Optimal Chemopreventive Regimens to Prevent Malaria and Improve Birth Outcomes in Uganda
Phase: PHASE3
Conditions: Malaria
Sponsor: Grant Dorsey, M.D, Ph.D.

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
 1 
 
 
 
 
Title: Optimal chemopreventive regimens to prevent malaria and improve birth 
outcomes in Uganda 
 
Short title: Optimizing IPTp in Uganda 
 
A UCSF/ IDRC COLLABORATION 
 
DMID Protocol Number: 
 
19-0014 
 
DMID Funding Mechanism:  
U01 AI141308-01A1 
 
Principal Investigators: 
 
Grant Dorsey, MD, PhD 
 
 
 
 
 
University California San Francisco 
 
 
 
 
 
 
Philip Rosenthal, MD 
 
 
 
 
 
University California San Francico 
 
 
 
 
 
 
Moses Kamya, MBChB, MMed, PhD 
 
 
 
 
 
Makerere University College of Health Sciences 
 
DMID Medical Officer: 
 
Dr. Greg Deye 
 
DMID Medical Monitor: 
 
Dr. Mohamed Elsafy 
 
DMID Clinical Project Manager: 
Walt Jones 
 
 
Protocol Version 8.0 
 
September 11, 2023 
 
 
 
 
* Ultrasound provided by Foundation for Society of Maternal Fetal Medicine 
 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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STATEMENT OF COMPLIANCE 
 
The study will be carried out in accordance with Good Clinical Practice (GCP) as required by the 
following: 
 
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
• ICH E6; 62 Federal Register 25691 (1997) 
• Ugandan National Council for Science and Technology 
• Completion of Human Subjects Protection Training 
• NIH Clinical Terms of Award 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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SIGNATURE PAGE 
 
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this study will be conducted according to all stipulations 
of the protocol, including all statements regarding confidentiality, and according to local legal 
and regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
Site Principal Investigator:  Professor Moses Kamya, MBChB, MPH, PhD 
 
Signed:  ______________________________________________ 
 
Name Prof Moses Kamya 
 
Title 
Professor and Dean, School of Medicine, Makerere University College of Health Sciences 
Executive Director, Infectious Diseases Research Collaboration (IDRC) 
 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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TABLE OF CONTENTS 
 
STATEMENT OF COMPLIANCE ........................................................................................................ 2 
SIGNATURE PAGE ........................................................................................................................... 3 
PROTOCOL TEAM ROSTER .............................................................................................................. 8 
LIST OF ABBREVIATIONS .............................................................................................................. 10 
CLINICAL PROTOCOL SYNOPSIS ................................................................................................... 11 
1. 
INTRODUCTION ................................................................................................................ 12 
1.1. 
Background and preliminary studies ..................................................................................... 12 
1.2. 
Rationale ............................................................................................................................. 18 
2. 
STUDY OBJECTIVES ........................................................................................................... 20 
2.1. 
Objective 1 .......................................................................................................................... 20 
2.2. 
Objective 2 .......................................................................................................................... 20 
2.3. 
Objective 3 .......................................................................................................................... 20 
3. 
STUDY DESIGN .................................................................................................................. 20 
4. 
SELECTION AND ENROLLMENT OF SUBJECTS .................................................................. 21 
4.1. 
Inclusion Criteria .................................................................................................................. 21 
4.2. 
Exclusion Criteria ................................................................................................................. 21 
4.3. 
Initial Screening ................................................................................................................... 22 
4.4. 
Study Enrollment Procedures and Baseline Evaluation .......................................................... 22 
5. 
STUDY TREATMENT .......................................................................................................... 23 
5.1. 
Treatment Group Assignments ............................................................................................. 23 
5.2. 
Treatment Allocation ........................................................................................................... 24 
5.3. 
Study Drug Dosing and Formulations .................................................................................... 24 
5.4. 
Blinding, Study Drug Administration, and Duration ............................................................... 25 
5.5. 
Study Drug Accountability .................................................................................................... 25 
6. 
SUBJECT MANAGEMENT .................................................................................................. 26 
6.1. 
Subject Follow-up ................................................................................................................ 26 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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6.2. 
Diagnosis and Management of Malaria ................................................................................. 26 
6.3. 
Management of Non-Malaria Illnesses ................................................................................. 27 
6.4. 
After Hours Visits ................................................................................................................. 28 
6.5. 
Routine Assessments ........................................................................................................... 28 
6.6. 
Delivery visit ........................................................................................................................ 30 
6.7. 
Postpartum visits. ................................................................................................................ 30 
6.8. 
Medical Care Outside the Study Clinic................................................................................... 30 
6.9. 
Duration of Follow-up and Criteria for Premature Study Withdrawal ..................................... 31 
6.10. 
Diagnostic and Laboratory Testing ........................................................................................ 31 
6.10.1. 
Microscopy ....................................................................................................... 31 
6.10.2. 
Clinical Laboratory Studies .............................................................................. 32 
6.10.3. 
Placental Studies .............................................................................................. 32 
6.10.4. 
Dried blood spots collected on filter paper .................................................... 33 
6.10.5. 
Quantitative PCR .............................................................................................. 33 
6.10.6  
Ex vivo drug sensitivity of malaria parasites ................................................... 34 
6.10.7   
Microbiome studies ......................................................................................... 34 
6.10.8  
Reproductive tract infections (RTIs) ................................................................ 37 
6.10.9  
 Drug sensitivity of urinary tract infection (UTI) pathogens and Neisseria 
gonorrhea ............................................................................................................ 38 
6.10.10  
Molecular markers of SP and DP resistance ................................................... 39 
6.10.11  
Pharmacokinetic assessments. ........................................................................ 40 
6.10.12  
Immunology Studies ........................................................................................ 41 
6.10.13  
Host red blood cell polymorphisms. ............................................................... 42 
6.10.14  
SARS-CoV-2 Testing. ......................................................................................... 43 
6.10.15 
Fetal Ultrasounds during and after febrile illness .......................................... 45 
6.10.16 
Sample storage for future use ......................................................................... 47 
6.11. 
Co-enrollment Guidelines ..................................................................................................... 48 
6.12. 
Management of Adverse Events Potentially Related to Study Drugs ...................................... 48 
6.12.1. 
Grade 1 or 2 Adverse Events ........................................................................... 48 
6.12.2. 
Grade 3 or 4 Adverse Events ........................................................................... 48 
7. 
MONITORING OF ADVERSE EVENTS AND MANAGEMENT ............................................. 49 
7.1 
Monitoring and Reporting of Adverse Events ........................................................................ 49 
7.1.1 
Definitions ........................................................................................................ 49 
7.1.2 
Identification of Adverse Events ..................................................................... 49 
7.1.3 
Reporting of Adverse Events ........................................................................... 50 
8. 
STATISTICAL CONSIDERATIONS ....................................................................................... 51 
8.1 
Hypothesis 1 ........................................................................................................................ 52 
8.1.1. Primary Outcome ............................................................................................................... 52 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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8.1.2 Analysis 
 .......................................................................................................................... 52 
8.2 
Hypothesis 2 ........................................................................................................................ 52 
8.2.1. Primary Outcome ............................................................................................................... 52 
8.2.2. Secondary Outcomes ......................................................................................................... 53 
8.2.3. Analysis 
 .......................................................................................................................... 53 
8.3 
Hypothesis 3 ........................................................................................................................ 53 
8.3.1. 
Outcomes for objective 3a .............................................................................. 53 
8.3.2. 
Outcomes for objective 3b .............................................................................. 54 
8.3.3. 
Analyses ........................................................................................................... 54 
8.4 
Sample size and power ......................................................................................................... 54 
8.5 
Data and Safety Monitoring Plan .......................................................................................... 55 
8.6 
Data and Safety Monitoring Board ....................................................................................... 55 
8.7 
Interim safety analysis ......................................................................................................... 57 
8.8 
Stopping rules ...................................................................................................................... 58 
9 
DATA COLLECTION AND MONITORING ........................................................................... 58 
9.1 
Record Keeping .................................................................................................................... 58 
9.2 
Data Quality Assurance and Monitoring ............................................................................... 58 
10. 
HUMAN SUBJECTS ............................................................................................................ 58 
10.1 
Subject Selection Criteria ..................................................................................................... 59 
10.2 
Risks and Discomforts .......................................................................................................... 59 
10.2.1 
Privacy ............................................................................................................. 59 
10.2.2 
Finger Pricks and Venipuncture ....................................................................... 59 
10.2.3 
Risks of Study Medications .............................................................................. 59 
10.3 
Treatment and Compensation for Injury ............................................................................... 66 
10.4 
Costs to the Subjects ............................................................................................................ 66 
10.5 
Reimbursement of Subjects .................................................................................................. 66 
10.6 
IRB Review and Informed Consent ........................................................................................ 66 
10.7 
Publication of Research Findings .......................................................................................... 67 
10.8 
Biohazard Containment ........................................................................................................ 67 
11. 
REFERENCES ................................................................................................................ 67 
12.  
APPENDICES ...................................................................................................................... 77 
Appendix A. Information Sheet ........................................................................................................ 77 
Appendix B.  Determination of Gestational Age ................................................................................ 78 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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Appendix C. Household survey ......................................................................................................... 79 
Appendix D. Administration of study drugs and placebos .................................................................. 83 
Appendix E. WHO Criteria for Severe Malaria ................................................................................... 84 
Appendix F. Uganda Ministry of Health Guidelines for Routine Care of Pregnant and Postpartum 
Women, and Newborns ....................................................................................................... 85 
Appendix G. Schedule of routine assessments and procedures in pregnant women ........................... 86 
Appendix H.    Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse 
Events (Corrected Version 2.1 - July 2017) ............................................................................ 88 
Appendix I. Pharmacokinetic Sub-Study .......................................................................................... 116 
 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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PROTOCOL TEAM ROSTER   
 
Investigators   
 
 
 
 
Grant Dorsey, MD, PhD, University of California, San Francisco 
 
Address:  San Francisco General Hospital, 995 Potrero Avenue, 
 
Building 30, Room 3420, UCSF Box 0811, San Francisco CA, 94110, U.S.A. 
Phone Number: +1-415-206-4680 
Fax Number:   +1-415-648-8425 
 
Email: grant.dorsey@ucsf.edu  
 
Philip Rosenthal, MD, University of California, San Francisco 
 
Address:  San Francisco General Hospital, 995 Potrero Avenue, 
 
Building 30, Room 3421, UCSF Box 0811, San Francisco CA, 94110, U.S.A. 
Phone Number: +1-415-206-8845 
Fax Number:   +1-415-648-8425 
 
Email: Philip.Rosenthal@ucsf.edu  
 
Stephanie Gaw, MD, PhD, University of California, San Francisco 
 
Address:  513 Parnassus Ave, 16HSE, San Francisco CA 94143, U.S.A. 
Phone Number: +1- 415-476-0535 
 
Email: Stephanie.Gaw@ucsf.edu 
 
Moses R. Kamya, MBChB, MMed, PhD, Makerere University College of Health Sciences 
 
Address:  Department of Medicine, P.O. Box 7072, Kampala, Uganda   
 
Phone Number:  +256-414-533200 
 
Fax Number:      +256-414-540524 
 
Email:  mkamya@infocom.co.ug 
 
Miriam Nakalembe, MBChB, MMed Ob/Gyn, Makerere University College of Health Sciences 
 
Address: Department of Medicine,  
P.O. Box 7072, Kampala, Uganda   
 
Phone Number: +256 753 857 433 (Mobile), +256 414 534 361 (Office) 
Fax +256 414 533 451 (AOGU Office)  
Email: ivuds@yahoo.com 
 
 
Abel Kakuru, MBChB, MSc, Infectious Diseases Research Collaboration 
Address: Infectious Diseases Research Collaboration,                                            
       
P.O. Box 7475, Kampala, Uganda 
 
 
 
 
 
      
Phone Number:  +256-414-530692  
 
 
                                
Fax Number:      +256-414-540524 
 
 
 
 
 
   
Email:  abelkakuru@gmail.com  
 
 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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Prasanna Jagannathan, MD, Stanford University 
Address: 300 Pasteur Drive, Lane Building, Suite 134, L133,  
Stanford, California  94305, U.S.A.      
Phone Number:  +1-650-724-5343 
Fax Number:      +1-650-498-9876 
 
 
 
                               
 
   
Email:  prasj@stanford.edu  
 
Francesca Aweeka, PharmD, University of California, San Francisco 
 
Address: 1001 Potrero Ave, SFGH 100, Rm 158, San Francisco, CA 94110, U.S.A. 
 
Phone Number: +1 415 476-0339 
 
Fax Number: +1 415 476-0307 
 
Email: fran.aweeka@ucsf.edu 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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LIST OF ABBREVIATIONS 
 
AE 
 
Adverse event 
ACT 
 
Artemisinin-based combination therapy 
AL 
 
Artemether-lumefantrine 
BV 
 
Bacterial vaginosis 
CAB 
 
Community advisory board 
CBC 
 
Complete blood cell count 
CHR 
 
Committee on Human Research 
CRF 
 
Case report form 
DP 
 
Dihydroartemisinin-piperaquine 
DAIDS  
National Institutes of Health Division of AIDS 
DMID  
National Institutes of Health Division of Microbiology and Infectious Diseases 
DSMB  
Data and Safety Monitoring Board 
ECG 
 
Electrocardiogram 
FDA 
 
Food and Drug Administration 
GBS 
 
Group B Streptococcus 
ICH 
 
International Conference on Harmonisation 
IDRC 
 
Infectious Diseases Research Collaboration 
IPT 
 
Intermittent preventive treatment 
IPTp 
 
Intermittent preventive treatment in pregnancy 
IRB 
 
Institutional review board 
IST 
 
Intermittent screening and treatment 
LLIN 
 
Long lasting insecticide treated net 
LBW 
 
Low birth weight 
LMP 
 
Last menstrual period 
MOH  
Ministry of Health 
MU 
 
Makerere University 
MU-SBSREC 
MU School of Biomedical Sciences Research and Ethics Committee 
NDA 
 
National Drug Authority 
NIH 
 
National Institute of Health 
qPCR  
Quantitative polymerase chain reaction 
RCT 
 
Randomized controlled trial 
RTIs 
 
Reproductive tract infections 
SAE 
 
Serious adverse event 
SNP 
 
Single nucleotide polymorphisms 
SGA 
 
Small-for-gestational age 
SP 
 
Sulfadoxine-pyrimethamine 
TMP-SMX 
Trimethoprim-sulfamethoxazole 
UCSF  
University California San Francisco 
UNCST  
Uganda National Council of Science and Technology 
UTI 
 
Urinary Tract Infection 
WHO  
World Health Organization 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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Clinical Protocol Synopsis   
Title 
Optimal chemopreventive regimens to prevent malaria and improve birth outcomes in Uganda 
Description 
Double blinded randomized controlled trial 
Study Objectives 
1) To compare the risk of adverse birth outcomes among pregnant women randomized to receive monthly IPTp with SP vs. DP 
vs. SP+DP.  
2) To compare safety and tolerability of IPTp regimens among pregnant women randomized to receive monthly IPTp with SP 
vs. DP vs. SP+DP.  
3) To compare risks of malaria-specific and non-malarial outcomes among pregnant women randomized to receive monthly 
IPTp with SP vs. DP vs. SP+DP. 
Participants and 
Sample Size 
2757 HIV-uninfected pregnant women  
Clinical Site 
The study will be conducted in Busia District, Uganda. A designated study clinic located within Masafu General Hospital will be 
open daily from 8:00 am to 5:00 pm and after-hours care will be available within the hospital complex.  
Selection Criteria 
Inclusion Criteria 
1. 
Viable singleton pregnancy confirmed by ultrasound 
2. 
Estimated gestational age between 12-20 weeks  
3. 
Confirmed to be HIV- uninfected by rapid test 
4. 
16 years of age or older 
5. 
Residency within Busia District of Uganda 
6. 
Provision of informed consent  
7. 
Agreement to come to the study clinic for any febrile episode or other illness and avoid medications given outside 
the study protocol 
8. 
Willing to deliver in the hospital 
Exclusion Criteria: 
1. 
History of serious adverse event to SP or DP 
2. 
Active medical problem requiring inpatient evaluation at the time of screening 
3. 
Intention of moving outside of Busia District Uganda 
4. 
Chronic medical condition requiring frequent medical attention 
5. 
Prior chemopreventive therapy or any other antimalarial therapy during this pregnancy 
6. 
Early or active labor (documented by cervical change with uterine contractions) 
7. 
Multiple pregnancies (i.e. twins/triplets) 
8. 
History of long QT syndrome 
Treatment 
assignment 
HIV-uninfected pregnant women will be randomized at the time of enrollment 
Treatment arms 
1. 
SP given every 4 weeks 
2. 
DP given every 4 weeks 
3. 
SP+DP given every 4 weeks 
Follow-up  
Study participants will be followed for all of their outpatient medical care in our study clinic. Routine assessments will be 
performed in the study clinic for all study participants every 4 weeks. Participants will be followed until 28 days postpartum. 
Primary study 
outcome 
The risk of having a composite adverse birth outcome defined as the occurrence of any of the following: 
• 
Spontaneous abortion (fetal loss at < 28 weeks gestational age) 
• 
Stillbirth (infant born deceased at > 28 weeks gestational age) 
• 
Low birth weight (< 2500 gm) 
• 
Preterm birth (< 37 weeks gestational age) 
• 
Small-for-gestational age (< 10th percentile relative to an external growth reference) 
• 
Neonatal death (death of infant within the first 28 days) 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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1. INTRODUCTION 
 
1.1. 
Background and preliminary studies 
 
Malaria in pregnancy: burden of disease and prevention. Malaria in pregnancy remains a 
major challenge in sub-Saharan Africa, where P. falciparum is the predominant species and 
transmission remains moderate to high in many areas.  In this setting a high proportion of 
infected women are asymptomatic, with low-level parasitemia often missed by microscopy or 
rapid diagnostic tests.1 These infections may lead to placental sequestration resulting in LBW 
due to preterm delivery and/or intrauterine growth restriction.2 Other complications of malaria 
in pregnancy include maternal anemia, spontaneous abortions, and stillbirths, which combined 
with LBW are estimated to cause over 100,000 fetal and neonatal deaths each year.3 
      
The only widely available tools for the prevention of malaria in pregnancy are LLINs and 
IPTp-SP. Resistance to the pyrethroid class of insecticides used in LLINs has become widespread 
throughout Africa, potentially compromising their efficacy and increasing reliance on 
antimalarial drugs for the prevention of malaria during pregnancy.4-6 SP was the first drug 
endorsed for IPTp, and it remains the only drug recommended by the WHO. In a Cochrane 
review of IPTp-SP vs. placebo from 6 randomized controlled trials conducted in Africa primarily 
in the 1990s before widespread SP resistance, IPTp-SP was associated with a 62% reduction in 
the risk of maternal parasitemia, 55% reduction in the risk of placental parasitemia, and 19% 
reduction in the risk of LBW.7 The meta-analysis of these trials was underpowered to evaluate 
the effects of IPTp-SP on preterm delivery and fetal/neonatal deaths, as these outcomes were 
not always measured and/or relatively uncommon. In a separate retrospective analysis of cross-
sectional datasets from 25 African countries from 2000-10, a combination of LLINs and IPTp-SP 
was associated with a 21% reduction in the risk of LBW and 19% reduction in the risk of 
neonatal mortality.8 In the early 2000s the WHO began recommending IPTp with at least 2 
doses of SP beginning in the second trimester, and subsequently placebo controlled trials of 
IPTp have been deemed unethical. However, given the emergence of SP resistance in Africa and 
the potential for shortening the period of post-treatment prophylaxis, a number of studies have 
looked at the impact of more frequent dosing of SP for IPTp. In a meta-analysis of 7 studies 
from Africa, 3 or more doses of SP was associated with a 49% reduction in the risk of placental 
malaria and a 20% reduction in the risk of LBW compared to 2 doses of SP.9 Interestingly, these 
differences were consistent across a wide range of prevalence of SP resistance markers. In 2012 
the WHO updated their recommendations, increasing the frequency of dosing of IPTp with SP 
to up to once a month, beginning as early in the 2nd trimester as possible.  
      
Despite the strong historical evidence for the benefits of IPTp-SP, there is concern that 
increasing spread of SP resistance will compromise its effectiveness. SP was abandoned as a 
recommended treatment for malaria in Africa in the early-2000’s and it has had limited or no 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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protective efficacy when used for chemoprevention in non-pregnant populations. In a 
randomized controlled trial from our group in Uganda, monthly SP provided no significant 
protection against malaria among children 6-24 months of age.10 In many parts of Africa 
(especially Eastern and Southern Africa), more than 90% of parasites harbor five dhfr/dhps 
mutations that have been associated with SP failure to clear existing infections and prevent 
new infections in pregnant women.11,12 Indeed, the presence of a sixth mutation (dhps 581G), 
which confers high-level SP resistance, is emerging in East Africa; this mutation will likely 
further compromise the efficacy of IPTp-SP.13-15 The spread of SP resistance has prompted the 
evaluation of alternative regimens for IPTp. In a randomized controlled trial conducted in 
Ghana, IPTp with amodiaquine alone or in combination with SP was poorly tolerated and did 
not improve outcomes compared to SP alone.16 In a multicenter randomized controlled trial, 
mefloquine provided in two dosing strategies lowered the risk of malaria during pregnancy 
compared to SP but did not improve birth outcomes and was poorly tolerated.17 In a 
multicenter randomized controlled trial, a combination of chloroquine and azithromycin also 
lowered the risk of malaria during pregnancy compared to SP but did not improve birth 
outcomes and was poorly tolerated due to vomiting.18 In a randomized controlled trial from 
Malawi, a combination of monthly SP plus two doses of azithromycin was not associated with 
significant improvement in the risks of LBW, preterm delivery, or placental malaria compared to 
monthly SP alone.19,20 In summary, results of studies evaluating alternatives to IPTp-SP using 
older antimalarial drugs have been disappointing and have not led to changes in policy 
recommendations.   
 
ACTs for the treatment and prevention of malaria in pregnancy. The ACT class of drugs offers 
an attractive alternative to SP for use in pregnancy. In a systematic review of parasitological 
efficacy for the treatment and prevention of falciparum malaria in pregnancy, placenta-positive 
rates were unacceptably high in a majority of SP trial arms and ACTs provided the lowest 
parasitological failure rates.21 The authors recommended that SP should no longer be used for 
treatment or prevention of malaria in pregnancy and that ACTs provide the most efficacious 
and safe alternative therapy. Several randomized controlled trials have shown that ACTs are 
safe and effective for the treatment of malaria during pregnancy22-25 and the introduction of DP 
as first line-treatment in Papua, Indonesia was temporally associated with a significant decrease 
in the risk of severe maternal anemia and LBW.26 Although the WHO has now endorsed ACTs as 
first-line therapy for malaria in the 2nd and 3rd trimesters of pregnancy, this is likely to have 
limited impact in endemic areas of Africa, where most infections during pregnancy are 
asymptomatic. This has stimulated interest in expanding the role of ACTs to IPTp. The most 
attractive option for IPTp is DP, given its excellent safety/efficacy profile and long post-
treatment prophylactic effect.  

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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IPTp with DP vs. SP. There have been 3 published RCTs evaluating DP for IPTp, one in Kenya 
and 2 from our group in Uganda (Table 1). The Kenyan study evaluated IPTp with SP or DP given 
at the time of each antenatal visit (median of 3 doses) and intermittent screening and 
treatment with DP, in which women were screened for parasitemia with a rapid diagnostic test 
(RDT) at each antenatal visit, and only given DP if parasitemia was detected.27 In Uganda we 
compared IPTp with SP given every 8 weeks with DP given every 8 or 4 weeks28 and then 
compared IPTp with SP or DP given every 4 weeks.29 In all three studies, DP was associated with 
significant reductions in the incidence of malaria, parasite prevalence, and parasitemia at 
delivery compared to SP (Table 1). Indeed, in our latest study from Busia District (the site of our 
proposed trial), IPTp with DP was associated with > 94% protective efficacy against these 
malaria specific outcomes. However, despite marked reductions in the burden of malaria during 
pregnancy, none of these studies demonstrated significant differences in the risks of adverse 
birth outcomes; indeed, mean birth weights were highest in the SP arms in all three studies 
(Table 1). Other key findings included: 1) Intermittent screening and treatment was ineffective 
in Kenya, as seen in studies of intermittent screening and treatment with DP in Malawi30 and AL 
in West Africa;31 the WHO recently concluded that intermittent screening and treatment should 
not be recommended.32 2) In Uganda, DP every 4 weeks was superior to DP every 8 weeks for 
several malaria specific outcomes. 3) In all 3 studies IPTp with DP was safe and as well tolerated 
as that with SP. Based on these data, the WHO Malaria Policy Advisory Committee recently 
concluded that DP is promising for IPTp, but that SP is still recommended in the absence of 
clear data showing improvements in birth outcomes with DP.32 
 
Table 1. Results of randomized controlled trials comparing IPTp with DP vs SP 
Location  
Drug 
Frequency of 
doses 
Enrolled 
Malaria 
incidencea 
Parasite 
prevalenceb 
Parasitemia  
at deliveryc 
LBWd 
Preterm 
birthd 
SGAd 
Mean birth 
weight (gm) 
Western 
Kenya27 
SP 
each visit 
515 
0.38 
19.1% 
10.2% 
4.4% 
4.7% 
5.9% 
3276 
DP 
each visit 
516 
0.06 
7.1% 
3.3% 
5.3% 
5.1% 
6.5% 
3188 
DP 
IST 
515 
0.53 
15.9% 
12.6% 
4.6% 
6.4% 
8.7% 
3234 
Tororo, 
Uganda28 
SP 
every 8 weeks 
106 
0.95 
40.5% 
19.4% 
13.3% 
8.2% 
20.4% 
2950 
DP 
every 8 weeks 
94 
0.31 
16.6% 
4.6% 
14.8% 
11.4% 
18.2% 
2904 
DP 
every 4 weeks 
100 
0 
5.2% 
2.1% 
7.2% 
4.1% 
18.6% 
2939 
Busia, 
Uganda29 
SP 
every 4 weeks 
391 
0.53 
30.8% 
12.5% 
8.2% 
7.3% 
13.2% 
3038 
DP 
every 4 weeks 
391 
0.03 
0.5% 
0.3% 
7.1% 
4.8% 
16.7% 
3018 
a Episodes of symptomatic malaria per person; b Detection of malaria parasites by microscopy (Kenya and Busia, Uganda) or LAMP 
(Tororo, Uganda); c Malaria parasites detected in maternal blood, placental blood, or placental tissue at the time of delivery; d Among 
live births 
     In summary, a clear pattern has emerged, with IPTp-DP being far superior to IPTp-SP in 
reducing the burden of malaria during pregnancy, but showing no clear benefit in improving 
birth outcomes. One logical explanation of this paradox, and the central hypothesis of this 
proposal, is that IPTp-SP improves birth outcomes independent of its limited antimalarial 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
 15 
 
activity, offering the potential for benefit in addition to that afforded by IPTp-DP when 
administered to pregnant women. 
 
Evidence for IPTp with SP improving birth outcomes independent of its antimalarial 
properties.  
 
IPTp-SP improves birth outcomes due to non-antimalarial effects. We conducted a meta-
analysis of individual level data comparing IPTp with DP vs SP, including only dosing regimens 
that were directly comparable for each study 
described above. Causal mediation analysis 
was used to distinguish antimalarial and non-
antimalarial effects based on placental malaria 
outcomes (Figure 1). In all 3 studies, SP was 
associated with a relative increase in birth 
weight due to non-antimalarial effects after 
adjusting for other mediators in the causal 
pathway (overall gain of 84 grams, 95% CI 41-
128), and DP was associated with a relative 
increase in birth weight due to antimalarial 
effects (overall gain of 46 grams, 95% CI 28-
66). These data support our central hypothesis and complement other studies that have shown 
positive impacts of IPTp-SP on LBW in areas where antimalarial resistance to SP is very 
common11,33 and areas where the prevalence of malaria infection is very low.34 
 
Potential impacts of SP on RTIs and birth outcomes. Treatable sexually transmitted pathogens, 
notably Chlamydia trachomatis, Neisseria gonorrhea, and Trichomonas vaginalis, are estimated 
to cause >350 million infections annually.35 These infections are associated with adverse birth 
outcomes, including fetal growth restriction, preterm birth, LBW, fetal loss, and neonatal 
death.36-39 Other infections also impact upon birth outcomes. Studies of association between 
colonization with GBS and preterm birth have yielded conflicting results,37 but true GBS 
infections are important causes of preterm birth, stillbirth, and puerperal sepsis, and maternal 
colonization is the main risk factor for early-onset neonatal GBS infection, a primary cause of 
neonatal sepsis.40 Thus, antibiotic prophylaxis to prevent early-onset GBS infection in high-risk 
deliveries is now standard practice in developed countries, but typically not in the developing 
world.41 Infection with Mycoplasma genitalum,42 related Mycoplasma and Ureaplasma 
species,43 and Candida,44 and UTIs37 may also be associated with preterm birth. BV, or 
overgrowth of vaginal bacteria other than lactobacilli, in particular Gardnerella, Prevotella, and 
Atopobium, is a strong predictor of preterm birth.45 BV is also associated with increased risk of 

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other RTIs associated with adverse birth outcomes, notably trichomonal, gonococcal, and 
chlamydial infections.46 A Cochrane review suggested that treatment of BV early in pregnancy 
may reduce the risk of preterm birth.47 Chorioamnionitis, caused by Ureaplasma, Mycoplasma, 
Gardnerella, Bacteroides, aerobic gram negative rods, and GBS, is present in an estimated 25% 
of preterm deliveries and strongly associated with adverse birth outcomes, including neonatal 
sepsis, fetal growth restriction, and death.48,49  
   
Our understanding of antibacterial activities of IPTp-SP is limited, but in a region of 
Zambia with widespread SP resistance in P. falciparum, increasing the dosing frequency of IPTp-
SP was associated with decreasing risk of RTIs and improved birth outcomes.34,50 One study 
showed at least intermediate activity of SP against multiple gram positive pathogens, including 
GBS.51 In a recent trial, IPTp with SP did not reduce recto-vaginal colonization with GBS, but this 
study used an outmoded regimen (SP twice during pregnancy).52 In predicting activity of SP 
against RTI pathogens, it is useful to consider the antifolate TMP-SMX, which is better studied 
and has a similar antibacterial spectrum. TMP-SMX is a primary therapy for many infections 
that may impact on birth outcomes in Africa, including common UTI pathogens, salmonellosis, 
brucellosis, melioidosis, and Q fever. Additional pathogens are likely impacted by antifolates; in 
the only available comprehensive review TMP-SMX was noted to be active against >75% of 
strains of C. trachomatis, N. gonorrhea, and GBS.53 Studies in HIV-infected individuals have 
suggested additional benefits for TMP-SMX related to anti-inflammatory activities and impacts 
on the gut microbiome.54  
 
Potential impacts of SP and DP on the gastrointestinal and vaginal microbiome. Non-malarial 
impacts of SP may be due in part to effects on the complex microbial communities found in and 
on the human body (the microbiota) or their aggregate genes and metabolic activities (the 
microbiome). Antibiotics have demonstrated remarkable effects independent of elimination of 
pathogenic microbes. These include the promotion of weight gain in agricultural animals,63 
improvement in severely malnourished children,64 and a survival advantage in healthy children 
receiving infrequent azithromycin to prevent trachoma.65,66 In rodents, antibiotics during 
pregnancy altered microbiome composition and promoted weight gain,67,68 but data in humans 
are limited. As expected, antibiotics with different spectra differ in their impacts on gut 
microbiota.69 Effects of metronidazole, which targets anaerobes, were more modest than those 
of vancomycin, which targets aerobic gram positives.70,71 Although studies of effects of SP or 
TMP-SMX are not available, extrapolation from results for vancomycin suggests that SP, which 
targets aerobic gram positive and gram negative organisms, would be expected to have 
profound impacts on the gut microbiome. Considering the vaginal microbiota, lactobacilli,72 
including vaginal isolates from pregnant women,73 were universally resistant to TMP-SMX, 
suggesting that SP will not foster loss of these organisms, which appear to be important for 
maintenance of vaginal homeostasis.74  

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     Pregnancy is accompanied by dramatic changes in the gut microbiota, including reduced 
individual richness (total number of species), increased between-subject diversity, increased 
abundance of the Proteobacteria and Actinobacteria phyla, and decreased abundance of 
Faecalibacteria and related genera.75 Gut microbiota may play a role in weight gain during 
pregnancy via increased absorption of glucose and fatty acids, increased adipocyte factor 
secretion, and induction of catabolic pathways.76,77 The vaginal microbiome also undergoes 
major changes during pregnancy including, in one small study, decreased overall diversity and 
increased abundance of Lactobacillus species.78 Lactobacilli normally dominate the vaginal 
microbiota, and their increase during pregnancy correlates with a decrease in the vaginal pH, 
creating a barrier against pathogenic bacteria.79,80 The fetus and placenta have until recently 
been considered sterile, but recent data suggest that bacteria reside in a healthy placenta, with 
demonstration of a unique placental microbiome,81 although these data are controversial.75 A 
recent study used 16S rRNA sequencing to demonstrate that chorioamnionitis is associated 
with marked changes in the placental microbiota that are in turn associated with adverse birth 
outcomes.82 Microbiomes differ among ethnic groups,79 between developed world and African 
populations,83 and depending on dietary intake,84 but impacts of interventions on the 
microbiota are not yet well characterized, especially in African populations. Considering the 
importance of changes in microbiota during pregnancy and marked impacts of antibiotics, it 
seems likely that SP alters the maternal microbiome in a manner that affects birth outcomes. 
     Very little information is available on impacts of the components of DP on the microbiome, 
but as these compounds do not have known antibacterial activity, their impact is anticipated to 
be modest. One study showed changes in the rat gut microbiome caused by artesunate, but 
this was in the context of long-term, high-dose therapy that led to cirrhosis, so likely of little 
relevance to IPTp-DP.85 While it is impossible to predict the specific changes associated with DP 
or SP, we expect that these will be greater with SP, and that, considering other evidence 
regarding antibiotic effects, these changes may mediate non-malarial benefits of SP.  
 
IPTp regimens and the selection of antimalarial drug resistance. Resistance to SP is well 
characterized, with 5 mutations in PfDHFR (51I, 59R, and 108N) and PfDHPS (437G and 540E) 
now common in much of Africa and associated with an intermediate level of resistance.55 
Additional mutations, notably pfdhfr 164L and pfdhps 581G, lead to high-level resistance; these 
were rare in older surveys, but recent studies,56,57 including our surveys in Uganda,58 have 
shown emergence of these mutations. Of concern is whether use of SP will select for mutations 
that mediate high-level resistance, rendering SP nearly worthless as an antimalarial. There have 
been important recent insights into resistance to both components of DP. Piperaquine is an 
aminoquinoline related to chloroquine and amodiaquine, but mechanisms of resistance are 
only partially shared with those agents. Mutations in two putative drug transporters, PfCRT and 
PfMDR1, are associated with decreased aminoquinoline sensitivity. For piperaquine, 

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associations are clearest with the pfdhfr 86Y mutation, which is selected by prior use of DP59 
and associated with decreased in vitro drug sensitivity.60 In southeast Asia, resistance to 
piperaquine has emerged, associated with increased plasmepsin gene copy number61,62 and a 
novel PfCRT mutation.63 These newly identified polymorphisms are uncommon in Uganda, and 
piperaquine sensitivity and DP efficacy remain excellent.60,64 Resistance to artemisinins has 
emerged in southeast Asia, mediated principally by mutations in the K13 gene propeller 
domain.65 K13 mutations are uncommonly detected in Ugandan samples,66 and these do not 
appear to be associated with altered in vitro sensitivity to DHA67 or clinical drug resistance.68 
Overall, resistance to DP has not clearly been identified in Uganda, but it is very important to 
study potential selection of resistance by IPTp-DP.  
 
Potential safety concerns for SP+DP. IPTp with SP has an excellent safety profile. Severe 
cutaneous reactions are well documented with SP, but are rare and primarily noted with long-
term prophylaxis in non-African populations. DP also has an excellent safety profile, with similar 
risks of adverse events for IPTp with SP or DP.27-29 The primary adverse effect noted with DP is 
dose dependent QT interval prolongation from piperaquine.69 However, in clinical trials of IPTp 
with DP, QT interval prolongation was transient and not associated with arrhythmias or 
cardiovascular symptoms.28,29,70 In a recent meta-analysis of almost 200,000 individuals who 
received DP, the risk of sudden unexplained death was not higher than baseline and the 
authors concluded that concerns about cardiotoxicity need not limit its use.71 In 
pharmacokinetic studies, our group and others reported that pregnancy decreases piperaquine 
and sulfadoxine exposure, with mixed results for pyrimethamine, suggesting no pregnancy-
associated increased risk, although data are limited.72-76 Piperaquine is metabolized by CYP 
P450 3A45 and 2C8.77-79  Sulfadoxine is cleared renally through glomerular filtration80,81 and 
pyrimethamine is primarily hepatically metabolized, with 15–30% excreted unchanged in 
urine.82 Available studies report no clinically relevant drug interactions for DP and SP, and there 
is no evidence that they alter the disposition of other drugs, except for one small study 
reporting increased exposure to halofantrine, a CYP P450 3A4 substrate, after a single dose of 
SP.81,83-86  
   Although no trials have studied SP+DP, we have shown that daily TMP-SMX+DP was as safe 
and well tolerated for IPTp as daily TMP-SMX alone among HIV infected pregnant women.70 To 
confirm that administration of SP+DP does not result in clinically relevant changes in 
piperaquine exposure, we will evaluate exposure to piperaquine at the onset of our trial.87 
 
1.2. 
Rationale 
 
Malaria in pregnancy remains a major challenge in Africa, where approximately 50 million 
women are at risk for P. falciparum infection during pregnancy each year.88 Among pregnant 

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women living in malaria endemic areas characteristic of much of Africa, symptomatic disease is 
uncommon, but infection with malaria parasites is associated with maternal anemia and 
adverse birth outcomes including abortions, stillbirth, preterm birth, LBW, and infant 
mortality.2 Thus, malaria in pregnancy causes an estimated 900,000 LBW deliveries and 100,000 
infant deaths each year.3,89 The WHO recommends the use of LLINs and IPTp-SP for the 
prevention of malaria in pregnancy in endemic areas of Africa. However, there is concern for 
diminishing efficacy of these interventions due to the spread of vector resistance to the 
pyrethroid insecticides used in LLINs and parasite resistance to SP.13,90 Thus, there is an urgent 
need for new strategies for the prevention of malaria in pregnancy and improving birth 
outcomes.  
     ACTs are now the standard treatment for malaria in Africa. DP is a fixed-dose ACT and an 
attractive alternative to IPTp-SP. DP is highly efficacious, and the long half-life of piperaquine 
provides at least 4 weeks of post-treatment prophylaxis.91,92 Recent randomized controlled 
trials from our group and others have shown that, compared to IPTp with SP, IPTp with DP 
dramatically reduced risks of maternal parasitemia and symptomatic malaria, maternal anemia, 
and placental malaria.27-29 In addition, IPTp with DP was as safe and well tolerated as IPTp with 
SP. Surprisingly, despite significant reductions in risks of malaria-specific outcomes afforded by 
DP in these studies, there were no differences between the SP and DP groups in risks of adverse 
birth outcomes, including LBW and preterm birth. Indeed, in 2015 a WHO Malaria Policy 
Advisory Committee concluded that, although IPTp with DP merits further study, SP should 
remain the recommended drug for IPTp until there is conclusive evidence that alternative 
regimens improve birth outcomes.93 Thus, the standard-of-care remains use of a poorly 
effective antimalarial to prevent malaria in pregnancy. 
The key question motivating this proposal is why IPTp with either SP or DP is associated 
with similar risks of adverse birth outcomes despite the far superior antimalarial activity of DP. 
The likely explanation is that SP, a broad-spectrum antibiotic, protects against non-malarial 
causes of LBW and preterm birth.94 In this regard, several studies reported that increased 
frequency of doses of IPTp with SP was associated with improved birth outcomes and a lower 
risk of RTIs, despite widespread SP resistance among malaria parasites.34,50 Our central 
hypothesis is that SP improves birth outcomes independent of its antimalarial activity and that 
IPTp with a combination of SP+DP will offer antimalarial and non-antimalarial benefits, thus 
providing superior prevention of adverse birth outcomes compared to either drug used alone. 
To test this hypothesis we will conduct a double-blinded randomized clinical trial in a rural area 
of Uganda with very high malaria transmission intensity where our group already has an 
established infrastructure for clinical research. 
 
 
 

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2. STUDY OBJECTIVES 
 
We will test the hypothesis that IPTp with SP+DP will significantly reduce the risk of adverse 
birth outcomes compared to IPTp with SP alone or DP alone. The specific study objectives are 
as follows: 
 
2.1. 
Objective 1 
 
To compare the risk of adverse birth outcomes among pregnant women randomized to 
receive monthly IPTp with SP vs. DP vs. SP+DP. We will conduct a randomized controlled trial 
in 2757 women to test the hypothesis that pregnant women who receive IPTp with SP+DP will 
have a lower risk of a composite outcome of LBW, preterm birth, small-for-gestational age, 
fetal loss or neonatal death compared to those who receive either SP or DP alone.  
 
2.2. 
Objective 2 
 
To compare safety and tolerability of IPTp regimens among pregnant women randomized to 
receive monthly IPTp with SP vs. DP vs. SP+DP. We will test the hypothesis that pregnant 
women who receive IPTp with SP+DP will have non-inferior risks of adverse events and 
measures of tolerability compared to those who receive either SP or DP alone. 
 
2.3. 
Objective 3 
 
To compare risks of malaria-specific and non-malarial outcomes among pregnant women 
randomized to receive monthly IPTp with SP vs. DP vs. SP+DP. We will test the hypotheses 
that a) pregnant women who receive IPTp with DP containing regimens will have a lower risk of 
maternal and placental malaria but a higher risk of P. falciparum markers of DP resistance 
compared to those who receive SP alone; and b) pregnant women who receive IPTp with SP 
containing regimens will have a lower risk of RTIs and a higher risk of P. falciparum markers of 
SP resistance compared to those who receive DP alone. Further, we will explore impacts of 
study drugs on the gut and vaginal microbiomes, as differences are likely to impact upon birth 
outcomes. 
 
3. STUDY DESIGN 
 
This will be a double-blinded randomized controlled phase III trial of 2757 HIV uninfected 
pregnant women. HIV uninfected women at 12-20 weeks gestation will be randomized in equal 

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proportions to one of three IPTp treatment arms: 1) SP given every 4 weeks, or 2) DP given 
every 4 weeks, or 3) SP+DP given every 4 weeks. SP or DP placebos will be used to ensure 
adequate blinding is achieved in the study as outlined in appendix D. Follow-up for the 
pregnant women will end 28 days after giving birth.  
 
We will recruit pregnant women presenting for routine care at antenatal clinics at 9 
government health facilities in Busia District, including Masafu General Hospital. Based on our 
previous study we estimate that ~200 women will be available for screening each month. 
Recruitment will take place in two phases. In phase 1, 300 participants will be enrolled over 2-3 
months. Phase 1 participants will undergo intensive safety and pharmacokinetic assessment. In 
phase 2, the remaining 2457 participants will be enrolled. After all participants enrolled in 
phase 1 have delivered, an interim analysis of safety data will be performed and presented to 
the DSMB as described in detail in the Protection of Human Subjects section and study 
protocol. If the DSMB approves, phase 2 of recruitment will continue until the target sample 
size has been reached. 
 
4. SELECTION AND ENROLLMENT OF SUBJECTS 
 
4.1. 
Inclusion Criteria 
 
1) Viable singleton pregnancy confirmed by ultrasound 
2) Estimated gestational age between 12-20 weeks  
3) Confirmed to be HIV uninfected by rapid test 
4) 16 years of age or older 
5) Resident of Busia District, Uganda 
6) Provision of informed consent  
7) Agreement to come to the study clinic for any febrile episode or other illness and avoid 
medications given outside the study protocol 
8) Willing to deliver in the hospital 
 
4.2. 
Exclusion Criteria 
 
1) History of serious adverse event to SP or DP 
2) Active medical problem requiring inpatient evaluation at the time of screening 
3) Intention of moving outside of Busia District, Uganda 
4) Chronic medical condition requiring frequent medical attention 
5) Prior chemopreventive therapy or any other antimalarial therapy during this pregnancy 
6) Early or active labor  
7) Multiple pregnancies (i.e. twins/triplets) 

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8) History of long QT syndrome 
 
4.3. 
Initial Screening 
 
We will recruit pregnant women presenting for routine care at the Masafu General Hospital 
antenatal clinic, presenting at local health centers within Busia District, or referred by the 
Uganda government voluntary health teams. Pregnant women will be approached about 
participating in the study and will be provided an information sheet about the requirements of 
the study (Appendix A).  If women are initially agreeable to screening for participation in the 
study and are < 20 weeks gestation by LMP, 16 years of age or older, and not known to be HIV 
infected, they will either be escorted to the study clinic or given an appointment to return at a 
later date.  At our study clinic, study physicians will assess for initial eligibility criteria through 
conversation with the woman (confirming the age of the woman, location of residence, 
agreement to come to the study clinic for any febrile episode or other illness and avoid 
medications given outside the study protocol, plan to deliver in the hospital, no history of 
serious adverse event to SP or DP, no active medical problems requiring inpatient evaluation at 
the time of screening, no intention of moving outside the study district, no chronic medical 
condition requiring frequent medical attention, no prior SP preventive therapy or any other 
antimalarial therapy during this pregnancy, and no history of long QT syndrome). Women who 
pass initial screening based on conversation with study physicians will undergo the following 
additional screening procedures: 1) rapid HIV testing to confirm HIV negative status, 2) 
ultrasound dating to confirm viable singleton pregnancy and gestational age of 12-20 weeks in 
conjunction with LMP (Appendix B), and 3) a pelvic examination to exclude early or active labor 
if clinically indicated. Women who are not eligible for the study will be referred back to their 
local antenatal clinic. 
 
4.4. 
Study Enrollment Procedures and Baseline Evaluation 
 
Informed consent will be obtained from women who pass initial screening. Study physicians will 
conduct the informed consent discussion in the study clinic. Informed consent will be 
conducted in the appropriate language and a translator will be used if necessary. The study will 
be described and consent obtained in one of 4 languages (Samia, Swahili, Luganda, or English). 
The consent forms will be translated into each language and back-translated into English to 
check for any loss or change of meaning. Following the informed consent discussion, pregnant 
women will be asked by the study physicians to sign a written consent form approved by the 
UCSF CHR, Makerere University School of Biomedical Sciences - Research and Ethics 
Committee, and the UNCST for their participation in the research study and a second approved 
consent form for the future use of biological specimens obtained during the course of the 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
 23 
 
study.  If the pregnant woman is unable to read or write, her fingerprint will substitute for a 
signature, and a signature from an impartial witness to the informed consent procedures will be 
obtained. 
 
Women will be enrolled in the study on the same day that they provide informed 
consent for participation in the research study.  On the day of enrollment, women will undergo 
a standardized history and physical examination, and have blood collected by venipuncture (15 
cc’s) for filter paper sample (for future molecular studies), routine baseline laboratory testing 
and storage.  Routine baseline laboratory testing will consist of a thick blood smear for malaria 
parasites (to be read later by laboratory technicians not involved in study participant care; will 
not be used to guide clinical care) and CBC measurement.  
Subjects will also provide samples for gut and vaginal microbiome studies. A clinician 
will collect a vaginal swab for microbiome analysis in a private area. The clinician will use an 
Endocervical Specimen Collection Kit, inserting the vaginal swab approximately 4-5 cm into the 
vagina for the collection. The swab will then be placed into the collection container and frozen 
in liquid nitrogen.  
The study participant will be provided with a stool collection container and can provide 
the stool sample either at the clinic or at home. If the sample is to be collected at home, the 
participant will be asked to bring the sample back to the clinic as soon as possible. The 
laboratory procedures for microbiome analysis are described in Section 6.10.7.  
Women who have history of fever in the previous 24 hours or a temperature > 38.0ºC 
(tympanic) will have a thick blood smear read urgently in the study clinic.  Women with history 
of fever in the previous 24 hours or a temperature > 38.0ºC (tympanic) and a positive blood 
smear will be diagnosed with malaria and treated as described in section 6.2. At the end of the 
enrollment visit all study participants will be given a LLIN and a household survey appointment 
will be scheduled within 2 weeks to collect household-level information on the use of bednets, 
house members, household characteristics, and GPS coordinates (Appendix C). 
 
5. STUDY TREATMENT 
 
5.1. 
Treatment Group Assignments 
 
An enrolled pregnant woman will be randomized to one of 3 treatment arms: SP given every 4 
weeks, DP given every 4 weeks or the combination of SP+DP given every 4 weeks. We will use a 
1:1:1 randomization scheme targeting 919 pregnant women in each treatment arm. A 
randomization list will be computer generated by a member of the project who will not be 
directly involved in the conduct of the study. The randomization list will include consecutive 
treatment numbers with corresponding random treatment assignments. Randomized codes will 
correspond to the 3 treatment arms using permuted variable sized blocks of 6 and 9. Sealed 

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 24 
 
copies of the original randomization list and documentation of the procedure used to generate 
the lists will be stored in the project administrative offices in San Francisco and Kampala. Prior 
to the onset of the study, a set of sequentially numbered, opaque, sealed envelopes will be 
prepared.  Each envelope will be marked on the outside with the treatment allocation number. 
The inside of the envelope will contain a piece of paper with the treatment allocation number 
and treatment group assignment along with a piece of carbon paper.  
 
 
 
5.2. 
Treatment Allocation 
 
On the day of enrollment, pregnant women will be referred to a study pharmacist responsible 
for treatment allocation. The study pharmacist will assign treatment arms as follows: 
1. Select next available envelope 
2. Note treatment number on the outside of the envelope 
3. Write date, time, and study number on the outside of the envelope 
4. Open envelope 
5. Remove form containing code for treatment arm and date, time, and study number 
(transferred to form via carbon paper inside of envelope) 
6. Store form in lockable file box in study pharmacy 
7. Record onto the treatment allocation master list the study number, enrollment date, 
treatment assignment code, treatment arm, and study medications to be given during 
pregnancy 
8. Store treatment allocation Master list in a lockable cabinet in study pharmacy 
9. Record treatment number in the study participant’s file 
 
5.3. 
Study Drug Dosing and Formulations 
 
During pregnancy, women will be given 1 of 3 treatment regimens: 1) SP given every 4 weeks 
during pregnancy, 2) DP given every 4 weeks during pregnancy, or 3) SP+DP given every 4 
weeks during pregnancy. Each treatment with SP will be given as a single dose consisting of 3 
full strength tablets. Each treatment with DP will consist of 3 full strength tablets given once a 
day for 3 consecutive days. In addition, placebos will be used to mimic the identical dosing 
strategy such that every 4 weeks women will receive two drugs on day 1 (SP and placebo or DP 
and placebo or SP and DP) followed by one drug on days 2 and 3 (DP or placebo). Two placebos 
will be used, one that mimics the appearance of SP and one that mimics the appearance of DP. 
Dosing schedules for each treatment regimen according to gestational age are presented in 
Appendix D. The study drugs, DP (Duo-Cotecxin) and SP (Kamsidar) will be supplied by Holley-

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 25 
 
Cotec, Beijing and Kampala Pharmaceutical Industries (KPI), Uganda, respectively. Holley-Cotec 
will manufacture the DP placebo of exact specifications as that of the active DP. The DP placebo 
will be exactly the same in color, size, shape and packaging as the active DP.  KPI will 
manufacture the SP placebo of exact specifications as that of the active SP. The SP placebo will 
be exactly the same in color, size, shape and packaging as the active SP. Details of the study 
drug formulations are included in Table 2. 
 
Table 2. Drug formulation and labeling   
Drug 
Formulations 
Trade name (Manufacturer) 
Sulfadoxine-Pyrimethamine (SP) 
500mg/25mg tabs 
Kamsidar (KPI) 
Dihydroartemisinin-Piperaquine (DP) 
40mg/320mg tabs 
Duo-Cotexin (Holley-Cotec) 
 
5.4. 
Blinding, Study Drug Administration, and Duration 
 
Administration of all study drugs will be double blinded such that study participants and study 
staff will be blinded to study treatments with the exception of the study pharmacist and 
pharmacy technician, who will not be involved with patient care or assessment of study 
outcomes. All doses of study drugs will be prepackaged by a study pharmacist and administered 
by a study nurse blinded to the study participant’s treatment regimen. All doses of SP (or SP 
placebo) administered will be directly observed in the clinic. For DP (or DP placebo), the first of 
the 3 daily doses will be directly observed in the clinic and the 2nd and 3rd daily doses will be 
administered at home using pre-packaged study drugs in opaque envelopes with dosing 
instructions written on the outside. For doses of study drugs administered in the clinic, if a 
study participant vomits the study drug within 30 minutes of administration, the drug will be re-
administered. For doses of study drugs administered at home, if a study participant vomits the 
study drug within 30 minutes of administration or study drug is lost, the study participant will 
be instructed to come to the study clinic as soon as possible where the study drug will be re-
administered/replaced. For pregnant women all doses of study drugs will be given between 16 
and 40 weeks gestation as outlined in Appendix D.   
 
5.5. 
Study Drug Accountability 
 
The study pharmacist will maintain complete records of all study drugs received in the study 
pharmacy.  Lot number and number of pills given to each study participant will be recorded. A 
registry of all study medication, current product labels, and Certificates of Analysis, provided by 
suppliers will be maintained within the regulatory binder for the study.  The date received, lot 
number, expiration date, and date used will be recorded for each of the study medications.  
Monthly inventory of all study medications will be conducted and a record log of investigational 
medications will be kept at the study clinic.  

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6. SUBJECT MANAGEMENT 
 
6.1. 
Subject Follow-up 
 
Pregnant women will be scheduled to be seen in the clinic every 4 weeks during their 
pregnancy and then 1 and 4 weeks following delivery. In addition, study participants will be 
instructed to come to the study clinic for all medical care and avoid the use of any outside 
medications. The study clinic will remain open 7 days a week from 8 a.m. to 5 p.m.  Pregnant 
women who are not seen on the day of their regularly scheduled visits will be visited at home 
and instructed to come to the clinic as soon as possible.  
 
Each time a study participant is seen in the clinic a standardized history and physical 
exam will be performed including temperature, pulse, weight, mid-upper arm circumference, 
and blood pressure measurement. Patients who are febrile (tympanic temperature > 38.0˚C) or 
report history of fever in the past 24 hours will have blood obtained by finger prick for a thick 
blood smear and have fetal ultrasounds performed. If the thick blood smear is positive, the 
patient will be diagnosed with malaria (Section 6.2).  If the thick blood smear is negative, the 
patient will be managed by study physicians for a non-malarial febrile illness (Section 6.3).  If 
the patient is afebrile and does not report a recent fever, a thick blood smear will not be 
obtained, except when following routine testing schedules (Section 6.5).  
 
6.2. 
Diagnosis and Management of Malaria 
 
Patients found to have malaria based on laboratory confirmation will have a second finger prick 
for hemoglobin measurement using a portable spectrophotometer (HemoCue), and collection 
of plasma. Patients with malaria may be asked to donate 5 cc of blood before treatment is 
initiated to allow studies of cultured malaria parasites. All episodes of malaria will be classified 
as uncomplicated or complicated based on the following criteria:   
 
Uncomplicated malaria (all of the following) 
1) Fever (> 38.0ºC tympanic) or history of fever in the previous 24 hours 
  
2) Positive thick blood smear 
3) Absence of complicated malaria 
 
Complicated malaria  
1) Clinical evidence of severe malaria (Appendix E)  
2) Positive thick blood smear 
 

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Episodes of malaria will also be classified into the following categories according to the timing 
of previous malaria episodes for treatment purposes: 
1. New episodes of malaria will be defined as any first episode during the study period or 
any episode occurring > 14 days after the diagnosis of a previous episode 
2. Treatment failures will be defined as any of the following: 
a. Complicated malaria occurring 1-14 days after the diagnosis of malaria 
b. Fever (> 38.0°C tympanic) or history of fever in the previous 24 hours with a 
parasite density > the parasite density of an episode of malaria diagnosed 2 days 
prior 
c. Fever (> 38.0°C tympanic) or history of fever in the previous 24 hours with a 
parasite density > 25% of the parasite density of an episode of malaria diagnosed 
3 days prior 
d. Fever (> 38.0°C tympanic) or history of fever in the previous 24 hours with a 
positive thick blood smear of any parasite density occurring 4-14 days after the 
diagnosis of a previous episode 
 
 
All patients diagnosed with new episodes of uncomplicated malaria will be prescribed 
AL, the recommended first-line treatment in Uganda for pregnant women. Patients with 
complicated malaria will be prescribed IV artesunate according to national malaria treatment 
guidelines. Patients with treatment failure within 14 days following treatment with AL will be 
prescribed pyronaridine-artesunate (Pyramax)  according to national malaria treatment 
guidelines. Patients with treatment failure within 14 days following treatment with Pyramax  
will be treated with quinine. Patients with treatment failure within 14 days following treatment 
with quinine will be treated with quinine plus clindamycin. Treatment of symptomatic malaria 
will have no impact on the administration of study drugs for IPTp.  
 
6.3. 
Management of Non-Malaria Illnesses 
 
Patients who are found to have illnesses other than malaria will receive standard-of-care 
treatment in the study clinic, according to standardized algorithms, or will be referred to the 
local hospital.  We will avoid the routine use of non-study medications with antimalarial 
activity, including antifolates (with the exception of assigned IPTp regimens) and macrolide or 
tetracycline antibiotics, when acceptable alternatives are available. In addition, drugs with 
known risk of torsades de pointes or CYP3A4 inhibitors (Appendix J and K) will be avoided if 
possible. If the study clinician deems that treatment with one of the drugs listed on Appendix J 
or K is required, the clinical management team will be consulted. During follow-up for non-
malarial illnesses, blood smears will be done at the discretion of the study physician if the 
subjects are febrile (tympanic temperature > 38.0˚C) or report history of fever in the past 24 

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hours.  If the blood smear is positive, the patient will be diagnosed with a new episode of 
malaria and managed per study protocol. We will be assessing for RTIs at delivery. For 3 
infections for which treatment is clinically indicated (C. trachomatis, N. gonorrhea, and T. 
vaginalis), women will be treated promptly, generally within one day of delivery, following 
standard guidelines for the treatment of these infections. 
 
 
6.4. 
After Hours Visits 
 
Pregnant women will be encouraged to come to the Masafu General Hospital maternity ward 
(open 24 hours a day) when urgent care is needed outside of study clinic hours. Pregnant 
women will be instructed to inform hospital personnel of their involvement in the study at the 
time of registration and to visit the study clinic on the following day. If a patient is diagnosed 
with uncomplicated malaria she will receive treatment from a hospital supply of AL and 
clinicians will be instructed to refer patients to our study clinic when it opens at 8 am the 
following day.  If a patient is diagnosed with severe malaria, she will receive I.V. artesunate 
following standard treatment guidelines. Patients with non-malarial illnesses will be managed 
at the discretion of the hospital staff.  Upon discharge, patients will receive follow-up at the 
study clinic as outlined above.   
 
6.5. 
Routine Assessments 
 
Routine assessments will be done in the clinic every 4 weeks (Appendix G). Study participants 
not seen in the clinic for routine visits will be visited at home and requested to come to the 
study clinic as soon as possible. Pregnant women will receive standards of care as designated in 
the Uganda MOH guidelines (Appendix F). Routine antenatal care will include screening and 
treatment for sexually transmitted infections; blood pressure assessment; urine dipstick for 
proteinuria; and prescription of iron, folate, multivitamins and mebendazole. During routine 
assessments subjects will be asked about visits to outside health facilities and the use of any 
medications outside the study protocol.  The study protocol will be reinforced with discussion 
regarding the need to come to the study clinic promptly upon the onset of any illness and to 
avoid use of outside medications.  Standardized assessment of adherence will also be done for 
study drugs administered at home and LLIN use.  A routine history and physical exam will be 
performed using a standardized clinical assessment form.  Blood will be collected by finger prick 
or phlebotomy (alternate routine visits) for thick smear, qPCR testing, CBC testing, immunology 
studies, and filter paper samples as described in Appendix G. Thick smears, qPCR testing, and 
collection of filter paper samples will be done every 4 weeks. Phlebotomy for CBC, for other 
laboratory tests to monitor for potential adverse events from study medications, for 

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immunology studies, and to collect blood for storage will be performed every 8 weeks.  For 
participants enrolled between 12-<18 weeks gestational age, blood will be collected by 
phlebotomy at the time of their week 20 routine visit. For participants enrolled between 18-20 
weeks gestational age, blood will be collected by finger prick at the time of their week 20 
routine visit. A repeat HIV test will be done at 28 weeks gestational age. If women are found to 
have become HIV-infected during pregnancy, they will be immediately referred for care 
following local prevention of mother-to-child transmission guidelines. If a pregnant woman 
reports a fever in the last 24 hours or the patient has a documented temperature > 38.0˚C 
tympanic, the patient’s thick blood smear will be read immediately and if positive the patient 
will be diagnosed and treated for malaria (see section 6.2). Thick blood smears other than those 
done when a woman has fever will not be used for clinical care of study participants.  Study 
drugs will be administered at the time of each routine visit as described in sections 5.3 and 5.4.  
A pregnancy ultrasound will be conducted at 24, 32 and 36 weeks gestational age to 
assess fetal size. Images and videos clips from the ultrasound will be saved, and read by trained 
study staff members who are not directly involved in patient care. A stool sample and a clinician 
collected vaginal swab for microbiome studies will also be collected at 32 weeks gestation, 
using similar procedures as at enrollment (Section 4.4, Section 6.10.7). If the study participant 
misses their 32 week appointment or delivers before the stool and vaginal samples can be 
collected, these samples will be collected as soon as possible after 32 weeks. 
 
For the 300 women enrolled in phase 1 of the study the following additional procedures 
will be conducted at 20, 28, and 36 weeks of gestation: 1) the administration of all 3 daily doses 
of study drugs will be directly observed in the clinic. 2) ECGs will be performed to measure the 
QTc interval just prior to the first dose and 2-6 hours after the third dose of study drugs. 3) 
Blood will be collected by phlebotomy just prior to performing each ECG to measure trough and 
peak piperaquine levels. These data will be used to assess for potential cardiotoxicity, impacts 
of SP on peak piperaquine drug levels, and other potential adverse events (which will be 
rigorously assessed in both phase 1 and phase 2). 
 
In a subset of women actively followed in the study after October 1st 2023, an additional stool 
sample will be collected 1 week after their week 28 gestational age routine visit to better assess 
potential changes to the stool microbiome shortly after peak concentrations of study drugs. 
Participants will be given a stool collection vial at the time of their week 28 gestational age 
routine visit with instructions to collect a stool sample at home 1 week later and then bring it as 
soon as possible to the study clinic. Participants will be provided transport reimbursement 
when they bring this stool sample to the study clinic but will not need to be seen by the study 
clinicians or have any additional evaluations.  This additional stool sample will be processed and 
analyzed as described in Section 6.10.7 
 

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6.6. 
Delivery visit  
 
Systems will be in place to facilitate a birth plan which will encourage women to come to the 
hospital for delivery, including access to transportation 24 hours a day. However, for women 
who are unable to travel to the hospital for delivery or choose to deliver at home, a study staff 
member will be driven to the home to follow study procedures.  Study staff will document 
details of the delivery, including date and time, type of delivery, blood pressure during labor, 
estimated blood loss and any maternal, obstetrical or neonatal complications. Study staff will 
document the infant’s Apgar score and birth weight with calibrated scales. Biological samples 
collected at the time of delivery will include maternal venous blood (for thick blood smear, filter 
paper samples, CBC, and storage of blood for immunology and other studies), cord blood (for 
thick blood smear, filter paper samples, hemoglobin measurement, and storage of blood for 
immunology and other studies), and placental tissue. A vaginal swab will be collected for the 
diagnosis of common infectious diseases (section 6.10.8). The GeneXpert/Endocerivcal 
Collection Kit will be used to collect the sample. The clinician will insert the swab 4-5 cm into 
the vagina, making a circular motion along the vaginal wall. If any infections are diagnosed, the 
participant will receive appropriate treatment after delivery.  Following delivery, neonatal care, 
as per national guidelines, will include polio and BCG immunization, ophthalmic tetracycline, 
and vitamin K.  
 
At the time of delivery, women will undergo repeat rapid HIV testing based on national 
guidelines. If women are found to have become HIV-infected during pregnancy, they will be 
immediately referred for care following local prevention of mother-to-child transmission 
guidelines.  
 
6.7. 
Postpartum visits.  
 
Women will be evaluated 1 and 4 weeks following delivery as part of routine care. Both visits 
will include an abdominal exam, syndromic management of RTIs, and follow-up on any 
obstetrical complications that occurred including evaluation of the neonate for any congenital 
abnormalities. Pelvic and breast exam will be done if clinically indicated. Contraceptive 
counseling, nutritional assessment and infant feeding and support will be provided.  
 
Summaries of all procedures done during routine visits in pregnant women are presented in 
Appendix G. 
 
6.8. 
Medical Care Outside the Study Clinic 
 

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We will provide routine medical care in our clinic free of charge, including medications, to the 
extent possible given resources available. Study participants will be reimbursed for costs of 
transportation to and from our clinic.  In addition, we will reimburse the cost of tests and drugs 
not available in the study clinic, referrals for study participants made by study physicians to 
other clinics and services, and after-hours hospital visits.  We anticipate reimbursing the cost of 
most diagnostic tests (including laboratory tests, X-rays, and ultrasounds) and medications 
resulting from these referrals, using available funds. However, reimbursement of all diagnostic 
tests and treatment recommended outside the study clinic cannot be guaranteed in all 
circumstances.  Decisions on reimbursement will be made by the study coordinator and the 
investigators, in conjunction with the funding agency if necessary.  
 
6.9. 
Duration of Follow-up and Criteria for Premature Study Withdrawal 
 
Study participants will be followed until their 4 week postpartum visit. Study participants will be 
prematurely withdrawn from the study for: 1) movement out of study area, 2) inability to be 
located for > 60 consecutive days, 3) withdrawal of informed consent, 4) inability to comply 
with the study schedule and procedures, 5) at the discretion of the site investigator if the study 
is not in the best interest of the participant, 6) subject judged by the site investigator to be at 
significant risk of failing to comply with the study protocol as to cause them harm or seriously 
interfere with the validity of study results, and 7) subject found to be HIV positive at the time of 
their 28 week gestational age routine visit. If a subject is withdrawn for reasons # 1-3, we will 
be unable to perform any additional study procedures.   If a subject is withdrawn for reasons # 
4-7, plans to obtain appropriate follow-up tests outside of the study will be individualized for 
each subject depending on the health status of the subject at the time of withdrawal and the 
willingness of the participant and his or her parent/guardian to proceed with additional testing.   
 
6.10. 
Diagnostic and Laboratory Testing 
 
Diagnostic and laboratory testing shall take place using accredited local laboratories, as 
described below. In the event that local capacity is not available for specialized testing, 
reasonable efforts will be made to build local capacity. In the event that local capacity is not 
available, samples will be exported with all required permissions and permits to external testing 
laboratories.   
 
6.10.1. 
Microscopy 
 
Thick blood smears will be stained with 2% Giemsa and read by experienced laboratory 
technologists.  Parasite densities will be calculated by counting the number of asexual parasites 

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per 200 leukocytes (or per 500 leukocytes, if the count is <10 asexual parasites/200 leukocytes), 
assuming a leukocyte count of 8,000/µl.  A blood smear will be considered negative when the 
examination of 100 high power fields does not reveal asexual parasites. Gametocytemia will 
also be determined from thick smears.  For quality control, all slides will be read by a second 
microscopist and a third reviewer will settle any discrepant readings. 
 
6.10.2. 
Clinical Laboratory Studies 
 
At enrollment and every 8 weeks during follow-up for pregnant women venipuncture blood 
samples will be collected for CBC testing. Additional venipuncture will be performed, as 
appropriate, for laboratory testing to evaluate non-malarial medical illnesses at the discretion 
of study physicians. Results will be made available to study physicians in a timely manner for 
patient management decision-making. Additional hemoglobin measurements will be performed 
each time a patient is diagnosed with malaria using a portable spectrophotometer (HemoCue, 
Angholm, Sweden), and results will be immediately available.  
 
6.10.3. 
Placental Studies 
 
For assessment of placental malaria, two 1 cm-wide full thickness biopsies, obtained about 5 
cm from the cord, will be obtained within 1 hour of delivery and placed in 10% neutral buffered 
formalin. Biopsy specimens will be embedded in paraffin wax, sectioned into 3 μm slices using a 
rotary microtome, fixed to glass slides, and dehydrated in sequential ethanol baths. Separate 
slides will be stained in 0.1% hematoxylin/1% eosin (H&E) for 5 and 1 min, respectively, or in 2% 
Giemsa for 30 min. Placentas will be graded into 5 categories using a standardized approach.95 
The presence of intervillous parasite-infected erythrocytes and of pigment in 
monocyte/macrophages or fibrin will be noted. Quantitative assessments of placental malaria 
will involve counting of 1000 intervillous blood cells under high power and determination of 
percentages of intervillous infected erythrocytes and monocyte/macrophages containing 
malarial pigment. Additional placental biopsies may be taken and preserved for genomic 
analysis. Briefly, any additional placental biopsies will be preserved in RNAlater (an RNA 
preservation agent) and stored per manufacturer’s directions, and/or flash frozen in liquid 
nitrogen and embedded in OCT mounting medium. For assessment of chorioamnionitis, 
placental membrane roll, umbilical cord, and chorionic plate/villous parenchyma specimens will 
be collected, dehydrated through an ethanol series, cleared in xylene, and embedded in 
paraffin wax blocks. A 3 μm thick section from each tissue block will be obtained using a rotary 
microtome, and sections will be mounted onto glass slides via a floatation water bath. The 
slides will be baked at 60oC for 30 min, de-paraffinized in xylene, hydrated through a series of 
ethanols, stained with H&E, dehydrated through an ethanol series, cleared with xylene, and 

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 33 
 
mounted with organic media. Slides will be examined by light microscopy, and chorioamnionitis 
diagnosed based on detection of neutrophils, based on a standardized diagnostic framework.96 
In addition, the chorioamniotic space will be sampled for genomic assessment; after delivery of 
the placenta, the chorion and amnion will be peeled apart,97 taking care to avoid 
contamination, the exposed surface will be swabbed, the swab placed in PBS, and sample flash 
frozen in liquid nitrogen for shipment and processing as described in Section 6.10.7.  Of note, 
our group already has expertise in all described techniques for studying placental 
histopathology.98 Cord blood and placental intravillous blood will be collected for immunology 
studies as described below.  
 
6.10.4. 
Dried blood spots collected on filter paper 
 
Each time a thick blood smear is obtained blood will also be collected onto filter paper. Samples 
will be collected by venipuncture or by finger-prick sampling. Filter paper (Whatman no 1, 
Whatman 3MM; Whatman, Maidstone, UK) will be pre-cut into individual squares and stapled 
to a thick card which will serve as its cover. Blood spots will be collected onto the filter paper in 
volumes of approximately 25 µl per blood spot (4 blood spots per sample). Filter paper samples 
will be labelled with bar codes on the covering cardboard and will be allowed to dry at ambient 
temperature and relative humidity before closing the card over the filter paper (like closing a 
matchbook).  Filter paper samples will be transported from the field in zip lock bags and placed 
into a stock card filter paper box for final storage at ambient temperature or at -20⁰C with a 
desiccant. Filter paper samples will be stored in Tororo or a central laboratory in Kampala. 
Molecular studies will include the extraction of DNA and/or RNA from filter paper, followed by 
characterization of parasite and host nucleic acid sequences using standard molecular 
procedures including PCR, DNA hybridization, restriction enzyme digestion, and/or nucleic acid 
sequencing.  Future molecular studies will be performed only for research purposes and will 
have no impact on the clinical management of study participants. 
 
 
6.10.5. 
 Quantitative PCR 
 
qPCR will be performed at the time of enrollment and at each routine visit (every 4 weeks). At 
each of these time points, 200 µL of whole blood will be used and DNA will be extracted using 
Qiagen spin columns or related methods. Extraction products will be tested for the presence 
and quantity of P. falciparum DNA via a highly sensitive qPCR assay targeting the multicopy 
conserved var gene acidic terminal sequence.99 This assay is now routinely performed by our 
group and demonstrates sensitivity and reproducible quantification to 0.1 parasites/µL. qPCR 

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 34 
 
for other potential pathogens may also be performed. These assays will be performed at our 
laboratory in Tororo where qPCR capacity is already established.  
 
6.10.6  
Ex vivo drug sensitivity of malaria parasites 
Blood will be collected from select subjects upon diagnosis of malaria for culture of infecting 
malaria parasites. After consent is obtained for this procedure, up to 5ml of blood will be drawn 
by venipuncture and collected in heparin containing tubes for spotting on filter paper and ex 
vivo culture of malaria parasites. Blood will be transported to our laboratory at Tororo District 
Hospital and drug susceptibilities assessed. Samples will be processed according to our 
previously published protocols, with detection of parasites based on SYBR green fluorescence 
or related methods.59,60 In brief, blood will be centrifuged, plasma and buffy coat removed, and 
erythrocyte pellets washed three times with RPMI 1640 medium. 200 µl aliquots of washed 
pellets will be added to 10 ml of RPMI 1640 medium supplemented with 25 mM HEPES, 0.2% 
NaHCO3, 0.1 mM hypoxanthine, 100 µg/ml gentamicin, 0.5% AlbuMAX II serum substitute, and 
serial dilutions of drugs of interest. Drug sensitivities will be determined with a 96-well 
microplate fluorescence assay with SYBR green I detection. In brief, plates will be maintained 
for 72h in a modular incubator, plates will be frozen and thawed, 100_µl from each well will be 
transferred to a black 96-well plate containing 100 µl/well SYBR green lysis buffer, plates will be 
incubated for 1 h in the dark at room temperature, and fluorescence will be measured with a 
plate reader. IC50s will then be derived by plotting percent growth against log drug 
concentration, and fitting the data by variable slope, sigmoidal curve fit. Studies of ex vivo drug 
sensitivities will have no impact on patient care.   
 
6.10.7   
Microbiome studies  
 
 
We will comprehensively compare the gut and vaginal microbiomes between our 3 treatment 
arms. We will begin with multiplex 16S rRNA gene amplicon sequencing of gut and vaginal 
microbiota and then advance to metagenomics sequencing to provide greater depth of analysis.  
 
Collection of samples for microbiota evaluation. Care is required to avoid undue degradation 
or contamination of samples, but studies of gut and vaginal microbiota have shown rather little 
impact of short term (2 weeks) storage, even at room temperature.106,107 All samples will be 
handled in the same manner, as inconsistent methods of collection and storage can impact on 
results106. Samples will be collected near the time of study enrollment (before first dose of 
study drug(s)) and at approximately 32 weeks gestation, before the scheduled monthly dose of 
study drug(s). We have chosen the 32 weeks gestation for the second time point because the 
majority of women, including those with preterm deliveries at <37 weeks gestation, will deliver 
after 32 weeks. Thus we will avoid collecting samples for microbiome studies during labor or 

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immediately after delivery, which may be contaminated. At enrollment and 32 weeks gestation, 
stool and vaginal swabs will be collected as described in section 4.4. Clinician collected rather 
than self-collected vaginal swabs were selected to reduce variability in longitudinal sample 
collection technique.  Samples (~ 1 ml stool and vaginal swab tips) will be placed in storage 
containers, labelled, snap frozen, and stored in liquid nitrogen. Liquid nitrogen is continuously 
available at Masafu General Hospital, delivered as needed from a supplier in Iganga, Uganda. 
We will take standard precautions to avoid contamination between samples or from the 
environment. However, samples with high concentrations of microbes, such as stool and 
vaginal samples, are less subject to misrepresentation of results due to contamination than are 
samples with low microbial concentrations.106  
 
Preparation of samples. Stool and vaginal swab samples will be shipped in liquid nitrogen to 
UCSF. For processing, samples will be thawed, placed promptly into lysis buffer (Wizard SV 96 
Genome DNA kit, Promega), and homogenized by beating for 5 min with beads of mixed size. 
The samples will then be centrifuged for 10 min, supernatants transferred to kit plates, and 
DNA purified according to kit instructions.  
 
Absolute abundance of bacterial DNA. For stool specimens, qPCR will be carried out in 
triplicate 10µL reactions with 200 nM 340F/514R primers with SYBRSelect for CFX Master Mix 
(Life Technologies). Absolute quantifications will be determined based against a standard curve 
of amplified E. coli MG1655 DNA,108 adjusting for dilutions during DNA extraction, 
normalization, and PCR reaction preparation, and dividing by the total fecal mass used for DNA 
extraction. Differences in microbiota between samples  from the 3 study arms will be assessed 
using non-parametric tests (Wilcoxon rank sum) and validated, in a representative subset of 
samples, by plating bacteria under anaerobic conditions and counting colony forming units 
and/or by counting cells with FACS, in both cases using established protocols.109  
 
Relative abundance of bacteria. To quantify relative abundances of bacterial taxonomic groups 
in stool and vaginal samples, variable region 4 rRNA will be amplified with barcoded 515F 
(GTGCCAGCMGCCGCGGTAA)/806R (GGACTACHVGGGTWTCTAAT) primers according to the 
methods of the Earth Microbiome Project (earthmicrobiome.org). Amplicons will be quantified 
with PicoGreen, pooled at equimolar concentrations, and purified. Libraries will then be 
quantified and sequenced with a 600 cycle MiSeq Reagent Kit (Illumina) with ~10% control PhiX 
DNA. Samples will be pooled such that the average number of reads/sample is >20,000.  
 
Relative abundance of fungi. Quantification of fungi may be particularly relevant, as by 
depleting bacteria SP may encourage fungal overgrowth. We will amplify the internal 
transcribed spacer (ITS), a region of the nuclear ribosomal RNA cistron shown to enable 

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successful identification across a broad range of fungal taxa.109,110 Barcoded amplicons will be 
cleaned, quantified, and pooled to achieve equal amounts of DNA per sample, as for 16S 
analysis. We will select gel purified 150-500 bp amplicons for sequencing, with ~10% PhiX DNA, 
on the MiSeq platform. Taxonomic assignments of ITS sequencing reads will be performed 
using the UNITE ITS database (unite.ut.ee) and an in-house database of curated ITS1 sequences 
from human-associated fungi.109  
 
Informatic analysis of 16S and fungal microbiota. Informatic methods will be based on those 
optimized by the Turnbaugh lab. Reads will be demultiplexed using QIIME111 before denoising 
and processing with DADA2.112 Taxonomy will be assigned using the DADA2 implementation of 
the RDP classifier using the DADA2 formatted training sets for SILVA123.113 A phylogenetic tree 
will be constructed using MUSCLE and MacQIIME using the FastTree algorithm with midpoint 
rooting. Samples with <10,000 reads will be filtered out. Diversity metrics will be generated 
using Vegan and Phyloseq, with principal coordinate analysis (PCoA) carried out with Ape. 
Principal component analysis (prcomp; PCA) will be carried out on centered log2-ratio 
normalized genus abundances calculated as Aclr=[log2(A1/ga), log2(A2/ga),… log2(An/ga),], where A 
is a vector of read counts with a prior of 0.5 added and ga is the geometric mean of all values of 
A. Cross sectional analysis of significant features will be carried out using ALDEx2114 using 256 
simulations and Benjamini-Hochberg corrected (for false discovery) Wilcoxon p-values (paired 
where appropriate; FDR<0.1 used to indicate statistical significance). Gene content based on 
16S rRNA gene sequencing data will be inferred using PICRUSt.115 KEGG Pathway enrichment 
will be carried out using clusterProfiler.116 Characterization of microbiota will enable 
comparison of gut and vaginal microbiota between members of different study arms and, 
within individuals, evaluation of changes in microbiota over the course of pregnancy. These 
comparisons will allow us to test the hypothesis that study regimens will differentially impact 
on microbiota, thereby affecting birth outcomes. 
 
Metagenomics. Metagenomic assessment (full genome sequencing) has multiple advantages 
over 16S analysis, including improved reliability, more in-depth analysis of bacterial species and 
strains, identification of other organisms (viruses and eukaryotic pathogens), and identification 
of specific features of interest in addition to taxonomic analysis (e.g. presence of drug 
resistance genes). However, due to simplicity and low cost, 16S analysis is still standard in 
microbiome analyses. Due to our cost considerations 16S bacterial and ITS fungal profiling will 
be conducted in years 2-4, and metagenomics assessment primarily in year 5 of this project. 
However, rapid reductions of costs for deep sequencing may enable earlier initiation of these 
studies. Aliquots of DNA analyzed by 16S/ITS sequencing will be fragmented by sonication and 
prepared for sequencing using the Illumina TruSeq PCR-free kit using our established 
protocols.109,117 Barcoded libraries will be pooled into groups of 10, and each pool will be 

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 37 
 
sequenced on a single lane of the Illumina HiSeq 2500 platform (or alternative platforms, as 
appropriate over time) at the UCSF Genomics Core Facility (15-20 Gbp per sample). Quality 
control will be performed in fastq-mcf,109,117 and host reads removed by mapping to the human 
genome with Bowtie.118 Filtered reads will be mapped to the KEGG database119 using 
RAPSearch2.120 Length-normalized read counts will be tallied for each KEGG orthology, with the 
exclusion of reads mapping to metazoans. Adjustment for average genome size will be done 
with MicrobeCensus.121 Differential abundance analysis will be done via the limma package in R, 
using precision weights obtained with voom.122 The differential abundance of KEGG orthologies 
will be further summarized at the module level using CAMERA.123 Hierarchical clustering will be 
performed using the “hclust” function in R. Multiple testing will be adjusted by controlling for 
false discovery rate. Shotgun metagenomic sequencing data will be further leveraged to 
identify bacterial species and strains using Metaphlan2124 and ConStrains.125 As noted above for 
16S and ITS analyses, metagenomic characterization will enable more in depth testing of our 
study hypothesis and also allow exploratory studies of impacts of study arms on specific 
features of microbiota including specific strains of interest, drug resistance determinants, and 
non-bacterial microbiota. 
 
6.10.8  
Reproductive tract infections (RTIs) 
 
To test the hypothesis that study interventions will offer differential impact upon RTIs, enrolled 
subjects will be evaluated for RTIs upon delivery. The following tests, based on state-of-the-art 
technology, will be conducted. Vaginal swab samples will be stored at -80°C, enabling additional 
molecular studies, eg for Mycoplasma, Ureaplasma, Candida, or other pathogens if deemed 
relevant over the course of the project. 
 
Sample collection for RTI analysis. Vaginal samples will be obtained during labor using the 
GeneXpert Vaginal/Endocervical Specimen Collection Kit. The swab will be inserted to ~4-5 cm, 
rotating against the vaginal wall 2-3 times, rolled on a glass microscope slide (for gram staining) 
and then placed into GeneXpert collection tubes containing transport reagent. Molecular 
testing will use GeneXpert qPCR technology, which is ideally suited for field use,100 and provides 
excellent sensitivity and specificity for diagnosis of these infections.101-103 
 
C. trachomatis, N. gonorrhea, T. vaginalis, and GBS. Assays will be conducted based on the 
manufacturer’s recommendations. Assay samples will be collected into GeneXpert transport 
reagent and then pipetted into assay cassettes. Assays will be performed and read, and quality 
control tested following GeneXpert recommendations, as explained in package inserts. 
 

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BV. This syndrome entails overgrowth of normal commensal bacteria by a range of other 
organisms. Gold standard diagnosis requires characterization of flora by gram stain of vaginal 
secretions. Slides obtained as noted above will be air-dried and gram stained. The Nugent 
score, the gold standard for evaluation of BV,104 will be used to score samples. In brief, trained 
microscopists (blinded to clinical information) will assess the presence of large gram-positive 
rods (principally lactobacilli; scored inversely 0-4), small Gram-variable rods (principally 
Gardnerella, scored 0 to 4), and curved Gram-variable rods (principally Mobiluncus, scored 0 to 
2). Scores of 0 to 3 will indicate normal findings, 4 to 6 intermediate findings, and 7-10 BV.105 Of 
note, new point-of-care technologies, measuring pH or sialidase activity are available, and if 
fully validated in the near future, a simpler assay might replace gram staining for diagnosis of 
BV. 
 
6.10.9  
Drug sensitivity of urinary tract infection (UTI) pathogens and Neisseria 
gonorrhea 
 
Drug sensitivity patterns for common bacterial pathogens are poorly characterized in Uganda. 
SP has activity against several bacterial pathogens including N. gonorrhea, and is thought to 
have a similar spectrum of activity as the related antifolate drug trimethoprim-
sulfamethoxazole (TMP-SMX). To test the hypothesis that SP increases the risk of detecting 
antifolate resistance among pathogenic bacteria isolated from women receiving SP for ITPp, we 
will culture urinary tract organisms and N. gonorrhea from study participants and determine 
sensitivities of these pathogens to commonly used antibiotics.  
 
Urinary tract infections. If a study participant presents to the clinic with symptoms of a UTI 
including dysuria, hematuria, suprapubic tenderness, and/or flank pain and a urine dipstick is 
positive for nitrites or leukocyte esterase, a sterile urine specimen will be collected. The woman 
will be treated empirically for UTI after specimen collection per Uganda National Guidelines. 
The urine specimen will be plated on Cystine Lactose Electrolyte Deficient or Oxoid Brilliance 
UTI or another appropriate culture agar. Gram stain will be obtained from colonies with 
sufficient growth, and suspected pathogens will be identified using conventional biochemical 
tests. The susceptibility of the isolates to select antibiotics (e.g. antibiotics prioritized by the 
Uganda National AMR Surveillance Program) will be determined by gradient strip (E-test or 
similar) or disk diffusion devices, and minimum inhibitory concentration (MIC) values will be 
based on the European Committee on Antimicrobial Susceptibility (EUCAST) guidelines. Results 
from the urine cultures and antibiotic sensitivities will be used for research purposes only. 
 

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Neisseria gonorrhea. If N. gonorrhea is detected by GeneXpert from a vaginal swab sample at 
delivery, a sterile cotton swab collected concurrently with the GeneXpert sample at delivery will 
be cultured for N. gonorrhea. This swab will be cultured on G.C. selective agar in a CO2 rich 
atmosphere. Any growth will be provisionally identified using an oxidase test and gram stain, 
and confirmed with N. gonorrhea specific antisera. The susceptibility of the isolates to select 
antibiotics (e.g. antibiotics prioritized by the WHO Global Antimicrobial Resistance Surveillance 
System database) will be determined by gradient strip devices (E-test or similar) or disk 
diffusion devices and minimum inhibitory concentration (MIC) values will be based on the 
EUCAST guidelines.  Results from the vaginal swab cultures and N. gonorrhea antibiotic 
sensitivities will be used for research purposes only. 
 
6.10.10  
Molecular markers of SP and DP resistance 
 
We will survey P. falciparum isolates identified during monthly screening or when study 
subjects present with symptomatic malaria for polymorphisms associated with altered 
sensitivity to SP or DP (Table 3). If malaria prevalence and incidence remain high, as expected, a 
randomly chosen subset of samples will be studied to avoid study of many more samples than 
needed to identify meaningful associations. Assessment of SNPs will initially utilize a multiplex 
ligase detection reaction fluorescent microsphere assay, utilizing the Luminex MAGPIX 
platform. This system is well-validated and used routinely in our labs at UCSF and Kampala for 
this purpose.126,127 In brief, DNA will be extracted from filter paper blood spots using Chelex, 
genes of interest will be amplified by nested PCR, and ligase detection reaction will be 
performed with allele-specific (5’ sequence complementing a sequence attached to a MagPlex-
Tag bead) and common (corresponding to a particular SNP and modified by 5’ phosphorylation 
and 3’ biotinylation) primers. A multiplex reaction will assess SNPs of interest, using established 
protocols (Table 4).126,128 Products will be hybridized to MagPlex-Tag beads, streptavidin-R-
phycoerythrin will be added, and the abundance of alleles will be determined by assessing 
fluorescence. Multiple K13 SNPs are associated with artemisinin resistance in SE Asia; these will 
be assessed by Sanger sequencing of the amplified propeller domain, as previously described.66 
Amplification of pfmdr1 (potentially important for piperaquine resistance, but rare to date in 
Africa)128 and plasmepsin genes (associated with piperaquine resistance in SE Asia),61,62 will be 
assessed by qPCR methods already standard in our laboratory.60,128 Consideration of additional 
polymorphisms may be appropriate over the course of this project, based on advances in our 
understanding of drug resistance. For broader consideration of parasite polymorphisms we are 
exploring deep sequencing approaches, in particular molecular inversion probes129 both to fully 
sequence genes of interest (Table 3) and to consider other potential resistance determinants, 
and these newer technologies will be incorporated as appropriate to best evaluate impacts of 
study regimens on parasite genomes.   

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6.10.11  
Pharmacokinetic assessments.  
 
Plasma drug concentrations for piperaquine, sulfadoxine, and pyrimethamine will be 
obtained to assess for potential drug-drug interactions between DP and SP in the combined 
treatment study arm, assess relationships between drug exposure and QT interval 
prolongation, and quantify drug-exposure response relationships for IPTp and clinical 
outcomes. During phase 1, all women will undergo venous plasma sampling to quantify 
piperaquine, sulfadoxine and pyrimethamine concentrations immediately prior to the first daily 
dose of study drugs (28-day trough) and 2-6 hours after the third daily dose of study drugs 
(corresponding with the piperaquine peak concentration) at 20, 28 and 36 weeks gestational 
age. ECG measurements will be paired with each plasma collection. Due to DHA’s short half-life, 
it is not responsible for the antimalarial protective efficacy of DP, and will only be measured 
after the 3rd daily dose. During phase 2, pre-treatment 28-day trough piperaquine, sulfadoxine, 
and pyrimethamine plasma concentrations may be quantified from a venous sample at weeks 
20, 28, 36, and at delivery, from a capillary stick at weeks 24, 32, and 40 or when malaria is 
diagnosed. Blood samples will be centrifuged to isolate plasma, flash frozen on site, and plasma 
will be stored and shipped to UCSF in liquid nitrogen or on dry ice. After unblinding by an 
individual not involved in patient care or data analysis, drug levels will be quantified at UCSF 
using tandem mass spectrometry (AB Sciex API5000) coupled with Shimadzu Prominence 
20ADXR UFLC pumps using established methods.  The lower limit of quantitation will be 0.5 
ng/mL for piperaquine, 0.5 ng/mL for dihydroartemisinin, 2.7 mcg/mL for sulfadoxine, and 10 
ng/mL for PYR.74,130 
Nonlinear mixed effects modeling will be used to analyze the sparse PK data. Previously 
developed population PK models for each drug during pregnancy will serve as Bayesian priors, 
and collected sparse PK data will be used to update and adapt the PK models. Covariate analysis 
will be conducted, including quantification of any effect of co-administered drugs on population 
PK parameters including relative clearance (CL/F), volumes of distribution (V/F) and relative 
bioavailability. Derived PK parameters for all drugs including area under the curve from 0 to 28 
days for piperaquine, sulfadoxine and pyrimethamine (AUC0-28) or AUC from 0 to 24 hours for 

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dihydroartemisinin. Cmax for pipearquine, and Ctrough for sulfadoxine, pyrimethamine, and 
piperaquine will also be reported for each study drug regimen as recommended by U.S. Federal 
Drug Administration (FDA) guidance. Absence of a drug- drug interaction effect will be defined 
as the 90% confidence interval for the PK parameters of the drug when administered in 
combination falls within 80 to 125% of the parameter when the drug is administered alone. 
Relationships between drug exposure and outcomes of interest including malaria parasitemia 
and prevalence of placental malaria and toxicity (QTc prolongation or other adverse events) will 
also be assessed. If a clinically significant drug-drug interaction is detected after the initial 
analysis of the first 300 participants, simulations of the population PK model will be conducted 
to predict optimal dosing of the drug and a dose adjustment will be considered for Phase 2. 
 
6.10.12  
Immunology Studies 
 
Protective immunity against the adverse effects of malaria in pregnancy increases with 
successive pregnancies. An important question is what impact interventions such as IPTp have 
on the acquisition of this gravidity-dependent immunity against malaria in pregnancy.  We will 
evaluate the malaria-specific immune response in women between our 3 treatment arms. 
Venipuncture blood samples collected in select subjects already undergoing phlebotomy, and 
cord and placental blood collected as above will be made available for immunology studies. Due 
to limitations in cost of processing and storage capacity, samples will not be collected for all 
individuals and/or all time points.  Approximately 15mls of blood will be collected into PAXgene 
RNA or standard blood collection tubes. Following collection, blood will be transported to our 
immunology laboratory located on the campus of Tororo District Hospital and separated into 
plasma and peripheral blood mononuclear cells (PBMCs), following standard protocols. Plasma 
will be stored at -80°C for future immunology studies, which may include measurement of 
levels of cytokines, antibodies, and other features related to the host immune 
response.   PBMCs will be isolated by Ficoll centrifugation and counted, and cells not used 
immediately for assays will be cryopreserved in liquid nitrogen to maintain viability. Fresh 
PBMCs will be used to evaluate the malaria-specific cellular immune response by flow 
cytometry, ELISPOT, surface phenotyping and intracellular cytokine staining, and other assays 
of immune function in our research laboratory in Tororo on the campus of Tororo District 
Hospital. Additional immunologic assays will be performed in research laboratories at the 
Central Public Health Laboratories in Butabika, Uganda. For assays requiring advanced 
instrumentation not available in Uganda (e.g. RNA sequencing, epigenetic profiling, time of 
flight mass cytometry), cryopreserved samples will be shipped to the University of California 
San Francisco or Stanford University.  
 

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6.10.13. Host red blood cell polymorphisms. 
 
Some inherited blood disorders are known to have a protective effect on human health, 
typically with protection in heterozygotes, but harmful effects in homozygotes. In Uganda, 
these polymorphisms are prevalent in some regions, with sickle cell trait (HbAS), alpha-
thalassemia, and the African variant G6PD A- present in 28%, 53%, and 29% of residents of 
Tororo District (a district adjacent to Busia). A1 Studies of associations between inherited blood 
disorders and malaria have largely focused on children. HbAS is known to protect against 
malaria, and particularly against severe malaria, among children.A2-3 The African variant G6PD 
A- was protective among homozygous males in some studies,A4 and alpha-thalassemia 
protected against severe malaria in some studiesA2 but not in others.A3 It is unknown if RBC 
polymorphisms convey antimalarial benefits in pregnant women, but there are data showing 
RBC polymorphisms associated with adverse pregnancy outcomes. Studies have reported a 
higher prevalence of low birth weight among women with alpha- and beta-thalassemia trait 
compared to women without these traits.A5-6 Studies have shown an increased risk of maternal 
mortality, pre-eclampsia, stillbirth, preterm birth and small-for-gestational age among 
pregnancies complicated with the sickle cell disease (HbSS) genotype compared to women 
without HbSS.A7 Associations between sickle cell trait and adverse outcomes have yielded 
conflicting results.A8-9 Malaria and adverse birth outcomes are common in Uganda, and 
understanding relationships between RBC polymorphisms and malaria and birth outcomes 
could allow risk stratifying pregnancies and targeting antimalarial or other pregnancy 
interventions.  
 
We will characterize genetic polymorphisms such as alpha thalassemia, hemoglobin variants, 
and G6PD A-, and assess their associations with maternal and birth outcomes. Maternal 
outcomes of interest will include clinical malaria, placental malaria, asymptomatic parasitemia, 
anemia and pre-eclampsia. Birth outcomes will include composite birth outcomes, as well as, 
low birth weight, preterm birth and small-for-gestational age. Molecular methods will be used 
to characterize host hemoglobin genotypes (including HbAA, HbAC, HbCC, HbSC, HbAS, HbSS), 
the alpha thalassemia 3.7kb deletion (wild type, heterozygous and homozygotes), G6PD A- 
(wild type, heterozygous and homozygotes) and possibly other genes using DNA extracted from 
filter paper blood spots which are collected at routine visits.A1  DNA will be extracted using 
Chelex, DNA for the genes of interest will be amplified by PCR with specific primers for each 
gene of interest, and polymorphisms will be characterized by dideoxy sequencing or analysis of 
gene fragments by electrophoresis after incubation with polymorphism-specific endonucleases. 
If an abnormal hemoglobin genotype is detected by the molecular assays and the participant 
remains under active follow-up during the study, the results will be confirmed by hemoglobin 
electrophoresis for sickle cell trait or sickle cell disease, clinically approved globin gene testing 

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for alpha thalassemia, and G6PD activity testing for G6PD A-. Results will be disclosed to the 
study participant by a study physician. 
 
Added References 
A1. Walakira A, Tukwasibwe S, Kiggundu M, et al. Marked variation in prevalence of malaria-
protective human genetic polymorphisms across Uganda. Infect Genet Evol 2017:281–287. 
A2. Mpimbaza A, Walakira A, Ndeezi G, et al. Associations between erythrocyte polymorphisms 
and risks of uncomplicated and severe malaria in Ugandan children: A case control study. PLoS 
One. 2018;13(9):1–15. 
A3. Kakande E, Greenhouse B, Bajunirwe F, et al. Associations between red blood cell variants 
and malaria among children and adults from three areas of Uganda: A prospective cohort study. 
Malar J 2020;19(1):1–9. 
A4. Guindo A, Fairhurst RM, Doumbo OK, Wellems TE, Diallo DA. X-linked G6PD deficiency 
protects hemizygous males but not heterozygous females against severe malaria. PLoS Med. 
2007; 4(3):516–522. 
A5. Vafaei H, Karimi S, Akbarzadeh Jahromi M, Asadi N, Kasraeian M. The effect of mother’s 
β-thalassemia minor on placental histology and neonatal outcomes. J Matern Neonatal Med 
2020; 0(0):1–8. 
A6. Charoenboon C, Jatavan P, Traisrisilp K, Tongsong T. Pregnancy outcomes among women 
with beta-thalassemia trait. Arch Gynecol Obstet 2016; 293(4):771–774. 
A7. Oteng-Ntim E, Meeks D, Seed PT, et al. Adverse maternal and perinatal outcomes in 
pregnant women with sickle cell disease: Systematic review and meta-analysis. Blood. American 
Society of Hematology; 2015. p. 3316–3325. 
A8. Bryant AS, Cheng YW, Lyell DJ, Laros RK, Caughey AB. Presence of the sickle cell trait 
and preterm delivery in African-American women. Obstet Gynecol 2007; 109(4):870–874. 
A9. Wellenstein WL, Sullivan S, Darbinian J, Ritterman Weintraub ML, Greenberg M. Adverse 
Pregnancy Outcomes in Women with Sickle Cell Trait. AJP Rep 2019; 9(4):E389. 
6.10.14. SARS-CoV-2 Testing. 
 
Rationale for SARS-CoV-2 testing in the DP-SP cohort 
 
SARS-CoV-2 has led to hundreds of millions of cases and millions of deaths worldwide,(1) yet 
the true extent of the SARS-CoV-2 epidemic in sub-Saharan Africa, including Uganda, remains 
unclear.(2) A recent study documented 12% SARS-CoV-2 seroprevalence of HCWs in Western 
Kenya, bordering Uganda(3), however this data was before the recent surge in SARS-CoV-2 
infections in Uganda which peaked in June 2021 at 32 daily new confirmed cases per million 
people (likely an underestimate due to limited testing).(4)  Pregnant women can be an 
important sentinel population for infectious diseases such as SARS-CoV-2, due to frequent 

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interactions with health systems. Further, pregnant women and their fetuses are particularly 
vulnerable to SARS-CoV-2, with infection during pregnancy being associated with placental 
perfusion abnormalities(5), preterm delivery(1), and low birth weight.(6) These are similar to 
effects seen from malaria infection during pregnancy, and it is as yet unknown if an interaction 
between malaria and SARS-CoV-2 co-infection during pregnancy would have a synergistic effect 
on adverse birth outcomes, or if, as has been postulated(7), one may mitigate the other, 
perhaps due to epitope cross-reactivity.(8) For these reasons, we are adding systematic SARS-
CoV-2 testing to the study protocol.  
Current SARS-CoV-2 testing algorithm in Busia, Uganda 
 
Current procedures for SARS-CoV-2 testing are as follows, according to ministry of health  
guidelines: Any participant who is suspected to have contracted or been exposed to SARS-CoV-
2 is referred to Masafu General Hospital for testing. If testing is available, a rapid diagnostic test 
is performed. If the rapid diagnostic test is negative, no further testing occurs. If the rapid 
diagnostic test is positive, then a confirmatory PCR test is performed. If positive, the participant 
would be managed according to local guidelines.(9) However, SARS-CoV-2 testing has been 
limited by availability of testing materials, and the true burden of COVID-19 in this population 
remains unknown; therefore we propose additional sample collection for systematic SARS-CoV-
2 testing in this clinical trial cohort.  
 
Additional sample collection 
 
Participants presenting either at routine or unscheduled visit with fever, history of fever, 
symptoms suggestive of SARS-CoV-2 infection (fatigue/malaise, nausea, vomiting, diarrhea, 
cough, headache, rhinorrhea, abdominal pain, loss of taste/smell), or exposure to a person 
suspected/diagnosed with COVID-19 within the past 14 days, will be referred for rapid antigen 
testing according to local protocols described above. In addition, selected participants will also 
have a nasopharyngeal swab specimen collected, frozen and stored in RNA preservative. As 
resources allow, these banked specimens will be tested later for SARS-CoV-2 PCR and other 
pathogens potentially causing febrile illness using nucleic acid amplification and deep 
sequencing-based methods. One experimental method involves RNA extraction, cDNA library 
synthesis, and meta-genomic next-generation sequencing, using a specific informatic pipeline 
to identify pathogens after filtering out human sequence information. These newer tests may 
be performed at UCSF, or at reference centers depending on availability of technology and 
funding for additional pathogen detection work.  These studies will be for research purposes 
only and will not affect patient care.  
 

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Additionally, serial testing of stored plasma samples will be used to assess for SARS-CoV-2 
seroconversion during pregnancy. Plasma will be obtained from blood spots already being 
collected as part of the approved study protocol, therefore no additional specimens will be 
collected for SARS-CoV-2 serology testing. Seroconversion will be determined as follows: 
plasma at time of delivery will be tested for SARS-CoV-2 antibodies; if positive at the time of 
delivery, stored plasma samples will be tested to determine whether seroconversion occurred 
during pregnancy. Stored specimens from the time of study enrollment in the first trimester will 
first be tested. If negative, then additional stored specimens from each routine visit during 
pregnancy will be tested, working backwards from the time of delivery, until a negative 
specimen is found, in order to determine the gestational age at time of seroconversion. 
 
Added References 
 
1. 
Martinez-Perez O, Prats Rodriguez P, Muner Hernandez M, Encinas Pardilla MB, Perez Perez N, 
Vila Hernandez MR, et al. The association between SARS-CoV-2 infection and preterm delivery: a 
prospective study with a multivariable analysis. BMC Pregnancy Childbirth. 2021;21(1):273. 
2. 
Nachega JB, Sam-Agudu NA, Budhram S, Taha TE, Vannevel V, Somapillay P, et al. Effect of SARS-
CoV-2 Infection in Pregnancy on Maternal and Neonatal Outcomes in Africa: An AFREhealth Call for 
Evidence through Multicountry Research Collaboration. Am J Trop Med Hyg. 2020. 
3. 
Etyang AO, Lucinde R, Karanja H, Kalu C, Mugo D, Nyagwange J, et al. Seroprevalence of 
Antibodies to SARS-CoV-2 among Health Care Workers in Kenya. Clin Infect Dis. 2021. 
4. 
Mathieu E, Ritchie H, Ortiz-Ospina E, Roser M, Hasell J, Appel C, et al. A global database of 
COVID-19 vaccinations. Nature Human Behaviour. 2021;5(7):947-53. 
5. 
Glynn SM, Yang YJ, Thomas C, Friedlander RL, Cagino KA, Matthews KC, et al. SARS-CoV-2 and 
Placental Pathology: Malperfusion Patterns Are Dependent on Timing of Infection During Pregnancy. Am 
J Surg Pathol. 2021. 
6. 
Lassi ZS, Ana A, Das JK, Salam RA, Padhani ZA, Irfan O, et al. A systematic review and meta-
analysis of data on pregnant women with confirmed COVID-19: Clinical presentation, and pregnancy and 
perinatal outcomes based on COVID-19 severity. J Glob Health. 2021;11:05018. 
7. 
Kalungi A, Kinyanda E, Akena DH, Kaleebu P, Bisangwa IM. Less Severe Cases of COVID-19 in Sub-
Saharan Africa: Could Co-infection or a Recent History of Plasmodium falciparum Infection Be 
Protective? Front Immunol. 2021;12:565625. 
8. 
Iesa MAM, Osman MEM, Hassan MA, Dirar AIA, Abuzeid N, Mancuso JJ, et al. SARS-CoV-2 and 
Plasmodium falciparum common immunodominant regions may explain low COVID-19 incidence in the 
malaria-endemic belt. New Microbes New Infect. 2020;38:100817. 
9. 
Revised National Guidelines for Management fo COVID-19, 3rd Edition. In: Health TRoUMo, 
editor. Kampala, Uganda2021. 
 
6.10.15 
Fetal Ultrasounds during and after febrile illness  
 
Rationale for additional ultrasounds during and after febrile illness in the DP-SP study cohort  
 
Malaria in pregnancy is responsible approximately 70% of fetal growth restriction (FGR) cases 
and 36% of preterm deliveries in high transmission areas (1).  Placental malaria is characterized 

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by the sequestration of infected RBCs in the intervillous spaces of the placenta and subsequent 
inflammatory response with infiltration of maternal monocytes/macrophages (2). However, the 
effect of malaria parasitemia on the uteroplacental and fetoplacental blood flow and its 
association with pregnancy outcomes remains unknown. Assessment of the vascular dynamics 
at multiple fetal vessels such as umbilical artery (UA) and middle cerebral artery (MCA) in 
addition to the estimated fetal weight via ultrasounds can provide a more comprehensive 
picture of fetal compensatory mechanisms to ongoing intrauterine insult during malaria in 
pregnancy (3).  
 
Doppler ultrasound allows for non-invasive real-time assessment of maternal-fetal vascular 
dynamics (3,4). Pregnancies complicated by FGR with abnormal UA Dopplers have decreased 
villous development and reduced vascularization, suggesting that aberrant fetoplacental 
vascular development is a mechanism for FGR (5,6). Alterations in UA or MCA Dopplers indicate 
increased placental insufficiency and the resultant fetal physiologic adaptation, respectively 
(3,4). Abnormalities in fetal Dopplers are associated with stillbirth and increased risk of adverse 
long term neurodevelopmental and cardiovascular outcomes (7,8,9). However, the onset and 
progression of these abnormalities after febrile illness remains unknown.  
 
Longitudinal monitoring of fetal blood flow by Doppler ultrasound is an integral part of optimal 
FGR management and reduces perinatal morbidity in high resource settings (4). The effect of 
infected RBC sequestration during acute malaria presenting with febrile illness in the 
intervillous space on the uteroplacental and fetoplacental blood flow is unknown. Dopplers 
studies are safe in pregnancy, relatively easy to perform, and can be performed using 
ultrasound machines used for growth assessment. Utilization of Doppler studies can aid in the 
diagnosis and management of FGR fetus at high risk of perinatal morbidity and mortality and 
may be a useful intervention to reduce perinatal mortality in LMIC. Fetal surveillance with 
Doppler studies in malaria-endemic areas is seldom accessible, despite the high incidence of 
FGR and malaria. For these reasons, we propose to study fetal vascular changes at the maternal 
and fetal interface in pregnancies affected by acute malaria presenting with febrile illness via 
ultrasounds in the DPSP cohort. 
 
Additional data collection:  
 
During febrile illness:  
We will perform additional fetal ultrasound assessments in study participants presenting with 
acute fever at the time of visit or within past 24 hours of their visit. Participants are instructed 
to present to the study clinic with fever, at which time they are evaluated for malaria with 
peripheral blood smear. We will perform a fetal ultrasound at the time of presentation for 
fever. We will then follow the patient approximately every 4 weeks at the time of previously 
scheduled routine assessments (see section 6.5) for additional ultrasound studies until delivery 
to determine if there are acute or chronic fetoplacental adaptations to malaria. For participants 
with abnormal ultrasound findings, weekly follow-up will be continued until > 2 subsequent 
studies are normal.  This data will not require and additional clinical visits and be used for 
research purposes only. Pregnancies will be otherwise managed per local guidelines.  

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Added References 
 
1.  
Katz J, Wu LA, Mullany LC, Coles CL, Lee ACC, Kozuki N, et al. Prevalence of Small-for-
Gestational-Age and Its Mortality Risk Varies by Choice of Birth-Weight-for-Gestation 
Reference Population. PLoS One [Internet]. 2014 Mar 18;9(3):e92074. Available from: 
https://doi.org/10.1371/journal.pone.0092074 
2.  
Zakama AK, Ozarslan N, Gaw SL. Placental Malaria. Curr Trop Med reports. 2020 Sep;1–
10.  
3.  
Abuhamad A, Martins JG, Biggio JR. Diagnosis and management of fetal growth 
restriction: the SMFM guideline and comparison with the ISUOG guideline. Vol. 57, 
Ultrasound in Obstetrics and Gynecology. 2021.  
4.  
Leavitt K, Odibo L, Nwabuobi C, Tuuli MG, Odibo A. The value of introducing 
cerebroplacental ratio (CPR) versus umbilical artery  (UA) Doppler alone for the 
prediction of neonatal small for gestational age (SGA) and short-term adverse outcomes. 
J Matern neonatal Med  Off J  Eur Assoc Perinat Med Fed Asia Ocean Perinat Soc Int Soc 
Perinat Obstet. 2021 May;34(10):1565–9.  
5.  
Chen C-P, Bajoria R, Aplin JD. Decreased vascularization and cell proliferation in placentas 
of intrauterine growth–restricted fetuses with abnormal umbilical artery flow velocity 
waveforms. Am J Obstet Gynecol. 2002;187(3):764–9.  
6.  
Jackson MR, Walsh AJ, Morrow RJ, Mullen JB, Lye SJ, Ritchie JW. Reduced placental 
villous tree elaboration in small-for-gestational-age pregnancies: relationship with 
umbilical artery Doppler waveforms. Am J Obstet Gynecol. 1995;172(2 Pt 1):518–25.  
7.  
Leitner Y, Fattal-Valevski A, Geva R, Eshel R, Toledano-Alhadef H, Rotstein M, et al. 
Neurodevelopmental outcome of children with intrauterine growth retardation: a 
longitudinal, 10-year prospective study. J Child Neurol. 2007;22(5):580–7.  
8.  
Crispi F, Miranda J, Gratacós E. Long-term cardiovascular consequences of fetal growth 
restriction: biology, clinical implications, and opportunities for prevention of adult 
disease. Am J Obstet Gynecol. 2018;218(2):S869–79.  
9.  
Ayoola OO, Omotade OO, Gemmell I, Clayton PE, Cruickshank JK. The impact of malaria 
in pregnancy on changes in blood pressure in children during their first year of life. 
Hypertension. 2014;63(1):167–72.  
 
 
6.10.16 
Sample storage for future use 
 
Certain biologic specimens (placenta, filter paper, whole blood, plasma, stool, vaginal fluid, 
nasopharyngeal swabs) will be stored in our laboratories for future laboratory studies, pending 
participant consent for future use of biological specimens obtained during the course of the 
study. Samples will generally be stored in our laboratory in Tororo, Uganda.  Certain specimens 
requiring long term freezer storage may be transferred for long-term storage to our laboratory 
in Kampala, Uganda (IDRC Molecular laboratory) or a specialized specimen bank with our 
collaborators at UCSF (Department of Medicine, 1001 Potrero Avenue, Building 3, Room 511).   

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With consent for future use, sample retention will generally be open ended. Samples that do 
not have consent for future use, as detailed above, will be destroyed upon completion of 
clinical studies. Samples will be destroyed following SOPs which comply with local regulations 
and guidance. 
 
6.11. 
Co-enrollment Guidelines  
 
The pregnant women from this study may be co-enrolled in observational studies.  They may 
not be co-enrolled in protocols that utilize concomitant study medications. Co-enrollment in 
other studies will be determined on a case-by-case basis by the protocol team. 
 
6.12. 
Management of Adverse Events Potentially Related to Study Drugs 
 
The following section outlines management of adverse events potentially related to study drugs 
(SP or DP) among pregnant women. Given the double-blinded nature of the intervention, study 
clinicians and study participants will not be aware of what study drugs are being administered. 
Therefore, all women will be considered potentially exposed to either study drug during the 
period between when study drugs are first given through completion of follow-up (4 weeks 
post-partum).  
 
6.12.1. 
Grade 1 or 2 Adverse Events 
 
Participants who develop grade 1 or 2 adverse events may continue study drugs.  The study 
clinicians will manage the grade 1 or 2 events according to standard practice.  
 
6.12.2. 
Grade 3 or 4 Adverse Events 
 
Management will be as follows: 
• Repeat observation or lab test within 72 hours of observation or of receiving lab results 
report. 
• Work-up to exclude other causes. 
• For grade 3 or non-life threatening grade 4 adverse events, subjects may continue taking 
study drugs pending clinic visit or repeat laboratory tests.  The study clinician has the option 
of immediately stopping the study drugs if the subject cannot be examined in clinic, if a 
repeat laboratory test cannot be performed within 72 hours, or if the clinician determines 
that the continuation of study drugs is unsafe while awaiting the clinic exam or test results.  
• For grade 4 life-threatening adverse events, study clinicians should hold study drugs 
pending laboratory confirmation. 

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• For all grade 3 or 4 adverse events confirmed by repeat clinical exam or laboratory test 
results, study drugs will be held, and laboratory tests will be repeated every 1-2 weeks, until 
the adverse event resolves to < grade 2 unless there is strong evidence that the adverse 
event is not related to either study drug.  
• If the adverse event persists at grade 3 or 4 for more than 28 days or recurs on re-challenge, 
and the adverse event is thought to be possibly related to one of the study drugs, the study 
drugs will be permanently discontinued.  
 
In the event that study drugs are permanently discontinued, study participants will remain in 
the study, following our intention-to-treat analysis approach. In the event that study drugs are 
permanently discontinued, study clinicians may become un-blinded to the study participant 
assigned treatment regimen if this knowledge may assist in the management of the grade 3 or 4 
adverse event(s) that lead to the permanent discontinuation of the study drugs. 
 
 
 
 
7. MONITORING OF ADVERSE EVENTS AND MANAGEMENT 
 
7.1 
Monitoring and Reporting of Adverse Events 
 
7.1.1 
Definitions 
 
An adverse event is defined as "any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product that does not necessarily have a 
causal relationship with this treatment" (ICH Guidelines E2A).  An adverse event can further be 
broadly defined as any untoward deviation from baseline health, which includes:  
• Worsening of conditions present at the onset of the study 
• Deterioration due to the primary disease 
• Concurrent illness 
• Events related or possibly related to concomitant medications  
(International Centers for Tropical Disease Research Network Investigator Manual, Monitoring 
and Reporting Adverse Events, 2003).    
 
7.1.2 
Identification of Adverse Events 
 
At each scheduled and unscheduled visit to the clinic, study clinicians will assess for adverse 
events in all pregnant women who have started study drugs, according to a standardized case 

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record form.  A severity grading scale, based on toxicity grading scales developed by DAIDS and 
DMID Toxicity Tables, will be used to grade severity of all symptoms, physical exam findings, 
and laboratory results (Appendix H).  All pregnant women, regardless of treatment arm, will be 
assessed using the same standardized case record form.  Adverse event monitoring will occur 
during the period when study drugs are given and up to 4 weeks post-partum.  
 
Data will be captured on the incidence of all adverse events, regardless of severity. For each 
adverse event identified and graded as severe or life threatening and felt to be possibly, 
probably or definitely related to study drugs, an adverse event report form will be completed.  
In addition, an adverse event form will be completed for all serious adverse events and 
unexpected events, regardless of severity. An adverse event report form will not be completed 
for events classified as mild or moderate (unless they are serious or unexpected), as mild and 
moderate symptoms are common and difficult to distinguish from signs and symptoms due to 
malaria and other common illnesses.  The following information will be recorded for all adverse 
experiences that are reported:  
1) Description of event  
2) Date of event onset 
3) Date event reported 
4) Maximum severity of the event 
5) Maximum suspected relationship of the event to study drugs (either SP or DP)   
6) Whether the event is a serious adverse event 
7) Initials of the person reporting the event 
8) Outcome  
9) Date event resolved 
 
7.1.3 Reporting of Adverse Events 
 
Guidelines for reporting of adverse events provided by DMID, UCSF CHR, and the FDA in the 
U.S. and the MU-SBSREC or MU-SOMREC, and NDA in Uganda will be followed as summarized 
in Table 4 below.  
 
Table 4. Guidelines for reporting adverse events 
Institution 
Type of Adverse Events 
When to Report 
UCSF-CHR 
External (off-site) Adverse Events are: 
• 
AEs that occur in study participants who 
are not enrolled at a UCSF or affiliated 
study site. These AEs occur at sites that 
are under the oversight of another IRB. 
• 
Not applicable 
MU-SBSREC or 
MU-SOMREC 
• 
All Serious and Unexpected events 
irrespective of relationship 
• 
Fatal or life-threatening events 
within 3 working days of awareness  
• 
All other SAEs within 7 calendar days 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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NDA 
• 
All Serious and Unexpected events 
irrespective of relationship 
• 
Within 7 calendar days of awareness 
FDA 
• 
Definitely, Probably or Possibly related 
AND BOTH Serious* AND Unexpected± 
• 
For fatal or life-threatening events, 
by telephone or fax within 7 
calendar days of first awareness 
• 
All other reportable events within 15 
calendar days of first awareness 
NIH/DMID 
• 
All Serious and Unexpected events 
irrespective of relationship 
• 
All SAEs must be submitted within 
24 hours of site awareness on an 
SAE form to the DMID 
pharmacovigilance contractor, at 
the following address: 
DMID Pharmacovigilance Group 
Clinical Research Operations and 
Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20817, USA 
 
SAE Hot Line: 1-800-537-9979 (US) or 1-
301-897-1709 (outside US) 
SAE FAX Phone Number: 1-800-275-7619 
(US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com  
 
 
SAE is any AE that results in any of the following outcomes:  
• Death,  
• Life-threatening adverse experience,  
• Inpatient hospitalization or prolongation of existing hospitalization,  
• Persistent or significant disability/incapacity,  
• Congenital anomaly/birth defect, or cancer, or  
• Any other experience that suggests a significant hazard, contraindication, side effect 
or precaution that may require medical or surgical intervention to prevent one of the 
outcomes listed above,  
• Event that changes the risk/benefit ratio of the study. 
 
Unexpected Adverse Event An adverse event is defined as being unexpected if the event 
exceeds the nature, severity, or frequency described in the protocol, consent form and 
investigator brochure (when applicable). An unexpected AE also includes any AE that meets any 
of the following criteria:  
• Results in subject withdrawal from study participation,  
• Due to an overdose of study medication, or  
• Due to a deviation from the study protocol  
 
8. STATISTICAL CONSIDERATIONS 
 
All analyses will be conducted using a modified intention-to-treat approach including all women 
randomized to therapy who have an evaluable outcome of interest. 

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8.1 
Hypothesis 1 
 
We will test the hypothesis that pregnant women who receive IPTp every 4 weeks with a 
combination of SP+DP will have a lower risk of a composite adverse birth outcome compared to 
those who receive either SP or DP alone. 
 
8.1.1. Primary Outcome 
 
The primary outcome will be the risk of having a composite adverse birth outcome defined as 
the occurrence of any of the following: 
Outcomes 
Definition 
Spontaneous abortion Fetal loss at < 28 weeks gestational age 
Stillbirth 
Infant born deceased at > 28 weeks gestational age 
LBW 
Live birth with birth weight < 2500 gm 
Preterm birth 
Live birth at < 37 weeks gestational age 
SGA 
Live birth with weight-for-gestational age < 10th percentile of 
reference population131 
Neonatal death 
Live birth with neonatal death within the first 28 days of life 
 
8.1.2 Analysis 
 
Primary analysis. Using a modified intent-to-treat approach, we will compare the prevalence of 
our primary outcome between the study arms using unadjusted log-binomial models. We will 
explore for any differences of potential confounders between the treatment arms and if 
necessary adjust our analysis using multivariate log-binomial models. 
 
Secondary analyses. We will compare the prevalence of our individual adverse birth outcomes 
using the same approach as our primary analysis.  
 
8.2 
Hypothesis 2 
 
We will test the hypothesis that pregnant women who receive IPTp with SP+DP will have non-
inferior risks of adverse events and measures of tolerability compared to those who receive 
either SP or DP alone. 
 
8.2.1. Primary Outcome 

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The primary outcome will be the incidence of any grade 3-4 AE or SAE per time at risk. Time at 
risk will begin the day study drugs are first given and will end when study participants reach 4 
week postpartum or early study termination. 
 
8.2.2. Secondary Outcomes 
 
Secondary outcomes will include the incidence of individual grade AEs, the incidence of AEs 
related to study drugs, vomiting following administration of study drugs, and measure of non-
adherence with study drugs.  
 
8.2.3. Analysis 
 
Primary analysis. Using a modified intent-to-treat approach, we will compare the incidence of 
our primary outcome between the study arms using a negative binomial regression model. 
Conclusions will be based on tests of non-inferiority (see section 8.4).  
 
Secondary analyses. We will compare the incidence of our other measures of safety using the 
same approach as our primary analysis. Comparisons of repeated measures of tolerability will 
be made using generalized estimating equations with a log-binomial regression model and 
robust standard errors.  
 
8.3 
Hypothesis 3 
 
For objective 3a we will test the hypotheses that pregnant women who receive IPTp regimens 
containing DP will have a lower risk of measures of malaria during pregnancy and at delivery 
but a higher risk of infection with malaria parasites containing mutations associated with DP 
resistance compared to those who receive SP alone. For objective 3b we will test the 
hypotheses that pregnant women who receive IPTp regimens containing SP will have a lower 
risk of RTIs, greater changes in their vaginal/intestinal microbiota, and a higher risk of infection 
with malaria parasites containing mutations associated with SP resistance compared to those 
who receive DP alone. 
 
8.3.1. Outcomes for objective 3a  
 
Outcomes 
Definition 
Placental malaria  
Detection of malaria parasites or pigment by histopathology 

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Incidence of malaria 
during pregnancy 
New episodes of fever plus positive blood smear per person time 
Prevalence of parasitemia 
during pregnancy 
Proportion of routine samples with asexual parasites detected by 
microscopy or qPCR 
Prevalence of anemia 
during pregnancy 
Proportion of routine hemoglobin measurements < 11 g/dL  
Prevalence of markers of 
DP resistance 
Proportion of parasite positive samples with molecular markers of 
DP resistance 
 
8.3.2. 
Outcomes for objective 3b 
 
Outcomes 
Definition 
Prevalence of RTIs at delivery 
Proportion of vaginal samples collected as the time of 
delivery positive for RTIs 
Changes in the vaginal/intestinal 
microbiota 
Measures of relative and absolute abundance of 
microorganisms 
Prevalence of markers of SP 
resistance 
Proportion of parasite positive samples with molecular 
markers of SP resistance 
 
8.3.3. Analyses 
 
Prevalence outcomes measured at a single time point will be compared using a log-binomial 
model. Prevalence outcomes involving repeated measures will be compared using generalized 
estimating equations with a log-binomial regression model and robust standard errors. 
Comparisons of malaria incidence will be made using a negative binomial regression model. 
 
8.4 
Sample size and power  
 
Our sample size calculation is based on testing the primary hypothesis for specific aim 1. Based 
on our previous study from the same site, the risk of the composite adverse birth outcome as 
defined for aim 1 was 22.6% in the SP and 25.1% in the DP arm among women meeting the 
eligibility criteria for this proposal. Given these estimates, we would need to enroll 2757 
women (assuming 15% loss to follow-up) to have 80% power (2-sided alpha = 0.05) to detect a 
25% or greater reduction in the risk of our composite adverse birth outcome in the SP+DP arm 
compared to either of the other arms. For the primary outcome of specific aim 2, we assume 
that the incidence of any grade 3-4 AE or SAE will be 0.37 per person year (SD=0.57) in the SP 

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arm and 0.18 per person year (SD=0.35) in the DP arm, based on our previous study. Given 
these estimates we will have 80% power (1-sided alpha = 0.025) for our non-inferiority margin 
to be set at 0.07 when comparing the SP+DP arm to the SP arm and at 0.03 when comparing 
the SP+DP arm to the DP arm. For the primary outcome of specific aim 3a, we assume that the 
risk of placental malaria will be 61% in the SP arm based on our previous study. Given our 
sample size, we will have 80% power (2-sided alpha = 0.05) to detect a 12% or greater relative 
reduction in the prevalence of malaria parasites at delivery in the SP+DP arm or the DP arm 
compared to the SP arm. 
 
8.5 Data and Safety Monitoring Plan 
 
The proposed study will conform to rigorous standard monitoring procedures, standardized 
reporting of adverse events (Adverse Event Report Forms are completed by study coordinators 
and sent immediately to the investigators), and regular review of the study by a DSMB. The PIs 
have primary responsibility for the overall conduct of the study, including the safety of human 
subjects. The PIs will ensure appropriate (1) conduct of the informed consent process (e.g. that 
informed consent is obtained before proceeding with study procedures); (2) enrollment of 
study subjects; (3) collection and analysis of data; (4) implementation of study procedures to 
ensure consistent monitoring of subjects for possible adverse events; (5) review of adverse 
events and reporting to the DSMB and the IRBs; and (6) maintenance of the privacy and 
confidentiality of study subjects. The PIs maintain ultimate responsibility for the project and for 
the safety of study participants.  The PIs will be in contact with the research team on a regular 
basis to review the progress of the study and address any human subject issues that occur. 
These discussions may involve adverse event prevention measures, recruiting of appropriate 
study subjects, research staff training on protection of human subjects, as well as occurrence of 
adverse events, unexpected incidents, or protocol problems. 
 
8.6 
Data and Safety Monitoring Board 
 
A DSMB will be established by the study team in cooperation with the sponsor to assess at 
intervals the progress of a clinical trial, safety data, and critical efficacy variables and 
recommend to the sponsor whether to continue, modify or terminate a trial. The DSMB will 
have written operating procedures and maintain records of all its meetings, including interim 
results; these will be available for review when the trial is complete. The DSMB will be a 
separate entity from the US and host-country IRBs. The independence of the DSMB is intended 
to control the sharing of important comparative information and to protect the integrity of the 
clinical trial from adverse impact resulting from access to trial information.  DSMB members will 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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not participate in the study as investigators and will not have conflicts of interest regarding the 
study or the investigational product. The composition of the DSMB will include at minimum: 
• DSMB Chair, having experience and expertise in clinical trials 
• Scientist with expertise in malaria and malaria in pregnancy 
• Biostatistician with expertise in clinical trials. 
 
A member of the sponsor, will be invited to attend and thus have access to un-blinded 
information, and control of dissemination of interim trial results within the sponsor 
organization. The DSMB will meet at least annually to review progress of the clinical trial and 
safety data.   
 
The DSMB will review the study for progress and safety. The PIs will provide information that 
will allow the DSMB to review and assess the following: 
• The research protocol, informed consent documents and plans for data safety 
and monitoring;   
• Periodic assessments of data quality and timeliness, participant recruitment, 
accrual and retention, participant risk versus benefit, and other factors that can 
affect study outcome;  
• Factors external to the study when relevant information, such as scientific or 
therapeutic developments, may have an impact on the safety of the participants 
or the ethics of the trial;   
• Study performance to make recommendations and assist in the resolution of 
problems;    
• The safety of the study participants;   
• The safety and scientific progress of the trial;  
• The continuation, termination or other modifications of the trial based on the 
observed beneficial or adverse effects of the treatment under study;   
• The confidentiality of the data and the results of monitoring; and  
• Any problems with study conduct, enrollment, sample size and/or data collection. 
 
The first meeting of the DSMB will take place prior to the initiation of the study to discuss the 
protocol and the Data Safety Monitoring Plan.  Meetings of the DSMB shall be held according to 
the plan outlined above. Meetings shall be closed to the public because discussions may 
address confidential patient data. Meetings may be convened as conference calls as well as in 
person. An emergency meeting of the Board may be called at any time should questions of 
patient safety arise. The DSMB may request the presence of study investigators at such 
meetings.  
 

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The study PIs will distribute study information to the DSMB at least 10 days prior to a scheduled 
meeting. The DSMB may request additions and other modifications to this information on a 
one-time or continuing basis. This information will consist of two parts: (1) information on 
study progress such as accrual, baseline characteristics, and other general information on study 
status and (2) any confidential data on study outcomes, including safety data. A formal report 
from the DSMB should be supplied to the PI within 6 weeks of each meeting. Each report 
should conclude with a recommendation to continue or to terminate the study. This 
recommendation should be made by formal majority vote. A recommendation to terminate the 
study should be transmitted to the PI, IRBs and NIH as rapidly as possible, by immediate 
telephone and fax if sufficiently urgent. In the event of a split vote in favor of continuation, a 
minority report should be contained within the regular DSMB report. 
 
 
8.7 Interim safety analysis 
 
Over the course of the trial, interim safety analyses will be performed when the first 300 
women enrolled have been followed through delivery (phase 1) and then every 6 months 
during phase 2 of the study. A standardized test statistic will be calculated for the incident rate 
ratio of significant adverse events (grade 3/4 & SAEs). If this statistic exceeds the nominal 
critical value calculated using the error spending function (Table 5), then a statistically 
significant result will have been achieved at the time of the analysis.  In that event, the sponsor 
will be notified and a report submitted for review by the DSMB. For the interim safety analyses, 
the study team will present information on recruitment and the results of interim safety 
analyses to the DSMB, which will review the data and recommend a course of action.  
 
Table 5. Schedule of interim safety analysis and boundaries to monitor study outcome   
Number of Evaluable Subjects Accrued   
Test Statistic 
Alpha 
Cumulative  
Alpha 
Lower Bound Upper Bound 
N=300 (phase 1) 
-5.37 
5.37 
0.0000001 0.0000001 
N=791 (phase 2) 
-3.71 
3.71 
0.0002069 0.000207 
N=1282 (phase 2) 
-2.97 
2.97 
0.002844 
0.003051 
N=1773 (phase 2) 
-2.54 
2.54 
0.009046 
0.0121 
N=2264 (phase 2) 
-2.25 
2.25 
0.01605 
0.02815 
N=2757 or 100% of accrual 
-2.04 
2.04 
0.02185 
0.05 
This analysis assumes α=0.05 (two-sided test), O’Brien-Fleming boundaries (DeMets error-
spending function) and 2757 trial participants. We will utilize Programs for Computing Group 
Sequential Boundaries Using the Lan-DeMets Method. 
 

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8.8 Stopping rules 
 
The DSMB will determine whether to stop the study for early evidence of intervention safety 
problems after a thorough review of interim data. Interim reports will provide cumulative 
enrollment figures and cumulative adverse birth outcomes, serious adverse events (classified 
according to grade), sorted by study arm. Brief clinical descriptions of key events will also be 
provided. The PIs will be responsible for immediately reporting to the funding agency any 
temporary or permanent suspension of the project and the reason for the suspension.  
 
9 
DATA COLLECTION AND MONITORING 
 
9.1  Record Keeping  
 
All clinical data will be recorded onto standardized CRFs by study clinicians.  Blood smear results 
will be recorded in a laboratory record book by the study laboratory technologists and then 
transferred to the case record forms by study coordinators, who will review the case record 
forms frequently for completeness and accuracy.  Other laboratory data (CBC, ALT 
measurements) will be entered into the CRFs and hard copies of the original results will be 
stored in a file. Data will be entered directly from CRFs into a computerized database or 
transferred from the CRFs onto standardized data extraction forms and then into a 
computerized database.  All computerized data will be double entered to verify accuracy of 
entry.  Electronic data including all study databases and supporting electronic documentation 
will be archived to large-scale digital tape on a daily basis.  On a monthly basis, a complete 
backup tape will be transported off-site to the Kampala Data Management Center for rotating 
secure storage.  
 
9.2 
Data Quality Assurance and Monitoring  
 
In order to ensure data quality, the study Data Manager will perform a quarterly data quality 
audit.  For this audit a 1% random sample of study forms entered into the data management 
system from the previous 2 weeks will be selected and compared for accuracy with the original 
case-report forms and source documents.  In addition, the study Data Manager will perform 
monthly reviews of the 100% double data entry data verification logs and the data 
management system audit trail log to identify potential data quality issues. The data will be 
owned by the IDRC-UCSF Research Collaboration. 
 
 
10. 
HUMAN SUBJECTS 

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10.1 
Subject Selection Criteria 
 
Study subjects will be HIV-uninfected pregnant women who meet our selection criteria and 
provide informed consent.  We plan to recruit only female Ugandan residents age 16 and 
above.   
 
10.2 
Risks and Discomforts 
 
10.2.1 Privacy 
 
Care will be taken to protect the privacy of subjects, as described in this protocol. However, 
there is a risk that others may inadvertently see patients’ medical information, and thus their 
privacy may be compromised.  
 
10.2.2 Finger Pricks and Venipuncture 
 
Risks of these procedures include pain, transient bleeding and soft-tissue infection. 
 
10.2.3     Risks of Study Medications 
 
10.2.3.1 
Risk of Sulfadoxine-Pyrimethamine 
 
Although technically a combination regimen, SP is generally considered a single antimalarial 
agent, as its success depends on the synergistic action of its two component inhibitors of folate 
synthesis. SP is currently the standard of care for IPTp throughout sub-Saharan Africa. Adverse 
reactions listed on the SP package insert are blood dyscrasias (agranulocytosis, aplastic anemia, 
thrombocytopenia), allergic reactions (erythema multiforme and other dermatological 
conditions), gastrointestinal reactions (glossitis, stomatitis, nausea, emesis, abdominal pain, 
hepatitis, diarrhea), central nervous system reactions (headache, peripheral neuritis, 
convulsions, ataxia, hallucinations), respiratory reactions (pulmonary infiltrates), and 
miscellaneous reactions (fever, chills, nephrosis); based on widespread experience with the 
drug, all of these reactions appear to be uncommon or rare with short-term therapeutic use. 
The best-documented severe adverse effects with SP are cutaneous reactions, primarily noted 
when SP was used for long-term chemoprophylaxis in non-African populations. Reported rates 
of serious reactions to SP in the UK, with long-term use for chemoprophylaxis, were 1:2100, 
with 1:4900 serious dermatological reactions and 1:11,100 deaths.132 Estimated rates of toxicity 
in the US were 1:5000-8000 severe cutaneous reactions and 1:11,000-25,000 deaths.133 Clinical 

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experience suggests that risks of severe toxicity are much lower with malaria treatment 
regimens in Africa.  
 
The WHO currently recommends IPTp with SP in areas with moderate-to-high malaria 
transmission.134 In a recent systematic review and meta-analysis of 7 trials from sub-Saharan 
Africa, IPTp with 3 or more doses of SP was associated with a higher birth weight and lower risk 
of low birth weight compared to 2 doses of SP. In addition there were no differences in the 
rates of serious adverse events between the two groups.  
 
10.5.3.2. Risk of Dihydroartemisinin-piperaquine 
 
Minyt and colleagues conducted a systematic review of DP efficacy and safety for treatment of 
malaria using data from 14 clinical trials involving adults and children.135 There were 2636 study 
participants treated with DP in 13 trials in which safety data were reported. Overall, DP was 
associated with fewer adverse events compared to comparator medications. The most common 
adverse events were dizziness, nausea and vomiting, though generally the medication was well-
tolerated by both adults and children (Table 6). Of note, the only serious adverse events in 
these 14 studies included 5 deaths (2 adults, 3 children) that were thought unrelated to DP.  
 
 
Table 6. Summary of adverse events following treatment with dihydroartemisinin-piperaquine 
 
 
Lwin et al conducted a randomized controlled trial of monthly vs. bimonthly DP IPT among 961 
adults at high risk of malaria at the Northwest border of Thailand.136 Overall, 69% of the 
participants included in the final analysis reported at least one adverse event. There was no 
difference in the proportion of those reporting at least one adverse event among participants in 

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the monthly vs. bimonthly vs. placebo arms. There was an increased risk of joint pain among 
participants randomized to the placebo arm, but otherwise there were no differences noted in 
adverse events by study arm. There was only one serious adverse event not related to the use 
of DP (Table 7).  In a recent systematic review and meta-analysis of repeated treatment, 
monthly DP was found to be effective, well tolerated, and safe.137 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 7. Frequency, incidence, and risk of the 20 most frequently reported adverse events  
 
 
Risks associated with DP during pregnancy. Preclinical animal studies 138-140 and clinical studies 
involving pregnant women 141,142 have not demonstrated significant safety concerns with the 
use of DP. In a recent study from Kenya, serious adverse events were less frequent in women 

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randomized to IPTp with DP compared to women randomized to IPTp with SP.28 In a recent 
study from our group comparing IPTp with 3 dose SP vs. 3 dose DP vs. monthly DP in Tororo 
Uganda, study drugs were well tolerated and there were no significant differences in the risk of 
adverse events.27 Vomiting occurred < 0.2% of the time after administration of study drugs with 
no differences between study arms (Table 8). There were no significant differences in the 
incidence of any adverse events apart from dysphagia, which was higher in the monthly DP arm 
compared to the 3 dose DP arm. All episodes of dysphagia were mild in severity and we are not 
aware of any previous reports of DP being associated with dysphagia. Only one grade 3-4 
adverse event was possibly related to study drugs; an episode of anemia which occurred both 
after the 1st and 2nd doses of monthly DP (study drug was subsequently withheld after the 2nd 
dose)(Table 8). Among 42 women who underwent ECG measurements at 28 weeks gestational 
age, all pre- and post-dosing QTc intervals were within normal limits (< 450 msec) and no 
clinical adverse events consistent with cardiotoxicity occurred during the course of the study. 
Median change in QTc intervals was greater in the 3 dose DP (20 msec) and monthly DP (30 
msec) arms compared to the 3 dose SP arm (5 msec), but these differences were not 
statistically significant. 
 
Table 8. Measures of safety and tolerability from IPTp study conducted in Tororo, Uganda 
Outcome 
Treatment arm 
 
3 dose SP 
3 dose DP 
Monthly DP 
Prevalence measures 
no./total no. (%) 
no./total no. (%) 
no./total no. (%) 
Vomiting following administration of study drugs 
 
 
 
   Observed after administration of 1st dose in clinic 
2/617 (0.32) 
0/542 (0) 
1/594 (0.17) 
   Reported after administration of 2nd or 3rd dose at home 
2/1222 (0.16) 
0/1067 (0) 
5/1180 (0.42) 
Incidence measures 
Events a 
Events a 
Events a 
Individual adverse events of any severity b 
 
 
 
   Abdominal pain 
172 (3.14) 
122 (2.52) 
132 (2.47) 
   Cough 
94 (1.72) 
71 (1.47) 
77 (1.44) 
   Headache 
90 (1.64) 
70 (1.45) 
78 (1.46) 
   Chills 
21 (0.38) 
14 (0.29) 
12 (0.22) 
   Diarrhea 
12 (0.22) 
10 (0.21) 
13 (0.24) 
   Malaise 
16 (0.29) 
9 (0.19) 
8 (0.15) 
   Dysphagia 
9 (0.16) 
2 (0.04) 
14 (0.26) c 
   Vomiting 
8 (0.15) 
8 (0.17) 
8 (0.15) 
   Nausea 
2 (0.04) 
4 (0.08) 
2 (0.04) 
   Urinary tract infection 
3 (0.05) 
2 (0.04) 
2 (0.04) 
   Anorexia 
2 (0.04) 
0 (0) 
4 (0.07) 
Individual grade 3-4 adverse events 
 
 
 
   Anemia 
12 (0.22) 
4 (0.08) 
6 (0.11) 
   Congenital anomaly 
2 (0.04) 
4 (0.08) 
0 (0) 
   Stillbirth 
1 (0.02) 
1 (0.02) 
1 (0.02) 
   Thrombocytopenia 
2 (0.04) 
0 (0) 
0 (0) 
   Threatened abortion 
1 (0.02) 
0 (0) 
0 (0) 
   Retained products of conception 
0 (0) 
1 (0.02) 
0 (0) 
   Preeclampsia 
0 (0) 
0 (0) 
1 (0.02) 
   Hypotension 
0 (0) 
0 (0) 
1 (0.02) 
   Pyelonephritis 
0 (0) 
1 (0.02) 
0 (0) 
   Respiratory distress 
0 (0) 
1 (0.02) 
0 (0) 

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All grade 3-4 adverse events 
18 (0.33) 
12 (0.25) 
9 (0.17) 
Grade 3-4 adverse events possibly related to study drugs 
0 (0) 
0 (0) 
1 (0.02) 
All serious adverse events 
6 (0.11) 
9 (0.19) 
4 (0.07) 
a Number of events (incidence per person year at risk) 
b Includes only those categories with at least five total events 
c P=0.02 comparing monthly DP with 3 dose DP 
 
More recently, data from a study our group completed in Busia, Uganda also provides evidence 
for the excellent safety and tolerability of IPTp with DP.29 In this study, 782 women were 
randomized to IPTp with SP or DP given every 4 weeks.  Vomiting occurred < 0.1% of the time 
after administration of study drugs, with no differences between study arms (Table 9). The 
mean change in the QTc interval after administration of study drugs was significantly higher in 
the DP arm compared to the SP arm (13 ms vs. 0 ms, p<0·0001) (Table 10). Within the DP arm, 
the mean change in the QTc interval was greater at 20 weeks (18 ms) compared to 28 weeks 
(12 ms, p<0·0001) and 36 weeks (10 ms, p<0·0001) gestation. There were seven events in which 
the QTc interval change was > 60 ms (all in the DP arm). No QTc intervals were > 500 ms. No 
episode of QTc prolongation was associated with arrhythmias, symptoms or clinical adverse 
events. There were no significant differences in the incidence of adverse events of any severity 
(Table 9). Among individual grade 3-4 adverse events, the incidence of congenital anomalies 
was higher in the DP arm (10 vs. 2 events, p=0·04). All congenital anomalies observed were 
considered minor and included 10 episodes of polydactyly (9 in the DP arm), a club foot (SP 
arm) and one infant born with a cleft palate and club foot (DP arm). Among ten grade 3-4 
adverse events deemed by the investigators to be possibly related to study drugs, nine were 
QTc prolongation (all in the DP arm) and one was anemia (SP arm).  
     These findings are consistent with a recent review from the WHO which concluded there 
were no significant differences in the risks of cardiotoxicity following treatment with standard 
doses of DP compared to other commonly used antimalarial drugs such as chloroquine and 
amodiaquine. Furthermore, the risks of cardiotoxicity with DP were felt to be similar for healthy 
volunteers and malaria patients.69 In a recent meta-analysis of almost 200,000 individuals who 
received DP, the risk of sudden unexplained death was not higher than baseline and the 
authors concluded that concerns about cardiotoxicity need not limit its use.71 
 

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Table 9. Measures of safety and tolerability from IPTp study conducted in Busia, Uganda 
Outcome 
Treatment arm 
p value 
Monthly SP  
Monthly DP 
Prevalence measures 
Prevalence 
Prevalence 
 
Vomiting following administration of study 
drugs 
   Observed after administration of 1st dose in 
clinic 
   Reported after administration of 2nd dose at 
home 
   Reported after administration of 3rd dose at 
home 
 
1/2057 (0·1%) 
1/2057 (0·1%) 
0/2057 (0%) 
 
6/2124 (0·3%) 
1/2124 (0·1%) 
1/2124 (0·1%) 
 
0·10 
0·98 
0·51 
Continuous measures 
Mean (SD) 
Mean (SD) 
 
Change in QTc (following 3rd dose – before 1st 
dose) in ms 
   All measurements (n=2003)    
   Week 20 gestational age measurements 
(n=697) 
   Week 28 gestational age measurements 
(n=677) 
   Week 36 gestational age measurements 
(n=629) 
 
0 (23) 
0 (23) 
0 (22) 
-1 (23) 
 
13 (23) 
18 (23) 
12 (22) 
10 (22) 
 
<0·0001 
<0·0001 
<0·0001 
<0·0001 
Incidence measures 
Events a 
Events a 
 
Individual adverse events b of any severity c 
   Abdominal pain 
   Cough 
   Headache 
   Pyuria 
   Diarrhea 
   Malaise 
   Vomiting 
   Chills 
 
822 (4·50) 
603 (3·30) 
586 (3·21) 
63 (0·34) 
62 (0·34) 
54 (0·30) 
44 (0·24) 
27 (0·15) 
 
800 (4·22) 
681 (3·59) 
604 (3·19) 
69 (0·36) 
66 (0·35) 
52 (0·27) 
45 (0·24) 
26 (0·14) 
 
0·34 
0·29 
0·87 
0·78 
0·90 
0·72 
0·96 
0·80 
Individual grade 3-4 adverse events b 
   Anemia 
   Proteinuria 
   Congenital anomaly 
   Thrombocytopenia 
   QTc interval prolongation 
   Stillbirth 
   Altered mental status 
   Elevated alanine aminotransferase 
   Respiratory distress 
   Hemorrhage 
   Complicated abortion 
 
28 (0·153) 
10 (0·055) 
2 (0·011) 
7 (0·038) 
0 (0) 
5 (0·027) 
0 (0) 
1 (0·005) 
1 (0·005) 
0 (0) 
0 (0) 
 
8 (0·042) 
6 (0·032) 
10 (0·053) 
3 (0·016) 
9 (0·047) 
2 (0·011) 
2 (0·011) 
1 (0·005) 
0 (0) 
1 (0·005) 
1 (0·005) 
 
0·001 
0·29 
0·04 
0·20 
0·004 
0·26 
0·50 
0·98 
0·50 
0·50 
0·50 
All grade 3-4 adverse events 
54 (0·295) 
43 (0·227) 
0·22 
All serious adverse events  
12 (0·066) 
19 (0·100) 
0·26 
Grade 3-4 adverse events possibly related to 
study drugs 
1 (0·005) 
9 (0·047) 
0·04 
a Number of events (incidence per person year at risk) 
b Individual adverse events were assessed and graded according to standardized criteria at every visit to 
the study clinic.143  
c Grade 1-4 adverse events. Includes only those with at least 50 total events 
 

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
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Although no clinical trials have studied SP+DP, we have shown that another antifolate 
combination, trimethoprim-sulfamethoxazole (TMP-SMX), given daily with monthly DP was as 
safe and well tolerated as daily TMP-SMX alone among 200 HIV infected pregnant women 
(Table 10).70 Available studies report no clinically relevant drug interactions for DP and SP, and 
there is no evidence that they alter the disposition of other drugs, except for one small study 
reporting increased exposure to halofantrine, a CYP P450 3A4 substrate, after a single dose of 
SP.81,83-86 In pharmacokinetic studies, our group and others have reported that pregnancy 
decreases piperaquine and sulfadoxine exposure, with mixed results for pyrimethamine, 
suggesting no pregnancy-associated increased risk, although data are limited.72-76 Piperaquine is 
metabolized by CYP P450 3A4 and 2C8.77-79  Sulfadoxine is cleared renally through glomerular 
filtration80,81 and pyrimethamine is primarily hepatically metabolized, with 15–30% excreted 
unchanged in urine.82  
 
Table 10. Measures of safety and tolerability comparing daily TMP-SMX alone vs daily TMP-
SMX + monthly DP during pregnancy 
Outcome 
Treatment arm 
P Value 
Daily TMP-SMX 
Daily TMP-SMX + monthly DP 
Prevalence measures 
n/N (%) 
 
Vomiting of study drugs 
   After administration of 1st dose in clinic 
   After administration of 2nd or 3rd dose at home 
 
0/482 (0%) 
0/944 (0%) 
 
1/468 (0.2%) 
2/915 (0.2%) 
 
0.49 
0.24 
Incidence measures 
Number of events  
(incidence per person year at risk) 
 
Individual adverse events of any severitya 
   Abdominal pain 
   Cough 
   Headache 
   Anemia 
   Malaise 
   Diarrhea 
   Chills 
   Thrombocytopenia 
   Vomiting 
   Anorexia 
 
85 (1.85) 
69 (1.50) 
39 (0.85) 
42 (0.91) 
27 (0.59) 
17 (0.37) 
11 (0.24) 
11 (0.24) 
7 (0.15) 
6 (0.13) 
 
74 (1.69) 
55 (1.25) 
43 (0.98) 
40 (0.91) 
24 (0.55) 
14 (0.32) 
11 (0.25) 
11 (0.25) 
7 (0.16) 
4 (0.09) 
 
0.57 
0.34 
0.51 
0.99 
0.80 
0.76 
0.91 
0.91 
0.94 
0.58 
Individual grade 3-4 adverse events 
   Anemia 
   Congenital anomaly 
   Stillbirth 
   Respiratory distress 
   Altered mental status 
   Elevated alanine aminotransferase  
 
10 (0.22) 
1 (0.02) 
0 (0) 
1 (0.02) 
0 (0) 
0 (0) 
 
7 (0.16) 
4 (0.09) 
1 (0.02) 
0 (0) 
1 (0.02) 
1 (0.02) 
 
0.44 
0.22 
0.99 
0.99 
0.99 
0.99 
All grade 3-4 adverse events 
12 (0.26) 
14 (0.32) 
0.74 
All serious adverse events 
4 (0.09) 
6 (0.14) 
0.55 
a Includes only those categories with at least ten total events 
 
 
 
 
 

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10.3 
Treatment and Compensation for Injury  
 
If a participant is injured as a result of being in this study, treatment will be available through 
Masafu General Hospital.  If there is need for compensation for injury as a result of 
participation in the study, this will be covered by insurance which is purchased for every study 
participant by IDRC.  Makerere University, UCSF, and DMID do not normally provide any other 
form of compensation for injury.   
 
10.4 
Costs to the Subjects  
 
There will be no cost to the participant for participation in this study. 
 
10.5 
Reimbursement of Subjects 
 
Participants will not be paid for their participation in the study. The study will provide all 
routine medical care, including evaluations, medications available in our clinic, and cost of any 
transportation free of charge. In addition, we will reimburse the cost of referrals made by study 
physicians to other clinics, including the resulting cost of the visit, most diagnostic tests 
(including laboratory test, X-rays, and ultrasounds), and medications, using available funds. 
However, reimbursement of all diagnostic tests and treatment recommended outside the study 
clinic cannot be guaranteed in all circumstances. 
 
10.6 
IRB Review and Informed Consent  
 
This protocol, all procedures and consent forms, and any subsequent modifications must be 
reviewed and approved by the IRBs of all the participating institutions in both the U.S. and in 
Uganda. This includes the UCSF CHR, the MU School of Medicine - Research and Ethics 
Committee (MU-SOMREC) or MU School of Biomedical Sciences - Research and Ethics 
Committee (MU-SBSREC), Stanford University Research Compliance Office, and the UNCST. 
 
All consent forms will be translated into the local languages (Samia, Swahili, Luganda, and 
English) and back-translated into English to ensure correct use of language. Consent forms will 
be read aloud to parents by trained study interviewers. The informed consent will describe the 
purpose of the study, all the procedures involved, and the risks and benefits of participation. 
Study physicians will ask parents/guardians of study participants to summarize the study and 
explain the reasons why they want to participate. Either a signature or a thumbprint (for 
participants who cannot read) will be acceptable to confirm informed consent for participation 
in the study.  

Optimizing IPTp in Uganda: Protocol Version 8.0, September 11, 2023  
 67 
 
 
 
10.7 
Publication of Research Findings 
 
The findings from this study may be published in a medical journal.  No individual identities will 
be used in any reports or publications resulting from the study. The researchers will publish 
results of the trial in accordance with NIH, UNCST, UCSF, Stanford University, and Makerere 
University guidelines.  
 
10.8 
Biohazard Containment 
 
As the transmission of HIV and other blood-borne pathogens can occur through contact with 
contaminated needles, blood, and blood products, appropriate blood and secretion precautions 
will be employed by all personnel involved in the drawing of blood, exposure to blood and 
secretions, and shipping and handling of all specimens for this study. We will follow the current 
guidelines set forth by the Centers for Disease Control and Prevention and the NIH.  All 
infectious specimens will be transported using packaging mandated in the Federal Code of 
Regulations, CDC 42 CFR Part 72. 
 
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12.  
APPENDICES 
 
Appendix A. Information Sheet  
A STUDY ON PREVENTING MALARIA IN PREGNANT WOMEN 
 
 
Makerere University in Uganda and the University of California, San Francisco in 
the United States are combining efforts in Busia district, Uganda to study new 
ways of using malaria drugs to prevent malaria in pregnant women.  
 
Malaria during pregnancy can have a harmful effect on you or your child. We 
want to study 2 different malaria drugs alone and in combination to see if they 
can be used to prevent malaria if taken during pregnancy. 
 
Our study clinic is located at Masafu General Hospital next to the antenatal clinic 
and is open every day from 8:00 am to 5:00 pm 
 
We want to enroll pregnant women who are at least 16 years old and follow them 
during pregnancy.  
 
Women will receive free medical care.  
 
We shall also give reimbursement for transport to and from our study clinic 
 
For more information, please come to our study clinic where our doctors will be 
happy to talk to you and see if you can be in the study. 
UCSF

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Appendix B.  Determination of Gestational Age 
 
Gestational age will be based on the first day of the last menstrual period (LMP) and the earliest 
available ultrasound performed at > 6 weeks gestation.  The estimated due date (EDD) is 
calculated as 280 days following the LMP. 
 
If the first available ultrasound is consistent with a gestational age of 6 weeks 0 days to 8 weeks 
and 6 days, and the ultrasound gestational age is within 5 days of that given LMP, then the LMP 
will be used to determine gestational age.  However, if the ultrasound gestational age differs 
from the LMP gestational age by more than 5 days, then the ultrasound will be used to 
determine gestational age. 
 
If the first available ultrasound is consistent with a gestational age of 9 weeks 0 days to 15 
weeks 6 days, and the ultrasound gestational age is within 7 days of that given LMP, then the 
LMP will be used to determine gestational age.  However, if the ultrasound gestational age 
differs from the LMP gestational age by more than 7 days, then the ultrasound will be used to 
determine gestational age. 
 
If the first available ultrasound is consistent with a gestational age of 16 weeks to 21 weeks 6 
days, and the ultrasound gestational age is within 10 days of that given by the LMP, then the 
LMP will be used to determine gestational age.  However, if the ultrasound gestational age 
differs from the LMP by more than 10 days, then the ultrasound will be used to determine 
gestational age. 
 
If the first available ultrasound is consistent with a gestational age of 22 weeks to 27weeks 6 
days, and the ultrasound gestational age is within 14 days of that given by the LMP, then the 
LMP will be used to determine gestational age.  However, if the ultrasound gestational age 
differs from the LMP by more than 14 days, then the ultrasound will be used to determine 
gestational age. 
 
If the first available ultrasound is consistent with a gestational age of 28 weeks or more, and the 
ultrasound gestational age is within 21 days of that given by the LMP, then the LMP will be used 
to determine gestational age.  However, if the ultrasound gestational age differs from the LMP 
by more than 21 days, then the ultrasound will be used to determine gestational age.  Care 
here should be taken to rule out intrauterine growth restriction. 
 
If the LMP is not known, then the earliest ultrasound performed at > 6 weeks will be used to 
determine gestational age. 
 
Participants must be between 12 weeks 0 days to 20 weeks 6 days of gestational age by best 
obstetric determination at the time of enrollment.  

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Appendix C. Household survey 
 
The household survey will be administered through a completely paperless QDS software 
system using hand-held tablet computers. A list of questions that will be used in the survey is 
provided below.  
 
Ques. No. 
Variable Name 
Question 
Section 1: Identification 
1 
VISDATE 
Date of final visit 
2 
STARTIME 
Start time of interview 
4 
BC 
Cohort Number 
3 
PPTIDA 
Study Participant ID 
5 
PPTID 
To ensure data integrity, please re-enter the Participant ID. 
7 
INTNUM 
Interviewer number 
8 
AGREE 
Are you going to conduct the interview with this household? 
Section 2: Household Characteristics 
9 
SWATER 
What is the main source of drinking water for members of your household? 
10 
OTHERSCS 
Specify other source of water 
11 
TFACLTY 
What kind of toilet facility do members of your household usually use? 
12 
OTHERFCY 
Specify other kind of toilet facilities 
13 
ELECTRIC 
Does your household have... 
 
...Electricity? 
14 
RADIO 
...Radio? 
15 
CASSETTE 
…Cassette player? 
16 
TV 
…Television? 
17 
MOBILE 
…Mobile phone? 
18 
PHONE 
…Fixed phone? 
19 
FRIDGE 
…Refrigerator? 
20 
TABLE 
…Table? 
21 
CHAIR 
…Chairs? 
22 
SOFA 
…Sofa set? 
23 
BED 
…Bed? 
24 
CUPBOARD 
…Cupboard? 
25 
CLOCK 
…Clock? 
26 
FUELTYPE 
What type of fuel does your household mainly use for cooking? 
27 
OTHERFUE 
Specify other type of fuel used 
28 
SENERGY 
What is the main source of energy for lighting in the household? 
29 
OTHERENG 
Specify other source of energy for lighting 
30 
MMFLOOR 
MAIN MATERIAL OF THE FLOOR  
  
 RECORD OBSERVATION. 
  
 MARK ONLY ONE. 
31 
OTHERMMF 
Specify other material of the floor 
32 
MMROOF 
MAIN MATERIAL OF THE ROOF. 
   
 RECORD OBSERVATION. 
   
 MARK ONLY ONE.  

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33 
OTHERMMR 
Specify other material of the roof 
34 
MMEWALLS 
MAIN MATERIAL OF THE EXTERIOR WALLS. 
   
 RECORD OBSERVATION. 
 
 MARK ONLY ONE.  
35 
OTHERMME 
Specify other material of the exterior walls 
36 
HHROOMS 
How many rooms in your household are used for sleeping? 
 
(INCLUDING ROOMS OUTSIDE THE MAIN DWELLING) 
 
If there are 15 or more rooms, enter 15 
37 
HHSPACES 
How many sleeping spaces like mats, mattresses, or beds are available in your 
household? 
 
If there are 25 or more sleeping places, enter 25 
38 
WATCH 
Does any member of your household own or have… 
 
 …A watch? 
39 
BICYCLE 
….A bicycle? 
40 
SCOOTER 
…A motorcycle or motor scooter? 
41 
CART 
…An animal-drawn cart? 
42 
CAR 
…A car or truck? 
43 
MBOAT 
…A boat with a motor? 
44 
NOMBOAT 
…A boat without a motor? 
45 
BANKACCO 
…A bank account? 
46 
NUMALAND 
How many acres of agricultural land do members of this household own? 
47 
DMARKT 
How far is it to the nearest market place? 
48 
HHMEALS 
Now I would like to ask you about the food your household eats. 
How many meals does your household usually have per day? 
49 
HHNUMT 
In the past week, on how many days did the household eat meat? 
50 
HHPSF 
How often in the last year did you have problems in satisfying the food needs 
of the household? 
51 
DHFCTY 
How far is it to the nearest health facility? 
52 
MTHFCTY 
If you were to go this facility, how would you most likely go there? 
53 
OTHERMTH 
Specify other means of transport to the health facility 
54 
PSPRAY 
At any time in the past 12 months, has anyone asked permission to come into 
your dwelling to spray the interior walls against mosquitoes? 
55 
GPSPRAY 
Did you grant them permission to spray the interior walls of your dwelling? 
56 
RGSPRAY 
What was the primary reason that you did not grant permission to spray the 
interior walls of your dwelling against mosquitoes? 
57 
OTHERRGS 
Specify other reasons for not granting permission to spray the interior walls of 
your dwelling against mosquitoes 
58 
TSPRAY 
How many months ago was the dwelling last sprayed? 
59 
WSPRAY 
Who sprayed the dwelling? 
60 
OTHERWSP 
Specify other people who sprayed the dwelling 
61 
DSPRAY 
Did you pay for the dwelling to be sprayed? 
62 
PPWALLS 
Since the spraying, have the walls in your dwelling been plastered or painted? 
63 
TPPWALLS 
How many months ago were the walls plastered or painted?  
 
64 
MSPRAY 
In the past 12 months, have you seen or heard any messages about spraying 
the interior walls of your dwelling against mosquitoes? 

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65 
MSGA 
Where did you hear or see message(s)? 
 
...Radio? 
66 
MSGB 
...TV? 
67 
MSGC 
...Newspaper/Leaflet? 
68 
MSGD 
…Health worker/CMD? 
69 
MSGE 
…Neighbor/Relative/Friend 
70 
MSGF 
...Community Leader? 
71 
MSGG 
…Village public address system 
72 
MSGH 
...Don't know 
73 
MSGI 
…Other 
74 
OTHERMSG 
Specify other 
75 
AHWKER 
Is there a community health worker (community medicine distributor/CMD, 
village health team/VHT, community own resource person/CORP) who 
distributes malaria medicines in your village or community? 
76 
AMCHWKER 
Does the community health worker currently have malaria medicines available? 
Section 3: Study Participants Sleeping Area Characteristics All questions in this section will be repeated with variable names entopen1, entcov1, etc. for 
each entryway and window in the study participant’s room  
77 
SRENTRY 
OBSERVATION: How many entryways into the room are there? 
78 
ENTOPN1 
OBSERVATION: Does it open to the outside? 
79 
ENTCOV1 
OBSERVATION: Is the entry way covered? 
80 
ETMM1 
OBSERVATION: Main material is the covering made of. 
81 
OTHCOV1 
Specify Other covering type 
82 
SRWINDOW 
OBSERVATION: How many windows are in the room? 
83 
WNDCOV1 
OBSERVATION: Is the window covered? 
84 
WNDOPN1 
OBSERVATION: Does the window open to the outside 
85 
SREAVES 
OBSERVATION: Does the room have eaves? 
86 
EAVESCOV 
OBSERVATION: If room has eaves, are the eaves covered? 
87 
EAVESOPN 
OBSERVATION: Do the eaves open to the outside? 
88 
AIRBRICK 
OBSERVATION: Does the room have airbricks? 
89 
AIRBRCOV 
OBSERVATION: If the room has airbricks, are the airbricks covered? 
90 
AIRBROPN 
OBSERVATION: Do the airbricks open to the outside? 
91 
AIRBRNUM 
OBSERVATION: How many airbricks are in the room? 
92 
SLEEP 
Where does the study participant usually sleep? 
93 
OTHERSL 
Specify other sleeping area 
94 
SRSLNUM 
Usually, how many people sleep in the same room as the study participant 
(excluding the study participant)? 
95 
SRSLNUM5 
How many of those people are under 5 years old (excluding the study 
participant)? 
96 
SLAREAS 
How many sleeping areas are in the room where the study participant sleeps? 
97 
SASLNUM 
How many people sleep in the same bed/sleeping area as the study participant 
under the mosquito net (excluding the study participant)? 
98 
SASLNUM5 
How many of those people are under 5 years old (excluding the study 
participant)? 
Section 4: Bednets All questions in this section will be repeated with variable names obs2, mnths2, where2, etc. for each mosquito net in the household 
(hhnumnet) 
99 
HHAMNETS 
IMMEDIATELY BEFORE Enrollment, did your household have any mosquito nets 
that can be used while sleeping? 
100 
HNUMNETS 
IMMEDIATELY BEFORE study enrollment, How many mosquito nets did your 
household have? 
101 
OBS1 
May I have a look at (all) the net(s) to establish the brand? 
102 
MNTHS1 
How many months ago did your household obtain the mosquito net? 
 

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103 
WHERE1 
Where did you get the mosquito net from? 
104 
SPCFRO1 
Specify other sources of the mosquito net 
105 
BRAND1 
OBSERVE OR ASK THE BRAND OR TYPE OF MOSQUITO NET. 
106 
OTHERB1 
Specify other brands or types of mosquito net 
107 
SMNET1 
Since you got the mosquito net, was it ever soaked or dipped in a liquid to 
repel mosquitoes or bugs? 
108 
TSMNET1 
How many months ago was the net last soaked or dipped? 
109 
SLPNET1 
Did anyone sleep under this mosquito net last night? 
110 
NUSED1A 
What are some of the reasons why this net was not used? 
 
...Too hot 
111 
NUSED1B 
...Don't like smell 
112 
NUSED1C 
...No mosquitoes 
113 
NUSED1D 
...Net too old/too many holes 
114 
NUSED1E 
...Net not hung 
115 
NUSED1F 
...Net too dirty 
116 
NUSED1G 
...Net no longer kill insects 
117 
NUSED1H 
...Don’t know 
118 
NUSED1I 
...Other 
119 
NTHUNG1 
If not hung, why not? 
120 
OTHRNT1 
Specify other reason why the net was not hung. 
Section 5: Interviewer Details 
122 
STOPTIME 
End time of interview 
123 
VSTATUS 
Result of Visit 
124 
OVSTATUS 
Specify other result 
125 
TOTVISIT 
Total number of visits 
126 
COMMENTS 
Interviewer's Comments 
 

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 83 
 
Appendix D. Administration of study drugs and placebos  
 
Timing of administration of study drugs during pregnancy 
Weeks of 
gestation 
Treatment arm 
SP every 4 weeks 
DP every 4 weeks 
SP + DP every 4 weeks 
16 
SP + DP placebo 
DP + SP placebo 
SP + DP 
20 
SP + DP placebo 
DP + SP placebo 
SP + DP 
24 
SP + DP placebo 
DP + SP placebo 
SP + DP 
28 
SP + DP placebo 
DP + SP placebo 
SP + DP 
32 
SP + DP placebo 
DP + SP placebo 
SP + DP 
36 
SP + DP placebo 
DP + SP placebo 
SP + DP 
40 
SP + DP placebo 
DP + SP placebo 
SP + DP 
 

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Appendix E. WHO Criteria for Severe Malaria  
 
Detection of malaria parasites by microscopy and any of the following: 
 
Cerebral malaria (unarousable coma not attributable to any other cause) 
Generalized convulsions (> 3 convulsions over 24 hours period) 
Severe normocytic anemia (Hb < 5 gm/dL) 
Hypoglycemia 
Metabolic acidosis with respiratory distress 
Fluid and electrolyte disturbances 
1. Acute renal failure 
2. Acute pulmonary edema and adult respiratory distress syndrome (ARDS) 
3. Circulatory collapse, shock, septicemia ("algid malaria") 
4. Abnormal bleeding 
5. Jaundice 
 

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Appendix F. Uganda Ministry of Health Guidelines for Routine Care of Pregnant and 
Postpartum Women, and Newborns 
 
In addition to receiving medical care as described above in the protocol, women and their 
newborns will receive standard routine prenatal and postpartum care according to Uganda 
Ministry of Health guidelines. These standard procedures are subject to availability at the local 
health care facilities.  
 
Routine antenatal care. Women enrolled in the study will receive routine care as designated in 
the Uganda Ministry of Health Guidelines. Routine antenatal care includes screening and 
treating for syphilis and syndromic management of sexually transmitted infections (STIs).  
Pregnant women will receive iron and folic acid supplementation. In addition, women will be 
given multivitamins that will be given once daily. In addition, women receive mebendazole 
500mg as a single dose as early as possible after the 1st trimester. Each antenatal visit also 
includes blood pressure assessment and urine dipstick for proteinuria.  
 
Routine intrapartum/delivery care. Routine delivery care for in-hospital births will include 
labor management by the midwifery staff and management of obstetrical complications as per 
Ministry of Health guidelines. Immediate postpartum infant care will include polio and BCG 
immunization, ophthalmic tetracycline, and vitamin K. 
 
Routine postpartum care.  All postpartum women will receive vitamin A supplementation 
(200,000 IU) immediately following delivery. Depending on clinical circumstances and based on 
local standard of care, women may receive 2 weeks of multivitamins twice a day. Common 
indications for postpartum multivitamins include anemia, postpartum hemorrhage and 
prolonged labor. Women will be seen at 1 week after delivery as per Ugandan standards of 
care. Women also undergo a 4-week postpartum visit as part of routine care. These visits 
include an abdominal exam, syndromic management of STIs, and follow-up on any obstetrical 
complications that occurred. In addition, women receive vitamin A at this visit, if not given 
immediately postpartum, and are continued on iron and folic acid supplementation. Pelvic and 
breast exam will be done if clinically indicated. Contraceptive counseling is performed at this 
visit as is a nutritional assessment and infant feeding and support.  
 
 

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Appendix G. Schedule of routine assessments and procedures in pregnant women 
Evaluations and Interventions 
Enrollment 
Weeks of gestation 
Delivery 
1 and 4 weeks 
postpartum 
16* 
20 
24 
28 
32 
36 
40 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
HIV testing1 
X 
 
 
 
X 
 
 
 
X 
 
Obstetrical ultrasound2 
X 
 
 
X 
 
X 
X 
 
 
 
Blood collected by phlebotomy 
for blood smear, qPCR, dried 
blood spots, CBC, plasma for 
PK, and immunology studies 
X 
 
X** 
 
X 
 
X 
 
X 
 
Blood collected by finger prick 
for blood smear, qPCR, plasma 
for PK, and dried blood spot 
 
X 
X** 
X 
 
X 
 
X 
 
 
Routine assessment in the 
study clinic3 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
Administration of study drugs 
 
X 
X 
X 
X 
X 
X 
X 
 
 
Collection of cord blood and 
placental blood/tissue 
 
 
 
 
 
 
 
 
X 
 
Labor and delivery 
documentation4 
 
 
 
 
 
 
 
 
X 
 
Collection of gut and vaginal 
samples for microbiome studies 
X 
 
 
 
 
X 
 
 
 
 
Collection samples for testing of 
RTIs 
 
 
 
 
 
 
 
 
X 
 
Standard Care 
Obstetrical exam5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Syphilis screening 
X 
 
 
 
 
 
 
 
 
 
Iron and Folic Acid 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Prenatal vitamins 
X 
 
 
 
 
 
 
 
 
 
Mebendazole6 
 
X 
 
 
 
 
 
 
 
Vitamin A7 
 
 
 
 
 
 
 
 
X 
 
Insecticide treated bednet 
X 
 
 
 
 
 
 
 
 
 
* Only if study subject enrolled prior to 16 weeks gestation 
** For participants enrolled between 12-<18 weeks gestational age, blood will be collected by 
phlebotomy at the time of their week 20 routine visit. For participants enrolled between 18-20 
weeks gestational age, blood will be collected by finger prick at the time of their week 20 
routine visit. 
 
Explanation of maternal schedule of events  
1. HIV test will be done at enrollment and documented. A repeat rapid HIV test will be done at 
28 weeks gestational age and delivery. HIV testing shall be done using standard rapid HIV-
testing algorithm.   
2. Ultrasound will be done to confirm intrauterine pregnancy and estimate gestational age at 
enrollment and to assess fetal growth at 24, 32 and 36 weeks gestational age. See Appendix 
B for dating criteria. 
3. Targeted physical exam will include anthropometric measurements (e.g. weight), mid-upper 
arm circumference, and vital signs (i.e. temperature, pulse, and blood pressure). 
Measurement of height at the enrollment visit only. 
4. Labor & Delivery documentation will include: Peripartum history, mode of delivery, Apgar 
scores (when available), weight, length, and head circumference of the child at birth, 
approximate gestational age, duration of labor, maternal blood pressure during labor, signs 

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of fetal distress (presence of meconium), summary of events in first days of life (including 
feeding, breathing patterns, jaundice, lethargy, or any additional abnormal findings), 
duration of admission if delivered in hospital. 
5. Obstetrical exam includes estimation of gestational age at study entry, fundal height 
measurement, fetal heart tones and urine dipstick for protein. A cervical exam will also be 
performed at screening and during antepartum study visits as clinically indicated. 
6. Mebendazole is typically given as 500mg as a single dose as early as possible after the 1st 
trimester (16 or 20 week visit). 
7. Vitamin A supplementation is dosed as 200,000 IU. 
 
 

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Appendix H.    Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric 
Adverse Events (Corrected Version 2.1 - July 2017) 
 
Citation:  
U.S. Department of Health and Human Services, National Institutes of Health, National Institute of 
Allergy and Infectious Diseases, Division of AIDS. Division of AIDS (DAIDS) Table for Grading the Severity 
of Adult and Pediatric Adverse Events, Corrected Version 2.1. [July 2017]. Available from: 
https://rsc.niaid.nih.gov/sites/default/files/daidsgradingcorrectedv21.pdfpdf 
 
Selected sections of the above document are listed here: 
 
Introduction 
The Division of AIDS (DAIDS) oversees more than 300 clinical trials domestically and 
internationally, which evaluate the safety and efficacy of therapeutic products, vaccines, and other 
preventive modalities. Adverse event (AE) data collected during these clinical trials form the basis for 
subsequent safety and efficacy analyses of pharmaceutical products and medical devices. Incorrect and 
inconsistent AE severity grading can lead to inaccurate data analyses and interpretation, which in turn 
can impact the safety and well-being of clinical trial participants and future patients using 
pharmaceutical products. 
Over the years, DAIDS scientific knowledge and experience have expanded, necessitating 
revisions of the DAIDS grading table, which serves as a guide for assessing the severity of AEs (including 
clinical and laboratory abnormalities) in participants enrolled in DAIDS-sponsored and -supported clinical 
trials. The Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events, 
Corrected Version 2.1 (July 2017) updates and replaces version 2.1 (March 2017). 
 
Instructions for Use 
 
General Considerations  
The Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events, 
Version 2.1 consists of parameters, or AEs, with severity grading guidance that are to be used in DAIDS 
clinical trials for safety data reporting to maintain accuracy and consistency in the evaluation of AEs. The 
term “severe” is not the same as the term “serious” in classifying AEs. The severity of a specific event 
describes its intensity, and it is the intensity which is graded. Seriousness, which is not graded, relates to 
an outcome of an AE and is a regulatory definition.  
 
Clinical sites are encouraged to report parameters in the DAIDS grading table as they are written to 
maintain data consistency across clinical trials. However, since some parameters can be reported with 
more specificity, clinical sites are encouraged to report parameters that convey additional clinical 
information. For example, diarrhea could be reported as neonatal diarrhea; seizures, as febrile seizures; 
and pain, as jaw pain.  
 
The DAIDS grading table provides an AE severity grading scale ranging from grades 1 to 5 with 
descriptions for each AE based on the following general guidelines:  
• 
Grade 1 indicates a mild event  
• 
Grade 2 indicates a moderate event  
• 
Grade 3 indicates a severe event  
• 
Grade 4 indicates a potentially life-threatening event  

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• 
Grade 5 indicates death (Note: This grade is not specifically listed on each page of the grading 
table).  
 
Other points to consider include:  
• 
Use age and sex values as applicable.  
• 
Unless noted, laboratory values are for term neonates. Preterm neonates should be assessed 
using local laboratory normal ranges.  
• 
Where applicable, Standard International (SI) units are included in italics.  
 
Selecting and Reporting a Primary AE Term  
When selecting a primary AE term to report, sites should select the term that best describes what 
occurred to the participant. For example, a participant may present with itching, urticaria, flushing, 
angioedema of the face, and dyspnea. If the underlying diagnosis is determined to be an acute allergic 
reaction, sites should report “Acute Allergic Reaction” as the primary AE term.  
 
Primary AE terms should be reported using the DAIDS Adverse Experience Reporting System (DAERS) 
only if they meet expedited reporting criteria. However, all primary AE terms should be reported using 
protocol-specific case report forms (CRFs). Because the reported information is stored in different 
databases (i.e., safety and clinical), sites should report primary AE terms using the same terminology for 
data consistency.  
 
When reporting using DAERS, other clinically significant events associated with a primary AE term that 
more fully describe the nature, severity, or complications of the primary AE term should be entered in 
the “Other Events” section. However, the severity grade for these events must be lower than or equal to 
the severity grade of the primary AE term. In the example above, dyspnea and angioedema of the face 
may be entered in the “Other Events” section, because they are more descriptive and provide additional 
information on the severity of the acute allergic reaction. However, their severity grades must be lower 
than or equal to the severity grade of the primary AE term of “Acute Allergic Reaction”. 
 
Differences exist in the reporting and recording of information (e.g., signs and symptoms, clinically 
significant events) in DAERS and CRFs. Therefore, sites should refer to their protocols and CRF 
requirements for further instructions. 
 
Grading Adult and Pediatric AEs 
When a single parameter is not appropriate for grading an AE in both adult and pediatric populations, 
separate parameters with specified age ranges are provided. If no distinction between adult and 
pediatric populations has been made, the listed parameter should be used for grading an AE in both 
populations. 
 
Reporting Pregnancy Outcomes 
In the Pregnancy, Puerperium, and Perinatal section, all parameters are pregnancy outcomes and should 
be reported using the mother's participant ID. If an infant is not enrolled in the same study as the 
mother, any identified birth defects should be reported using the mother's participant ID. However, if an 
infant is enrolled in the same study as the mother or in another study, any identified birth defects 
should be reported using the infant's participant ID. Sites should refer to the applicable network 
standards for reporting abnormal pregnancy outcomes on the CRFs. 
 
Determining Severity Grade for Parameters between Grades 

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If the severity of an AE could fall in either one of two grades (i.e., the severity of an AE could be either 
grade 2 or grade 3), sites should select the higher of the two grades. 
 
Laboratory Values 
General. An asymptomatic, abnormal laboratory finding without an accompanying AE should not be 
reported to DAIDS in an expedited timeframe unless it meets protocol-specific reporting requirements. 
Sites should refer to the applicable network standards for reporting abnormal laboratory findings on the 
clinical case report forms. 
 
Values below Grade 1. Any laboratory value that is between the ULN and grade 1 (for high values) or the 
LLN and grade 1 (for low values) should not be graded or reported as an AE. Sites should consult the 
Manual for Expedited Reporting of Adverse Events to DAIDS, Version 2.0 and their protocol when 
making an assessment of the need to report an AE. 
 
Overlap of Local Laboratory Normal Values with Grading Table Ranges. When local laboratory normal 
values fall within grading table laboratory ranges, the severity grading is based on the ranges in the 
grading table unless there is a protocol-specific grading criterion for the laboratory value. For example, 
“Magnesium, Low" has a grade 1 range of 1.2 to < 1.4 mEq/L, while a particular laboratory’s normal 
range for magnesium may be 1.3 to 2.8 mEq/L. If a study participant’s magnesium laboratory value is 1.3 
mEq/L, the laboratory value should be graded as grade 1. 
 
Estimating Severity Grade for Parameters Not Identified in the Grading Table 
The functional table below should be used to grade the severity of an AE that is not specifically 
identified in the grading table. In addition, all deaths related to an AE are to be classified as grade 5. 
 
PARAMETER  
GRADE 1MILD  
GRADE 2 
MODERATE  
GRADE 3 SEVERE  
GRADE 4 
POTENTIALLY LIFE-
THREATENING  
Clinical adverse 
event NOT identified 
elsewhere in the 
grading table  
Mild symptoms 
causing no or 
minimal interference 
with usual social & 
functional activities 
with intervention not 
indicated  
Moderate symptoms 
causing greater than 
minimal interference 
with usual social & 
functional activities 
with intervention 
indicated  
Severe symptoms 
causing inability to 
perform usual social 
& functional activities 
with intervention or 
hospitalization 
indicated  
Potentially life-
threatening 
symptoms causing 
inability to perform 
basic self-care 
functions with 
intervention 
indicated to prevent 
permanent 
impairment, 
persistent disability, 
or death 
 
 
 

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Appendix I. Pharmacokinetic Sub-Study 
 
Pharmacokinetics of Dihydroartemisinin-Piperaquine (DP) with Sulfadoxine-Pyrimethamine 
(SP) in Pregnant Women receiving DP, SP or DP and SP as Chemoprevention for Malaria 
 
1. INTRODUCTION 
 
1.1. Background 
 
Pregnancy is associated with reduced dihydroartemisinin (DHA), piperaquine (PQ), 
sulfadoxine (SDX) and pyrimethamine (PYR) pharmacokinetic exposure compared to non-
pregnant adults. Although dosing of antimalarials is based on non-pregnant adults, 
physiological changes in pregnancy affect drug exposure, including reduced absorption, 
increased distribution, diminished protein binding, and altered clearance.B1-2 In addition, 
increased hepatic blood flow and CYP activity during pregnancy increases metabolism of many 
drugs.B3-4 In our evaluations of IPTp with DP, compared to non-pregnant adults, pregnant 
women exhibited 30-40% decreased pharmacokinetic exposure to both DHA and PQ,B5  and the 
lower exposure to PQ led to increased risk of malaria parasitemia and adverse birth outcomes 
during pregnancy.B6 In African women receiving IPTp with SP, SDX exposure was also lower 
during pregnancy than post-partum.B7 In another African study, clearance during pregnancy 
was increased 3-fold for SDX, but decreased 18% for PYR compared to that in non-pregnant 
adults;B8 similar findings were reported from Papua New Guinea.B9  
 
A drug-drug interaction between SP and DP is unlikely, but clinical confirmation is needed. No 
data are available on the pharmacokinetics or efficacy of concurrent administration of DP and 
SP for IPTp. There is no evidence that the components of these drugs alter metabolism or renal 
clearance. One small study reported increased exposure to halofantrine, a CYP3A4 
substrate,B9,B10 after a dose of SP,B11 but others reported no clinically relevant interactions with 
SP.B12-14 We have also shown that daily trimethoprim-sulfamethoxazole, a related antifolate 
combination, given with DP was as safe and as well tolerated as trimethoprim-
sulfamethoxazole alone for IPTp among HIV infected pregnant women.B15 In addition, others 
have shown that DP administered as malaria treatment among women receiving SP effectively 
treats malaria and does not cause additional toxicity.  However, as protective efficacy is tightly 
linked to maintaining protective concentrations of PQ for DP, and even small decreases in PQ 
exposure could impact antimalarial efficacy, characterization of any potential drug interactions 
between PQ, SDX and PYR are needed. 
 
1.2. Preliminary Studies: 
 
Pharmacokinetic Studies 
 
We have established the infrastructure to enroll pregnant women into pharmacokinetic (PK) 
studies at our study site.B5,B16 Our intensive PK studies in pregnant populations have shown that 
compared to non-pregnant Ugandan women, pregnancy is associated with a 27% reduction in 

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area under the curve (AUC) from 0-8 h for DHA, and a 40% reduction in AUC from 0-21 d for 
PQ.B5 These data allowed us to propose optimal DP regimens during pregnancy which improved 
time above malaria protective PQ concentrations. We were also able to identify a critical drug-
drug interaction between PQ and efavirenz which caused further reductions in PQ exposure for 
women with HIV who received efavirenz.  
 
To expand our understanding of DP and SP individually, and in combination, a designated PK 
study room has been established at the clinical trial site specifically for intensive PK studies. 
This will allow subjects to remain comfortable for up to eight hours. All PK sample handling is 
carried out in our onsite research laboratory where plasma samples are adequately stored at -
20⁰C immediately after collection and then transferred from Busia to Tororo to -80⁰C to await 
shipment to UCSF for analysis.  In addition to this proposed study in pregnant women, prior PK 
investigations have included a) artemether-lumefantrine in HIV-uninfected and HIV infected 
children,B17 b) DP in HIV-uninfected infants,B18 and c) DP in pregnant women with and without 
HIV.B5    
 
Our studies are supported through our state-of-the-art pharmacology laboratory, the Drug 
Research Unit, located at UCSF with >35 years of experience in pharmacology where specialized 
methods for antimalarials are continuously developed and improved to support field 
investigations.B19  
 
1.3. Rationale 
 
This study is designed to directly address the pharmacology of DP and SP, alone and in 
combination, among pregnant women at high risk of malaria in Busia District, Uganda.  Results 
will inform dosing guidelines for DP and SP alone and in combination during pregnancy.  
Traditionally, studies have focused on non-pregnant adults, largely ignoring the effects of 
pregnancy and concomitant drugs on drug disposition. This study utilizes state-of-the-art PK 
designs and drug assay methods. The intensive PK design in a relatively small number of women 
is needed to address these focused PK questions for PQ, SDX, and PYR. 
 
Rationale for this study is: 
• Dosing guidelines for pregnant women have historically relied on studies in non-pregnant 
adults even though pregnancy impacts how drugs are handled by the body. 
• If DP and SP provide additive or even synergistic benefit for birth outcomes, it will be 
important to understand how each drug should be dosed for safety and efficacy during 
pregnancy. 
• PK studies of antimalarials in vulnerable populations and drug interaction studies are 
deemed a high priority by the Worldwide antimalarial resistance network (WWARN) and 
National Institutes of Allergy and Infectious Diseases to reduce the threat of ACT drug 
resistance and treatment failure. 
• An intensive PK design yielding a precise estimate of drug exposure (i.e. AUC) permits 
robust comparisons informing treatment guidelines and policy for pregnant women living in 
malaria endemic settings. 

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2. PHARMACOLOGY OBJECTIVES:  
 
2.1. Primary Objectives 
 
• To evaluate the PK exposure of PQ, SDX, and PYR during pregnancy and 
investigate the potential drug-drug interaction between PQ and SP.  90 pregnant 
women (approximately 30 each in DP, SP, or DP+SP IPTp arms) will undergo 
intensive PK evaluations over 21 days for PQ, SDX and/or PYR at 28 weeks 
gestation. We hypothesize that there will be no changes to drug exposure of PQ, 
SDX or PYR when DP is given with SP compared to each drug being given 
separately.  
 
2.2. Secondary Objectives 
 
• To quantify relationships between the PK of DP and SP and toxicity.  
In addition to PK data described above, the 90 pregnant women will be assessed at 
each study visit for adverse events that may be drug related. We will assess 
associations between drug concentration or area under the curve and any grade 3 
or 4 adverse event including anemia. We hypothesize that variable exposure to 
each drug in the context of standard dosing will not be associated with an 
increased risk for adverse events. 
 
 
3. STUDY DESIGN 
 
This will be a PK sub-study carried out to reach a sample size of 90 HIV-uninfected pregnant 
women from the DP+SP study.  The PK sub-study will be carried out at the time of study drug 
administration at 28 weeks of gestation and sampling specific for the PK sub-study will continue 
until 23 days after dosing. If a woman is lost to follow up during the PK sub-study, additional 
women will be enrolled to reach the 90 women with PK data at all time points (>90 women may 
be enrolled in the study to reach this goal). The design is an intensive study that entails multiple 
blood collections in a relatively small sample of women in order to accurately estimate the area 
under the concentration time curve (AUC) for a) SDX and PYR prior to the 1st dose on day 1 and 
then following to 23 days post-dosing, and b) PQ prior to the 3rd daily dose of a 3 day regimen 
and then following to 23 days after the first dose (e.g. all women will undergo the same PK 
sampling regardless of which arm they are assigned to since study staff and subjects are 
blinded). 
  
DP and SP PK: Women in the DP only or SP only arms will serve as the control group for the 
DP+SP arm for the primary outcome of assessing a drug interaction between DP+SP.  
 

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Sample size: A combined enrollment of 90 women, to achieve approximately 30 from each IPTp 
arm will complete the intensive PK evaluations. All participants and on-site study personnel will 
remain blinded regarding the assigned treatment group for all participants of this PK sub-study. 
Any pregnant woman who meet eligibility criteria will be approached to participate in the PK 
study until enrollment for the PK sub-study is complete. 
 
4. PK SUB-STUDY SELECTION AND ENROLLMENT 
 
4.1. Inclusion Criteria 
 
• Enrollment in DP+SP IPTp study 
• Provision of informed consent for the PK sub-study 
• Willingness to undergo intensive PK sampling, starting with the 1st dose of study 
drugs scheduled for 28 weeks gestation and continuing through day 23 after the 
first dose. 
 
4.2. Exclusion Criteria 
• Receipt of any medications affecting CYP450 metabolism (as per section 4.3) within 
14 days of the scheduled intensive PK evaluation. 
• Hemoglobin < 8.0 g/dL at the start of the PK sub-study 
• Prior treatment for clinical malaria within 14 days of the scheduled intensive PK 
evaluation 
 
4.3. Disallowed Medication Guidelines 
 
The following medications are disallowed within 14 days prior to the intensive PK evaluations.  
The medications are in addition to disallowed medications for the parent study: 
• 
Carbamazepine 
• 
Ketoconazole 
• 
Phenobarbital 
• 
Phenytoin 
• 
Rifabutin  
• 
Rifampin  
• 
Grapefruit juice should be avoided during the study due to its potential effects 
on CYP3A4. 
 
 
4.4. Study Enrollment Procedures 
 
Women may be approached for this PK sub-study at any point after women enroll in the parent 
study and will be provided information about the study.  Consent will be obtained at or before 
28 weeks gestation.  Prior to obtaining consent, women will be assessed for eligibility which 
includes determining that they are not on any disallowed medications, their hemoglobin was 

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≥8.0 g/dL at enrollment, and they have not had malaria diagnosed within 14 days of their 28-
week study visit.   
 
As done for the primary study, study physicians will conduct the informed consent discussion 
for the PK sub-study in the study clinic in the appropriate language and an interpreter will be 
used if necessary. Following the informed consent discussion, participants will be asked by the 
study physicians to sign a research participation informed consent form specific for the PK sub-
study, approved by the UCSF Committee for Human Research (UCSF CHR), MU School of School 
of Biomedical Sciences - Research and Ethics Committee (SBS-REC), and the Uganda National 
Council for Science and Technology (UNCST). If the participant is unable to read or write, her 
fingerprint will substitute for a signature, and a signature from a witness to the informed 
consent procedures will be obtained as is done for the parent study.  
 
5. PK SUB-STUDY PROCEDURES AT 28 WEEKS GESTATION 
 
• Women will begin the study at their routine visit for 28 weeks gestation and will receive 
their assigned 3-day treatment course per their blinded assignment in parent study: DP + SP 
placebo or SP + DP placebo or DP + SP. Women will be instructed to come to the study clinic 
at a specified time for directly observed administration of the study drug for all doses. In all 
cases, the exact time of each daily dose of study drug will be recorded by study staff.   
 
• On the first and third dosing days, an indwelling catheter will be placed in a vein to allow for 
serial PK sampling.  A single venous sample (0.5 ml) will be collected just prior to each daily 
dose.  After doses on the first and third days, additional blood samples (0.5 ml each) will be 
collected at 0.5, 1, 2, 3, 4, 6 and 8 hours after the doses are administered. Women will 
remain in the clinic until completion of the 8-hour blood sample, the catheter will be 
removed and women will be discharged home. 
 
• On the second day of dosing, participants will come to clinic for a pre-drug venous or 
capillary sample collection followed by administration of study drug (with precise times 
recorded) 
 

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• In addition to the blood samples described above, women will return to the clinic for PK 
sample collections 72 h after the first study drug dose and on days 7, 9, 16, and 23 (Figure 
1). The PK sample at 72 hours post first dose will be collected by venous stick (0.5 ml) and 
capillary stick (200 μl) to permit correlation of venous and capillary plasma drug 
concentrations for PQ, SDX and PYR.  Samples on days 7, 9, 16 and 23 can either be 
collected via venous (0.5 mL) or capillary stick (200 μl each) and processed for plasma. The 
method and precise time of collection (venous or capillary) will always be recorded. 
 
• Women will be provided modest compensation for their time. 
 
• Samples from 0.5-24 hours on the first day of dosing will be used to determine SDX and PYR 
concentrations. Samples from 0.5-8 hours the 3rd day will be used to determine PQ 
concentrations, and for all remaining samples concentrations of all three drugs will be 
quantified. 
 
• Subjects with malaria infection during the 21 days of the PK study will provide an additional 
PK sample via venous (0.5 mL) or capillary (200 μL) collection at malaria diagnosis.  
 
 
• After blood is collected, plasma will be separated immediately and aliquoted into cryovials. 
Plasma samples will be initially placed at -20⁰C, then transported to the Tororo Lab within 
24 hours for storage in -80⁰C freezers. Samples will be shipped to the UCSF Drug Research 
Unit on dry ice or liquid nitrogen for drug concentration quantification.   
 
 
 
6. ANALYTICAL METHODS.  PQ, SDX and PYR will be measured in plasma using optimized 
methods validated in the UCSF Drug Research Unit with all methods utilizing liquid 
chromatography tandem mass spectrometry.  A non-study staff member will be made 
aware the study drug assignments for the PK sub-study participants to allow quantification 
 
 
Figure 1. PK sub-study clinical visits 
SP/placebo
DP/placebo
DP/placebo
DP/placebo
SDX, PYR, PQ measured
SDX, PYR measured
PQ measured
0 h
24 h
48 h
72 h
7 d
9 d
16 d
23 d
8 h
8 h
*Time not to scale to allow for full visualization of sampling

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of relevant analytes in the sample.  This information will not be shared with other study 
investigators until unblinding has occurred. 
 
6.1. Blood Volumes 
The total blood volumes collected from intensive PK study participants during the 23 days 
will be no more than 20 mL. 
 
7. MISSED DOSES OF STUDY DRUG 
 
Study drugs will be re-dosed if vomiting occurs within 30 minutes of administration.  At the 
time of the third daily treatment dose, PK sampling will only continue if the subject can be re-
dosed with a full dose and vomiting occurred ≤30 minutes post-administration of DP.  The re-
dosing will be carefully noted on the study case report forms.  Participants in the PK sub-study 
must have taken all 3 daily doses successfully in order to participate in the intensive PK 
sampling. 
 
8. STATISTICAL CONSIDERATIONS 
 
Primary Analysis:  We will evaluate the PK exposure of PQ, SDX, and PYR in women who receive 
DP, SP or DP+SP at 28 weeks gestation for women enrolled the PK sub-study.  PK parameters of 
PQ, SDX and PYR will be estimated using non-compartmental analysis via the linear up-log 
down trapezoidal rule in conjunction with first-order input using WinNonlin 6.0 (Pharsight 
Corporation, Mountain View, USA). PK parameters, including elimination rate constant (λz) and 
half-life (t1/2) will be estimated with t1/2 calculated as ln2/ λz. The AUC will be estimated as 
the sum of AUClast (AUC to the end of the sampling period) and AUC last-∞ (AUC from the end of 
sampling to infinity (∞)). If PK parameters are found to have skewed distributions and data 
transformations do not induce symmetry, rank-based tests will be used.  
 
 
Comparison Groups 
 
Primary 
1. To evaluate the PK exposure of PQ, 
SDX, and PYR during pregnancy and 
determine if there is a drug-drug 
interaction between PQ and SP. 
 
 
Pregnant women receiving monthly DP+SP in 
the 2nd and 3rd trimesters of pregnancy vs 
pregnant receiving DP only or SP only during 
the 2nd and 3rd trimesters of pregnancy. 
 

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Secondary 
1. To quantify relationships between the PK 
exposure of DP and/or SDX/PYR and 
toxicity.  
Pregnant women who only receive SP will be 
the control group for women receiving DP or 
DP+SP. Pregnant women who only receive DP 
will serve as the control group for women 
receiving SP or DP+SP.   
 
8.1. Outcome Measures 
 
• Primary outcome  
 
Primary outcome measurement is AUC for PQ, SDX and PYR. 
 
• Secondary outcomes 
 
Grade 3 and 4 adverse events. 
 
8.2. Sample Size and Power for primary outcome. Using measures of observed mean AUC 
and standard deviations from our prior studies and those published in the literature 
and assuming a group sample size of 30 subjects with complete data for each study 
drug regimen, this study will achieve 80% power to detect a difference in mean AUC 
between groups of 23% for PQ and 30% for SDX, and 19% for PYR with a significance 
level (alpha) of 0.05 using a two-sided two-sample t-test (coefficients of variation [CV] 
for AUC for PPQ=35%, DHA=38%, sulfadoxine=40%, pyrimethamine=25%). 
 
 
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