ASSESSING THE EFFECTS OF MYCOTOXIN EXPOSURE ON THE MATERNAL-FETAL DYAD

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1000601
• Grant No.: 2013-67011-21116
• Project No.: NYC-399575
• Proposal No.: 2013-03169
• Program Code: A7101
• Project Start Date: Sep 1, 2013
• Project End Date: Aug 31, 2015
• Grant Year: 2013

Project Director
Smith, L. E.

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Div. of Nutritional Sciences

Non Technical Summary
Aflatoxins pose a threat to the safety of the US and global food supply, and may adversely affect the health of mothers and the health and development of their children. The overall goal of the proposed research is to inform public health and food safety policy in the United States and internationally by describing the effect of aflatoxin exposure on the health and wellbeing of mothers and their infants. This pre-doctoral fellowship research proposal specifically addresses the AFRI Challenge Area Food Safety and the AFRI foundational program Food Safety, Nutrition and Health by focusing on satisfying human food needs while enhancing the quality of life for society as a whole. Understanding the effects of aflatoxin is important in mitigating risks to vulnerable populations as US and global agriculture strives to meet food and feed needs. We will explore the relationship between maternal mycotoxin exposure, maternal anemia and birth outcomes by assessing aflatoxin exposure during pregnancy in 4800 rural Zimbabwean mothers. The proposed research will generate new knowledge of the biological effects of aflatoxin exposure during pregnancy to inform the development of domestic and international food safety policy, as well as interventions to improve maternal and child health. This research supports US priorities in agriculture, food safety, nutrition and health, and additionally supports the Feed the Future goal to sustainably reduce global poverty and hunger and the Centers for Disease Control, March of Dimes and National Institutes of Health focus on pre-term birth.

Animal Health Component

0%

Research Effort Categories

Basic

0%

Applied

100%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
723	6099	1170	70%
723	6099	1010	10%
723	6099	1150	20%

Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
6099 - People and communities, general/other;

Field Of Science
1170 - Epidemiology; 1010 - Nutrition and metabolism; 1150 - Toxicology;

Keywords

aflatoxin

anemia

fetal development

maternal and child health

mycotoxin

preterm birth

Goals / Objectives
Career Goals 1) Develop research skills bridging basic laboratory biological science and applied population-level field studies 2) To further develop my grant writing skills 3) To gain additional experience implementing community-based intervention trials Research Goals 1) To describe the distribution of aflatoxin exposure in pregnant women in rural Zimbabwe 2) To test the hypothesis that aflatoxin exposure is associated with maternal anemia 3) To test the hypothesis that aflatoxin exposure is associated adverse birth outcomes including fetal loss, IUGR, and pre-term birth

Project Methods
I plan to explore the relationship between maternal mycotoxin exposure, maternal anemia and birth outcomes by assessing AF exposure at two timepoints during pregnancy in 4800 rural Zimbabwean mothers. The biomarker for aflatoxin exposure, the aflatoxin-albumin adduct represents a 2-3 month exposure window. The first timepoint (3 months) will capture exposure during the first trimester of pregnancy and the second timepoint (7 months) will capture exposure during the 2nd and 3rd trimesters of pregnancy, allowing investigation of the effect of the level of exposure as well as the effect of timing of exposure during gestation. This research will be nested within the SHINE trial which aims to determine the independent and combined impact of improved sanitation/hygiene (i.e., the WASH Intervention) and improved infant feeding (i.e., the nutrition intervention) on linear growth and anemia in 4800 children from birth through 18 months in a cohort of mothers enrolled during early pregnancy. The project will be a community-based, cluster-randomized trial among all households in which a woman becomes pregnant during the recruitment period. The project will be conducted in two rural districts (Chirumanzu and Shurugwi) comprising a total population of ~180,000 where the primary livelihood is subsistence farming. Only 7% of Zimbabwean women are classified as underweight, yet stunting and micronutrient deficiencies are common in this population. AFB1-albumin ELISA assay will be validated and conducted on 4800 pregnant women at 10-12 weeks gestation and 7 months gestation at the Zvitambo laboratory in Harare, Zimbabwe. We will use the procedure previously described by Wild et al. In brief, we will extract albumin from 50 µL plasma and quantify AFB1-albumin by competitive ELISA against an AFB1-albumin standard. A random sample of maternal samples at each timepoint (200 samples total) will be selected to conduct LC-MS validation at the AiBMST. LC-MS quantification of AFB1-albumin will follow the procedure described by Scholl et al. I propose to analyze potential determinants and outcomes of AF exposure at two timepoints during pregnancy representing exposure during all trimesters. Seasonality, harvest practices and geographical location will be assessed as potential determinants of AF exposure. Primary outcomes include maternal anemia (assessed by hemoglobin), miscarriage, chronic IUGR (short-for-gestational-age), acute IUGR (small-for-gestational-age), spontaneous pre-term birth (birth before 37 weeks) and stillbirth. Additionally I will describe the variation of exposure in this population (Aim1). Aims 2 and 3 will test for associations between exposure and adverse outcomes. I will consider covariates (including dietary diversity, food security, socioeconomic status (SES) randomization group, HIV status, dietary diversity, iron status, malaria and other maternal infections) as potential confounders and/or effect modifiers. Aim 1: To describe the distribution of Aflatoxin in Pregnant Women in Rural Zimbabwe: My first step will be to describe patterns of exposure across seasons as well as potential patterns in gestation (10/12 weeks vs. 7 months) and geographical variation across the study districts. I will explore potential cross-sectional associations between AF exposure, mother's health status, post-harvest practices, socio-economic status, dietary diversity and food security utilizing the comprehensive baseline survey that will be conducted in all study participants.. Aim 2: Test of the hypothesis that aflatoxin exposure is associated with maternal anemia: I will test this hypothesis using multivariate regression models, with hemoglobin/anemia as the outcome variable and mycotoxin exposure at baseline as the independent variable along with covariates. Maternal anemia will be defined as Hemoglobin < 120 g/L assessed using the Hemocue hemoglobinometer at baseline. Aim 3: Test of the hypothesis that aflatoxin is associated with IUGR and adverse birth outcomes: I further hypothesize that the association will depend on the timing of exposure. Aflatoxin exposure during the first trimester could lead to miscarriage as well as both short-for-gestational age and small-for-gestational age. Aflatoxin exposure during later pregnancy could cause acute IUGR (small-for-gestational age), spontaneous preterm birth and stillbirth. First, I will investigate whether there is an association between AF exposure and birthweight treated as a continuous variable. Second, I will use multinomial logistic regression including outcomes of normal birth, short-for-gestational age, small-for-gestational age, miscarriage, stillbirth and pre-term birth modeled as dichotomous variables.

Progress 09/01/13 to 08/31/15

Outputs
Target Audience:During the first reporting period, I communicated research findings and implications to academic audiences, policy and government and development agencies. Main audiences and activities included: Cornell University- Gave presentation to a broad audience of academics and students at the Cornell IGERT Symposium: Food systems and poverty reduction titled, "Seasonal Variation in Aflatoxin Exposure in Rural Zimbabwe." Zimbabwe Ministry of Health and Childcare- Provided a written report of preliminary research findings to the Ministry titled, "Preliminary Report: Aflatoxin exposure among pregnant women participating in the SHINE study in Shurugwi and Chirumanzu districts" DFID- Presentation to DFID program reviewers about the importance of measuring mycotoxin exposure in the context of the SHINE trial. FAO- Presentation to FAO country representatives about the importance of measuring mycotoxin exposure in the context of the SHINE trial. Discussion about the implications of the current research for informing future interventions to decrease mycotoxin exposure in vulnerable populations. USAID- Presentation to USAID local mission and US representatives about the importance of measuring mycotoxin exposure in the context of the SHINE trial. Provided written report of preliminary research findings titled, "Assessment of Maize and Groundnut Harvest and Post-harvest Practices in Rural Zimbabwe". Invited webinar presentation titled, "Going beyond Nutrition to Understand Health Growth and Development." delivered to 150 researchers and programmers around the world. EU- Provided written report of preliminary research findings titled, "Assessment of Maize and Groundnut Harvest and Postharvest Practices in Rural Zimbabwe". Continued discussions about providing technical expertise to NGO's based in Zimbabwe on programmatic issues related to mycotoxins. During the current project period, I have continued communicating with these stakeholders. We convened a meeting with the Zimbabwean Food and Nutrition Council, Ministry of Health and Childcare and the Ministry of Agriculture to discuss mycotoxin mitigation in Zimbabwe. We prepared a special report on harvest practices for the Food and Nutrition Council's publication. Through this partnership, we convened a project plan that was funded by the EU to explore aflatoxin exposure and health effects from basic science to policy. In addition, I gave a presentation to the Bill and Melinda Gates Foundation on progress and future plans. Changes/Problems:The only change was that the delay in research led to the research goal completion being delayed. Instead, the research agenda in this fellowship period have focused on biomarker development, measurement of the distribution of exposure, and measurement of potential biological pathways by which aflatoxin exposure could cause adverse birth outcomes. What opportunities for training and professional development has the project provided?Guest lectures at the University of Zimbabwe Medical School on breastfeeding and infant and young child feeding. Presentations to a variety of government (Ministry of Agriculture, Ministry of Health and Childcare, and Food and Nutrition Council) international development organizations (USAID, DFID, FAO, EU) on the importance of mycotoxins and preliminary data on exposure in Zimbabwe. Organized and participate in the Cornell Mycotoxin Working Group which brings together faculty and graduate students from multiple disciplines working on mycotoxin-related issues. Participated in the preliminary planning for a trial assessing the effects of mycotoxin mitigation on child growth. Mentored other graduate students on research design and analysis. How have the results been disseminated to communities of interest?As summarized in the section of targeted audiences, I have disseminated preliminary research results and implications of the research to Cornell University, the Zimbabwean Ministry of Health and Child Care, DFID, FAO, USAID and the EU. This information has been disseminated through a series of presentations meetings and written reports. Additionally research findings have been synthesized in a manuscript that I am preparing for publication this year. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Develop research skills bridging basic laboratory biological science and applied population-level field studies a. Quality control for biological sample and anthropometry collection. Worked with a team to identify errors along data collection and identify errors. b. Methods development and assay optimation for ELISA assays to measure C-reactive protein, alpha-1-acid glycoprotein, insulin-like growth factor 1, intestinal fatty acid binding protein and soluable CD14. Coordinated an experiment to test the effect of time and volumen of urine collection on the lactulose mannitol test. 2) To further develop my grant writing skills a. First authored 3 grant proposals to the USAID (Bureau for Food Security), NIH (Environmental Health Sciences- R21), and EU to support measurement of maternal mycotoxin exposure, its associated relationships with gut health, inflammation, and adverse birth outcomes in pregnant women in Zimbabwe. The EU funding also supports translation of research findings into policy. 3) To gain additional experience implementing community-based intervention trials a. Through this fellowship, I have had the opportunity to work for more than 2 years in the context of the SHINE trial in Zimbabwe on all facets of the study from laboratory analysis, research data collection, intervention development and data analysis. I have also had the opportunity to participate in a planning grant to develop a mycotoxin mitigation trial to examine the effects of removing mycotoxins from the environment on child growth. Research Goals 1) To describe the distribution of aflatoxin exposure in pregnant women in rural Zimbabwe a.We have successfully measured AFM1 in 3000 pregnant women in our study population. I have drafted a manuscript assessing the determinants of aflatoxin exposure. The analysis shows significant temporal and spatial variation. Spatial variation is associated with elevation and rainfall. Additionally dietary practices are predictive of aflatoxin exposure. 2) To test the hypothesis that aflatoxin exposure is associated with maternal anemia Due to delays in development of a method to measure aflatoxin-lysine, this objective has been delayed. In the meantime, I have been testing associations with biomarkers that would indicate an inflammatory pathway that could lead to anemia. 3) To test the hypothesis that aflatoxin exposure is associated adverse birth outcomes including fetal loss, IUGR, and pre-term birth We are currently working on shipping samples outside of Zimbabwe to measure aflatoxin-lysine. We have identified 261 miscarriages and 78 stillbirths to date. We have a baseline maternal blood sample on 150 miscarriages and 60 stillbirths. Although this project will be completed outside of the terms of the fellowship, the fellowship allowed me to leverage more funding from other stakeholders to complete the project.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Smith LE, Prendergast AJ, Turner P, Mbuya MN, Mutasa K, Kembo G, Stoltzfus RJ and the SHINE Trial Group (2015). The potential role of mycotoxins as a contributor to stunting in the SHINE Trial. Clinical and Infectious Diseases. **(suppl *):S***-**. In press.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Desai A, Smith LE, Mbuya M, Chigumira A, Fundira D, Tavengwa NV, Malaba TR, Majo F, Humphrey JH, Stoltzfus RJ and the SHINE Trial Group (2015). The SHINE Trial infant feeding intervention: Pilot study of effects on maternal learning and infant diet quality in rural Zimbabwe. Clinical and Infectious Diseases. **(suppl *):S***-**. In press.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: SHINE Trial Study Group (2015). "A cluster randomized trial of the independent and combined effects of water, sanitation/hygiene and infant feeding on infant growth and anemia in rural Zimbabwe: The SHINE study rationale, design and methods." Clinical and Infectious Diseases **(suppl *):S***-**.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Smith LE, Stasiewicz M, Hestrin R, Morales L, Mutiga S, and Nelson RJ (2015). Environmental Drivers of Spatial Variability in Mycotoxin Accumulation in Maize in Kenya. African Journal of Food, Agriculture, Nutrition and Development. In press.
• Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Smith LE, Prendergast AJ, Turner, P, Humphrey, JH, and Stoltzfus RJ. Aflatoxin During Pregnancy: A potential cause of maternal anemia and adverse birth outcomes. Environmental Health Perspectives.

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: During this reporting period, I have worked to communicate resarch findings and implications to academic audiences, policy and government and development agencies. Cornell University- Gave presentation to a broad audience of academics and students at the Cornell IGERT Symposium: Food systems and poverty reduction titled, "Seasonal Variation in Aflatoxin Exposure in Rural Zimbabwe." Zimbabwe Ministry of Health and Childcare- Provided a written report of preliminary research findings to the the Ministry titled, "Preliminary Report: Aflatoxin exposure among pregnant women participating in the SHINE study in Shurugwi and Chirumanzu districts" DFID- Presentation to DFID program reviewers about the importance of measuring mycotoxin exposure in the context of the SHINE trial. FAO- Presentation to FAO country representatives about the importance of measuring mycotoxin exposure in the context of the SHINE trial. Discussion about the implications of the current research for informing future interventions to decrease mycotoxin exposure in vulnerable populations. USAID- Presentation to USAID local mission and US representatives about the importance of measuring measuring mycotoxin exposure in the context of the SHINE trial. Provided written report of preliminary research findings titled, "Assessment of Maize and Groundnut Harvest and Post-harvest Practices in Rural Zimbabwe". Invited webinar presentation titled, "Going beyond Nutrition to Understand Health Growth and Development." delivered to 150 researchers and programmers around the world. EU- Provided written report of preliminary research findings titled, "Assessment of Maize and Groundnut Harvest and Post-harvest Practices in Rural Zimbabwe". Continued discussions about providing technical expertise to NGO's based in Zimbabwe on programmatic issues related to mycotoxins. Changes/Problems: One major change has been the decision to rely upon mass spectrometry to measure aflatoxin-albumin. We were unable to use the existing ELISA methodology because the method was not reliable. That has led to considerable delays and increases in the funds necessary to conduct the research. We also decided to measure another biomarker, aflatoxin M1 in urine to describe the seasonal variation in exposure in this population. We have requested for an extension of funds from year 1 to assist in paying for this change to the project. It is currently under review. To my knowledge, this does not dictate any additional reporting requirements. What opportunities for training and professional development has the project provided? Guest lecture at the University of Zimbabwe Medical School titled, "Infant and Young Child Feeding Practices: Implications for Practitioners" Organize the Cornell Mycotoxin Working Group meetings which bring together faculty and graduate students from several disciplines working on mycotoxin-related issues. Presented to a variety of international development organizations (USAID, DFID, FAO, EU) and the Zimbabwean Ministry of Health and Child Welfare on the importance of assessing mycotoxins. How have the results been disseminated to communities of interest? As summarized in the section of targeted audiences, I have disseminated preliminary research results and implications of the research's to Cornell University, the Zimbabwean Ministry of Health and Child Care, DFID, FAO, USAID and the EU. This information has been disseminated through a series of presentations meetings and written reports. More detail on the specific activities is included in the previous section of this report. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we hope to finalize methodology to measure aflatoxin-albumin. This will allow us to conduct analyses surrounding adverse pregnancy outcomes including maternal anemia and birth outcomes. In addition, I will continue to work with the trial and learn about research and laboratory methods. I will also continue to pursue opportunities to enhance my teaching abilities. We plan to submit several papers during the next year to peer-reviewed journals for publication.

Impacts
What was accomplished under these goals? Career Goals: 1. Develop research skills bridging basic laboratory biological science and applied population-level field studies a. Quality control for biological sample and anthropometry collection. Worked with the research team to identify errors along the data collection and correct errors. b. Methods development for measurement of aflatoxin-albumin and aflatoxin M1. Worked at validating an ELISA methodology for assessing both metabolites. Succeeded at measurement of aflatoxin M1. Due to difficulties and the unreliability of the aflatoxin-albumin ELISA, we will not be able to use. We are corrently working on developing a LCMS method for measurement of aflatoxin-albumin. 2. To further develop my grant writing skills a. Successfully wrote and received funding from USAID to support measurement of maternal exposure to mycotoxins. b. Submitted NIH R21 grant titled, "Investigating the Distribution of Mycotoxin Exposure During Pregnancy and Its Association with Adverse Birth Outcomes." Proposal ranked at 5%. Awaiting notice of funding decision. 3. To gain additional experience implementing community-based intervention trials. a. For the past year, I have been working in the context of the SHINE study on all facets from laboratory analysis to research data collection to additional intervention development. I have gained a well-rounded working knowledge of the complexities in a large community-based trial. Research Goals 1. To describe the distribution of aflatoxin exposure in pregnant women in rural Zimbabwe. a. We have successfully measured aflatoxin M1 in urine in 1500 pregnant women in our study population. Initial analysis suggests significant seasonal variation. Dietary diversity and food security also seem to be important predictors of exposure. 2. To test the hypthesis that aflatoxin exposure is associated with maternal anemia- No progress to date 3. To test the hypothesis that aflatoxin exposure is associated adverse birth outcomes including fetal loss, IUGR, and pre-term birth. a. We are currently designing a case-control study to investigate the relationship between aflatoxin exposure and pregnancy loss.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Smith LE, Prendergast AJ, Turner P, Humphrey JH and Stoltzfus RJ. Aflatoxin During Pregnancy: A potential cause of maternal anemia and adverse birth outcomes. Environmental Health Perspectives.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Wu F, Stoltzfus RJ, Gong YY, and Smith LE. WP4: Human Studies in Children and Impact of Growth and Development. IARC, 2014.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Smith LE, Rukobo S, Mutasa K, Prendergast AJ, Mbuya M, Humphrey JH and Stoltzfus RJ. Seasonal Variation in Aflatoxin Exposure in Rural Zimbabwe. Presented at Cornell IGERT Symposium on Food Systems and Poverty Reduction.
• Type: Other Status: Other Year Published: 2014 Citation: Smith LE. Assessment of Maize and Groundnut Post-Harvest Practices in Rural Zimbabwe. Report submitted to BMGF and USAID.