for women and < 14 µg/dl for men, and plasma ferritin < 12 µg/l. The results indicated in the Table 2 (see attached file) show that the plasma ferritin decreased significantly in the RUTF group at 9 weeks, but not in the Control group. Most of the patients in both groups were not suffering from iron deficiency, or iron deficiency anemia suggesting that the etiology of anemia among our patients was not iron deficiency. The possibility that others mechanisms including the direct effect of HIV on erythropoiesis were discussed in the manuscript, section/discussion, page 13, line 2-4 (ref 36 and 37 of the manuscript). Another explanation is Tuberculosis infection (McDermid et al., 2013), which was the leading opportunistic infection in our patients. We now corrected this missing by citing the paper of McDermid et al. (2013) in the section/discussion, page 13, line 3. Tuberculosis treatment after 9 weeks could be related to the resolving of anemia in the Control group. Nevertheless, Hb increased more significantly in the supplemented group who had a higher iron intake than the Control group, indicating that the combination of Tuberculosis treatment and the supplement containing several nutrients, iron included was better to improve the anemic status of the PLWH. Iron supplementation has been suspected to potentially have negative effects on HIV infection (Jacobus DP, 1996). These negative effects are due to iron toxicity in which excessive iron intake may disrupt the homeostasis of oxidative stress (which is high among people living with HIV). In this study, iron intake from the supplement was very low (2.9 mg/day) and was not expected to have similar negative effects when high dose of iron such as 30 or 60 mg/day were administrated. This sentence will be insert in the discussion, page 13, line Minor comments Reviewer #2: Abstract lines 9-10 Comparison with control? We agree with the reviewer, in the section/abstract, page 2, line 7-11, and we propose this sentence After 9 weeks of supplementation, body weight, and fat-free mass increased significantly by +11% (p=0.033), and +11.8% (p=0.033) in the RUTF group, but not in the Control group, while percentage body fat was comparable between both groups (p=0.888).

5 P 4 line RUTF also contains phytates. We agree, in the section/ Introduction, page 4, line 12 13, the sentence is corrected as following It is also possible that a low efficiency of zinc absorption from the supplement had occurred due to the presence of phytates in the RUTF and the millet porridge. P 5 lines RUTF was given at 100 g/day whereas standard sachets are 92 g (500 kcal). Explanation? P 6 line 6. Same comment: 92 g or 100 g? Indeed, the sachets of RUTF are 92 g, but during this study, we didn t us the sachet, we weighed exactly 100 g of RUTF for each meal. P 11 the last sentence on line 25 continuing on p 12 is not clear. We agree, in the section/discussion, page 12, line 2-6, we modified the sentence as follow In this study, the standard hospital diet as well as diets provided by the relatives, even fortified with a vegetable soup were unable to cover the daily nutrient intake of the patients. Improving the diet with a high-energy dense fortified supplement, consisting of 100 g RUTF mixed with rice porridge increased significantly the energy, vitamins (B complex vitamins except for biotin, C, D, E, and minerals intake (zinc, selenium, cooper, calcium, potassium, phosphorus, magnesium). P 12 line 8. Better say "daily recommended intake" instead of "requirements". Check the whole text. P 12 line 14: same comment: recommended intake not recommended requirement. We accept your suggestion. In the section/discussion, page 12, line 12 and 19, and in the whole document, we replaced daily requirements by daily recommended intake. 1