Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation

Najeeha T Iqbal, Sana Syed, Kamran Sadiq, Marium N Khan, Junaid Iqbal, Jennie Z Ma, Fayaz Umrani, Sheraz Ahmed, Elizabeth A Maier, Lee A Denson, Yael Haberman, Monica M McNeal, Kenneth D R Setchell, Xueheng Zhao, Shahida Qureshi, Lanlan Shen, Christopher A Moskaluk, Ta-Chiang Liu, Omer Yilmaz, Donald E Brown, Michael J Barratt, Vanderlene L Kung, Jeffrey I Gordon, Sean R Moore, S Asad Ali, Najeeha T Iqbal, Sana Syed, Kamran Sadiq, Marium N Khan, Junaid Iqbal, Jennie Z Ma, Fayaz Umrani, Sheraz Ahmed, Elizabeth A Maier, Lee A Denson, Yael Haberman, Monica M McNeal, Kenneth D R Setchell, Xueheng Zhao, Shahida Qureshi, Lanlan Shen, Christopher A Moskaluk, Ta-Chiang Liu, Omer Yilmaz, Donald E Brown, Michael J Barratt, Vanderlene L Kung, Jeffrey I Gordon, Sean R Moore, S Asad Ali

Abstract

Background: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority.

Methods: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community.

Discussion: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals.

Trial registration: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Keywords: Childhood undernutrition; Duodenal biopsies; Endoscopy; Environmental enteropathy; Gut barrier function; Low- middle income countries; Mucosal gene expression; Small intestinal microbiota.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Conceptual framework for hypothesis testing in SEEM. The severity of clinical phenotypes in Matiari children with wasting and suboptimal response to nutritional rehabilitation will highly correlate with the histopathological appearance of duodenal biopsies; duodenal and fecal dysbiosis; perturbation of duodenal gene expression profiles; systemic biochemical profiles; and children’s genotypes. The image in the top right panel demonstrates the histological changes observed in the small intestine as environmental enteropathy progresses. Note: L:R lactulose:rhamnose ratio, EE environmental enteropathy, GI gastrointestinal, HLA Human Leukocyte Antigen
Fig. 2
Fig. 2
SEEM Data Collection Process. Note: CHW community health workers, UGI upper GI, WHZ weight for height Z score
Fig. 3
Fig. 3
SEEM transcriptome/genetics/biomarkers/microbiome framework at the time of endoscopy. a Is a detailed description of how samples will be collected throughout the study process from the birth cohorts; b Shows the groups, samples, and planned analyses at the time of endoscopy. Note for a: WHZ Weight-for-Height Z score, RF  Random Forest, EGD Esophagogastroduodenoscopy, infants* = 0 — 3 months old. Note for b: SEEM Study of Environmental Enteropathy and Malnutrition, CCHMC Cincinnati Children’s Hospital Medical Center, IBD Inflammatory Bowel Disease, CBC complete blood count, CRP C-Reactive Protein, EE Environmental Enteropathy, * = with a preference to enroll children under 5 years of age
Fig. 4
Fig. 4
Framework of data flow in SEEM. Description of how data will be transferred between institutions and a summary of the samples/analyses conducted at each institute. Note: AKU Aga Khan University, L:R Lactulose:Rhamnose ratio, Bx biopsy, EE Environmental enteropathy, CCHMC Cincinnati Children’s Hospital Medical Center, HLA Human Leukocyte Antigen, UVA University of Virginia, ‘omics multiomics, H&E  Haemotoxylin and Eosin, IHC Immunohistochemistry, EEDBI Environmental Enteric Dysfunction Biopsy Initiative, WUPAX Washington University Digital Pathology Exchange, GF Germ Free

References

    1. Campbell DI, Elia M, Lunn PG. Growth faltering in rural Gambian infants is associated with impaired small intestinal barrier function, leading to endotoxemia and systemic inflammation. J Nutr. 2003;133(5):1332–1338. doi: 10.1093/jn/133.5.1332.
    1. Solomons NW. Environmental contamination and chronic inflammation influence human growth potential. J Nutr. 2003;133(5):1237. doi: 10.1093/jn/133.5.1237.
    1. Chacko CJ, Paulson KA, Mathan VI, Baker SJ. The villus architecture of the small intestine in the tropics: a necropsy study. J Pathol. 1969;98(2):146–151. doi: 10.1002/path.1710980209.
    1. Keusch GT. Subclinical malabsorption in Thailand. I. Intestinal absorption in Thai children. Am J Clin Nutr. 1972;25(10):1062–1066. doi: 10.1093/ajcn/25.10.1062.
    1. Lindenbaum J, Gerson CD, Kent TH. Recovery of small-intestinal structure and function after residence in the tropics. I. Studies in peace corps volunteers. Ann Intern Med. 1971;74(2):218–222. doi: 10.7326/0003-4819-74-2-218.
    1. Fagundes Neto U, Martins MC, Lima FL, Patricio FR, Toledo MR. Asymptomatic environmental enteropathy among slum-dwelling infants. J Am Coll Nutr. 1994;13(1):51–56. doi: 10.1080/07315724.1994.10718371.
    1. Galpin L, Manary MJ, Fleming K, Ou CN, Ashorn P, Shulman RJ. Effect of Lactobacillus GG on intestinal integrity in Malawian children at risk of tropical enteropathy. Am J Clin Nutr. 2005;82(5):1040–1045. doi: 10.1093/ajcn/82.5.1040.
    1. Goto R, Panter-Brick C, Northrop-Clewes CA, Manahdhar R, Tuladhar NR. Poor intestinal permeability in mildly stunted Nepali children: associations with weaning practices and Giardia lamblia infection. Br J Nutr. 2002;88(2):141–149. doi: 10.1079/BJN2002599.
    1. Thurnham DI, Northrop-Clewes CA, McCullough FS, Das BS, Lunn PG. Innate immunity, gut integrity, and vitamin a in Gambian and Indian infants. J Infect Dis. 2000;182(Suppl 1):S23–S28. doi: 10.1086/315912.
    1. Campbell DI, Murch SH, Elia M, Sullivan PB, Sanyang MS, Jobarteh B, et al. Chronic T cell-mediated enteropathy in rural west African children: relationship with nutritional status and small bowel function. Pediatr Res. 2003;54(3):306–311. doi: 10.1203/01.PDR.0000076666.16021.5E.
    1. Campbell DI, McPhail G, Lunn PG, Elia M, Jeffries DJ. Intestinal inflammation measured by fecal neopterin in Gambian children with enteropathy: association with growth failure, Giardia lamblia, and intestinal permeability. J Pediatr Gastroenterol Nutr. 2004;39(2):153–157. doi: 10.1097/00005176-200408000-00005.
    1. Trehan I, Shulman RJ, Ou CN, Maleta K, Manary MJ. A randomized, double-blind, placebo-controlled trial of rifaximin, a nonabsorbable antibiotic, in the treatment of tropical enteropathy. Am J Gastroenterol. 2009;104(9):2326–2333. doi: 10.1038/ajg.2009.270.
    1. Syed S, Ali A, Duggan C. Environmental enteric dysfunction in children. J Pediatr Gastroenterol Nutr. 2016;63(1):6–14. doi: 10.1097/MPG.0000000000001147.
    1. Syed Sana, Yeruva Sunil, Herrmann Jeremy, Sailer Anne, Sadiq Kamran, Iqbal Najeeha, Kabir Furqan, Ahmed Kumail, Qureshi Shahida, Moore Sean R., Turner Jerrold, Ali S. Asad. Environmental Enteropathy in Undernourished Pakistani Children: Clinical and Histomorphometric Analyses. The American Journal of Tropical Medicine and Hygiene. 2018;98(6):1577–1584. doi: 10.4269/ajtmh.17-0306.
    1. WHO. Children: reducing mortality 2017 Updated October 2017. Available from: .
    1. Levels and trends in child malnutrition; key findings of the joint malnutrition estimates 2017 edition [internet]. 2017 Available from: . Cited 04/09/201.
    1. Arndt MB, Richardson BA, Ahmed T, Mahfuz M, Haque R, John-Stewart GC, et al. Fecal markers of environmental enteropathy and subsequent growth in Bangladeshi children. Am J Trop Med Hyg. 2016;95(3):694–701. doi: 10.4269/ajtmh.16-0098.
    1. Guerrant RL, Leite AM, Pinkerton R, Medeiros PH, Cavalcante PA, DeBoer M, et al. Biomarkers of environmental enteropathy, inflammation, stunting, and impaired growth in children in Northeast Brazil. PLoS One. 2016;11(9):e0158772. doi: 10.1371/journal.pone.0158772.
    1. Korpe PS, Petri WA., Jr Environmental enteropathy: critical implications of a poorly understood condition. Trends Mol Med. 2012;18(6):328–336. doi: 10.1016/j.molmed.2012.04.007.
    1. Naylor C, Lu M, Haque R, Mondal D, Buonomo E, Nayak U, et al. Environmental enteropathy, Oral vaccine failure and growth faltering in infants in Bangladesh. EBioMedicine. 2015;2(11):1759–1766. doi: 10.1016/j.ebiom.2015.09.036.
    1. Iqbal NT, Sadiq K, Syed S, Akhund T, Umrani F, Ahmed S, Yakoob MY, Rahman N, Qureshi S, Xin W, Ma JZ, Hughes M, Ali SA. Promising Biomarkers of Environmental Enteric Dysfunction: A Prospective Cohort study in Pakistani Children. Sci Rep 2018;8(1):2966. 10.1038/s41598-018-21319-8. PubMed PMID: ; PubMed Central PMCID: .
    1. Syed S, Dinallo V, Iqbal NT, Di Iorio L, Di Fusco D, Guleria S, et al. High SMAD7 and p-SMAD2,3 expression is associated with environmental enteropathy in children. PLoS Negl Trop Dis. 2018;12(2):e0006224. doi: 10.1371/journal.pntd.0006224.
    1. Syed S, Iqbal NT, Sadiq K, Ma JZ, Akhund T, Xin W, et al. Serum anti-flagellin and anti-lipopolysaccharide immunoglobulins as predictors of linear growth faltering in Pakistani infants at risk for environmental enteric dysfunction. PLoS One. 2018;13(3):e0193768. doi: 10.1371/journal.pone.0193768.
    1. Sullivan PB, Marsh MN, Mirakian R, Hill SM, Milla PJ, Neale G. Chronic diarrhea and malnutrition--histology of the small intestinal lesion. J Pediatr Gastroenterol Nutr. 1991;12(2):195–203. doi: 10.1097/00005176-199102000-00010.
    1. Ramakrishna BS, Venkataraman S, Mukhopadhya A. Tropical malabsorption. Postgrad Med J. 2006;82(974):779–787. doi: 10.1136/pgmj.2006.048579.
    1. Peterson KM, Buss J, Easley R, Yang Z, Korpe PS, Niu F, et al. REG1B as a predictor of childhood stunting in Bangladesh and Peru. Am J Clin Nutr. 2013;97(5):1129–1133. doi: 10.3945/ajcn.112.048306.
    1. Haberman Y, Tickle TL, Dexheimer PJ, Kim MO, Tang D, Karns R, et al. Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature. J Clin Invest. 2014;124(8):3617–3633. doi: 10.1172/JCI75436.
    1. Bragde H, Jansson U, Jarlsfelt I, Soderman J. Gene expression profiling of duodenal biopsies discriminates celiac disease mucosa from normal mucosa. Pediatr Res. 2011;69(6):530–537. doi: 10.1203/PDR.0b013e318217ecec.
    1. Hart SN, Therneau TM, Zhang Y, Poland GA, Kocher JP. Calculating sample size estimates for RNA sequencing data. J Comput Biol. 2013;20(12):970–978. doi: 10.1089/cmb.2012.0283.
    1. Unicef. National guidelines for the management of acute malnutrition among children under five and pregnant and lactating women:community based management of acute malnutrition (CMAM). Available from: .
    1. Hill ID, Fasano A, Guandalini S, Hoffenberg E, Levy J, Reilly N, et al. NASPGHAN clinical report on the diagnosis and treatment of gluten-related disorders. J Pediatr Gastroenterol Nutr. 2016;63(1):156–165. doi: 10.1097/MPG.0000000000001216.
    1. Wit JM, Himes JH, van Buuren S, Denno DM, Suchdev PS. Practical application of linear growth measurements in clinical research in low- and middle-income countries. Horm Res Paediatr. 2017;88(1):79–90. doi: 10.1159/000456007.
    1. Bill and Melinda Gates Foundation . Environmental enteropathy in Zambia: biomarkers defined by pathogenesis. 2012.
    1. Mahfuz M, Das S, Mazumder RN, Masudur Rahman M, Haque R, Bhuiyan MMR, et al. Bangladesh environmental enteric dysfunction (BEED) study: protocol for a community-based intervention study to validate non-invasive biomarkers of environmental enteric dysfunction. BMJ Open. 2017;7(8):e017768. doi: 10.1136/bmjopen-2017-017768.
    1. Wierdsma NJ, Peters JH, Peters JH, van Bokhorst-de van der Schueren MA, Mulder CJ, Metgod I, van Bodegraven AA. Bomb calorimetry, the gold standard for assessment of intestinal absorption capacity: normative values in healthy ambulant adults. J Hum Nutr Diet. 2014;27(Suppl 2):57–64. doi: 10.1111/jhn.12113.
    1. Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014;370(14):1287–1297. doi: 10.1056/NEJMoa1311194.
    1. Yu DH, Gadkari M, Zhou Q, Yu S, Gao N, Guan Y, et al. Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome. Genome Biol. 2015;16:211. doi: 10.1186/s13059-015-0763-5.
    1. van Nieuwenhoven MA, Geerling BJ, Deutz NE, Brouns F, Brummer RJ. The sensitivity of the lactulose/rhamnose gut permeability test. Eur J Clin Investig. 1999;29(2):160–165. doi: 10.1046/j.1365-2362.1999.00421.x.
    1. van Wijck K, Verlinden TJ, van Eijk HM, Dekker J, Buurman WA, Dejong CH, et al. Novel multi-sugar assay for site-specific gastrointestinal permeability analysis: a randomized controlled crossover trial. Clin Nutr. 2013;32(2):245–251. doi: 10.1016/j.clnu.2012.06.014.
    1. Dorshow RB, Hall-Moore C, Shaikh N, Talcott MR, Faubion WA, Rogers TE, et al. Measurement of gut permeability using fluorescent tracer agent technology. Sci Rep. 2017;7(1):10888. doi: 10.1038/s41598-017-09971-y.
    1. Denno DM, VanBuskirk K, Nelson ZC, Musser CA, Hay Burgess DC, Tarr PI. Use of the lactulose to mannitol ratio to evaluate childhood environmental enteric dysfunction: a systematic review. Clin Infect Dis. 2014;59(Suppl 4):S213–S219. doi: 10.1093/cid/ciu541.
    1. Smith HE, Ryan KN, Stephenson KB, Westcott C, Thakwalakwa C, Maleta K, et al. Multiple micronutrient supplementation transiently ameliorates environmental enteropathy in Malawian children aged 12-35 months in a randomized controlled clinical trial. J Nutr. 2014;144(12):2059–2065. doi: 10.3945/jn.114.201673.
    1. Weisz AJ, Manary MJ, Stephenson K, Agapova S, Manary FG, Thakwalakwa C, et al. Abnormal gut integrity is associated with reduced linear growth in rural Malawian children. J Pediatr Gastroenterol Nutr. 2012;55(6):747–750. doi: 10.1097/MPG.0b013e3182650a4d.
    1. Blanton L. V., Charbonneau M. R., Salih T., Barratt M. J., Venkatesh S., Ilkaveya O., Subramanian S., Manary M. J., Trehan I., Jorgensen J. M., Fan Y.-m., Henrissat B., Leyn S. A., Rodionov D. A., Osterman A. L., Maleta K. M., Newgard C. B., Ashorn P., Dewey K. G., Gordon J. I. Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children. Science. 2016;351(6275):aad3311–aad3311. doi: 10.1126/science.aad3311.
    1. Subramanian S, Huq S, Yatsunenko T, Haque R, Mahfuz M, Alam MA, et al. Persistent gut microbiota immaturity in malnourished Bangladeshi children. Nature. 2014;510(7505):417–421. doi: 10.1038/nature13421.
    1. Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et al. Human gut microbiome viewed across age and geography. Nature. 2012;486(7402):222–227. doi: 10.1038/nature11053.
    1. Kau AL, Planer JD, Liu J, Rao S, Yatsunenko T, Trehan I, et al. Functional characterization of IgA-targeted bacterial taxa from undernourished Malawian children that produce diet-dependent enteropathy. Sci Transl Med. 2015;7(276):276ra24. doi: 10.1126/scitranslmed.aaa4877.
    1. Syed S, Duggan CP. Risk factors for malnutrition and environmental enteric dysfunction-you really are what you eat. J Pediatr. 2016;178:7–8. doi: 10.1016/j.jpeds.2016.08.005.
    1. Gevers D, Kugathasan S, Denson LA, Vazquez-Baeza Y, Van Treuren W, Ren B, et al. The treatment-naive microbiome in new-onset Crohn's disease. Cell Host Microbe. 2014;15(3):382–392. doi: 10.1016/j.chom.2014.02.005.
    1. Morgan XC, Tickle TL, Sokol H, Gevers D, Devaney KL, Ward DV, et al. Dysfunction of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol. 2012;13(9):R79. doi: 10.1186/gb-2012-13-9-r79.
    1. de Onis M, Onyango AW, Borghi E, Siyam A, Nishida C, Siekmann J. Development of a WHO growth reference for school-aged children and adolescents. Bull World Health Organ. 2007;85(9):660–667. doi: 10.2471/BLT.07.043497.
    1. llumina. Infinium OmniExpressExome-8 Kit [Available from: . Accessed 8 Aug 2018.
    1. Menendez-Corrada R, Nettleship E, Santiago-Delpin EA. HLA and tropical sprue. Lancet. 1986;2(8517):1183–1185. doi: 10.1016/S0140-6736(86)92195-1.
    1. Goodrich JK, Waters JL, Poole AC, Sutter JL, Koren O, Blekhman R, et al. Human genetics shape the gut microbiome. Cell. 2014;159(4):789–799. doi: 10.1016/j.cell.2014.09.053.
    1. Palma GD, Capilla A, Nova E, Castillejo G, Varea V, Pozo T, et al. Influence of milk-feeding type and genetic risk of developing coeliac disease on intestinal microbiota of infants: the PROFICEL study. PLoS One. 2012;7(2):e30791. doi: 10.1371/journal.pone.0030791.
    1. Wacklin P, Tuimala J, Nikkila J, Sebastian T, Makivuokko H, Alakulppi N, et al. Faecal microbiota composition in adults is associated with the FUT2 gene determining the secretor status. PLoS One. 2014;9(4):e94863. doi: 10.1371/journal.pone.0094863.
    1. Schumann M, Siegmund B, Schulzke JD, Fromm M. Celiac disease: role of the epithelial barrier. Cell Mol Gastroenterol Hepatol. 2017;3(2):150–162. doi: 10.1016/j.jcmgh.2016.12.006.
    1. Huttonhower Lab. Galaxy webserver The Huttenhower Lab - Department of Biostatistics, Harvard T.H. Chan School of Public Health [Available from: ]. Accessed 28 Mar 2018.
    1. Friedman JH. Stochastic gradient boosting. Comput Stat Data Anal. 2002;38(4):367–378. doi: 10.1016/S0167-9473(01)00065-2.
    1. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B Methodol. 1995;57(1):289–300.
    1. Trapnell C, Salzberg SL. How to map billions of short reads onto genomes. Nat Biotechnol. 2009;27(5):455. doi: 10.1038/nbt0509-455.
    1. Bray NPH, Melsted P, Pachter L. Near-optimal RNA-Seq quantification with kallisto. 2015.
    1. Roberts A, Pachter L. Streaming fragment assignment for real-time analysis of sequencing experiments. Nat Methods. 2013;10(1):71–73. doi: 10.1038/nmeth.2251.
    1. Chen J, Bardes EE, Aronow BJ, Jegga AG. ToppGene suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res. 2009;37(Web Server issue):W305–W311. doi: 10.1093/nar/gkp427.
    1. Kaimal V, Bardes EE, Tabar SC, Jegga AG, Aronow BJ. ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems. Nucleic Acids Res. 2010;38(Web Server issue):W96–102. doi: 10.1093/nar/gkq418.
    1. Saito R, Smoot ME, Ono K, Ruscheinski J, Wang PL, Lotia S, et al. A travel guide to Cytoscape plugins. Nat Methods. 2012;9(11):1069–1076. doi: 10.1038/nmeth.2212.
    1. Beam AL, Kohane IS. Big data and machine learning in health care. JAMA. 2018;319(13):1317–1318. doi: 10.1001/jama.2017.18391.
    1. Ribeiro MT, Singh S, Guestrin C. Why should i trust you?: Explaining the predictions of any classifier. In: Proceedings of the 22nd ACM SIGKDD international conference on knowledge discovery and data mining: ACM; 2016. p. 1135–44.
    1. LeCun Y, Bengio Y, Hinton G. Deep learning. Nature. 2015;521(7553):436–444. doi: 10.1038/nature14539.
    1. Zeiler MD, Fergus R. European conference on computer vision. In: Springer; 2014. Visualizing and understanding convolutional networks; pp. 818–833.
    1. Zeiler MD, Krishnan D, Taylor GW, Fergus R. 2010 IEEE Computer Society Conference on Computer Vision and Pattern Recognition, CVPR. 2010. Deconvolutional networks; p. 7.
    1. Selvaraju RR, Cogswell M, Das A, Vedantam R, Parikh D, Batra D. Grad-CAM: visual explanations from deep networks via gradient-based localization: arXiv preprint arXiv; 2016. p. 161002391.
    1. Amadi B, Fagbemi AO, Kelly P, Mwiya M, Torrente F, Salvestrini C, et al. Reduced production of sulfated glycosaminoglycans occurs in Zambian children with kwashiorkor but not marasmus. Am J Clin Nutr. 2009;89(2):592–600. doi: 10.3945/ajcn.2008.27092.
    1. Joint Commission International. JCI Accredited Organizations [Available from: ]. Accessed 30 Apr 2018.
    1. The Aga Khan University Hospital. World Class Quality Health Services [Available from: .]. Accessed 30 Apr 2018.
    1. Di Segni A, Braun T, BenShoshan M, Barhom SF, Saar EG, Cesarkas K, Squires JE, Keller N, Haberman Y. Guided protocol for fecal microbial characterization by 16S rRNA-amplicon sequencing. J Vis Exp. 2018;19(133):e56845.
    1. International Committee of Medical Journal Editors (ICMJE). Defining the Role of Authors and Contributers [Available from: ]. Accessed 28 Mar 2018.

Source: PubMed

Patrocinadores e Colaboradores

Condições médicas

Intervenções de drogas

3
Se inscrever