"LAMPREG TRIAL" Active Case Detection and Treatment of Malaria in Pregnancy Using LAMP Technology: A Pragmatic Randomized Multi-Center Diagnostic Outcomes Trial
Phase: NA
Conditions: Malaria, Pregnancy Malaria, Malaria in Pregnancy
Sponsor: University of Calgary

University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
ClinicalTrials.gov Identifier: NCT03754322                                                                       v.1.1 29 March 2020 
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                                              “LAMPREG TRIAL” 
Active case detection and treatment of malaria in pregnancy using LAMP technology: 
A pragmatic randomized Multi-Center diagnostic outcomes trial 
 
 
 
• Principal Investigator from University of Calgary (UC), Calgary, Canada: 
o Dylan R. Pillai, MD, PhD, Professor 
• Principal Investigator from Armauer Hansen Research Institute (AHRI), Addis Ababa. 
Ethiopia: 
o Abebe Genetu Bayih, PhD, Director General 
• Co-Investigator from Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia  
o Rediet Fikru Gebresenbet, MD, MPH  
• Co-Investigator from Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia     
o Zelalem Mekonnen Bekele, MD, MSc 
 
• Co-Investigator from Jimma University, Jimma, Ethiopia 
o Delenasaw Yewhalaw, PhD 
 
• Co-Investigator from Amhara Public Health Institute (APHI), Bahir Dar, Ethiopia 
o Banchamlak Tegegne, MSc 
  
 
 
 v.1.1 29 March 2020 

University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
ClinicalTrials.gov Identifier: NCT03754322                                                                       v.1.1 29 March 2020 
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Table of Contents 
 
Table of Contents .......................................................................................................................................... 2 
SUMMARY ..................................................................................................................................................... 4 
1. INTRODUCTION ......................................................................................................................................... 5 
1.1. 
Background ................................................................................................................................... 5 
1.2. Literature Review ............................................................................................................................... 6 
2. 
SIGNIFICANCE OF THE STUDY ............................................................................................................... 8 
4. OBJECTIVES ............................................................................................................................................. 12 
4.1. General objective ............................................................................................................................. 12 
4.2. Specific objectives ........................................................................................................................... 13 
5. MATERIALS AND METHODS .................................................................................................................... 13 
5.1. Study area......................................................................................................................................... 13 
5.2. Study design and period ................................................................................................................... 13 
5.3. Population ........................................................................................................................................ 16 
5.3.1. Study population ....................................................................................................................... 16 
5.4. Inclusion and exclusion criteria ....................................................................................................... 17 
5.5. Variables .......................................................................................................................................... 17 
5.5.1. Primary outcome ....................................................................................................................... 17 
5.5.2. Independent variables ............................................................................................................... 17 
5.5.3 Other endpoints .............................................................................................................................. 18 
5.6. Sample size and sampling techniques .............................................................................................. 18 
5.7. Data collection and laboratory methods ........................................................................................... 18 
5.7.1. Socio-demographic and clinical data ........................................................................................ 18 
5.7.2. Blood sample collection and processing ................................................................................... 19 
5.7.3. Malaria microscopy and RDT ................................................................................................... 19 

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5.7.4. Loop-mediated isothermal amplification (LAMP) ................................................................... 20 
5.8. Statistical Analysis Plan ................................................................................................................... 20 
5.9. Data analysis and interpretation ....................................................................................................... 20 
5.10. Benefits and beneficiaries of the proposed study ........................................................................... 21 
5.11. Dissemination of findings .............................................................................................................. 21 
5.12. Ethical Considerations ................................................................................................................... 21 
6.0. WORK PLAN .......................................................................................................................................... 26 
7.0 English Version of Questionnaire ..................................................................................................... 27 
8.0 References …………………………………………………………………………………..29 
 
 
 
 
 
 
 
 
 
 
 
 

University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
ClinicalTrials.gov Identifier: NCT03754322                                                                       v.1.1 29 March 2020 
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SUMMARY 
Background: Malaria is one of the major public health problems in sub-Saharan Africa. It 
contributes significantly to maternal and fetal morbidity and mortality in affected countries.  
Objective: The aim of this study is to evaluate the impact of enhanced case detection using 
molecular testing (LAMP) on maternal and infant morbidity and mortality in a prospective study 
design. 
Method: A pragmatic randomized control diagnostic trial will be conducted from October 2020 
until October 2021 in pregnant mothers at sites in Ethiopia. Both symptomatic and asymptomatic 
first and early second trimester pregnant women will be included in the study and individually 
randomized to either standard of care or enhanced cased detection arms using LAMP for malaria. 
Women (n=2583) will be enrolled during a twelve-months period encompassing the peak 
transmission seasons and then followed until delivery. In the standard of care arm, venous blood 
sample will be collected from each study participant and the presence of Plasmodium infection 
will be diagnosed by microscopy in symptomatic patients. Pregnant women who test positive for 
malaria will be referred and treated for malaria with quinine or artemisinin combination therapies 
(ACTs) as per national guidelines. In the intervention arm, mothers who are symptomatic or 
asymptomatic will be tested by a commercially available CE-approved LAMP malaria test and 
microscopy/RDT for malaria at each clinic visit and treated if positive by any test. Pregnant 
mothers who require treatment will be referred and treated with either quinine or artemisinin 
combination therapy (ACTs) as per national guidelines. The primary outcome is the proportion of 
deliveries with low birth weight based on WHO definition, with secondary outcomes of:(i)absolute 
birth weight; (ii) maternal hemoglobin;(ii) neonatal hemoglobin at birth;(iv) neonatal mortality; 
(v) stillbirth; and (vi) prematurity in each arm of the study. 
Result Dissemination plan: The results of this study will be presented at national and international 
scientific conferences. It will also be published in a reputable journal.  
Work plan and budget: This study will be conducted from October 2020 to October 2021. The 
study is funded by Grand Challenges Canada, FIND, AHRI, University of Calgary, with support 
from test manufacturers (Human Diagnostics, Wiesbaden, Germany and Meridien Diagnostics, 
Cincinatti, USA) 
Key words:  Malaria, LAMP, Pregnancy 

University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
ClinicalTrials.gov Identifier: NCT03754322                                                                       v.1.1 29 March 2020 
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1. INTRODUCTION 
1.1. Background 
 
In 2017 about 219 million cases of malaria occurred worldwide and approximately 435,000 people 
died of the disease globally according to the World Health Organization.  The sub-Saharan region 
experienced around 92% of these deaths [1]. Mortality is concentrated around several high-risk 
groups, including pregnant women and infants. In Africa, 30 million women living in malaria-
endemic areas become pregnant each year. Up to 200,000 newborn deaths each year result from 
malaria in pregnancy. Malaria infection has an adverse impact on pregnancy in endemic countries. 
Diagnosis of malaria in pregnancy is limited by microscopy and RDT both with poor sensitivity. 
Malaria, particularly due to P. falciparum, in pregnant women increases the risk of maternal death, 
miscarriage, stillbirth and neonatal death. In areas of seasonal malaria transmission, pregnant 
women are three times more likely to suffer from severe malaria as compared to non-pregnant 
counterparts. The World Health Organization (WHO) recommends prompt diagnosis and 
appropriate treatment of malaria. However, diagnostics are lacking in detecting low-level 
infections typically found in pregnant women because malaria parasites are sequestered in the 
placenta and not present in the peripheral blood in large quantities.  
It is well documented that sequestration in the placenta leads to adverse outcome for the mother 
and infant especially related to anemia and birth weight [2,3]. Both anemia and low birth weight 
are predictors of poor outcomes in infancy for the child. Malaria in pregnancy therefore affects not 
only the current generation but the next, hobbling cognitive development and thus economic 
productivity. In other parts of sub-Saharan Africa, the issue is so profound mothers are exposed to 
antimalarials during pregnancy without testing to prevent malaria. But this comes at a cost since 
empiric use of anti-malarial drugs contributes to resistance and may lead to side effects. 
The National Strategy to eliminate malaria in Ethiopia and other sub-Saharan countries in the next 
10 years will demand highly sensitive diagnostics in patients with such low-level infections. Our 
group has validated a DNA based test for malaria called "LAMP" and shown up to 30% greater 
detection of malaria in pregnant women who are symptomatic and even greater than 90% detection 
when screening “asymptomatic” women. We are now actively seeking to scale up this heath care 

University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
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technology to determine the true impact across Ethiopia with key partners. Meaningful impact will 
be measured by determining health outcomes for mother and child, especially reduction in infant 
anemia and improvement of birth weight. 
 
1.2. Literature Review 
 
There are many different types of tests available for malaria. Microscopy of stained blood smear 
and rapid diagnostic techniques (RDTs) are the most widely used laboratory methods. Molecular 
tests for malaria such as polymerase chain reaction (PCR) are highly sensitive but not feasible in 
resource-limited settings due to high capital equipment costs, laboratory infrastructure needs, 
reagent supply chain, and specialized training. The process of the PCR is also time-consuming 
resulting in delays in reporting the results to clinicians which, in turn, compromises timely 
management of patients. Traditionally, laboratory diagnosis has relied on the identification of 
parasites in a peripheral blood film using either Giemsa, Wright, or Field stain[4,5]. Microscopy 
is an accurate tool but requires well-trained laboratory technicians. In experienced hands, the limit 
of detection is about 50 parasites/µL[5]. Rapid diagnostic tests (RDTs) are alternatively used for 
the diagnosis of malaria in all health facilities or through rural health extension and outreach. RDTs 
are easier to perform and used for screening of malaria in remote areas where electricity and other 
resources are limited [6,7]. However, microscopy and RDTs cannot reliably detect lower-density 
parasitemia (<100 parasites/µL)[5]. As most adverse outcomes caused by malaria are because of 
wrong, late, or unavailable diagnosis, there is a need to find a new alternative diagnostic tool for 
field diagnosis of malarial infection [8]. 
 
Loop-mediated isothermal amplification (LAMP) is a potential point of care test (POCT) that 
provides an alternative to microscopy and RDTs [9–11]. It is a molecular method, which in 
comparison to PCR is cheaper, simpler, and faster, overcoming three major disadvantages of PCR. 
The LAMP test is a nucleic acid amplification method that relies on a strand-displacement DNA 
polymerase which also retains reverse transcriptase activity. The principal merit of this method is 
that no denaturation of the DNA template is required, and thus, the LAMP reaction can be 

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ClinicalTrials.gov Identifier: NCT03754322                                                                       v.1.1 29 March 2020 
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conducted under isothermal condition [12]. It is low cost, requires no electricity, provides rapid 
results and can be performed by minimally trained health workers [13]. Studies have found that 
LAMP has a comparable sensitivity and specificity to PCR, and is superior to microscopy and 
RDTs [14]. The method can detect parasitaemia as few as 1 parasite/μl of blood or lower, below 
the detection limit of microscopy or RDTs [12,15]. This is particularly important in pregnant 
women, where sequestration of parasites in the placenta is associated with low level parasitemia 
in peripheral blood [3]. This would lead to cases of chronic malaria going undetected. Although 
microscopy is considered as a gold-standard method, several nucleic acid amplification techniques 
have been evaluated for the diagnosis of malaria in the general population and also in pregnant 
women. Rantala and colleagues (2010) compared a real-time PCR assay and conventional 
microscopy for the detection of P. falciparum in Malawi.  Of the 475 women, P. falciparum was 
detected in 11 (2.3%) by microscopy and in 51 (10.7%) patients by real-time PCR, thus questioning 
the accuracy of microscopy in this population. 
Our group conducted a cross-sectional study in North Gondar, Ethiopia in 2014 to evaluate the use 
of LAMP in combination of a non-instrumented nucleic acid amplification (NINA) heater for the 
diagnosis of malaria. Using nested PCR as reference, the sensitivity and specificity of the primary 
NINA-LAMP assay were 96.8% (83.2% - 99.5%) and 84.3% (71.4% - 92.9%), respectively for 
detection of Plasmodium genus. Microscopy demonstrated sensitivity and specificity of 93.6% 
(78.5% - 99.0%) and 98.0% (89.5% - 99.7%), respectively for the detection of Plasmodium 
parasites. Post-hoc repeat NINA-LAMP analysis showed improvement in diagnostic accuracy, 
which was comparable to nested PCR performance and superior to microscopy for detection at 
both the Plasmodium genus level and P. falciparum parasites[10]. Recently, our group also showed 
the usefulness of LAMP for the diagnosis of malaria in pregnant women with 100% sensitivity 
achieved in a cohort of 87 women diagnosed with malaria [9]. What remains to be demonstrated 
is that gains in sensitivity with LAMP translate to improved outcomes for mother and child. 
Malaria contributes very significantly to maternal anemia and fetal mortality [16]. Pregnant 
women are three times more likely to suffer from severe malaria compared to the non-pregnant 
control population. Pregnant women infected with malaria usually have more severe symptoms 
and outcomes, with higher rates of miscarriage, intrauterine demise, premature delivery, low-birth-
weight neonates, and neonatal death. Chronic non-fatal infections also lead to complications with 

University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
ClinicalTrials.gov Identifier: NCT03754322                                                                       v.1.1 29 March 2020 
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the main problems being maternal anemia, intra-uterine growth retardation [2] and low birth 
weight.  
In sub-Saharan Africa, anemia reportedly accounts for about 20% of all maternal deaths [17]. It is 
estimated that in areas where malaria is endemic, around 19% of infant low birth weights are due 
to malaria and 6% of infant deaths are due to low birth weights caused by malaria [18]. Low birth 
weight is thought to be the single biggest risk factor for neonatal and infant mortality [16].These 
complications are so commonplace and severe that most countries in sub-Saharan offer 
intermittent presumptive therapy (IPT) to pregnant women, but resistance rates to the drug used 
(sulfadoxine-pyrimethamine) are very high and compliance with the full 3 doses during pregnancy 
poor. Other interventions include insecticide treated nets (ITN) and effective educational outreach 
programs [19]. 
Low-level parasitemia also have a role in disease in the non-pregnant population. Recent studies 
have pushed for asymptomatic malaria to be termed chronic malaria instead, with a view that even 
low level parasitemia can lead to severe health and economic consequences[20]. Additionally, as 
malaria incidence continues to fall, asymptomatic carriers with a low level parasitemia become 
more important in reducing transmission. They are thought to contribute to the malaria reservoir 
which leads to ongoing transmission by mosquitoes. Identifying and treating this group will be 
essential if the fight to eradicate malaria is to succeed. 
An accurate parasite-based diagnosis of malaria is essential for proper treatment of the individual 
patient. Reliably excluding the diagnosis is equally valuable, because this will guide the clinician 
to consider an alternative diagnosis, which can be lifesaving [21]. A correct diagnosis is also 
important for public health, because avoidance of inappropriate antimalarial treatment will reduce 
costs and helps prevent the spread of drug resistance.  
2. 
SIGNIFICANCE OF THE STUDY 
 
Malaria in pregnancy often results in high degree of morbidity and mortality of the pregnant 
mother and the fetus. Early and accurate diagnosis of subclinical infections will be critical to 
malaria elimination and specifically the goals of the World Health Organization to reduce the  

University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
ClinicalTrials.gov Identifier: NCT03754322                                                                       v.1.1 29 March 2020 
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burden of disease by 90% before 2030. This goal can only be achieved using highly sensitive 
methods such as LAMP that are capable of detecting subclinical infections with very low 
parasitemia. Currently both Giemsa stained blood film microscopy and RDT are the only 
laboratory methods that are used to diagnose malaria both in pregnant mothers and the general 
population. This leaves a big gap in the detection of low-level infections and asymptomatic malaria 
due to the documented lack of sensitivity of the aforementioned methods. This, in turn, predisposes 
pregnant mothers to malaria-related complications that endangers the life of the mother and the 
fetus. In this study, we propose that the use of a highly sensitive LAMP technique will enable us 
to detect more asymptomatic Plasmodium infections in pregnant women. This consequently results 
in early treatment of the pregnant mothers and may avert maternal and fetal morbidity and 
mortality.  This study is of particular importance in Ethiopia where IPT is not used for pregnant 
women and therefore accurate screening is paramount. 
 
3. 
PILOT DATA 
At the time of writing, the phase 1 proof of concept retrospective study is complete. Phase 1 data 
are shown in Table 1 and 2 demonstrating the superior performance of LAMP technology 
compared to traditional methods. In particular, LAMP was able to detect up to 30% more malaria 
infected women than traditional methods using passive case detection (i.e., only symptomatic 
women).  
 
 
 
 
 
 
 
 
 
 

University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
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Table 1: Demographic and clinical features of pregnant women enrolled in the phase 1 
retrospective evaluation of LAMP (commercial) versus traditional malaria diagnostic methods. 
 
Variable 
Frequency* 
Positive 
N (%) 
Negative 
N (%) 
Age    18-24 
25-30 
31-35 
36+ 
3   (13.0) 
5   (10.9) 
1 (7.1) 
1   (25.0) 
20 (87.0) 
41 (89.1) 
13 (92.9) 
3  (75.0) 
Previous malaria history 
 
 
Yes 
No 
5 (11.4)              
5 (11.6)              
39 (88.6) 
38 (88.4) 
Trimester 
 
 
First 
Second 
Third 
2 (13.3)              
5  (13.9)             
3  (8.3)               
13 (86.7) 
31 (86.1) 
33 (91.7) 
Gravidity 
 
 
Primigravidae 
Multigravidae 
2 (9.1)                
8 (12.3)              
20 (90.9) 
57 (87.7) 
                          * As determined by PCR.  
 
Table 2: Performance of LAMP compared to microscopy and RDT in symptomatic pregnant 
women (N=87, Gondar, NW Ethiopia), phase 1 retrospective study. 
Method 
Sensitivity % (95%CI) 
Specificity % (95%CI) 
Microscopy 
90 (66.3-100) 
98.7 (96.5-100) 
RDT 
70 (33.8–100) 
97.4 (92.9–100) 
LAMP 
100 (100) 
93.5 (86.5-100) 
 
In phase 2 proof of concept, we compared ELUCIDx and a commercial LAMP assay to traditional 
diagnosis (RDT and microscopy) in both asymptomatic and symptomatic pregnant women in a 

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prospective fashion at three antenatal clinics. Our most complete data to date is from Jawi Health 
Centre in Amhara Region during the 2018 peak transmission season. In this study population, 28 
of 34 pregnant women (mean age 24.2 years) who presented to the antenatal clinic were 
asymptomatic, 26 of 34 were in the first trimester, and 13 of 34 were primigravida. Malaria 
positivity amongst asymptomatic women was 3/34 (8.8%) by microscopy, 1/34 (2.9%) by RDT, 
12/34 (35.3%) by commercial LAMP, and 12/34 (35.3%) by ELUCIDx, and 11/34 (32.3%) by 
qPCR. The sensitivity of microscopy in pregnant women in this study population was 25.0% (95% 
CI 5.49 - 57.19%), RDT 8.33% (0.21 - 38.48)), commercial LAMP 81.82% (48.22 - 97.72), 
ELUCIDx 100.00% (73.54 -100.00) and qPCR 91.67% (61.52 - 99.79) using a consensus gold 
standard. These data suggest that ultrasensitive diagnostics like LAMP are able to detect malaria 
far more frequently than traditional diagnostic methods when coupled to active case detection of 
symptomatic and asymptomatic pregnant women. The marked decrease in sensitivity for 
microscopy and RDT in phase 2 as compared to phase 1 is directly linked to the low level of 
infection found in asymptomatic pregnant women. We predict that greater detection of malaria in 
pregnancy with LAMP-based active case detection will lead to better critical outcomes such as 
LBW in pregnancy in the full-scale LAMPREG trial.  
 
Table 3: Performance of LAMP compared to microscopy and rapid diagnostic tests (RDT) in 
asymptomatic and symptomatic pregnant women (N=34, Jawi, Amhara Region), phase 2 proof of 
concept prospective study with active case detection. 
Method 
Sensitivity % (95%CI) 
Specificity % (95%CI) 
Microscopy 
25.00 (5.49 - 57.19) 
100.00 (84.56 - 100.00) 
RDT 
8.33 (0.21 - 38.48) 
100.00 (84.56 - 100.00) 
Meridian LAMP (competitor) 
81.82 (48.22 - 97.72) 
86.96 (66.41 - 97.22) 
ELUCIDx LAMP 
100.00 (73.54 -100.00) 
100.00 (84.56 - 100.00) 
qPCR 
91.67 (61.52 - 99.79) 
100.00 (84.56 - 100.00) 
 

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In additional preliminary data from Gojeb, Southwest Ethiopia, our study team has shown that 
treating pregnant women positive by LAMP has benefits in terms of fetal hemoglobin and birth 
weight, with the former reaching statistical significance (n=200, p<0.05). This is important data 
which requires confirmation in a full-scale trial across several epidemiological settings in Ethiopia. 
Table 4: Outcome measures in pregnant women (n=200) randomized to LAMP versus standard of 
care microscopy/RDT in Gojeb, Ethiopia.  
Outcome 
measure 
LAMP Arm Mean (n=150) SOC Arm Mean (n=50) 
p Value 
(t test) 
Birth Weight (g) 3059 
2965 
0.1478 
Mat Hgb 1st 
visit (g/dL) 
12.11 
12.01 
0.4981 
Mat Hgb 2nd 
visit (g/dL) 
11.68 
11.63 
0.7211 
Mat Hgb 3rd 
visit (g/dL) 
11.33 
11.24 
0.4606 
Mat 
Hgb 
at 
birth (g/dL) 
11.51 
11.55 
0.771 
Fet Hgb at birth 
(g/dL) 
13.1 
12.84 
0.04286 
4. OBJECTIVES 
4.1. General objective 
 
To assess the impact of LAMP in the diagnosis of malaria in pregnancy and its potential role in 
reducing mortality and morbidity attributable to malaria. We hypothesize that the additional 
sensitivity of LAMP coupled with active case detection in detecting malaria in pregnancy will 
result in additional cases being identified and treated.  

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4.2. Specific objectives 
 
1. To evaluate the impact of LAMP versus microscopy/RDT for the detection of malaria in 
pregnant mothers in terms maternal and infant morbidity and mortality. 
2. To evaluate the impact of enhanced case detection of malaria in pregnancy by screening 
asymptomatic mothers at each antenatal visit until delivery. 
3. To determine the impact of treating LAMP-positive asymptomatic and symptomatic 
pregnant women as compared to standard of care. Treatment is per national guidelines. 
5. MATERIALS AND METHODS 
5.1. Study area 
The study will be conducted at sites across several sites in Ethiopia to obtain sufficient enrolment 
and spanning different transmission settings based on epidemiological data provided by the 
Federal Ministry of Health. The study sites selected are Chisabay, Hamusit and Andasa Health 
centers; northwest Ethiopia, from southwest Ethiopia; Bonga area (Uffa Health Center and Bonga 
(GebreTsadik Shawo) General Hospital ) and one site from Gambela (Lare Health center); in which 
some are sites where preliminary data were gathered.  In Ethiopia, malaria is characterized by its 
seasonality where the peak transmission season is from October to December with a second peak 
in June.  P. falciparum and P. vivax are the predominant species in the area [22]. Residents often 
live in non-substantive accommodation and despite a scale up in preventative measures in 2005 
including ITN distribution, they are at risk of malaria.  
5.2. Study design and period 
 
The study is prospective diagnostic study of malaria in pregnant women. The goal is to determine 
whether: (i) LAMP provides a clinically measurable benefit compared to current first line 
diagnostic test of Giemsa-stained microscopy and whether (ii) enhanced case detection of 
asymptomatic mothers with LAMP has added value in terms of outcomes. We hypothesize that 
addition of LAMP to one arm will be of greater benefit than microscopy alone due to additional 
LAMP sensitivity. We further hypothesize that enhanced case detection by screening 
asymptomatic mothers at each antenatal visit will be of additional value in treating malaria. Both 

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symptomatic and asymptomatic first and second trimester mothers will be included in the study 
and individually randomized to one of two arms: standard of care or enhanced cased detection 
arms using LAMP for malaria. Mothers will be enrolled during a twelve-months period from  
October 2020 to October 2021and then followed until 28 days after delivery. Given the rate of 
pregnant mothers at the locations, we anticipate that the required minimum of 2583 mothers will 
be enrolled in the study during the study period. In the first standard of care arm, venous blood 
sample will be collected from each study participant and the presence of Plasmodium infection 
will be diagnosed by microscopy in symptomatic patients. Pregnant women who test positive for 
malaria will be referred and treated for malaria with quinine or ACTs as per national guidelines. 
In the second (test) arm, mothers whether symptomatic or asymptomatic will be tested by a 
commercially available CE-approved LAMP malaria test (Meridien Biosciences, Illumigene 
Malaria M kit, Cincinatti, USA) and Human Diagnostics LoopAMP (Wiesbaden, Germany) at 
each clinic visit in addition to RDT/microscopy. The commercial LAMP tests can distinguish P. 
falciparum and P. vivax and treatment will be given according to national guidelines. The purpose 
of doing all tests in the intervention arm is to determine how many additional cases LAMP 
identified. The primary outcome is the proportion of deliveries with low birth weight, with 
secondary outcomes of: (i) absolute birth weight; (ii) maternal hemoglobin; (ii) neonatal 
hemoglobin at birth (Standard Hgb testing from peripheral blood or finger prick); (iv) neonatal 
mortality; (v) stillbirth; and (vi) prematurity in each of the two arms. Additionally, signs of current 
or past Plasmodium sequestration in the placenta will be evaluated. 
 
 
 
 
 
 
 
 
 
 
 

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Figure 1: Flowchart summarizing randomized trial  
 
 
There will be a total of at least five antenatal visits (mandatory). Emergencies (medical 
consultation outside the planned visit) will be recorded for each included woman. Visit 1 and 2 
may be combined for mothers who present in the second trimester. The following table summarizes 
the procedures to be conducted at each visit. 
 
 
 
 

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Table 5: Actions to be taken at each visit to the health center by a pregnant mother 
Visit # Time frame 
Aim 
Action for the study 
1 
Visit at health 
care center 
Inclusion 
Enrolment and randomisation 
2 
26 
weeks 
gestation 
Follow up 
Malaria testing as per inclusion arm 
(2mL venous blood on EDTA) 
+ANC regular care 
Maternal Hgb 
3 
32 
weeks 
gestation 
Follow up 
Malaria testing as per inclusion arm 
(2mL venous blood on EDTA) 
+ANC regular care 
Maternal Hgb 
4 
36-38 
weeks 
gestation 
Follow up 
Malaria testing as per inclusion arm 
(2mL venous blood on EDTA) 
+ANC regular care 
Maternal Hgb 
5 
Delivery 
Delivery visit 
Foetal Hb 
Maternal Hb 
Malaria testing 
Birth weight + size 
Weeks of pregnancy - preterm? 
Placenta blood 
Death 
6 
28 days after 
birth 
Néonatal 
assessment visit 
Newborn weight/              
 hemoglobin/ 
death/ 
other 
 
5.3. Population 
5.3.1. Study population 
The study population will comprise pregnant women 18 years of age and above, in the first and 
second trimester, both symptomatic and asymptomatic, attending antenatal clinics during the study 
period. Gestational age will be determined using the mother’s last normal menstrual period and by  

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measuring symphysis fundal height. Women will be assisted to determine their last normal 
menstruation with detailed calendar including local events reminder. Those who don’t remember 
their last normal menstruation will not be included in the study. As described in the WHO 
guidelines, in high-transmission settings, where levels of acquired immunity tend to be high, P. 
falciparum infection is usually asymptomatic in pregnancy. Yet, parasites may be present in the 
placenta and contribute to maternal anemia even in the absence of documented peripheral 
parasitemia. Both maternal anemia and placental parasitemia can lead to low birth weight, which 
is an important contributor to infant mortality. In high-transmission settings, the adverse effects of 
P. falciparum infection in pregnancy are most pronounced for women in their first pregnancy. In 
low-transmission settings women of reproductive age have relatively little acquired immunity to 
malaria, malaria in pregnancy is associated with anemia, an increased risk of severe malaria, and 
it may lead to spontaneous abortion, stillbirth, prematurity and low birth weight. We hypothesize 
that both symptomatic and asymptomatic cases of malaria occur in pregnant women. Patients who 
are positive by LAMP and negative by microscopy will be consented for treatment based on the 
evidence that LAMP has greater sensitivity.  
5.4. Inclusion and exclusion criteria 
Pregnant women in their first or second trimester (until 28 weeks of pregnancy) presenting to the 
health centers will be enrolled in the study and randomized to one of two arms as above. Informed 
written consent will be obtained from participating women based on the level of literacy. Pregnant 
women in the third trimester,  those mothers with severe malaria,  those who are taking or have 
taken anti-malaria medication three weeks prior to study commencement, those with severe anemia 
with hemoglobin of less than 8 g/dL, pregnant mothers who are severely unwell and mothers who 
are classified as having high risk pregnancy will be excluded from the study. Women who do not 
remember the date of their last menstruation will be excluded. 
5.5. Variables 
5.5.1. Primary outcome 
✓ Proportion with low birth weight (<2500gm) 
5.5.2. Independent variables 
✓ Maternal hemoglobin during pregnancy and at delivery 

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✓ Fetal hemoglobin 
✓ Actual birth weight (gm) 
✓ Stillbirths 
✓ Premature births 
✓ Neonatal death 
5.5.3 Other endpoints 
Performance of LAMP, RDT, Microscopy, compared to qRT-PCR (gold standard) including 
sensitivity, specificity, PPV, NPV. Signs of Plasmodium infection: current (with presence of 
asexual parasites in placental blood), or past infection (presence of malaria pigment). 
5.6. Sample size and sampling techniques 
 
The sample size calculation for this study is based on detecting a difference in the proportion of 
deliveries with a low birth weight (LBW), which was felt to be the most clinically important 
outcome variable. The prevalence of deliveries with a LBW in Ethiopia was estimated to be 17.3%.  
An alpha of 0.05 was selected with an allocation ratio of 3:1 in favor of the enhanced case detection 
arm. The study will be powered to detect an absolute difference in the proportion of deliveries with 
a LBW of 5%, so a proportion in the enhanced case detection arm of 12.3% compared to 17.3% in 
the standard of care arm. Using a continuity correction and an attrition rate of 20%, a total sample 
size of 2583 is required to achieve 80% power. 
5.7. Data collection and laboratory methods 
5.7.1. Socio-demographic and clinical data 
Socio-demographic characteristics and clinical data will be collected using interview-based 
questionnaire. Questionnaire will be developed in English and translated to Amharic and then 
translated back to English to maintain its consistency.     
Written informed consent will be obtained from study participants and data collected will be 
anonymized. Data will only be used for the purpose of this trial. Participants who test positive for 
malaria will be referred for medical consultation in the ante-natal clinic to ensure appropriate 

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treatment. LAMP testing has been shown to be superior in sensitivity and specificity to Giemsa 
microscopy and RDTs.  
The participants will then be followed up for the duration of their pregnancy and their hemoglobin 
will be measured when they attend for delivery. The fetal birth weight will be recorded as an 
additional end point and any complications will be documented. 
5.7.2. Blood sample collection and processing 
For microscopy, two milliliters of venous blood will be collected from each study participant using 
EDTA anticoagulant test tube. Soon after venous blood collection, a drop of blood will be taken 
for RDT test and another two separate drops of blood will be placed on frosted microscope slides 
to prepare thin and thick blood films. For LAMP, two milliliters of venous blood will be collected 
from each study participant using EDTA anticoagulant test tube. LAMP will be performed at the 
respective health centers based on the manufacturer’s recommendation (Meridian Diagnostics, and 
Human Diagnostics). Dried blood spot (DBS) samples will also be collected from all individuals 
by using two drops of blood placed on a Whatmann filter paper. DBS will be stored in AHRI for 
further testing if required. DBS will also be sent to the University of Calgary, Canada (cf MTA) 
for ultrasensitive malaria technique (qRT PCR) and parasitic whole genome sequencing. For 
placental infection assessment, placental blood will be collected on anticoagulant after delivery. 
 
5.7.3. Malaria microscopy and RDT 
Blood film will be air-dried at room temperature and the thin blood film will be fixed with absolute 
methanol. Then, blood film will be stained with 10% Giemsa solution for 10 minutes and examined 
by experienced laboratory technician. The presence of Plasmodium will be ruled out if no parasites 
are observed after examination at least 100 microscopic fields with hundred (100x) objective. 
Parasitemia will be estimated in thick film by counting the number of asexual parasites along with 
200 white blood cells (WBC) or 500 WBC if the parasite count is less than 10 parasites per 200 
WBC. A total of 8,000/µl white blood cells count will be considered for the determination of 
parasitemia. At delivery, placental blood will be collected with anticoagulant and immediately 
dried as a smear on a microscope slide. Placental blood smear will be air dried then stained using 

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10% Giemsa (as for regular peripheric blood samples). Presence of asexual parasites or malaria 
pigment will be recorded.  
 
5.7.4. Loop-mediated isothermal amplification (LAMP) 
Since patients testing positive by LAMP will be treated for malaria, a CE-marked test (approved 
for human use in Europe) will be used to diagnose patients. We will use the Meridien Biosciences, 
Illumigene Malaria M kit (Cincinatti, USA) and Eiken LoopAMP Malaria (Human Diagnostics, 
Wiesbaden, Germany). The test will be conducted exactly according to manufacturer’s 
recommendations. The tests are both run to detect P. falciparum and P. vivax. A third 
investigational LAMP test (ELUCIDx, Calgary, Canada) will be run only for data gathering and 
method validation purposes. 
 
5.8. Statistical Analysis Plan 
Raw data will be entered in SPSS version 20 software and analyzed using STATA version 13. 
Mothers with twins will be excluded from the analysis of birth outcomes. The sensitivity, 
specificity and predictive values will be calculated for microscopy and LAMP versus a gold 
standard of PCR. Statistical analysis of epidemiological variables and malaria positivity will be 
determined using univariate binomial regression. Variables found to be statistically significant 
with initial analysis will then characterized through multivariate analysis regression analysis. Risk 
ratios for primary outcome variables for intervention against standard of care arm will be 
calculated. Subgroup analysis will be done for potential confounders, and to compare positivity by 
LAMP and microscopy in the intervention arm. 
5.9. Data analysis and interpretation 
Raw data will be entered in SPSS version 20 software. The sensitivity, specificity, predictive 
values and kappa coefficient will be determined using SISA online statistical software. 
 

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5.10. Benefits and beneficiaries of the proposed study 
Based on previous studies performed by our group, molecular diagnostics such as LAMP is 
now considered imperative in the malaria elimination plan for Ethiopia. Our group conducted 
a community survey of asymptomatic residents of Gambella, Ethiopia using LAMP and 
confirmed that over 50% of infected are missed by microscopy and RDT in this high 
transmission setting. These so-called asymptomatic individuals were anemic (lower 
hemoglobin) compared to uninfected persons in this survey suggesting there is a consequence 
to parasite carriage. Malaria in pregnancy has been shown to lead to maternal anemia, intra-
uterine growth restriction and reduced fetal birth weight. In sub-Saharan Africa, anemia 
reportedly accounts for about 20% of all maternal deaths [17] and low fetal birth weight is 
thought to be the single biggest risk factor for neonatal and infant mortality. We aim to 
demonstrate that improved detection and then treatment of low level parasitemia translates 
into a clinical benefit for both mother and child. This proof of clinical benefit may lead to the 
increased use of LAMP testing in the resource limited setting and pave the way for more 
research in the area. LAMP methodology technology transfer will occur in the course of this 
study. Staff from the local study sites will be trained in LAMP technology. The study aims to 
show the benefits of active case detection using a reliable highly sensitive malaria point of 
care diagnostic test. In the long term, we hope that the technology and protocol will be 
considered at the national level and implemented in the local laboratories. However, this will 
depend on funding from FMOH and support from multilateral agencies.  
 
5.11. Dissemination of findings 
The results of this study will be presented at national and international scientific conferences. The 
result of the study will be communicated to the Ministry of Health of Ethiopia and the regional 
health bureaus where the study will be conducted. Dissemination workshop will be done after the 
completion of the study. Study findings will also be submitted to internationally reputable 
scientific journals. 
5.12. Ethical Considerations 
Ethical clearance will be obtained from Institutional Research Ethics Board (IRB) of the Armauer 
Hansen Research Institute in Ethiopia, the Conjoint Health Research Ethics Board (CHREB) 

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University of Calgary, and from National Ethical Review Committee of Ethiopia. Consultation 
and permission to conduct this study will also be obtained from regional Health Offices as 
appropriate. 
 
In each LAMPREG study center, medical officer will be informed of the study and trained on 
study inclusion procedure, including informed consent collection. Only trained staff will be 
allowed to proceed to inclusion. All eligible women consulting for ANC in a LAMPREG study 
center will be made aware of the study by the trained medical officers. Women interested in the 
study will be informed of the protocol by the medical officer as per Protocol Information Sheet. 
The fact that women agreeing or not to participate in the study will not impact the quality of the 
care received will be emphasized. Medical officers will be vigilant to give eligible women enough 
time and a safe space to decide whether they agree or not to participate in the LAMPREG study. 
No coercion or undue influence will be imposed on women. Written informed consent will be 
obtained from every study participant by the trained medical officer, as per Informed Consent 
sheet. For women who are illiterate, particular attention will be given in ensuring that each point 
of the protocol and consent form are well understood. For these women, signature on the consent 
sheet will be by either initials or inked fingerprint. Medical officers will inform women that they 
can withdraw their participation from the study at any time, and that doing so will not affect the 
medical care provided to them. All women will be free to be accompanied or not by a person of 
their choice during each visit. 
 
To ensure confidentiality about participants information, anonymous typing will be used, and any 
personal identifier of participants will not be written. Samples will be identified solely with patient 
inclusion number. Information for the LAMPREG study will be collected at the time of the study 
and directly anonymized. Discussions between pregnant women and medical staff regarding study 
protocol, inclusion, randomization, study withdrawn and follow up visit will be held in a 
confidential space to ensure that women are not subject to social or staff pressure. 
In case of adverse events during the study, women will be referred to a hospital and the event will 
be notified to study investigator. Contact of study investigators are available to women through 
inclusion forms. Women having any concerns regarding study procedure will be welcome to ask 
precisions to medical officers at any time during the LAMPREG trial. Study investigators will 
monitor adverse events from each study site on a monthly basis and take appropriate measures if 
needed. Since the LAMPREG trial is integrated with regular ANC visit, women participation in 
the study will not be noticeable by other community members, ensuring confidentiality. 

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The LAMPREG trial intervention arm consists of asymptomatic women screening for malaria 
during each ANC visit with highly sensitive techniques. The major added risks are due to the blood 
sampling. All material used will be single-used sterile needles. Medical staff will be vigilant on 
communication and will be explaining the steps of the study to the participants. Women tested 
positive for malaria will be administered treatment according to national guidelines pending on the 
gestational age.  Before treatment administration, women will be explained the results of the 
LAMP test and the potential benefits of treatment. 
 
At delivery, women are particularly vulnerable. The LAMPREG trial staff will respect women 
privacy during delivery. LAMPREG data collection and sampling will not affect the care given to 
the women and the newborn during delivery, and absolute priority will be given to the mother and 
child health and safety. For placenta blood collection, study staff will remind women about the 
procedure before sampling, and women will be allowed to withdraw consent. 
 
Pregnant women who do not consent to participate in the study will be tested for malaria using 
standard Giemsa microscopy, ensuring that they receive prompt treatment, as per Ethiopian 
national guidelines. Pregnant women who are severely unwell requiring admission to the health 
center for monitoring or referred to a hospital will be excluded from the study. Only CE-marked 
commercially available diagnostic tests for LAMP (Meridien Biosciences, Illumigene Malaria M 
kit, Cincinatti, USA) will be used in this study. These kits are able to identify both P. falciparum 
and P. vivax to the species level. Treatment will be according to national guidelines for 
symptomatic and asymptomatic women positive for Plasmodium.  Quinine has been widely used 
for MiP treatment. The main adverse effect of quinine are cinchonism (with tinnitus (38%-85%), 
vomiting, dizziness, nausea and anorexia), quinine related hypoglycemia (17%) and QT 
prolongation (1%) [23]. Abortion risk is considered as very low for quinine [24] at the normal 
dosage.  
Artemisinin derivatives (in combination with partner drug such as lumefantrine) showed similar 
profiles in terms of stillbirth, congenital abnormalities and risk of miscarriage in women exposed 
to ACT during pregnancy compared to standard intermittent preventive treatment (IPT) for malaria 
[23]. Despite animal preclinical studies highlighting a risk of embryotoxicity and teratogenicity 
(musculoskeletal/cardiovascular defects) [25], a recent meta-analysis study did not show a 
difference in pregnant women treated with ACT compared to quinine, even in the embryo sensitive 

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period (6-12 weeks of gestational age) [26]. Two studies report ECG monitoring of pregnant 
women treated by artemether lumefantrine [27,28] and showed a QT prolongation in 2% of treated 
women. Several studies have reported the safety of artemether lumefantrine during the first 
trimester of pregnancy in 1693 women, and no increase in miscarriage or congenital abnormalities 
where observed compared to standard of care [29–32]. Recently, the PREGACT trial compared 
four artesunate combined therapy in the second and third trimester of pregnancy in women positive 
for P. falciparum infection regardless of symptoms. No differences were observed in terms of 
treatment associated negative birth outcomes [33].  
Regarding chloroquine, a cohort of 169 pregnant women treated by chloroquine in non-malaria 
endemic area did not show newborn abnormalities compared to placebo [34]. Recently, a study 
conducted in Malawi to evaluate the use of chloroquine for intermittent preventive treatment did 
not show any adverse effect in the 600 treated women compared to the standard of care IPT 
(sulfadoxine pyrimethamine) [35]. 
In regards of the recent literature and publications related to quinine, artemether lumefantrine and 
chloroquine during pregnancy, there is no major risk for treatment of women presenting 
asymptomatic Plasmodium spp infection. Importantly, Plasmodium spp infection, even 
asymptomatic and submicroscopic, has impact on birth outcomes.  
Asymptomatic malaria is the presence of Plasmodium spp infection in peripheral blood in the 
absence of any symptom of the disease. During pregnancy, peripheral parasitemia are particularly 
low due to sequestration of infected erythrocytes in the placenta villosities [36,37]. The 
sequestration of the parasite in the placenta originates the negative birth outcomes associated with 
MiP : low birth weight, maternal anemia, abortion and stillbirth [38]. Estimation recall that 20% 
of stillbirth and 11% of all newborn death are direct consequences of MiP [39]. Both P. falciparum 
and P. vivax induce adverse pregnancy outcome. P. falciparum pathogenicity in MiP is well 
described [37,40] while the pathophysiology of P. vivax infection during pregnancy needs yet to 
be elucidated [41,42].  
It is clearly demonstrated that pregnant women presenting asymptomatic or submicroscopic 
Plasmodium infection present more anemia than uninfected women [43]. A recent longitudinal 
study combining Plasmodium peripheral detection and placenta histology at delivery showed that 

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even women with only submicroscopic infection have an increased risk of placental malaria 
compared to women without any parasitemia [44]. 
For the above-mentioned reason, Plasmodium infection, even asymptomatic, present a risk for the 
mother health and fetal development. 
 
Expectant mother safety is a priority during the LAMPREG trial. After Plasmodium spp infection 
diagnostic, final treatment choice will be at the discretion of the physician assessing the patient. 
Any pre-existing condition will be taken in consideration.  
For any adverse event, women will be given the contact of the local study coordinator. Community 
Health Worker will be sensitive to the study. At the following ANC visit, women will be 
encouraged to report any side effect or concern to the study staff. Each local study site coordinator 
will summarize any adverse event on a monthly basis and transmit the corresponding information 
to the national coordinator. Proper pharmacovigilance notification will be operated to the health 
authorities if necessary. 
 
The LAMPREG clinical trial aims to evaluate the impact of active detection of both sub 
microscopic symptomatic MiP and asymptomatic (microscopic and submicroscopic) MiP. In this 
trial, even asymptomatic and submicroscopic MiP will be treated for Plasmodium spp infection as 
if they were uncomplicated MiP cases. The proposed used drugs fall under the drugs approved by 
Ethiopia Food, Medicine and Health Care Administration and Control Authority and have been 
proven safe during pregnancy. Importantly, the benefits of the treatment overpass the risk due to 
treatment induction. Information sheet and informed consent were modified to provide information 
regarding the treatment risk. Importantly, drugs prescription will be at the discretion of the 
physician assessing the women, and women safety will be the main priority of the medical staff in 
charge of the study.  

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6.0. WORK PLAN 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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7.0 English Version of Questionnaire 
Patient inclusion questionnaire 
 
This questionnaire records both socio-demographic and clinical characteristics. For each question 
please give the answer carefully and clearly. The participant’s name is not included in the 
questionnaire and their information will be kept completely anonymous and confidential. Their 
answers will be kept only by the study investigators and will not be distributed to anyone else. 
 
Socio-demographic characteristics  
 
1.1. 
ID no. of participant: ______________________ 
1.2. 
Age: _______ (Years) 
1.3. 
Address: 
 
_________________________________________________________________ 
1.4. 
Are you currently pregnant?   
 
 
 
 
A. Yes     B. No 
1.5. 
How many weeks pregnant are you?                                                           ______________ 
1.6. 
How many pregnancies have you had before this one?                               ______________ 
1.7. 
Have you had any miscarriages or stillbirths before? 
 
A. Yes     B. No 
1.8. 
If yes, how many? 
 
 
 
 
 
            ______________ 
1.9. 
Do you spend most your time indoor or outdoor? 
A. Yes     B. No  
1.10. Has your house undergone indoor residual spraying in the last 3 months? 
A. Yes     B. No  
1.11. Do you sleep under insecticide treated nets? 
 A. Yes     B. No  
1.12. Has anyone in your household been diagnosed with malaria in the last month? 
 A. Yes     B. No  
1.13. What is your level of education? 
None Primary 
 
Secondary 
Post-secondary 
 
 

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Clinical characteristics  
 
1. 
Does the participant have any of the following symptoms or signs? 
 
Fever   
 
 
 
 
 
 
 
A. Yes     B. No            
Chills   
 
 
 
 
 
 
 
A. Yes     B. No            
Sweats  
 
 
 
 
 
 
 
A. Yes     B. No            
Headache  
 
 
 
 
 
 
 
A. Yes     B. No            
Muscle pains   
 
 
 
 
 
 
A. Yes     B. No            
Nausea  
 
 
 
 
 
 
 
A. Yes     B. No            
Vomiting  
 
 
 
 
 
 
 
A. Yes     B. No  
General malaise           
 
 
 
 
 
A. Yes     B. No 
Diarrhea  
 
 
 
 
 
 
 
A. Yes     B. No 
 
 
2. 
Does the participant have an axillary temperature greater than 37.5 degrees Celsius?  
A. Yes     B. No 
3. 
Does the participant have any signs of severe malaria? 
A. Yes     B. No 
4. 
Has the participant taken any anti-malarial medication in the last three weeks? 
A. Yes     B. No 
 
 
 
 
 
 
 
 
 

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University of Calgary Conjoint Health Research Ethics Board (CHREB) number: REB17 – 1335 
ClinicalTrials.gov Identifier: NCT03754322                                                                       v.1.1 29 March 2020 
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