Combined Effects of RTS,S Vaccination and PBO Nets on Malaria Infection and Transmission in Malawi
Conditions: Malaria, Malaria Vaccine, Insecticide-treated Bednets
Sponsor: Michigan State University

Project 004: Combined effects of RTS,S vaccination (malaria vaccine) and  piperonyl butoxide (PBO) nets on malaria 
infection and transmission. Version 2.0: 12 October 2019 
 
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Intransigence of malaria in Malawi: Understanding hidden reservoirs, 
successful vectors, and prevention failures 
 
Project 004: Combined effects of RTS,S vaccination and PBO nets on 
malaria infection and transmission in Malawi 
 
 
Principal Investigator 
  
Don P. Mathanga, MBBS, MPH, PhD1 
 
 
 
 
 
 
 
Co-Investigators 
 
Miriam K. Laufer MD MPH2 
Clarissa Valim, MD, MSc, SM, ScD3 
Themba Mzilahowa, BSc, MSc, PhD1 
Mark L. Wilson, ScD4 
Peter A. M. Ntenda. BSc, MSc, PhD1 
Edward D. Walker, PhD5 
 
Participating Academic Institutions  
1Malaria Alert Centre-Communicable Diseases Action Centre, College of Medicine,  
University of Malawi, Blantyre, Malawi 
2Center for Vaccine Development and Global Health, University of Maryland School of Medicine, 
Baltimore, USA 
3Dept. Of Global Health, Boston University, School of Public Health, Boston, MA, USA  
4Blantyre Malaria Project, College of Medicine, University of Malawi, Blantyre, Malawi  
4Dept. of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, USA 
5Dept. of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA 
 
 

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Contact information: Don Mathanga, MBBS, MPH, PhD, Malaria Alert Centre, University of 
Malawi College of Medicine, Private Bag 360, Blantyre 3, Malawi. Phone: +265-1-870-145 
Fax: 265-1-876-217. E-mail: dmathang@mac.medcol.mw 
 
 
 
Institutions under whose umbrella the research project will be conducted: 
I. 
Malaria Alert Centre, University of Malawi, College of Medicine 
 
II. 
Michigan State University 
 
III. 
University of Maryland School of Medicine 
 
IV. 
Boston University 
 
V. 
University of Michigan 
 
 
 

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CONTENTS 
1. LIST OF ABBREVIATIONS ........................................................................................................................ 4 
2. EXECUTIVE SUMMARY ........................................................................................................................... 6 
3. BACKGROUND INFORMATION .............................................................................................................. 7 
3.1 Significance of the problem .............................................................................................................. 7 
3.2 Novel interventions to prevent malaria ........................................................................................... 7 
3.3 Malaria vaccine ................................................................................................................................. 8 
3.4 Malaria in Malawi ............................................................................................................................. 8 
3.5 Preliminary Studies ........................................................................................................................... 9 
4. JUSTIFICATION………. .............................................................................................................................. 9 
5. OBJECTIVES………………….. .................................................................................................................... 10 
5.1 Broad Objective .............................................................................................................................. 10 
5.2 Specific Objectives .......................................................................................................................... 10 
6. METHODS……………………. ..................................................................................................................... 11 
6.1 Study Design ................................................................................................................................... 11 
6.2 Study Location ................................................................................................................................ 12 
6.3 Study Population ............................................................................................................................ 12 
6.3.1 Study period ................................................................................................................................ 13 
6.3.2 Sample size …………………………………………………………………………………….13 
6.4 Participant recruitment and study procedures .............................................................................. 14 
6.5 Analysis Plan 16 
6.5.1 Data handling and record keeping .............................................................................................. 17 
6.6 Results presentation and dissemination ........................................................................................ 18 
7. 7.ETHICAL CONSIDERATIONS............................................................................................................... 19 
7.1 General Principles ........................................................................................................................... 19 
7.2 Informed Consent ........................................................................................................................... 19 
7.3 Possible risks and benefits to study participants ........................................................................... 20 
7.3.1 Potential Risks ............................................................................................................................. 20 
7.3.2 Known Potential Benefits ............................................................................................................ 20 
8. POSSIBLE CONSTRAINTS ...................................................................................................................... 21 
9. REQUIREMENTS……. ............................................................................................................................ 21 
10. TRAINING PROVIDED ........................................................................................................................... 22 
11. REFERENCES……………….. ...................................................................................................................... 23 
12. BUDGET ................................................................................................................................................ 26 

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1. LIST OF ABBREVIATIONS 
ACT 
Artemisinin Combination Therapy 
CDC 
Centers for Disease Control and Prevention 
COMREC 
College of Medicine Research Ethics Committee 
CRF 
Case Report Form 
CSP 
Circumsporozoite protein 
DBS 
Dried Blood Spot 
DMC 
Data Management Core 
EIR 
Entomologic Inoculation Rate 
ELISA 
Enzyme-Linked Immunosorbent Assay 
EPI 
Expanded Programme on Immunization 
F1 mosquitoes 
First generation mosquito progeny 
GCP 
Good Clinical Practice 
GPS 
Global Positioning System 
HC 
Health Center (or Centre) 
HH 
Household 
HIV 
Human Immunodeficiency Virus 
HLC 
Human Landing Catch 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
IEC 
Independent or Institutional Ethics Committee 
IRB 
Institutional Review Board 
ITN 
Insecticide-Treated Net 
LLIN  
Long-Lasting Insecticide-Treated Net 
LC50 
Single dose that kills 50% of test mosquitos 
MoH 
Ministry of Health 
MOP 
Manual of Procedures 
mRDTs 
Malaria rapid diagnostic tests 
NMCP 
National Malaria Control Programme 

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ICEMR 
International Center for Excellence in Malaria Research 
ITNs 
Insecticide-treated bed nets 
MDHS 
Malawi Demographic and Health Survey 
MSU 
Michigan State University 
NCST 
National Commission for Science and Technology 
NIAID 
National Institute of Allergy and Infectious Diseases  
NIH 
National Institutes of Health 
OPD 
Outpatient Department 
PBO nets 
Piperonyl butoxide-impregnated nets 
PCR 
Polymerase Chain Reaction 
PI 
Principal Investigator 
PID 
Participant Identification Number 
RTS,S 
Central repeat region of Plasmodium falciparum 
circumsporozoite protein (CSP); T-cell epitopes of the CSP; 
and the hepatitis B surface antigen (HBsAg). 
SAE 
Serious Adverse Event 
SES 
Socio-Economic Status 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
SP 
Sulfadoxine-Pyrimethamine 
SSA 
Sub-Saharan Africa 
WBC 
White Blood Cell 
WHO 
World Health Organization 
 
 

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2. EXECUTIVE SUMMARY 
Introduction: The decline in malaria incidence has stalled globally and incidence is increasing in 
some high transmission settings of sub-Saharan Africa (SSA), including Malawi. The situation is 
worsening despite the scale-up of previously effective interventions, raising concerns that the 
impact of current malaria control and prevention strategies maybe compromised.  
Problem: There is an urgent need for innovative approaches to malaria control and Malawi is 
currently positioned to assess two of the most promising new interventions. The Malawi Ministry 
of Health (MOH) is launching large scale projects to evaluate a new formulation of insecticide-
treated bed nets with a chemical synergist, piperonyl butoxide (PBO), designed to enhance the 
insecticidal effect of pyrethroids and the new malaria vaccine RTS,S/ ASO1 (RTS,S). In an effort 
to gain the most information from these, interventions Malawi’s National Malaria Control 
Programme (NMCP) have invited the Malawi International Center for Excellence in Malaria 
Research (ICEMR) to evaluate the effectiveness of the two interventions (alone and in 
combination) on malaria prevalence and transmission.  
Objective: In this proposed implementation study, we propose to assess the impact of PBO nets 
and RTS,S vaccine on Plasmodium infection prevalence and transmission.  
Study type and methodology: We will enroll children in a prospective cohort study in which the 
follow-up will be at the 2nd, 4th, and 6th month. Using malaria Rapid Diagnostic Tests (mRDTs), all 
participants will be screened for malaria infection at enrollment. Participants with with positive 
mRDTs will will be treated with a full, weight appropriate treatment course of dispersible 
artemether-lumefantrine (LA) by government staff. The mRDTs and treatment of mRDT positive 
children will be repeated at the final visit. Similarly, all participants will be examined for hemoglobin 
levels at enrollment and at every two-month follow-up visits using Hemoglobin Color Scale (HCS). 
Children in households that are scheduled to receive both PBO nets and RTS,S vaccine will be 
compared to children in households that are not scheduled to receive either of these interventions. 
The outcomes of interest will be malaria infection prevalence, gametocyte prevalence and 
entomological evidence of the force of infection. 
Results dissemination:  Results from this study will be shared with malaria national programs in 
Malawi and globally, through conference presentations and peer-reviewedpublications, to 
contribute to the development of policy in rolling out these new interventions. 
 
 
 

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3. BACKGROUND INFORMATION  
3.1 Significance of the problem 
Malaria remains a significant global health problem, with an estimated 216 million cases 
worldwide in 2016 (WHO, 2017). At present, the main approaches to malaria control are 
insecticide-treated bed nets (ITNs) and prompt diagnosis with effective treatment. Malaria-related 
morbidity and mortality declined globally over the past decade, however, this progress has 
stagnated. Indeed, five million more malaria cases occurred globally in 2016 than in 2015, with 
the greatest increases occurring in countries like Malawi (WHO, 2017). This increased incidence 
arose despite continued use of previously effective interventions, raising fears about the future 
usefulness of current malaria control activities, and highlights the urgent need for innovative 
approaches to malaria control. In Malawi, two new interventions will be introduced over the period 
of surveillance supported by the Malawi International Center of Excellence in Malaria Research 
(ICEMR): a new formulation of bed nets that is designed to manage insecticide resistance and a 
pilot study of RTS,S/ASO1 vaccination in young children. The Malawi ICEMR has a truly unique 
opportunity to evaluate the implementation and impact on Plasmodium transmission of these two 
interventions. 
Implementation and interactions of RTS,S and bed nets: Both the RTS,S vaccine and PBO 
nets have been studied as part of large clinical trials. RTS,S vaccine has been evaluated in the 
setting of high ITN use (Agnandji, 2011) and PBO nets have been studied with and without indoor 
residual spraying. Impacts of these interventions, when implemented on a large-scale 
programmatic level, must be evaluated for their individual and combined effects, and their cost-
effectiveness against other potential interventions. The combined effects of these interventions 
could be enhanced or diminished by interactions. For example, if young children receiving the 
RTS,S vaccine are also from households more likely to use ITNs, then vaccine effectiveness 
could be relatively more. Or it may be that parents of those receiving malaria vaccine believe their 
children have become protected against malaria illness, and therefore become less vigilant in 
shielding their children from mosquito bites. Perhaps people's perceptions of PBO nets (benefits 
over conventional nets or concerns about the added synergist) will alter their use compared to 
conventional ITNs. The significance of our study lies in our ICEMR's ability to evaluate both of 
these major new interventions, and systematically collect evidence about the impact of RTS,S 
vaccination and PBO nets on the incidence of Plasmodium infection. 
 
3.2 Novel interventions to prevent malaria 
The use of piperonyl butoxide (PBO) nets: Pyrethroids are the only class of insecticide 
approved for use in ITNs, but resistance among the Anopheles vectors in Africa is widespread. In 
Malawi, pyrethroid resistance is present in the two major malaria vectors, An. funestus and An. 
arabiensis, and has recently become more severe (Mzilahowa, 2016). Increased prevalence and 
intensity of pyrethroid resistance is a key contributor to the observed failure of current ITNs to 
protect against Plasmodium infection in our setting and elsewhere. We recently demonstrated 
that pyrethroid-based ITNs in southern Malawi provided only modest or no protection against 
infection and disease (Mathanga, 2015; Buchwald, 2017) yet no alternative insecticide is currently 
available. However, the addition of a chemical synergist, piperonyl butoxide (PBO), is designed 
to enhance the insecticidal effect of pyrethroids even in the face of resistance. PBO inhibits the 
enzyme that detoxifies the pyrethroid, allowing the pyrethroid to act on the mosquito. A recent 
cluster-randomized trial in Tanzania showed that use of PBO nets compared to conventional 
pyrethroid nets produced 44% and 33% protective efficacy in the first and second year, 
respectively. The impact of PBO net use was also detectable in key entomological measures 

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including Anopheles density, sporozoite rate and entomological inoculate rates (Protopopoff, 
2018). Following these promising preliminary results, Malawi's National Malaria Control Program 
(NMCP) will lead a PBO-net mass distribution campaign in six districts, presenting us with the 
opportunity to study the effectiveness of these nets in the context of real-world program setting. 
The significance of this opportunity lies in our ability to carefully evaluate the impact on 
Plasmodium transmission and also investigate accompanying changes in pyrethroid resistance 
to better understand the mechanism of protection. 
3.3 Malaria vaccine 
A highly effective malaria vaccine remains elusive, but the RTS,S vaccine has progressed through 
the clinical development stages. RTS,S is a subunit vaccine that includes a portion of the 
circumsporozoite protein (CSP) co-expressed with Hepatitis B surface antigen combined with 
ASO1 adjuvant. The Phase 3 trial of three doses administered to 5-17-month-olds confirmed 
moderate protection, with overall efficacy estimates of 50.4% against clinical malaria and 34.8% 
against severe malaria after three doses (Agnandji, 2011). Efficacy, which waned over time, was 
marginally improved by boosting at 18 months. The European Medicines Agency adopted a 
positive scientific opinion of the vaccine for use outside of the European Union. The WHO has 
created the Malaria Vaccine Implementation Program (MVIP) and selected Malawi as one of the 
sites to explore the feasibility, efficacy and safety of RTS,S vaccination in the context of routine 
use. In addition, when exploring RTS,S feasibility and safety in routine health delivery, little 
evidence exists regarding how RTS,S vaccine may affect Plasmodium infection and transmission. 
An analysis of several RTS,S trials suggested 70-90% protection against microscopy-defined 
infection (Penny, 2015), but this does not consider submicroscopic, PCR-detected Plasmodium 
infections containing gametocytes infectious to mosquitoes. The significance of our proposed 
studies is that RTS,S vaccination may reduce human infection prevalence and transmission to 
mosquitoes. Thus, RTS,S vaccine may offer indirect protection and decrease infection in other 
age groups. Alternatively, if vaccination permits low-density Plasmodium infections that do not 
lead to disease and treatment-seeking, vaccinees may actually serve as sources of added 
household transmission. The Malawi Ministry of Health (MOH) has invited our ICEMR to help 
analyze impacts of RTS,S vaccination on Plasmodium transmission and malaria incidence. 
Knowledge of these outcomes is critical to future role out of the vaccine. 
3.4 Malaria in Malawi 
Malaria remains a major public health problem in Malawi. Recent surveys suggest that malaria 
infection risk exists throughout Malawi, with the highest risk being along the hotter, wetter and 
more humid low-lying regions, and the lowest in the highland areas (Kazembe, 2007). Despite 
the scale-up of ITNs in certain locations, there has been no corresponding reduction in malaria 
risk and morbidity (Roca-Feltrer, 2012; Okiro, 2013; Sisya, 2015). From 2000 to 2010, the number 
of people living in hyper-endemic conditions actually increased by 37% (MOH-KEMRI, 2013) 
whilst the number of reported cases of malaria increased from 3.7 million in 2005 to 6.8 million in 
2010, an increase of 46% in a population that grew by 14.1% during the same time period. Malaria 
morbidity burden also remained high in hospital settings (Okiro, 2013). Further, the incidence of 
malaria ranged from 200 - 600 cases per 1,000 populations per district in 2013, with 9.2 million 
courses of artemisinin combined treatment (ACT) dispensed in 2013 alone. Yet during this time 
frame, the proportion of under-five children and pregnant women who slept under an ITN the 
previous night reportedly increased from 25% and 8% to 67% and 62%, respectively, by 2013. 
Although few data reflecting actual transmission exist, these numbers suggest that the intensity 
of P. falciparum transmission may actually have increased despite a decade of intensified nation-
wide investment in malaria control and prevention. 
 

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3.5 Preliminary Studies 
Evaluation of effectiveness of large-scale bed 
net projects. Beginning in the early 2000s and 
continuing into the current decade, we analyzed the 
effectiveness of ITN (Lindblade, 2015; Mathanga, 
2015) programs in Malawi. In a 2012 study, the 
incidence of infection with P. falciparum was 30% 
lower in ITN users than non-users despite the 
presence of pyrethroid resistance in the An. 
funestus population (Lindblade, 2015). In parallel, 
there was no reduction in cases of malaria in 
children presenting with fever to clinics who used 
bed nets compared to matched controls not using 
ITNs, suggesting no effect of bed net use in contrast 
to a previous systematic review that showed 
reductions of 50-62% in malaria episodes across 
different transmission settings (Mathanga, 2015), 
highlighting the need for more effective interventions 
across different transmission intensities.  
Insecticide resistance in Malawi. In support of the government campaigns to roll out ITNs, we 
coupled analyses of insecticide resistance in vector populations to describe what the IR analyses 
were coupled to (Mzilahowa, 2008; Riveron, 2015). Results revealed a spectrum of phenotypic 
and genotypic resistance attributes and in particular, showed that many populations of An. 
funestus were resistant to presently used pyrethroids used in ITNs in Malawi. We have shown 
that mortality rates realized by exposing wild-caught Anopheles to seven different ITN types, 
including two dually-treated nets (Olyset plus and PermaNet 3.0) were very low, indicating that 
the An. funestus strains were resistant (Fig. 1). Mortality of mosquitoes was complete (100%) 
only when they were exposed to the top-panel of PermaNet 3.0, which was treated with high 
doses of deltamethrin and piperonyl butoxide (PBO), a synergist. This study provides an 
opportunity to measure the impact of PBO nets at the population level in an area of differing 
insecticide resistance levels and in the context of other interventions. 
4. JUSTIFICATION  
Despite massive scale-up of malaria control interventions in Malawi, especially ITNs, malaria 
disease burden still remains high. This study will assess the impact of two new interventions 
being introduced into the Malawi national malaria control program. The outstanding research 
question is 
a)  What is the impact of RSS,S vaccine on P. falciparum prevalence and transmission?  
The focus of past RTS,S clinical trials has always been the protective efficacy against clinical 
disease. Given the large-scale cluster-randomized introductions of the vaccines, and the existing 
ICEMR community-based surveillance infrastructure, we will evaluate the effect of RTS,S 
vaccination on the prevalence of P. falciparum infection in the vaccinated children and their 
household members and also on various measures of transmission. This critical information has 
never previously been explored in evaluating malaria vaccine effectiveness. In addition, the use 
of serological markers will generate measures of transmission for individuals on a finer scale than 
entomological measures alone and will allow controlling for exposure level in the analysis. 
Accurate estimations of effectiveness in prior vaccination studies have been hampered by the 
lack of measures of individual exposure.  

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b) Does the use of PBO nets decrease exposure to malaria infection and limit 
transmission? Although PBO nets are likely to have more potent insecticide activity, only one 
study has been published to date that shows that the implementation of PBO nets decreases 
human malaria infection (Protopopoff 2018). 
 
c) What are the combined effects of RTS,S vaccine and PBO nets?  
While it is possible that the impact of RTS,S vaccine plus the impact of PBO nets is additive, 
human behavior, parasite dynamics and vector responses may lead to either synergistic or 
antagonistic effects when they are combined. The factorial study design proposed in this 
application allows the evaluation of the impact and interaction of these two interventions. 
 
Findings from this study will have implications for policy makers since understanding both the 
direct and indirect, population-level benefits of malaria vaccination and PBO nets will be critical 
when countries decide whether to adopt these intensive interventions. Hence the data obtained 
in this study will be used to enhance program effectiveness, both at national and international 
level. This study will be divided into two phases. This protocol is particularly focused on detailing 
the first phase of the study, although it briefly summarizes the proposal of the second phase of 
the study. 
 
5 OBJECTIVES 
5.1 Broad Objective 
To assess the combined impact of RTS,S (malaria) vaccination and PBO nets on Plasmodium 
infection and transmission, and how they interact when they are introduced together.  
5.2 Specific Objectives 
The specific objectives for the study are as follows: 
1. To estimate the impacts of PBO nets and RTS,S vaccine on the prevalence and 
transmission of Plasmodium infection. (Phase 1). 
2. To assess the feasibility of evaluating the impact of RTS,S vaccine and PBO nets 
on the prevalence and transmission of Plasmodium infection in a larger scale 
study. (Phase 2). 

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6 METHODS 
6.1 Study Design 
This will be a prospective cohort study. This is a preliminary study to determine if we can detect 
an impact of RTS,S vaccine combined with PBO nets on parasitological, entomological and/or 
serological evidence of infection and transmission. If this preliminary study yields significant 
results, we will have the opportunity to apply for funding from our sponsor (US National Institutes 
of Health) for a much larger study that will assess the impact of each of these interventions 
individually and how they interact with each other. If we are approved to conduct this multi-arm 
study, we will submit a new protocol to COMREC and the MSU IRB for review and approval. 
Using malaria and vaccine registers, we will establish community-based cohorts of children age-
eligible to receive RTS,S vaccinationfrom the two extreme groups: i.e. Nyambi health centre in 
Machinga and Kalembo health centre in Balaka. The Machinga district, is undergoing 
implemetation of both RTS,S vaccination and PBO nets (RTS,S and PBO interventions). Balaka 
households do not receive RTS,S vaccination and only receive conventional nets arms (Table 1). 
We will enroll children of the age that would be eligible for vaccination and all of the children over 
nineteen months and under ten years of age in their house. We will conduct interviews and collect 
specimens at enrolment and at 2, 4 and 6 months, as described below. During these visits, data 
onindividual, household, social, and behavioral determinants, immunization status, ITN use will 
be collected.  
Table 1: Overview of study design and methods 
Specific Objective 1: To estimate the impact of PBO nets and RTS,S vaccine on Plasmodium infection 
prevalence and transmission in Malawi. 
Study design: Cohort study  
Phase 1: 3,000 children in total:  1000 age-eligible children: 250 children age-eligible for RTS,S vaccination [7 to 
18 months]  in each of the two sites and in two separate cohorts 
                                                         2000 siblings:  500 siblings (up to two children >18 months and ≤10 years old 
from the same households as the vaccine-eligible children) in each of two sites and in two separate cohorts   
Methods  
Data sources 
Key variables/ outcome  
Phase 1:  Recruited from 
Nyambi Machinga health 
centre – in catchment 
areas that receive RTS,S 
vaccination and PBO nets. 
Recruited from Kalembo 
health centre in Balaka – in 
catchment areas that 
receive conventional nets 
and no malaria 
vaccination.   
• 
Interviews of 
parent/guardians 
participants 
• 
Blood samples 
• 
Captured mosquitos  
• 
Human reporting about Nets 
• 
Finger prick blood 
specimens 
• 
Plasmodium falciparum infection  
• 
Gametocyte presence 
• 
Anopheles spp. abundance 
• 
Anopheles gravidity 
• 
Anopheles sporozoite rate  
• 
Net usage 
• 
Immunization status 
• 
Serological markers 
 

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6.2 Study location 
The study will be carried out in Machinga and Balaka Districts, in Southern Malawi. Machinga 
District (14.55 S, 35.40 E), located ~120 km northeast of Blantyre, is a poor rural district. Malaria 
transmission is intense and year-round, peaking during the rainy season (November through 
May). Between July 2013 and June 2014, an estimated 162,500 (1-year risk of 28%) malaria 
cases were reported and accounted for 24% of the total Outpatient Department (OPD) attendance 
(the total population of Machinga District is ~572,000 people). In 2015, malaria annual incidence 
was 367 cases/1,000 individuals. The use of ITNs is widespread in the district; findings from the 
2015-2016 MDHS reported that 66.3% of the individuals and 80.5% of under-five children slept 
under an ITN in the previous night.  
Balaka District (15.01° S, 35.01° E), forms a boundary with Machinga and is also a poor, rural 
district. Malaria is generally high in the district with intense, year-round transmission that also 
peaks during the rainy season. Between July 2013 and June 2014, malaria cases were accounted 
for 29% of the total OPD attendance (the total population of Balaka District is ~372,000 people). 
In 2015, malaria annual incidence was 310 cases/1,000 population. Findings from 2015-2016 
MDHS indicated that 67.5% of the population and 87.3% of under-five children slept under an ITN 
in the previous night.  
The timing for this proposal is optimal as we will be able to take advantage of the Malawi 
government-funded nationwide ITN distribution campaign (2018) and the RTS,S evaluation 
project (2019). In November 2018, Malawi undertook a country-wide bed net campaign, 
distributing nets to each household with a target of one net for every two people. In Machinga 
district, PBO nets were distributed, while neighboring Balaka District conventional pyrethroid-
treated bed nets were given. About eight months later, both districts are part of the vaccination of 
infants and young children with the RTS,S vaccine, where children in selected clusters will receive 
RTS,S whilst children in some clusters of the same district would act as controls. The Malawi 
Ministry of Health has asked our ICEMR team, through the College of Medicine Malaria Alert 
Centre, to support the evaluation of RTS,S vaccination, thus providing a perfect opportunity to 
assess the combined effects of these interventions.  
6.3 Study Population 
 
Children whose age is between 7 and 18 months, which indicates they could receive at least 3 
RTS,S doses, will be eligible to participate. Following informed consent from parents/guardians, 
up to two children who are 18 months but ≤10 years of age, from the same household as the 
vaccine-eligible child, will also be enrolled. Only households whose families intend to stay in the 
area for at least six months and whose eligible children will also be expected to use an assigned 
health center for child vaccinations will be eligible to participate. 
Inclusion/Exclusion Criteria 
To be eligible for the cohort, participants must meet all the following inclusion criteria:  
• 
Children aged 7 to 18 months of age (age-eligible for at least 3 doses of RTS,S doses) 
OR being one of not more than two children living in the household of an enrolled age-
eligible child and being >18 mos and < 10 years of age.   
• 
Not on cotrimoxazole prophylaxis for HIV infection 
• 
Weight >5 kg 
• 
Permanent residence of Health Centre (HC) catchment area  
• 
Residence within 10 km from the HC 
• 
Written informed consent from parent/guardian for the child to participate in the study 

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Non-residents of the catchment area and visitors to the study area will be excluded because the 
study requires follow-up for at least 6 months and access to interventions such as conventional, 
PBO nets and malaria vaccination.  
6.3.1 Study period 
We will conduct two consecutive cohort studies with 500 children in the desired age group in each 
arm, and we will also study up to two children >18 mos and < 10 years of age (anticipated one or 
two per household) in each household to obtain preliminary data about the impact on transmission 
in the household (Table 3). Children will be identified and enrolled after obtaining parental 
consent. They will undergo interviews and one finger-prick blood sample collection every two 
months for six months (2, 4, and 6 months) after enrollment. Household entomological sampling 
will occur during the rainy season. 
Table 2: Timing of Phase 1 study visits and specimen collection  
Timeline 
Activity  
May-November 2019                  Finalizing research plan and approval by IRBs (CoM and MSU) 
November 2019 
Community consultation with community leaders, Staff recruitment 
and training, Developing practical arrangements and SOPs and 
developing the data management system 
December 2019 
Cohort recruitment 
January- 2019-May2020 
Cohort follow up (round 1) 
May 2020-October 2020 
Cohort recruitment and follow up (round 2) 
November 2020-March 2021 
Data analysis and assessment of Go/No-Go criteria 
6.3.2 Sample size 
Our sample size estimates are based on previous high and low transmission season surveys 
conducted during the first ICEMR (2010–2017) in areas similar to the clusters that will be selected 
for this study. Based on these data, we anticipate that 13%-18% of households will contain a child 
7-18 months old. The risk of infection in this age group is estimated to be 21% (30% high and 
20% low transmission seasons). In children ≤10 years of age the risk of infection is estimated to 
be 31% (46% in the high and 32% in the low transmission seasons). The within-cluster correlation 
is estimated at 0.004 (0.05 in the high and 0.00 in the low transmission seasons) and 0.14 (0.22 
in the high and 0.06 in the low transmission seasons) for children aged 7-18 months and ≤10 
years of age, respectively. Based on these data, by studying the 25 clusters with 20 households 
with a child 7-18 months per intervention arm, we will analyze 500 children aged 7-18 months and 
a similar or larger number of older children. For power calculations, we assumed that these 
proportions represented the risk of incident cases of infection. 
This proposed sample size will allow us to estimate moderate effectiveness of the combined 
interventions (versus no intervention) with 80% power in 2-sided tests, accounting for within-
cluster correlation, and with α-level of 0.05. For instance, based on the cross-sectional estimations 
of individual risk of malaria infection obtained at enrollment of each cohort, we will be able to 

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detect the effectiveness of 34% (36% and 33% when stratifying by season) in children 7-18 
month-old (Hayes, 2009). In a repeated measure analysis, with within-subject correlation of 1.5 
to 2 times larger than within-cluster correlation, we will be able to detect effectiveness of 34-37% 
in children 7-18 month old (Liu, 2002). 
For entomological assessments, 100 households from each of the two arms will be visited for 
entomological analysis including Anopheles abundance, female mosquito parity, and source of 
blood meal and sporozoite prevalence. Based on our previous experience, we have found an 
average of one Anopheles mosquito per household sampled (although with very heterogeneous 
distribution), and 75-95% human-biting prevalence. 
6.4 Participant recruitment and study procedures 
Participant recruitment. Prior to recruiting participants, households in which children have 
received RTS,S and PBO nets will be identified. (PBO+, RTS,S+). Addtionally, through 
vaccination records, we will identify households in Balaka where children who received the full 
scheme of Pentavalent (Diphtheria-pertussis-tetanus; Hepatitis B; Haemophilus influenzae type 
b) vaccine and have received a standard bed net live. In Nyambi catchment area, by the end of 
September, an estimated 260 children are expected to be vaccinated with RTSS vaccine. Thus, 
to achieve an adequate sample size for the significant inferential statistics, children will be enrolled 
from the Health Centre and tracked back in the community. Children will be identified and RTS,S 
information will be extracted from the registers by the research team. With the help from the health 
surveillances Assistants (HSAs) and community health volunteers (CHVs), RTS,S age-eligible 
children (7-18 months old) will be tracked using information obtained from the registers back to 
their villages and households where children will be enrolled and followed up for the designated 
study period. In the Kalembo catchment area, the same process that will be employed in Nyambi 
will be adopted. However, to make the cohort more comparable, using under two EPI vaccine 
register, children who completed three doses of Pentavalent (Diphtheria-pertussis-tetanus; 
Hepatitis B; Haemophilus influenzae type b) vaccine and are in RTS,S vaccine age-eligible will 
be considered in Kalembo.  
In both catchment areas, up to two children in the household aged >18 months and < 10 years 
old residing with the vaccine age-eligible children will also be offered the opportunity to enroll. All 
invited children will be screened by a study nurse for eligibility using a screening form. During the 
screening process, the details of the study procedures that will take place in the cohort will be 
described in detail. For children who meet the eligibility criteria, parents/guardians will then be 
asked whether they are willing to have their child participate in the study and those that agree will 
be asked to sign the consent form. A copy of the signed consent form will be given to the head of 
the parent/guardian. All enrolled will be given a study identification number. Only children from 
the families intending to stay in the area whose parents provide written informed parental consent 
will be considered for enrollment. In addition, consent will be sought from the parent/guardian for 
entomological activities in the household.  
 
 

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Table 3: Overview of study procedures 
Visit Type 
Home 
Visit/enrollment 
Two-monthly 
visits:  
Central location 
near home 
 
Study procedure 
 
 
Potentially eligible participants identified 
through 
satellite 
imagery 
and 
predetermined clusters. Households 
are visited and those with eligible 
children identified 
X 
 
Obtain informed consent to participate 
in the study 
X 
 
Child 
demographic 
and 
HH 
socioeconomic information 
X 
 
Through a finger prick, collect blood 
sample and DBS for quantitative PCR, 
serum 
separation, 
antibody 
quantification, preservation of RNA, and 
storage – done on every two months 
follow up for entire 6 months. 
X 
X 
Structured questionnaire interview  
X 
X 
ITN use and vaccination status 
X 
X 
Participants reminded of their visit date 
X 
X 
Household 
mosquito 
collection 
for 
testing 
 
One time 
Hemoglobin measurement 
X 
X 
Malaria rapid diagnostic test 
X 
Final visit 
only 
 
At enrollment, we will conduct household and individual interviews. In this initial household 
interview, we will collect data on hemoglobin status, SES, household construction, and on ITN 
use (PBO or conventional net). Through visual inspection of each bed net to determine if it is a 
PBO or conventional net, we will also identify which other household members aged ≤10 years of 
age who slept under each bed net and with respective frequency. We will also document 
vaccination status including RTS,S vaccine administrations. See details of proposed study 
procedures at the enrollment and every two months follow up for six months in the community. 
See Table 3 above.   
Individual participant’s visits. Every two months through the sixth month, participants in the cohort 
will be invited to the nearest central location for data and specimen collection. A structured 
questionnaire will be administered to collect information on demographics, net utilization and 
vaccination status). At each visit, we will review the target child’s health passport to document all 
vaccinations, including the standard EPI vaccines through 9 months of age and the 15-month 
measles vaccine. We will also document the dates of all RTS,S vaccine administrations.         

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Human blood samples and testing.  
Clinical testing: To establish the study cohort, potential participants will be screened by the study 
team to assess for their eligibility. At enrollmentthe cohort will be screened for malaria using 
Malaria Rapid Diagnostic Tests (The SD BIOLINE Malaria Ag P.f/Pan (HRP-II)™ rapid diagnostic 
test RDT)) and those participants who test positivewill be treated with a full, weight appropriate 
treatment course of dispersible artemether-lumefantrine (LA) by government Staff. The RDT and 
treatment of RDT positive children will be repeated at the final visit.   
Hemoglobin levels from each participant will also be examined at enrollment and at every two-
month follow-up visits. Parents of children with a haemoglobin level under 11 g/dl will be advised 
to take the child to a health facility for follow-up care and will be given a referral letter with the 
haemoglobin reading to show to staff at the health facility by the research Team. 
Molecular analyses: At enrollment and at every two-month visit, we will collect finger-prick blood 
samples from all enrolled participatants. These specimens will be used to assess P. falciparum 
infection by quantitiative PCR. Then the specimens will be stored for later determination of 
gametocyte-specific gene expression and gametocyte density. Serum will be collected to assess 
for serological evidence of exposure to infection and response to vaccination. 
Household mosquito collection and testing. Vector populations will be sampled from 100 study 
households (from RTS,S age-eligible households) in both intervention arms. The selection of the 
households for mosquito collection will be done during the recruitment of age-eligible children.  In 
each catchment area CDC miniature light traps will be set in sleeping rooms to capture host-
seeking vectors indoors. We will also sample blood-fed and gravid mosquitoes by indoor 
aspiration and exit trap sampling. Sampling will occur at each house on four consecutive nights 
during the last four weeks of the rainy season to optimize catch numbers. Light traps will be set 
up in a single functional bedroom in each of the households. Local personnel in each catchment 
area will be trained to carry out these procedures.  
Species of captured adult mosquitoes will be determined by visual examination under microscopy 
and PCR-based assays using procedures well established in our laboratories. Blood-fed mosquito 
abdomens will be tested for the host species that were fed upon using a PCR method based on 
the vertebrate mitochondrial cytochrome B gene. The separated thorax and head of each female 
will be tested for salivary gland P. falciparum sporozoites using standard ELISA or PCR to 
estimate infection prevalence. For non-blood fed mosquitoes, we will assess their parity status by 
dissection and examination of mosquito ovaries. 
6.5 Analysis Plan 
To describe the study population, evaluate potential confounding, and identify sources of selection 
bias, baseline information will be summarized and compared across children who do/do not 
comply with either RTS,S vaccination, or with PBO nets, or with both. These analyses will be 
primarily conducted at an individual level, but to assess robustness of results, comparisons will 
also be conducted at cluster levels. Analysis at individual levels will be performed using random-
effect models to account for the within  subject correlation. Linear, logistic, and over-dispersed 
Poisson models will be used to analyze continuous, categorical, and count variables, respectively. 
Baseline information will also be assessed for children ≤10 years old at individual- and household-
level through nested random effects accounting for correlation of subjects within households.  
 
For analysis of effectiveness on prevalence and transmission, separate models will be fitted to 
estimate total and direct effectiveness (Hayes, 2009; Hallaron, 2010) in children aged 7-18 
months at six time points. Analysis will be primarily done at the individual level, with intervention 

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groups compared through nested random effect models (accounting for correlation across time). 
A subgroup analysis will include children who will have been vaccinated within 3 months from 
Month (M) 1. Models will be logistic for the occurrence of infection and presence of gametocytes, 
and overdispersed Poisson or zero-inflated for gametocyte density. In all analysis, the linearity of 
association of the outcome with predictors will be evaluated through semiparametric regression 
and, if necessary, predictors will be categorized or transformed. In secondary analyses, all 
incident events will be assessed in individual-level analysis through negative binomial models 
(corresponding to discrete proportional hazard multiple event models) with nested random effects. 
We will also explore comparisons of the number of new infection events per follow-up month in 
over-dispersed Poisson models. Since adherence to vaccination (post-randomization variable) 
can introduce selection bias, we will also explore estimating total and direct effectiveness using 
causal inference models.  
 
To examine antibodies of exposure, two approaches will evaluate the association between levels 
of exposure antibodies and intervention groups: 1) antibody levels will be summarized using 
partial least square regression (PLS); 2) levels of individual antibodies will be used. Summary 
scores and antibody levels will be compared over time between the two groups through joint 
outcome (shared parameter) models (Verbeke, 2010). Moreover, joint outcome models will be fit 
for positivity of responses and a zero-inflated model will be fit for the breadth of responses 
(number of antibodies that are positive in a subject).  
 
For analysis of effectiveness in all household members (in Phase 1, children aged >18 months 
and < 10 years old), indirect effectiveness at the individual level at four-time points will be 
estimated. Also, in nested random effect models for individual analysis, we will account for 
correlation across visits, within the household. The household-level analysis will be conducted by 
summarizing outcomes in the household, and also in nested random effect models, using the 
same link functions described above. 
For entomological outcomes, analysis of effectiveness will be performed comparing intervention 
groups through zero inflated negative binomial models with random effects, including as offsets 
hours of capture effort or number of captured mosquitoes, as appropriate. Outcome variables will 
include abundance of Anopheles, sporozoite rates, blood-meal originated from humans, and 
female parity in households. 
 
6.5.1 Data handling and record keeping 
Data for the survey will be collected through questionnaires programmed onto computer tablets. 
Electronic records (in the form of structured questionnaires) will be made from each data-
producing contact between the study participants and the study team. These source data are de-
identified (each sheet containing date, participant number, household number and signature of 
the data collector). Questionnaires will be administered to household owners by enumerators. 
Every day after the fieldwork the questionnaires will be uploaded loaded to a secure central 
database. The team leader/ data manager will immediately check for errors. Additionally, periodic 
reports will check for errors in data capture. The database will be managed at the Data 
Management Core at the College of Medicine in Blantyre. All documentation regarding the 
participants will be identified with appropriate participant codes, both on paper and in computer 
files. Samples will be de-identified and contain only a sample identification code. The names will 
only appear on informed consent forms and a separate coding list that will be secured stored, 
separately from the database. Only the Principal Investigators and designated study personnel 

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will have access to the code list linking study identification numbers with the subject identifiable 
information.  
The investigators shall maintain the records of their copies of the questionnaires and regulatory 
documents (informed consents, ethical approval) with all records kept in a secure place. Clinical 
information will not be released without written permission of the subject, except as necessary for 
monitoring. 
6.6 Results presentation and dissemination 
The results of this study will be presented through tables, graphs and powerpoint presentations. 
If maps are used to display information about malaria prevalence, household GPS data will be 
aggregated at the village or cluster level such that individual households are not identifiable. 
Locally, the results will be discussed with the villages in the study area, the health centers 
involved, Machinga and Balaka District Health Office and Assembly. The main findings will also 
be published in international peer-reviewed journals and the Malawi Medical Journal, and through 
presentations at local, regional or international conferences/seminars. Results will also be 
presented to the College of Medicine Research and Ethics Committee (COMREC) and the 
National Malaria Control Program since they develop and implement policy as well as other 
stakeholders. 
Data will be collected prospectively and this data will be of use to the Malawi National Malaria 
Control Program, NIH and other malaria control partners in Malawi and beyond. Data from studies 
will be made available to researchers, policymakers and the wider community at no cost to 
promote dialogue on the results, encourage further analysis, inform new research questions and 
disseminate information to the community. This data sharing plan is designed to enable access 
to as much data as possible while protecting the human subject identity and ensuring proper use. 
 
De-identification of data:  
Under this proposal, we will de-identify all the data intended for broader use and variables which 
could be used to deductively disclose the identity of individual subjects. Examples of data that will 
be removed include names, date of births or attendance at clinics, medical record numbers, GPS 
location etc. Only the PI and designated personnel in Malawi will have access to the link between 
study and sample identification number and the names and medical record numbers of study 
participants.  Only staff and investigators with authorization from the  
 
Data Sharing:  
All de-identified data will be made available through NIH supported portals for wide dissemination 
and access. All de-identified human clinical and epidemiological data will be available through the 
ClinEpiDB repository whilst genomic data from humans (if any) and mosquitoes will be made 
available through controlled access to the EuPathDB repository. Any data shared will be 
subjected to conditions of use governing access to the public release data, including restrictions 
against attempting to identify study participants, destruction of the data after analyses are 
completed, reporting responsibilities, restrictions on redistribution of the data to third parties, and 
proper acknowledgment of the data resource.  
Data Submission and Release Timeline: 
Research data from a completed study will be shared with the scientific community and other 
interested organizations within a period of one year from completion of the study when the data 
has been thoroughly cleaned and a draft report has been written. Data will be sent to the 
repositories no later than the closure date for the grant.  
 

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7 ETHICAL CONSIDERATIONS 
7.1 General Principles 
Strict measures will be taken by the study investigators and study staff members to ensure subject 
confidentiality. The study protocol, documentation, data and all other information generated will 
be kept in strict confidence. Each participant will be assigned a unique Participant Identification 
Number (PID) that will be used for data entry and analysis linked to the different Case Report 
Forms (CRFs). Records will only be accessible to the data entry team, the individual conducting 
data entry checks as part of the dual entry system and the authorized study investigators. All 
consent forms containing identifiable information will be kept in a locked cabinet available only to 
the Principal Investigator.  All data will be stored in and analyzed using password-protected, 
encrypted secure computers. No information or data of the study participants will be released to 
any unauthorized third party without prior approval of the study participant and appropriate IRBs. 
If maps are used to display information about malaria prevalence, household GPS data will be 
kept in a separate file (to which only the PI and designated personnel involved in data captures 
will have access). 
7.2 Informed Consent 
Consent will be sought from all collectors and study participants. Informed consent will be initiated 
by the study staff prior to anyone agreeing to participate in the study. The study staff members 
will introduce themselves and explain the purpose of the study to the parent/guardian. In this 
study, a guardian or caretaker will be defined as any adult of legal age with a blood relation to the 
child (such as a grandparent, sister or aunt) or a legal guardian who is involved in taking care of 
the child. The parent/guardian of potentially eligible children will then be asked to read the 
informed consent in the preferred language either in Chichewa (the local language) or in English. 
The informed consent will be read and explained verbally in Chichewa to those who are unable 
to read. This verbal explanation will be attended by an impartial witness who will also sign the 
consent form. If no impartial witness is available, a member of the study staff other than the person 
administering consent may serve as a witness and sign the document. Study staff members will 
then answer any questions that the parent/legal guardian of the child participant may have, 
following which they will be asked to sign the informed consent form. For those who cannot write, 
a thumbprint will be placed on the form, with the signature and the date of the witness. A copy of 
the consent form will be given to the parent/guardian. All consent forms will be IRB approved in 
Malawi and the USA.   
Study participation will be completely voluntary and participants will be informed that they may 
withdraw consent at any time throughout the course of the study without penalty. The rights and 
welfare of participants will be protected by emphasizing to the parent/guardian that the quality of 
the participant’s medical care will not be adversely affected if they decline participation. If the 
parent/guardian decides to withdraw consent, the participant/child will be able to continue to 
receive health care at the government clinics and hospitals or wherever they choose.  
Future Use of Stored Specimens: 
The informed consent form for this study has a section that will offer participants a choice about 
the use of study specimens that remain after the study procedures have been completed. Any 
use of study samples that is outside the scope of this protocol will be submitted for prior review 
and approval by the appropriate IRBs when the need arises. Only blood samples collected during 
finger pricks will be stored for possible future use either as dry blood spots on filter paper or as 
plasma or serum in cryovial tubes.  

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7.3 Possible risks and benefits to study participants 
7.3.1 Potential Risks 
Fingerprick blood sampling:  All participating children will be screened for Plasmodium infection 
during monthly visits, and for disease during unscheduled visits to the HC when they have any 
illness. Plasmodium infection screening will be done through a routine monthly sample taken for 
dried blood spots (DBS). Blood samples for all these procedures will be done through a finger 
prick which may cause participants temporary discomfort. Moreover, blood specimens collected 
through finger prick will be used to measure serological markers. However, this will also represent 
a negligible volume of blood collected though finger prick. To prevent any discomfort and risks 
associated with finger pricks, we will use optimal lancets and will appropriately clean the area 
before the extraction of blood. 
Breach of confidentiality:  All data collected during the survey is strictly confidential. There is 
however a small risk of breach of confidentiality during the study with inadvertent access of 
personal health information to non-study personnel, Efforts directed to reduce this risk will be a 
priority by having all study data coded. Each participant will be given a unique Participant 
Identification number (PID), and all electronic data will be linked to this PID. Directions to the 
house of the participant will be linked to the PID. It will however, be accessible by the home 
interviewer only during home interviews and by Data Managers to address queries. All field data 
collection will be done on PDAs that will be password protected and accessible only to the relevant 
study personnel staff. Data recorded in the PDA will be transferred to the main data base at the 
end of each day.  
All electronic data will be stored in password-protected computers in a locked room in the data 
management Core section (DMC). The password protected database files will only be accessible 
to designated data entry personnel and study investigators. Backup copies of the database files 
will be maintained in a separate hard disk that will be kept in a locked filing cabinet at DMC. The 
dataset mapping PID to subject identifiatble information including names and directions to the 
households will be stored separately from the research database and only the PI and the essential 
research team will be granted access to this dataset.  
Compensation:  In accordance with the Malawi National Commission for Science and Technology 
(NCST) circular dated 22nd August 2019, we will reimburse the parent/guardian (household) with 
MK 2,000 for the travelling costs each time she/he comes with participants to the central location 
for blood sample collection and interviews. The amount of money to be reimbursed is an estimated 
average cost for participants who travel to and from the disignated central location. 
7.3.2 Known Potential Benefits 
This study will enroll children aged 7 months to 10 years within two government HC catchment 
areas. In Malawi, this age group is at greatest risk of malaria infection and its complications. The 
participants will benefit from being intensively followed-up during the six months specifically at (0, 
2, 4, and 6 months) study period, and by having mRDT results provided and treatment provided, 
if indicated at 1 and 6 months. The research team will give the participants health advice and refer them 
for treatment at the local health center. The study team will support the local health center with laboratory 
services and drugs so that all participants will be guaranteed access to laboratory diagnostics of 
malaria and or antimalarial treatment where necessary. The individuals who participate in this 
study will have access to prompt diagnosis and treatment of malaria when appropriate. We are 

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going to perform mRDTs to participants at recruitment and 6 months. Similarly, we will also test 
participants for anemia at recruitment and at all the follow up months. By participating in this study, 
the children will have access to high-quality and personalized medical attention. In the case of 
life-threatening emergencies, the study team will use their vehicle to transfer the study participants 
to the nearest government or private hospital. The minimal risks of this study, associated with 
finger pricks and the breaches of confidentiality are outweighed by these benefits. In addition, the 
study will provide support to the health centre in the study with resources mainly in malaria 
diagnosis and treatment which will benefit the community at large, not just research participants. 
In addition, the results of this study will help to maximize the implementation and impact of malaria 
prevention and control measures throughout Malawi. Each enrolled houslehold will be given 
K2000 for each study visit as compensation for their transport cost during enrolment and monthly 
visits.    
8 POSSIBLE CONSTRAINTS 
The main possible problems with participation are hesitation over providing a small volume of 
blood specimens through a finger prick. Extensive sensitization before and during the survey 
should significantly mitigate this possibility. In ICEMR, we have successfully conducting the 
collection of blood specimens through finger prick without notable resistance from communities. 
9 REQUIREMENTS 
For the evaluations of study participants, each study site will have the regular HC-based team 
and field teams. The regular HC teams will be comprised of government employed clinical, 
nursing and laboratory staff, and ICEMR-employed epidemiological field teams of a nurse, 
enumerator and a village field assistant. The study nurses and enumerators will receive training 
in human subjects’ research, entomological sampling, and data collection using electronic data 
capture devices. The HC based team will be responsible for passive disease surveillance from 
participants referred by the field teams during monthly visits and/or from participant’s non-
scheduled visits to the facilities at any other time during the follow-up. Responsibilities assigned 
to the study team members will be stipulated in standard operating procedures (SOPs) and will 
be conducting in-depth interviews. Entomological assessments will be conducted by a different 
team comprised of an entomology research assistants and field technicians.  
A data team lead by Data Managers will be responsible for receiving, archiving and processing 
all incoming data according to standard operating procedures for local storage and transfer to 
collaborating institutions. All data will be collected using electronic devices. The nurse, 
enumerator and an entomology research assistant will be using laptop computers to access the 
REDCap electronic data capture system for data collection. The REDCap system will be accessed 
using the internet, however, if internet connectivity is lost, an offline version of REDCap installed 
on the laptops will be used. The field worker will use the tablet computer to collect data.  
Other requirements include local transportation (a vehicle, motorcycles, and bicycles will be 
procured), office administration (computers, office supplies, space rentals), laboratory and 
pharmaceutical consumables, including kits for Luminex assays and calibration reagents for the 
Luminex machine in the laboratory of Malawi, and protective wear. Details are highlighted in the 
budget below. 

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10 TRAINING PROVIDED 
The study offers training to 1 Malawian post-doctoral research fellow from the College of 
Medicine, University of Malawi who will lead the project implementation and will supervise core 
local staff. Other PhD students/post-docs include: 
• 
Jimmy Vareta (molecular epidemiology of malaria transmission) 
• 
Christopher Stanley (biostatistician with expertise in longitudinal analysis) 
• 
Rex Mbewe (impact of PBO nets on entomological indicators) 
 
 

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in field populations of Anopheles quadriannulatus and Anopheles arabiensis in Malawi: evidence 
for larval selection. Medical and veterinary entomology. 2008;22(3):258-63. 
 
Mzilahowa T, Chiumia M, Mbewe RB, et al. Increasing insecticide resistance in Anopheles 
funestus and Anopheles arabiensis in Malawi, 2011-2015. Malar J. 2016;15(1):563. 
doi:10.1186/s12936-016-1610-1.  
 
Ochomo EO, Bayoh NM, Walker ED, et al. The efficacy of long-lasting nets with declining 
physical integrity may be compromised in areas with high levels of pyrethroid resistance. Malar 
J. 2013;12(1):1. doi:10.1186/1475-2875-12-368. 
 
Okiro EA, Kazembe LN, Kabaria CW, Ligomeka J, Noor AM, Ali D, et al. Childhood malaria 
admission rates to four hospitals in Malawi between 2000 and 2010. PloS one. 
2013;8(4):e62214. 
 
Penny MA, Pemberton-Ross P, Smith TA. The time-course of protection of the RTS,S vaccine 
against malaria infections and clinical disease. Malar J. 2015;14:437. doi:10.1186/s12936-015-
0969-8.  
 
Protopopoff N, Mosha JF, Lukole E, et al. Effectiveness of a long-lasting piperonyl butoxide-
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Project 004: Combined effects of RTS,S vaccination (malaria vaccine) and  piperonyl butoxide (PBO) nets on malaria 
infection and transmission. Version 2.0: 12 October 2019 
 
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World Malaria Report 2017.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Project 004: Combined effects of RTS,S vaccination (malaria vaccine) and  piperonyl butoxide (PBO) nets on malaria infection and transmission. Version 2.0: 12 
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12 BUDGET 
 
Item Description 
Posts 
Salary (USD) 
Salary (MK) 
Effort 
Total Salaries 
(USD) 
 Total Salaries 
(MK)  
Totals (USD) 
Totals (MK) 
A 
Study Staff Salaries 
  
  
  
  
  
  
212,510.00  
  
155,132,300.00  
  
Post Title / Personnel 
No. 
Posts 
 Salary/post 
/month  
 Salary/ post 
/month  
Effort in 
mos/Yr 
 Total 
Salaries/Yr   
  Total 
Salaries/Yr     
  
  
Field-based staff 
  
  
 
  
  
 
  
 
1 
Grant administrator - finance and 
admin 
1 
4,000 
 2,920,000.00  
0.50 
2,000.00 
     1,460,000.00  
  
  
2 
Grant administrator - HR extended to 
all 5 years 
1 
4,000 
 2,920,000.00  
1.00 
4,000.00 
     2,920,000.00  
  
  
3 
Procurement Coordinator extended to 
all 5 years 
1 
1,200 
    876,000.00  
1.00 
1,200.00 
        876,000.00  
  
  
4 
Project Coordinator 
1 
2,500 
 1,825,000.00  
6.00 
15,000.00 
   10,950,000.00  
  
  
5 
Data Manager 
1 
3,000 
 2,190,000.00  
4.80 
14,400.00 
   10,512,000.00  
  
  
6 
Nurses (2 in year1 and 2) Epi 
2 
1,000 
    730,000.00  
12.00 
12,000.00 
     8,760,000.00  
  
  
7 
Enumerators (2 in yr1 and 2 yr 2) Epi 
2 
690 
    503,700.00  
12.00 
8,280.00 
     6,044,400.00  
  
  
8 
4 Ento field workers (6 months in yr 2) 
4 
690 
    503,700.00  
24 
16,560.00 
   12,088,800.00  
  
  
9 
1 lab technician (12 months) EPI 
1 
0 
                 -   
12 
0.00 
                     -    
  
  
10 
1 lab technician (12 months in yr 2) 
Ento 
1 
1,000 
    730,000.00  
0 
0.00 
                     -    
  
  
11 
1 lab tech, Molecular Core (SA1) 
1 
1,500 
 1,095,000.00  
6 
9,000.00 
     6,570,000.00  
  
  
12 
1 Driver 
1 
570 
    416,100.00  
6 
3,420.00 
     2,496,600.00  
  
  
13 
Undergrad Intern 
1 
500 
    365,000.00  
36 
18,000.00 
   13,140,000.00  
  
  
  
Sub-total for Field-based Staff 
  
  
                 -     
103,860.00 
   75,817,800.00  
  
  
  
Post Doctoral Associates 
  
  
                 -     
  
                     -    
  
  

Project 004: Combined effects of RTS,S vaccination (malaria vaccine) and  piperonyl butoxide (PBO) nets on malaria infection and transmission. Version 2.0: 12 
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1 
EPI Postgraduate (TBR)  
1 
3,000 
 2,190,000.00  
12 
36,000 
   26,280,000.00  
  
  
2 
Ento Postgraduate (TBR) 
1 
0 
                 -   
0 
0 
                     -    
  
  
3 
EPI Research Assistant (TBR) 
1 
1,200 
    876,000.00  
6 
7,200 
     5,256,000.00  
  
  
4 
Ento Research Assistant (TBR) 
1 
1,200 
    876,000.00  
6 
7,200 
     5,256,000.00  
  
  
5 
Ento Research Assistant 
1 
1,200 
    876,000.00  
  
0 
                     -    
  
  
  
Subtotal Post-docs salary 
  
  
                 -   
  
50,400.00 
   36,792,000.00  
  
  
  
Graduate Students stipend/ pay  
  
  
                 -   
  
  
                     -    
  
  
1 
EPI Intern (TBR)  
1 
1,000 
    730,000.00  
12 
12,000.00 
     8,760,000.00  
  
  
  
Subtotal Grad students stipend/ 
pay 
  
  
                 -   
  
12,000.00 
     8,760,000.00  
  
  
  
Sub-total support staff 
  
  
                 -   
  
  
                     -    
  
  
  
Professional staff 
  
  
                 -     
  
                     -    
  
  
1 
Overall PI 
1 
12,000 
 8,760,000.00  
2.50 
30,000.00 
   21,900,000.00  
  
  
2 
Entomological Co-PI 
1 
6,500 
 4,745,000.00  
2.50 
16,250.00 
   11,862,500.00  
  
  
  
Sub-total professional staff 
  
  
 
  
46,250.00 
   33,762,500.00  
  
  
  
Total salary support    
  
 
  
212,510.00 
 155,132,300.00  
  
  
 
 
  
B 
Travel Costs 
  
  
 
  
  
 
    5,510.00       4,022,300.00  
C 
Office Rental Costs 
  
  
 
  
  
 
    3,744.00       2,733,120.00  
D 
CLINICAL SUPPLIES (for sample 
collection and analysis for 12 
months)  
  
  
 
  
  
 
67,300.00  
   
 
 49,129,000.00  
E 
Equipment 
  
  
 
  
  
 
  24,828.00     18,124,440.00  
  
Total Direct Cost  
  
  
 
  
  
 
313,892.00  
 
229,141,160.00  
  
F&A, College of Medicine (8%)* 
  
  
 
  
  
  
  23,125.12     16,881,337.60  
  
Total 
  
  
 
  
  
  
337,017.12   246,022,497.60  

Project 004: Combined effects of RTS,S vaccination (malaria vaccine) and  piperonyl butoxide (PBO) nets on malaria infection and transmission. Version 2.0: 12 
October 2019 
 
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