Effect of supplemental nutrition in pregnancy on offspring’s risk of cardiovascular disease in young adulthood: Long-term follow-up of a cluster trial from India

Background Undernutrition during intrauterine life and early childhood is hypothesised to increase the risk of cardiovascular disease (Developmental Origins of Health and Disease Hypothesis), but experimental evidence from humans is limited. This hypothesis has major implications for control of the cardiovascular disease epidemic in South Asia (home to a quarter of world’s population), where a quarter of newborns have low birth weight. We investigated whether, in an area with prevalent undernutrition, supplemental nutrition offered to pregnant women and their offspring below the age of 6 years was associated with a lower risk of cardiovascular disease in the offspring when they were young adults. Methods and findings The Hyderabad Nutrition Trial was a community-based nonrandomised controlled intervention trial conducted in 29 villages near Hyderabad, India (1987–1990). Protein-calorie food supplement was offered daily to pregnant and lactating women (2.09 MJ energy and 20–25 g protein) and their offspring (1.25 MJ energy and 8–10 g protein) until the age of six years in the 15 intervention villages, but not in the 14 control villages. A total of 1,826 participants (949 from the intervention villages and 877 from the control villages, representing 70% of the cohort) at a mean age of 21.6 years (62% males) were examined between 2009 and 2012. The mean body mass index (BMI) of the participants was 20 kg/m2 and the mean systolic blood pressure was 115 mm Hg. The age, sex, socioeconomic position, and urbanisation-adjusted effects of intervention (beta coefficients and 95% confidence intervals) on outcomes were as follows: carotid intima-media thickness, 0.01 mm (−0.01 to 0.03), p = 0.36; arterial stiffness (augmentation index), −1.1% (−2.5 to 0.3), p = 0.097; systolic blood pressure, 0.5 mm Hg (−0.6 to 1.6), p = 0.36; BMI, −0.13 kg/m2 (−0.75 to 0.09), p = 0.093; low-density lipoprotein (LDL) cholesterol, 0.06 mmol/L (−0.07 to 0.2), p = 0.37; and fasting insulin (log), −0.06 mU/L (−0.19 to 0.07), p = 0.43. The limitations of this study include nonrandomised allocation of intervention and lack of data on compliance, and potential for selection bias due to incomplete follow-up. Conclusions Our results showed that in an area with prevalent undernutrition, protein-calorie food supplements offered to pregnant women and their offspring below the age of 6 years were not associated with lower levels of cardiovascular risk factors among offspring when they were young adults. Our findings, coupled with evidence from other intervention studies to date, suggest that policy makers should attach limited value to cardiovascular health benefits of maternal and child protein-calorie food supplementation programmes.

height and maternal leg length in particular are used as proxy measures of adverse nutritional and socioeconomic conditions in their childhood.
Study plan: An initial enumeration exercise to account for the current available numbers of subjects in the birth cohort (F1) in both intervention and control groups of the Hyderabad Nutrition Trial will be carried out. A sampling list of these subjects will then be used to carry out a retracing exercise through home visits in the villages by trained field investigators to enumerate the numbers of parents (F0) alive in both groups. We will also attempt to record in this visit the number, age and date of birth of the siblings of the index subject/subjects in the Nutrition trial. (Appendix 1 -Tracking Form). Parents and siblings will then be invited to participate in the current study.
Examination: All participants will undergo an interview to record information on health, diet, nutrition and physical activity besides anthropometric measures, blood pressure, body composition measurements (TANITA) and pulmonary function assessment. Blood samples will be collected and analysed to measure glucose, insulin, cholesterol, triglycerides and liver function. Saliva samples will be collected and stored for future genetic studies. All subjects will also be invited to a screening examination at NIN where DXA scanning will be used to measure body fat and central-body fat. Noninvasive measures of coronary disease risk will be made.

Timetable:
The fieldwork is expected to take 18 months to complete (Jan 2010 to June 2011), with a further 3 months to analyse the data and write-up the results (study end date October 2011).

Study Staff and their Responsibilities
The team will consist of Project Manager, Field manager, three SRFs, Fourteen fieldworkers, three biochemists, one DXA technician (NIN staff member), two data entry operators, and four clinic helpers.

Project Manager
• Overall responsibility for the performance of the team • Deliver recruitment targets • Assist with training of staff • Ensure data quality and completeness • Coordinate day-to-day running of the study (plan activities of other staff, surveys, and clinics) • Supervise data entry, data back-up, data storage (electronic and paper), data validation and verification • Supervise record keeping • Procure equipment and consumables on time, and ensure their maintenance (except for DXA scanner) • Develop links with the village leaders and Anganwadi staff.
• Report to the local PI at NIN and PI at Bristol.

Field Manager
• Overall responsibility for day-to-day management of field work and teams and coordinate smooth functioning of all components of fieldwork • Ensure recruitment targets • Primarily responsible for establishing contact with the Village leaders / Panchayat officials, securing permissions as appropriate and earmarking clinic site towards end of work in previous site. This should allow smooth moving over of the team from site to site without loss of time between villages.
• Conduct household visits with the field staff and motivate participation of subjects.
• Ensure daily calibration of equipment and appropriate measures when required • Ensure data quality, completeness and accuracy • Responsible for checking all equipment are in working condition and take suitable action when required • Keep an inventory of all consumables and procure all requisite material on time • Daily mechanism of reporting fieldwork to the Project Manager and together working on any logistic issues in the field work, including co-ordinating staff suitably in case of absenteeism so work is not affected in the field.
• Report to the local PI and PI at Bristol.

Medical officer (Senior research fellow) At NIN
• Complete the medical proforma, where incomplete.
• Measure augmentation index and pulse wave velocity when the fieldworker is not available.
• Assist in DEXA studies, where necessary.
• Communicate results of medical examination (blood pressure) and offer advice to the participants • Manage any medical emergencies or referrals that may be required • Accompany the field team in case need arises, as when Field doctor is absent.

Field Medical officer (Senior research fellow)
• Accompany the field team to the village clinics • Complete the medical proforma.
• Collect/assist in blood sample collection in case biochemist has difficulty or is unavailable • Assist in Pulmonary Function Tests, where necessary.
• Communicate results of medical examination (blood pressure, Hemoglobin and fasting glucose) and offer advice to the participants • Manage any medical emergencies or referrals that may be required • Overall assistance in field clinic for other measurements.

Fieldworkers (X12)
• Arrange appointments for participants • Explain nature of study to participants • Provide information sheets to participants • Send reminders to participants • Organise clinics, maintain equipment and records • Involved in all aspects of the fieldwork including questionnaire completion, anthropometry, blood pressure, body composition (TANITA) and Pulmonary function measurement. • Collect participants and transport to NIN and back for NIN component of study.
• Report to the Project Manager • Along with the field manager and village helper, will be responsible for household visits and motivation of subjects to attend the clinics.

Fieldworkers (X2)
• Collect participants and transport to NIN and back for NIN component of study.
• Measure augmentation index and pulse wave velocity • Involve in all aspects of the fieldwork including questionnaire completion, anthropometry, blood pressure, body composition (TANITA) and Pulmonary function measurement if needed. • Report to the Project Manager Field Biochemist (2) • Accompany the field team to village clinics, collect blood samples, centrifuge in the field and ensure timely and accurate processing/analyses of samples • Analyse for glucose and haemoglobin if the NIN Biochemist is not available • Maintain equipment and necessary supplies of consumables • Assist biochemist at NIN in arranging transport of samples and quality control with the coordinating laboratory in Delhi • Report to the Project Manager and Dr Ruby Gupta/ Dr. Vipin Gupta in Delhi Biochemist NIN (1) • Store the samples collected in the field with the help of Field Biochemist and clinic helper • Analyse for glucose and haemoglobin on the same day.
• Give the results to the Field Manager.
• Maintain equipment and necessary supplies of consumables • Arrange transport of samples and sample dispatch sheets and quality control with the coordinating laboratory in Delhi • Report to the Project Manager and Dr Ruby Gupta/ Dr. Vipin Gupta in Delhi

DXA technician (NIN staff)
• Conduct whole body, lumbar spine, and hip scan.
• Conduct analyses to estimate adiposity and abdominal adiposity from whole body scan • Report to the Project Manager and local PI

Data Manager ( 2 )
• Data entry and check data completion, maintain databases • Enter data from DXA and TANITA printouts into database • Enter census codes on the questionnaires using special software • Generate and print labels and lists for fieldwork.
• Maintain records and paperwork, progress spreadsheets, electronically transmit the required files at timely intervals • Report to the Project Manager.

Field Clinic helpers (2)
• Set up clinics, instruments and organise the flow of participants and arrange refreshments • Help and support the activities of the team

NIN Clinic helpers (2)
• Set up clinics, instruments and organise the flow of participants and arrange refreshments.
• Help the DXA technician during the scans.
• Help and support the activities of the team • Help in cleaning the lab glassware.

General Outline of Plan for Fieldwork and Data Collection
The APCAPS work will involve the conduct of clinics in each of 29 villages which formed the base of studies conducted since 1987 -90, as part of the Hyderabad Nutrition Trial. Children from these study households who were enrolled in the original study are currently being followed up as part of the Hyderabad DEXA study. They form the index subjects. Parents and siblings of these index subjects will be invited to participate in the APCAP study, along with the DEXA subjects.
After carrying out a household -level survey to track the availability of household members, a detailed action plan will be charted out to conduct clinics in each of these villages. Preliminary visits to establish a rapport with the subjects and communicate with the Village Head/ Gram Panchayat and obtain his/her/their permission to conduct the clinics in a suitable, convenient and centrally located site will be done by the Field Manager and field workers.
These subjects will also undergo DEXA studies for body composition and vascular studies at the NIN location.
A detailed protocol is set out for each phase of the study -Tracking exercise, Clinics, NIN arm of study and transport of biochemical samples. 8. Fieldworker continues down the list until everyone in the village who is eligible has been visited, and has either been contacted or refused participation or cannot be identified. 9. The information collected from this exercise is entered into a spreadsheet and a weekly count of villages covered with number of households and status sheet of participants available in each household for the APCAPS is prepared.
10. This information will then be used to generate a participant list for the APCAPS.

Participant Information Sheet -APPENDIX 2
At the time of first contact, the fieldworker reads the participant information sheet (translated into Telugu) to the participant and answers any questions.
The fieldworker makes sure he has covered all the points in the checklist for participation.
Benefits of the study (to be communicated to the potential participant by the Field staff at first contact, and while inviting to clinic for participation).
Tests will be conducted for the following, free of cost: • Blood pressure • DXA scan to measure body fat • Heart and blood vessel function (Augmentation Index, Pulse Wave Velocity, Carotid Intima Media thickness) • Lung function • Blood tests, including glucose You will be told if a problem is detected and will be given your blood tests For examination, please remember the following: • Be on time for the clinic.
• You will be reimbursed for your time and food is provided • Come fasting overnight, unless you are pregnant or diabetic • Avoid metal on clothes. Preferably do not wear gold or silver jewellery • No calcium for 2 days before hand • Avoid alcohol and fatty food one day before • Do not smoke from the evening before the examination • For women, come within 10 days of last menstrual period unless ceased childbearing • Bring proof of ID-household ration card, voter ID or other • Bring any medication that you use

Informed consent. Completed by research assistants -APPENDIX 3
One of the field workers who ever is the field in charge for that village, will explains the study using the prepared information sheet and takes consent. He/she must make sure that the participants have been able to ask any questions or address any concerns. The Telugu versions are also available for those subjects who prefer to sign this version.
Protocol for Village Clinic: 1. Village clinics will be carried out alternately in the intervention and control villages so as to cover all households in the list in each village selected for study at a time.
2. Appointments will be given in the selected village to about 6 households using the participant list generated by the Field Manager / Field-in-charge. The parents and all siblings of the index subject from the DEXA study will be invited to participate in APCAPS, along with the index DEXA subject.
3. Inclusion Criteria: All mothers who participated in the Hyderabad Nutritional Trial in either Intervention or Control arm, their spouses and all children born to them, will be included in the study.
4. Exclusion Criteria: There are no overall exclusion criteria as all the members of the family are invited to participate. Only in some cases as outlined below, some participants will be excluded for some components of the study.
i. Subjects with structural limb / spinal deformities will be excluded from the anthropometry protocol, but encouraged to complete other sections of the protocol.
ii. Children below 10 years will be excluded from blood sampling protocol.
iii. Subjects who are mentally disabled, Deaf & Dumb can be excluded from Questionnaire, but encouraged to complete other sections of the protocol with the help of a family attendant.
iv. Pregnant women will be excluded from the DEXA examination.
5. The field staff will visit them 2-3 days before, explain the nature and purpose of the study and hand out the Participant information sheet. Any questions the subjects may have at this point are answered. Appropriate instructions for fasting overnight are given. A log book will be also maintained and the fieldworker records information on how many subjects were contacted and whether they have agreed to attend. Reasons for non-attendance and refusal are also noted. 6. The field staff will also enlist the help of the Anganwadi teacher and village helper to remind the subjects of the clinic in the village.
7. The Field Manager or DEO supervises and prepares all the questionnaire folders, sampling kits, etc with appropriate labels that have been printed with barcodes and sent from the SANCD office by Dr. Vipin Gupta / Ms. Gagandeep in New Delhi. 8. On the morning of the clinic, the team consisting of the Field Manager, one Medical Officer (SRF), 6 Field workers, one Biochemist and the clinic helper will leave in the morning with all necessary equipment from the Project office at NIN. A checklist of equipment to be transported for the clinics is maintained and checked before departure. 9. On arrival at the clinic site in the village, the equipment is set up by the clinic helper and the field staff and daily calibrations are carried out.
10. The biochemist sets up the field centrifuge and ensures sampling kits are in place and ready. 11. The clinic helper meanwhile also ensures that the contact person in the village has arrived with the subjects for the day. He will visit the households if necessary to remind the subjects scheduled for the day. The Field Manager and one Field -in-charge will also conduct household visits to motivate participation at the beginning and end of each day.
12. All subjects who attend the clinic are then requested to sign the Consent Forms after ensuring all information in the Participant information sheet is understood and any outstanding questions are answered.
13. The clinic helper arranges for breakfast for all subjects in the meantime.
14. Height, weight, blood pressures are recorded. After ensuring the subjects have fasted overnight, the biochemical and saliva samples are collected. The biochemist then centrifuges the samples while the saliva samples are stored in the ice box. In the event that there is a power shutdown, either a generator is used to ensure continuous supply or the biochemist will have to return to NIN to process the samples and carry out analyses of HB and fasting glucose. The biochemist will ensure the proper storage of samples in the freezers at NIN. A set protocol will be followed for regular transfer of samples to the Central lab at SANCD in Delhi. Where subjects refuse blood sampling, an attempt at explaining and reassurance is tried but no subject will be pressurised for the same. Instead, he/she is encouraged to complete other measurements and the questionnaire. 15. Body composition measurements using TANITA are carried out. Subjects are then provided breakfast. They will then move sequentially through the process of answering the questionnaire, undergo remaining anthropometry measurements and pulmonary function tests. 16. Accelerometers for recording Physical activity are also given to the subjects and appropriate instructions are provided about use and precautions. 17. At the completion of all requisite tests, a check is made to confirm completion of the questionnaire and all procedures. 18. The participant is then thanked for their time and participation, reimbursement is provided and appropriate vouchers with signatures are retained for the same. The ID proof is checked, details to be noted such as school registration / voter ID/ pan card / ration card number in the questionnaire.
The ID proof will then be scanned electronically using a scanner and the jpeg image will be saved under the subject ID. If two or more subjects from the same family have a single ID, duplicate copies of the scanned image will be made and renamed with the respective subject ID. 19. Appointments are provided as and when possible for the subjects to travel to NIN for the DEXA and vascular studies.
20. When all the subjects have been covered for the day, the field staff checks the questionnaires, files the folders and the clinic helper dismantles and stores all the equipment in the vehicle for return to NIN. Where possible and if a safe and secure location is available, anthropometry and TANITA equipment as well as the centrifuge and generator are stored in the site of the village clinic for use during the remaining clinics in the village.
21. The Field Workers with the help of the Field Manager and Field-in-charge prepare the participant list and folders for the next day. The Biochemist prepares the vene puncture material, sampling kits with appropriate labels and transport box for the next day. 22. Appropriate phone calls and reminders are made to subjects attending the clinic next day. One phone call is always made to the Anganwadi Teacher/helper/contact person identified in the village who will then ensure attendance at the clinic in the morning. 23. The Field Manager will spend much of the day planning and organising fieldwork and ensuring overall smooth running of the fieldwork, participant attendance, data collection and complete measurements. 24. The Database person (DEO) will assist and support the Project Manager, manage the database, and enter data simultaneously as far as possible with the data collection. 25. A smooth mechanism for data checking, and transfer to the data manager at Bristol needs to be in place once data entry actually begins.

Exclusion of pregnant women from DXA scanning
People are exposed to a very low dose of radiation when they undergo a DXA scan. This means that we must ensure that pregnant women do not undergo DXA scanning.
Steps to ensure pregnant women are not screened using DXA.
1) Fieldworker visits female subjects together with Anganwadi worker two weeks before planned appointment.
2) Anganwadi worker asks subject about last menstrual period and regularity of period and estimated start of next menstrual period.
3) Female is given tentative appointment for 1-10 days after next menstrual period 4) One day before the appointment the female is visited by fieldworker and Anganwadi worker to ask about the start of the last menstrual period. If last menstrual period was not in the last 9 days, they are given a new appointment.
5) Women who are pregnant are given appointments for other components of the study in the same ways as the men, but are not given DXA scans.

Blood Sampling
Blood samples will be taken by the biochemist. The Medical officer will assist / help the biochemist whenever required. For all subjects a fasting sample will be taken. Pregnant women and people with diabetes will not be asked to fast, but blood samples will still be taken. Blood samples will not be taken for children less than 10 years old.
Answers to the following questions are recorded before the blood sampling.

Labelling
Bar code labels will be used to label all the sample tubes, storage vials, boxes, questionnaire and all the paper documents of the project. Labels with barcodes will be printed in SANCD, Delhi and will be dispatched to NIN, Hyderabad. Kindly intimate the SANCD staff well in advance so that labels could be printed and posted well in advance to avoid last hour rushes and back logs.

SANCD, New Delhi
As soon as the subject will arrive in the field clinic, the subject ID will be recognized from the village list and the corresponding labels will be identified. The box sheets, report forms and reimbursement sheets need not be labeled. There will be 24 labels for each subject in the following sequence: Protocol for Blood sampling

Preparation of Subjects:
• Inform your subject at least two days before about sample collection.
• To come after 10-12 hours of fasting.
• Inform the 'Place' and 'Venue' of sample collection 2. Prepare kits with the following contents for sampling:

Blood sample collection
• Draw blood from the subject in the recumbent position, where possible. Make the subject sit for 10 min. before starting sample collection. • Keep ready one holder, one needle, one red-capped vacutainer, one grey-capped vacutainer and two purple-capped vacutainers.
• Attach needle to the holder.
• After applying the tourniquet insert the needle into the vein. (Avoid application of tourniquet for more than one minute) • Insert red-capped vacutainer. Collect blood up to the capacity.
• Take out the red-capped vacutainer and insert grey-capped vacutainer. Ensure that the tube is filled to the mark.
• Take out the grey-capped vacutainer and insert purple-capped vacutainers (2). Ensure that the tube is filled to the mark.
• Mix grey capped vacutainer (containing fluoride) and purple capped vacutainer (containing EDTA) by inversion for 7-8 times (Do not shake vigorously) and keep them in an ice-bucket (at 4 degree temperature).
• Allow the blood in red-capped vacutainer (Plain tube) to stand for 30 min at room temperature and allow blood to clot. Centrifuge the tube at 3500 rpm for 15 min.
• Hemoglobin estimations to be performed simultaneously with fresh blood from EDTA vacutainer.
It is important that serum is separated as soon as possible and at no cost later than 45 minutes after blood collection.
• Aspirate the serum using the given plastic pipette into three labelled storage vials provided (Vials labelled as 'S' for serum) • Similarly centrifuge the grey-capped vacutainers for 15 minutes at 3500 rpm. Aspirate the plasma and transfer into three labelled vials provided in the kit (Vials labelled as 'P' for Plasma) • Ensure that the storage vials are closed tightly.
• Do not centrifuge the purple-capped vacutainer.

• Mix well by inversion and Estimate Hemoglobin
• Centrifuge the tubes at 3500 rpm for 15 min • Transfer EDTA plasma into two storage vials labelled 'EP'. One vial of 'P' should be used for glucose estimation without much delay.
• Needle should be destroyed by needle destroyer and vacutainers collected as biowaste material should be sent to incinerator.

Storage Daily Activity
Properly labeled vacutainers and storage vials should reach NIN, Hyderabad at the end of each field and should be stored at -20 O C in the deep freezer. The labelled questionnaire should be stored at the A-7 flat systematically in the order of subject recruitment and data manager should be well versed with their storage sequence. After data entry the questionnaires should be stored back in the same order. The storage vial containing fluoride plasma aliquot which is used for estimating fasting glucose level should be stored back in the deep freezer in the appropriate storage box along with its other aliquots after use as soon as possible. The location of storage vials in the boxes should be recorded and stored with the lab and field technician. Field manager will be responsible to overview and supervise all these daily activities from time to time. The village leader ( FW) will be responsible to report the total number of samples collected on each day to the field manager and confirm the completion of the daily activities mentioned above.

At the end of completing one village
After the field work of one village is complete (after reaching atleast 80% response rate), all the questionnaires and samples (saliva tubes, vacutainers, storage vial) will be finally arranged in such a manner that samples can be transferred on dry ice to SANCD, New Delhi via reliable courier service. The exact procedure given below should be followed to arrange the samples of a village. Field manager and project manager will be responsible to supervise and conduct these activities to ensure the completeness and precision of the sample information.

Questionnaires
After the completion of one village (data entry should continue simultaneously), all the questionnaires will be arranged serially (in the order of family ID) by the data manager along with the NIN helper under the supervision of field manager and project manager.
Confirm the subject ID applied on the questionnaires using the information from the village list, reimbursement sheet and daily worksheets (made by field technician and data manager) to check any missing questionnaires.
Separate all the questionnaires into four lots based on the subject type.
After separating all the questionnaires of DXA index children (subject ID starting with 1), their sibs (subject ID starting with 3), the mothers (subject ID starting with 5) and the fathers (subject ID starting with 6); arrange them in increasing order of their subject ID's.
After the four lots are arranged serially, all the questionnaires will be arranged according to the village list, i.e., in order of their family Id in the order of father, mother, sibs and DXA subjects. This step will be performed after insuring the completeness of data in each questionnaire.
All the subject ID (in the order stated in step 3) will then be noted on the dispatch sheets to make a record of total number of subjects covered on the village along with their other essential details.

Vacutainers
Once the serum is separated, the red capped plain 4 ml vacutainers can be discarded at NIN in appropriate manner. After the completion of one village, the lab technician will arrange and pack all the grey and purple capped vacutainers with the help of field technician, field helper and other field workers under the supervision of field manager and project coordinator. The grey capped 2 ml sodium fluoride coated vacuatiners containing whole blood will be permanently stored in NIN while the purple capped 3ml and 6 ml EDTA coated vacutainers containing whole blood will be transferred to SANCD, New Delhi.
Separate all the vacutainers in four lots based on the subject type. All the grey and purple capped vacutainers of one individual or subject should be kept together.
After separating all the vacutainers of DXA index children (subject ID starting with 1), their sibs (subject ID starting with 3), the mothers (subject ID starting with 5) and the fathers (subject ID starting with 6); arrange them in increasing order of their subject ID's.
After the four lots are arranged serially on the thermocol stands, all the purple capped vacutainers of the members of one family will be tied together with the rubber band and will be then kept in a transparent plastic lock bags. Keep the list of the village handy to confirm the subject ID's of the members of each family.
While tying with the rubber band, note the missing vacutainers and other comments regarding the purple capped vacutainers in the appropriate column of the dispatch sheet.
Repeat steps 3 and 4 for each family, such that different bundles representing families are made and kept in the plastic bags.
Once the plastic bag is full note down the village name, date of packing, packet number and the list of family ID of the vacutainers kept in that bag on a sheet of paper and place it inside the plastic bag such that information is visible from outside.
Lock the plastic bag and pack it with the cello tape. Take care not to over full the plastic bags to avoid its tearing.
Repeat Steps 3-6, till all the purple capped vacutainers are packed.
Make a record of total number of purple capped vacutainers of a village.
Repeat the same procedure (steps 3 to 9) for the grey capped vacutainers. The missing vacutainers and other comments regarding the grey capped vacutainers will be recorded in the NIN record sheet while repeating step 4.

Storage Vials
After the completion of one village, the lab technician will arrange and pack all the storage vials in the storage boxes with the help of field technician, field helper and other field workers under the supervision of field manager and project coordinator. Two storage vials each of serum, fluoride plasma and EDTA plasma will have to be transferred to SANCD, New Delhi and one storage vial each of serum, fluoride plasma and EDTA plasma will be stored permanently at NIN, Hyderabad in the deep freezer.
Information regarding the location of each subject's storage vials should be with both the field and lab technician as the vials will be stored inside the boxes in the order of date of collection.
The storage vials ( in the order of 'S', 'P' and 'EP') will be rearranged according to the increasing order of family ID by consulting the village list such that members of one family are stored together (father, mother, sibs, DXA subject) as done in case of saliva tubes and vacutainers.
Three aliquots will be permanently stored in three different boxes; such that each box has only one storage vial of serum, fluoride plasma and EDTA plasma of an individual. Out of these, two boxes will be transferred to SANCD, New Delhi and remaining one box will be permanently stored in NIN, Hyderabad.
The fluoride plasma aliquot used for fasting glucose estimates will be kept back in the NIN storage box.
Note down the details of the samples on the box sheet along with the volumes with the help of previous records given by field technician as soon as the storage box is full.
Label the box properly as NIN or SANCD box along with the village name and box number with the help of adhesive tape and cello tape.
Simultaneously, record any missing information and other comments in the appropriate columns of dispatch sheet and NIN record sheet.
Record the total number of boxes to be transferred to SANCD, New Delhi and also to be permanently stored at NIN, Hyderabad.

FLOW CHART
Protocol for blood collection and sampling at zero time in fasting sample  6. Once the plastic bag is full note down the village name, date of packing, packet number and the list of family ID of the tubes kept in that bag on a sheet of paper and place it inside the plastic bag such that information is visible from outside. 7. Lock the plastic bag and pack it with the cello tape. Take care not to over full the plastic bags to avoid its tearing. 8. Repeat Steps 3-6, till all the saliva tubes are packed.
Make a record of total number of saliva tube packets of a village

Sample Transfer
All the samples will be transferred to SANCD, New Delhi from time to time (depending on the temporary storage capacity available in NIN, Hyderabad) by the lab and field technicians with the help of field helper and other field workers under the supervision of field manager and project coordinator. Please inform the SANCD staff well in advance to make arrangements for sample transfer. While all the samples are being finally packed by the courier service providers, all the details regarding the sample transfer (number of boxes, number of packets, etc.) will be recorded by the field manager and mailed to SANCD lab in-charge. The soft copies of the NIN box sheets and NIN record sheets will also be mailed to SANCD lab in-charge for reference.

CLINIC QUESTIONNAIRE
Clinic questionnaire completed by fieldworker • Filled in biro.
• It is only permissible to leave a section blank if it is in a shaded region.
• If a section is blank then cross through section.

Guide for conducting structured interviews
Structured interviews capture vital information. The aim of a structured interview is to measure facts and people's attitudes accurately and in such a way that if one were to repeat it at another time one would get the same answer.
It is, therefore, very important to get the structured interview right. Respondents need to be able to provide truthful answers which reflect their lives. Careful and precise interviewing techniques are essential to ensure the collection of complete, standardised, unbiased and accurate data. Much research has been undertaken to identify what works in getting the best from a structured interview and what does not.
For example: • Interviewers who adopt a business like manner (wearing a dark suit) are less likely to gain trustresponders tend not to give truthful answers.
• Young interviewers obtain less reliable results than older interviewers.
• Male interviewers obtain less full information from male respondents than female interviewers and vice versa.
Response and accuracy is likely to be increased if the interviewer looks happy, appears positive. It is likely to be decreased if the interviewer looks tense. Matching up respondents with interviewers is particularly difficult. It is, therefore, vital that the questions in a structured interview are asked in the same way and that the responses are recorded in the same way by the interviewer.
The following provide essential steps and aids to help conduct the structured interview.

The interviewer
Foremost the interviewer needs to: • Know and understand what the study is about-inside out.
• Understand the questionnaire

Good interviewers
• Are sensitive and trustworthy • Have an ability to establish a rapport with a wide range of people • Are friendly and positive • Are good listeners and do not interrupt people before they have finished speaking • Are committed and persevering • Have the ability to adopt a neutral manner (showing neither approval or disapproval) • Have legible handwriting • Are adept at leaving the respondent happy • Are good planners • Are able to ensure and maintain confidentiality • Dress neutrally-suitable for any kind of home/clinic • Have good intuition about when it is appropriate to approach respondents or not.

The interview
The interviewer must approach potential respondents in a positive manner in order to encourage them to participate. The critical moment is when the interviewer introduces themselves.
Always present your ID Card. Be honest about the study's aims and let them know how long the interview will take. Always provide respondents with details about the study and information on who to contact if/ should they want to.
Find a place where the interview can be conducted in private without interruptions.
Most respondents want to be seen in the best possible light and will want to give answers that reflect this. They will want to answer in such a way as to please the interviewer. The reliability of the information collected will be dependent on the rapport and satisfactory relationship established during the introduction. If a respondent feels anxious or uneasy they may be less likely to want to provide personal information.
When asking questions, the interviewer must always • be sensitive to the needs of the respondent; e.g. not sitting in sunlight, comfortable seats etc.
• be encouraging • use the exact words printed in the a questionnaire, and in the exact order: changing words or sentence orders will introduce serious bias to the study • speak in a non-judgmental manner • express polite interest • read the questions and pre-coded answers out in reasonable volume and speed ensuring the respondent has heard and understood the question • look at the respondent after each question to pick up on any visual clues of embarrassment or unease • accurately record the answer on the questionnaire NEVER • appear surprised or disapproving • express opinions or beliefs • make assumptions about respondents likely answers • hurry the respondent for answers • ask questions in a biased or leading way • ring/circle a category that comes nearest to the respondent's reply. If the code does not exist, record the exact words used by the respondent in their response • allow long silences to become embarrassing • apologise for asking personal or embarrassing questions If the interviewer appears hesitant, reluctant, unconfident, negative or thinks a question is too personal, then the respondent will be influenced and will decline to give accurate answers. In fact they will encourage negative responses.
It is important to maintain the respondent's interest and motivation throughout the interview.
It is common for respondents to go off the topic and talk about other issues not relevant in the questionnaire. The interviewer has to be skilled to bring respondents back to the point. If this is done firmly from the onset, further problems are less likely to recur. The following phrases may be helpful: "This is important information that we can cover a little later-can we now focus on" "What you say is most interesting but could I just ask you now on" Another problem with respondents who go off the topic is that they may have imparted some information that is useful in the questionnaire. Asking them again may give rise to irritability. BUT, it is very important never to assume the answer. To avoid potential irritability-prefix the question with: "I know you have already mentioned this, but can I just check" "Now you have already said something about this, but I would like to ask you this question".
It is never a good idea to break off the interview-it compromises the rapport and relationship already established. It is important to ensure that the respondent is fully aware of how long they are committing themselves to the interview.
If interviewers are seriously worried about a respondent (e.g. someone admitting suicidal thoughts or an elderly person being abused), the interviewer should offer to put the respondent in contact with a suitable professional. If accepted, ensure that this is put in writing and signed by the respondent. If the respondent refuses, only contact a professional if it is seen as a case of emergency.

Probing and Prompts
A probe is a stimulus which is used to obtain information form respondents who experience discomfort, hesitation, or feel unhappy to reveal information about themselves. Directive probing techniques are acceptable if one is eliciting factual information. Here it is important to motivate respondents in an undemanding and understanding tone. The aim is to encourage respondents to give accurate information. Probes listed below may help. "Can you tell me more about" "In what way" "Can you describe" "what sort of (office work do you do)" "Before writing your answer down, can I just check?"

Third Parties
Caution would be exercised when other people want to sit in on the interview. The presence of a third party will always influence the respondent and will lead to biased answers. This is best avoided.

Hesitancy, misunderstanding and non-response
Some respondents may seem hesitant in answering questions and the interviewer could affirm this with "there is no right or wrong answer on this-we just trying to get your ideas. Some will ask the interviewer for their opinion and here it is important not to give opinions but to explain that it is the respondent's opinion that matters. Some may want to answer with a "don't know" and this can have many different meanings E.g. It may mask misunderstanding of the question, (see next paragraph), may not want to impart the information (use probes), or may genuinely not know. It is important to record such information on the questionnaire.
When respondents genuinely do not understand, they may ask the interviewer to explain it. In this case, the interviewer should repeat the question and if no answer is forthcoming make a note of it on the questionnaire. It is important not to succumb explaining the question. Interviewers will give different explanations evoking different answers.
If a respondent is very reluctant to answer a question, it may help if the interviewer • Confirms the information given is confidential • That replies will be aggregated in tables so that individual responses cannot be identified.
If the respondent still feels unwilling to respond, a note should be made of it and the next question pursued.
Ultimately, if respondents refuse to take part or answer any question, their wishes must be respected. The interviewer should apologise for the inconvenience. Most interviewers achieve 8/10 response rate.
Often a respondent will change their minds and a follow-up letter may help.

End of the interview
Interviewers must leave their respondents in a positive frame of mind. After the interview, interviewers must: • Check the questionnaire to ensure all parts are completed A hostel here refers to any institutional household (group of unrelated persons taking their meal from a common kitchen). Persons living in a common building but not taking their meals from a common kitchen are classed as single/family type as appropriate. Common kitchen refers to joint cooking, not just sharing of a room used as kitchen).

6.2
What is the material used in the construction of the house? 1=Kutcha 2=Semi-pucca 3=Pucca Kutcha (made from mud, thatch, or other low quality material); Semi-pucca (partly low quality and high quality material); Pucca (high quality material used throughout including roof, walls, floor)

6.3a.
What is the main source of lighting for your household? 3= I am extremely anxious or depressed 9. 6 We have drawn a scale on which the best state you can imagine is marked 100 and the worst state you can imagine is marked 0. Please indicate on this scale how good or bad your own health is today, in your opinion % Show them the scale. We want to know how they rate their own health

Depression
Over the last 2 weeks, how often have you been bothered by any of the following problems?
1=Not at all 2=Several days 3=More than half the days 4=Nearly every day

9.7
Little interest or pleasure in doing things

9.8
Feeling down, depressed, or hopeless

9.9
Trouble falling or staying asleep, or sleeping too much

9.10
Feeling tired or having little energy

9.11
Poor appetite or overeating

9.12
Feeling bad about yourself, or that you are a failure, or have let yourself or your family down

9.13
Trouble concentrating on things, such as reading the newspaper or watching television

9.14
Moving or speaking so slowly that other people could have noticed. Or the opposite -being so fidgety or restless that you have been moving around a lot more than usual

9.15
Thoughts that you would be better off dead, or of hurting yourself in some way

9.16
In the last 4 weeks, have you had an anxiety attack -suddenly feeling fear or panic

9.20
During your last bad anxiety attack, did you have symptoms like shortness of breath, sweating, your heart racing or pounding, dizziness or faintness, tingling or numbness, or nausea or upset stomach?

9.21
If you checked off any problems on this questionnaire so far, how difficult have these problems made it for you to do your work, take care of things at home, or get along with other people?
1=Not difficult at all 2=Somewhat difficult 3=Very difficult 4=Extremely difficult In the last 4 weeks, how much have you been bothered by any of the following problems?
1=Not bothered 2=Bothered a little 3=Bothered a lot

9.22
Worrying about your health

9.23
Your weight or how you look

9.24
Difficulties with husband/wife, parents, or other relatives

9.25
The stress of taking care of children, parents or other family members

9.26
Stress at work outside of home or at school

9.27
Financial problems or worries

9.28
Having no one to turn to when you have a problem

9.29
Something bad that happened recently

9.30
Thinking or dreaming about something terrible that had happened to you in the past -like your house being destroyed, a severe accident, being hit or assaulted, or being forced to commit a sexual act Ask to recall over last month Physical activity questionnaire

INSTRUCTIONS FOR COMPLETION OF PAQ
The physical activity questionnaire aims to assess the habitual physical activity of the individual, by ascertaining recall over the one week.

PHYSICAL ACTIVITY QUESTIONNAIRE
Ask the subject, how many hours he/she spends working (Q1). This will be easy to ascertain if the person has regular working hours, as is the case with a factory worker on the shop floor. On the other hand executives may tend to work irregular hours and you may have to resort to asking them "On average, how many hours do you work?" Once you have determined how many hours they spend at work, ask them how many hours they spend in 'standing', 'sitting', and 'walking'. Please use prompt by listing the sample activities provided under these headings in the questionnaire. The distribution of time spent, will depend on the occupation of the individual. Executives for instance may spend a substantial amount of their time sitting. Manual workers in a factory may spend substantial times 'walking' or on activities more strenuous than walking.
Ask whether the participant participated in each activity listed under the category "more strenuous than walking". If they participated in the activity, record frequency and duration.

In case you travelled for a long duration to reach this place, or stayed back in this place for a few days, please recall the activities of the week before you left to this place.
The first questions are about your work/college. This includes paid jobs, working in your farm, study/training, any volunteer work or college activities.
Do not include unpaid work you might do around your home, like housework, garden work, and caring for your family. I will ask you about these later.

11.9
During the last week, on how many days did you travel to places on a bicycle except to and from work?

11.10
During the last week, on how many days did you did you travel to places by walking except to and from work ?

days [mts]
Now I am going to ask you some questions about how you spent your time, apart from work outside of the home over the LAST WEEK

Sports / games / exercise
Now think about all the physical activities that you did in the last 7 days solely for sport, exercise of leisure. Please do not include any activities you have already mentioned.

INSTRUCTIONS FOR COMPLETION OF FFQ
The aim of the questionnaire is to obtain the average food intake of an individual over a one year period.
The questionnaire, after entry into a computer, will provide details of consumption of various nutrientsenergy, carbohydrates, fat, proteins, vitamins and minerals. Entry of each food item correctly is essential as different foods provide different nutrients. Please use the portion size provided as they guide the subject in reporting the quantity he / she consumes.

COMPONENTS OF THE QUESTIONNAIRE:
The questionnaire comprises two components:

FOOD LIST
The food items are grouped under the following headings to help the subject recall the foods better: • Cereals • Lentils/Dhals/Gravies

DIET RELATED QUESTIONS:
This comprises a set of questions aimed at eliciting further information on the food pattern of an individual.
Information on the type of oil used for cooking of food, coconuts used for cooking, type of milk used, usage of vitamins and mineral tablets/capsules and any special diet adhered to are recorded in this section.

ADMINISTERING THE QUESTIONNAIRE:
Please familiarise yourself with the food frequency questionnaire before administering it.

INTERVIEWING THE SUBJECT:
Some of the food items may not be familiar to the subject. If the subject is not aware of the food item, please proceed to the next item in the list.
Please place the standard serving sizes e.g. katori etc. in front of the subject before you start the food frequency questionnaire. This will help the subject to estimate the food quantities that he/she is reporting.

PORTION SIZES:
Standard Then proceed to ask the questions and make entries in the method specified below to obtain and document information in the food frequency questionnaire: 9. Use general items where ever specific foods are not mentioned. For instance, coconut biscuits, cream biscuits will be entered under "sweet biscuits". Similarly, groundnuts, cashew nuts and other type of nuts will be entered under "nuts".
10. If individuals consume a combination of preparations eg. paneer mutter masala OR mixed groundnut and coconut chutney, enter the food under that item which is in greater proportion.
11. For fruits and vegetables ask the subject if they consume that food only in season or year round. If seasonal put a cross for the item in the appropriate column.
For example if a person eats one banana a day throughout the year and 1 apple a day when apples are in season, your entry will be:

FOOD LIST DESCRIPTIONS:
All the food items listed in the food list in the FFQ have been described in the following section, so that the interviewers understand the foods items listed. All food items have been listed and described in the same order listed in the questionnaire. Pickle: Includes all types of pickles used. 14.4

CEREALS
Papad: Refers to papads fried in oil. Papads are thin crisp sun-dried disc shaped wafers of dhal or cereal flour. 14.5 Coconut chutney: Refers to chutneys prepared with mainly coconut. 14.6 Groundnut chutney: Refers to chutney prepared with mainly groundnut. 14.7 Tomato chutney: Refers to chutney prepared with mainly tomato.

Includes all fresh milk given by vendors and supplied by milk booths. Whole milk can be only cow's milk OR milk with a combination of cow's milk and buffalo milk OR buffalo milk. It also includes whole milk powders and long life milk available in the market. Products available:
Red packet government dairy milk Heritage Jersey Nestle, Amul and Vijaya liquid whole milk and whole milk powder Skimmed milk Includes whole milk from which cream has been removed at home and the liquid skimmed milk available in the market.

Anthropometry Protocol
The methodology for anthropometrical assessments was adapted from standard reference texts on the subject.
General principles • A digital weighing machine (Seca scales) with an accuracy of 100 gms will be used to measure weight. • Height will be measured by a portable plastic stadiometer with a base plate, accurate to 1 mm (Seca Leicester height measure).
• For sitting height, a standard table (of known height) high enough for the legs of the participant to dangle over the edge when seated will be used.
• A skinfold calliper accurate to 0.2 mm (Holtain skinfold calliper;) will be used to measure the thickness of the skinfolds.
• Lengths and circumferences will be measured with a non-stretch metallic tape with a narrow blade and a blank lead-in (ADE metallic tape; ).
• The measurements (where relevant) will be made on the left side. If for some reason this is not possible, measurements should be taken on the opposite side and this change recorded.
• Measurements should be conducted in normal indoor clothing (basic clothes minus shoes, sweaters, socks, etc).
• Weight and height will be recorded twice, circumferences twice and skinfolds readings will be taken three times.

Notes:
(a) Extreme care should be taken to avoid digit preference when taking the recordings (b) Any problems at the time of measurement that can affect their accuracy (such as those due to physical deformities, postural problems or bandages on the limbs) are recorded. However, in many cases the problem may be partially rectifiable e.g. removal of bandage in case of one. A common problem is that of hunching in case of older subjects. If so, check if maximum of the back is in contact with the wall. As the inadequate measurements cannot be used, it is strongly advisable to try and address the problem rather than record inadequate measurements.

Weight
The weighing machine should be placed on the most level part of the floor and calibrated at the start of every clinic. At the time of each reading, the scale should be turned again to ensure that the monitor reads 'zero'. The participant should be asked to stand on the scale reasonably straight and looking ahead. Weight should be recorded only when the reading on the monitor had settled. Avoid taking the reading as the participant is coming off the weighing machine.

Height and sitting height
The stadiometer should be set up on the most level part of the floor and calibrated at the start of every clinic. Where the floor is not level (as detected with the help of a spirit level), a suitable standard wooden block with appropriate wedges set into place should be used to deal with the faulty floor. The stadiometer is then placed on this block and measurements done as stated below.
The participant should be asked to stand on the stadiometer, while the observer checks the following points:

Waist circumference
The participant should be asked to stand straight with feet close together and abdomen relaxed, weight evenly balanced on both feet, and the arms hanging loosely at the sides. The measurement should be carried out on the bare skin. The observer should face the participant and place the tape around the participant, in a horizontal plane, at the level of the natural waist, which is the narrowest part of the abdomen between the ribs and the iliac crest (top of the hip bone), as seen from the anterior aspect. With the participant breathing out gently looking straight ahead, the reading should be taken at the end of a normal expiration.

General notes for measuring circumferences:
Circumferences should be recorded with the zero end of the tape held in the left hand above the remaining part of the tape held by the right hand. The plane of the tape should be perpendicular to the long axis of that part of the body, and parallel to the floor. For all circumference measurements, the tape should not be pulled too loose or too tight, held snugly around the body part but not indenting it. In some individuals there may be gaps between the tape measure and the skin, in such cases attempting to reduce the gap by increasing the tension of the tape is not recommended.

Hip (buttock) circumference
The measurement is carried out with the participant wearing normal indoor clothing. The tape measure is applied to the widest part of the buttock. This is ensured by the observer squatting at the side of the participant so that the level of the maximum extension of the buttocks can be seen. Attention should be paid that the hip muscles of the participant are not contracted, and the tape is horizontal and not compressing the skin.

Mid-arm circumference
The

Calf circumference
The calf circumference is measured with the participant standing, evenly balanced, with the feet shoulder width apart. The measurement should be taken on bare skin. The tape is positioned horizontally around the calf and moved up and down to locate the maximum circumference in a plane perpendicular to the long axis the calf. The observer should bend down at the time of the reading to avoid errors due to parallax. The level at which the measurement is taken should be marked at the medial aspect of the calf (inner border).

Head circumference
The head circumference is measured with the subject standing. Added objects, for example, pins are removed from the hair. The measurer stands facing the left side of the adult. The tape is passed around the head and positioned so that large amounts of cranial hair (braids) are excluded. Anteriorly, the tape is placed just superior to the eyebrows and posteriorly it is placed so that the maximum circumference is measured. The tape is pulled tightly to compress hair. The measurement is recorded to the nearest 0.1 cm.

Chest circumference
The chest circumference is measured with the subject breathing normally. The tape should be held horizontally at the level of the nipples, passing over the lower scapular angle. The arm of the subject may be raised before fixing the tape around the chest, but after that the arms should rest normally, by the side, while taking the measurement. In case of females, another circumference at the base of the xiphoid process, horizontal to the thorax may be taken.

Chest girth inspiration:
The subject should be allowed to draw his breath in as much as possible and note the highest value.

Chest Girth Expiration:
The subject should be asked to breathe out and the least value is noted.

General notes for measuring skinfolds:
The sites should be marked before taking these readings. Palpation of the site prior to measurements helps familiarise the participant with contact in the area. The callipers should be held in the right hand, while the thumb and the index finger of the left hand are used to elevate a double fold of skin and subcutaneous adipose tissue about 1 cm proximal to the site at which the skinfold will be measured. Place the thumb and the index finger on the skin about 3 inches apart on a line perpendicular to the long axis of the skinfold, and gently pull the skinfold away from the body by drawing the thumb and index finder towards each other. The fold needs to be grasped firmly, and the amount of tissue must be sufficient to form a fold with approximately parallel sides. Care must be taken so that only skin and adipose tissue are elevated (but not the muscle). The calliper head should perpendicular to the skinfold (with the dial facing), placed halfway between the crest and base of the fold. Gently and fully release the calliper pressure (the release of the pressure should be gradual to avoid discomfort). The pinch should be maintained for 5 seconds before taking the measurement. It is important to keep this timing accurate as otherwise the reading will vary. Errors due to parallax should be avoided by correct positioning over the dial (this may involve standing on something to be at the right height).

Triceps skinfold thickness
The triceps skinfold is measured in the midline of the posterior aspect of the arm, over the triceps muscle,

Biceps skinfold thickness
Biceps skinfold thickness is measured as the thickness of a vertical fold raised on the anterior aspect of the arm, over the belly of the biceps muscle. The skinfold is raised 1 cm superior to the line marked for the

Subscapular skinfold thickness
The lowermost tip of the scapula is identified. If it is difficult to appreciate this, the participant should be asked to place the back of his/her hand on the lumbar region. The medial border of the scapula is followed downwards until the inferior angle is felt. Once it is identified, the participant was asked to relax with arms hanging by the side before a mark is applied to the skin immediately below the lower most tip (angle) of the scapula. The skinfold is picked up obliquely above the mark with the fold slightly inclined downward and laterally (at about 45 degree to horizontal), in the natural cleavage of the skin. The calliper jaws are applied below the fingers, such that the marked cross is at the apex of the fold. Readings should be taken after 5 seconds.

Suprailiac skinfold thickness
The suprailiac skinfold is measured in the anterior axillary line immediately superior to the iliac crest. The

Calf medial skinfold thickness
The participant stands with the foot of the side being measured on a platform, so that the knee and hip are flexed to about 90 degrees. The skinfold is measured at the level of the maximum calf circumference, already marked as before. From a position in front of the participant, the observer raises a skinfold parallel to the long axis of the calf on its medial aspect, when viewed from the front, at a level slightly proximal to the marked site. The calliper head should be perpendicular to the skinfold with the dial facing up at the marked point on the medial border of the calf.

Grip strength
1. Before this test is administered, the handle of the dynamometer must be adjusted for the size of each individual subject. The handle should fit comfortably in the hand with enough allowance for a good grip. Record the setting found on the inside gauge if follow-up testing is to occur.

2.
Place the subjects arm at their side keeping it away from the body with the elbow bent slightly (approximately 20 0 ). Illustrate the use of the instrument to the subject prior to testing.

3.
The test is to be administered with dominant hand first and then with the non-dominant hand. The examiner should be confident that the subject's maximum grip strength is being measured. Emphasis on "squeeze as hard as you possible can" and other forms of encouragement may be necessary for maximum effect. Allow three trials with each hand, right and left hand alternately, but introduce a brief pause of about 10 to 20 seconds between each trial to avoid excessive fatigue.

4.
Record the amount of strength registered at each trial. If the difference between the scores of each is within 3 kilograms, (considering the scores of each hand separately) the test is complete. If a difference of more than 3 kilograms is noted, the test is repeated after a sufficient rest period. Calculate the averages for each hand separately. It is important that the dials be returned to the "0" position after every trial. Readings are taken to the nearest whole kilogram. A digital thermometer (model name, company, country) will be used to measure room temperature. BP will be measured in right upper arm in sitting position.
Note: All those expected to be involved in the BP measurements should go through the instruction manual beforehand to familiarise themselves with the issues, possible problems and care of the equipment.

Steps in blood measurement
1) Check the subject has not undertaken following activities for at least 30 minutes preceding the examination: strenuous exercise, eating, drinking of anything other than water, smoking or drugs that affect the BP. Ask the subject to sit on the chair with right arm on the table. Important: Upper arm (cuff when applied) should be at the level of the heart, neither too high nor low. If required use a pillow or some other support to ensure this.
2) Apply the correct size cuff. This should be determined by the arm circumference (see above), which should be read from the anthropometry section. You should also keep a measuring tape handy to check in case the subject has not been to the anthropometry section (in case you do measure the arm circumference for assessment of the cuff size, avoid the temptation to note it in the anthropometry datasheet at this stage). The green coloured band (indicating the centre of the bladder) should be positioned 1-2 cm above the elbow joint on the inside of the arm. Close the cuff with the fabric fastener. The green area of the cuff must cover the brachial artery. The cuff should not be too tight or too loose; it should fit snugly so that it is just possible to fit two fingers between the arm and the cuff. Do not place the cuff on thick clothes or roll up the sleeve if it is too tight. Important: The sleeve of the dress should not constrict the blood flow in the arm. If so (either because of tight fitting shirt/blouse or rolling up of tight sleeve), take steps to address this (e.g. asking the subject to take off shirt over that arm or in case of female subject provide them with a loose gown to change into).
3) After the cuff is applied, wait for five minutes before taking the first reading. The subject should be made to relax during this time by making them sit comfortably, asking them to breathe easily and to relax the body and arm, and explaining the procedure. Stress that they should not move, talk or touch the device during measurement. This period should also be used to note down information in the datasheet: researcher code and initials, BP apparatus number, cuff size used, and arm side on which the measurement is being made. Important: The BP apparatus will give accurate readings at temperature range of 10 -40 degree Celsius. If room temperature is outside this range, take steps to address this (use room heater if too cold or cooler or table fan with cloth soaked in water if cooler not available if too warm).

4)
Press the START button on the machine. Wait for the machine to complete the measurement. This is confirmed by the flashing of a single downward arrow symbol. When all the air has been released the 'heart' symbol on the display panel and the readings are displayed for the next five minutes. Note down the systolic and diastolic blood pressure and the pulse rate. Important: If the subject moves or talks during the measurement or the measurements appear inaccurate or the symbol 'E' (indicating error) is displayed on the screen, repeat the measurement.

5)
Wait for 1 minute before taking the second reading so that blood circulation can resume. This can be ensured by presence of 'heart' symbol on display panel. If you need to interrupt a measurement for any reason, you can do so by pressing the ON/OFF button. Record the second set of readings on the datasheet. Note down any problems (such as subject appearing too anxious despite reassurance or if the readings had to be taken on the left arm for some reason). Switch off after measurement to conserve batteries. The monitor will also switch off automatically after five minutes. .

Spirometry test Pulmonary Function Assessment (PFTs)
The PFTs will be measured using the Card Guard SpiroPro device-a portable spirometer that accurately measures lung ventilatory functions during Vital Capacity (VC) and Forced Vital Capacity (FVC) tests. The system records the Volume-time and Flow-volume curves.
Step 1. Before administering the test, explain to the participant that you are going to ask them to do a breathing test, which involves them breathing into a tube. Ask if they are happy with this. If they say yes, then proceed with the questionnaire. If they say no, then explain test further and attempt to persuade them.
Step 2. Administer respiratory function questionnaire (below). If any of questions 23.1 to 23.8 is "yes" do not proceed with test.
Step 3. Check person has passed urine before test. 4.
Step 4. Check weight and height without shoes are recorded. 5.
Step 6. Check participant is sitting upright on chair with both feet flat on floor.
Describe spirometry test to participant. Say to the participant: "After breathing normally, slowly blow out until your lungs are empty. Then take a big deep breath in filling your lungs completely. Place the mouthpiece inside mouth and close lips tightly around the mouthpiece Blow out as hard and as fast as you can. Keep going until you cannot push anymore air out." 8.
Step 8. Fieldworker demonstrates test with own mouthpiece. a) Move the switch to 'BLOW' b) Attach nose clip with new piece of plastic c) Demonstrate the test 9.
Step Repeat test for Blows 2 and 3 13.
Step 13. Switch off unit and then back on again. Repeat test at least 2 more times and write down the FEV1 and FVC for blows 1, 2 and 3. Check if test is acceptable after each blow.
Criteria for stopping the test. 14.
Step Repeat blows until • Both of the above criteria are met • Or, a total of eight tests have been performed • Or, the subject cannot continue 15.
Step 15. Discard mouthpiece at end of test.
Potential problems which may affect test: • Not putting mouthpiece inside mouth (needs to go between the teeth but not so they bite on it) • Embarrassment and laughing during test which makes effort not be maximal • Oral phobia so refuses to put it in their mouth or cannot open mouth wide enough to do the test • Stress incontinence in women so effort will not be maximal. Make sure people pass urine before the test to try and stop this happening.
Other important points: • Fieldworkers must write their name on their mouthpiece and keep it for the duration of the day.
• Fieldworkers must write down only the FEV and FVC (do not record PEF and PER when they move the switch to VIEW position) • Check spare batteries are available

Spirometry flow chart
Instructions for Accelerometer 1. Check and set time on the computer so that accelerometer takes up the same time.
2. Accelerometer to be given when available to consenting subjects among parents and siblings.
3. Initialize the accelerometer right after download of the previous recording (no need to set a starting time) 4. Put belt on the subject at the end of the clinic itself. 5. Instruct subjects to keep the accelerometer on for 5 days and return. 6. Instruction sheet to explained to all the subjects and then given. 7. For illiterate subjects, sheet completed on return of the accelerometer by asking subject (approximate times should be okay). 8. Accelerometer to be given to those keen and reliable. 9. Give preference to repeats if available, at the same time not to wait.
10. Ensure that the accelerometer sheet is completed-record start/end times, times taken off and details of travel if person has travelled for more than 2 hours.

[The above to be filled by the team and needs to be entered at the end of clinic but could be different if subject insists on starting later at home in which case subject could fill it].
Date of final accelerometer removal: _______________________________

[To be filled by team/subject and team to confirm the timings with the subject once again when they return the accelerometer].
Please ask the subject to keep the accelerometer on at all times, including during sleep. If they need to remove the accelerometer at any point, please ask them to fill the information in on the sheet below.

Body Composition Measurements using TANITA (pictures to be referred from manual Appendix for each step) Body Composition/Bio -Impedance
Step1: Turn on the Power: Press the ON/OFF key Step 2: Enter Clothes Weight: Enter the approximate weight of clothes worn by the participant using the numerical keys Step 3: Select the Body Type: Select the body type from Standard Male, Standard Female, Athletic Male and Athletic Female Step 4 : Enter Age: Step 5 : Enter Height Step 6 : "STEP ON" while flashing arrow will appear next to "STEP ON" Start Measurement: Ask the participant to step on the Weighing Platform with bare feet in a stable position without bending the knees so they touch the electrodes Taking Measurement: Make sure heels are placed on the posterior electrodes, and the front parts of the feet are in contact with the anterior electrodes Measure the Impedance: When the grips are grasped with both hands, "0000" will appear at the bottom of the display and the impedance measurement will begin. The "0000" marks will disappear one by one during the measurement; after five full cycles, the measurement will be complete.
The participant should not step off the Weighting Platform until the "0000" symbols disappear completely.

Measurement is completed:
Once the body weight and impedance measurements have been completed, the overall body fat percentage will be shown at the bottom of the display and a buzzer will sound.
The printer should be ON such that the measurement results are printed out.
When You Continue to Measure: After printing is completed, go back to step 3. Follow the same procedure mentioned above.
Finish Measurement: Press the "ON / OFF key and turn off the power.
The output record for each subject will be entered immediately in the appropriate section of the questionnaire and the record also attached to the questionnaire booklet for future reference if required.

What you SHOULD DO
• Ask the participant to remove the heavy outer clothing like coat, shawl, pull-over etc.
• Ask the participant to hold both arms straight down while taking measurements

NIN Component of study
All parents and children will be invited to participate in the NIN component of the study. Consenting subjects will be brought to NIN for undergoing DEXA and vascular measurements. A subset of parents and siblings will also undergo the pulmonary function tests as a small validation exercise for the measurements obtained in the field using Card-Guard Spiro-Pro. One SRF to conduct carotid IMT scan and help in DEXA scan and two FW's to conduct the vicorder component and two clinic helpers will be stationed at NIN.

Sampling Frame
The APCAPS Phase I study will cover 20 villages, 10 in Intervention and 10 in the Control arm. The sampling list will be the list of 1826 households that have been followed in the Hyderabad DEXA study. The villages in each arm will be paired by number of households and a random selection of 10 villages with their closest pair in size will be chosen for the study. This will result in about 700 households in each arm. i.e about 1400 households for the total study.

January 2011
From January 2011 onwards, there will be two teams working independently in the field. A training workshop will be conducted in early January to ensure standardised methods of measurement and complete understanding of the entire protocol so that both teams work in an identical manner to reach the requisite target recruitment by June/July 2011.
Each team will consist of one Medical Officer (JRF), 6 research Assistants, one laboratory technician, and one clinic helper. The Project Manager and Field Manager will ensure overall coordination and functioning of fieldwork in the two arms. One laboratory technician will be appointed at NIN and will work for the latter part of the day and analyse all samples for haemoglobin and blood glucose at the NIN lab on the same day as collection. In the event of absenteeism of one or the other Medical Officer/technician, the Project manager and Field manager will ensure alternate arrangements so that field clinics are not disrupted. 27. Enter a "1" into the DXA box on front of questionnaire.

DXA Measurements
28. Once all the participants for the day have been scanned, print out the results of the total body, lumbar spine and hip scans for each participant. These will be collected by the data entry officer each day.
29. On Monday, Wednesday and Friday afternoons, carry out the L1-L4 and L2-L4 calculations twice for each participant. Print the forms for each participant and staple together. These forms will be collected and entered into the database by the Data Entry Operator.
30. At the end of each week, save all the study files for that week to CD. This will be collected by the Data Entry Operator.  -Do not press too hard (e.g. if participant coughs or complains).
-Make sure not to take videos by accident as these cannot be processed -Do not clean probe with spirits or other agents. Gently wipe with soft cotton.
-Make sure probe is dry when it is stored.
-If the ultrasound machine loses power and you cannot get the laptop to recognise it, unconnect all the cables and restart the laptop. Reconnect the cables-you may need to use a different USB port on the laptop for the connection. 5. Get the subject to lie down in the supine position. Explain to the subject about the two cuffs (neck and thigh). Check that the subject is comfortable with the cuffs.
6. Place 10cm blue cuff as high as possible on the thigh, making sure it is not too tight or too loose. The tube on the thigh cuff should be pointing towards the head and should be in line with the femoral artery.
7. Feel the carotid pulse and place the 2.5cm cuff (white) attached to the black Velcro neck band on to the neck (right carotid), making sure it is not too tight and not too loose (you should be able to fit 1-2 fingers inside the band). The white pressure pad should be on the inside of the cuff with one end starting in the middle of the neck and the other (end with the tube) over the carotid area. Make sure that the tube is pointing downwards.
8. Connect the red lead to the proximal (neck) cuff and the blue lead to the distal (thigh) cuff.
9. Ensure that the subject has been resting in the supine position for TEN MINUTES before the PWV reading 10. Take the subject's blood pressure 3 times and record the values on the questionnaire. 11. Measure path lengths in cm with tape measure and enter into questionnaire. Explain to the subject about the two Vicorder cuffs (brachial and thigh).Check that the subject is comfortable with the cuffs. 5. Place a 10cm blue cuff as high as possible on the thigh, making sure it is not too tight or too loose. The tube on the thigh cuff should be pointing towards the head and should be in line with the femoral artery. Attach the tube of this cuff to the blue Vicorder lead. 6. Place a 10cm blue cuff around the upper arm with the tube pointing downwards. Attach the tube of this cuff to the red Vicorder lead. 7. Measure the distance from the top of the brachial cuff to the top of the femoral cuff with a tape measure. 8. In the quick launch tab on the right hand side of the Vicorder programme, select PWA. This will bring up the study folder. 9. Enter the subject details in the study folder: Study Name (APCAPS), Patient ID, First name, Last name, Sex, DOB (to change DOB you need to highlight each section of the date and use the up and down arrow keys) 10. For the proximal site, select right brachial from the drop down menu. For the distal site, select right femoral from the drop down menu. 11. Enter the following into the study folder: a. Aortic path length (distance between cuffs) b. Systolic blood pressure (use the third reading) c. Diastolic blood pressure (use the third reading) 12. Click on OK. This will bring up the data capture screen. 13. Press F3. The screen should now split in half, with the top half for the brachial readings and the bottom for the thigh readings. 14. Inform the subject that you are about to inflate the cuffs and that they will feel some pressure on the thigh and upper arm. 15. Press the SPACEBAR to inflate the cuffs. 16. Wait until there have been several successive screens of uniform waves in both windows and the values in the grey boxes have stabilised.
17. Press the SPACEBAR to capture the data. 18. To save the data: a) Press ENTER. b) Browse to find the Brachial PWA spreadsheet in the Real study PWV folder. Select this and Click OK. A message will appear asking if you want to overwrite this file, click Yes. 19. Repeat steps 14-17 twice more so that you capture 3 readings for each subject.
NOTE: For the second and third readings for a subject, the readings will automatically be saved to the same spreadsheet when you press ENTER.

Quality Control
To check the quality of the waveforms, for one subject per day, the images of the PWV waveforms should be recorded. To do this: 1. After saving a recording, press F4 to export the data. Browse to the qc Brachial PWA folder in the real study PWV folder. 2. Save the image as a jpeg and name it with the patient ID, name and number of the recording eg 1,2,3

VICORDER INSTRUCTIONS
Plug in VICORDER to laptop with main USB / 8-way cable.
Load up software onto the laptop once prompted to do so.
On the main screen, click on the START menu in the bottom left hand corner.
Then go to SETTINGS.
Then go to CONTROL PANEL.
In Control Panel, switch to CATEGORY VIEW if you are in Classic View. Once in Category view, select PERFORMANCE & MAINTENANCE.
In Performance and Maintenance, select SYSTEM at the bottom of the window and then select HARDWARE, then select DEVICE MANAGER.
In Device Manager, scroll down the list until you find the USB driver name with an exclamation mark next to it. If you cannot see this, then double click on the USB name (Universal Serial Bus Controllers).

Previously when this software has been loaded problems arose with the USB <-> Serial
Right click on the USB name which has the exclamation mark next to it and select UPDATE DRIVER. Files for the two separate medical measurements need to be copied onto the CD.

Reimbursement of participants
Distribution of reimbursement to participants is the responsibility of the Field Manager / Project Manager, and is undertaken at the end of the day when all examinations have been completed.
The level of reimbursement was arrived at using the following principles. Understanding of these principles may be useful at times for clarification, or in unusual circumstances where an appropriate level needs to be calculated. However, these do not need to be routinely explained to the participant, and reimbursement should be offered, as far as possible, on a lump sum basis using the suggested scales. In case there is doubt about the correct scale, please err on the higher side remembering that participants are volunteers, under no obligation to participate.
The APCAPS subjects are reimbursed for their time and loss of wages while attending the day clinic at the village and for attending the clinics at NIN for DEXA and vascular studies. Total ------>

Proof of identity :
It is important that some proof of identity is available for the subject that confirms the relationship and place of residence of the subject. This requirement will be explained in the invitation letter. Examples of proof of identity include the ration card, voting card, work identity card, driving or other license, or letter from the village head. The subjects will be requested to bring a photocopy along with the original. The photocopy should be checked against the original and retained for record. If the subject brings the original but forgets the photocopy, the study team should get a photocopy made locally (the money for this can be claimed back). Where the subject forgets to bring a proof along, they should still be recruited, but only after asking suitable questions to confirm the identity. This will have to be done in a very careful manner to avoid upsetting the participants. In such cases the participants should be asked to post a photocopy of the proof after their return home, and they should be chased up for this if necessary. Where no proof becomes available, this fact should be recorded in the database.

Component completed
This section should be completed at the end just before the subject departs, generally at the time of reimbursement

MEDICAL REFERRAL
Who should be referred?
• Medical help can be obtained from the Medical Officer in the field team.
-Complaining of possibly serious medical conditions • Chest pains on exertion, breathlessness, severe pain -Wanting a second opinion for known medical conditions • Discuss with SRF and negotiate with Referral hospital/Medical college . Abnormal results and Referral: All reports will be communicated to the subjects. For any abnormal results based on the clinical examination and laboratory results like anaemia, high blood pressure or sugar etc. the subject will be seen by medical officer and if required referred to the local medical college hospital/referral hospital.
First aid: The doctor will provide first aid as needed or refer the subject to the local medical college hospital as needed. In our scenario, we are most likely to encounter cases as: Heat exhaustion: If you suspect heat exhaustion, get the person out of the sun into a shady location. Lay the person down and elevate the feet slightly. Loosen or remove person's clothing. Cool the person by spraying him or her with cool water and fanning and have the person drink cool water. Monitor the person carefully. If person shows symptoms and signs of a heatstroke like temperature greater than 104 F, fainting, confusion or seizures, the person should be immediately referred. Fainting If a subject looks or feels faint during the procedure, it should be discontinued. The subject should be asked to place their head between their knees. They should subsequently be asked to lie down. If they are happy for the test to be continued after a suitable length of time, it should be done so with the subject supine and the circumstances should be recorded. They may wish to discontinue the procedure at this point, but willing to give the blood sample at a later time.
Needle stick injuries The wound should be encouraged to bleed. The wound should be washed with soap and warm water, if available. Other hand cleaner may be used if water is not available.

Bruises
For any bruises following the blood sampling, elevate the injured area and apply ice or cold pack for 30 to 60 minutes at a time for a day or two after the injury.

INVENTORY AND CARE OF EQUIPMENT
All the equipment will have to be handled with care. It is the responsibility of the Field Manager to ensure at the end of the day when clinics are over; field workers who are carrying out the anthropometric measurements will pack the equipment properly and keep in a safe place. In case of any damage to the instruments or loss of instruments, this will have to be communicated to Delhi/Bristol or NIN immediately so that a replacement can be made.
An inventory of the general items as well as biochemistry items will have to be maintained by the centre and sent to Delhi/Bristol and NIN at the end of the month. Any item which needs to be procured will have to be informed well in advance.

Communications-
• Every day the Project Manager will check the mail once in the morning and once at the end of the day.
Mails which require immediate attention should be given high priority. All such mails must be replied by the evening of the day and even if the mentioned task cannot be done immediately, this should be communicated with the likely date of completion.
• Wherever deadlines have been specified, it is important that these are met. If for some unavoidable reasons it is not possible to complete a task in time, then this should be communicated well ahead of time so that a backup plan can be thought of.
• The must have a date diary-a large size one with a page to date. This is to make note on that day of the diary of the things to be done. Once any action is completed, it can be cancelled off from the list. With the diary you can also plan ahead for actions.
• Matters related to biochemistry, database and finances should be addressed to the concerned person and copied to the Project Manager.
• All urgent / important mails need to be acknowledged.
• Anything sent through post (photocopy of questionnaires, bills, attendance etc.) should be preceded by a mail to keep the other centre informed. Again receipt of anything should also be acknowledged by mail.

Daily Calibration
-All instruments which are being used for measurements in the clinics have to be calibrated on all the days when clinics are held. -As far as possible, readings for the calibration to be taken on the same 2 fieldworkers everyday.
-Calibration has to be done early morning on arrival at the clinics before the subjects come.
-This has to be entered into a calibration sheet provided and then saved into the PC at the end of the day.
-Calibration needs to be done on active instruments only i.e., the one being currently used. In cases where both instrument is being used at the same time, then calibrations to be done for both.
-For the calibration of Card Guard Spiro Pro readings are recording on two fieldworkers and the readings are saved in an excel file.
-These readings need to be reviewed at the weekly meetings and if there is a very wide variation in the readings, then this needs to be communicated to the coordinating centre for appropriate action.
-For calibration of the callipers, measurements of a thin and thick book will suffice.

Monthly Calibration
Besides daily calibration, monthly calibration of the instruments needs to be done. The procedure for this is as below: Weighing Machine: -This can be done by comparing against standard weights starting from 5kg and above.
Stadiometer: -The metallic tape can be used for this and readings to be taken at 75 and 150 cms on the stadiometer.
Callipers: -Same as the daily calibrations, i.e., using 2 books with different thickness.
BP apparatus: -Comparing it against a sphygmomanometer. It is important however to remember that the same sphygmomanometer to be used throughout the study. Spirometer: -The Card-Guard SpiroPro should also be calibrated once a month using a standard three litre calibration syringe. Method -. With the Spirometer in zero position, and using an appropriate mouthpiece or connector, the three litre calibrating syringe will be emptied into the spirometer.
This procedure will be repeated three times, with the spirometer returning to zero position after each syringe check. The spirometer should read within +/-three percent of the syringe volume.
After the calibration check, the research staff will perform upto eight maneuvers on himself/herself.
The same person will do the calibration each time. The graphs are saved and readings entered into an excel sheet and reviewed by the coordinating center every month. Any marked variations will mean that the device needs to be checked and appropriate action taken.

FAQs
What if a woman (or in some cases men e.g. if elderly) refuses to travel alone?
• In such cases additional payment (50% extra) may be made for an accompanying person.
However, the accompanying person is not to be included in the study. If the accompanying person wishes to have him or herself checked, a short examination (or blood pressure measurement) may be considered to ensure cooperation of the family; however, the data will not be recorded.
What if the subject arrives to the clinic but is not completely fasting?
• As long as the subject has not eaten or drank anything other than water in the last 4 hours, the sample should be collected and the subject recruited.
What if a woman is pregnant or may be pregnant?
• If there is any possibility that a woman may be pregnant, ask her to take a pregnancy test.
Women who are pregnant undergo all the questionnaires and tests in the same way but do not undergo screening through DXA. • Women who are pregnant are not asked to fast before the examination clinic.

Reporting System
This will be a regular reporting system from NIN to Delhi/Bristol.

NIN to Delhi / Bristol.
The Project Manager is required to send the weekly reports to Delhi/Bristol by Wednesday afternoons. Reports for a week will contain data through the last Wednesday to the current Tuesday. The weekly reports to be send to Delhi/Bristol are: -Weekly summary report -Fieldworker performance sheet -Database copies At the end of each month (on the last Wednesday of the month along with the weekly reports), Field Manger and Project Manager are required to note the following: inventory of the general items inventory of biochemistry items * Each research assistants will also maintain the daily progress report which can be reviewed by the team leader every week at the weekly meeting on Friday. ** Every Friday there is a phone call between the Project Manager and PI at LSHTM so as to keep them abreast with the activities carried through the month and also discussing any issues/problems requiring solution.
IMPORTANT: All reports to be prepared by the Data Entry Operator and reviewed by the / Project Manager before sending it to Bristol.
At the end of each month the Project Manager is required to send the attendance signed by the Principal Investigator to Mr I Alex / Ms Lata by the 25 th of each month.
The statement of Expenditure along with all supporting documents is required to be sent to Mr I Alex / Ms Lata biweekly.
Format for Statement of Expenditure:

DATA ENTRY
The database construction will be done in Bristol; set-up, operation and administration will be carried out at NIN by the Project Manager in consultation with Pete Shiarly at Bristol.
Two data entry operators will be involved in simultaneous data entry at NIN and data quality control procedures will be followed as outlined below. Copies of the database will be sent at regular intervals to the coordinating centre.

Data quality of the questionnaires
Every week when the team meets, the Field Manager / Project Manager picks 2 questionnaires filled in the current week by one interviewer. The Field Manager / Project Manager needs to go through the questionnaire, question by question with the entire team, to identify incomplete/missing entries and any errors. The purpose of this is to rectify and clear any doubts which the interviewers may still have.

Data quality of the database
For the data quality of the database, the Filed Manager needs to compare the questionnaires with the same entered into the database. For this, 10% of the questionnaires per week, already entered into the database, could be checked. A blank questionnaire is kept at hand when doing this exercise and errors/missing data identified in the database can then be noted as tally lines against the particular question in the blank questionnaire. For e.g, if there is an error in Qs. 3.1 of the questionnaire, then a tally line needs to be marked against the question. This exercise has to be repeated every week so that by the end of the month, n questionnaires would have been covered. Then the tally lines in the blank questionnaire can be added up for all the n questionnaires and this adds up to the total number of errors and gives us the numerator for the error rate.
Error rate = Total number of tally lines Total number of filled fields in the questionnaire x n

1) All the questionnaires filled should be filed appropriately.
2) For completed questionnaires pending for data entry, one separate box can be maintained. All questionnaires in this box should be filed according to ID. For easy management, boxes with ranges of IDs starting in a series. 3) Once the completed questionnaires have been checked by the Field Manager these will be filed by ID in the filing cabinet draw "For Data Entry". 4) After data entry the questionnaires are filed by ID in the filing cabinet draw "Entered Questionnaires" 5) The photocopies of the ID will also be filed by ID in the filing cabinet draw "IDs".

Clinics
For the clinics, 20 file folders will be maintained. On arrival of a subject to the clinic, a file folder containing the following will be handed over to him/her by the fieldworker who is receiving them. The file folder for the participant will contain the following: This file folder will then be handed to the Field Manager at the end of the clinic procedures who will then check for missing/incomplete data. If everything is complete, this is to be piled in the box pending for data entry. The empty file folders will then be use for the next batch of subjects. The photocopies of the ID will also be filed.

PARTICIPANT INFORMATION SHEET Andhra Pradesh Children And Parents Study (APCAPS) Nutritional challenges, abdominal adiposity and type 2 diabetes in Indians: Parental and offspring cardiometabolic risk -a Transgenerational Extension of the Hyderabad Nutrition Trial
Purpose of the study Researchers at the National Institute of Nutrition in Hyderabad and at the London School of Hygiene and Tropical Medicine are interested in understanding the effects of poor nutrition and environmental circumstances during pregnancy and childhood, on the chances of diabetes and heart disease occurring in the offspring. You have been chosen for this study as you/your wife/your mother participated in an earlier study conducted by National Institute of Nutrition. At this time, some but not all the participants were provided with extra food with the help of the Anganwadi. We are trying to know from this research whether the chances of getting heart diseases have been reduced in the children of women who got extra food. These comparisons will help to predict future needs for health services in India which will benefit planning. The research will also help in understanding the health effectsboth positive and negative-of nutritional supplementation which may lead to ideas for preventing bad outcomes. Finally, the study provides an opportunity for you to gain important information about your health status.

Questions and concerns
You are being invited to participate in this medical research study. Kindly read this information sheet attentively. If you are not clear about anything or there is any uncertainty, then you are free to ask any questions when you receive a visit from the study staff. Sign the consent letter only when you are able to understand the nature of this study fully along with your rights as a participant. You are free to discuss it with anybody, whose consultation is important to you.

Voluntary participation
It is entirely your decision to participate in the study. If you want to discontinue at any point of time, you are free to leave this study without stating any reason. Your medical care will not be affected by your decision.

What does it mean to participate?
Participation in this study involves answering some questions about your general and medical habits, having body measurements taken, your blood drawn (15ml), saliva (spits) sample taken and a short medical examination, which includes some measures of your heart and a breathing test. You will have a special type of X-ray taken to measure your body fat. Your answers are confidential and will be used only for the study.
You will be required to give a blood sample You will be asked to fast overnight before the visit in which you give your blood samples. During the visit we will ask you to donate a small sample of your blood. Trained personnel will draw the blood. The supplies used for drawing blood will be safe and sterile and used only once and the supplies will be destroyed after use. The blood you give will be used for research purposes only. Any blood that is left over after the test will may be used for further test related to medical research including tests to find out whether any diseases run in the family.

Follow up in the future
The present research does not require the research team to see you again. However, important information can only be gained by linking your current health and life style to what happens to you in the future. Therefore, we would like to invite you to continue to participate in the future if you wish.
Benefits from the study You will get a medical examination by an experienced team, including doctors. You will undergo the following tests: blood pressure, blood glucose for diabetes, haemoglobin, DXA scanning to measure fat levels, three tests on your heart and blood vessel function (arterial stiffness, pulse wave velocity and carotid intima media thickness), respiratory function and saliva sampling for genetic studies. Counselling will be given to you based on the results of the medical examination and blood tests. You will be given your blood results.
By participating in this study you will help researchers gain a clearer understanding of how nutritional supplementation has an effect on diseases like obesity and diabetes. Your participation will also help them understand how lifestyle, physical activity and dietary habits affect your health.

Risks of participating in the study
We do not expect that you will incur any risks by participating in this study. Blood drawing may cause a small amount of discomfort, but it is only temporary.

Financial costs
You will not incur any costs as a result of your participation in this study. Your travel fare along with any other expenses incurred and loss of daily wages towards time spent for our study will be reimbursed. Refreshments will also be provided.

Confidentiality
If you decide to take part in the study, all details provided by you will be kept confidential and it will only be made available to investigators related to this study. Information will be stored in password protected computer in Hyderabad and in London. The results will be published in research magazines and reports. However, the names and details of the study subjects will not be disclosed and you will not be recognized from them.

Funding & Coordinating agency
The funds for this study are being provided by the Wellcome Trust, a major UK based research charity.

Ethical Review
The study proposal has been approved by the London School of Hygiene & Tropical Medicine and the National Institute of Nutrition.

Contact for further information
If you require any further information or need to clarify some issue, you can contact any of our study team members at National Institute of Nutrition, Hyderabad What your signature means Your signature on the next page means that you understand the information given to you about the study. If you sign the form it means that you agree to join the study. You will be provided a copy of this patient information sheet to keep with your records.
• The purpose of this study was explained to me in my own language.
• I have been given the opportunity to ask questions and reply was given for all the questions to my satisfaction.
• I have been informed by the investigators about the process including the nature, objective and known likely inconveniences related to this study and I have understood them. • My medical data are strictly confidential and I only authorise the persons, involved in the research, identified by the sponsor or health authorities to consult about the same.
• By signing this form, I give my free and informed consent to take part in this study as outlined in the information sheet and this consent form. Specifically, I agree to being interviewed, examined and having (15 ml) blood drawn. I agree to my information, including results of blood tests, to be used in research.
• I give permission for any blood that is left over after the tests to be stored and used for further laboratory tests for medical research • I understand that for all practical purposes I may not gain anything by participating in the study though in the long run it may be beneficial to the community.
• I understand that I can withdraw from the study at any point without giving any reasons and withdrawing from the study will not affect me in any way.
• I understand that I will receive a very small radiation dose from the DXA scan, so I shouldn't have this scan if I am pregnant. • I have been given the opportunity to ask questions and reply was given for all the questions to my satisfaction.
• I have been informed by the investigators about the process including the nature, objective and known likely inconveniences related to this study and I have understood them.
• My medical data are strictly confidential and I only authorise the persons, involved in the research, identified by the sponsor or health authorities to consult about the same.
• By signing this form, I give my free and informed consent to take part in this study as outlined in the information sheet and this consent form. Specifically, I agree to being interviewed, examined, having saliva samples taken and blood drawn (15ml) for necessary tests. I agree to my information, including results of blood tests, to be used in research.
• I understand that the saliva sample and blood sample may be used for genetic research aimed at understanding genetic and epigenetic influences on diseases.
• I understand that for all practical purposes I may not gain anything by participating in the study though in the long run it may be beneficial to the community.
• I understand that I can withdraw from the study at any point without giving any reasons and withdrawing from the study will not affect me in any way.

Andhra Pradesh Children And Parents Study (APCAPS) Nutritional challenges, abdominal adiposity and type 2 diabetes in Indians: Parental and offspring cardiometabolic risk -a Transgenerational Extension of the Hyderabad Nutrition Trial
Purpose of the study Researchers at the National Institute of Nutrition in Hyderabad and at the London School of Hygiene and Tropical Medicine are interested in understanding the effects of poor nutrition and environmental circumstances during pregnancy and childhood, on the chances of diabetes and heart disease occurring in the offspring. You have been chosen for this study as you/your wife/your mother participated in an earlier study conducted by National Institute of Nutrition. At this time, some but not all the participants were provided with extra food with the help of the Anganwadi. We are trying to know from this research whether the chances of getting heart diseases have been reduced in the children of women who got extra food. These comparisons will help to predict future needs for health services in India which will benefit planning. The research will also help in understanding the health effects -both positive and negative-of nutritional supplementation which may lead to ideas for preventing bad outcomes. Finally, the study provides an opportunity for you to gain important information about your health status.

Questions and concerns
You are being invited to participate in this medical research study. Kindly read this information sheet attentively. If you are not clear about anything or there is any uncertainty, then you are free to ask any questions when you receive a visit from the study staff. Sign the consent letter only when you are able to understand the nature of this study fully along with your rights as a participant. You are free to discuss it with anybody, whose consultation is important to you.

Voluntary participation
It is entirely your decision to participate in the study. If you want to discontinue at any point of time, you are free to leave this study without stating any reason. Your medical care will not be affected by your decision.

What does it mean to participate?
Participation in this study involves answering some questions about your general and medical habits, having body measurements taken, your blood drawn (15ml), saliva (spits) sample taken and a short medical examination, which includes some measures of your heart and a breathing test. You will have a special type of X-ray taken to measure your body fat. Your answers are confidential and will be used only for the study.
You will be required to give a blood sample You will be asked to fast overnight before the visit in which you give your blood samples. During the visit we will ask you to donate a small sample of your blood. Trained personnel will draw the blood. The supplies used for drawing blood will be safe and sterile and used only once and the supplies will be destroyed after use. The blood you give will be used for research purposes only. Any blood that is left over after the test will may be used for further test related to medical research including tests to find out whether any diseases run in the family.

Follow up in the future
The present research does not require the research team to see you again. However, important information can only be gained by linking your current health and life style to what happens to you in the future. Therefore, we would like to invite you to continue to participate in the future if you wish.
Benefits from the study You will get a medical examination by an experienced team, including doctors. You will undergo the following tests: blood pressure, blood glucose for diabetes, haemoglobin, DXA scanning to measure fat levels, three tests on your heart and blood vessel function (arterial stiffness, pulse wave velocity and carotid intima media thickness), respiratory function and saliva sampling for genetic studies. Counselling will be given to you based on the results of the medical examination and blood tests. You will be given your blood results.
By participating in this study you will help researchers gain a clearer understanding of how nutritional supplementation has an effect on diseases like obesity and diabetes. Your participation will also help them understand how lifestyle, physical activity and dietary habits affect your health.

Risks of participating in the study
We do not expect that you will incur any risks by participating in this study. Blood drawing may cause a small amount of discomfort, but it is only temporary.

Financial costs
You will not incur any costs as a result of your participation in this study. Your travel fare along with any other expenses incurred and loss of daily wages towards time spent for our study will be reimbursed. Refreshments will also be provided.

Confidentiality
If you decide to take part in the study, all details provided by you will be kept confidential and it will only be made available to investigators related to this study. Information will be stored in password protected computer in Hyderabad and in London. The results will be published in research magazines and reports. However, the names and details of the study subjects will not be disclosed and you will not be recognized from them.

Funding & Coordinating agency
The funds for this study are being provided by the Wellcome Trust, a major UK based research charity.

Ethical Review
The study proposal has been approved by the London School of Hygiene & Tropical Medicine and the National Institute of Nutrition.

Contact for further information
If you require any further information or need to clarify some issue, you can contact any of our study team members at National Institute of Nutrition, Hyderabad What your signature means Your signature on the next page means that you understand the information given to you about the study. If you sign the form it means that you agree to join the study. You will be provided a copy of this patient information sheet to keep with your records. (For the examination today we will ask you to undertake the following: interview, measurement of body size, DXA scan and a visit with the doctor. We will also ask you to give up to two blood samples. The examination will last until the afternoon.)

Nutritional challenges, abdominal adiposity and type 2 diabetes in Indians: Parental and offspring cardiometabolic risk -a Transgenerational Extension of the Hyderabad Nutrition Trial
• I am free to participate or not to participate in this study.
• The purpose of this study was explained to me in my own language.
• I have been given the opportunity to ask questions and reply was given for all the questions to my satisfaction.
• I have been informed by the investigators about the process including the nature, objective and known likely inconveniences related to this study and I have understood them. • My medical data are strictly confidential and I only authorise the persons, involved in the research, identified by the sponsor or health authorities to consult about the same. • By signing this form, I give my free and informed consent to take part in this study as outlined in the information sheet and this consent form. Specifically, I agree to being interviewed, examined and having (15 ml) blood drawn. I agree to my information, including results of blood tests, to be used in research. • I give permission for any blood that is left over after the tests to be stored and used for further laboratory tests for medical research • I understand that for all practical purposes I may not gain anything by participating in the study though in the long run it may be beneficial to the community. • I understand that I can withdraw from the study at any point without giving any reasons and withdrawing from the study will not affect me in any way. • I understand that I will receive a very small radiation dose from the DXA scan, so I shouldn't have this scan if I am pregnant. • I have been given the opportunity to ask questions and reply was given for all the questions to my satisfaction.
• I have been informed by the investigators about the process including the nature, objective and known likely inconveniences related to this study and I have understood them. • My medical data are strictly confidential and I only authorise the persons, involved in the research, identified by the sponsor or health authorities to consult about the same. • By signing this form, I give my free and informed consent to take part in this study as outlined in the information sheet and this consent form. Specifically, I agree to being interviewed, examined, having saliva samples taken and blood drawn (15ml) for necessary tests. I agree to my information, including results of blood tests, to be used in research. • I understand that the saliva sample and blood sample may be used for genetic research aimed at understanding genetic and epigenetic influences on diseases. • I understand that for all practical purposes I may not gain anything by participating in the study though in the long run it may be beneficial to the community. • I understand that I can withdraw from the study at any point without giving any reasons and withdrawing from the study will not affect me in any way. • I have been given a copy of the information sheet and consent form to keep. Thinking or dreaming about something terrible that had happened to you in the past -like your house being destroyed, a severe accident, being hit or assaulted, or being forced to commit a sexual act Now I will ask you questions relating to your usual sleep patterns. .
[l] 27.13 If unable to obtain satisfactory spirometry (check one):