Durban Diabetes Study:A Study of the Epidemiology of Diabetes Mellitus in Urban South Africans of African Descent (DDS)
Durban Diabetes Study:A Study of the Epidemiology of Diabetes Mellitus in Urban South Africans of African Descent (DDS)
Type 2 diabetes (T2D) is an emerging epidemic in sub-Saharan Africa, with an estimated
 prevalence of 6%. With around seven million cases of T2D in 2000, it is anticipated that over
 18 million Africans will have the disease by 2030. In South Africa the prevalence of T2D in
 people of African descent has been reported to be between 3-10%. However, there have been
 limited studies on diabetes epidemiology in South Africans using currently employed World
 Health Organization (WHO) criteria.

 To assess the burden of T2D and associated risk factors in South Africa, we are establishing
 the Durban Diabetes Study (DDS) - a population-based cross-sectional study in the city of
 Durban (the eThekwini municipality) to be undertaken in 1,200 participants of African
 descent. In-depth health questionnaire responses, biophysical measurements and blood and
 urine samples will be gathered from these participants. These data will allow researchers to
 estimate the population prevalence of T2D and associated risk factors in the region. The
 infrastructure created for this cross sectional study has the potential to serve as a strong
 framework for future research initiatives and public health interventions within the region.
STUDY OBJECTIVES

 -  To estimate the prevalence of diabetes mellitus and associated cardiometabolic and
 infectious risk factors in this population

 -  To provide aetiological insights into the variation in cardiometabolic and infectious
 risk factors in adults using both population genetic and epidemiological approaches

 -  To compare diagnostic tests for diabetes in this population

 -  To create a unique framework for building a large scale cross sectional study in an
 African population to examine a wide range of health indices-and lay the groundwork for
 additional long-term studies

 -  to inform health policy and public health programmes aimed at addressing the rise in
 non-communicable diseases (NCDs) in South Africa, which may also shape public health
 strategies in other African countries

 STUDY DESIGN

 The DDS involves a number of stages:

 Stage 1: Geographical sampling frame The eThekwini Municipality (city of Durban) is selected
 as the initial sampling frame and broken down into nine planning unit clusters (PUCs),
 representing nine townships (Umlazi, Inanda, KwaMashu, Ntuzuma, Mpumalanga Complex, Cato
 Manor, Clermont, Lamontville and Chesterville) totalling a population of approximately
 1,378,750 individuals. Streets are randomly selected in each PUC proportional to the PUC size
 and the proportion of formal and informal housing in each PUC. Households are selected by
 systematic cluster sampling of homes located on the randomly selected streets.

 Stage 2: Community awareness Once local ethics approval is obtained from the BioMedical
 Research Ethics Committee at the University of KwaZulu-Natal (UKZN) and the support of the
 KwaZulu-Natal Provincial Department of Health is obtained, the local health authorities and
 community leaders from each of the nine PUCs, will be contacted for approval.

 Stage 3: Participant enrolment Once the selected PUC areas have been sensitised to the study
 (end of Stage 2), they are visited again by the DDS team. During this stage the DDS team
 visits households in the selected neighbourhoods and all family members who are of African
 descent, not pregnant and aged 18 years or older are invited to participate in the study.
 Consent is obtained from residents agreeing to participate. All residents are invited to
 attend a central venue (i.e. local community centre/school) the following week where the DDS
 team collects health and lifestyle questionnaire information, performs simple biophysical
 measures and collects blood and urine samples. While recruiting participants in the
 community, the DDS team uses geographical information system (GIS) mapping techniques to
 gather accurate geographical and boundary data on the selected PUC areas.

 Stage 4: Data and sample collection, testing and data analysis Data and sample collection are
 undertaken on specific mornings at a central venue. This is important as participants are
 required to fast overnight for an oral glucose tolerance test (OGTT). Questionnaire and
 biophysical data from the study are collected, stored electronically, and analysed. Blood and
 urine samples are transported to the University of KwaZulu-Natal in Durban for storage.
 Laboratory tests are done at a private laboratory. Some samples are sent to specialist
 centres in the UK, Europe, the USA and other countries for genomic analyses.

 Stage 5: Feedback of results to study participants Starting eight weeks after sample
 collection, participant results will be available at the DDS feedback office. All OGTT and
 lipid results will be posted back to participants, unless they choose not to receive them. To
 receive all other results (e.g. HIV, hepatitis C virus [HCV], liver function tests)
 participants must make an appointment at the DDS feedback office to collect their results.
 This is optional, and participants who do not wish to collect their results can choose not to
 contact the DDS feedback office. If appropriate, participants collecting results that are
 abnormal or potentially harmful will be advised to visit their local clinic or hospital for
 further investigation and management as part of their standard clinical care.

 DATA AND SAMPLE COLLECTION A. Collection of samples

 1. Blood sample overview Only fasted participants are eligible for complete blood sample
 collection. Non-fasted participants are only eligible for the collection of the 10 ml
 plain serum and the four ethylenediaminetetraacetic acid (EDTA) blood samples. Aseptic
 venepuncture is performed by a registered research nurse via a closed evacuated blood
 collection system, using standardised procedures to minimise the risk of infection..

 2. Blood samples required Eleven blood tubes are collected from each fasted participant: a
 10 ml plain serum tube, two 10 ml EDTA whole blood tubes, two 2 ml EDTA whole blood
 tubes and, for the OGTT, three 2 ml sodium fluoride (NaF) tubes and three 2 ml plain
 serum blood tubes. Thus, a maximum of 46 ml venous blood (approximately three
 tablespoons of blood) is taken from each participant. For non-fasted participants, only
 a 10 ml plain serum sample and the four EDTA whole blood samples are collected. Since
 linked anonymous HIV testing is undertaken as part of this study, the study nurse is
 trained in HIV counselling and follows relevant national guidelines.

 For fasted participants, the 75 g OGTT will be performed as recommended by WHO.
 Categories of glucose tolerance will be according to the WHO 1998 Classification.

 The 10 ml plain serum tube is used to test blood for infectious diseases and
 cardiometabolic biomarkers. The first 10 ml EDTA whole blood tube is shipped to the UK
 for genetic analysis. The second 10 ml EDTA whole blood tube undergoes plasma extraction
 for HCV viral load testing and RNA extraction. The two 2 ml EDTA whole blood tubes are
 used for HbA1c testing and full blood count, respectively.

 Linked anonymous HIV testing As part of this study, consenting participants will be
 tested for HIV via linked anonymous testing. This will be done in accordance with
 national guidelines.

 3. Urine sample collection A spot (untimed) urine sample will be taken for measuring
 albumin-to-creatinine ratio and urinary sodium and potassium.

 B. Collection of questionnaire data Once appropriate consent is obtained and blood sample
 collection has begun, participants move to the questionnaire station. The questionnaire used
 in this study is an electronic questionnaire (EQ) and is an adaptation of the standardised
 and validated WHO STEPwise approach to Surveillance (STEPS) tool designed for the collection
 of chronic disease risk factors, including socio-demographic indices, lifestyle, infectious
 disease risk factors and cardiometabolic history.

 C. Collection of biophysical measurements

 Study participants move to the biophysical measurements station which are done using
 calibrated and validated equipment. The following measures are taken using standardised
 protocols:

 -  Height and weight (used to calculate body mass index)

 -  Waist circumference and hip circumference (used to calculate waist-hip ratio)

 -  Mid upper-arm circumference

 -  Blood pressure and heart rate

 BLOOD SAMPLE ANALYSES

 All blood samples are assayed for infectious and cardiometabolic biomarkers at a private
 laboratory in Durban. Confirmatory HIV tests will be done. Below is a list of laboratory
 methods used for serological and biochemical tests:

 -  Plasma glucose: Enzymatic method, based on hexokinase and glucose-6-phosphate
 dehydrogenase enzymes

 -  Serum creatinine: Based on the reaction of picric acid with creatinine in an alkaline
 medium

 -  Serum lipids:

 -  Cholesterol: Enzymatic method, using cholesterol esterase and cholesterol oxidase
 conversion, followed by a Trinder endpoint

 -  Triglycerides: Based on the Fossati three step enzymatic reaction with a Trinder
 endpoint. The single reagent procedure quantifies the total triglycerides including
 the mono- and di-glycerides and the free glycerol fractions

 -  HDL: Enzymatic reaction based on the procedures developed by Izawa, Okada and
 Matsui

 -  LDL: Calculated using the Friedewald formula or enzymatic method

 -  Lipoprotein (a): Turbidometric (Beckmann)

 -  HbA1c: High performance liquid chromatography. Instrument: BioRad Variant II

 -  HIV: 4th generation ELISA (Abbott Architect)

 -  Hepatitis B and C: ELISA

 DNA and RNA extraction will take place at the University of KwaZulu-Natal, the University of
 Cambridge, the Wellcome Trust Sanger Institute, or an authorised third party facility in the
 UK. The study coordinator is responsible for shipping samples to the UK for DNA and RNA
 extraction. Purified DNA and RNA is then sent to the Wellcome Trust Sanger Institute, UK, for
 genetic analysis.

 GENETIC ANALYSIS

 1. Overview Given the need for large scale automated pipelines and next-generation Illumina
 sequencing and genotyping technology, all genotyping and sequencing is done at the
 Wellcome Trust Sanger Institute, UK (http://www.sanger.ac.uk/). The Wellcome Trust
 Sanger Institute is an international genome centre with extensive experience and
 expertise in handling DNA and RNA samples from global genetic research initiatives,
 including blood and samples from several countries in SSA. A material transfer agreement
 has been devised for the shipment of biological samples to the UK.

 2. Extraction and quantification of DNA and RNA DNA and RNA are extracted at the University
 of KwaZulu-Natal, the University of Cambridge, the Wellcome Trust Sanger Institute or by
 an authorised third party service provider in the UK, e.g. GenProbe, Manchester.
 Purified DNA or RNA is then sent to the Wellcome Trust Sanger Institute. All extracted
 nucleic acid is quantified by either gel electrophoresis or picogreen (for DNA samples)
 or ribogreen (for RNA samples) and quantified relative to standards of known
 concentration. All nucleic acid samples are stored at the Wellcome Trust Sanger
 Institute in secure -80 ˚C freezers.

 3 Genotyping and sequencing Current genome-wide human diversity arrays do not sufficiently
 capture the high levels of genetic diversity and population structure found in African
 populations, limiting their utility. Therefore, we will use next-generation sequencing,
 genotyping arrays and imputation to help determine the association between human genetic
 variation in the study population and quantitative risk factors and diseases and viral
 diversity. All genotyping and sequencing is done at the Wellcome Trust Sanger Institute. In
 addition, because of the massive storage and curation requirements for the genomic data from
 next-generation sequencing method, all raw genomic data is stored at Wellcome Trust Sanger
 Institute, UK.

 4 Data transfer, storage and management All raw genomic data will be held at the Wellcome
 Trust Sanger Institute, UK and the European Genome-phenome Archive (EGA) at the European
 Molecular Biology Laboratory-European Bioinformatics Institute (EBI), UK.
Inclusion Criteria:

 -  African descent

 -  aged 18 years or older

 -  not pregnant

 -  currently residing in Durban (eThekwini Municipality)

 Exclusion Criteria:

 -  non- African descent

 -  pregnant

 -  not currently residing in Durban (eThekwini Municipality)