Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

A thesis submitted to the

Graduate School

of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

MASTER OF PUBLIC HEALTH (M.P.H.)

in Epidemiology

in the Division of Public Health Sciences

of the Department of Environmental Health

of the College of Medicine

by

Arielle G. Hernandez

Bachelor of Arts (B.A.), University of Kansas, 2011

Committee: Ranjan Deka, Ph.D.

Aimin Chen, M.D. Ph.D.

Russell E. Ware, M.D. Ph.D.

Patrick T. McGann, M.D. M.S.

Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

ABSTRACT

Background

Sickle cell disease (SCD) contributes substantially to childhood mortality in sub-Saharan

Africa. In the Republic of Uganda, the number of annual SCD births and the geographical distribution of the disease burden are unknown. A partnership between the Uganda Ministry of

Health, Makerere University College of Health Sciences, and Cincinnati Children’s Hospital

Medical Center was established to design and conduct the Uganda Sickle Surveillance Study.

Objective

To describe the current prevalence and distribution of SCT and SCD in Uganda. The goals were to establish a partnership between academia and government, build local sickle cell laboratory capacity and determine the feasibility of testing a high-volume of samples for SCD, and create an accurate geospatial map of the SCD burden across Uganda.

Methods

A Sickle Cell Laboratory was constructed and outfitted, and local personnel were recruited and trained. Hemoglobin electrophoresis was performed by isoelectric focusing on dried blood spots already collected in the Ministry of Health’s Early Infant Diagnosis Program from HIV-exposed children over a one-year period, to identify and quantify the presence of sickle cell trait (SCT), SCD, and other hemoglobin variants.

ii Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Results

A total of 99,243 dried blood spots were tested between February 2014 and March 2015, with 97,631 results successfully obtained. The national prevalence of SCT was 13.3%, ranging from 4.6% in the South Western region to 19.8% in the East Central region, and the SCD prevalence was 0.7%, ranging from 0.2% in the South Western region to 1.5% in the East

Central region. SCT was detected in all districts, with the highest prevalence being 23.9% in

Alebtong district. Analysis by region revealed that the observed SCT prevalence positively correlated with published malaria prevalence. SCD was less common in children older than 12 months and those who were HIV-positive, consistent with early mortality and co-morbidity.

Conclusions

A successful North-South partnership joined academia and government to design and conduct an integrated national surveillance study, and to build local sickle cell laboratory capacity through training and program monitoring and evaluation. These successful strategies have positioned the Ministry of Health to begin to address the country’s high disease burden, and will motivate and prioritize efforts for improving the management of SCD in Uganda.

iii Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

iv Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

ACKNOWLEDGMENTS

There are many people who contributed to the story of the Uganda Sickle Surveillance

Study and the completion of this thesis.

First and foremost, I want to thank my mentor, Dr. Russell Ware. From day one, he has given me his time, support, and trust. Dr. Ware’s belief in me has been the ultimate motivator, and I am so grateful for the autonomy that demands creativity, cultivates profound learning, and pushes personal thresholds. I thank him for being a devoted teacher and for encouraging me to develop and invest in my own sickle cell education. I have come to deeply appreciate the complexities and far-reaching implications of this cause and research that I will dedicate my career to. The past four years have been a steep and rapid learning curve, and I am looking forward to many more years of working with and learning from Dr. Ware.

I also want to thank my other committee members. I am very thankful to Dr. Patrick

McGann, who is such a great teacher and friend who I look up to. Thank you to my Academic

Advisor, Dr. Ranjan Deka, who has shared his expertise to foster my interest in population genetics and has always shown me great support and kindness. I also want to thank Dr. Aimin

Chen, for his support throughout the MPH program.

I am very grateful to my Ugandan colleagues for a wonderful research partnership and friendships that make the thousands of miles between Uganda and Ohio feel so much closer.

Their dedication to their work and country is incredibly inspiring to witness.

Finally, I want to thank my family for being a constant source of love and inspiration all my life.

v Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

TABLE OF CONTENTS

Abstract ...... ii

Acknowledgments...... v

List of Tables, Images, and Figures ...... viii

I. Introduction ...... 1

i. Background ...... 1

ii. Problem Statement ...... 3

iii. Approach ...... 5

II. Methods...... 6

i. Partnership...... 6

ii. Study Design ...... 7

iii. Sickle Cell Laboratory ...... 8

iv. Data Management and Analysis ...... 17

v. Monitoring and Evaluation ...... 18

III. Results ...... 19

i. Partnership ...... 19

ii. Sickle Cell Laboratory ...... 21

iii. Program Assessment ...... 23

iv. Surveillance Study ...... 25

v. Dissemination of Results ...... 40

IV. Discussion ...... 41

vi Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

i. Goals ...... 42

ii. Strengths, Limitations, and Challenges ...... 44

iii. Significance to Public Health ...... 50

iv. Direction for Future Planning and Research ...... 52

V. Institutional Review Board/Ethics Committee Approval ...... 55

VI. References ...... 56

VII. Vita ...... 60

Appendix A: Isoelectric Focusing (IEF) Hemoglobin Electrophoresis Procedure ...... 61

Appendix B: Uganda Sickle Surveillance Study Program Assessment Plan ...... 76

Appendix C: Uganda Sickle Surveillance Study Program On-Site Assessment Report ...... 83

Appendix D: Burden of sickle cell trait and disease in the Uganda Sickle Surveillance Study

(US3): a cross-sectional study ...... 96

Appendix E: Is integrating sickle cell disease and HIV screening logical? ...... 103

Appendix F: Prevalence and Mapping of Sickle Cell Trait and Disease in Uganda ...... 106

Appendix G: School of Medicine Research Ethics Committee at Makerere University ...... 134

Appendix H: Cincinnati Children’s Hospital Institutional Review Board ...... 137

vii Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

LIST OF TABLES, IMAGES, AND FIGURES

Tables

I. Table 1: Hemoglobin Letter Categories for IEF Analysis ...... 17

II. Table 2: Regional Hemoglobin Results ...... 26

III. Table 3: District Hemoglobin Results...... 27

IV. Table 4: Regional Correlation Between SCT and Malaria in Uganda...... 36

V. Table 5: SCT and SCD Prevalence by Age and HIV Status...... 37

Images

I. Image 1: Wallac DBS Puncher System in Sickle Cell Laboratory ...... 12

II. Image 2: Automatic 3.2 mm Sample Punch by Wallac DBS Puncher ...... 12

III. Image 3: Pipetting Eluted Samples into Sample Template on IEF Gel ...... 13

IV. Image 4: Migration of Samples on Electrophoresis Unit...... 14

V. Image 5: Crop and Enlargement of Right Side of an IEF Gel Depicting Band Patterns

for N, T, S and Standard Control ...... 17

VI. Image 6: US3 team at Opening of Sickle Cell Laboratory and Launch of the Study

...... 22

VII. Image 7: “Break the Silence” Banner at US3 Final Result Reveal and Public Sickle

Cell Testing Launch ...... 41

Figures

VIII. Figure 1: District Map of the Prevalence of SCT in Uganda ...... 33

viii Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

IX. Figure 2: Regional Map of the Prevalence of SCT and Malaria in Uganda ...... 35

X. Result Reporting and Follow-up on Infants with SCD at Health Facilities ...... 39

ix Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

INTRODUCTION

Background

Sickle cell disease (SCD) refers to a group of genetic blood disorders that affects hemoglobin, the protein molecule in erythrocytes (red blood cells) that is responsible for carrying oxygen throughout the body. SCD is caused by a mutation in the beta-globin gene on chromosome 11, due to a single nucleotide polymorphism of adenine to thymine at the sixth codon position that results in an amino acid substitution of glutamic acid for valine.1 The beta- globin gene encodes production of beta-globin protein, a subunit of the hemoglobin molecule.2

Normal adult hemoglobin (HbA) is a tetramer that consists of four protein subunits of two alpha- globin gains and two beta-globin chains. The mutation in the beta-globin chain that creates the abnormal variant sickle hemoglobin (HbS) causes the hemoglobin molecule to become unstable due to the hydrophobic nature of valine compared to the hydrophilic glutamic acid. After delivering oxygen to the body’s tissues, the deoxygenated HbS tetramer quickly forms a polymer within the erythrocytes; this act of “sickling” in erythrocytes results in long, inflexible tactoids that irreversibly distort the cell membrane into the distinct crescent or “sickle” shape.

The most common and severe form of SCD is sickle cell anemia (SCA), where individuals inherit the HbS allele from each parent, resulting in the homozygous genotype of

HbSS that manifests with hemolytic anemia, acute vaso-occlusion, chronic organ damage, and early mortality. Other SCD genotypes include the compound heterozygous HbS with beta- thalassemia (sickle-beta thalassemia), HbS with HbC (HbSC disease), and others. Individuals who inherit a single copy of the mutated HbS allele in combination with a normal HbA allele

1 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study have the heterozygous HbAS genotype and do not have SCD; they are called carriers and have

SCT, which has no phenotypic characteristics.

The sickle gene arose independently in different geographical locations around the world that are now or have been endemic for malaria.3 This observation led to the “malaria hypothesis”4 that suggests the prevalence of SCD in high-pressure malaria regions reflects a survival advantage that the sickle gene offers,3,5 allowing the sickle allele to reach high frequencies in these areas. The sickle gene is thus a classic example of a balanced genetic polymorphism, as it confers positive selection for heterozygous carriers (HbAS, SCT) who are relatively protected against Plasmodium falciparum malaria, but negative selection for homozygotes (HbSS, SCA) who very often die from malarial infection.6 Distinct beta-globin haplotypes can be assigned to the HbS allele, which reflect the regions in Sub-Saharan Africa,

India, and the Arab peninsula where the mutation arose. Most African-Americans have ancestry originating from West or Central Africa, which includes the Senegal, Benin, or Central African

Republic haplotypes.

SCD most commonly presents within the first one to two years of life with acute and life- threatening complications, such as increased susceptibility to infections, painful vaso-occlusive events, and chronic hemolytic anemia. Without adequate early diagnosis and treatment, SCD is associated with a very high risk of death within the first five years of life. This is especially true in developing countries, where infection is the leading cause of death for children with SCD.

Almost inexplicably, SCD is rarely mentioned alongside other common causes of childhood mortality, which thus constitutes it as a silent killer of children in developing countries. Only

2 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study with proper diagnosis and treatment can the global burden of SCD be adequately recognized and managed.

Problem Statement

SCD is one of the most commonly inherited hematological disorders, affecting millions of people worldwide. More than 75% of the annual 400,000 global SCD births occur within sub-

Saharan Africa, with the number projected to climb another 30% by 2050.5 It is estimated that

50-90% of affected children with SCD in Africa will die before age five years.7 Most countries with the highest burden of SCD have limited health resources and infrastructure, and there are scant data describing SCD within their borders. Furthermore, the lack of knowledge about SCD among both healthcare providers and the general population limits the potential for early and accurate diagnosis, which significantly contributes to the high early, and often preventable, mortality associated with the disease.

Overshadowed by well-publicized and well-funded infectious pandemics such as malaria, tuberculosis, and HIV, SCD persists as an unrecognized and unaddressed health issue on the

African continent. In 2006 and again in 2010, the WHO issued reports highlighting the serious public health implications of SCD in Africa, acknowledging the inadequate national SCD policies and plans, and making a call for interventions to identify the scope of the problem in each country and to address awareness, disease prevention, and early detection.8,9 The aim of the

2010 WHO SCD strategy for the African Region was reduction of incidence, morbidity and mortality, which outlined specific interventions for member states and partners, including strengthening laboratory and diagnostic capacity and supplies with nationwide coverage;

3 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study initiating and enhancing SCD surveillance, early identification, and screening; provision of comprehensive healthcare management and affordable medicines for SCD patients; genetic counselling, testing, and advocacy; creation or strengthening of national SCD programs within the framework of non-communicable disease and child health agendas; program monitoring and evaluation; and fostering partnerships and resource mobilization from internal and external sources to support these interventions. The WHO further proposed that these interventions be guided by the principles of country ownership, leadership, and cultural sensitivity; integrated evidence-based and prevention approaches that are cost-effective and accessible; and partnership and team building. This WHO strategy stands bold and explicit, yet there remains a lack of policies and programs in high-burden countries focused on SCD prevention and management.

The underappreciation of SCD as a public health problem in these countries limits progress toward achieving new UN Sustainable Development Goal 3, Good Health and Wellbeing, which aims to end preventable deaths of newborns and children under five years of age by 2030.10

In the Republic of Uganda, a small East African country, rough estimates suggest that up to 20,000 babies are born with SCD annually, but information on these estimates and the geographic distribution of SCD within the country rely primarily upon outdated and limited data.

In 1949, a study by Lehmann and Raper reported the prevalence of SCT among different tribes compared with their physical anthropology in Uganda. They found the differences to be statistically significant, ranging from low prevalence (<5%) among Hamitic tribes, to more than

20% for many of the Nilotic tribes in the north, and a wide range among Bantu tribes with

Bamba being the highest at 45% in the west.11 A more recent but limited study of Uganda in

2010 suggested estimates much lower, reporting a prevalence of 3% to 17% for SCT and 0% to

4 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

3% for SCD in five districts in Eastern and Western Uganda.12 While both studies document the burden of SCD in Uganda, neither provides widespread geographic coverage of the county or adequate sample sizes for generalizability. Current epidemiological data are necessary to focus resources and interventions in high-burden areas, and to provide evidence-based guidance to develop and implement effective national sickle cell strategies for comprehensive primary care and management programs.

Approach

The Uganda Sickle Surveillance Study (US3) was the first national-level surveillance study of SCD to be conducted in a sub-Saharan African country. The objective of this study was to describe the current prevalence and distribution of SCT and SCD in Uganda. The short-term goals were to establish an effective partnership between academia and government, build local sickle cell laboratory capacity and determine the feasibility of testing a high-volume of samples for SCD, and create an accurate geospatial map of the SCD burden across the country. The long- term goals were to develop and implement national sickle cell strategies, including newborn screening, education and training of healthcare providers, public awareness, and early preventive therapeutic interventions.

We hypothesized that there is a large burden of SCD in Uganda with substantial geographic variability in the prevalence of disease across the country. US3 planned to test up to

100,000 dried blood spots (DBS) for SCT and SCD by isoelectric focusing (IEF) hemoglobin electrophoresis and to use this data to generate geospatial maps and to perform demographic analyses. The epidemiological data generated from the study will inform the Ministry of Health

5 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

(MOH) about the current prevalence and distribution of SCD, guiding evidence-based recommendations to address the sickle cell burden within the country.

METHODS

Partnership

The Uganda MOH’s overarching goals for child health are to scale-up and sustain beneficial and cost-effective services to reduce under-five infant and neonatal mortality rates in the country. With support from the Director General of Health Services, the MOH commissioned accurate up-to-date data on SCD in the country to be able to prioritize its limited resources for targeted sickle cell interventions and improvements to primary care, as well as to provide evidence to leverage health policy agendas and mobilize funding for developing sustainable services, such as newborn screening and early preventive treatment.

The MOH enlisted Cincinnati Children’s Hospital Medical Center (CCHMC) Division of

Hematology for technical and clinical expertise and extensive research experience to help guide a large-scale cross-sectional study. CCHMC developed a study team to provide overall planning and programmatic support, as well as to conduct technical training and monitoring and evaluation. Funding for US3 was provided by the Cincinnati Children’s Research Foundation.

Makerere University College of Health Sciences (MakCHS) was recruited as the local academic body for study consultation and local ethics and regulatory management. MakCHS includes the medical and public health schools, and is affiliated with the Mulago Hospital Sickle

Cell Clinic.

6 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

The Central Public Health Laboratories (CPHL) is a unit under the National Disease

Control Program of the MOH and is located in the capital city of Kampala. The CPHL provides laboratory support for disease surveillance, coordinates health laboratory services, assists in developing policy and guidelines, and conducts training and implementation of quality assurance schemes for laboratories throughout the country. The CPHL is home to the Early Infant

Diagnosis (EID) program for prevention of mother-to-child transmission of HIV that was initiated in 2006 with support from the Centers for Disease Control and Prevention.

Approximately 100,000 HIV-exposed infants are identified and tested each year through the EID program, which utilizes a National Sample Transport System for coverage across the country.13

The CPHL provided space and personnel for a new Sickle Cell Laboratory, as well as the existing infrastructure of the EID program and National Sample Transport System for surveillance sample collection and data management.

Study Design

US3 was designed to be a cross-sectional study of the prevalence and distribution of SCT and SCD in Uganda using all DBS collected in the MOH’s EID Program from HIV-exposed children over a one-year period. Hemoglobin electrophoresis was performed by IEF on consecutive DBS samples at the CPHL to identify the presence of SCT, SCD, and other hemoglobin variants.

7 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Sickle Cell Laboratory

The WHO 2010 report on SCD in the African region included recommendations for developing and strengthening of laboratory facilities for surveillance and accurate diagnostics, as part of the comprehensive strategy for reducing the SCD burden. This specific intervention included the improvement of laboratory management at all levels of the health system, screening of newborns, disease surveillance, training of health professionals, and development of protocols.9

One of the primary goals of the US3 study was to build and develop local sickle cell laboratory capacity that will enable sustainable long-term sickle cell testing and research in

Uganda. To accomplish this goal, the first step was to establish and outfit physical space at the

CPHL for sickle cell testing and to provide initial and ongoing technical training and support to laboratory personnel. Next, the quality and accuracy of laboratory testing and data management was to be monitored for program improvement and, finally, the feasibility of high-volume testing for SCD was to be assessed to prepare for scale-up to targeted and possibly national newborn screening capabilities.

Set-up and Equipment

The CPHL served as the study setting for US3. The space that was provided for the

Sickle Cell Laboratory underwent physical renovations to construct countertops and shelving units, input plumbing and electrical systems, add air conditioning to reduce heat and humidity, and to overall create an adequate and productive work environment.

8 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

IEF is a common and robust method for hemoglobin identification for DBS specimens, including those for infants that have high amounts of fetal hemoglobin (HbF). Equipment for the

IEF procedure was selected, purchased by CCHMC, donated to the CPHL, and set-up in the

Sickle Cell Laboratory with the assistance of the CCHMC technical team. The IEF set-up includes many robust and durable pieces of equipment. The Wallac DBS Puncher automatically punches DBS samples into microtitration plates for efficient and consistent sample preparation.

Multiphor II Electrophoresis Units connect to programmable power supplies, to obtain high voltage fields, and circulating water baths, to maintain required cooling and temperature control, to run samples on one-dimensional gels (RESOLVE® Neonatal Hemoglobin Test Kit,

PerkinElmer, Inc.). Cold storage of gels and reagents used a purchased refrigerator, and long- term sample storage was in a -20°C freezer.

Personnel and Training

The CPHL recruited six full-time personnel for the Sickle Cell Laboratory, which included one Laboratory Manager/Technologist, three Laboratory Technicians, one Data Entry

Clerk, and one Data Manager/Administrator.

The team from CCHMC was responsible for initial and ongoing training of the laboratory personnel. Prior to on-site training at the CPHL, the CCHMC team developed a step-by-step technical protocol (Appendix A) and visual electronic presentation of the IEF procedure, as well as educational materials on hemoglobins and result interpretation. On-site training took place in

February 2014 for a total of 10 days, prior to the official launch of US3. The Laboratory

Technologist and Laboratory Technicians learned about the study and how to set-up and operate

9 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study the IEF equipment. They were then guided through each step of the IEF process in parts, then through the entire sequential IEF process repeatedly for the course of the training days. This training approach enabled each laboratory based personnel to learn every step of the process for cross-coverage and task delegation. By the end of the 10 day training period, each laboratory personnel was able to independently perform the entire IEF process from start to finish.

For on-going remote training and troubleshooting, the laboratory personnel and CCHMC team met on a weekly basis by Skype throughout the study. At the end of each week, scored gels were scanned and sent to the CCHMC team for assessment of gel and sample quality and score accuracy. Any inquiries, resolutions, and recommendations discussed in the meetings were documented and disseminated to personnel and CPHL leadership for reference and training purposes.

Data Collection

As part of the EID program, blood samples were routinely collected from HIV-exposed infants by heel stick or finger stick on a standard DBS card. The DBS cards were labeled with the name of the health facility where the sample was collected, the date of collection, the infant’s name and date of birth, and a unique identifying number (exposed infant number). Each health facility completed a Dispatch Form with the demographic information transcribed from all of the

DBS cards collected, as well as each infant’s address and telephone number for follow-up result reporting. The DBS cards and associated Dispatch Form were packaged per established EID protocol and transported by motorbike to hubs at the sub district level and then shipped by courier to the CPHL in Kampala.

10 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

When the samples were received at the CPHL, all demographic information provided on the Dispatch Forms was entered into the EID electronic database. Each DBS card had a unique barcode that is enacted to link the exposed infant number with the demographic information in the database. The samples were then sent to the EID laboratory for testing by viral polymerase chain reaction (PCR). Within one week, the same DBS samples were made available to the

Sickle Cell Laboratory. HIV positive samples were temporarily withheld for retesting and were later made available to the Sickle Cell Laboratory.

Laboratory Techniques

SCD can be diagnosed starting at birth by examining the hemoglobin patterns present in the blood, which will indicate the presence of abnormal HbS and the lack of the normal HbA.

Each different hemoglobin variant has its own unique isoelectric point, which reflects the amino acid composition and overall electrical charge. IEF utilizes these minor differences between Hb variants and involves the separation of globin chain proteins according to their isoelectric points in a stabilized pH gradient.

When the samples arrived in the Sickle Cell Laboratory, each DBS card was scanned with a barcode reader then punched (3.2 mm diameter) by the automatic Wallac DBS Puncher into the corresponding well of a 96-well microtitration plate in a pre-programmed pattern. Each complete plate was comprised of 80 individual hemoglobin sample punches and eight empty wells for hemoglobin standard controls. The puncher generated a worklist from the DBS card barcode scans to reflect the sample locations and identifying numbers in the 96-well plate. A

11 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study solvent (Hb Elution Solution, PerkinElmer, Inc.) was added to each well to elute hemoglobins from the sample punches.

Image 1: Wallac DBS Puncher System in Sickle Cell Laboratory

Image 2: Automatic 3.2 mm Sample Punch by Wallac DBS Puncher

A precast agarose gel containing RESOLVE Ampholytes, which establishes a pH gradient appropriate for hemoglobin analysis, was prepared on the electrophoresis unit surface with an apparatus of wicks saturated with anode or cathode buffer and sample application templates. The samples were pipetted into the templates on the gel and hemoglobin standard

12 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study controls were pipetted into the template after every tenth sample. An electrical current was applied to the gel causing hemoglobin variants to migrate to their unique isoelectric point on the gel. (When an individual variant’s isoelectric point equals zero, it becomes stationary or

“focused” and forms a discrete band at predicted and reproducible migration locations on the gel.

These bands can be visually compared to standard controls that are used as pattern markers for hemoglobin variant analysis and to assess assay reproducibility.)

Image 3: Pipetting Eluted Samples into Sample Template on IEF Gel

13 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Image 4: Migration of Samples on Electrophoresis Unit

When all of the hemoglobins had been separated and focused, the gel was then fixed with trichloroacetic acid and stained using a staining solution (JB-2 Staining System, PerkinElmer,

Inc.) for optimal visualization of bands. The gels were placed in a gel dryer for several hours or overnight. Dry gels were used for result interpretation and were later stored with the corresponding puncher worklist and independently scored worklists in a sheet protector and placed in a binder only after final scores were determined. Binders were organized by testing date in a secured file cabinet.

Result Interpretation and Reporting

US3 study samples were primarily from children six weeks to 18 months of age. When scoring the IEF gel results, there were four expected hemoglobin patterns that appeared

14 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study differently depending on the infant’s age. These patterns include normal, SCT, SCD, or other variant hemoglobin patterns. In many samples, HbF was observed with the highest amount in newborns. HbF is gradually replaced by HbA or HbS over the first six to 12 months of life.

Normal newborns produce approximately 60-85% HbF and 15-40% HbA.14 Both HbF and HbA are included in this normal hemoglobin pattern, referred to as “FA.” HbF should gradually disappear between age six and 12 months such that a normal hemoglobin pattern after age one year contains almost exclusively HbA. This adult hemoglobin pattern was referred to as

“A.”

The hemoglobin pattern for SCT in newborns includes HbF, HbA, and HbS, referred to as “FAS.” After the first year of life, children with SCT will have predominantly HbA and HbS.

This adult pattern of SCT was referred to as “AS.”

SCA is the most common form of SCD. Most commonly, SCD is referred to as HbSS disease, but can include the combination of both HbS with HbC (HbSC) or other variant hemoglobins. In newborns, hemoglobin patterns including HbF and HbS (without the presence of HbA) will indicate SCA, referred to as “FS.” Similarly, the presence of HbF, HbS, and HbC

(without the presence of HbA) also represents a form of SCD, referred to as “FSC.”

Variant hemoglobin is any hemoglobin that is not the common HbA, HbF, HbS, or HbC, and was labeled as “X” to indicate variant hemoglobin. For example, if a sample had both HbF and HbA, but also had a band in between the location of HbS and HbC, the score of this sample was “FAX” to indicate the presence of HbF, HbA, and this variant “X” hemoglobin. The combination of HbX and HbS, absent of HbA, represents a form of SCD and is referred to as

“FSX” or “SX.”

15 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

In the US3 study, the process of result interpretation required that two laboratory personnel independently score each gel by transcribing the hemoglobin band pattern observed for each sample onto the accompanying worklist (e.g., FAS). A third laboratory personnel independently scored the gel with observed band patterns and compared it with the previous scores, sometimes performing a tiebreak, to determine the final band score. The third scorer translated the hemoglobin band patterns into one of four letter categories: N for normal, T for trait (SCT), S for sickle (SCD), or V for variant (Table 1). The letters recorded onto the worklist by the third scorer served as the final result score that was sent to the Data Entry Clerk for input into the US3 database.

Initially, all samples scored as SCT or SCD were retested with a second DBS punch and run again on IEF to verify result accuracy. Due to the high degree of sample accuracy, soon, only samples scored as SCD were retested. Indeterminate samples were also retested with a second

DBS punch in an attempt to obtain a valid result. If the second test was also deemed indeterminate, samples were considered invalid and recorded as such in the US3 database.

Result Report Forms for each sample tested were prepared and printed at the CPHL and disseminated back to the health facilities through the National Sample Transport System. The forms included an explanation of the result and basic care instructions. Infants identified with

SCD were advised to seek medical care and to receive their full series of pneumococcal vaccination and penicillin prophylaxis promptly at a local health facility.

16 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Table 1: Hemoglobin Letter Categories for IEF Analysis

SCORING RESULT Hb A Hb S Hb F Hb X

Normal (N) Present None Variable None

Trait (T) Present Present Variable None

Sickle (S) None Present Variable Variable

Variant (V) Present None Variable Present

Image 5: Crop and Enlargement of Right Side of an IEF Gel Depicting Band Patterns for

N, T, S and Standard Control

Data Management and Analysis

The EID electronic database recorded the infant age, location by region and district, and

HIV status among other demographic factors during sample reception at the CPHL. US3 results were entered based on the final letter score into an analogous parallel database (Microsoft

17 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Access). The CCHMC team helped to ensure data quality with periodic database review for outlier or missing data. The US3 and EID databases were merged to link results and demographic data, as well as for simultaneous result reporting. The database also had the ability to create and run specific queries for data cleaning and analysis purposes.

The overall prevalence of SCT, SCD, and hemoglobin variants was determined by calculating proportions among all 10 regions and 112 districts. Linear regression was used to compare regional prevalence of SCT and published data regarding microscopy-positive malaria.15 Additional comparative analyses between groups by age and HIV status were done using the chi-squared test, odds ratios (OR), and 95% confidence intervals (95% CI) to investigate early mortality and comorbidities. A geospatial district map of Uganda was created using graphic design software (Adobe Illustrator) with a gradient color scheme to illustrate the prevalence and geographic distribution of SCT throughout the country. A regional geospatial map set visualized the SCT and malaria comparative data side-by-side with gradient colors.

Monitoring and Evaluation

Study operations were routinely monitored and optimized remotely and during periodic on-site visits by the CCHMC team to ensure quality and accurate laboratory procedures, study results, and data output through ongoing training and a program assessment.

At the study half point, a formal on-site program assessment was developed and conducted by CCHMC (Appendix B). The assessment served several purposes to ensure the data collected were high quality and accurate to meet the end goals of the study, which included evaluation of the efficiency of the overall study operations, to document the logistics of and

18 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study adherence to laboratory operations and procedures, to assess the quality and accuracy of study data, to review data entry and result reporting, and to provide recommendations for improvements and to determine the next steps for expanding program capabilities. A formal program assessment report was disseminated to laboratory personnel and CPHL leadership

(Appendix C).

RESULTS

Partnership

The MOH recognized the fundamental need for current and accurate data on the magnitude of the SCD burden in Uganda to be able to garner national political and financial support, yet needed the research skills to be able to inform and facilitate a well-designed large- scale surveillance study. To achieve this, the committed local leadership of the MOH acquired

North and South academic collaborators with expertise and research experience in SCD to establish a multi-disciplinary partnership with the goals generating critical data and building targeted capacity within the existing healthcare infrastructure.

The MOH initiated and coordinated the US3 partnership, and served as a valuable implementation partner through government action, such as providing access to samples for the study by waiving informed consent for all samples collected and tested as part of US3. To help increase awareness and prepare for routine monitoring and care of children with SCD, the MOH modified the existing Child Health Card (the individually held health care record) to include information on SCD testing and newborn screening. As the leader of the sustainability of SCD

19 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study programs in the country, the MOH will continue to play an essential role in setting priority interventions by developing national sickle cell plans and strategies, and allocating current resources, including the availability of penicillin, anti-malarial prophylaxis, and pneumococcal vaccines for affected children, and mobilizing internal and external funding. Furthermore, the

MOH will need to gain political support from other government bodies, particularly the Uganda

National Drug Authority for provision of essential SCD treatments, such as hydroxyurea therapy, and then garner support externally from WHO, UNICEF, and other international groups.

The academic partners, CCHMC and MaKCHS, together had the role of developing the epidemiologic study design; ensuring that the cross-sectional design would be feasible, ethical, and achievable in the provided setting; contributing to enhancing human and infrastructural capacity; and providing quality data with appropriate statistical methods for analysis.

Additionally, each academic partner contributed unique strengths. CCHMC had technical expertise in sickle cell testing methodologies and training, as well as recent experience developing a newborn hemoglobinopathy screening program in the Republic of Angola.16

CCHMC is a leading pediatric hospital in the United States with a scientific research arm, the

Cincinnati Children’s Research Foundation (CCRF), which supports collaborative efforts to conduct innovative laboratory and clinical research to improve care for children. The CCRF provided funding to the Division of Hematology to support US3, which was allocated to the construction of the Sickle Cell Laboratory, purchase and donation of equipment and reagents, laboratory personnel payroll, and other study expenses. MaKCHS has a highly regarded and established local Ethics Committee that evaluated the integrity and cultural acceptability of the design, as well as offered input on educational aspects of the study, such as result reporting and

20 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study healthcare provider training. CCHMC’s Comprehensive Sickle Cell Center and Mulago

Hospital’s Sickle Cell Clinic in Kampala provided different perspectives and an exchange of knowledge on sickle cell clinical care, which will be used to introduce and improve standards and access to management and treatment of patients with SCD.

From inception, US3 has been a collaborative effort based on mutual agreement and benefit. Prior to the start of the study, a Memorandum of Agreement (MOA) was created between the MOH and CCHMC; this signed document set out terms regarding the responsibilities and accountability necessary for all parties to work together towards the common goals of the study. The MOA is not a legal document enforceable in court in any jurisdiction, but rather relies on the trust and good will of the collaborating parties. The nature of this agreement was a meaningful choice and best fit for US3 to enable transparency, dialogue, creativity, flexibility, and mutual respect to establish a truly balanced global partnership.

Sickle Cell Laboratory

In February 2014, the Sickle Cell Laboratory completed construction, was fully-equipped and fully-staffed, and began operations five to six days a week. On-site training by the CCHMC team introduced a workflow of four gels per day (320 samples per day, 1,600 samples per week).

Initially, all SCT and SCD samples were retested for result confirmation. The local laboratory personnel developed confidence in their IEF techniques over the first several months, which was apparent in their ability to identify technical issues and make recommendations for troubleshooting, which led to improved gel quality and scoring accuracy evaluated during remote review and training with the CCHMC team.

21 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

By the end of March 2014, workflow increased to 6-8 gels per day (480-640 samples per day; 2,400-3,200 samples per week), which was steadily maintained throughout the remainder of the study. This workflow allowed the Sickle Cell Laboratory to close the backlog of samples from the EID laboratory and match the daily intake of DBS cards at the CPHL along with repeats of SCD and inadequate samples, to meet good standard laboratory screening practice. Due to the cross-coverage training approach, overall efficacy of laboratory processes were improved with delegation of laboratory responsibilities by having rotating individuals tasked with loading and running the gels, punching samples, or scoring gels.

Image 6: US3 Team at Opening of Sickle Cell Laboratory and Launch of the Study

Pictured left to right: Munirah Namutebi (CPHL Sickle Cell Data Entry Clerk), Arielle

Hernandez (CCHMC Program Coordinator), Priscilla Khaniza (CPHL Sickle Cell Laboratory

Technician), Iga Tadeo (CPHL EID Laboratory Technician), Raymond Mugabe (CPHL Sickle

Cell Laboratory Technologist), Mercy Nabunya (CPHL Sickle Cell Laboratory Technician),

Stephen Aeko (CPHL Sickle Cell Laboratory Technician), Isaac Ssewanyana (CPHL EID

Laboratory Manager), Thad Howard (CCHMC Laboratory Manager)

22 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Program Assessment

The first on-site program assessment was conducted in June 8-14, 2014. The assessment covered the study from the start date of February 14, 2014 to June 2, 2014, during which 26,621 samples had been collected, scored, and entered into the US3 database. The study was evaluated in four study periods: period 1 and 2 represented start-up phase I and II, respectively, which focused on the first two months of the study, and period 3 and 4 represented the active phases I and II. A random selection of gels per period were assessed for accuracy between independent scorers, number of samples scored per gel, accuracy of interpretation of hemoglobin band patterns, accuracy of interpretation of band scores into letter categories, accuracy of repeat samples, accuracy between scores and remote review, and accuracy and completeness of data entry. The overall study operations and laboratory processes were also described and assessed.

The accuracy between independent scorers averaged 97% agreement for all assessment periods, beginning strongly with 96.3% agreement in phase 1. The samples scored per gel averaged 79.4 valid samples out of 80 per gel (99.3%), which was an indication that the DBS sample yield was significant and that the test had been set-up correctly and run at the appropriate equipment settings. This resulted in high quality gels with distinguishable bands that were able to be scored, maximizing testing output. Accuracy between repeat scores (SCT, SCD, and indeterminate samples) increased gradually over the periods from 67.4% agreement in phase 1 to

77.4% agreement in phase 4. Comparison of scores between the laboratory personnel and

CCHMC team during review were consistently high, averaging 97.7% agreement for all assessment periods.

23 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

In an effort to eliminate discrepancies between scorers and repeat samples, the Sickle

Cell Laboratory was recommended to continue to meet weekly with the CCHMC team by Skype for band interpretation and improving their overall scoring practices for the remainder of the study. An increase in workflow from four to five gels was observed, and a plan was put in place to continue to six or more gels through optimizing laboratory roles and delegation of tasks.

Adherence to the IEF protocol was excellent, and the laboratory personnel were encouraged to continually update the protocol with any procedure changes from troubleshooting outcomes and tailoring to the existing laboratory environment. The goal with revisiting and updating the protocol was to document laboratory standard operating procedures and create standard training manuals for sickle cell testing in Uganda. There was also a need to improve upon communication between the laboratory and data entry to ensure correct and complete data collection, such as bridging quality assurance checks through routine querying of the database, and defining unique markers for samples for data entry and gel and sample storage purposes.

The overall outcome of the assessment was very positive. The progressed skill set of the laboratory personnel and the regular remote training by the CCHMC team made for proficient laboratory operations early on in the study. The program assessment concluded that the Sickle

Cell Laboratory was in a great position to complete the study successfully and to confidently progress into program expansion, including pilot newborn screening and other research initiatives, such as DNA-based confirmatory testing.

24 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Surveillance Study

Between February 2014 and March 2015, a total of 99,243 consecutive DBS cards were collected and tested in the Sickle Cell Laboratory. The sample population was balanced with respect to gender, and almost all samples (99.3%) were collected before 18 months of age with a median age of 2 months. A total of 97,631 (98.4%) samples were successfully scored and analyzed from all ten regions in Uganda. There were 1,612 (1.6%) samples with an invalid result that were excluded from the final analysis. Half of the regions had over 10,000 samples, which includes Central 1, Kampala, Mid Northern, Mid Western, and South Western regions. Table 2 summarizes the hemoglobin result proportions across the 10 regions.

25 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Table 2: Regional Hemoglobin Results

REGION SEX NORMAL TRAIT DISEASE VARIANT MEDIAN (IQR) AGE (m) (total) (boys/girls/NA [%]) (%) (%) (%) (%) Central 1 2 12757 1896 69 34 7127(48.3)/ 7430(50.4)/ 199(1.3) (14756) (2-8) (86.5) (12.8) (0.5) (0.2) Central 2 2 9442 1566 94 33 5364(48.2)/ 5661(50.8)/ 110(1.0) (11135) (2-8) (84.8) (14.1) (0.8) (0.3) East Central 3 5131 1306 96 48 3275(49.8)/ 3243(49.3)/ 63(1.0) (6581) (2-12) (78.0) (19.8) (1.5) (0.7) Kampala 2 11499 1835 90 42 6670(49.5)/ 6607(49.1)/ 189(1.4) (13466) (2-8) (85.4) (13.6) (0.7) (0.3) Mid Eastern 3 4014 752 60 39 2342(48.1)/ 2455(50.5)/ 68(1.4) (4865) (2-11) (82.5) (15.5) (1.2) (0.8) Mid Northern 3 10028 2445 160 127 6220(48.7)/ 6401(50.2)/ 139(1.1) (12760) (2-12) (78.6) (19.2) (1.3) (1.0) Mid Western 2 11418 1431 64 32 6271(48.4)/6507(50.3)/167(1.3) (12945) (2-10) (88.2) (11.1) (0.5) (0.2) North East 2 3676 702 46 34 2201(49.4)/ 2204(49.4)/ 53(1.2) (4458) (2-10) (82.5) (15.7) (1.0) (0.8) South Western 3 12979 631 23 16 6543(47.9)/ 6896(50.5)/ 201(1.5) (13649) (2-11) (95.1) (4.6) (0.2) (0.1) West Nile 2 2534 415 14 53 1566(51.9)/ 1406(46.6)/ 44(1.5) (3016) (2-12) (84.0) (13.8) (0.5) (1.8) All 2 83478 12979 716 458 47579(48.7)/ 48810(50.0)/ 1242(1.3) (97631) (2-9) (85.5) (13·3) (0.7) (0.5)

26 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

SCT and SCD Prevalence

The national prevalence of SCT was 13.3%, ranging from 4.6% in the South Western region to 19.8% in the East Central region. The SCD prevalence was 0.7%, with a range of 0.2% to 1.5% in the South Western and East Central regions, respectively. With the carrier frequency of 13.3%, the percentage of the population born with HbSS is predicted to be 0.4%. The observed higher SCD prevalence than expected could be due to positive assortive mating within the population, where mating occurs more frequently between individuals with the same genotype resulting in more homozygotes than predicted by the Hardy-Weinberg model.

All districts in Uganda had SCT. Table 3 summarizes the hemoglobin result proportions among all 112 districts. Eight districts, all within the South Western region, had <5% prevalence with the smallest proportion in the Mitooma district at 2.5%. There were eight districts with SCT prevalence >20% located in the East Central, Mid Western, and Mid Northern regions. The highest prevalence rate was at 23.9% in Alebtong district in the Mid Northern region. Figure 1 is a district map of Uganda that illustrates the prevalence of SCT in 112 districts.

Table 3: District Hemoglobin Results

NORMAL TRAIT DISEASE VARIANT DISTRICT REGION TOTAL (%) (%) (%) (%) Bukomansimbi Central 1 579 85.8 13.1 0.7 0.3

Butambala Central 1 417 85.4 13.2 1.2 0.2

Gomba Central 1 414 86.0 12.3 1.4 0.2

Kalangala Central 1 563 86.0 13.3 0.5 0.2

Kalungu Central 1 922 85.5 14.1 0.4 0.0

Lwengo Central 1 773 88.2 11.4 0.4 0.0

Lyantonde Central 1 609 91.5 8.4 0.0 0.2

Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Masaka Central 1 2298 86.2 13.0 0.6 0.3

Mpigi Central 1 1043 87.4 12.0 0.4 0.2

Rakai Central 1 2223 83.7 15.9 0.3 0.1

Ssembabule Central 1 601 90.5 9.0 0.2 0.3

Wakiso Central 1 4314 86.7 12.5 0.4 0.3

Buikwe Central 2 1665 82.1 16.7 0.9 0.3

Buvuma Central 2 328 84.1 14.3 1.2 0.3

Kayunga Central 2 692 81.5 17.1 1.3 0.1

Kiboga Central 2 719 84.3 14.7 0.6 0.4

Kyankwanzi Central 2 324 84.3 14.8 0.3 0.6

Luweero Central 2 1530 84.6 13.9 1.2 0.3

Mityana Central 2 1226 85.2 13.5 0.7 0.7

Mubende Central 2 2032 88.4 10.9 0.5 0.2

Mukono Central 2 1568 85.9 13.3 0.6 0.1

Nakaseke Central 2 546 84.8 14.3 0.7 0.2

Nakasongola Central 2 505 81.4 16.2 2.0 0.4

Bugiri East Central 605 77.9 20.2 1.3 0.7

Buyende East Central 314 80.3 17.5 1.6 0.6

Iganga East Central 932 77.9 19.6 1.5 1.0

Jinja East Central 1690 78.0 19.5 1.7 0.8

Kaliro East Central 280 78.9 19.3 1.8 0.0

Kamuli East Central 655 77.4 20.5 1.4 0.8

Luuka East Central 259 78.4 20.1 0.8 0.8

Mayuge East Central 796 77.6 19.7 1.8 0.9

28 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Namayingo East Central 739 77.9 20.6 1.2 0.3

Namutumba East Central 311 76.5 21.9 0.6 1.0

Kampala Kampala 13466 85.4 13.6 0.7 0.3

Budaka Mid Eastern 167 81.4 17.4 0.6 0.6

Bududa Mid Eastern 163 91.4 7.4 0.6 0.6

Bukwo Mid Eastern 29 86.2 13.8 0.0 0.0

Bulambuli Mid Eastern 196 91.3 8.2 0.5 0.0

Busia Mid Eastern 522 78.2 19.0 1.9 1.0

Butaleja Mid Eastern 187 78.6 16.6 2.7 2.1

Kapchorwa Mid Eastern 157 88.5 9.6 0.6 1.3

Kibuku Mid Eastern 197 77.2 19.8 2.0 1.0

Kween Mid Eastern 34 94.1 5.9 0.0 0.0

Manafwa Mid Eastern 294 88.1 10.9 0.7 0.3

Mbale Mid Eastern 1019 85.2 13.3 1.0 0.5

Pallisa Mid Eastern 312 82.1 16.3 0.3 1.3

Sironko Mid Eastern 250 88.8 9.6 0.8 0.8

Tororo Mid Eastern 1338 77.9 19.6 1.6 0.9

Agago Mid Northern 888 81.3 17.0 1.0 0.7

Alebtong Mid Northern 351 71.2 23.9 2.0 2.8

Amolatar Mid Northern 450 80.9 17.1 0.9 1.1

Amuru Mid Northern 320 79.7 17.2 1.6 1.6

Apac Mid Northern 938 78.6 19.2 1.8 0.4

Dokolo Mid Northern 382 79.1 19.1 1.3 0.5

Gulu Mid Northern 3014 77.9 19.9 1.0 1.2

29 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Kitgum Mid Northern 1046 80.9 17.7 0.7 0.8

Kole Mid Northern 438 76.7 19.9 1.8 1.6

Lamwo Mid Northern 543 79.6 18.6 0.4 1.5

Lira Mid Northern 1778 78.0 19.8 1.6 0.6

Nwoya Mid Northern 532 77.6 19.5 1.3 1.5

Otuke Mid Northern 431 77.7 19.7 2.3 0.2

Oyam Mid Northern 982 77.7 19.9 1.3 1.1

Pader Mid Northern 667 80.7 17.5 1.2 0.6

Buliisa Mid Western 152 75.7 21.7 2.0 0.7

Bundibugyo Mid Western 309 76.1 21.7 1.9 0.3

Hoima Mid Western 1568 85.5 13.3 0.8 0.4

Kabarole Mid Western 3038 90.0 9.5 0.2 0.3

Kamwenge Mid Western 969 93.8 6.0 0.1 0.1

Kasese Mid Western 1215 89.5 10.2 0.2 0.0

Kibaale Mid Western 1604 88.6 10.3 0.7 0.3

Kiryandongo Mid Western 516 84.1 14.9 0.2 0.8

Kyegegwa Mid Western 796 89.9 9.2 0.5 0.4

Kyenjojo Mid Western 1763 89.6 10.0 0.3 0.1

Masindi Mid Western 803 82.8 15.7 1.4 0.1

Ntoroko Mid Western 212 84.4 15.6 0.0 0.0

Abim North East 180 81.1 17.8 1.1 0.0

Amudat North East 34 85.3 14.7 0.0 0.0

Amuria North East 572 80.6 17.1 1.7 0.5

Bukedea North East 273 85.7 12.8 0.0 1.5

30 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Kaabong North East 114 93.0 7.0 0.0 0.0

Kaberamaido North East 516 82.2 16.9 0.6 0.4

Katakwi North East 490 82.2 16.3 0.4 1.0

Kotido North East 90 86.7 12.2 1.1 0.0

Kumi North East 361 83.1 14.1 1.1 1.7

Moroto North East 102 84.3 14.7 0.0 1.0

Nakapiripirit North East 64 93.8 6.3 0.0 0.0

Napak North East 116 87.9 12.1 0.0 0.0

Ngora North East 210 79.0 19.5 0.5 1.0

Serere North East 451 80.3 17.3 2.2 0.2

Soroti North East 885 81.2 16.2 1.5 1.1

Buhweju South Western 142 96.5 3.5 0.0 0.0

Bushenyi South Western 1211 95.6 4.2 0.1 0.1

Ibanda South Western 864 94.1 5.7 0.2 0.0

Isingiro South Western 1157 94.3 5.4 0.3 0.1

Kabale South Western 1076 97.3 2.6 0.0 0.1

Kanungu South Western 745 96.0 3.5 0.4 0.1

Kiruhura South Western 916 93.8 5.7 0.3 0.2

Kisoro South Western 361 95.6 4.2 0.3 0.0

Mbarara South Western 3012 94.2 5.5 0.1 0.1

Mitooma South Western 397 97.5 2.5 0.0 0.0

Ntungamo South Western 1315 95.7 4.0 0.1 0.2

Rubirizi South Western 360 90.8 7.8 1.1 0.3

Rukungiri South Western 1191 96.1 3.8 0.0 0.2

31 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Sheema South Western 902 95.3 4.4 0.1 0.1

Adjumani West Nile 239 81.2 15.5 0.4 2.9

Arua West Nile 973 84.6 12.5 0.2 2.7

Koboko West Nile 217 82.9 15.7 0.0 1.4

Maracha-Terego West Nile 93 90.3 7.5 0.0 2.2

Moyo West Nile 161 85.7 13.0 0.0 1.2

Nebbi West Nile 810 80.2 17.4 1.2 1.1

Yumbe West Nile 212 90.1 9.0 0.0 0.9

Zombo West Nile 311 88.1 10.9 0.3 0.6

32 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Figure 1: District Map of the Prevalence of SCT in Uganda

Other Hemoglobin Variants

The national prevalence of hemoglobin variants other than HbS was 0.5%, ranging from

0.1% in the South Western region to 1.8% in the West Nile region. Five districts had a variant prevalence >2%, with the highest proportion of 26 variants in 973 samples (2.7%) in Arua

33 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study district located in the West Nile region. The IEF patterns for variant hemoglobins identified in the study were not all the same and could not be specifically characterized by this technique.

A selection of variant DBS samples were transferred back to CCHMC for DNA-based testing by Taqman, PCR, and Sanger Sequencing methods. In the preliminary analysis, a specific pattern was found of a previously described variant hemoglobin: Hb Stanleyville II (alpha chain variant, HBA2:c.[237C>A;237C>G]).17 Further identification of other common hemoglobin variants and examination of their geographic distribution can help provide a better understanding of the health burden of hemoglobinopathies in the country, and research regarding the hematological and clinical effects of other variant hemoglobins, especially in combination with

HbS, is important for care purposes.

SCT and Malaria

Almost 70 years ago, J. B. S. Haldane first proposed the “malaria hypothesis” on the protection from malaria afforded by the sickle gene.4 Since then, much evidence has accumulated to support that areas of high sickle gene frequencies are coincident with currently and historically high levels of malaria, due to the process of natural selection for the sickle gene.

A recent study confirmed this association at the global level by compiling available data on sickle cell gene frequencies and comparing them to the historical endemicity and distribution of malaria. This study showed a visual overlap in the global distribution of the two conditions in areas with high malaria pressure, most notably in sub-Saharan Africa, the Middle East, and

India.18

34 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

To investigate this association within Uganda, a visual and statistical comparison of the regional prevalence of SCT and malaria was made. Figure 2A illustrates the regional SCT prevalence based on US3, with the highest prevalence in the East Central and Mid Northern regions. Figure 2B illustrates malaria prevalence based on the Uganda Bureau of Statistics 2009 malaria indicator survey data (the percentage of children 0-59 months of age with positive microscopy for malaria), ranging from 4.9% in Kampala and 11.6% in the South Western regions to 62.5% in the Mid Northern region.15 These maps visualize concordance between SCT and malaria prevalence in Uganda, and the linear regression of numerical data by region (Table

3) resulted in a strong positive correlation (r2=0·69, p=0.026).

Figure 2: Regional Map of the Prevalence of SCT and Malaria in Uganda

A B

35 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Table 4: Regional Correlation Between SCT and Malaria in Uganda

TRAIT MALARIA REGION (%) (%) Central 1 12.8 39.1

Central 2 14.1 50.7

East Central 19.8 56.2

Kampala 13.6 4.9

Mid Eastern 15.5 37.5

Mid Northern 19.2 62.5

Mid Western 11.1 42.7

North East 15.7 40.0

South Western 4.6 11.6

West Nile 13.8 45.7

Early Mortality in SCD

The association between SCD and early mortality in children in sub-Saharan Africa is frequently cited.6,7,19,20,21,22 SCD is estimated to account for 6.4% of under-five deaths across

Africa;6 however, in countries like Uganda that experience higher HbS allele frequencies and lower overall childhood mortality, SCD may contribute up to 15% of the country’s under-five mortality rate.22 To investigate this association, a cross-sectional age analysis on children ≤18 months was performed. Of the total number of samples collected, 98,519 (99.3%) were from children 18 months and less, of which 96,927 of which had valid results.

The prevalence of SCD was age-stratified into three groups as shown in Table 5, which revealed a decrease in the prevalence of SCD with increasing age. Specifically, the OR at age

36 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

12.1-18.0 months, compared to age 0.0-6.0 months, was 0.79 (95% CI 0.64-0.97), which is consistent with early mortality for babies born with SCD in Uganda.

The prevalence of SCD compared to HIV status (Table 5) identified co-morbidity with early mortality for HIV in babies with SCD. The OR of having SCD among HIV-positive infants, compared to HIV-negative infants, was 0.60 (95% CI 0.40-0·91). The low odds ratio highlights an important potential co-morbidity between SCD and HIV that results in early death.

This observation is not fully supported by the current published medical literature, where the interaction between SCD and HIV has been described in different ways, including SCD reducing risk and slowing progression of HIV, or that HIV worsens SCD, or even that SCD and HIV synergistically increase risk for certain disease complications.23 This gap in knowledge is critical and our observations warrant prospective investigation of the overlap of two important diseases that heavily impact the same geographical areas.

Table 5: SCT and SCD Prevalence by Age and HIV Status

TRAIT DISEASE TOTAL (%) (%) Age (m) 8802 521 0.0 – 6.0 66670 (13.20) (0.78) 1758 87 6.1 – 12.0 13182 (13.34) (0.66) 2326 105 12.1 – 18.0 17075 (13.62) (0.61) HIV Status 12293 693 Negative 92024 (13.36) (0.75) 672 23 Positive 5080 (13.23) (0.45)

37 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Result Reporting and Follow-up on Infants with SCD

The Sickle Cell Laboratory conducted a telephone survey for brief information on result reporting to health facilities and follow-up information regarding all of the 716 infants identified with SCD in US3. The survey revealed that 689 (96%) of the SCD results were confirmed to be received by the health facilities (Figure 3A). Of these results received, all but 13 infants (676,

94%) were known to be under care at health facilities, while 40 infants (6%) had a status of unknown (Figure 3B). Of all the infants identified with SCD, 676 were reported to still be living, five were presumed to have died, and 27 had a status of unknown (Figure 3C). The survey does not provide details about the care or status of the infants, which supports the need for sickle cell care guidelines and long-term follow-up of affected children.

38 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Figure 3: Result Reporting and Follow-up on Infants with SCD at Health Facilities

A

B

C

39 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Dissemination of Results

US3 concluded on March 31, 2015, and on April 16, 2015 the final US3 data and maps were revealed to local scientific and academic groups and to government and industry stakeholders prior to public dissemination and publication of the results. The formal scientific symposium took place at Makerere University in Kampala as a platform for critical review and discussion that was presided over by the MOH’s Director General of Health Services with support from the Honorable State Minister for Health and Primary Health Care.

Following the symposium was a large public event on the main grounds of the university to launch national sickle cell testing services. Thousands of local community members were in attendance and voluntary collection of samples for sickle cell screening was offered on site for testing in the Sickle Cell Laboratory. The theme of the public launch was “Break the Silence,” named by local US3 partners based in Uganda, which appropriately captured the increased awareness of SCD and the commitment to services and treatment of the disease as an outcome of the study result reveal.

The US3 manuscript, entitled “Burden of sickle cell trait and disease in the Uganda

Sickle Surveillance Study (US3): a cross-sectional study,” (Appendix D) was recently accepted by The Lancet Global Health, a peer-reviewed global health specific medical journal, and published with an Editor’s Choice in the March 2016 issue. The publication was featured in a commentary in the same issue, “Is integrating sickle cell disease and HIV screening logical?,”(Appendix E) which spoke highly of the novel US3 model of integrating screening programs to provide useful data for health services and its reproducibility in other sub-Saharan

African countries with EID programs. The article further made the call for African countries and

40 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study leaders to support newborn screening programs for SCD, especially coupled with public education and providing the necessary care and treatment to those affected, to make an impact on preventing early death.

Image 7: “Break the Silence” Banner at US3 Final Result Reveal and Public Sickle Cell

Testing Launch

DISCUSSION

SCD is a worldwide public health issue requiring a collaborative approach that represents the global North and South to enhance international support and cooperation in the exchange of science, technology, and knowledge focused on SCD. Weatherall and colleagues made the case for the mutual benefit of developing such partnerships for sickle cell research, stating that these interactions would improve clinical and diagnostic facilities to benefit the management of patients with SCD in the developing world, and will simultaneously provide a better understanding of mechanisms for phenotypic diversity and the role of genetics and environment in disease course to improve care in developed countries.24 UN Sustainable Development Goal

17, Revitalizing the Global Partnership for Sustainable Development, makes the urgent call for

41 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study global, national, and regional partnerships between governments, the private sector, and civil society built upon principles and values, and a shared vision and goals.10 The hope with these partnerships is that they will mobilize and shift transformable resources that will help deliver on sustainable objectives. US3 represents a best practice model for a global North-South partnership for SCD, especially meeting the UN Sustainable Development Goal 17 target of enhancing international support for implementing effective and targeted capacity-building in developing countries to support sustainable programs.10 The success of the US3 partnership was due to all partners being equitably involved in the research process, as well as had the advantage of a multi-disciplinary collaboration between academia and government that benefited from the unique strengths of each party to ultimately combine knowledge with action.

Goals

The Uganda MOH commissioned the US3 in response to the WHO directives set out in the 2010 SCD strategy for the African Region, which had the same mission aimed at reduction of childhood mortality, and specifically to reduce the burden of SCD. The US3 study included short-term goals to reflect the priority interventions of the WHO strategy, specifically focused on partnerships, research, and capacity building.9

The first short-term goal of US3 was to establish a working partnership between academia and government to design and execute a national surveillance study of SCD in Uganda.

To accomplish this aim, a partnership between CCHMC, the MOH, and MakCHS was established to plan and implement a sickle cell surveillance study. A Memorandum of

Agreement was created and executed, and regular in-person and remote operations and strategic

42 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study planning meetings built and maintained relations among all partners to streamline study responsibilities and goals. The result was a strong and sustainable long-term working partnership between academia and government that supported US3 to generate crucial policy-informative data in Uganda.

The second short-term goal was to build and develop local sickle cell laboratory capacity and determine the feasibility of high-volume testing for SCD. The first designated Sickle Cell

Laboratory was constructed and outfitted at the CPHL in Kampala, where initial and ongoing technical laboratory training and support was provided by CCHMC. A mid-study program assessment was conducted to monitor the quality and accuracy of laboratory testing procedures and data management, which assisted with program improvements and assessed the feasibility of high-volume testing for SCD. Building and developing local sickle cell laboratory capacity will enable sustainable long-term sickle cell testing and research in Uganda.

The third short-term goal was to generate current and accurate epidemiological evidence regarding the prevalence and distribution of SCD across Uganda. US3 tested almost 100,000 dried blood spots for SCT and SCD by IEF hemoglobin electrophoresis and used these data to generate detailed geospatial maps and to perform demographic analyses. The study documented a large burden of SCD in Uganda, with geographic variability in the prevalence of disease across the country. In addition, novel co-morbidities were identified that should be tested prospectively in follow-up studies.

The long-term goals of US3 are to guide the development and implementation national sickle cell strategies, beginning with the MOH issuing a national SCD plan to set standards of care and to establish cost-effective and evidence-based planning in response to the current

43 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study burden of SCD in the country that has been confirmed and documented by the study. The epidemiological evidence obtained in US3 will be critical for this goal, by providing accurate data regarding the prevalence and distribution of SCT and SCD in all regions and districts of the country.

Strengths, Limitations, and Challenges

Strengths

There were several major strengths of the US3 study design and results. The first was the efficient and cost-effective design that was embedded within the existing infrastructure the EID program and the National Sample Transport System. Since EID adopted simplified DBS collection technology in 2006 and centralized the testing of DBS at the CPHL in 2010, the program has drastically improved sample management and transport for overall health system strengthening, especially increasing access to diagnostic and treatment monitoring services and shortening the turn-around times. A retrospective study by CPHL to assess implementation of the

National Sample Transport System at piloted hubs described an increase in EID sample testing volumes, as well as a 46.9% decrease in overall collection to result turn-around time (46 to 26 days) and a further 46.2% reduction with introduction of SMS printers (26 to 14 days).

Furthermore, the cost-effectiveness of combining operations through central coordination was anticipated to save the EID program 1.2 million USD in a four year period on sample transport alone, in comparison to the previous decentralized sample collection and testing system.13 The benefits offered by the National Sample Transport System and centralized testing made US3

44 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study possible, and is now also available to support growing prevention and treatment programs for

SCD in the country.

A second strength of the study was the study sample volume and distribution. Use of the

National Sample Transport System provided a large representative cohort across the country, within a short timeframe to make the cross-sectional study possible. DBS samples were collected from each district allowing for detailed analysis at both the district and regional level and for a national depiction of the burden of SCT and SCD. Testing nearly 100,000 samples provided an accurate description of the burden within the country, and allowed identification of high-burden districts that warrant close attention in subsequent studies.

A third strength was that the MOH provided a waiver of informed consent for all US3 samples, which made it possible to repurpose DBS cards collected in the EID program, as well as eliminated costs, resources, and time that would be needed for introduction of a new health service and training at health facilities across the country. This early decision by MOH was critical to launching the US3 study, because it obviated the need for individual consent processes for sample collection and testing.

A fourth strength was that the study established local sickle cell laboratory capacity by constructing and outfitting physical space, conducting initial and ongoing technical training of personnel, and providing thorough monitoring and evaluation for quality and accuracy of sample testing and data management. The Sickle Cell Laboratory demonstrated efficiency in their ability to complete up to 6-8 gels per day during a majority of the study. Shifting now toward newborn screening, this workflow design, with the current personnel and equipment, has the potential to test more than 200,000 samples annually. Doubling personnel and employing a second shift

45 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study would bring this number to more than 400,000 samples annually, which would potentially cover

25% of the 1.6 million annual births in Uganda. The development of local sickle cell laboratory capacity has provided the MOH with a national sickle cell testing and reference laboratory with the necessary infrastructure and experience to achieve targeted newborn screening, with the ability to scale-up testing services and even establish satellite testing sites in high-burden regions.

The final strength of the study is that it demonstrated a highly successful North-South partnership between academia and government that devised methods and strategies to generate evidence-based sickle cell data to inform national policy. Overall, US3 is an effective research model that can be exported to other sub-Saharan African countries with EID infrastructure or similar programs. The EID program funded by the Center for Disease Control and Prevention is well-established in many Western and Central African countries,25 and repurposing DBS for simultaneous sickle cell testing should be possible in other countries with a high burden of SCD including Angola, Democratic Republic of Congo, Nigeria, Kenya, Tanzania, and others.

Limitations

There were also several limitations of the US3 study. The first limitation was that all

DBS samples were collected through the EID program from HIV-exposed infants, presenting a potential systematic bias between the inheritance of SCT or SCD and maternal HIV status. There is no evidence to support this bias, however, and the US3 data document that the prevalence of

SCT in HIV-positive and HIV-negative infants was almost identical at about 13% (Table 5). To

46 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study address this possible confounding influence, it will be necessary to test non-HIV exposed infants to confirm the prevalence of SCT and SCD by region and district.

The second limitation was that the cross-sectional study design did not allow collection of longitudinal data. The Sickle Cell Laboratory was able to receive brief follow-up information on all infants identified with SCD, but the nature of the telephone survey is not completely reliable and did not fulfill the need to track affected infants over time. Longitudinal data will be important to document the preliminary identification of age-related mortality for children with

SCD, which has major implications for external international health funding organizations.

A third limitation was the inability to ensure that all infants identified with SCD in the study received optimal clinical care, due to the current lack of sickle cell care guidelines and knowledge among healthcare providers. During the study, preliminary results provided strong evidence of high SCT and SCD prevalence areas within the country, which prompted the US3 partnership to begin to create education and training protocols for healthcare providers to work with high-burden districts to support future pilot sickle cell newborn screening programs. In addition, the EID Dispatch Forms were updated to include sickle cell IEF as a separate testing option. Newborn samples collected at all health facilities can now be sent for testing as part of the EID program, or for sickle cell screening only, or both. The CPHL also has created a separate database from the US3 database to record and report newborn samples. A neighboring room to the Sickle Cell Laboratory was provided by the CPHL and renovated to increase space for additional IEF equipment and storage. With an additional equipment and reagent donation form

PerkinElmer, the testing capacity was doubled to be able to accommodate the growing screening efforts.

47 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Challenges

The first major challenge faced by US3 was the ethical dilemma of whether or not to report results on samples under waived consent. There is currently little to no access to care for many patients with SCD in Uganda, which begins with inadequate knowledge among healthcare providers to diagnose and appropriately treat SCD or to provide sufficient education on the significance of carrier status. The MOH’s waiver of consent was approved by the local Ethics

Committee on the condition that all sickle cell results were provided to the health facilities, just as is currently done with the HIV results. The importance of this bold decision was that it was taken under ethical consideration and ultimately made by the country’s own public health leaders, in an effort to make sickle cell testing part of routine primary care in Uganda. Although newborn screening and diagnostic testing, parental education, and comprehensive care are not accessible at this time, simple early interventions exist for affected infants identified in US3, such as penicillin prophylaxis and necessary childhood vaccines provided at no cost by the

MOH.

Another challenge for US3 was the diagnostic limitations of the IEF approach used in the

Sickle Cell Laboratory. IEF has been well-documented to be a robust methodology for newborn hemoglobinopathy screening and is the most commonly used method in the United States, but does require specific collection cards, specialized technological training, and a notification system since results are obtained several weeks after collection. Expanding screening for sickle cell disease in currently designated sickle cell clinics has obvious benefits, but screening portions of the general population for sickle trait could also be beneficial and will likely grow with

48 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study increased awareness. However, use of IEF equipment without context of proper sample collection, training, and notification can cause issues with accurate reporting. In addition, IEF interpretation is challenging in the context of blood transfusions, which is a common treatment for SCD patients where they receive healthy (HbA) donor blood, since transfused blood causes the IEF hemoglobin pattern to appear as “AS” (SCT), as opposed to the true SCD diagnosis of

“SS”. Another challenge of IEF is to characterize specific variant hemoglobin based on their IEF pattern alone. Variant hemoglobins in the homozygous and heterozygous state, especially in combination with HbS, may be clinically significant and require additional testing methods for accurate diagnosis.

The major challenge of program sustainability is primarily financial. Due to the MOH’s constrained budget, funding for SCD programs will continue to be a challenge as they compete with other emerging non-communicable disease and infectious disease efforts. US3 has provided the MOH with evidence for priority resource allocation for sickle cell interventions. Of the 112 districts, eight districts have a documented SCT prevalence >20% and are all located within three of the 10 regions within the country, and only 16 districts contain half of all SCD burden.

Identification of high-burden districts allows the encouraging possibility that limited financial input can deliver high impact sickle cell efforts, such as targeted newborn screening, education, public awareness campaigns, and clinical care in these areas, until health policy and funding can deliver sustainable sickle cell services nationally.

49 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Significance to Public Health

SCD is a largely unrecognized, neglected, and increasing public health issue affecting millions of people around the world.22 Developing countries have the largest occurrence of SCD births, with an estimated 230,000 annual HbSS births in sub-Saharan Africa.18 In Uganda, the youngest US3 cohort of ≤6 months had a SCT prevalence of 13.2% and SCD prevalence of

0.8%, which leads to the estimate of at least 15,000 SCD births per year in the country. Many developing countries, including Uganda, are experiencing an epidemiological shift where under- five mortality rates are decreasing, due to improved nutrition and infectious disease control, but the relative contribution of genetic and other non-communicable conditions to early mortality is increasing. Affected babies with SCD, who would previously have died early in life, may now be surviving long enough to present for diagnosis and are in need of care and treatment.24,26 The circumstance of higher sickle allele frequencies and lower childhood mortality from other conditions means that SCD will contribute more to the childhood mortality of countries like

Uganda with a high burden of SCD.22 The expected significant increase in the number of patients with SCD, coupled with the lack of appreciation of the disease burden, poses a serious global health issue that warrants immediate attention.

Interventions such as newborn screening, which allows early initiation of care and treatment, have had significant impact on improving the quality of life and survival for individuals with SCD in high resource countries.27 In the United States and some European countries, newborn screening identifies affected but asymptomatic babies, allowing for early parental education and initiation of important treatments such as penicillin prophylaxis and pneumococcal immunizations before severe, life-threatening disease complications arise.22 These

50 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study same interventions can be achieved in Africa, where routine newborn screening does not currently exist, but where programs and improvements to basic medical care will benefit the greatest number of individuals with SCD.28 In Uganda, simple interventions, such as penicillin and anti-malarial prophylaxis along with pneumococcal vaccinations, can be specifically administered to patients with SCD as part of early preventive care. The implementation of a prospective sickle cell newborn screening program will greatly increase identification of affected individuals and, therefore, must be linked with comprehensive training for healthcare providers regarding fever, pain, and emergency management of SCD, as well as providing parental education on basic home care, such as maintaining hydration and checking for fever and spleen palpation.29 Broadened public awareness and genetic counseling also will help overcome myths and misconceptions about SCD, and help explain the risks of the carrier state to both parents and children.

Appropriate and potentially available disease-modifying therapies for SCD include blood transfusions, which are used to alleviate anemia and acute vaso-occlusive complications, and hydroxyurea as a once-daily oral medication to increase fetal hemoglobin and help improve the laboratory and clinical complications of SCD. In the United States, hydroxyurea is the recommended treatment for patients with SCD and is listed on the WHO’s Model List of

Essential Medicines for Children,29 but is not yet widely available in many countries for treatment of SCD, including Uganda, due to the lack of clinical evidence of and experience with its use for SCD. There are several ongoing prospective research studies underway through the

CCHMC Division of Hematology that are generating critical data about the safety, efficacy, feasibility, and benefit of hydroxyurea for children with SCD. The REACH study (Realizing

51 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Effectiveness Across Continents with Hydroxyurea) is a prospective open-label dose escalation clinical trial that will treat 600 children total in four African countries, including the Democratic

Republic of Congo, Kenya, Angola, and Uganda.31,32 NOHARM (Novel use Of Hydroxyurea in an African Region with Malaria) is a clinical trial that additionally looks to determine the malaria incidence and severity in children with SCA treated with hydroxyurea versus placebo, in ~200 randomized children at the Mulago Hospital Sickle Cell Clinic.33 REACH and NOHARM will address gaps in the knowledge and help develop local expertise about treatment for SCD with the use of hydroxyurea, with the long-term goal of making it widely available for patients with SCD across sub-Saharan Africa.

Finally, the US3 evidence of the current high-burden of SCD in Uganda should help create the necessary urgency around the diagnosis and management of SCD in the country. As non-communicable disease movements are growing in importance within the global health sphere, the MOH is poised to prioritize SCD within the country’s non-communicable disease agenda to begin efforts for next step planning for SCD in Uganda.

Direction for Future Planning and Research

The direction for the future of SCD in Uganda will be guided by the National Strategic

Sickle Cell Plan to be developed by the MOH. The Programme for the Prevention and Control of

Non-Communicable Diseases is a unit within the MOH’s Department of Community Health that is growing in importance due the recent non-communicable disease epidemic in Uganda, and has mentioned SCD as a priority area where there is a need for current data. US3 has helped to ensure that the strategic plan will be based on the most updated understanding of the burden of

52 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study disease, and has poised the MOH with local laboratory capacity for testing and research to support sustainable sickle cell programs within the framework of the national non-communicable disease agenda. The overarching principles for the national response to SCD should be evidence- based planning and integrated, comprehensive approaches that can be applied to specific goals for addressing the burden of disease, such as reducing morbidity and mortality among individuals with SCD, and reducing the incidence of SCD in the general population. The proposed next steps for Uganda to achieve these goals include targeted newborn screening efforts in the highest burden districts; training of healthcare providers, with the goal of establishing regional and district sickle cell clinics that provide penicillin and anti-malarial prophylaxis, family education, and immunizations; research on the safety, dosing, and benefits of hydroxyurea; and creation of a national sickle cell awareness strategy that includes among others, pre-marital testing and counseling aimed at lowering the sickle cell burden in Uganda.34

Future research in SCD to further describe the burden in Uganda should include prospective cohort studies that link testing to care to determine incidence, causes of mortality, and to investigate the potential co-morbidity between HIV and SCD. Further data regarding the use of hydroxyurea for SCD can add to current safety and efficacy trials to further incentivize action to provide access to this transformative treatment for patients in Uganda and across

Africa. The focus on laboratory based research that can contribute to clinical care includes characterizing SCD and other variant hemoglobin samples using DNA-based technology to determine the frequency of important known genetic modifiers such as alpha thalassemia trait and G6PD deficiency.

53 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

SCD is a neglected subject of research and funding, and the magnitude of this global issue has yet to be appreciated on the public health front. US3 has provided vital data to realize the current state of SCD in Uganda, and has answered the calls of the WHO and UN to develop meaningful international partnerships for capacity building and strategic planning on the principles of evidence-based research and sustainability, to eliminate the disparity of this important growing health issue, and to save and improve the lives of thousands of individuals with SCD in Uganda.

54 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

INSTITUTIONAL REVIEW BOARD/ETHICS COMMITTEE APPROVAL

The US3 protocol (Appendix F) was approved by the School of Medicine Research

Ethics Committee at Makerere University (Ref No. 2012-138, Appendix G) and the Uganda

National Council for Science and Technology in Kampala. The study was also approved by the

CCHMC Institutional Review Board (Ref No. 2013-4576, Appendix H) and was funded by the

CCRF.

55 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

REFERENCES

1. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. The

human genome browser at UCSC. Genome Res 2002 Jun;12(6):996-1006.

2. GeneCards (HBB). Retrieved from http://www.genecards.org/cgi-

bin/carddisp.pl?gene=HBB

3. Gabriel A, Przybylski J. Sickle-cell anemia: A Look at Global Haplotype

Distribution. Nature Education 2010;3(3):2.

4. Haldane JBS. The rate of mutation of human genes. Heredity 1949; 35(suppl):267–73.

5. Piel FB, Patil AP, Howes RE, et al. Global epidemiology of sickle haemoglobin in

neonates: a contemporary geostatistical model-based map and population estimates.

Lancet 2013;381:142–51.

6. Piel FB, Hay SI, Gupta S, Weatherall DJ, Williams TN. Global burden of sickle cell

anaemia in children under five, 2010-2050: modelling based on demographics, excess

mortality, and interventions. PLoS Med 2013;10(7):e1001484.

7. Grosse SD, Odame I, Atrash HK, Amendah DD, Piel FB,Williams TN. Sickle cell

disease in Africa: a neglected cause of early childhood mortality. Am J Prev Med

2011;41:S398–405.

8. World Health Organization. Sickle-cell anaemia. Report A59/9. WHO: Geneva,

Switzerland, 24 April 2006.

9. World Health Organization. Sickle-cell disease: A strategy for the WHO African Region.

Report AFR/RC60/8. WHO: Geneva, Switzerland, 22 June 2010.

56 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

10. United Nations. Transforming our world: the 2030 Agenda for Sustainable Development.

A/RES/70/1. UN: New York, New York, 21 October 2015.

11. Lehmann H, Raper AB. Distribution of the sickle-cell trait in Uganda, and its

ethnological significance. Nature 1949;164:494-5.

12. Okwi AL, Byarugaba W, Ndugwa CM, Parkes A, Ocaido M, Tumwine JK. An up-date

on the prevalence of sickle cell trait in Eastern and Western Uganda. BMC Blood Disord

2010 Jun 23;10:5.

13. Kiyaga C, Sendagire H, Joseph E, et al. Uganda’s new national laboratory sample

transport system: a successful model for improving access to diagnostic services for early

infant HIV diagnosis and other programs. PLoS One 2013;8:e78609.

14. Henney MM and Ware RE. (2015). Chapter 20: Sickle Cell Disease. In Nathan DG (Ed.),

Nathan and Oski’s Hematology and Oncology of Infancy and Childhood Volume One

(pp. 675-714). Philadelphia, PA: Elsevier Saunders.

15. Uganda Bureau of Statistics (UBOS) and ICF Macro. Uganda malaria indicator survey

2009. Calverton, MD: UBOS and ICF Macro, 2010.

16. McGann PT, Ferris MG, Ramamurthy U et al. A prospective newborn screening and

treatment program for sickle cell anemia in Luanda, Angola. Am J Hematol

2013;88(12):984-989.

17. Globin Gene Server. Retrieved from http://globin.cse.psu.edu/

18. Piel FB, Patil AP, Howes RE, et al. Global distribution of the sickle cell gene and

geographical confirmation of the malaria hypothesis. Nature Commun 2010;1:104.

57 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

19. Aygun B, Odame I. A global perspective on sickle cell disease. Pediatr Blood Cancer

2012 Aug;59(2):386-90.

20. Weatherall DJ. The inherited diseases of hemoglobin are an emerging global health

burden. Blood 2010;115:4331–36.

21. McGann PT. Sickle cell anemia: an underappreciated and unaddressed contributor to

global childhood mortality. J Pediatr 2014;165:18–22.

22. Ware RE. Is sickle cell anemia a neglected tropical disease? PLoS Negl Trop Dis 2013

May 30;7:e2120.

23. Owusu EDA, Visser BJ, Nagel IM, Mens PF, Grobusch MP. The interaction between

sickle cell disease and HIV infection: a systematic review. Clin Infect Dis 2015;60:612-

26.

24. Weatherall D, Hofman K, Rodgers G, Ruffin J, Hrynkow S. A case for developing North-

South partnerships for research in sickle cell disease. Blood 2005 Feb 1;105(3):921-3.

25. Global HIV/AIDS at CDC – Countries Where We Work. Retrieved from

http://www.cdc.gov/globalaids/Global-HIV-AIDS-at-CDC/countries/default.html

26. Williams TN, Weatherall DJ. World distribution, population genetics, and health burden

of the hemoglobinopathies. Cold Spring Harb Perspect Med 2012;2:a011692.

27. Quinn CT, Rogers ZR, McCavit TL, Buchanan GR (2010) Improved survival of children

and adolescents with sickle cell disease. Blood 2010;115(17):3447–52.

28. Makani J, Williams TN, Marsh K. Sickle cell disease in Africa: burden and research

priorities. Ann Trop Med Parasitol 2007 Jan;101(1):3-14.

58 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

29. Evidence-Based Management of Sickle Cell Disease: Expert Panel Report, 2014.

Retrieved from http://www.nhlbi.nih.gov/health-pro/guidelines/sickle-cell-disease-

guidelines

30. WHO Model Lists of Essential Medicines. Retrieved from

http://www.who.int/medicines/publications/essentialmedicines/en/

31. McGann PT, Tshilolo L, Santos B, Tomlinson GA, Latham T, Aygun B, et al.

Hydroxyurea therapy for children with sickle cell anemia in sub-Saharan Africa:

Rationale and design of the REACH trial. Pediatr Blood Cancer 2016;63(1):98-104

32. Realizing Effectiveness Across Continents with Hydroxyurea (REACH). Retrieved from

https://clinicaltrials.gov/ct2/show/NCT01966731

33. Novel use Of Hydroxyurea in an African Region with Malaria (NOHARM). Retrieved

from https://clinicaltrials.gov/ct2/show/NCT01976416?term=NOHARM&rank=1

34. Ndeezi G, Kiyaga C, Hernandez AG, et al. Burden of sickle cell trait and disease in the

Uganda Sickle Surveillance Study (US3): a cross-sectional study. Lancet Glob Health

2016 Mar;4(3):e195-200.

59 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

VITA

Arielle Gail Hernandez was born on May 25, 1989 in Hollywood, Florida. Arielle graduated from Cinco Ranch High School in Katy, Texas in 2007 and received a Bachelor of

Arts in English and Peace and Conflict Studies from the University of Kansas in Lawrence,

Kansas in 2011.

After graduation, Arielle worked at the Baylor College of Medicine Texas Children’s

Hospital Center for Global Health in Houston, Texas and in 2013 she joined CCHMC’s Division of Hematology in Cincinnati, Ohio as Program Coordinator for US3. In this position, she has had an integral role in overall strategic study planning and operations, such as building partnerships; drafting and updating legal documents; creating study budgets and monitoring funds; purchasing and inventory of laboratory equipment and supplies; learning laboratory techniques in order to create and conduct onsite and remote training on technical protocols; writing and conducting the program assessment; and data reporting and analysis.

Arielle will graduate in Spring 2016 with a Master of Public Health in Epidemiology from the University of Cincinnati College of Medicine in Cincinnati, Ohio. In Fall 2016, she will continue to a Doctor of Philosophy program in Epidemiology from the University of Texas

Health Science Center at Houston School of Public Health in Houston, Texas.

60 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix A: Isoelectric Focusing (IEF) Hemoglobin Electrophoresis Procedure

61 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Isoelectric Focusing (IEF) Hemoglobin Electrophoresis Procedure

ABBREVIATIONS

DBS: Dried Blood Spot IEF: Isoelectric Focusing EID: Early Infant Diagnosis pI: Isoelectric Point Hb: Hemoglobin SCA: Sickle Cell Anemia HbA: Adult (normal) hemoglobin SCD: Sickle Cell Disease HbF: Fetal hemoglobin SCT: Sickle Cell Trait HbS: Sickle hemoglobin US3: Uganda Sickle Surveillance Study

PURPOSE

The purposes of this protocol include the following:

 To provide an overview of the US3 Study and the method of hemoglobin identification by isoelectric focusing (IEF).  To provide detailed step-by-step instructions of the IEF hemoglobin electrophoresis procedure using the RESOLVE Hemoglobin Kit.  To review the significance of various hemoglobin patterns and to describe how to identify infants and children with sickle cell trait and sickle cell disease.

OVERVIEW

Sickle cell disease (SCD) is an autosomal recessive disorder of hemoglobin (Hb) caused by a mutation in the beta-globin gene that led to an abnormal sickle hemoglobin (HbS). When one copy of the beta-globin sickle mutation is inherited, a person has sickle cell trait (SCT, HbAS). The inheritance of two copies of the beta-globin sickle mutation results in sickle cell anemia (SCA, HbSS). SCD is a group of blood disorders all characterized by the predominance of HbS, which causes deformities and instability of red blood cells, hemolytic anemia, and many associated acute and chronic medical problems. Without adequate early diagnosis and treatment, SCD is associated with a very high risk of death within the first five years of life, especially from infection and anemia. Based on older studies and population estimates, SCD is likely a very common, yet underappreciated, public health problem for infants and children in Uganda.

The Uganda Sickle Surveillance Study (US3) is a cross-sectional study designed to determine the geographic prevalence of sickle cell trait and sickle cell disease in Uganda. Dried blood spots (DBS) are routinely collected from HIV exposed children aged six weeks to 18 months from all districts of Uganda as part of the Early Infant Diagnosis (EID) program of the Uganda Ministry of Health and the Central Public Health Laboratories. This study will use these same DBS specimens collected in the EID program to determine the hemoglobin patterns. The objective of this study is to develop detailed geospatial maps of the distribution of SCD in Uganda, which will be made available to the Ministry of Health for planning screening strategies coupled with

62 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study integrated community-based SCD care and treatment programs, with a long-term goal of developing a national sickle cell strategy.

IEF ASSAY

SCD can be diagnosed easily at birth by examining the hemoglobin patterns present in the blood, which will indicate the presence of HbS and the lack of the normal adult hemoglobin (HbA).

IEF is a common and robust method for Hb identification, particularly for infant DBS specimens. Each different Hb variant has its own unique isoelectric point (pI). IEF utilizes these minor differences and involves the separation of globin chain proteins according to their isoelectric points in a stabilized pH gradient. In this assay, hemoglobins are eluted from DBS specimens and samples are run on an agarose gel containing RESOLVE Ampholytes, which establishes a pH gradient appropriate for Hb analysis. An electrical current is applied to the gel causing hemoglobin variants to migrate to their unique pI on the gel. When an individual variant’s pI equals zero, it becomes stationary or “focused” and forms a discrete band at predicted and reproducible migration locations on the gel. These bands can be visually compared to standard controls that are loaded into the gel after every tenth Hb sample.

When all of the hemoglobins have been separated and focused, the gel is then fixed with trichloroacetic acid (TCA) and stained using a staining solution for optimal visualization of bands. Results for each sample are then scored and entered into the US3 database.

GENERAL INFORMATION

Safety:

 Wear lab coat and gloves throughout the entire procedure.  Refer to equipment safety and maintenance procedures. Other:

 Do not use any reagents past their expiration date.  Store DBS cards in a dry and refrigerated location for best quality.  Agarose IEF Gels are to be stored in a refrigerator at 4-10°C. DO NOT allow gels to freeze. Handle gels with care.  Special care should be taken to prevent contamination of samples.

63 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

EQUIPMENT

Multiphor II Electrophoresis Unit Gel Dryer Rocking Platform Wallac DBS Puncher Circulating Water Bath Computer for Wallac DBS Puncher Programmable Power Supply Scanner for Wallac DBS Puncher

MATERIALS

RESOLVE Hemoglobin Kit: Pipettes P20:1-20 µL P200: 20-200 µL  Agarose IEF Gel P1000: 200-1000 µL  Anode Solution Pipette Tips Yellow: 1-200 µL  Cathode Solution Blue: 200-1000 µL  Hb Elution Solution Automatic Pipette  IEF Electrode Wicks Serological Pipettes (10 mL)  Blotting Papers Sample Application Templates  Blotting Strips 96-Well Plates Stain and Rinse Trays HbFASC Control Kit Graduated Cylinder (100 mL) JB-2 Stain Solution Kit Amber Bottles (500 mL) Trichloroacetic acid (TCA) (500 g or 1 kg) Forcepts

CONTROL AND REAGENTS

FASC Control is a standardized reagent that produces sharp bands for HbF, HbA, HbS and HbC. The FASC control is used with each gel as a control and as a placeholder to provide a reference for sample location:

 Each FASC control kit contains vials of control as a dry red protein and vials of a clear reconstitution buffer solution. Store kit in refrigerator.  Each vial makes 1 mL FASC control solution. One vial of FASC control is enough for 200 wells. Store FASC control vials in the refrigerator. To make 1 mL FASC control: Add 1 mL reconstitution buffer (use P1000 pipette) directly to the vial containing the FASC control dry red protein. Pipette up and down in the vial until you have a deep red solution. Label the vial with the date of preparation. Control solution should be used for longer than two weeks after being reconstituted.

Trichloroacetic Acid (TCA) is used to fix the hemoglobin proteins in the gel for subsequent staining, analysis and long-term storage. 10% TCA is used for the IEF procedure. 200 mL of

Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

10% TCA should be made fresh daily from the stock solution of 100% TCA. This daily 10% TCA can be used to fix multiple gels on a given day, but should be properly discarded at the end of each day. Instructions for preparation of both 100% and 10% TCA are as follows (pay close attention to whether you have a 500 g or 1 kg bottle of TCA powder):

**Warning: TCA is a highly toxic chemical and may be hazardous if inhaled, ingested, or if it comes in contact with the skin or eyes. Gloves must be worn when handling TCA. If TCA gets on the skin or in the eyes, wash thoroughly and immediately with water and remove any contaminated clothing and wash before reuse.**

To make a 500 mL bottle of 100% TCA stock: Add 227 mL filtered water (use 100 mL graduated cylinder) to a 500 g bottle of TCA powder. Mix well. Store this 100% TCA stock at room temperature.

To make a 1 L bottle of 100% TCA stock: Add 454 mL filtered water (use 100 mL graduated cylinder) to the 1 kg bottle of TCA. Mix well. Store 100% TCA at room temperature.

To make 200 mL 10% TCA from the 100% TCA stock: In an amber bottle, combine 180 mL water (use 100 mL graduated cylinder) and 20 mL of the 100% TCA stock (use auto pipette and 10 mL serological pipette). Mix well. Rinse the amber bottle for reuse.

JB-2 Stain Solution is used to stain the hemoglobin bands after fixation to enable more accurate identification of the hemoglobin pattern:

 Each JB-2 stain solution kit contains the following: 1) stain concentrate; 2) stain buffer; and 3) stain activator. Store this kit at room temperature.  The staining solution should be made fresh daily and discarded at the end of the day. Each fresh batch can be used to stain multiple gels on a given day. Make 200 mL stain solution fresh daily. **The staining solutions are light sensitive and should be stored in provided plastic bottles in a cabnet or covered area. The prepared stain solution will go bad when exposed to light and should be mixed in an amber bottle!**

To make 200 mL stain solution, mix in amber bottle: 140 mL filtered water (use 100 mL graduated cylinder) 36 mL stain concentrate (use auto pipette and 10 mL serological pipette) 20 mL stain buffer (use auto pipette and 10 mL serological pipette) 4 mL stain activator (use auto pipette and 10 mL serological pipette)

65 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

IEF STEP-BY-STEP PROCEDURE

Procedure Overview: 1. Punch DBS Specimens into 96-Well Plate (using Wallac DBS Puncher) 2. Elute Hemoglobin from DBS Specimens 3. Gel Preparation and Apparatus Set-up 4. Load Samples and Run Gel (using Multiphor II IEF Electrophoresis Unit) 5. Fix Gel (using TCA) 6. Stain Gel (using JB-2 staining solution) 7. Dry Gel 8. Score Samples and Store Gel 1. Punch DBS Specimens into 96-Well Plate (using Wallac DBS Puncher) 1. Turn on the puncher and the computer. 2. Turn on the circulating water bath and set the temperature to 12°C. 3. On the right side on the puncher display, select SLAVE. 4. On the computer desktop, click on “Wallac DBS Puncher” icon to open the software. 5. In the computer software window under “Select Protocol” click on US3. This protocol is programmed to skip spaces for AFSC controls (see Figure 1). 6. In the computer software window click START PUNCHING. 7. A message will pop up on the computer screen asking you to load the 96-well plate. On the puncher select OK to allow the plate carrier to move to loading position. 8. Record the gel number and date on the end of the blank 96-well plate with a permanent marker. This will help to identify the 96-well plate in subsequent steps. 9. Lift the puncher safety lid and securely place the 96-well plate in the plate carrier with A1 in the top right corner position. 10. On the puncher select OK to return the plate carrier with the 96-well plate for punching. 11. The computer screen will show a layout of the samples to be punched. A1 should be labeled as an AFSC control and punching should automatically begin in well A2. 12. Select a DBS card for punching and, using the barcode reader, scan the card’s barcode. 13. Hold the DBS card under the punching head and align to an area of saturated blood sample, then press the trigger or step on the foot plate to make a 3.2 mm punch. The first punch will drop into well A2. The puncher will automatically move to the next well location (A3) for the next punch. The computer will not allow multiple punches to be collected in the same well. 14. Repeat steps 12 and 13 until the 96-well plate is complete. When the puncher reaches the end of each row it will automatically move to the next well location in the next row (A1- A12 to next row B1-B12, etc.) to continue punching.

NOTE: At any time you can review what has been punched by selecting the CHECK button on the puncher and the 96-well plate will be presented allowing you to see all of the wells. The previous punch position is remembered for when you want to continue.

66 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Select RESUME on the puncher to allow the 9-well plate return to the next punch position.

15. When the last punch is made into the 96-well plate (into well H4), on the computer screen click PLATE COMPLETED. Each gel hold 80 samples and 8 FASC controls (88 total wells used). Do not punch any DBS samples into H5-H12. 16. On the puncher select END to determine the end of punching and then select YES to confirm the end of punching. 17. On the puncher, select EJECT to move the plate carrier with the completed 96-well plate to the unloading position. 18. Lift the puncher safety lid and remove the 96-well plate from the plate carrier and then select OK to return the empty plate carrier to its original position. 19. On the computer screen, a message will appear indicating that you have punched all the plates to this worklist. Click OK. To punch next set of DBS samples into a new 96-well plate, repeat steps 6-19. 20. The worklist should automatically print once you click the final “OK” in the computer software. The date and well plate number will be displayed at the top of the worklist. Sample punches will be listed by well location and card number or control/standard (STD). Figure 1: Use 96-Well Plate Format

In the first set of 44 wells (A1 – D8), four wells are designated for standard control (FASC) and 40 wells are designated for samples. Wells A1 – D8 will be loaded into Sample Application Template 1. In the second set of 44 wells (D9 – H4), four wells are designated for standard control (FASC) and 40 wells are designated for samples. Wells D9 – H4 will be loaded into Sample Application Template 2. Eight wells (H5 – H12) will be left empty. There will be eight standard control wells total and 80 sample wells total per 96-well plate.

1 2 3 4 5 6 7 8 9 10 11 12 START A FASC FASC

B FASC

C FASC

D FASC

E FASC

F FASC

G FASC Leave Leave Leave Leave Leave Leave Leave Leave H STOP Empty Empty Empty Empty Empty Empty Empty Empty

67 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

2. Elute Hemoglobin from DBS Specimens 1. Remove the bottle of Hb Elution Solution from the refrigerator. 2. Pipette 25 µL Hb Elution Solution (use P200 pipette and yellow pipette tip) into each well. Ensure that each sample is covered by elution solution. Do not touch the DBS punches with the pipette tip. If you do, change pipette tips. 3. Allow the samples elute for 30 minutes at room temperature while preparing the gel. Or the samples can elute overnight in the refrigerator at 4°C. 3. Gel Preparation and Apparatus Set-up 1. Clean the surface of the electrophoresis plate with water, and then dry using a paper towel. 2. Remove the gel from the wrapper and inspect for damage or blemishes. Remove the top and bottom trays from the gel and set aside trays for later use. Carefully peel the plastic from the top of the gel. 3. Pipette ~2 mL filtered water (use P1000 pipette and blue pipette tip) onto the center of the electrophoresis plate. 4. Place the gel in the center of the electrophoresis plate by holding the diagonal edges of the gel to form a parabola shape and slowly lowering until flat on the plate, and water is spread evenly across the entire gel without air bubbles (see Figure 2).

Figure 2: Gel Placement on Electrophoresis Plate

5. Remove any excess water from the sides of the gel using a paper towel. 6. Gently place one sheet of blotting paper over the entire gel for three seconds then pick back up. Avoid over drying the gel. 7. Remove three IEF wicks, the Anode Solution bottle, and the Cathode Solution bottle from the RESOLVE Hemoglobin Kit. 8. Set two of the wicks on one of the empty gel trays and saturate with 3-4 mL of Anode Solution per wick (use P1000 pipette and blue pipette tip). If necessary, gently blot dry with blotting strip such that wicks are fully saturated but not dripping. 9. Place the two anode wicks on the gel in a straight line using the “2” and “16” markers on the electrophoresis plate. Ensure good contact with the gel by running your finger across the entire length of the wick. Wash or change gloves.

68 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

NOTE: It is extremely important that Anode and Cathode solutions DO NOT come into contact with each other. WASH OR CHANGE GLOVES to prevent mixing solutions on the wicks.

10. Set one wick on the other empty gel tray and saturate with 3-4 mL Cathode Solution (use P1000 pipette and blue pipette tip). If necessary, gently blot with blotting strip such that the wick is fully saturated but not dripping. 11. Place the cathode wick in a straight line in the middle of gel using the “9” marker on the electrophoresis plate. Ensure good contact with the gel by running your finger across the entire length of the wick. Wash or change gloves. 12. Position one sample application template on either side of the cathode wick using the parabolic technique. Ensure good contact with the gel and no air bubbles. Gently pat dry with the blotting strip to avoid leaking of samples.

Figure 3: Final Gel Set-up

4. Load and Run Gel (using Multiphor II IEF Electrophoresis Unit): 1. Load 5 µL of each hemolysate (use P20 pipette and yellow pipette tip per sample) into sample application template 1 starting at the farthest end of the template (starting from the back of the electrophoresis unit). Skip the wells designated for FASC controls. Repeat for sample application 2.

69 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

2. Load 5 µL FASC control into designated control wells (use P20 pipette and yellow pipette tip per sample). There should be 8 controls loaded per gel (4 into each template).

NOTE: It is important to be consistent with how samples are loaded each time into the wells. Be careful to remember which sample you are loading into each well.

3. When all samples have been loaded, place the glass plate with electrode holders over the gel. If it is necessary to realign the electrode holders, untighten and slide the electrode holders along the glass plate until they are aligned evenly over the wicks. Tighten the electrode holders in place and lower the glass plate into position on the gel box. Ensure that the metal wires are in direct contact with the anode and cathode strips along the entire length of the gel. 4. Connect the electrode leads from the electrode holders to the electrophoresis unit. Two anode electrode leads have red tips and are connected to the two outside electrode holders at the back of the glass plate. One cathode electrode lead has a black tip and is connected to the center electrode holder at the front of the glass plate. a. Connect both of the anode electrode leads into the sockets at the back right of the electrophoresis unit. b. Connect the cathode electrode lead into the socket at the front left of the electrophoresis unit. 5. Place safety lid firmly on gel box. 6. Connect the two leads from the lid to the power supply. Connect the red lead to the red outlined outlet (+) on the power supply and connect the black lead to the blue outlined outlet (-) on the power supply. 7. Program the power supply for 1200 V, 100 mA, 30 W (60 W if running 2 gels), for 90 minutes. 8. On the power supply press RUN. 9. After 15 minutes, on the power supply press PAUSE. Disconnect the lid leads from the power supply and remove the lid, then disconnect the electrode leads from the electrophoresis unit and remove the glass plate from the gel box. Carefully remove the application templates from the gel and blot away any excess moisture using a paper towel. 10. Reposition the glass plate on gel box and reconnect the electrode leads to the electrophoresis unit, and reposition the lid on the gel box and reconnect the lid leads to the power supply. On the power supply press RUN to resume and run for the remaining 75 minutes. 11. At the end of the run, on power supply press STOP. Disconnect the leads from the power supply and remove the lid. Disconnect electrode leads from the electrophoresis unit and remove the glass plate from the gel box. 12. Turn off the circulating water bath. 13. All parts should be rinsed thoroughly with filtered water. Sample application templates can be washed (using alcohol if available) and reused.

70 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

5. Fix Gel 1. Make 200 mL 10% TCA (see page 4). 2. Carefully remove the wicks from the gel. Mark the gel by making scissor cuts to the corners of the gel (1 cut for 1st gel, 2 cuts for 2nd gel, etc.) with a single or largest cut indicating the A1 position of first sample application template. Carefully remove the gel from the electrophoresis plate. 3. Rinse the gel by adding 200-300 mL filtered water to a tray. Submerge the gel in water and place the tray on rocking platform for 5 minutes. 4. Fix the gel by adding 200 mL of 10% TCA to a tray. Submerge the gel in TCA and place the tray on the rocking platform for 10 minutes. 5. Rinse the gel by adding 200-300 mL filtered water to a tray. Submerge the gel in water and place the tray on the rocking platform for 15 minutes. NOTE: It is important to keep track of samples on the gel by being aware of how gel is placed in the tray. Place the single or largest cut on the gel at the spout of the tray each time, indicating the A1 position of the first sample application template.

6. Stain Gel 1. Make 200 mL JB-2 Stain Solution (see page 3). 2. Stain the gel by adding 200 mL JB-2 Stain Solution to a tray. Submerge the gel in stain solution, cover the tray with aluminum foil, and place the tray on the rocking platform for 8-10 minutes. 3. Rinse the gel by adding 200-300mL filtered water to a tray. Submerge the gel in water and place the tray on the rocking platform for 12 minutes. Repeat 5 more times adding new filtered water each time. The background of the gel should be clear. Repeat more rinses if the gel appears to be over-stained. NOTE: It is important to ensure that when fixing, staining, and rinsing the gel that these steps are completed for the full amount of time that is instructed. This will produce more distinct bands on the gel and prevent the need to repeat samples.

7. Dry Gel 1. Place the gel in the dryer. 2. Set the dryer timer for 4 hours at 50-70°C. 3. Store dry gel in sheet protector with puncher worklist and scoring worksheet.

8. Score and Store Gel 1. Read the sample and score the results. Abbreviations:

F = fetal hemoglobin, hemoglobin F, HbF A = adult hemoglobin, hemoglobin A, HbA

71 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

A2 = hemoglobin A2, HbA2 S = sickle hemoglobin, hemoglobin S, HbS C = hemoglobin C, HbC X = Unknown hemoglobin, variant hemoglobin

US3 study samples are primarily from children aged six weeks to 18 months. Fetal hemoglobin (HbF) will be present in some samples. In young children, age-dependent levels of HbF are observed, with the highest amount of HbF present in newborns. HbF is gradually replaced by HbA or HbS over the first two years of life. Throughout the course of this study, four hemoglobin patterns are expected to be seen. These patterns will appear differently based upon the infant’s age. Figure 6 is an easy reference for describing the significance of these patterns in respect to the age of the patient. In all cases, the scoring result should list the amount of each Hb seen on the gel, from most to least. 1. “Normal” hemoglobin patterns are expected to be seen in a large majority of the samples tested in this study, estimated at 80%. At birth, a “normal” pattern includes both HbF and HbA. This pattern is referred to as HbFA or simply “FA.” HbA2 is a normal hemoglobin that begins to appear at about age six months. Between age 6 months and 2 years, the normal hemoglobin pattern will include HbF, HbA and small amounts of HbA2. HbF should then slowly disappear such that a normal hemoglobin pattern after two years of age includes only HbA and HbA2. This adult hemoglobin pattern is referred to simply as “A.”

2. Sickle cell trait will likely represent about 10-20% of the samples tested in the US3 study. In newborns, the hemoglobin pattern consistent with sickle cell trait includes the presence of HbF, HbA, and HbS. This pattern is referred to as “FAS.” As above, HbA2 will appear after six months of age such that a hemoglobin pattern FAS + A2 will be seen for infants 6 months – 2 years with sickle cell trait. After two years, children with sickle cell trait will have HbA, HbS and HbA2. This “adult” pattern of sickle cell trait is referred to as “AS.”

3. Sickle cell disease: SCA (HbSS) is the most common type of sickle cell disease that is expected to be seen throughout the US3 study. It is expected that at least one sample with sickle cell disease should be seen in every 1-2 gels. Sickle cell disease most commonly referred to as HbSS disease, but also includes the combination of both HbS and HbC (HbSC). In infants, hemoglobin patterns including HbF and HbS without the presence of HbA will indicate sickle cell anemia, referred to as FS. Similarly, the presence of HbF, HbS, and HbC (without the presence of HbA) will also represent sickle cell disease, referred to as FSC. As above, HbA2 will also appear at about six months of age for children with sickle cell disease. Children with sickle cell disease are more likely to continue with low levels of HbF even beyond two years. Figure 5 indicates the full range of hemoglobin patterns that indicate sickle cell disease.

72 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

4. Variant hemoglobin patterns will be rare, but will likely be seen throughout the course of the study. Any hemoglobin that is not the common HbA, HbF, HbS, HbC, or HbA2 should simply be labeled as “X” to indicate a Hb variant. For example, if a sample clearly has both HbF and HbA, but also has a band in between the location of HbS and HbC, you would score this sample “FAX” to indicate the presence of HbF, HbA, and this variant “X” hemoglobin.

Figure 5: IEF Gel Results

73 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Figure 6: IEF Scoring Table

2. Score sample results on scoring worksheet (see Figure 7). Hemoglobin is reported in order of amounts found in blood and should be written as follows: A normal FA normal (newborn or young children with fetal hemoglobin) AS sickle cell trait FAS sickle cell trait (newborn or young child with fetal hemoglobin) S sickle cell anemia (HbSS disease) FS sickle cell anemia (newborn or young child with fetal hemoglobin) SC sickle cell HbSC disease FSC sickle cell HbSC disease (newborn or young child with fetal hemoglobin) X unknown pattern

3. Store gel in sheet protector with puncher worklist and well plate worksheet. Initial and date worksheet. File in binder. 4. Record results in database. NOTE: Any samples that with an IEF pattern that suggests sickle cell disease should be repeated to confirm. Any samples that are contaminated or did not produce clear results should be repeated.

74 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Figure 7: Scoring Worksheet

75 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix B: Uganda Sickle Surveillance Study Program Assessment Plan

76 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

UGANDA SICKLE SURVEILLANCE STUDY (US3)

PROGRAM ASSESSMENT PLAN

DOCUMENT TYPE: Uganda Sickle Surveillance Study (US3) Program Assessment Plan

PREVALENCE AND MAPPING OF SICKLE CELL TRAIT AND PROTOCOL: SICKLE CELL DISEASE IN UGANDA

PRINCIPAL Grace Ndeezi MBChB MMed PhD INVESTIGATORS: Russell Ware MD PHD

Sarah Kiguli MBChB MMed MHPE Charles Kiyaga MBLSM CO- INVESTIGATORS Jane Ruth Aceng MBChB MMed MPH Jesca Nsugwa, MBChB, MMed, PhD Deogratias Munube, MBChB, MMed Sickle Cell Laboratory/US3 Team STUDY PERSONNEL: Thad Howard MS, Laboratory Manager Arielle Hernandez, Program Coordinator

STUDY DURATION: Eighteen (18) months

Sickle Cell Laboratory, Central Public Health Laboratories (CPHL), Kampala, Uganda STUDY LOCATION: Division of Hematology, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, Ohio

77 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

1. STUDY INFORMATION

1.1. Study Title: Uganda Sickle Surveillance Study (US3) 1.2. Design: A cross-sectional study designed to determine and map the geographic prevalence of sickle cell trait and sickle cell disease in Uganda

2. STUDY GOALS

2.1. Short-term goals  To build local sickle cell laboratory capacity  To determine the feasibility for testing a high-volume of samples for sickle cell trait (SCT) and sickle cell disease (SCD)  To document laboratory start-up, operations, and procedures to develop formal standardized documents  To identify research opportunities within the surveillance study cohort

2.2. Long-term goals  To produce prevalence estimates for SCT and SCD  To generate geospatial distribution maps of the prevalence of SCT and SCD across Uganda  To inform the Uganda Ministry of Health about the current burden of disease and to assist in the development of a comprehensive national sickle cell strategic plan  To initiate research projects related to the SCT and SCD cohorts

3. PROGRAM ASSESSMENT PURPOSE AND GOALS

3.1. To evaluate the efficiency of the overall study operations 3.2. To document the logistics of and adherence to laboratory operations and procedures 3.3. To assess the quality and accuracy of study data 3.4. To review data entry and result reporting 3.5. To determine the next steps for expanding the US3 surveillance study toward a pilot newborn screening program

78 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

4. PROGRAM ASSESSMENT PLAN

4.1. Program Assessment

The program assessment of US3 will consist of continuous review and support of overall operations and procedures to ensure the data collected are high quality and accurate to meet the end goals of the study and to prepare for expansion of the US3 surveillance study toward a screening program. The program assessment of US3 will take place in two forms in an on- going manner: (1) continuous remote assessment and (2) periodic on-site study assessment.

4.2. Overview of Continuous Remote Assessment Intervals and Approach

The remote program assessment of the following items is comprised of continuous review and support.

 Review and support via bi-weekly US3 Steering Committee Meeting of CCHMC and CPHL leadership, by Skype  Review and support via weekly, and more often as needed, US3 Laboratory Meeting of CCHMC and lab personnel, by Skype  Other correspondence as needed, by Skype and email

4.3. Overview of Periodic On-site Assessment Intervals and Approach

The on-site program assessment of the following items will be comprised of periodic on-site visits.

4.3.1 A random sample of a total number of gels from one workday from four different study periods will be selected, approximately one month apart. Of this random sample, approximately 1500 samples, the following will be assessed:

 Documentation of scoring process (three independent scores with initials and dates)  Discrepancy rate between three independent scores and final resolution  Number of samples scored per gel and number indeterminate (P) samples per gel  Review and document notes/comments related to P samples

79 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

 Accurate interpretation of hemoglobin patterns into scores (% accuracy)  Accurate interpretation from final band score to final letter score of N, T, S or V (% accuracy)  Discrepancy rate of repeat sample scores and final resolution  Discrepancy rate of scores and final resolution from remote review  Accurate and complete entry of final scores into database

4.3.2. A review of all positively identified sickle cell disease samples will occur. For each sample scored as having sickle cell disease (S), the following will be assessed:

 Confirm band score and final letter score (final letter score of S includes hemoglobin SS disease, HbSS, as well as any sickle cell variants, HbSV)  Repeat sickle cell disease sample, confirm discrepancy rate of score and final resolution

4.3.3. Review of laboratory processes. The following components will be observed and assessed:

 Process and timeline for Sickle Cell Laboratory to receive samples from Early Infant Diagnosis (EID) Laboratory  Sickle Cell Laboratory workflow process  Process and timeline for Sickle Cell Laboratory to deliver final scores for entry into database  Process and timeline for final score entry into database  Process and timeline for result reporting to health facilities

4.3.4. Review of laboratory operations. The following components will be observed and assessed:

 Laboratory capacity and workflow  Laboratory adherence to established protocols  Laboratory protocols are maintained and up-to-date for reference and training purposes  Determination and performance of troubleshooting  Performance of best laboratory practices and techniques  Proper use of stored reagents in the lab and refrigeration unit

80 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

 Review of monitoring, storage, and documentation of inventory (e.g. temperature logs, regular inventory checks)  Unmet needs or limitations of current lab operations

4.3.5. Review of database. The following database components will be observed and assessed:

 Observe data entry process  Review and document data entry fields related to the sickle cell isoelectric focusing (IEF) results (e.g., age, region, district, and result)  Review and document database reporting related to the study (e.g., results report form, monthly data reporting)

4.4. On-site Assessment Visits: Scope and Components

On-site assessment visits will be scheduled with the site leaders, site team, and program coordinator. Data queries will be resolved and documented appropriately during the assessment visit when possible.

Upon completion of the on-site assessment visit, the program coordinator will prepare a follow up report that will include a summary of items reviewed, a listing of findings, action items, and overall recommendations. During the on-site assessment visit, the program coordinator will meet with the site leaders and study team to discuss any issues noted and to provide additional training as indicated by the visit findings. This report will be completed and made available to the team at Cincinnati Children’s and to the site within two (2) weeks.

5. FUTURE PLANNING

The program assessment will help provide on-going program review and support to help to establish the next steps for the US3 surveillance study.

5.1. Expanding screening program efforts  Documenting the Sickle Cell Laboratory operations and procedures to develop formal standardized documents for reference and training  Increase laboratory workflow (e.g., increase efficiency and/or personnel)  Increase laboratory capacity (e.g., laboratory infrastructure, increase equipment)

81 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

 Planning of pilot newborn screening program for patients with SCD to include sample collection, follow-up, and care and treatment within specific regions of the country

5.1.2. Further confirmatory testing  Assess capacity and most effective method for further confirmatory testing to further identify and diagnose sickle cell variants or other hemoglobin variants

5.1.3. Research  Determine associations with malaria and anemia prevalence within Uganda  Identify early mortality based on SCT and SCD ratios by age  Estimate effects of co-morbidity with HIV on survival  Determine the prevalence of genetic modifiers within the SCD cohort  Identify variants observed in the surveillance study cohort

82 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix C: Uganda Sickle Surveillance Study Program On-Site Assessment Report

83 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

UGANDA SICKLE SURVEILLANCE STUDY (US3)

PROGRAM ON-SITE ASSESSMENT REPORT

Uganda Sickle Surveillance Study (US3) Program On-Site DOCUMENT TYPE: Assessment Report Prevalence and Mapping of Sickle Cell Trait and Sickle Cell STUDY NAME: Disease in Uganda Sickle Cell Laboratory, Central Public Health Laboratories SITE NAME: (CPHL), Kampala, Uganda

VISIT NO: On-Site Assessment Visit 01

VISIT DATE: 8 June – 20 June 2014

REPORT DATE: 21 July 2014

REVIEWER: Arielle Hernandez, Program Coordinator

Charles Kiyaga, Co-Investigator SITE STAFF PRESENT AT Isaac Ssewanyana, CPHL Lab Manager VISIT: Sickle Cell Laboratory Staff: Raymond Mugabe, Stephen Aeko, Mercy Nabunya, Priscilla Khaniza, Munirah Nmutebi ANTICIPATED NEXT ON-SITE February 2015, at the 12-month mark ASSESSMENT DATE:

84 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

I. Summary of Assessment Visit:

Since the opening of the Sickle Cell Laboratory and the launch of US3 in February 2014, the study has picked up quickly and is experiencing early successes, such as sickle cell result reporting to health facilities and telling preliminary data. This is all thanks to the hard work and support from the team at the CPHL.

It is impressive how quickly the lab techs have picked up on the lab procedures. Their dedication and attention to the work has allowed them to identify any technical issues and to work together with the Cincinnati team to carefully troubleshoot and continue to learn. Many of the lab techs came in with existing lab experience, which has allowed all the moving parts of establishing a new lab to come together easily (e.g., protocol adherence, inventory checks, temperature logs).

The lab workflow has been steadily maintained, but there is room to improve efficiency when it comes to delegating lab responsibilities, which will allow for an increase in workflow. There is also the need to improve communication between the lab component and the data entry component by bridging some quality assurance checks to continue to ensure correct and complete data entry.

The overall outcome of the assessment is very positive. The Sickle Cell Lab is in a great position to complete the study successfully and to confidently progress into program expansion.

II. Summary of Attachments and Items Reviewed:

A. US3 Program Assessment Plan (Version 1.0, 5 June 2014) The US3 Program Assessment Plan is available as an attachment. This report satisfies the periodic on-site assessment and future planning portions of the plan.

B. US3 On-Site Assessment Data Report The US3 On-Site Assessment Data Report is available as an attachment and will be referenced throughout this report. The data report captures the gel review portion of the plan. Highlights in red will identify important discrepancies or other findings.

85 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

C. Study Periods and Samples Reviewed This report covers the study from the start date of 14 February 2014 (first gel run) to 6 June 2014. The total number of samples run and scored to the date of 6 June 2014 is 26,621 samples. The chart below represents the four study periods that have been identified as significant study phases considered in this report.

Study Dates Description Period (2014) 13 Feb – Start-Up Phase I: workflow of 4 gels per day; repeating all SCT, 1 6 March SCD, variant, and other indeterminate samples Start-Up Phase II: Increased workflow to 5 gels per day; 7 March – 2 repeating all SCT, SCD, variant, and other indeterminate 20 March samples 21 March – Active Phase I: Maintaining workflow of 5 gels per day; only 3 7 May repeating indeterminate samples 8 May – Active Phase II: Maintaining workflow of 5 gels per day; 4 18 June repeating S and indeterminate samples

III. Assessment Findings:

Gel review by random selection per study phase (US3 Assessment Plan, Section 4.3.1)

A. Period 1, Start-Up Phase I (Gels 1 – 4 from 18 February 2014)

1. Site Scoring Process a) All gels were initialed and dated by the individual responsible for running the gel for that day. b) When reviewing the binders, only gel 1 had worksheets from all three scorers, which were dated and initialed. Gels 2-4 only had gel and worksheet with final scores, which were not initialed. c) Score summaries at the bottom of the worksheet were not always consistent with the scores that were counted during review (see data report). d) The lab techs receive feedback on scoring via remote review with the Cincinnati team; if there are any discrepancies they review amongst themselves to determine a final score for the sample.

86 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

e) For this set, the interpretation from band scores into final letter scores was an issue a variant band score that is often documented with the band score of FASX, but was given a letter score of V. It has been recommended that these samples be repeated next to a standard to confirm presence of S band or V band. If the S band is confirmed as present, then this sample would need to have a final letter score of T.

2. Percent Accuracy a) Independent Scoring: Independent scoring was not documented for all gels. For gel 1, the agreement rate between scorers was 96.25% agreement. NOTE: Different band scores that resulted in a different final letter score were documented as discrepancies, as well as different band scores that resulted in same final letter score. b) Samples Scored: For all four gels, the average number of samples that scored was 79.5/80 or 99.37% samples scored. c) Repeat Scoring: Repeats from gel 2 were unable to be located. The agreement rate of the available repeat scores was 67.38% agreement. d) Remote Review Scoring: For the purpose of the assessment, the remote review is only of the initial run of the gel set. In this study period, samples underwent an initial run and review/remote review and possibly a repeat run a review/remote review. The outcome of the remote review for this gel set was 97.45% agreement.

3. P Sample Observations The P samples that were unable to be scored had no visible bands due to little to no hemoglobin elution from the sample. A lack of hemoglobin elution is possibly due to a technical error or poor sample. It has been recommended that the lab tech increase the sample load into template or add an additional manual punch and repeat the sample. If there is still no hemoglobin elution and no visible bands after troubleshooting, then the sample can be excluded.

4. Data Entry Observations The worksheets were dated with the puncher automated date, and the gels were dated with date of run. Data entry used the date on puncher worksheet when entering final result into database.

87 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

B. Period 2, Start-Up Phase II (Gels 1 – 5 from 14 March 2014)

1. Site Scoring Process a) Every gel was accompanied with three worksheets total: two worksheets from independent scorers and one worksheet from a final independent scorer who interpreted the band scores into final letter scores. These final letter scores are provided to data entry. b) Gel was dated with the same date as the worksheet. c) There were no lab tech initials documented on gels. Lab tech initials were present on puncher worksheet indicating who punched the gel, but this is not always the same person who runs the gel. It is recommended that all lab techs complete and document any procedure they perform with a date and initial. d) Score summaries at bottom of worksheet were miscounted for gel 1 (see data report). e) All the gels were scored by the same two scorers.

2. Percent Accuracy a) Independent Scoring: The agreement rate between scorers for all five gels was 98.5% agreement. b) Samples Scored: For all five gels, the average number of samples scored was 79.4/80 or 99.25% samples scored. c) Repeat Scoring: The Agreement rate of repeat scores for the set was 74.44% agreement. d) Remote Review Scoring: The outcome of remote review for this set was 96.75% agreement.

3. P Sample Observations The P samples that were unable to be scored had no visible bands due to little to no hemoglobin elution from the sample. Recommendations for troubleshooting P samples were applied. Another cause of the P samples was a mix up of samples, which was noted with a date and initial and the samples were repeated.

4. Data Entry Observations

88 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

a) Data entry used initial run date for repeat sample results instead of repeat run date. Data entry needs to determine and communicate which run date they will consistently use for repeat samples. b) Gel dated with same date as worksheet. All gels were initialed. Worksheet with final scores noted who the final reviewer was since the result report form has final reviewer’s initials.

C. Period 3, Active Phase I (Gels 1 – 5 from 22 April 2014)

1. Site Scoring Process a) Gels were dated as 23 April (the date the gel was run instead of worksheet date). This date was directly written on the gels and was used during the remote review. In the previous study period, lab techs were dating gels with the same date and the puncher worksheet date. It is important to be consistent with dating the gel the same as the worksheet to ensure data entry knows which date to use when inputting results. This was addressed during time of on-site assessment. b) Still seeing the FASX band score with letter score of V, but the appropriate band score is FAX (V). This has been addressed during time of on-site assessment, as well as during weekly lab meetings. The recommendation remains that that these samples be repeated next to a standard to confirm presence of S band or V band.

2. Percent Accuracy a) Independent Scoring: The agreement rate between scorers for all five gels was 96.75% agreement. b) Samples Scored: The average number of samples that were scored was 79.4/80 or 99.25% samples scored. c) Repeat Scoring: No repeats for this gel set. d) Remote Review Scoring: The outcome of the remote review for this gel set was 97.75% agreement.

3. P Sample Observations No new observations.

4. Data Entry Observations

89 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

When reviewing final data entry, some of the gels were input with the 22 April date and others with the 23 April date. This could have an effect on the results matching up the lab numbers and dates makes it difficult to locate a sample for reference or review. This was addressed during time of on-site assessment.

D. Period 4, Active Phase II (Gels 1 – 5 from 4 June 2014)

1. Site Scoring Process a) Repeating all S, variant, and indeterminate samples. b) Gels dated with same date as worksheet. c) Initials and dates present on all gels. d) Score summaries at the bottom of the worksheet for gel 5 were not consistent with the scores that were counted during review (see data report).

2. Percent Accuracy a) Independent Scoring: The Agreement rate between scorers for all five gels was 96.5% agreement. b) Samples Scored: For all five gels, the average number of samples that were able to be scored was 79.4/80 or 99.25% samples scored. c) Repeat Scoring: The agreement rate of repeat scores for the set was 77.44% agreement. d) Remote Review Scoring: Remote review was not completed for this set. The outcome of the available remote review was 98.75% agreement.

3. P Sample Observations No new observations.

4. Data Entry Observations No new observations.

Gel review of all positively identified SCD samples (US3 Assessment Plan, Section 4.3.2)

From 14 February 2014 to 6 June 2014, a total of 141 S samples have been identified and entered into the database. For the purpose of the assessment, a list of all of the positively

90 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

identified S samples was exported directly from the database. 138 of the 141 were found and assessed during the gel review. Please reference the data report for complete review of the positively identified SCD samples. Highlights in red note important discrepancies or other findings.

IV. Reviewing of Operational and Laboratory Processes

A. Operational Processes (US3 Assessment Plan, Section 4.3.3)

1. Sample Receipt Processes

a) When the samples first arrive at the CPHL, they undergo EID lab processes. Demographic information for each sample is entered into database and then sent to the EID lab for HIV testing in the same day. b) EID repeat samples are removed from sample packets. Sample packets can then be picked up and brought to the Sickle Cell Lab in the same day. EID repeat samples may be picked up later for sickle cell testing, as they are not excluded from study. c) Samples picked up from the EID Lab are brought into the Sickle Cell Lab to be punched on the same day and then run on a gel the following day. d) Currently, the Sickle Cell Lab is not at the same sample pace as the EID Lab, as they are about 6 – 8 gels (480 – 649) behind. e) The Sickle Cell Lab runs and scores gels on the same day, and final scores are available to data entry at the end of each day. f) Data entry varies from 2 – 3 days to a week behind on entry of sickle cell results. This allows for further review of scores with remote review recommendations that are provided by the Cincinnati team weekly. g) Data entry coordinates simultaneous result reporting of EID and sickle cell test results back to the health facilities. h) Result reporting back to health facilities is dependent on data entry, which also varies from 2 – 3 days to a week before results are sent back out to health facilities.

B. Laboratory Processes (US3 Assessment Plan, Section 4.3.4)

1. Capacity and Workflow

91 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

a) To make up the Sickle Cell Lab team, there are currently: 4 full-time lab techs, 1 full-time data entry, and 1 part-time data manager/administrator. b) The Sickle Cell Lab quickly increased lab workflow from running 4 gels per day to running 5 gels per day. The lab techs continue to maintain the workflow of running 5 gels per day. The lab techs feel comfortable with this workflow as they are continuing to learn, troubleshoot, and work on their techniques (such as loading samples, mixing reagent, etc.). c) Each lab tech has the role of loading and starting the run of a gel per day. Each lab tech is also designated to more specific lab responsibilities for the day, such as doing the day’s punching, or monitoring the fix/stain/wash of multiple gels, or scoring multiple gels.

2. Adherence to Existing Lab Protocols

a) The lab techs were trained with the US3 lab protocol and training materials created and provided by the Cincinnati team. b) The US3 lab protocol was updated and reformatted into the standard CPHL protocol format. c) US3 lab meetings are held weekly between the Sickle Cell Lab team and the Cincinnati team. All troubleshooting and comments have been documented and disseminated to all team members in the lab meeting minutes. d) At this time, the US3 lab protocol has not been updated. It is recommended that the Sickle Cell Lab team and the Cincinnati team work to update the US3 lab protocol with any procedure changes made since the start of the study. It is then encouraged that the protocol is reviewed and updated on a regular basis.

3. Physical Space Considerations a) Refrigerator and freezer in Sickle Cell Lab are monitored and documented twice a day, once in the morning and once in the afternoon. b) Reagents and consumables are able to be organized and stored in current lab space. Gel boxes not in use are kept in the CPHL cold room. Temperature documentation of cold room is encouraged.

92 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

c) Supply inventory moved from weekly updates to biweekly updates. Supply inventory updates have revealed that the supplies are being consumed at an appropriate and predictable rate. d) The current Sickle Cell Lab space cannot accommodate any additional personnel or equipment.

C. Database

1. Reviewer Observation of Data Entry a) Data entry for sickle cell results takes place in a separate data room just outside the Sickle Cell Lab. This data room is also the storage space for the gel binders, as well as any other Sickle Cell Lab related documents (e.g., training materials, equipment manuals, office supplies, etc.) b) The Sickle Cell Lab has a designated staff member for data entry. This person is able to communicate right away with any queries to the lab techs. c) Data entry staff is not involved with any known quality assurance (QA) measures, but it is recommended that this become a part of the data entry role to add another level of QA. d) The database had been built and operated in Microsoft Access. Sickle cell results are not added to the EID database tables directly, but are instead entered in a separate table and later merged with the EID database for result reporting purposes. e) Sickle cell results are entered based on the final scoring worksheet that is provided by the Sickle Cell Lab. The scores on the final worksheet have been interpreted into the final letter scores, and the final letter scores are entered into the database. f) The database has the ability to program and run specific queries on both the separate sickle cell result table and the merged EID/sickle cell table (e.g., ages and results, region and results, etc.).

V. Recommendations and Future Planning

A. Overall Recommendations for Current Activities 1. The Sickle Cell Lab has maintained the workflow of 5 gels per day which has allowed the lab techs to grow confident in their abilities and techniques. It is

93 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

recommended that Sickle Cell Lab team and the Cincinnati team develop a plan to increase workflow to 6 gels per day. The benefits would include keeping up to pace with the EID samples per day, as well as preparing the lab for the larger volumes of samples they will encounter as the US3 program grows. 2. The Sickle Cell Lab should continue to work with the Cincinnati team on band interpretation and improving their overall scoring practices. Together, the Sickle Cell Lab team and the Cincinnati team will continue to meet weekly to review any scoring queries and to identify practices that will help with the scoring process. It is also encouraged that both teams work to document these queries and their resolutions for reference and training purposes. 3. The Sickle Cell Laboratory should update the US3 Lab Protocol, as well as maintain and revisit the protocol regularly. This document is being developed as a standard training manual for sickle cell labs in Uganda. 4. The Sickle Cell Lab should work to organize daily lab tech roles to help improve lab workflow, such as developing a work chart for daily lab responsibilities per person. 5. The Sickle Cell Lab should implement more QA checks to bridge the lab component and the data entry component. Lab techs and data entry staff should work together to maintain unique samples that can be identified consistently in gel records and in the database, such as making a decision together on how to consistently date worksheets and gels and the final results in the database. Lab techs and data entry should also improve overall communication, such as data entry raising queries to lab techs if samples do not have complete results (especially concerning repeat samples).

B. Future Activities and Next Steps

1. Screening Program The preliminary data of the US3 study has provided early insight into the prevalence and distribution of sickle cell trait and disease by region. Strong evidence of high prevalence areas has allowed for identification of four sites that are existing EID hubs and that operate on the hub sample transport system. The sites/hubs of Lira and Gulu have been identified for the first phase of the roll out of pilot newborn screening programs. The sites/hubs of Jinja and Kampala have been identified for the second phase of the roll out of pilot newborn screening programs.

94 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

2. Further Confirmatory Testing a) A Material Transfer Agreement between the CPHL and CCHMC and an amendment to the US3 protocol regarding further confirmatory testing will be developed and submitted for approval to the Uganda National Council for Science and Technology (UNCST). b) SCD samples and variant samples will be tested in the lab of the US3 Principal Investigator, Dr. Russell Ware, at Cincinnati Children’s Hospital Medical Center for further characterization of the US3 samples. This characterization includes: (1) investigation of known genetic modifiers of SCD samples (2) further investigation of variant hemoglobin samples for proper identification c) Proposed methods for further confirmatory testing will include US-based analysis of these samples to determine the best lab techniques and molecular biological methods needed to accomplish the work. The best methods will be exported to the CPHL to build molecular capacity as an extension of the Sickle Cell Lab.

3. Future Research Opportunities N/A at this time.

95 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix D: Burden of sickle cell trait and disease in the Uganda Sickle Surveillance Study

(US3): a cross-sectional study

96 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

97 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

98 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

99 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

100 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

101 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

102 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix E: Is integrating sickle cell disease and HIV screening logical?

103 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

104 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

105 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix F: Prevalence and Mapping of Sickle Cell Trait and Disease in Uganda

106 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

PREVALENCE AND MAPPING OF SICKLE CELL TRAIT AND DISEASE IN UGANDA

Principal Investigators: Grace Ndeezi Russell E. Ware

Presented to the School of Medicine Research and Ethics Committee Makerere University College of Health Sciences Kampala, Uganda September, 2014

Protocol Version 3.0

107 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

PREVALENCE AND MAPPING OF SICKLE CELL TRAIT AND DISEASE IN UGANDA

Table of Contents LIST OF APPENDICES ...... 109 LIST OF INVESTIGATORS ...... 109 ABSTRACT ...... 111 INTRODUCTION ...... 112 Background and Literature Review ...... 112 Problem Statement and Justification ...... 115 METHODS ...... 116 Overview ...... 116 Study design ...... 116 Study setting ...... 117 DATA COLLECTION ...... 119 Procedures ...... 120 Sample size ...... 122 Data Management and analysis ...... 123 POTENTIAL BENEFITS ...... 123 POTENTIAL RISKS ...... 124 INSTITUTIONAL REVIEW BOARDS (IRB) APPROVAL ...... 124 STRENGTHS ...... 124 LIMITATIONS ...... 125 REFERENCES ...... 126 APPENDIX ...... 128

108 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

PREVALENCE AND MAPPING OF SICKLE CELL TRAIT AND DISEASE IN UGANDA

LIST OF INVESTIGATORS

Principal Investigator Grace Ndeezi, MBChB, MMed, PhD Associate Professor Department of Pediatrics & Child Health College of Health Sciences Makerere University

Co - Principal Investigator Russell E. Ware MD PhD Professor of Pediatrics Director, Division of Hematology Associate Director, Global Health Center Cincinnati Children’s Hospital Medical Center Cincinnati OH 45229

Co-Investigators Sarah Kiguli, MBChB, MMed, MHPE Associate Professor of Pediatrics Head of Department Department of Pediatrics & Child Health College of Health Sciences Makerere University

Charles Kiyaga, MBLSM. Director, Early Infant Diagnosis (EID) Laboratory, National Coordinator EID Program Ministry of Health, Uganda

Jane Ruth Aceng , MD MBChB, MMED, MPH Director General Health Services Ministry of Health, Uganda

Jesca Nsugwa, MBChB, MMed, PhD Assistant Commissioner for Health Ministry of Health, Uganda

109 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Deogratias Munube, MBChB, MMed Pediatrician Sickle Cell Clinic Department of Pediatrics & Child Health Mulago Hospital

110 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

ABSTRACT

Introduction: It is estimated that over 250,000 babies are born with sickle cell disease (SCD) annually in sub-Saharan Africa, and only 10% - 50% of them survive beyond five years of age. Data describing the magnitude of the sickle cell problem are lacking in most African countries. The available data on prevalence were mainly from older studies and small numbers of hospitalized patients. In Uganda, approximately 25,000 children are born with SCD but 70-80% die before their 5th birthday. Lehmann and Raper found ‘sicklaemia’ prevalence of 0.8% and 45% in the Sebei and Bambaa ethnic groups, respectively. A recent study found a SCT and SCD prevalence of 3% - 19% and 0% - 3%, respectively but this study addressed only 5 of Uganda’s 111 districts and used a small convenience sample of children aged 6 – 60 months. The objective of this study is to determine the prevalence and map out the burden of SCT and SCD in Uganda.

Methods: We propose a cross-sectional study of sickle cell trait (SCT) and disease in all districts of Uganda using dried blood spots (DBS) collected from HIV exposed babies over a 12-month period. About 90,000 DBS samples collected from HIV exposed children from all districts of Uganda from February 2014 to March 2015 will be analyzed. The study will be conducted at the Central Public Health (CPHL) in Kampala where the samples from across the country are collected for PCR testing. We will perform hemoglobin analysis using isoelectric focusing (IEF). Data analysis will be done using STATA 11.0 (College Station, TX, USA). The overall prevalence and the prevalence by district of SCT and SCD will be determined. Geospatial mapping will be performed using a specialized software program to produce a map illustrating the prevalence of SCT and SCD throughout the country, and allow associations between sickle cell trait/disease with either malaria prevalence or HIV co-morbidity. Further analysis of sickle cell disease or hemoglobin variants will be conducted using HPLC and DNA-based techniques.

111 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Utility: This study will result in detailed and up-to-date geospatial maps of the distribution of the sickle cell gene in Uganda which will be availed to the Uganda MOH for planning integrated community-based SCD care and treatment programs that are adapted to local burden. These will provide the necessary data and stimulus for the Uganda MOH to scale up development of a national strategy and SCD program.

INTRODUCTION

The United Nations1, World Health Organization (WHO)2, and African Union3 recently declared sickle cell disease (SCD) to be a major public health problem, particularly for sub-Saharan Africa. The WHO has further recognized that SCD contributes substantially to under-five childhood mortality in Africa, further noting that SCD impedes efforts toward achieving Millennium Development Goals 4 and 54. Finally, WHO challenged countries with high SCD prevalence to develop and implement a national SCD control program within the context of their national health strategic plan by the year 20204.

Background and Literature Review

Sickle cell disease, characterized by severe anemia, susceptibility to serious and life-threatening infections, sporadic painful events, and progressive organ failure affects individuals who inherit genes that code for abnormal hemoglobin from both their parents. It is estimated that over 250,000 babies are born annually in sub-Saharan Africa with sickle cell disease5, and only 10% - 50% of them survive beyond five years of age6. Presumably, most deaths result from bacterial infections, malaria, and anemia7,8. Despite causing much death and suffering, statistics that describe the magnitude of the sickle cell problem across different regions are lacking in most African countries. The available data on prevalence, morbidity and mortality were mainly derived from older studies and small numbers of hospitalized patients, and hence unreliable. Health system planners need accurate information to judiciously allocate scarce resources.

112 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Typical of autosomal recessive conditions with high genetic penetrance, the prevalence of the benign heterozygous ‘carrier’ condition - sickle cell trait (SCT) - determines incidence of the disease. According to a recent document from the World Health Organization (WHO), 10% - 40% of the population of equatorial Africa has the SCT4. This level of SCT corresponds to a homozygous disease incidence of 1% - 3% of all newborns. These general estimates, obtained from limited surveys in Africa are imprecise and cannot be generalized across entire populations. Furthermore, the well-documented variation in prevalence of SCT between and within countries also challenges the accuracy and utility of these figures. In the most often quoted study of SCT from Uganda, Lehmann and Raper found ‘sicklaemia’ prevalence of 0.8% and 45% in the Sebei and Bambaa ethnic groups, respectively9. Conversely, a recent study of Uganda found a SCT and sickle cell disease prevalence of 3% - 19% and 0% - 3%, respectively10. This latest study addressed only five of Uganda’s 111 districts and used a small convenience sample of consenting children aged 6 – 60 months. These results are limited by the relatively small sample size and restricted geographic coverage of the country.

At current under-five mortality levels, one in every seven Ugandan children does not survive to their fifth birthday11. SCD may account, therefore, for an astounding 12% - 15% of the country’s under-five mortality if 2.5% of newborns in Uganda have SCD, assuming 70% - 80% of them die before 5 years of age. Since deaths due to SCD mostly occur in children under 5 years old, efforts to save lives must include early diagnosis and treatment. In addition to early death, SCD also causes profound adverse effects among surviving children on their education and future employment with resulting loss of productivity. As death due to infections declines in Uganda, the economic and health burden of non-communicable diseases such as SCD will undoubtedly increase proportionately and consume significant medical and social resources.

Despite infrastructure limitations, small newborn screening programs in developing countries have proven to be feasible in Ghana12, Benin13, Nigeria14, and the Democratic Republic of Congo.15 These urban maternity-based programs have enrolled only a small number of

113 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study potentially affected children partly because most babies in developing countries are born at home or in the countryside. Comparable to resource-rich countries, early diagnosis followed by active management of affected children decreased mortality in Benin and Jamaica13,16. Pilot newborn screening data from the Republic of Angola have documented a high SCD incidence; and successful location and retrieval of affected babies has allowed early intervention including pneumococcal prophylaxis and insecticide-treated bed nets17.

We propose a cross-sectional study of sickle cell trait and disease in all 111 districts of Uganda using dried blood specimens collected from HIV (human immunodeficiency virus) exposed babies over a 12-month period. Uganda’s early infant diagnosis (EID) program currently collects dried blood spots (DBS) from HIV-exposed infants from 6 weeks of age for PCR (polymerase chain reaction) testing. In an effort to reduce turnaround time and thus enhance the ‘preventing mother-to-child transmission’ (PMTCT) program, the Ministry of Health (MOH) reorganized the EID program in July 2011. All DBS are now sent to the central EID laboratory at CPHL in Kampala for PCR testing via an elaborate system of transport hubs scattered throughout Uganda. Currently, the EID laboratory receives all DBS specimens collected across the country. At the EID laboratory, DNA PCR is done for HIV and the results are printed and entered onto the dispatch form. The results are sent with the dispatch form back to the dispatch point. The dispatch point ensures that the results are sent back to the EID care point (center for comprehensive EID care services in the health facility). Caregivers come to the EID care point, which is one clearly-identified place to retrieve results and seek continued care for their infant.

For this study, we will perform hemoglobin analysis in collaboration with the EID laboratory, using isoelectric focusing (IEF). Names, subject’s age (month and year of birth), district of residence and other personal identifiers will be recorded from the dispatch forms to allow the tracing of the children after the tests are done. Since the EID program receives at least 90,000 specimens yearly from all the districts, and inheritance of the sickle cell gene is not reportedly

114 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study influenced by HIV exposure, this study is expected to result in detailed, up-to-date, and accurate geospatial maps of the distribution of the sickle cell gene in Uganda. Results of the SCD test will be printed out and attached to the DNA PCR results. We shall use the existing EID system to deliver the results to the caregivers. The report from this study will be submitted to the Ministry of Health (MoH). Using the report from this study, we hope that the MoH will develop a national SCD screening program and improve the existing care and treatment programs, which will be rolled out depending on the regional burden of disease.

Problem Statement and Justification

In Uganda, approximately 25,000 children are born with sickle cell disease but 70-80% die before their second birthday18. There are no newborn screening programs in the country and children are often diagnosed after development of a crisis. Due to lack of newborn screening and lack of pre-marital genetic counseling, individuals with the SCT and SCD are intermarrying leading to an increase in the incidence of sickle cell trait and disease. However, there have not been national prevalence studies conducted to see if there is a change in the prevalence from the previous studies9,10. As stated above, most figures quoted are based on the 1949 study by Lehmann.

In Uganda, health care has been decentralized from a national referral hospital to regional referral hospitals, district hospitals, health center (HC) IVs, HC III and HC II. However, there is only one SCA clinic located in the national referral hospital in Mulago with no treatment centers in the regional hospitals. With lack of a screening program, diagnosis is usually made late and many children are lost before they are diagnosed. This study that will test samples from across the country will be able to map out the burden of sickle cell throughout the country. Such population-based geomaps will be invaluable to the Uganda MOH for planning integrated community-based SCD care and treatment programs that are adapted to local needs.

115 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Additionally, this study will improve the laboratory technical capacity and provide a foundation for a larger newborn screening program administered through the EID program. Most importantly, these data will provide the necessary data and stimulus for the Uganda MOH to scale up development of a national strategy and SCD program. Similar to HIV, newborns identified to have sickle cell disease via this screening mechanism can be tracked and referred to community clinicians for active management and life-saving interventions.

Research Question What is the burden of sickle cell anemia in Uganda? Objectives 1. To determine the prevalence of SCT and SCD in Uganda 2. To map out the burden of SCT and SCD in Uganda. 3. To obtain follow-up data on babies with SCD identified in the surveillance study 4. To explore associations between SCT and SCD with malaria and HIV 5. To characterize hemoglobin variants identified during surveillance 6. To identify genetic modifiers of SCD such as alpha-thalassemia trait or G6PD deficiency, using DNA-based technology

METHODS

Overview All DBS samples collected across Uganda as part of the HIV-exposed infant program over a 12- month period will be analyzed. These DBS samples are now being shipped to the central EID laboratory for HIV testing, and we will perform additional screening for sickle cell trait and disease using these same samples.

Study design This study will use a cross-sectional design to determine the prevalence of sickle cell trait and disease in all districts in Uganda. Study samples to be analyzed will be dried blood spots, which have been collected from HIV exposed children from 111 districts of Uganda.

116 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Inclusion criteria: All DBS samples collected from HIV exposed infants from all districts of Uganda from approximately February 2014 to March 2015 will be included.

Exclusion criteria: Repeat samples on the same individuals during the study period will be excluded.

Study setting Cincinnati Children’s Hospital Medical Center The study will be conducted in collaboration with Cincinnati Children’s Hospital Medical Center (CCHMC), one of the top pediatric hospitals in the United States. Dr. Ware (co-Investigator) has recently relocated to CCHMC, which will provide equipment and technical expertise to perform these studies. CCHMC staff will work in close collaboration with the Central Public Health Laboratories (CPHL), where the Early Infant Diagnosis (EID) Laboratory is located and all the DBS samples from the country are sent and tested.

The Central Public Health Laboratories (CPHL) The Central Public Health Laboratories is a unit under the National Disease Control Program of the Ministry of Health. The CPHL provides laboratory support for disease surveillance and feeds into the Health Management Information System (HMIS) database at the Ministry of Health resource center. It is also in-charge of coordinating health laboratory services in the country, developing policy/guidelines, training and implementation of quality assurance schemes for laboratories. The CPHL will provide space for a sickle cell screening unit where the equipment for sickle cell screening will be placed.

Department of Pediatrics and Child Health (DPCH), Makerere University and Sickle Cell Clinic, Mulago Hospital

117 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

The Department of Pediatrics and Child Health mission is “To become a centre of excellence in child health through service delivery, training and research in Uganda and beyond”. The Department is composed of four admission firms of which includes a Hematology ward which caters for children with SCA; the Acute care unit; the Malnutrition Unit and the Special Care baby Unit. In addition, there are specialized outpatient clinics which include the Sickle cell Clinic. The Sickle Cell Clinic is one of the clinics run by the DPCH and all children currently diagnosed with SCA in Uganda receive health care related services from the clinic. The clinic has been in existence for the last 30 years and it has a total of over 8,000 registered clients with SCA. It is primarily a children’s clinic but also provides services for adults with SCA. It also runs as a Day Care Centre for clients with acute sickle cell crisis.

Cincinnati Children’s Hospital: Division of Hematology and the Global Health Center The mission of Cincinnati Children’s Hospital is to improve child health and transform delivery of care through full integrated, globally recognized research, education, and innovation. Specific diseases and conditions that are associated with under-five child mortality are targeted to provide a focused effort with maximal impact, as part of the long-term vision of being the leader in improving child health. An important long term goal is to create self-sustaining facilities in resource limited settings to reduce the impact and burden of targeted childhood diseases.

Ministry of Health, Child Health Division The over-arching goal of the Ministry of Health (MOH) headquarters, Child Health division is to scale-up and sustain high, effective coverage of a priority package of cost-effective interventions to be able to reduce under-five infant and neonatal mortality rates. The core functions of the MOH headquarters are: policy analysis, formulation and dialogue; strategic planning; setting standards and quality assurance; resource mobilization; advising other ministries, departments and agencies on health-related matters; capacity development and technical support supervision; provision of nationally coordinated services including health

118 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study emergency preparedness and response and epidemic prevention and control; coordination of health research; and monitoring and evaluation of the overall health sector performance. A policy framework for monitoring and research is in place, and it recognizes the ministries contribution to coordinated priority setting of the national research agenda, mobilizing funding for research and translating research into policy. The child health division has developed a mother child health card, an individually held record, which includes information on newborn screening for sickle cell disease, among others. The Ministry will play an essential role in the provision of the samples for the study and the rolling out of newborn sickle cell screening.

Consent: We shall seek waiver of consent since Sickle cell testing is now considered a routine screening test by the MOH.

DATA COLLECTION

Early Infant Diagnosis (EID) is essential in the prevention of mother to child to transmission (PMTCT) and identifying infants and children infected with HIV so they can receive treatment early. The Dried Blood Spot (DBS) for DNA PCR is a feasible method that has enabled collection and testing of samples from across the country using a Hub transport network. When exposed infants are identified, blood is collected using a prick onto the DBS card. The DBS card is clearly labeled with the infant’s name, date of collection, age/date of birth (DOB), sex, name of facility and unique exposed number (exp number). The specimens are allowed to dry before packing. After drying, the DBS cards are packaged following appropriate protocols. The PCR DBS Dispatch Form is filled in and sent with the DBS package. Information collected on the Dispatch Form includes the name of the Health Unit, name of the district, name of the sender and their telephone number, date of collection, infant name, exp number, sex, age in months, caregiver telephone number, entry point clinic, whether it is 1st or 2nd PCR and information on whether child is breastfeeding and the antiretroviral therapy the mother or infant is taking. It also includes the site return address and any comments. The package is then sent to the testing laboratory located at CPHL.

119 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

The National Sample and Result Transport Network The Ministry of Health set up a comprehensive national transport network which involves setting up local networks centered around regional, district hospitals and health facilities, called hubs. Each hub is given a motorbike and a sample transporter who visits 20-30 health facilities within 20-40km radius around the hub, taking all referred samples from each facility and delivers results on a weekly basis. The hub then further refers highly specialized tests like DNA PCR tests. Currently there are 19 operational hubs serving about 600 health facilities and the MoH plans to have 100 hubs operating by December 2012 (Hub Map in Appendix D). The MOH organizes hub meetings every quarter. This study will work in collaboration with the EID program by attending the launches and meetings where we shall be able to introduce the sickle cell study to the EID focal people who are usually in attendance at these meetings. (Letter from the EID National coordinator in Appendix F).

Procedures

IEF procedure: Perkin Elmer instrumentation will be used for standardized isoelectric focusing tests. The methodology is according to manufacturer’s directions. It involves a small (~5 mm) punch from the DBS into a 96-well plate, followed by chemical elution of hemoglobin and electrophoresis using pre-cast gels. Newborn sickle genotype results with normal hemoglobin (HbA) and sickle hemoglobin (HbS) are typically FA (normal), FAS (trait), or FS (disease) although other variants including HbC may be detected. Calls will be made on each sample by trained technicians but also reviewed by supervisory personnel. Cincinnati Children’s Hospital Medical Center will provide the laboratory equipment and support needed for this project to the CPHL.

120 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

After completing the required HIV tests, EID staff will send the DBS samples to the sickle cell laboratory. The laboratory technician will identify the DBS samples that meet the criteria for this study. A small (~5 mm) punch will then be collected from the DBS for the purpose of hemoglobin analysis. Demographic information - sex, month of birth, date of blood collection, location (District and Health Unit) will be abstracted from the Dispatch Form and recorded for each sample. This information will be entered directly into a web-based electronic data capture system on a computer located at the CPHL. All study procedures including identification of eligible specimens, recording of demographic information, and hemoglobin testing by IEF will be conducted in the premises of the laboratory.

A separate result will be printed out and attached to the HIV results that will be sent out to the health facility via the hub transport network. The EID focal person will give the result to the parents or caregivers and will provide a flyer with information on sickle cell disease. They will also link the child found with SCD to the nearest health facility with a physician or pediatrician where they will be able to get appropriate care (linkage form attached).

Additional objectives are proposed, based on preliminary results of the US3 surveillance data: 1. Clinical follow-up of the babies with sickle cell disease, using the EID infrastructure. The health facilities with positive samples will be contacted, to learn whether or not the families have been notified, and if the babies have received intervention such as pneumococcal immunization. 2. Correlations between sickle cell trait/disease and malaria will be explored, as well as the comorbidity of HIV with sickle cell trait/disease. Existing data regarding the geospatial distribution of malaria and HIV across the country will be compared to the current data about the prevalence and distribution of sickle cell trait/disease across Uganda. Micromapping efforts will determine how closely the prevalence of malaria is associated with that of sickle cell trait.

121 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

3. About 1.0% of the specimens reveal hemoglobin variants that require further characterization. This will be done by HPLC analysis and followed by specific DNA testing, in collaboration with Cincinnati Children’s Hospital. The identity of the variant hemoglobins will be determined by HPLC electrophoresis whenever possible (e.g., Hb Stanleyville), but it is likely that the alpha and beta globin genes will need to be sequenced at the DNA level to identify the genetic variants. Cincinnati Children’s Hospital will determine the best methods of identifying these variants and then provide the equipment, reagents, and technical expertise to perform this analysis at CPHL. 4. The samples with sickle cell disease will be further characterized using DNA-based technology to determine the importance of known genetic modifiers such as alpha thalassemia trait, G6PD deficiency, and others. This work will also be done in collaboration with Cincinnati Children’s Hospital, using their previously published methods of DNA extraction followed by PCR-based identification of specific genetic variants.19 As before, the Cincinnati team will first determine the best methods and then bring this technology to CPHL for local performance.

Sample size We plan to test approximately 90,000 DBS samples i.e. the estimated number of specimens processed yearly from all the districts. The sample size is an estimate since this study involves testing of available DBS collected between approximately February 2014 to March 2015. For the additional research objectives listed above, the sample size will be determined by the surveillance results. For example, correlations with sickle cell trait and malaria will depend on the number of samples that test positive for sickle cell trait, which is approximately 10% of all tested specimens. For laboratory analysis of hemoglobin variants, the number of samples is about 1.0% so we estimate that 500-1000 samples will be analyzed. Finally, for analysis of genetic modifiers of sickle cell disease, the number of samples is again close to 1.0% so determined by the prevalence data but approximately 500-1000 samples total.

122 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Data Management and analysis A web-based electronic data capture system will be designed on a computer located at the CPHL. This database will allow internet-based data entry and storage. Cincinnati Children’s Hospital will work with CPHL to provide the database support for this project. The data manager at the sickle cell clinic will work with the data officers to enable capacity building. Projected data fields will include patient sex, month of birth, date of collection, date of testing, location (District, and Health Unit), and sickle genotype result.

Information collected will be entered directly into this system. The system will be designed with appropriate controls and validation checks. The computer and the database will be password protected and daily backup of the data will be done. The study coordinator will be responsible for checking the completeness of the data daily.

Data analysis: Analysis will be done using STATA 11.0 (College Station, TX, USA).

Statistical analyses: Descriptive analysis of the data will be done to describe the study population by determining frequencies. The overall prevalence of SCT and SCD will be determined by calculating the proportions of those with SCT and SCD respectively. The prevalence of SCT and SCD will then be determined by district. Comparative analysis between groups will be investigated using the chi-square test. Standard reports of prevalence by district and results in time intervals (e.g. <3 months and >12 months) will be calculated. Additional geospatial mapping will be performed using a specialized software program. The end result will be a detailed map of Uganda with each region and district, illustrating representation of the prevalence of SCT and SCD throughout the country.

POTENTIAL BENEFITS

123 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Parents and caregivers will be able to find out at an early age whether their children have sickle cell disease or trait and therefore will be able to seek appropriate care for their children. The benefit to society will be a comprehensive prevalence study of sickle cell trait and disease using samples collected from all 111 districts of Uganda. This study will provide valuable information about SCD in Uganda; in particular, the study will provide information on the sickle cell disease burden in the different regions, which should inform treatment plans and policy decisions in the future.

POTENTIAL RISKS

We do not anticipate any risk to individual subjects who contribute DBS samples for this study.

INSTITUTIONAL REVIEW BOARDS (IRB) APPROVAL

This proposal was originally approved by the School on Medicine Research and Ethics Committee (SOMREC) Makerere College of Health Sciences on 14th August 2012, with annual renewals in 2013 and 2014. We here seek approval of the amendments herein; to add additional study objectives based on the preliminary results. We intend to seek similar approval from the Uganda National Council of Science and Technology (UNCST). The amended protocol will also be submitted to the IRB for Cincinnati Children’s Hospital.

STRENGTHS

One of the strengths of this study is that the DBS samples are all available in one central place at the CPHL where the laboratory is going to be located.

There is also a large sample size available for this study since samples are collected from all over the country, which provides a representative sample and therefore the study will be able to provide region-specific information on the burden of SCT and SCA.

124 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

The location of the sickle cell lab at CPHL, a structure of the MOH, will allow easy roll-out into a national screening program at the completion of the prevalence study.

LIMITATIONS

All the DBS samples to be used for the study are samples collected from HIV exposed infants and children. However, there is no evidence that HIV-exposed children are any different from the non-exposed children in as far as SCT and SCD are concerned, therefore the sample will still be representative.

125 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

REFERENCES

1. UN General Assembly. Recognition of sickle-cell anaemia as a public health problem, 2008 (A/RES/63/237).

2. WHO. Sickle-cell anemia. Report by the Secretariat A59/6. World Health Organization. Fifty- ninth world health assembly. 2006.

3. African Union. Documents Assembly/AU/Dec. 73–90 (V), Assembly/AU/Decl. 1–3 (V) and Assembly/AU/Resolution 1 (V), 2005.

4. WHO. Sickle-cell disease: a strategy for the WHO African Region. AFR/RC60/8. World Health Organization. Regional Office for Africa, 2010.

5. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: an increasing global health problem. Bull. World Health Organ. 2001;79(8):704-712.

6. Weatherall D, Akinyanju O, Fucharoen S, Olivieri N, Musgrove P. Inherited Disorders of Hemoglobin. In: Jamison D, Breman J, Measham A, et al, eds. Disease Control Priorities in Developing Countries. Second Edition. New York: The World Bank and Oxford University Press; 2006:663 – 80.

7. McAuley CF, Webb C, Makani J, et al. High mortality from Plasmodium falciparum malaria in children living with sickle cell anemia on the coast of Kenya. Blood. 2010;116(10):1663 -1668.

8. Van-Dunem JCC, Alves JGB, Bernardino L, et al. Factors associated with sickle cell disease mortality among hospitalized Angolan children and adolescents. West Afr J Med. 2007;26(4):269-273.

9. Lehmann H, Raper AB. Distribution of the sickle-cell trait in Uganda, and its ethnological significance. Nature. 1949;164(4168):494.

126 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

10. Okwi AL, Byarugaba W, Ndugwa CM, et al. An up-date on the prevalence of sickle cell trait in Eastern and Western Uganda. BMC Blood Disord. 2010;10:5.

11. Uganda Demographic and Health Survey 2006. Uganda Bureau of Statistics, Kampala Uganda. Macro International Inc. Calverton, Maryland, USA; 2007.

12. Ohene-Frempong K, Oduro J, Tetteh H, Nkrumah F. Screening newborns for sickle cell disease in Ghana. Pediatrics. 2008;121:S120-S121.

13. Rahimy MC, Gangbo A, Ahouignan G, Alihonou E. Newborn screening for sickle cell disease in the Republic of Benin. J. Clin. Pathol. 2009; 62(1):46-48.

14. Odunvbun ME, Okolo AA, Rahimy CM. Newborn screening for sickle cell disease in a Nigerian hospital. Public Health. 2008; 122(10):1111-1116.

15. Tshilolo L, Aissi LM, Lukusa D, et al. Neonatal screening for sickle cell anaemia in the Democratic Republic of the Congo: experience from a pioneer project on 31 204 newborns. J. Clin. Pathol. 2009;62(1):35-38.

16. Lee A, Thomas P, Cupidore L, Serjeant B, Serjeant G. Improved survival in homozygous sickle cell disease: lessons from a cohort study. BMJ. 1995; 311(7020):1600-1602.

17. McGann P, Macosso P, de Oliveira V, et al. Operational successes and challenges of a pilot newborn screening program for sickle cell anemia in the Republic of Angola. Abstract for CDC meeting. 2012.

18. Serjeant GR, Ndugwa CM: Sickle cell disease in Uganda: A time for action. East African Medical Journal 2003, 80:383-87.

19. Sheehan VA, Luo Z, Flanagan JM, Howard TA, Thompson BW, Wang WC, Kutlar A, and Ware RE for the BABY HUG Investigators: Genetic modifiers of sickle cell anemia in the BABY HUG cohort: influence on laboratory and clinical phenotypes. Am J Hematol 2013, 88(7):571-76.

127 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

APPENDIX

Appendix A - PCR Dried Blood Spot Dispatch Form

128 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix B – Referral Form

129 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix C – Chart Abstraction Form

PREVALENCE AND MAPPING OF SICKLE CELL TRAIT AND DISEASE IN UGANDA

CHART ABSTRACTION FORM

Date Completed: ______/______/______(dd/mm/yyyy)

Person completing form: ______

1. Batch No. ______

2. Health Unit: ______

3. District: ______

4. Date of collection: ______/______/______(dd/mm/yyyy)

5. Date sample dispatched: ______/______/______(dd/mm/yyyy)

6. Date sample received: ______/______/______(dd/mm/yyyy)

7. Gender (M/F): ______

8. Age (in months): ______

9. Date sample processed: ______/______/______(dd/mm/yyyy)

10. Test Result

FA □ FAS □ SS □

130 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix D – Map of Uganda showing Hubs and their regional coordination

131 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix E – Uganda Ministry of Health Approval

132 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix F – Letter from EID National Coordinator

133 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix G: School of Medicine Research Ethics Committee at Makerere University

134 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

135 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

136 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

Appendix H: Cincinnati Children’s Hospital Institutional Review Board

137 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

138 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

139 Effective strategies used to describe and address the burden of sickle cell disease in the

Republic of Uganda: The Uganda Sickle Surveillance Study

140