Impact of supplementation of a package of iron folic acid (IFA) with probiotics on hemoglobin levels in elderly with mild to moderate anemia: Protocol for a double blind randomized controlled trial (PRobiotics in Anemia Trial for Healthy Ageing Management - PRATHAM)

Impact of supplementation of a package of iron folic acid (IFA) with probiotics on hemoglobin levels in elderly with mild to moderate anemia: Protocol for a double blind randomized controlled trial (PRobiotics in Anemia Trial for Healthy Ageing Management - PRATHAM) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Study protocol Impact of supplementation of a package of iron folic acid (IFA) with probiotics on hemoglobin levels in elderly with mild to moderate anemia: Protocol for a double blind randomized controlled trial (PRobiotics in Anemia Trial for Healthy Ageing Management - PRATHAM) Farhad Ahamed, Semanti Das, Sunom Merab Lepcha, Abhishek Jaiswal, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4320990/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Anemia is a major health concern of the elderly with iron deficiency anemia and anemia of chronic diseases being the two major causes of anemia. Probiotics help in anemia correction in several ways. It increases oral iron absorption by directly modulating immunity, eliciting an anti-inflammatory response, reducing ferric iron to bioavailable ferrous iron, and enhancing iron uptake by enterocytes. This is especially relevant in old age, as aging is associated with age-related gradual attrition of protective organisms in gut microbiota. As there are no studies on the efficacy of probiotics supplementation in anemia management in the elderly, a study has been planned to determine the impact of supplementation of a therapeutic package of Iron and Folic Acid (IFA) and probiotics on hemoglobin levels in elderly with mild to moderate anemia in comparison to IFA only. Methods This community-based randomized controlled trial will be conducted in the Kalyani Municipality area of Nadia district, West Bengal, India. Individuals aged 60 years and above with mild to moderate anemia will be randomized into the intervention (IFA and probiotics) and control group (IFA and placebo gelatin capsules containing ORS). The medications will be self-administered. The calculated sample size is 100 in each arm. Block randomization will be done. Participants will be followed up weekly for 12 weeks. Discussion Changes in hemoglobin, ferritin, hepcidin, C-reactive protein (CRP), composition, and diversity of the gut microbiome from baseline to 12 weeks in both groups will be assessed. Ethics and dissemination: Ethical approval was obtained from the Institutional Ethics Committee of AIIMS Kalyani (Ref. No: IEC /AIIMS / Kalyani / Meeting/ 2023 / 020). Trial Registration Number: The trial was registered in Clinical Trials Registry-India (CTRI) (CTRI/2023/09/057830) on 19.09.2023 Protocol version: Version: 1.0 Anemia elderly iron supplementation probiotics randomized controlled trial Gut microbiome Figures Figure 1 Background Population aging is an unavoidable demographic transition. The rapid increase of the elderly population (aged 60 years and above) has become a global phenomenon. By 2050, the proportion of elderly population in the word is expected to rise to 22% from 12% of the total population in 2015 and 80% of this population is expected to belong to low and middle-income countries. 1 In India, while the general population grew by 18% and 12.4% in each decade from 2001; the elderly population grew by 36% in each of the last two decades. India's elderly population count is projected to rise by 56 million in the next decade. 2 Anemia is a common, multi factorial condition among elderly. The prevalence of anemia increases with age, representing an important health problem among older individuals in India and in the world. 3 – 5 Unfortunately, many symptoms of anemia like weakness, fatigue, and shortness of breath are often misdiagnosed as normal phenomena in elderly individuals. Anemia of chronic disease (ACD) and nutritional anemia especially due to iron deficiency (IDA) are the two most common causes of anemia in the elderly population and these two conditions are often found to coexist. 6 Anemia has been reported to be associated independently with decreased functional ability/physical function, increased dementia, and increased risk of falls, morbidity, and mortality in the elderly population. 7 – 9 Thus, anemia among the elderly population affects their health-related quality of life and increases their mortality. Host-microbiota metabolic crosstalk has been suggested to play an important role in systemic iron homeostasis. 10 It increases the expression of DMT1 (iron transporter in the gut) and DcytB (Ferric reductase) (via the HIF-2α pathway) located at the enterocyte apical brush border membrane which reduces ferric iron (III) to absorbable ferrous iron (II). 11 , 12 These two factors ensure better iron absorption in the gut and reduce the negative side effects of oral iron supplementation. In conditions of less iron absorption from orally administered IFA tablets, the excess unabsorbed iron passes through the colon and gets involved in the Fenton & Haber-Weiss reaction, which not only alters composition of intestinal flora but also increases the inflammatory factors, adding to any existing background inflammatory conditions which is common in this age group. 13 The protective organisms of gut microbiota are believed to play a prominent role in increasing the bio-availability of dietary iron by decreasing the amount of iron binding compounds (MUC5AC) in the gut. 14 It has now been established that gut microbiota in elderly individuals suffer gradual decline in beneficial microorganisms ( Firmicutes and Acenobacteria ) and increase of facultative anaerobic bacteria ( Proteobacteria ). 15 Reduced dentition, chewing strength and loss of appetite also lead to limited intake of food ingredients which support the microbial diversity. 16 These changes are responsible for a decrease in short chain fatty acid (SCFA) production. SCFA protect against pathogens and exert anti-inflammatory properties including upregulation of anti-inflammatory cytokines. 17 Thus, host microbiota interaction plays an important role in iron absorption and utilization for heme synthesis. Intact gut microbiota has also been found to reduce iron absorption (gut microbial metabolites 1,3-diaminopropane (DAP) and reuterin) in iron excess body state and store excess iron via ferritin regulation thereby preventing harmful effect of iron. 10 Probiotics are live microbial feed supplements which beneficially affect the host animal by improving gut microbial balance. 18 Probiotics supplementation has shown promising results in improving hemoglobin level when combined with oral iron supplementation 19 through immunomodulation, anti-inflammatory response, formation of bioavailable ferrous form by reduction of ferric iron, enhanced iron uptake by enterocytes and replenishment of the gut microbiota with good bacteria ( Lactobacillus and Bifidobacterium ). 20 Further, probiotics protect the gut microbiota from harmful effects of unabsorbed oral iron. 21 In relation to the above observations, we hypothesize that supplementation of probiotics along with IFA will have positive effect on hemoglobin level in elderly without harming the gut microbiota and thereby improve their quality of life. Few studies have been done so far to see the effect of probiotics on iron absorption and iron status. 19 Although very few studies have seen the effect of supplementation of IFA and probiotics together, majority of them were not adequately powered to see the effect. The current study aims to determine the impact of supplementation of a therapeutic package of IFA and probiotics (intervention group) on hemoglobin level (from base line to 12 weeks) in elderly with mild to moderate anemia in comparison to IFA only (control group). It shall also assess the impact on ferritin concentration, hepcidin level, CRP level, incidence of acute infections, composition and diversity of the gut microbiome from base line to 12 weeks in both the groups. Methods Trial design : This will be a community based, placebo controlled, double-blind, randomized controlled trial. Groups will be parallel; allocation ratio will be 1:1 and the framework will be superiority. Eligibility criteria : Individuals aged 60 years and above, a permanent resident of the area and expected to stay in the area for another six months, who consent to participate in the study shall be eligible for the study. Individuals screened to have mild to moderate anemia and who shall not have taken any probiotic or antibiotics in the past 30 days prior to enrolment will be included in the study. Individuals with no anemia (Male: Hemoglobin ≥ 13gm/dL and Female: Hemoglobin ≥ 12gm/dL), severe anemia (Hemoglobin < 8gm/dL), known history of gastrointestinal & metabolic disorders, chronic renal failure, history of receiving blood donations in last six months and regularly consuming nutritional supplements (Iron, folic acid, Vitamin B12, Vitamin D and other multivitamins and minerals), diagnosed pancreatitis, hypersensitivity to any ingredient in the supplement and individuals who shall have participated in any other clinical trial in the preceding 30 days will be excluded from the study. Operational definitions Anemia Hemoglobin level < 13gm/dL in male and hemoglobin level < 12gm/dL in female will be considered as anemia. Hemoglobin level < 8 gm/dL will be considered as severe anemia in the both the sexes. Iron Deficiency Anemia (IDA) Anemia related to absolute iron deficiency (iron deficiency anemia, IDA) will be characterized by a decreased serum ferritin level ( 100 µ g/mL) and raised CRP (≥ 0.5 mg/dL). Compliance Individuals who shall have a cumulative consumption ≥ 80% of both the tablets (IFA) and capsules (probiotics/placebo) at the end of the study period will be considered as compliant to the therapy. Intervention The eligible individuals will be randomized into intervention and control group. In the intervention arm, study participants will receive 60 mg of elemental oral iron and 500mcg of folic acid as IFA tablets and capsules containing probiotics daily for three months. The control group will receive 60 mg of elemental oral iron and 500mcg of folic acid as IFA tablets and placebo gelatin capsules containing ORS daily for 12 weeks. Probiotics will contain Streptococcus , Bifidobacterium and Lactobacillus . The tablets will be manufactured in certified laboratories. Modifications - If any individual develops side effects associated with IFA supplementation like excessive vomiting even after consuming the medicine two hours after a major meal and without any other obvious reason, or major side effects related to probiotics, such as gastro-intestinal symptoms (nausea, abdominal pain, flatulence, bloating, unpleasant taste in the mouth, changes in stool, incontinence), flu-like or coryza like symptoms and unexplained arthralgia, fatigue, stress, headache and depression, he/she will be advised to discontinue the medicine. Adherence -An individual will be considered loss to follow-up if he/she could not be contacted on two repeated visits with a minimal interval of one week, has shifted the residence to other place permanently, died during the course of trial or could not be available for blood collection at the end of three months of trial will be considered as loss to follow-up. Concomitant care - Participants requiring any other medical advice will be referred to AIIMS, Kalyani for further management. Outcomes- Primary outcome: To determine the change in hemoglobin level from base line to 12 weeks in elderly with mild to moderate anemia in intervention and control groups. Secondary outcomes : To determine the change in serum ferritin level from base line to 12 weeks in elderly with mild to moderate anemia in intervention and control groups. To determine the change in hepcidin level from base line to 12 weeks in in elderly with mild to moderate anemia in intervention and control groups. To determine the change in CRP level from base line to 12 weeks in elderly with mild to moderate anemia in intervention and control groups. To determine the difference in incidence of acute infections from baseline to 12 weeks in elderly with mild to moderate anemia in intervention and control groups. To compare the composition and diversity of the gut microbiome in elderly among the intervention and control groups at the baseline and end line. The study design is outlined in the form of a flowchart in Fig. 1 . The details of the study are described subsequently. Sample size Assuming a superiority margin of 0.4 gm/dL, mean (SD) observed difference of 0.7 gm/dL between the two groups before and after the intervention 22 , power 80%, alpha error of 5%, and attrition rate of 10%, we need to recruit 100 participants in each arm giving a total sample size of 200. The sample size was calculated using STATA version 17. Recruitment- The study setting being the urban field practice area of a medical college, the principal investigator has the list of households with elderly population. Out of 22,100 households, currently there are 2112 households which have elderly individuals. The prevalence of mild to moderate anemia among the study age group has been estimated around 60% in our earlier study. 23 First, 1000 households will be selected randomly using computer generated random numbers. If more than two elderly individuals reside in a single household, one of them will be selected using the lottery method. The randomly selected elderly individuals will then be screened for hemoglobin level using HemoCue 301 device. This device has very high sensitivity and specificity as compared to the gold standard method. 24 Individuals who will be screened as mild or moderately anemic and fulfilling other inclusion criteria will be requested for blood samples (5 ml). The blood sample will be drawn aseptically from the ante-cubital vein of the non-dominant hand of each participant and will be stored in plain vial. The sample will be carried to the institute on the same day. Serum will be separated from the blood sample at the laboratory of the department of Biochemistry in centrifuge machine at 3000 rpm. Written informed consent will be taken at the time of enrolment and again at the time of blood sample collection. Randomization : Before the start of the intervention, a third person not related to the study shall conduct block randomization (with a block size of four) in 1:1 ratio dividing the intervention and control arms in equal numbers. He/she will sort and pack the similar looking tablets and capsules into pouches which will be serially numbered according to the random sequence generated. The serial numbered pouches will be subdivided into tablets of 30 (IFA) and capsules of 30 (probiotics/placebo gelatin capsules containing ORS) each so that compliance can be measured on monthly basis. The investigator will be handed over only serial numbered pouches containing IFA tablets and probiotics/placebo gelatin capsules with ORS. The original content inside each pouch will only be known to the third person who generated the random sequence and did packaging. This will ensure allocation concealment and since the tablets/capsules which will be used in the study will look similar in all aspects, both the investigator and the study participants will be blinded. Treatment arms Intervention group Study participants in this group will receive 60 mg of elemental oral iron and 500mcg of folic acid as IFA tablets and capsules containing probiotics daily for three months. The participants will be asked to take the probiotics capsules thirty minutes before breakfast and IFA capsules one hour before lunch. The field investigators will visit every study participant on weekly basis to record the side-effects. Compliance will be calculated on monthly basis. Participant consuming 80% of the doses at the end of each month would be considered as compliant. Participants developing signs of gastric discomfort would be advised to take IFA tablets 2 hours after meals. If the participant develops any other untoward events, complete evaluation would be done and required modifications in treatment regimens would be made. Control group Study participants in this group will receive 60 mg of elemental oral iron and 500mcg of folic acid as IFA tablets and placebo gelatin capsules containing ORS daily for 12 weeks. The participant will be asked to take the placebo capsules before breakfast and IFA capsules one hour before lunch. The field investigators will visit every study participant on a weekly basis to record the side-effects. Compliance will be calculated on monthly basis. Participant consuming 80% of the doses at the end of each month would be considered as compliant. Participants developing signs of gastric discomfort would be advised to take IFA tablets 2 hours after meals. If the participant develops any other untoward events, complete evaluation would be done and required modifications in treatment regimens would be made. Drugs The drugs required for the study are; 1. Iron and folic acid tablets containing 60 mg of elemental iron and 500 µg of folic acid; 2. Probiotics containing ( Streptococcus , Bifidobacterium and Lactobacillus ) 3. Placebo gelatin capsules containing Oral Rehydration Salt (ORS) similar to intervention drug in size, shape and colour. The tablets will be manufactured in certified laboratories. Data collection Baseline socio-demographic information will be collected using pre-tested semi structured interview schedule from all included individuals. The hemoglobin level will be measured by HemoCue 301 using capillary blood. Five millilitre (5 ml) of blood will be withdrawn aseptically from ante-cubital vein from each participant and will be stored in plain vial. The sample will be carried to the centralized institute on the same day. Serum will be separated from the blood sample at the laboratory of the department of Biochemistry in centrifuge machine at 3000 rpm. The separated serum will be stored at -20 0 C. Initially serum hepcidin, ferritin and CRP will be assessed. All the test will be done at the department of Biochemistry. All the tools will be calibrated before and during estimation of hematological and biochemical parameter. The laboratory has their own internal and external quality control measures. The participants will be provided with two sterile containers (with and without RNALater) to collect at least 2 grams of stool samples in each container. The containers will be transported at 4˚C to microbiology laboratory of AIIMS Kalyani and then frozen at -80˚C. The isolation of DNA and RNA will be done. 16S rRNA amplicon sequencing will be performed using Illumina MiSeq platform and analysed using the MGnify pipeline. The Genome Taxonomy Database (GTDB-Tk) will be used for bacterial taxonomic identification. Field investigators will visit study participants on weekly basis to collect data on side-effects. They will also provide the drugs to the study participants on monthly basis and collect the pouches given in the earlier visit. Compliance on monthly basis. Each participant will be followed-up for 12 weeks. The field investigators will be blinded about the group (intervention/control) of the study participants. The Hb level will be measured using HemoCue 301 after the completion of intervention. Five millilitres (5 ml) of blood will be drawn aseptically from each participant and stored in plain vial and will transport, processing, and finally storage as done for the baseline samples. This stored serum will be used for measurement of serum Hepcidin, Ferritin and CRP at the laboratory of the department of Biochemistry. The technique for biomarker (Ferritin, CRP, and hepcidin) and gut microbiome diversity assessment, calibration, internal quality, and external quality assessment will be similar as stated for the baseline samples. The team which will analyse the sample will be unaware about the group (intervention/control) of the study participants. (Refer to Fig. 1 ) We will be assessing any side-effects with routine visits every week. Participants developing any adverse effect will be treated at AIIMS, Kalyani, West Bengal, India. Study tools Hemoglobin will be measured using HemoCue 301 instrument. Hepcidin will be measured in ELISA method. CRP will be measured in bio-chemical autoanalyser. Ferritin will be measured using chemiluminescence technique. 16S rRNA amplicon sequencing will be performed using Illumina MiSeq platform and analysed using the MGnify pipeline. Study Duration The study will be completed in a duration of 12 months. The project will have three phases namely i) Preparatory Phase (3 months), ii) Implementation Phase (6 months) and iii) Data Analysis and Final Report Writing Phase (3 months). Preparatory phase Recruitment of staff, project manual development, procurement of survey logistics, development of study questionnaire, training of field staff on data collection, quality control and sample collection will be done. The packaging of drugs and random sequence generation by a third person not related to the design, conduct or analysis of the study will also be done. Implementation phase Screening, enrolment, randomization, intervention and biochemical analysis of the sample will be done during this phase. Data analysis The data will be collected in Epicollect 5 mobile based app and exported in Microsoft Excel version 13. The data will be analysed with the help of STATA 17 statistical software. Categorical data will be presented as percentage (%). Normally, distributed data will be presented as means and standard deviation or 95% confidence interval (CI). Pearson’s Chi-square test will be used to evaluate differences between groups for categorical variables. Student’s t -test for independent samples will be used for comparison between groups with continuous variables and paired t -test will be used for comparison of before and after changes. Both per-protocol and intention to treat analysis will be done. Changes in richness and evenness, Faith’s phylogenetic diversity (PD), and Shannon diversity will be measured between month 0 and 6 and compared to the placebo group using a Wilcoxon Rank Sum Test and a Holm-Bonferroni method with family-wise error rate P-value correction. Weighted UniFrac,Bray-Curtis dissimilarity, and unweighted UniFrac will be used for beta diversity analysis to identify any structural changes in the gut microbiome of trial participants. Microbiome differential abundance will be assessed. A “p” value ≤ 0.05 will be considered as statistically significant. Discussion This study protocol describes a randomized controlled trial to assess the impact of supplementation of a package of iron folic acid (IFA) with probiotics on hemoglobin levels in elderly with mild to moderate anemia. Correction of anemia in the elderly is slower. Thus, only iron supplementation may not be effective in improving iron levels. Host-microbiota has been hypothesized to play an important role in regulating iron absorption through immunomodulation, anti-inflammatory response, formation of bioavailable ferrous form by reduction of ferric iron, enhanced iron uptake by enterocytes and replenishment of the gut microbiota with good bacteria. The need of the hour, especially in a country with a huge burden of anemia, is to improve the response to iron supplementation. Co-supplementation of iron folic acid tablets with probiotics have shown promising results. The study shall involve iron folic acid supplementation to mild and moderately anemic participants aged more than 60 years in one group and iron and folic acid and probiotic supplementation in the other and following them up for 12 weeks. The outcome shall involve assessing for changes in hemoglobin levels as the primary outcome and changes in ferritin, CRP and hepcidin level as the secondary outcome. Frequency of acute infections and changes in the gut microbiome shall also be measured. Abbreviations IFA- Iron folic acid CRP-C-reactive protein ACD-Anemia of chronic disease IDA-Iron Deficiency Anemia SCFA - Short chain fatty acid GTDB-Tk -Genome Taxonomy Database CI- confidence interval Declarations Ethics approval and consent to participate: AIIMS, Kalyani Ethics Committee clearance will be obtained and the guidelines pertaining to the participant information sheet (PIS) and Participant informed consent form (PICF) will be followed. Participant Information sheet will be given to each study participant. PIS will be in the local language (Bengali) and will be provided and read out to each study participant before their recruitment. A written informed consent will be obtained from each study participant willing to participate in the study, by the field investigator. Participants with requiring any other medical advice will be referred to AIIMS, Kalyani for further management. Participants will be provided with the following information a) Expected duration of participation in study, b) The benefits to be expected from the research to the study participants, c) Any risk to the study participant associated with the study, d) Maintenance of confidentiality of records. Participant will have freedom to participate or withdraw from study at any time without penalty or loss of benefits to which the study participant would otherwise be entitled. The trial results will be communicated with the participant through publication in a reputed journal and through health talk with the communities. Any changes in the protocol during the trial will be informed in advance to the ethical committee of AIIMS, Kalyani, and the same will also be communicated with the Journal after the publication of the initial protocol. Consent for publication: Consent obtained directly from patient(s). Availability of data and materials: The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Trial Registration Number: The trial was registered in Clinical Trials Registry-India (CTRI) (CTRI/2023/09/057830) on 19.09.2023 Competing interests: The authors declare that they have no competing interests. Funding: The project will be funded by the Department of Health Research, Ministry of Health and Family Welfare, Government of India under extramural grants (R.11014/07/2023-GIA/HR) Authors’ contributions: FA is the guarantor, drafted the manuscript and is the corresponding author. FA, SD, SML, SP, KG, MS, AB, and CMH developed the trial concept and drafted the protocol manuscript. FA developed the trial concept and revised the manuscript critically for important intellectual content. All listed authors approved the final version of the manuscript for publication and agreed to be accountable for all aspects of the work. Acknowledgement: Not applicable References Ageing and health [Internet]. [cited 2024 Jan 23]. Available from: https://www.who.int/news-room/fact-sheets/detail/ageing-and-health Elderly in India 2021.pdf [Internet]. [cited 2024 Jan 23]. 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Malesza IJ, Bartkowiak-Wieczorek J, Winkler-Galicki J, Nowicka A, Dzięciołowska D, Błaszczyk M, et al. The Dark Side of Iron: The Relationship between Iron, Inflammation and Gut Microbiota in Selected Diseases Associated with Iron Deficiency Anaemia-A Narrative Review. Nutrients. 2022 Aug 24;14(17):3478. Sandberg AS, Önning G, Engström N, Scheers N. Iron Supplements Containing Lactobacillus plantarum 299v Increase Ferric Iron and Up-regulate the Ferric Reductase DCYTB in Human Caco-2/HT29 MTX Co-Cultures. Nutrients. 2018 Dec 8;10(12):1949. Salazar N, Valdés-Varela L, González S, Gueimonde M, de Los Reyes-Gavilán CG. Nutrition and the gut microbiome in the elderly. Gut Microbes. 2017 Mar 4;8(2):82–97. Gut microbiota: Changes throughout the lifespan from infancy to elderly | Request PDF [Internet]. [cited 2024 Jan 23]. Available from: https://www.researchgate.net/publication/223317175_Gut_microbiota_Changes_throughout_the_lifespan_from_infancy_to_elderly Asarat M, Apostolopoulos V, Vasiljevic T, Donkor O. Short-Chain Fatty Acids Regulate Cytokines and Th17/Treg Cells in Human Peripheral Blood Mononuclear Cells in vitro. Immunol Invest. 2016;45(3):205–22. Zakrzewska Z, Zawartka A, Schab M, Martyniak A, Skoczeń S, Tomasik PJ, et al. Prebiotics, Probiotics, and Postbiotics in the Prevention and Treatment of Anemia. Microorganisms. 2022 Jun 30;10(7):1330. Vonderheid SC, Tussing-Humphreys L, Park C, Pauls H, OjiNjideka Hemphill N, LaBomascus B, et al. A Systematic Review and Meta-Analysis on the Effects of Probiotic Species on Iron Absorption and Iron Status. Nutrients. 2019 Dec 3;11(12):2938. Rusu IG, Suharoschi R, Vodnar DC, Pop CR, Socaci SA, Vulturar R, et al. Iron Supplementation Influence on the Gut Microbiota and Probiotic Intake Effect in Iron Deficiency—A Literature-Based Review. Nutrients. 2020 Jul 4;12(7):1993. Tolkien Z, Stecher L, Mander AP, Pereira DIA, Powell JJ. Ferrous sulfate supplementation causes significant gastrointestinal side-effects in adults: a systematic review and meta-analysis. PLoS One. 2015;10(2):e0117383. Busti F, Campostrini N, Martinelli N, Girelli D. Iron deficiency in the elderly population, revisited in the hepcidin era. Front Pharmacol. 2014;5:83. Debnath A, Rehman T, Ghosh T, Kaur A, Ahamed F. Prevalence of Anemia Among Elderly Population Residing in an Urban Area of West Bengal: A Community-Based Cross-Sectional Analytical Study. Indian Journal of Community Medicine. 2022 Dec;47(4):604. Yadav K, Kant S, Ramaswamy G, Ahamed F, Jacob OM, Vyas H, et al. Validation of Point of Care Hemoglobin Estimation Among Pregnant Women Using Digital Hemoglobinometers (HemoCue 301 and HemoCue 201+) as Compared with Auto-Analyzer. Indian J Hematol Blood Transfus. 2020 Apr;36(2):342–8. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4320990","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Study protocol","associatedPublications":[],"authors":[{"id":297198963,"identity":"4a068df9-8661-4bac-8ddc-93a6b5110bfd","order_by":0,"name":"Farhad Ahamed","email":"data:image/png;base64,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","orcid":"","institution":"AIIMS Kalyani","correspondingAuthor":true,"prefix":"","firstName":"Farhad","middleName":"","lastName":"Ahamed","suffix":""},{"id":297198969,"identity":"485222c3-651d-4ceb-8eff-6884964a874c","order_by":1,"name":"Semanti Das","email":"","orcid":"","institution":"AIIMS Kalyani","correspondingAuthor":false,"prefix":"","firstName":"Semanti","middleName":"","lastName":"Das","suffix":""},{"id":297198973,"identity":"e9661347-8cb9-48d6-a731-22e1d5d0b690","order_by":2,"name":"Sunom Merab Lepcha","email":"","orcid":"","institution":"AIIMS Kalyani","correspondingAuthor":false,"prefix":"","firstName":"Sunom","middleName":"Merab","lastName":"Lepcha","suffix":""},{"id":297198977,"identity":"95ffe9ff-7630-4ee8-9485-6579695d2597","order_by":3,"name":"Abhishek Jaiswal","email":"","orcid":"","institution":"ESI Medical College and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Abhishek","middleName":"","lastName":"Jaiswal","suffix":""},{"id":297198981,"identity":"b6f23123-30a7-40c2-b39b-0a12cb68d754","order_by":4,"name":"Sarika Palepu","email":"","orcid":"","institution":"AIIMS Kalyani","correspondingAuthor":false,"prefix":"","firstName":"Sarika","middleName":"","lastName":"Palepu","suffix":""},{"id":297198984,"identity":"df77d0e6-d412-42b7-ac39-6b45675dd44e","order_by":5,"name":"Kalyan Goswami","email":"","orcid":"","institution":"AIIMS Kalyani","correspondingAuthor":false,"prefix":"","firstName":"Kalyan","middleName":"","lastName":"Goswami","suffix":""},{"id":297198987,"identity":"d52566fa-df0e-4017-8340-faaee3ca6159","order_by":6,"name":"Mallika Sengupta","email":"","orcid":"","institution":"AIIMS Kalyani","correspondingAuthor":false,"prefix":"","firstName":"Mallika","middleName":"","lastName":"Sengupta","suffix":""},{"id":297198990,"identity":"0d686483-68e3-4ab9-96bc-170da6e1dcf9","order_by":7,"name":"Arkapal Bandyopadhyay","email":"","orcid":"","institution":"AIIMS Kalyani","correspondingAuthor":false,"prefix":"","firstName":"Arkapal","middleName":"","lastName":"Bandyopadhyay","suffix":""},{"id":297198993,"identity":"3f1f0399-1a3f-4a03-9432-16255cf3a741","order_by":8,"name":"Chowdhury Mobaswar Hossain","email":"","orcid":"","institution":"Maulana Abul Kalam Azad University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Chowdhury","middleName":"Mobaswar","lastName":"Hossain","suffix":""}],"badges":[],"createdAt":"2024-04-25 02:38:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4320990/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4320990/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56016053,"identity":"788bebe6-ca22-4411-9f7c-b9da5a737cef","added_by":"auto","created_at":"2024-05-07 15:15:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":731782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eStudy flow chart\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4320990/v1/bab1e7044b0c6ab711775ab6.png"},{"id":63804181,"identity":"897c0e25-94a0-4d70-9e22-ae41c64a9cbb","added_by":"auto","created_at":"2024-09-02 13:27:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1288586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4320990/v1/3e0c270d-d0e4-45ab-bef4-c5c09938d839.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of supplementation of a package of iron folic acid (IFA) with probiotics on hemoglobin levels in elderly with mild to moderate anemia: Protocol for a double blind randomized controlled trial (PRobiotics in Anemia Trial for Healthy Ageing Management - PRATHAM)","fulltext":[{"header":"Background","content":"\u003cp\u003ePopulation aging is an unavoidable demographic transition. The rapid increase of the elderly population (aged 60 years and above) has become a global phenomenon. By 2050, the proportion of elderly population in the word is expected to rise to 22% from 12% of the total population in 2015 and 80% of this population is expected to belong to low and middle-income countries.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e In India, while the general population grew by 18% and 12.4% in each decade from 2001; the elderly population grew by 36% in each of the last two decades. India's elderly population count is projected to rise by 56\u0026nbsp;million in the next decade. \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAnemia is a common, multi factorial condition among elderly. The prevalence of anemia increases with age, representing an important health problem among older individuals in India and in the world. \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Unfortunately, many symptoms of anemia like weakness, fatigue, and shortness of breath are often misdiagnosed as normal phenomena in elderly individuals. Anemia of chronic disease (ACD) and nutritional anemia especially due to iron deficiency (IDA) are the two most common causes of anemia in the elderly population and these two conditions are often found to coexist.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Anemia has been reported to be associated independently with decreased functional ability/physical function, increased dementia, and increased risk of falls, morbidity, and mortality in the elderly population.\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Thus, anemia among the elderly population affects their health-related quality of life and increases their mortality.\u003c/p\u003e \u003cp\u003eHost-microbiota metabolic crosstalk has been suggested to play an important role in systemic iron homeostasis.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e It increases the expression of DMT1 (iron transporter in the gut) and DcytB (Ferric reductase) (via the HIF-2α pathway) located at the enterocyte apical brush border membrane which reduces ferric iron (III) to absorbable ferrous iron (II). \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e These two factors ensure better iron absorption in the gut and reduce the negative side effects of oral iron supplementation. In conditions of less iron absorption from orally administered IFA tablets, the excess unabsorbed iron passes through the colon and gets involved in the Fenton \u0026amp; Haber-Weiss reaction, which not only alters composition of intestinal flora but also increases the inflammatory factors, adding to any existing background inflammatory conditions which is common in this age group. \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e The protective organisms of gut microbiota are believed to play a prominent role in increasing the bio-availability of dietary iron by decreasing the amount of iron binding compounds (MUC5AC) in the gut.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIt has now been established that gut microbiota in elderly individuals suffer gradual decline in beneficial microorganisms (\u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eAcenobacteria\u003c/em\u003e) and increase of facultative anaerobic bacteria (\u003cem\u003eProteobacteria\u003c/em\u003e).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Reduced dentition, chewing strength and loss of appetite also lead to limited intake of food ingredients which support the microbial diversity. \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e These changes are responsible for a decrease in short chain fatty acid (SCFA) production. SCFA protect against pathogens and exert anti-inflammatory properties including upregulation of anti-inflammatory cytokines.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Thus, host microbiota interaction plays an important role in iron absorption and utilization for heme synthesis. Intact gut microbiota has also been found to reduce iron absorption (gut microbial metabolites 1,3-diaminopropane (DAP) and reuterin) in iron excess body state and store excess iron via ferritin regulation thereby preventing harmful effect of iron. \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eProbiotics are live microbial feed supplements which beneficially affect the host animal by improving gut microbial balance.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Probiotics supplementation has shown promising results in improving hemoglobin level when combined with oral iron supplementation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e through immunomodulation, anti-inflammatory response, formation of bioavailable ferrous form by reduction of ferric iron, enhanced iron uptake by enterocytes and replenishment of the gut microbiota with good bacteria (\u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e). \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Further, probiotics protect the gut microbiota from harmful effects of unabsorbed oral iron. \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn relation to the above observations, we hypothesize that supplementation of probiotics along with IFA will have positive effect on hemoglobin level in elderly without harming the gut microbiota and thereby improve their quality of life.\u003c/p\u003e \u003cp\u003eFew studies have been done so far to see the effect of probiotics on iron absorption and iron status. \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Although very few studies have seen the effect of supplementation of IFA and probiotics together, majority of them were not adequately powered to see the effect.\u003c/p\u003e \u003cp\u003eThe current study aims to determine the impact of supplementation of a therapeutic package of IFA and probiotics (intervention group) on hemoglobin level (from base line to 12 weeks) in elderly with mild to moderate anemia in comparison to IFA only (control group). It shall also assess the impact on ferritin concentration, hepcidin level, CRP level, incidence of acute infections, composition and diversity of the gut microbiome from base line to 12 weeks in both the groups.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eTrial design\u003c/b\u003e: This will be a community based, placebo controlled, double-blind, randomized controlled trial. Groups will be parallel; allocation ratio will be 1:1 and the framework will be superiority.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEligibility criteria\u003c/b\u003e: Individuals aged 60 years and above, a permanent resident of the area and expected to stay in the area for another six months, who consent to participate in the study shall be eligible for the study. Individuals screened to have mild to moderate anemia and who shall not have taken any probiotic or antibiotics in the past 30 days prior to enrolment will be included in the study. Individuals with no anemia (Male: Hemoglobin\u0026thinsp;\u0026ge;\u0026thinsp;13gm/dL and Female: Hemoglobin\u0026thinsp;\u0026ge;\u0026thinsp;12gm/dL), severe anemia (Hemoglobin\u0026thinsp;\u0026lt;\u0026thinsp;8gm/dL), known history of gastrointestinal \u0026amp; metabolic disorders, chronic renal failure, history of receiving blood donations in last six months and regularly consuming nutritional supplements (Iron, folic acid, Vitamin B12, Vitamin D and other multivitamins and minerals), diagnosed pancreatitis, hypersensitivity to any ingredient in the supplement and individuals who shall have participated in any other clinical trial in the preceding 30 days will be excluded from the study.\u003c/p\u003e\n\u003ch3\u003eOperational definitions\u003c/h3\u003e\n\u003cp\u003e \u003cstrong\u003eAnemia\u003c/strong\u003e \u003cp\u003eHemoglobin level\u0026thinsp;\u0026lt;\u0026thinsp;13gm/dL in male and hemoglobin level\u0026thinsp;\u0026lt;\u0026thinsp;12gm/dL in female will be considered as anemia. Hemoglobin level\u0026thinsp;\u0026lt;\u0026thinsp;8 gm/dL will be considered as severe anemia in the both the sexes.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIron Deficiency Anemia (IDA)\u003c/strong\u003e \u003cp\u003eAnemia related to absolute iron deficiency (iron deficiency anemia, IDA) will be characterized by a decreased serum ferritin level (\u0026lt;\u0026thinsp;30 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL) in combination with low serum CRP levels (\u0026le;\u0026thinsp;0.5 mg/dL).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAnemia of Chronic Disease (ACD)\u003c/strong\u003e \u003cp\u003eACD will be defined by high ferritin levels (\u0026gt;\u0026thinsp;100 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL) and raised CRP (\u0026ge;\u0026thinsp;0.5 mg/dL).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompliance\u003c/strong\u003e \u003cp\u003eIndividuals who shall have a cumulative consumption\u0026thinsp;\u0026ge;\u0026thinsp;80% of both the tablets (IFA) and capsules (probiotics/placebo) at the end of the study period will be considered as compliant to the therapy.\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eIntervention\u003c/h3\u003e\n\u003cp\u003eThe eligible individuals will be randomized into intervention and control group. In the intervention arm, study participants will receive 60 mg of elemental oral iron and 500mcg of folic acid as IFA tablets and capsules containing probiotics daily for three months. The control group will receive 60 mg of elemental oral iron and 500mcg of folic acid as IFA tablets and placebo gelatin capsules containing ORS daily for 12 weeks. Probiotics will contain \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e. The tablets will be manufactured in certified laboratories.\u003c/p\u003e \u003cp\u003e \u003cb\u003eModifications\u003c/b\u003e- If any individual develops side effects associated with IFA supplementation like excessive vomiting even after consuming the medicine two hours after a major meal and without any other obvious reason, or major side effects related to probiotics, such as gastro-intestinal symptoms (nausea, abdominal pain, flatulence, bloating, unpleasant taste in the mouth, changes in stool, incontinence), flu-like or coryza like symptoms and unexplained arthralgia, fatigue, stress, headache and depression, he/she will be advised to discontinue the medicine.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdherence\u003c/b\u003e-An individual will be considered loss to follow-up if he/she could not be contacted on two repeated visits with a minimal interval of one week, has shifted the residence to other place permanently, died during the course of trial or could not be available for blood collection at the end of three months of trial will be considered as loss to follow-up.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConcomitant care\u003c/b\u003e - Participants requiring any other medical advice will be referred to AIIMS, Kalyani for further management.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eOutcomes-\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003ePrimary outcome:\u003c/h2\u003e \u003cp\u003eTo determine the change in hemoglobin level from base line to 12 weeks in elderly with mild to moderate anemia in intervention and control groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSecondary outcomes\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine the change in serum ferritin level from base line to 12 weeks in elderly with mild to moderate anemia in intervention and control groups.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine the change in hepcidin level from base line to 12 weeks in in elderly with mild to moderate anemia in intervention and control groups.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine the change in CRP level from base line to 12 weeks in elderly with mild to moderate anemia in intervention and control groups.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo determine the difference in incidence of acute infections from baseline to 12 weeks in elderly with mild to moderate anemia in intervention and control groups.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTo compare the composition and diversity of the gut microbiome in elderly among the intervention and control groups at the baseline and end line.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe study design is outlined in the form of a flowchart in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The details of the study are described subsequently.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSample size\u003c/strong\u003e \u003cp\u003eAssuming a superiority margin of 0.4 gm/dL, mean (SD) observed difference of 0.7 gm/dL between the two groups before and after the intervention \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, power 80%, alpha error of 5%, and attrition rate of 10%, we need to recruit 100 participants in each arm giving a total sample size of 200. The sample size was calculated using STATA version 17.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRecruitment-\u003c/h2\u003e \u003cp\u003eThe study setting being the urban field practice area of a medical college, the principal investigator has the list of households with elderly population. Out of 22,100 households, currently there are 2112 households which have elderly individuals. The prevalence of mild to moderate anemia among the study age group has been estimated around 60% in our earlier study.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e First, 1000 households will be selected randomly using computer generated random numbers. If more than two elderly individuals reside in a single household, one of them will be selected using the lottery method. The randomly selected elderly individuals will then be screened for hemoglobin level using HemoCue 301 device. This device has very high sensitivity and specificity as compared to the gold standard method.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIndividuals who will be screened as mild or moderately anemic and fulfilling other inclusion criteria will be requested for blood samples (5 ml). The blood sample will be drawn aseptically from the ante-cubital vein of the non-dominant hand of each participant and will be stored in plain vial. The sample will be carried to the institute on the same day. Serum will be separated from the blood sample at the laboratory of the department of Biochemistry in centrifuge machine at 3000 rpm. Written informed consent will be taken at the time of enrolment and again at the time of blood sample collection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRandomization\u003c/b\u003e: Before the start of the intervention, a third person not related to the study shall conduct block randomization (with a block size of four) in 1:1 ratio dividing the intervention and control arms in equal numbers. He/she will sort and pack the similar looking tablets and capsules into pouches which will be serially numbered according to the random sequence generated. The serial numbered pouches will be subdivided into tablets of 30 (IFA) and capsules of 30 (probiotics/placebo gelatin capsules containing ORS) each so that compliance can be measured on monthly basis. The investigator will be handed over only serial numbered pouches containing IFA tablets and probiotics/placebo gelatin capsules with ORS. The original content inside each pouch will only be known to the third person who generated the random sequence and did packaging. This will ensure allocation concealment and since the tablets/capsules which will be used in the study will look similar in all aspects, both the investigator and the study participants will be blinded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTreatment arms\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eIntervention group\u003c/strong\u003e \u003cp\u003eStudy participants in this group will receive 60 mg of elemental oral iron and 500mcg of folic acid as IFA tablets and capsules containing probiotics daily for three months. The participants will be asked to take the probiotics capsules thirty minutes before breakfast and IFA capsules one hour before lunch. The field investigators will visit every study participant on weekly basis to record the side-effects. Compliance will be calculated on monthly basis. Participant consuming 80% of the doses at the end of each month would be considered as compliant. Participants developing signs of gastric discomfort would be advised to take IFA tablets 2 hours after meals. If the participant develops any other untoward events, complete evaluation would be done and required modifications in treatment regimens would be made.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eControl group\u003c/strong\u003e \u003cp\u003eStudy participants in this group will receive 60 mg of elemental oral iron and 500mcg of folic acid as IFA tablets and placebo gelatin capsules containing ORS daily for 12 weeks. The participant will be asked to take the placebo capsules before breakfast and IFA capsules one hour before lunch. The field investigators will visit every study participant on a weekly basis to record the side-effects. Compliance will be calculated on monthly basis. Participant consuming 80% of the doses at the end of each month would be considered as compliant. Participants developing signs of gastric discomfort would be advised to take IFA tablets 2 hours after meals. If the participant develops any other untoward events, complete evaluation would be done and required modifications in treatment regimens would be made.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDrugs\u003c/strong\u003e \u003cp\u003eThe drugs required for the study are; 1. Iron and folic acid tablets containing 60 mg of elemental iron and 500 \u0026micro;g of folic acid; 2. Probiotics containing (\u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e) 3. Placebo gelatin capsules containing Oral Rehydration Salt (ORS) similar to intervention drug in size, shape and colour. The tablets will be manufactured in certified laboratories.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eData collection\u003c/strong\u003e \u003cp\u003eBaseline socio-demographic information will be collected using pre-tested semi structured interview schedule from all included individuals.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe hemoglobin level will be measured by HemoCue 301 using capillary blood. Five millilitre (5 ml) of blood will be withdrawn aseptically from ante-cubital vein from each participant and will be stored in plain vial. The sample will be carried to the centralized institute on the same day. Serum will be separated from the blood sample at the laboratory of the department of Biochemistry in centrifuge machine at 3000 rpm. The separated serum will be stored at -20\u003csup\u003e0\u003c/sup\u003eC. Initially serum hepcidin, ferritin and CRP will be assessed. All the test will be done at the department of Biochemistry. All the tools will be calibrated before and during estimation of hematological and biochemical parameter. The laboratory has their own internal and external quality control measures. The participants will be provided with two sterile containers (with and without RNALater) to collect at least 2 grams of stool samples in each container. The containers will be transported at 4˚C to microbiology laboratory of AIIMS Kalyani and then frozen at -80˚C. The isolation of DNA and RNA will be done. 16S rRNA amplicon sequencing will be performed using Illumina MiSeq platform and analysed using the MGnify pipeline. The Genome Taxonomy Database (GTDB-Tk) will be used for bacterial taxonomic identification.\u003c/p\u003e \u003cp\u003eField investigators will visit study participants on weekly basis to collect data on side-effects. They will also provide the drugs to the study participants on monthly basis and collect the pouches given in the earlier visit. Compliance on monthly basis. Each participant will be followed-up for 12 weeks. The field investigators will be blinded about the group (intervention/control) of the study participants.\u003c/p\u003e \u003cp\u003eThe Hb level will be measured using HemoCue 301 after the completion of intervention. Five millilitres (5 ml) of blood will be drawn aseptically from each participant and stored in plain vial and will transport, processing, and finally storage as done for the baseline samples. This stored serum will be used for measurement of serum Hepcidin, Ferritin and CRP at the laboratory of the department of Biochemistry. The technique for biomarker (Ferritin, CRP, and hepcidin) and gut microbiome diversity assessment, calibration, internal quality, and external quality assessment will be similar as stated for the baseline samples. The team which will analyse the sample will be unaware about the group (intervention/control) of the study participants. (Refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWe will be assessing any side-effects with routine visits every week. Participants developing any adverse effect will be treated at AIIMS, Kalyani, West Bengal, India.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStudy tools\u003c/strong\u003e \u003cp\u003eHemoglobin will be measured using HemoCue 301 instrument. Hepcidin will be measured in ELISA method. CRP will be measured in bio-chemical autoanalyser. Ferritin will be measured using chemiluminescence technique. 16S rRNA amplicon sequencing will be performed using Illumina MiSeq platform and analysed using the MGnify pipeline.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStudy Duration\u003c/strong\u003e \u003cp\u003eThe study will be completed in a duration of 12 months. The project will have three phases namely i) Preparatory Phase (3 months), ii) Implementation Phase (6 months) and iii) Data Analysis and Final Report Writing Phase (3 months).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePreparatory phase\u003c/strong\u003e \u003cp\u003eRecruitment of staff, project manual development, procurement of survey logistics, development of study questionnaire, training of field staff on data collection, quality control and sample collection will be done. The packaging of drugs and random sequence generation by a third person not related to the design, conduct or analysis of the study will also be done.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eImplementation phase\u003c/strong\u003e \u003cp\u003eScreening, enrolment, randomization, intervention and biochemical analysis of the sample will be done during this phase.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eData analysis\u003c/strong\u003e \u003cp\u003eThe data will be collected in Epicollect 5 mobile based app and exported in Microsoft Excel version 13. The data will be analysed with the help of STATA 17 statistical software. Categorical data will be presented as percentage (%). Normally, distributed data will be presented as means and standard deviation or 95% confidence interval (CI). Pearson\u0026rsquo;s Chi-square test will be used to evaluate differences between groups for categorical variables. Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for independent samples will be used for comparison between groups with continuous variables and paired \u003cem\u003et\u003c/em\u003e-test will be used for comparison of before and after changes. Both per-protocol and intention to treat analysis will be done. Changes in richness and evenness, Faith\u0026rsquo;s phylogenetic diversity (PD), and Shannon diversity will be measured between month 0 and 6 and compared to the placebo group using a Wilcoxon Rank Sum Test and a Holm-Bonferroni method with family-wise error rate P-value correction. Weighted UniFrac,Bray-Curtis dissimilarity, and unweighted UniFrac will be used for beta diversity analysis to identify any structural changes in the gut microbiome of trial participants. Microbiome differential abundance will be assessed. A \u0026ldquo;p\u0026rdquo; value\u0026thinsp;\u0026le;\u0026thinsp;0.05 will be considered as statistically significant.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study protocol describes a randomized controlled trial to assess the impact of supplementation of a package of iron folic acid (IFA) with probiotics on hemoglobin levels in elderly with mild to moderate anemia. Correction of anemia in the elderly is slower. Thus, only iron supplementation may not be effective in improving iron levels. Host-microbiota has been hypothesized to play an important role in regulating iron absorption through immunomodulation, anti-inflammatory response, formation of bioavailable ferrous form by reduction of ferric iron, enhanced iron uptake by enterocytes and replenishment of the gut microbiota with good bacteria. The need of the hour, especially in a country with a huge burden of anemia, is to improve the response to iron supplementation. Co-supplementation of iron folic acid tablets with probiotics have shown promising results.\u003c/p\u003e \u003cp\u003eThe study shall involve iron folic acid supplementation to mild and moderately anemic participants aged more than 60 years in one group and iron and folic acid and probiotic supplementation in the other and following them up for 12 weeks. The outcome shall involve assessing for changes in hemoglobin levels as the primary outcome and changes in ferritin, CRP and hepcidin level as the secondary outcome. Frequency of acute infections and changes in the gut microbiome shall also be measured.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIFA- Iron folic acid\u003c/p\u003e\n\u003cp\u003eCRP-C-reactive protein\u003c/p\u003e\n\u003cp\u003eACD-Anemia of chronic disease\u003c/p\u003e\n\u003cp\u003eIDA-Iron Deficiency Anemia\u003c/p\u003e\n\u003cp\u003eSCFA\u003cstrong\u003e-\u003c/strong\u003e Short chain\u0026nbsp;fatty acid\u003c/p\u003e\n\u003cp\u003eGTDB-Tk -Genome Taxonomy Database\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCI- confidence interval\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAIIMS, Kalyani Ethics Committee clearance will be obtained and the guidelines pertaining to the participant information sheet (PIS) and Participant informed consent form (PICF) will be followed. Participant Information sheet will be given to each study participant. PIS will be in the local language (Bengali) and will be provided and read out to each study participant before their recruitment. A written informed consent will be obtained from each study participant willing to participate in the study, by the field investigator. Participants with requiring any other medical advice will be referred to AIIMS, Kalyani for further management. Participants will be provided with the following information a) Expected duration of participation in study, b) The benefits to be expected from the research to the study participants, c) Any risk to the study participant associated with the study, d) Maintenance of confidentiality of records. Participant will have freedom to participate or withdraw from study at any time without penalty or loss of benefits to which the study participant would otherwise be entitled.\u003c/p\u003e\n\u003cp\u003eThe trial results will be communicated with the participant through publication in a reputed journal and through health talk with the communities. Any changes in the protocol during the trial will be informed in advance to the ethical committee of AIIMS, Kalyani, and the same will also be communicated with the Journal after the publication of the initial protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eConsent obtained directly from patient(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial Registration Number:\u0026nbsp;\u003c/strong\u003eThe trial was registered in Clinical Trials Registry-India (CTRI) (CTRI/2023/09/057830) on 19.09.2023\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe project will be funded by the Department of Health Research, Ministry of Health and Family Welfare, Government of India under extramural grants (R.11014/07/2023-GIA/HR)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u0026nbsp;\u003c/strong\u003eFA is the guarantor, drafted the manuscript and is the corresponding author.\u003c/p\u003e\n\u003cp\u003eFA, SD, SML, SP, KG, MS, AB, and CMH developed the trial concept and drafted the protocol manuscript. FA developed the trial concept and revised the manuscript critically for important intellectual content. All listed authors approved the final version of the manuscript for publication and agreed to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgeing and health [Internet]. [cited 2024 Jan 23]. Available from: https://www.who.int/news-room/fact-sheets/detail/ageing-and-health\u003c/li\u003e\n\u003cli\u003eElderly in India 2021.pdf [Internet]. [cited 2024 Jan 23]. Available from: https://mospi.gov.in/sites/default/files/publication_reports/Elderly%20in%20India%202021.pdf\u003c/li\u003e\n\u003cli\u003eVadakattu SS, Ponday LR, Nimmathota A, Nagalla B, Kondru DS, Undrajavarapu P, et al. Prevalence of Nutritional Anemia and Hyperhomocysteinemia in Urban Elderly. Indian J Clin Biochem. 2019 Jul;34(3):330\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eGupta A, Ramakrishnan L, Pandey RM, Sati HC, Khandelwal R, Khenduja P, et al. Risk factors of anemia amongst elderly population living at high-altitude region of India. J Family Med Prim Care. 2020 Feb 28;9(2):673\u0026ndash;82. \u003c/li\u003e\n\u003cli\u003eMcLean E, Cogswell M, Egli I, Wojdyla D, de Benoist B. Worldwide prevalence of anaemia, WHO Vitamin and Mineral Nutrition Information System, 1993-2005. Public Health Nutr. 2009 Apr;12(4):444\u0026ndash;54. \u003c/li\u003e\n\u003cli\u003eGuralnik JM, Eisenstaedt RS, Ferrucci L, Klein HG, Woodman RC. Prevalence of anemia in persons 65 years and older in the United States: evidence for a high rate of unexplained anemia. Blood. 2004 Oct 15;104(8):2263\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eEisenstaedt R, Penninx BWJH, Woodman RC. Anemia in the elderly: current understanding and emerging concepts. Blood Rev. 2006 Jul;20(4):213\u0026ndash;26. \u003c/li\u003e\n\u003cli\u003eDenny SD, Kuchibhatla MN, Cohen HJ. Impact of anemia on mortality, cognition, and function in community-dwelling elderly. Am J Med. 2006 Apr;119(4):327\u0026ndash;34. \u003c/li\u003e\n\u003cli\u003eHong CH, Falvey C, Harris TB, Simonsick EM, Satterfield S, Ferrucci L, et al. Anemia and risk of dementia in older adults: findings from the Health ABC study. Neurology. 2013 Aug 6;81(6):528\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eDas NK, Schwartz AJ, Barthel G, Inohara N, Liu Q, Sankar A, et al. Microbial Metabolite Signaling Is Required for Systemic Iron Homeostasis. Cell Metabolism. 2020 Jan 7;31(1):115-130.e6. \u003c/li\u003e\n\u003cli\u003eSchwartz AJ, Das NK, Ramakrishnan SK, Jain C, Jurkovic MT, Wu J, et al. Hepatic hepcidin/intestinal HIF-2\u0026alpha; axis maintains iron absorption during iron deficiency and overload. J Clin Invest. 2019 Jan 2;129(1):336\u0026ndash;48. \u003c/li\u003e\n\u003cli\u003eXu MM, Wang J, Xie JX. Regulation of iron metabolism by hypoxia-inducible factors. Sheng Li Xue Bao. 2017 Oct 25;69(5):598\u0026ndash;610. \u003c/li\u003e\n\u003cli\u003eMalesza IJ, Bartkowiak-Wieczorek J, Winkler-Galicki J, Nowicka A, Dzięciołowska D, Błaszczyk M, et al. The Dark Side of Iron: The Relationship between Iron, Inflammation and Gut Microbiota in Selected Diseases Associated with Iron Deficiency Anaemia-A Narrative Review. Nutrients. 2022 Aug 24;14(17):3478. \u003c/li\u003e\n\u003cli\u003eSandberg AS, \u0026Ouml;nning G, Engstr\u0026ouml;m N, Scheers N. Iron Supplements Containing Lactobacillus plantarum 299v Increase Ferric Iron and Up-regulate the Ferric Reductase DCYTB in Human Caco-2/HT29 MTX Co-Cultures. Nutrients. 2018 Dec 8;10(12):1949. \u003c/li\u003e\n\u003cli\u003eSalazar N, Vald\u0026eacute;s-Varela L, Gonz\u0026aacute;lez S, Gueimonde M, de Los Reyes-Gavil\u0026aacute;n CG. Nutrition and the gut microbiome in the elderly. Gut Microbes. 2017 Mar 4;8(2):82\u0026ndash;97. \u003c/li\u003e\n\u003cli\u003eGut microbiota: Changes throughout the lifespan from infancy to elderly | Request PDF [Internet]. [cited 2024 Jan 23]. Available from: https://www.researchgate.net/publication/223317175_Gut_microbiota_Changes_throughout_the_lifespan_from_infancy_to_elderly\u003c/li\u003e\n\u003cli\u003eAsarat M, Apostolopoulos V, Vasiljevic T, Donkor O. Short-Chain Fatty Acids Regulate Cytokines and Th17/Treg Cells in Human Peripheral Blood Mononuclear Cells in vitro. Immunol Invest. 2016;45(3):205\u0026ndash;22. \u003c/li\u003e\n\u003cli\u003eZakrzewska Z, Zawartka A, Schab M, Martyniak A, Skoczeń S, Tomasik PJ, et al. Prebiotics, Probiotics, and Postbiotics in the Prevention and Treatment of Anemia. Microorganisms. 2022 Jun 30;10(7):1330. \u003c/li\u003e\n\u003cli\u003eVonderheid SC, Tussing-Humphreys L, Park C, Pauls H, OjiNjideka Hemphill N, LaBomascus B, et al. A Systematic Review and Meta-Analysis on the Effects of Probiotic Species on Iron Absorption and Iron Status. Nutrients. 2019 Dec 3;11(12):2938. \u003c/li\u003e\n\u003cli\u003eRusu IG, Suharoschi R, Vodnar DC, Pop CR, Socaci SA, Vulturar R, et al. Iron Supplementation Influence on the Gut Microbiota and Probiotic Intake Effect in Iron Deficiency\u0026mdash;A Literature-Based Review. Nutrients. 2020 Jul 4;12(7):1993. \u003c/li\u003e\n\u003cli\u003eTolkien Z, Stecher L, Mander AP, Pereira DIA, Powell JJ. Ferrous sulfate supplementation causes significant gastrointestinal side-effects in adults: a systematic review and meta-analysis. PLoS One. 2015;10(2):e0117383. \u003c/li\u003e\n\u003cli\u003eBusti F, Campostrini N, Martinelli N, Girelli D. Iron deficiency in the elderly population, revisited in the hepcidin era. Front Pharmacol. 2014;5:83. \u003c/li\u003e\n\u003cli\u003eDebnath A, Rehman T, Ghosh T, Kaur A, Ahamed F. Prevalence of Anemia Among Elderly Population Residing in an Urban Area of West Bengal: A Community-Based Cross-Sectional Analytical Study. Indian Journal of Community Medicine. 2022 Dec;47(4):604. \u003c/li\u003e\n\u003cli\u003eYadav K, Kant S, Ramaswamy G, Ahamed F, Jacob OM, Vyas H, et al. Validation of Point of Care Hemoglobin Estimation Among Pregnant Women Using Digital Hemoglobinometers (HemoCue 301 and HemoCue 201+) as Compared with Auto-Analyzer. Indian J Hematol Blood Transfus. 2020 Apr;36(2):342\u0026ndash;8. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anemia, elderly, iron supplementation, probiotics, randomized controlled trial, Gut microbiome","lastPublishedDoi":"10.21203/rs.3.rs-4320990/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4320990/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnemia is a major health concern of the elderly with iron deficiency anemia and anemia of chronic diseases being the two major causes of anemia. Probiotics help in anemia correction in several ways. It increases oral iron absorption by directly modulating immunity, eliciting an anti-inflammatory response, reducing ferric iron to bioavailable ferrous iron, and enhancing iron uptake by enterocytes. This is especially relevant in old age, as aging is associated with age-related gradual attrition of protective organisms in gut microbiota. As there are no studies on the efficacy of probiotics supplementation in anemia management in the elderly, a study has been planned to determine the impact of supplementation of a therapeutic package of Iron and Folic Acid (IFA) and probiotics on hemoglobin levels in elderly with mild to moderate anemia in comparison to IFA only.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis community-based randomized controlled trial will be conducted in the Kalyani Municipality area of Nadia district, West Bengal, India. Individuals aged 60 years and above with mild to moderate anemia will be randomized into the intervention (IFA and probiotics) and control group (IFA and placebo gelatin capsules containing ORS). The medications will be self-administered. The calculated sample size is 100 in each arm. Block randomization will be done. Participants will be followed up weekly for 12 weeks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChanges in hemoglobin, ferritin, hepcidin, C-reactive protein (CRP), composition, and diversity of the gut microbiome from baseline to 12 weeks in both groups will be assessed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and dissemination: \u003c/strong\u003eEthical approval was obtained from the Institutional Ethics Committee of AIIMS Kalyani (Ref. No: IEC /AIIMS / Kalyani / Meeting/ 2023 / 020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial Registration Number: \u003c/strong\u003eThe trial was registered in Clinical Trials Registry-India (CTRI) (CTRI/2023/09/057830) on 19.09.2023\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtocol version: Version: \u003c/strong\u003e1.0\u003c/p\u003e","manuscriptTitle":"Impact of supplementation of a package of iron folic acid (IFA) with probiotics on hemoglobin levels in elderly with mild to moderate anemia: Protocol for a double blind randomized controlled trial (PRobiotics in Anemia Trial for Healthy Ageing Management - PRATHAM)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-07 15:15:12","doi":"10.21203/rs.3.rs-4320990/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"302a9fb2-6498-462e-9fd7-6508044fcc5f","owner":[],"postedDate":"May 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T13:19:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-07 15:15:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4320990","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4320990","identity":"rs-4320990","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}