RESEARCH ARTICLE
Sensitive Detection of Asymptomatic and Symptomatic Malaria
with Seven Novel Parasite-Specific LAMP Assays and Translation
for Use at Point-of-Care
Kenny Malpartida-Cardenas,a,b Nicolas Moser,b Felix Ansah,c Ivana Pennisi,a Diana Ahu Prah,c,d Linda Eva Amoah,c,e
Gordon Awandare,c Julius Clemence R. Hafalla,d,e Aubrey Cunnington,a Jake Baum,f,g Jesus Rodriguez-Manzano,a
Pantelis Georgioub
aDepartment of Infectious Disease, Faculty of Medicine, Imperial College London, London, United Kingdom
bDepartment of Electrical and Electronic Engineering, Faculty of Engineering, Imperial College London, London, United Kingdom
cWest African Centre for Cell Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Legon, Ghana
dDepartment of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom
eImmunology Department, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana
fDepartment of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, United Kingdom
gSchool of Biomedical Sciences, University of New South Wales Sydney, Sydney, Australia
Jesus Rodriguez Manzano and Pantelis Georgiou are both senior authors.
ABSTRACT Human malaria is a life-threatening parasitic disease with high impact in the
sub-Saharan Africa region, where 95% of global cases occurred in 2021. While most malaria
diagnostic tools are focused on Plasmodium falciparum, there is a current lack of testing
non-P. falciparum cases, which may be underreported and, if undiagnosed or untreated,
may lead to severe consequences. In this work, seven species-specific loop-mediated
isothermal amplification (LAMP) assays were designed and evaluated against TaqMan quan-
titative PCR (qPCR), microscopy, and enzyme-linked immunosorbent assays (ELISAs).
Their clinical performance was assessed with a cohort of 164 samples of symptomatic
and asymptomatic patients from Ghana. All asymptomatic samples with a parasite load
above 80 genomic DNA (gDNA) copies per mL of extracted sample were detected
with the Plasmodium falciparum LAMP assay, reporting 95.6% (95% confidence interval
[95% CI] of 89.9 to 98.5) sensitivity and 100% (95% CI of 87.2 to 100) specificity. This
assay showed higher sensitivity than microscopy and ELISA, which were 52.7% (95% CI
of 39.7 to 67%) and 67.3% (95% CI of 53.3 to 79.3%), respectively. Nine samples were posi-
tive for P. malariae, indicating coinfections with P. falciparum, which represented 5.5% of
the tested population. No samples were detected as positive for P. vivax, P. ovale, P. knowl-
esi, or P. cynomolgi by any method. Furthermore, translation to the point-of-care was
demonstrated with a subcohort of 18 samples tested locally in Ghana using our hand-
held lab-on-chip platform, Lacewing, showing comparable results to a conventional fluo-
rescence-based instrument. The developed molecular diagnostic test could detect asymp-
tomatic malaria cases, including submicroscopic parasitemia, and it has the potential to
be used for point-of-care applications. Editor Jian Li, Hubei University of Medicine
IMPORTANCE The spread of Plasmodium falciparum parasites with Pfhrp2/3 gene deletions Copyright © 2023 Malpartida-Cardenas et al.
This is an open-access article distributed under
presents a major threat to reliable point-of-care diagnosis with current rapid diagnostic the terms of the Creative Commons
tests (RDTs). Novel molecular diagnostics based on nucleic acid amplification are needed Attribution 4.0 International license.
to address this liability. In this work, we overcome this challenge by developing sensitive Address correspondence to Jesus Rodriguez-
tools for the detection of Plasmodium falciparum and non-P. falciparum species. Furthermore, Manzano, j.rodriguez-manzano@imperial.ac.uk.
The authors declare no conflict of interest.
we evaluate these tools with a cohort of symptomatic and asymptomatic malaria patients
Received 20 December 2022
and test a subcohort locally in Ghana. The findings of this work could lead to the imple- Accepted 18 April 2023
mentation of DNA-based diagnostics to fight against the spread of malaria and provide Published 9 May 2023
reliable, sensitive, and specific diagnostics at the point of care.
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
KEYWORDS nucleic acid amplification, malaria, diagnostics, point-of-care
Human malaria is a life-threatening parasitic disease that affected an estimated 247million people worldwide in 2021. In 2020, malaria deaths increased by 10% compared
to in 2019 and declined slightly to 619,000 deaths in 2021 (1, 2). There is an unmet need to
implement point-of-care (POC) digital diagnostics for the detection of Plasmodium falcipa-
rum and non-P. falciparum cases to reduce the burden of this disease and report progress
toward global technical strategic goals. To date, eight species have been identified that
infect humans: (i) Plasmodium falciparum, (ii) Plasmodium vivax, (iii) Plasmodium ovale walli-
keri, (iv) Plasmodium ovale curtisi, (v) Plasmodium malariae, (vi) Plasmodium knowlesi, (vii)
Plasmodium cynomolgi, and (viii) Plasmodium simium. The first five are specific to humans,
with P. ovale wallikeri and P. ovale curtisi considered distinct sympatric Plasmodium species
that can only be differentiated at the DNA level (3). The last three species, P. knowlesi, P. cyno-
molgi, and P. simium, have been known to infect primate hosts. However, natural transmission
by anophelines from primates to humans has recently been reported, and malaria is consid-
ered a zoonotic disease in this instance (4).
Current methods for malaria diagnostics include microscopy, antigen detection, and
nucleic acid amplification. However, species-specific detection could be challenging by micros-
copy due to the similar morphology of the parasites (5, 6), and the lower levels of parasitemia
of non-P. falciparum species may limit their detection with antigen-based methods. Nucleic
acid amplification tests (NAATs) are the most promising tools for species-specific malaria diag-
nostics due to the high specificity and sensitivity they offer (7–9). PCR assays for the detection
of P. falciparum, P. vivax, P. malariae, P. ovale, or P. knowlesi have been reported. (10–15).
However, they require long turnaround times or the use of expensive primer modifica-
tions and fluorophores (16). In addition, these methods require expensive, sophisticated
equipment and trained/skilled personnel, which is hampering their translation to the POC.
As an alternative to PCR, loop-mediated isothermal amplification (LAMP) is a promising
candidate for molecular testing outside the laboratory (17). LAMP species-specific assays
have been reported mostly targeting the conserved 18S rRNA gene or mitochondrial genes
(18–25). However, targeting conserved genes increases the likelihood of interspecies cross-
reactivity.
In this work, we report seven species-specific LAMP assays for the discrimination of
human-infective Plasmodium species. Their performance was evaluated with 164 clinical
samples from Ghana using TaqMan quantitative PCR (qPCR) as a reference and also perform-
ing traditional diagnostic methods, such as microscopy and antigen-based detection.
Translation into POC diagnostics was achieved by combining the LAMP assays with our
in-house-developed lab-on-chip (LoC) platform called Lacewing. A schematic outline of the
study conducted is presented in Fig. 1.
RESULTS
LAMP assays for the detection of human-infective Plasmodium species. LAMP assays
were designed to discriminate seven human-infective Plasmodium species with high sensitivity
and specificity: (i) P. ovale wallikeri and curtisi (duplex, LAMP-PoRBP2), (ii) P. malariae (LAMP-
PmK13), (iii) P. vivax (LAMP-PvAT2), and (iv) P. cynomolgi (LAMP-PcyAT2). Assays for the specific
detection of P. falciparum (26), P. knowlesi (25), and Plasmodium pan-genus (23) were included
from previous studies. Sequences of selected target genes were collated from PlasmoDB and
NCBI (sequences are provided in Table S1, and the accession numbers are in Table S2 in the
supplemental material). The LAMP-PoRBP2 assay consisted of a duplex assay targeting a con-
served region of the reticulocyte binding protein 2 (RBP2) gene, which is only present in
P. ovale and P. vivax (27). If the duplex assays are used independently, they can discriminate
the two species based on differential time-to-positive (TTP) values (Fig. S1). The LAMP-PmK13
assay was designed based on the kelch 13 gene, the same gene used for P. falciparum detec-
tion (26), leveraging the differences due to their high divergence (27). Lastly, the LAMP-PvAT2
and LAMP-PcyAT2 assays were designed using a-tubulin 2 ortholog genes based on the
numerous mismatches found with respect to other Plasmodium species (18).
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
FIG 1 Schematics of the conducted study. (A) Data stratification of the studied cohorts and methods used in this work. (B) Workflow for
lab-on-chip diagnostics: nucleic acid extraction from blood, nucleic acid amplification using the lab-on-chip platform, real-time data
visualization through a smartphone application, and diagnostic outcome.
Analytical sensitivity was evaluated using synthetic DNA. Standard curves are shown in
Fig. 2A to G, with a correlation R2 of 95 to 99%. TTP values were below 20 min, with a limit
of detection of 10 to 100 copies per reaction. Details of the LAMP assays are shown in
Table 1, and primer sequences are provided in Table S3. Analytical specificity was assessed
in silico based on multiple sequence alignment using the MUSCLE algorithm (Fig. S2).
Primer sets were highly specific, with no cross-reactivity (primers did not anneal to sequen-
ces of other Plasmodium species or had a minimum of 4 mismatches per primer). Available
clinical isolates (26) of P. falciparum, P. ovale wallikeri, P. malariae, P. vivax, and P. knowlesi
were tested with the species-specific LAMP assays and the LAMP-Pan18S assay. As shown
in Fig. 2H, the species-specific LAMP assays amplified only their target isolate, and the
LAMP-Pan18S assay detected all.
Performance of LAMP assays in Ghanaian children with symptomatic and asymp-
tomatic P. falciparum parasitemia. Seventy samples from symptomatic and asymptom-
atic Ghanaian children were collected in Obom, Ghana. DNA was extracted, and samples
were tested with our species-specific LAMP assays, a LAMP assay targeting the human
housekeeping gene ACTB (LAMP-ACTB) (28), and two TaqMan qPCR assays (12) targeting
the 18S rRNA gene of P. falciparum (PCR-Pf) and Plasmodium pan-genus (PCR-Pan). Primer
sequences are detailed in Table S3. This cohort was also analyzed by microscopy and enzyme-
linked immunosorbent assay (ELISA) targeting Plasmodium and P. falciparum, respectively.
Based on the symptoms and microscopy results for P. falciparum identification, data were
stratified into (i) symptomatic, (ii) asymptomatic with detectable parasitemia, (iii) asymp-
tomatic with submicroscopic parasitemia, and (iv) uninfected. Distribution of the TTPs and
parasite density according to this stratification is shown in Fig. 3A, and details are summar-
ized in Table 2. Performance against TaqMan PCR within these 4 groups is detailed as follows.
The LAMP-PfK13 assay had 100% (95% confidence interval [95% CI] of 63.1 to 100%) sensitiv-
ity (SEN), 100% (95% CI of 78.2 to 100%) specificity (SPE), and 100% (95% CI of 85.2 to 100%)
accuracy (ACC) within the symptomatic group. Results within the asymptomatic group with
detectable parasitemia were 86.40% (95% CI of 65.1 to 97.1%) SEN, 100% (95% CI of 78.2
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
FIG 2 Analytical sensitivity and specificity of species-specific LAMP assays. (A) P. falciparum standard curve (LAMP-PfK13). (B) P. vivax standard curve (LAMP-PvAT2).
(C) P. malariae standard curve (LAMP-PmK13). (D) P. ovale curtisi standard curve (LAMP-PoRBP2), (E) P. ovale wallikeri standard curve (LAMP-PoRBP2). (F) P. cynomolgi
standard curve (LAMP-PcyAT2). (G) P. knowlesi standard curve (LAMP-Pk18S). The highest concentration available for P. knowlesi was 106 copies per mL. (H) Cross-
reactivity of species-specific LAMP assays using clinical isolates (P. falciparum [Pf], P. malariae [Pm], P. ovale wallikeri [Pow], P. vivax [Pv], and P. knowlesi [Pk]). We have
included the TTP values obtained at 50 copies per reaction in A and B. This concentration was not detected in D, F, and G.
to 100%) SPE, and 91.9% (95% CI of 78.1 to 98.3%) ACC. Lastly, 80.77% (95% CI of 60.7 to
93.5%) SEN, 100% (95% CI of 78.2 to 100%) SPE, and 87.8% (95% CI of 73.8 to 95.9%) ACC
was achieved within the asymptomatic group with submicroscopic parasitemia, demon-
strating the higher SEN of the LAMP assay over microscopy. Results are summarized in
Table S4.
Correlation of PCR and LAMP in Ghanaian children with symptomatic and asymp-
tomatic P. falciparum parasitemia. The results obtained with TaqMan PCR were used as a
reference to classify the samples into three categories (high, medium-low, and low) based on
cycle threshold (CT) values (Table S5). This three-category division (high, medium-low, and
low) matched with the distribution of positives based on the syndromic approach. The ratios
of symptomatic to asymptomatic patients were 50% to 50% within the category “high”, 4%
to 96% within the category “medium-low” and 0% to 100% within the category “low”.
Using PCR-Pf results as the gold standard, microscopy had an overall 52.7% (95% CI
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
TABLE 1 Summary of the assays used in this work
Limit of detection Gene
Assay (copies/reaction)c Gene abbreviation TTP (min) Tm (°C) Reference
LAMP-PfK13 50 Kelch 13 K13 ,15a 78 26
LAMP-PmK13 10 Kelch 13 K13 ,15a 79 This study
LAMP-PoRBP2-w 10 Reticulocyte binding protein 2 RBP2 ,15a 78 This study
LAMP-PoRBP2-c 100 Reticulocyte binding protein 2 RBP2 ,15a 77 This study
LAMP-PvTUBA2 50 Alpha tubulin 2 TUBA2 ,15a 87 This study
LAMP-PcyTUBA2 100 Alpha tubulin 2 TUBA2 ,15a 85 This study
LAMP-Pk18S 100 18S rRNA 18S ,15a 83 25
LAMP-Pan18S 50 18S rRNA 18S ,20 85 23
PCR-Pan 1 18S rRNA 18S 40 cycles NAb 12
PCR-Pf 1 18S rRNA 18S 40 cycles NAb 12
aTTP of lowest detectable DNA concentration. It is recommended to run the assay for 20 min.
bNA, not applicable.
cReaction volume of 5mL.
of 39.7 to 67%) SEN, 86.7% (95% CI of 59.5 to 98.3%) SPE, and 60% (95% CI of 48.3 to 72%)
ACC. ELISA targeting the P. falciparum histidine-rich protein 2 (PfHRP2) antigen had 67.3%
(95% CI of 53.3 to 79.3%) SEN, 86.7% (95% CI of 59.5 to 98.3%) SPE, and 71.4% (95% CI of 59.4
to 81.6%) ACC. Further details are shown in Fig. 3B and are summarized in Tables S5 and S6.
The assay LAMP-PfK13 detected 100% of asymptomatic children in the “high” and
“medium-low” categories (n = 35), that is, samples presenting more than 80 genomic DNA
(gDNA) parasite copies per mL. Furthermore, 83% of all asymptomatic cases (n = 47) were
detected with this LAMP assay. In this cohort, the LAMP-PfK13 assay had 87.3% (95% CI of
75.5 to 94.7%) SEN, 100% (95% CI of 78.2 to 100%) SPE, and 90% (95% CI of 80.5 to 95.9%)
ACC; the LAMP-Pan assay had 90.9% (95% CI of 80.1 to 97%) SEN, 93.3% (95% CI of 68.1 to
99.9%) SPE, and 91.4% (95% CI of 82.3 to 96.8%) ACC. No samples were detected as positive
for P. vivax, P. ovale, P. malariae, P. knowlesi, or P. cynomolgi. Further details are shown in
Fig. 3C, Table 3, Table S7, and Fig. S3. The higher SEN and SPE obtained with the LAMP
assay are more apparent in the “high” and “medium-low” categories; where microscopy
detection achieved 87.5% SPE and 43.8% SEN and antigen detection achieved 93.8% SPE
and 71.9% SEN. LAMP-PfK13 achieved 100% SPE and 90.6% SEN, and LAMP-Pan achieved
100% SPE and 96.9% SEN.
Evaluation of LAMP assays with a cohort from Cape Coast Ghana. A second cohort
of 94 samples collected from Cape Coast (Ghana) was tested with our species-specific LAMP
assays, LAMP-ACTB assay, and PCR-Pf and PCR-Pan TaqMan assays. The LAMP-PfK13 assay
had a SEN of 85.4% (95% CI of 75.8 to 92.2%), SPE of 100% (95% CI of 73.5 to 100%), and
ACC of 87.2% (95% CI of 78.8 to 93.2%). The LAMP-Pan assay had 80.7% (95% CI of 70.6 to
93.3%) SEN, 100% (95% CI of 71.5 to 100%) SPE, and 83% (95% CI of 73.8 to 90%) ACC.
Further details are shown in Fig. 3D, Table 4, Table S8, and Fig. S3. In addition, the SEN values
obtained with the LAMP assays in the “high” and “medium-low” categories (parasitic load
above 80 gDNA copies per mL) were above 95%; samples belonging to the “low” category
with parasitic loads above 40 gDNA copies per mL were also detected with both LAMP
assays. The LAMP-PmK13 assay detected 9 samples, all of them also positive by PCR-Pan
and PCR-Pf, indicating coinfections and representing 5.5% of the studied subpopulation.
No samples were detected as positive for P. vivax, P. ovale, P. knowlesi, or P. cynomolgi by any
method.
All samples included in this study (n = 164) were positive by the LAMP-ACTB assay,
which was used as an extraction control to verify the human origin of the samples and
the quality of extracted DNA. ACTB concentration was calculated based on a standard
previously built with a positive control. The homogeneous distribution of the ACTB concen-
tration across the different categories, including the uninfected samples, indicated optimal
quality of the extraction (Fig. S4A). Clinical sensitivity and specificity of the overall study with
the LAMP assays (LAMP-PfK13 and LAMP-Pan18S) are summarized in Tables S9 and S10 and
Fig. S4B to D.
Translation into a POC LoC device. A subset of 14 P. falciparum-positive samples
and 4 negative samples from the 164 samples were tested on our LoC platform to
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
FIG 3 Evaluation of samples from Ghana. (A) Three-dimensional distribution of samples (cohort 1, N = 70) stratified according to symptoms, where the x axis
represents parasite density log10 (parasites/mL), the y axis represents PCR TTP (cycles), and the z axis represents LAMP TTP (min). Dots at “0” indicate that the sample
was not detected by the corresponding method. (B) Box and whisker plot showing the distribution of P. falciparum-positive samples detected by microscopy and
ELISA across parasitic loads (H, high; M-L, medium-low; L, low). (C) Box and whisker plot showing the distribution of P. falciparum-positive samples detected by PCR-
Pf and LAMP-PfK13 in cohort 1 across parasitic loads (H, high; M-L, medium-low; L, low). (D) Box and whisker plot showing the distribution of P. falciparum-positive
samples detected by PCR-Pf and LAMP-PfK13 in cohort 2 across parasitic loads (H, high; M-L, medium-low; L, low). Each dot represents a sample; horizontal lines in
the boxes indicate medians; lower and upper edges of boxes indicate the interquartile range, and whiskers are less than 1 times the interquartile range.
demonstrate the reliability of a portable device for nucleic acid amplification. In compari-
son to fluorescence-based instruments, Lacewing relies on electrochemical detection (pH
measurements are transduced into electrical signals), which allows label-free real-time moni-
toring without visual inspection. Furthermore, portability and a digital record of the test le-
verage its future use as a POC device. Indeed, its transferability and flexibility to accom-
modate LAMP assays positions this platform as a promising device for digital diagnostics
in limited-resource settings and front-line testing (29, 30). As shown in Fig. 4A, amplifica-
tion curves were obtained with our LoC platform (eLAMP). TTP values were extracted
based on the Cy method (26, 31). Samples were tested in parallel in a conventional real-
time fluorescence-based instrument (qLAMP). TTP values are summarized in Table S11.
Differences between the data obtained with each device are shown in the Bland-Altman
plot in Fig. 4B. The distribution of the TTP values was comparable (difference within
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
TABLE 2 Data stratification of cohort 1 using as a reference microscopy and TaqMan PCR assays (N = 70)
PCR-Pan CT (cycles) PCR-Pf CT (cycles) Parasite density (parasite/mL)
Category median (IQR1, IQR3) median (IQR1, IQR3) median (IQR1, IQR3) Sample (n)
Symptomatic 18.36 (17.79, 19.07) 20.68 (20.07, 21.43) 13,943 (4,428.8, 20,814) 8
Asymptomatica 24.37 (21.73, 26.28) 27.01 (24.37, 29.07) 1,152.32 (240, 1,410) 21
Asymptomaticb 26.07 (23.05, 28.22) 28.86 (25.79, 31.12) NAc 26
Uninfected Negative 15
Total 70
aAsymptomatic with detectable parasitemia. IQR, interquartile range.
bAsymptomatic with submicroscopic parasitemia.
cNA, not applicable.
the 95% limits of agreement) across instruments, indicating the reliability of the pro-
posed device.
DISCUSSION
The high prevalence of asymptomatic malaria cases (32) and the high rate of misdiagnosis
highlight the need of highly sensitive molecular-based methods for diagnosis at the POC.
In 2020, malaria cases and deaths increased by 6% and 12%, respectively, compared to in
2019 (1), which was related to disruption in diagnostics, prevention, and treatment during
the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In 2021, malaria
cases increased, and the number of deaths did not decrease significantly. Improvement of cur-
rent diagnostic strategies, such as microscopy and rapid diagnostic tests (RDTs), to detect sub-
clinical cases and non-P. falciparum infections (33) which are currently underreported (34, 35),
could be achieved using molecular digital diagnostics.
To date, there are no commercially available rapid tests based on molecular methods
for the specific detection of all non-P. falciparum species. Most distributed tests detect pan-
genus or P. falciparum and rely on antigen-specific detection through lateral flow dipsticks
(LFDs). Only Eiken chemical (Tokyo, Japan) has deployed a kit (Loopamp malaria pan/Pf/Pv
detection kit) for pan-genus, P. falciparum, and P. vivax detection using LAMP. However,
laboratory-based instruments are still required to perform the test. Target product pro-
files for malaria diagnostics should not only meet the REASSURED criteria from the WHO
(36) but also focus on the detection of submicroscopic, low-parasite-density-infected popula-
tions and species-specific identification for appropriate treatment guidance (37). We describe
here the first LAMP assay for the detection of the zoonotic species P. cynomolgi, which
may be of clinical relevance at the POC where this species and other zoonotic species
(e.g., P. knowlesi) are present. Their use for prospective studies may add valuable infor-
mation about their epidemiological distribution. In addition, we developed the first LAMP
assays for the discrimination between P. ovale wallikeri and P. ovale curtisi. Due to the com-
mon prescription (primaquine) for the dormant liver stage of the P. ovale species, we pro-
posed the use of the assays in duplex format. Diagnostics of non-P. falciparum malaria
could significantly reduce a reservoir for transmission (38). In the studied cohorts, all the
positive cases were P. falciparum, including 9 P. malariae coinfections, which corresponded
to 5.5% of the population assessed, a percentage that agrees with other studies conducted in
Ghana (32, 39).
TABLE 3 Evaluation of samples from Obom (cohort 1) with LAMP-PfK13 and TaqMan PCR-Pf (N = 70)
PCR-Pf CT DNA conc.
Categorya (cycles) (copies/reaction)b Sample (n) TPa (n) FNa (n) TNa (n) FPa (n) SENa (%) SPEa (%) ACCa (%)
H ,24 .2.34
103 16 16 0 100
M 24 to 29 2.34
 103 to 8.03
 101 27 27 0 100
L $29 #8.03
101 12 5 7 41.7
N Not detected 15 15 0
Total 70 48 7 15 0 87.3 100.0 90.0
aTP, true positives; FN, false negatives; TN, true negatives; FP, false positives; H, high; M-L, medium-low; L, low; U, uninfected; /, Not applicable.
bDNA concentration (copies/reaction) was calculated based on PCR-Pf CT values using 1 microliter (mL) of sample in a final reaction volume of 10 microliter (mL).
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
TABLE 4 Evaluation of samples from Cape Coast (cohort 2) with LAMP-PfK13 and TaqMan PCR-Pf (N = 94)
PCR-Pf CT DNA conc.
Categorya (cycles) (copies/mL)b Sample (n) TPa (n) FNa (n) TNa (n) FPa (n) SENa (%) SPEa (%) ACCa (%)
H ,24 .2.34
 103 40 40 0 100.0
M 24 to 29 2.34
 103 to 8.03
 101 23 22 1 95.7
L $29 #8.03
 101 19 8 11 42.1
N Not detected 12 12 0
Total 94 70 12 12 0 85.4 100 87.2
aTP, true positives; FN, false negatives; TN, true negatives; FP, false positives; H, high; M-L, medium-low; L, low; U, uninfected; /, Not applicable.
bDNA concentration (copies/reaction) was calculated based on PCR-Pf CT values using 1 microliter (mL) of sample in a final reaction volume of 10 microliter (mL).
Detection of low-level parasitemia in symptomatic and asymptomatic carriers is required
for epidemiological research and the identification of hot spots for malaria control interven-
tions (37). We demonstrated a SEN of 100% when testing samples from asymptomatic
malaria cases with concentrations above 80 gDNA copies per mL and a SEN of 80.77%
in asymptomatic cases with submicroscopic parasitemia. These results are higher to
ultrasensitive RDTs, such as Alere ultrasensitive malaria Ag P.f (Abbott) RDT (targeting
PfHRP2), which achieved a SEN between 55.5% and 73%, as reported in Danwang et al.
(40) and Acquah et al. (41). In another study from Yeung et al. (42), this ultrasensitive
RDT had a SEN of between 53.8% and 60% compared to qPCR in a low-transmission
setting targeting asymptomatic P. falciparum infections. Although deletions in PfHRP2/
3 have been widely reported, their prevalence in sub-Saharan Africa remains low (43).
We also performed a PfHRP2 ELISA because it is an indicator of total parasite biomass,
and its performance reflects the current diagnostic tools available in this region.
Aiming to translate the developed LAMP assays into the POC, we demonstrated as
proof of concept the detection of 14 P. falciparum-positive samples with Lacewing, our
handheld device. Portable molecular-based digital diagnostics is required to identify
asymptomatic malaria cases in limited-resource settings and reduce the burden of this
disease. Other platforms, such as the “noninstrumented nucleic acid amplification” (NINA)
heater, enable molecular diagnostics to be performed at the POC (44). This platform allows
electricity-free, constant, and hermetic incubation to run LAMP reactions. However, this
instrument only provides endpoint and, therefore, qualitative results. Our LoC platform
performs real-time amplification and provides quantitative data via a dedicated app con-
nected via Bluetooth. This study presents a molecular diagnostic test with the potential to
be used with a portable LoC device for P. falciparum and non-P. falciparum digital diagnostics.
Although there are some limitations to this study, this introduces a promising technology for
malaria control and surveillance. The sample size was small and may not reflect actual preva-
lence of Plasmodium species in Ghana. Also, the developed LAMP assays should be further
validated with clinical samples from patients infected with other non-P. falciparum species.
A future study could be performed in another setting where multiple species coexist (45).
FIG 4 Evaluation of samples in a portable LOC platform in Ghana. (A) Amplification curves of clinical
samples (n = 18) using our lab-on-chip platform and LAMP assay for P. falciparum detection. (B) Bland-
Altman plot showing in the y axis the TTP difference between the LOC and real-time instrument and in
the x axis the mean TTP values with both instruments. Dashed lines represent 95% limits of agreement,
being calculated as the mean difference (bias) 6 1.96 times the standard deviation (SD).
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
Lastly, to evaluate a truly sample-to-result device at the POC, a low-cost and simple sample
preparation module has to be integrated or easily combined with the LoC platform. (28)
Future work will address these limitations, providing a reliable molecular-based test for POC
malaria diagnostics.
MATERIALS ANDMETHODS
Ethical approval statement. Collection of samples in Obom between November 2018 and 2019
was approved by the ethical committees of the Noguchi Memorial Institute for Medical Research, University of
Ghana (number 024/14-15), and the London School of Hygiene and Tropical Medicine (ID numbers 14322,
15684, and 17257). Written informed consent was obtained from parents or guardians, and assent was appro-
priately received from the children before they were enrolled in this study (46). Sample collection in Ewim
Polyclinic in Cape Coast (Ghana) between the period of July 2018 and December 2019 was approved by
the ethics committees of the Ghana Health Service (GHSERC005/12/17), the Noguchi Memorial Institute for
Medical Research, University of Ghana (NMIMR-IRB CPN 077/17-18), and the Kintampo Health Research
Centre (KHRCIEC/2018-10). All participants and/or guardians of participants gave written informed consent
before recruitment (16).
Clinical samples. Seventy samples (cohort 1) were collected from children aged 6 to 12 years who
were symptomatic, asymptomatic, or uninfected with malaria parasites. The children were recruited between
November 2018 and 2019 in Obom, a malaria hyperendemic subdistrict located in Accra, Ghana. Peripheral
blood samples (5 mL) were collected into EDTA and PAXgene tubes before any antimalarial treatment and
were transported at 4°C to the laboratory for investigation (46). A total of 94 samples (cohort 2) were col-
lected from subjects with suspected malaria. Study participants were recruited at Ewim Polyclinic in Cape
Coast (Ghana) between the period of July 2018 and December 2019. A volume of 5 mL of venous blood
was collected, and 200 mL of sample was used for DNA extraction with the QIAamp DNA minikit (Qiagen,
Manchester, UK) following instructions from the manufacturer. DNA was eluted in a total volume of 100 mL
of elution buffer and stored at220°C.
Synthetic samples. Synthetic DNA gBlocks containing the target genes of interest of P. falciparum,
P. ovale curtisi, P. ovale wallikeri, P. malariae, P. vivax, and P. cynomolgi were purchased from Thermo Fisher
Scientific (USA). Sequence details and reference accession numbers are detailed in Tables S1 and S2 in the
supplemental material.
Microscopy and antigen detection. Samples in cohort 1 were analyzed by microscopy and enzyme-
linked immunosorbent assay (ELISA). Microscopic examination of parasite density was performed by two
independent microscopists blinded to each other’s results. Quantification was performed by counting
numbers of infected red blood cells relative to white blood cells (WBCs) and assuming a WBC count of
8,000 permL; at least 500 fields were examined before a smear was classified as negative. The PfHRP2 enzyme
immunoassay (malaria antigen CELISA; CeLLabs, Australia; https://www.cellabs.com.au/malaria) was performed
on plasma as an indicator of total body parasite load of P. falciparum through the detection of the PfHRP2-spe-
cific antigen using plasma samples and following the manufacturer’s instructions (46).
LAMP primer design. LAMP assays were designed using the software Primer Explorer version 5.0 (http://
primerexplorer.jp/lampv5e/index.html) based on the sequences retrieved from the NCBI GenBank database
(https://www.ncbi.nlm.nih.gov/genbank/) and PlasmoDB. Sequences were aligned using the MUSCLE algo-
rithm (47) in Geneious 10.0.5 software (48), and primers were designed to specifically detect the target DNA
without interspecies cross-reactivity. All primers were purchased from Integrated DNA Technologies (IDT)
and rehydrated in Tris-EDTA (TE) buffer at 100 mM. The LAMP assay specific to the human housekeeping
gene ACTB, LAMP-ACTB, was used as reported in reference 28. Primer sequences are shown in Table S3.
LAMP reaction conditions. LAMP reactions were performed in duplicate with a final volume of 5 mL
per reaction. Each reaction contained the following: 0.5 mL of 10
 custom isothermal buffer (pH 8.5 to 9),
0.3 mL of MgSO4 (100 mM stock), 0.28 mL of dNTPs (25 mM stock), 0.3 mL of bovine serum albumin (BSA;
20 mg/mL stock), 0.8mL of betaine (5 M stock), 0.5mL of 10
 or 20
 LAMP primer mix (with final concentra-
tions per reaction of F3/B3 0.25 or 0.5 mM, LF/LB 1 or 2 mM, and FIP/BIP 2 or 4 mM), 0.13 mL of SYTO9 dye
(20 mM stock), 0.13 mL of NaOH (0.2 M stock), 0.03 mL of Bst 2.0 DNA polymerase (120 kU/mL stock), 1 mL of
sample, and enough nuclease-free water (Invitrogen) to bring the volume to 5 mL. Reactions were loaded
into 96-well plates and were performed at 63°C for 30 min using a LightCycler 96 real-time PCR system
(LC96; Roche Diagnostics). One melting cycle was performed at 0.1°C/s from 63°C up to 97°C for validation of
the specificity of the amplified products. LAMP-PmK13, LAMP-PoRBP2, and LAMP-ACTB primer mixes were
prepared at 20
; the rest of the assays used in this study were prepared at 10
.
PCR conditions. TaqMan PCRs were performed in duplicate with a final volume of 10mL per reaction
using the GoTaq Probe qPCR kit from Promega. Each reaction contained the following: 5 mL of 2
 GoTaq
Probe qPCR master mix, 1mL of PCR-Pan assay primer/probe mix, 1mL of PCR-Pf assay primer/probe mix, 1mL
of sample, and enough nuclease-free water to bring the volume to 10mL. Final concentrations per reaction of
the primer mix were forward primer 0.4mM, reverse primer 0.4mM, and probe 0.2mM. Thermal cycling condi-
tions were adapted from Kamau et al. (12) as follows: 1 cycle of 2 min at 95°C, 45 cycles of 15 s at 95°C, and
1 min at 60°C.
Analytical sensitivity and specificity of the developed LAMP assays. Analytical sensitivity was
evaluated using 10-fold serial dilutions of synthetic DNA ranging from 107 to 10 copies permL. The highest con-
centration available for P. knowlesi was 106 copies per mL. In the case that 102 copies per mL was detected but
10 copies per mL was not, a 2-fold dilution to a final concentration of 50 copies per mL was also tested.
Dilutions were performed using nuclease-free water. Each condition was run in triplicate using the LC96 real-
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LAMP Assays for Malaria Diagnostics at Point-of-Care Microbiology Spectrum
time instrument. Analytical specificity was performed by testing available clinical isolates (26) of P. falciparum,
P. ovale wallikeri, P. malariae, P. vivax, and P. knowlesi, which were previously screened with the TaqMan PCR-
Pan assay, and their calculated concentrations were P. falciparum 1.3
 105 copies/mL, P. malariae 7.7
 102 cop-
ies/mL, P. ovale wallikeri 2.2
 103 copies/mL, P. vivax 6.5
 101 copies/mL, and P. knowlesi 6.8
 104 copies/mL.
LoC platform. The LoC system consisted of (i) a portable platform powered by a rechargeable bat-
tery that contains a Peltier heating module and Bluetooth for data transfer to a smartphone application, (ii) a
disposable ion-sensitive field-effect transistor (ISFET) microchip (4,368 sensors, 2 
 4 mm) for pH sensing dur-
ing nucleic acid amplification, (iii) a microfluidic chamber for containment of the reaction solution (top sealed
with PCR tape), which is in contact with the chip through a reference Ag/AgCl electrode, and (iv) a smartphone
application (AndoridOS) for real-time data visualization, geotagging, and cloud connectivity for data storage
(Amazon web services) (26, 29, 49–51).
Statistical analysis. Time-to-positive (TTP) data are presented as mean TTP 6 standard deviation.
P values were calculated by Welch’s unequal variance two sample t test or one-way analysis of var-
iance (ANOVA) with a Fisher least significant difference (LSD) means comparison in Origin 2019 (v9.6).
A Bland-Altman test was performed in GraphPad using the built-in function for it and calculating the
difference versus the average with automatic 95% limits of agreement. A P value equal to 0.05 was
considered the threshold for statistical significance.
Study design and sample size. The samples used in this study were previously tested, and, there-
fore, samples were known to be positive or negative (16, 46). Consequently, the following calculations (52)
were performed to estimate the number of samples required to evaluate the LAMP-PfK13 assay and LAMP-
Pan assay,
1:962pð12 pÞ
n$ ;
x2
where p is the suspected sensitivity, and x is the desired margin of error. We define the true-positive rate (sensi-
tivity) as the proportion of P. falciparum- or Plasmodium-positive samples that are correctly detected by the
LAMP assays compared to the gold standard TaqMan PCR-Pf and PCR-Pan assays, respectively. We suspected
the sensitivity and specificity to be 80% with a desired margin of error of 10%. With these conditions, the num-
ber of required samples was 61.47 (rounded up to 62), and we tested a total 164 samples.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 6.4 MB.
ACKNOWLEDGMENTS
We declare no competing interests.
This work was supported by Research England Global Challenges Research Fund and the
Centre for Antimicrobial Optimisation (CAMO) at Imperial College London. We acknowledge
EPSRC HiPEDS CDT (EP/L016796/1 to K.M.-C.), EPSRC Doctoral Prize Fellowship (EP/T51780X/1
to K.M.-C.), Wellcome Investigator Award (100993/Z/13/Z to J.B.), World Bank African Centers
of Excellence Grant (ACE02-WACCBIP to G.A.), Wellcome/African Academy of Sciences
DELTAS grant (DEL-15-007/107755/Z/15/Z to G.A.), The Royal Society University Research
Fellowship (UF0762736/UF120026 to J.C.R.H.), and Challenge Grant (CH160018 to J.C.R.H.)
for supporting this work.
Authors K.M.-C., P.G., and J.R.-M. are affiliated with the NIHR Health Protection Research
Unit (HPRU) in Healthcare Associated Infections and Antimicrobial Resistance at Imperial
College London in partnership with the UK Health Security Agency (previously PHE), Imperial
Healthcare Partners, the University of Cambridge, and the University of Warwick.
Conceptualization: K.M.-C., P.G., J.R.-M., A.C., and J.B. Investigation: K.M.-C., N.M., F.A., I.P.,
and D.A.P. Data curation: K.M.-C. and N.M. Resources: K.M.-C., J.R.M., A.C., J.B., F.A., D.A.P.,
L.E.A., J.C.R.H. Formal analysis and software: K.M.-C. and N.M. Methodology: K.M.-C. and N.M.
Project administration: K.M.-C. Funding acquisition: J.B., A.C., P.G., J.R.-M., K.M.-C., and G.A.
Writing original draft: K.M.-C. Writing review and editing: all authors.
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