The Journal of Molecular Diagnostics, Vol. 23, No. 10, October 2021jmdjournal.orgDevelopment of Cooperative Primer-Based
Real-Time PCR Assays for the Detection of
Plasmodium malariae and Plasmodium ovale
Felix Ansah,*y Jonathan Suurbaar,*y Derrick Darko,* Nsoh G. Anabire,*yz Samuel O. Blankson,* Bright K.S. Domson,*
Alamissa Soulama,*y Paulina Kpasra,y Jersley D. Chirawurah,*y Lucas Amenga-Etego,* Prosper Kanyong,*x
Gordon A. Awandare,*y and Yaw Aniweh*From the West African Centre for Cell Biology of Infectious Pathogens* and the Department of Biochemistry,y Cell and Molecular Biology, College of Basic
and Applied Sciences, University of Ghana, Legon, Accra, Ghana; the Department of Biochemistry and Molecular Medicine,z School of Medicine, University
for Development Studies, Tamale, Ghana; and Flexmedical Solutions Ltd.,x Eliburn Industrial Park, Livingston, United KingdomAccepted for publicationC
T
h
July 14, 2021.
Address reprint requests to
Gordon A. Awandare, Ph.D.,
or Yaw Aniweh, Ph.D.,
WACCBIP, University of
Ghana, P.O. Box LG 54,
Legon-Accra, Ghana. E-mail:
gawandare@ug.edu.gh or
yaniweh@ug.edu.gh.opyright ª 2021 Association for Molecular
his is an open access article under the CC B
ttps://doi.org/10.1016/j.jmoldx.2021.07.022Plasmodium malariae and Plasmodium ovale are increasingly gaining public health attention as the
global transmission of falciparum malaria is decreasing. However, the absence of reliable Plasmodium
species-specific detection tools has hampered accurate diagnosis of these minor Plasmodium species. In
this study, SYBR Greenebased real-time PCR assays were developed for the detection of P. malariae and
P. ovale using cooperative primers that significantly limit the formation and propagation of primers-
dimers. Both the P. malariae and P. ovale cooperative primer-based assays had at least 10-fold lower
detection limit compared with the corresponding conventional primer-based assays. More important,
the cooperative primer-based assays were evaluated in a cross-sectional study using 560 samples ob-
tained from two health facilities in Ghana. The prevalence rates of P. malariae and P. ovale among the
combined study population were 18.6% (104/560) and 5.5% (31/560), respectively. Among the
Plasmodium-positive cases, P. malariae and P. ovale mono-infections were 3.6% (18/499) and 1.0% (5/
499), respectively, with the remaining being co-infections with Plasmodium falciparum. The study
demonstrates the public health importance of including detection tools with lower detection limits in
routine diagnosis and surveillance of nonfalciparum species. This will be necessary for comprehensively
assessing the effectiveness of malaria interventions and control measures aimed toward global malaria
elimination. (J Mol Diagn 2021, 23: 1393e1403; https://doi.org/10.1016/j.jmoldx.2021.07.022)Supported by the National Institute for Health Research (NIHR) Global
Health Research program 16/136/33, using aid from the UK Government,
World Bank African Centres of Excellence for Development Impact-West
African Center for Cell Biology of Infectious Pathogens and Non-
Communicable Diseases grant (ACE IMPACT - WACCBIP-NCDs;
G.A.A.), and Wellcome/African Academy of Sciences Developing Excel-
lence in Leadership, Training and Science (DELTAS) Africa grant DEL-15-
007 (G.A.A.).
The views expressed in this publication are those of the authors and not
necessarily those of the NIHR or the UK Department of Health and Social
Care, World Bank, Wellcome, or African Academy of Sciences.
Disclosures: None declared.Human malaria is a life-threatening disease caused by five
distinct Plasmodium species (namely, Plasmodium falcipa-
rum, Plasmodium malariae, Plasmodium ovale, Plasmo-
dium vivax, and Plasmodium knowlesi).1 Among these
species, P. falciparum and P. vivax are the most prevalent
and cause the most severe forms of the disease.2,3 However,
with the general global reduction in falciparum malaria,
significant attention has been drawn toward the minor
Plasmodium species: P. malariae and P. ovale.4,5 Although
these less prevalent species are generally associated with
benign malaria,6,7 recent reports have implicated them in
major disease burden, such as severe anemia, acute respi-
ratory distress syndrome, and acute renal failure.8,9 There-
fore, the availability of reliable tools for timely and accuratePathology and American Society for Investiga
Y-NC-ND license (http://creativecommons.orgdiagnosis of nonfalciparum malaria is necessary to inform
appropriate treatment and effective management.tive Pathology. Published by Elsevier Inc.
/licenses/by-nc-nd/4.0).
Ansah et alCurrent methods for malaria diagnosis include micro-
scopy, rapid diagnostic tests, and nucleic acidebased
amplification tests (NAATs).10 However, because of the
morphologic similarities among Plasmodium species and
the low parasite densities of P. malariae and P. ovale spe-
cies in clinical isolates, both microscopy and rapid diag-
nostic tests remain unsatisfactory for routine detection of
nonfalciparum Plasmodium species.6,11,12 Efforts to address
this diagnostic gap led to the development of highly sensi-
tive and specific NAATs, including PCR and loop-mediated
isothermal amplification.12,13
Several NAATs, involving the use of TaqMan probes and
SYBR Green, have been developed for the detection of
nonfalciparum species.14e17 Although these NAATs have
improved sensitivity, lower detection limits, and higher
specificity compared with microscopy and rapid diagnostic
tests,13 the formation and propagation of primers-dimers
remains a major sensitivity and specificity limiting factor,
especially at low target concentration.18,19 Attempts to
address primers-dimers over the years led to the develop-
ment of cooperative primers, which is the first technology
that simultaneously curbs primer-dimer formation and
propagation.20 The cooperative primers were shown to
significantly limit primer-dimer formation and propagation
up to 2.5 million-fold compared with the conventional
primers.20
A cooperative primer-based real-time assay [real-time
quantitative PCR (qPCR)] has been developed for the
detection of P. falciparum, and the assay was shown to
have lower detection limit relative to its corresponding
conventional primer-based assay.20 In this study, SYBR
Greenebased qPCR assays were developed for the detection
of P. malariae and P. ovale using cooperative primers that
target the 18S ribosomal rRNA genes.
Materials and Methods
Ethical Approval
The study obtained ethical clearance from the ethics com-
mittees of the Ghana Health Service (GHSERC005/12/17),
the Noguchi Memorial Institute for Medical Research
(Institutional Review Board Certified Protocol Number 077/
17-18), and the Kintampo Health Research Center
(KHRCIEC/2018-10). A written informed consent was ob-
tained from all participants and/or from parents or guardians
of participants.
Development of P. malariae and P. ovale Cooperative
Primer-Based qPCR Assays
The 18S rRNA gene sequences of P. falciparum
(XR_002273101.1), P. malariae (M54897.1), P. ovale cur-
tisi (KF696371.1), P. ovale wallikeri (KF696364.1), and P.
vivax (XR_003001225.1) were retrieved from the National
Center for Biotechnology Information database and aligned1394using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/
clustalo, last accessed May 12, 2021). Conserved genomic
regions were selected for the design of the cooperative
primers. Each cooperative primer consisted of a low
melting temperature short primer and a capture sequence
connected by two units of hexaethylene glycol (spacer
18). The process of annealing and extension of the
cooperative primer has been previously described.20 At-
tempts to develop assays consisting of both forward and
reverse cooperative primers were unsuccessful for both P.
malariae and P. ovale. Because neither the forward nor the
reverse conventional primers, when used alone, would
produce detectable primers-dimers,21 a cooperative primer
was paired with a conventional primer in both the P.
malariae and P. ovale assays (Table 1). For P. ovale
cooperative primer, one wobble base was introduced into
capture sequence to ensure perfect complementarity to the
two P. ovale subspecies: P. ovale curtisi and P. ovale
wallikeri. The performance of the cooperative primers was
compared with parallel assays containing the capture
sequence of each cooperative primer adopted as the con-
ventional primer and paired with the other conventional
primer that was used in the cooperative assays (Table 1).
The assays were compared using 10-fold serially diluted
MRA-179 and MRA-180 plasmids (ATCC, Manassas, VA)
for P. malariae and P. ovale, respectively, with concentra-
tions ranging from 106 to 100 copies/mL. All primers used in
the study were synthesized by Biosearch Technologies
(Petaluma, CA).
SYBR GreeneBased qPCR Assays
The P. malariae and P. ovale SYBR Greenebased qPCR
assays were performed on the QuantStudio5 system
(Applied Biosystems, Waltham, MA). All reactions were
performed in a total volume of 15 mL containing 1 Luna
Universal qPCR Master Mix (New England BioLabs,
Hitchin, UK), 250 nmol/L of each of the cooperative and
the conventional primers, and 3 mL of the template DNA.
The cycling conditions for both assays consisted of 3
minutes at 95C, followed by 45 cycles of 15 seconds at
95C, 40 seconds at 50C, and 40 seconds at 60C. The
specificity of the qPCR products was determined by
analyzing the amplicons using the melting curves and on
1.5% agarose gel. The resulting gel was processed using the
Amersham Imager 600 (GE Healthcare Life Sciences,
Chicago, IL).
Analytical Specificity and Limit of Detection
The analytical specificity of the assays was determined
using the National Center for Biotechnology Information
Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/
primer-blast, last accessed on May 12, 2021).
Experimental specificity was also determined using
genomic DNA of P. falciparum, P. malariae, P. ovale,jmdjournal.org - The Journal of Molecular Diagnostics
qPCR Detection of Nonfalciaprum Species
Table 1 Sequence of Oligonucleotides for Real-Time Quantitative PCR Assays
Assay Target Primer code Sequence
Cooperative Plasmodium malariae PlasmoF 50-TTATGAGAAATCAAAGTCTTTGGGTT-30
MalR3_Coop 50-AAAACATTCTAATATTTTAATCA[Sp18][Sp18]GGGAAAAGAACGT-30
Cooperative Plasmodium ovale OvaF_Coop 50-CTGYTCTTTGCATTCCTTAT[Sp18][Sp18]GCTTAGACAATA-30
Plasmo2* 50-AACCCAAAGACTTTGATTTCTCATAA-30
Conventional P. malariae PlasmoF 50-TTATGAGAAATCAAAGTCTTTGGGTT-30
MalR3 50-AAAACATTCTAATATTTTAATCA-30
Conventional P. ovale Ova_F 50-CTGYTCTTTGCATTCCTTAT-30
Plasmo2* 50-AACCCAAAGACTTTGATTTCTCATAA-30
Plasmodium falciparum Pf_stRNA_Fy 50-AAGTAGCAGGTCATCGTGGTT-30
Pf_stRNA_Ry 50-TTCGGCACATTCTTCCATAA-30
*Primer sequence has been previously published by Rougemont et al.15
yPrimer sequence has been previously published by Heinberg et al.22
[SP18], spacer 18.and P. vivax. The limit of detection and efficiency were
determined using a 10-fold serial dilution of MRA-179
and MRA-180 plasmids for P. malariae and P. ovale,
respectively. Each plasmid was diluted to obtain 106, 105,
104, 103, 102, 101, and 100 copies/mL in tris-ethylenedi-
amine tetraacetic acid buffer. All assays were performed in
triplicate.
Clinical Samples
To validate the cooperative primer-based assays for the
detection of P. malariae and P. ovale in clinical isolates,
whole blood samples were obtained from individuals
who presented with suspected malaria at the Ewim Poly-
clinic in Cape Coast (n Z 178) and the Richard Novati
Catholic Hospital in Sogakope (n Z 382) between
December 2017 and December 2018. Cape Coast is located
in the Central Region of Ghana (Google Map Plus Code:
4Q74þQ9 Cape Coast), whereas Sogakope is located in
the Volta Region of Ghana (Google Map Plus Code:
2H5QþC9 Sogakope). Both study sites are meso-endemic
for malaria, with all-year malaria transmission that peaks
during the June-July rainy season.23e25 Venous blood was
collected from individuals who consented to participate in
the study.
Detection of P. falciparum, P. malariae, and P. ovale in
Clinical Isolates
Genomic DNA was purified from 200 mL of the venous
blood using the QIAamp DNA Mini Kit (Qiagen, Man-
chester, UK) following instructions from the manufacturer.
DNA was eluted in a total volume of 100 mL using elution
buffer provided by the manufacturer (Qiagen). The purified
genomic DNA was stored at 20C until ready for mo-
lecular analysis. Identification of Plasmodium species was
first performed by microscopy. Thick and thin blood smears
were prepared at the time of blood sample collection and
stained with 10% Giemsa for microscopy examination. The
number of parasite-infected red blood cells was determinedThe Journal of Molecular Diagnostics - jmdjournal.orgper 500 white blood cells. Parasite count per microliter of
blood was determined using the standard leukocyte count of
8000 leukocytes per microliter of blood, as previously
described.26 The P. malariae and P. ovale were detected
using the SYBR Green cooperative primer-based assays
described earlier in this study, whereas P. falciparum
detection was performed using a previously described
SYBR Green qPCR protocol with primers targeting P. fal-
ciparum seryl-tRNA synthetase (PF3D7_0717700).22 The
specificity of the qPCR amplicons was determined using
melt curve analysis. The resulting CT values for positive
samples were used to estimate parasite copy number per
microliter using standard curves obtained from 10-fold
serially diluted plasmids.
Statistical Analysis
Data analysis was performed using IBM SPSS Statistics
version 26 (International Business Machines Corporation,
Armonk, NY), GraphPad Prism version 8.0.2, (GraphPad
Software Inc., San Diego, CA), and Microsoft Excel 2016
Software (Microsoft Corporation, Redmond, WA). Probit
analysis was used to estimate the limit of detection of the
assays at 95% CI. Statistical significance for the proportion
of positive samples between the two study sites was deter-
mined using c2 test or Fisher exact test, as appropriate.
Parasite load across three or more groups was compared
using the Kruskal-Wallis test, and where differences were
observed, pair-wise comparisons were conducted using the
U-test. Statistical significance for all analyses was consid-
ered at P < 0.05.Results
Analytical Sensitivity, Specificity, and Limit of
Detection
SYBR Greenebased qPCR assays for the detection of
P. malariae and P. ovale were developed using cooperative
primers. Analysis of amplicons using melt curves (Figure 1,1395
Ansah et al
Figure 1 The specificity and detection limits of Plasmodium malariae and Plasmodium ovale assays. Assays were performed using Plasmodium falciparum
(Pf), P. malariae (Pm), P. ovale curtisi (Po curtisi), P. ovale wallikeri (Po wallikeri), and Plasmodium vivax (Pv) genomic DNA. A: Melt curve for P. malariae
(blue). B: Melt curves for P. ovale curtisi (green) and P. ovale wallikeri (orange) assays. Other colors represent the P. falciparum, P. vivax, and nontemplate
control (NC). C: Separation of the resulting P. malariae real-time quantitative PCR (qPCR) amplicons on 1.5% agarose gel. D: Separation of the resulting qPCR
amplicons for P. ovale subspecies on 1.5% agarose gel. Expected amplicon sizes for P. ovale curtisi and P. ovale wallikeri were 528 and 521 bp, respectively.
E: The detection limits for P. malariae (blue curve) and P. ovale (red curve) assays were determined using a 10-fold serial dilution of plasmids. Plasmid
concentrations were log-transformed and then analyzed using probit analysis. The probability of detection was plotted against the plasmid copy numbers/mL
of the DNA. Molecular weight marker (MM) shown in bp.A and B) and gel electrophoresis (Figure 1, C and D)
showed that both assays were specific to the selected 18S
rRNA genomic region with no cross-reactivity to other1396Plasmodium species. The melting temperature values of the
assays were 85.60  0.46C and 79.44  0.17C for P.
malariae and P. ovale, respectively (Table 2). Using 10-foldjmdjournal.org - The Journal of Molecular Diagnostics
qPCR Detection of Nonfalciaprum Species
Table 2 Real-Time Quantitative PCR Assay Details and Efficiencies
Assay Slope Intercept R2 Efficiency, % Amplicon length, bp Melting temperature, C*
Plasmodium malariae e4.4 39.10 0.99 68.1 373 85.60  0.46
Plasmodium ovale e4.0 38.73 0.99 76.9 528y, 521z 79.44  0.17
*Mean melting temperature for technical replicates.
yExpected qPCR amplicon length for P. ovale curtisi.
zExpected qPCR amplicon length for P. ovale wallikeri.serially diluted plasmids, the detection limits estimated at
95% confidence level for the P. malariae and P. ovale as-
says were 1.0 plasmid copy/mL (95% CI, 0.94e1.06) and
1.0 plasmid copy/mL (95% CI, 0.96e1.04), respectively
(Figure 1E). The amplification efficiencies of the assays
were 68.1% and 76.9% for P. malariae and P. ovale,
respectively (Table 2).Comparison of the Cooperative and Conventional
Primers
To evaluate the performance of the cooperative primer-
based assays, the cooperative primers were compared with
their corresponding conventional primers using plasmids.
The CT values observed for the conventional primer were
relatively lower compared with its corresponding coopera-
tive primers (Table 3). Despite this observation, the coop-
erative primer-based assays detected as low as 1.0 copy/mL,
whereas the conventional primer-based assays had a detec-
tion limit of 10.0 copies/mL for both the P. malariae and
P. ovale assays (Table 3). Separation of the resulting qPCR
amplicons on a 1.5% agarose gel showed primers-dimers as
the concentration of P. malariae and P. ovale decreased for
the conventional assays (Figure 2). On the contrary, no
observable primers-dimers were generated for both the
P. malariae and P. ovale cooperative primer-based assays,
even at the lowest concentration of 1.0 copy/mL (Figure 2).
Taken together, the data suggest that the cooperative primer-
based assays have 10-fold lower limit of detection compared
with using conventional primers.Table 3 Comparison of CT Values for Cooperative and Conventional Pr
Plasmodium malariae
Plasmid copies/mL Conventional Coopera
106 9.83  0.07 11.15 
105 13.57  0.18 17.53 
104 16.96  0.39 21.90 
103 21.22  0.38 27.22 
102 25.31  0.22 33.00 
101 27.35  0.09 38.28 
100 Negative 41.94 
Data are expressed as means  SD.
The Journal of Molecular Diagnostics - jmdjournal.orgPrevalence of P. falciparum, P. malariae, and P. ovale
among Study Participants
The prevalence of P. falciparum, P. malariae, and P. ovale
among study participants (n Z 560) (Table 4) was assessed
by microscopy and SYBR Greenebased qPCR assays. The
overall prevalence rates of Plasmodium species infection by
microscopy were 67.4% (120/178) and 66.8% (255/382) in
Ewim and Sogakope, respectively (Figure 3A). Using qPCR,
the prevalence rates of Plasmodium species infection in
Ewim and Sogakope were 91.6% (163/178) and 88.0% (336/
382), respectively (Figure 3A). For species identification
by microscopy, the prevalence rates of P. falciparum,
P. malariae, and P. ovale for the combined study population
were 66.4% (372/560), 2.5% (14/560), and 1.1% (6/560),
respectively (Figure 3B). As expected for qPCR, higher
prevalence was observed for each of the three Plasmodium
species compared with microscopy. The prevalence rates of
P. falciparum, P. malariae, and P. ovale using qPCR were
85.6% (479/560), 18.5% (104/560), and 5.5% (31/560),
respectively (Figure 3B).
A total of 3.2% (18/560) of the participants were found to
be negative by both microscopy and qPCR for all the three
Plasmodium species. For discrepancies between microscopy
and qPCR, 7.5% (42/560) of the microscopy-positive par-
ticipants were qPCR negative for the three Plasmodium
species. Also, 29.8% (167/560) of the qPCR-positive par-
ticipants were undetected by microscopy. Among the
qPCR-positive participants who were undetected by mi-
croscopy, 76.6% (128/167), 2.4% (4/167), and 1.8% (3/167)
were found to harbor mono-infections for P. falciparum,imer Assays
Plasmodium ovale
tive Conventional Cooperative
0.15 12.94  0.01 13.70  0.04
0.28 16.60  0.21 17.38  0.07
0.36 21.46  0.11 22.65  0.05
0.25 26.11  0.15 27.46  0.13
0.45 28.15  0.13 29.65  0.14
0.36 32.75  0.16 34.20  0.29
0.54 Negative 38.77  2.15
1397
Ansah et al
Figure 2 Comparison of cooperative and conventional real-time quantitative PCR (qPCR) assays. Assays were performed using serially diluted Plasmodium
malariae (MRA-179) and Plasmodium ovale (MRA-180) plasmids. The qPCR amplicons were separated on 1.5% agarose gel. Molecular weight marker (MM)
shown in bp.P. malariae, and P. ovale, respectively, whereas the
remaining 19.2% (32/167) harbored mixed infections of two
or three Plasmodium species.
Among the qPCR-positive cases, P. falciparum, P.
malariae, and P. ovale mono-infections in Ewim wereTable 4 Demographic Characteristics of Study Participants
Study site
Characteristic Ewim Sogakope Total
Sample size, n 178 382 560
Sex, n (%)
Female 94 (52.8) 177 (46.3) 271
Male 84 (47.2) 205 (53.7) 289
Age group, years, n (%)
5 44 (24.7) 93 (24.3) 137
6e10 56 (31.5) 79 (20.7) 135
11e20 45 (25.3) 97 (25.4) 142
21e40 13 (7.3) 69 (18.1) 82
>41 20 (11.2) 44 (11.5) 64
Hemoglobin, g/dL* 10.6 (0.2) 10.3 (0.1)
*Data presented as mean (SEM).
139874.2% (121/163), 3.7% (6/163), and 3.1% (5/163), respec-
tively (Figure 3C). In Sogakope, the prevalence rates of P.
falciparum, P. malariae, and P. ovale mono-infections were
74.7% (251/336), 1.8% (6/336), and 0.0% (0/336), respec-
tively (Figure 3C). There were no statistically significant
differences in the distribution of P. falciparum (P Z 0.91)
and P. malariae (P Z 0.20) mono-infections between the
two sites. The proportions of P. falciparum/P. malariae
mixed infection in Ewim and Sogakope were 14.7% (24/
163) and 17.9% (60/336), respectively, whereas P. falcip-
arum/P. ovale, P. malariae/P. ovale, and P. falciparum/P.
malariae/P. ovale mixed infections were all <5.0% in both
study sites (Figure 3C). Study participants were further
strati ed into ve age groups, as previously described14fi fi ;
however, participants aged 5 years were classified as one
group, and the prevalence of Plasmodium species was
determined. The P. falciparum infection was highest among
the age group of 6 to 10 years, whereas P. malariae
infection was highest among age group of 11 to 20 years
(Figure 3D). The P. ovale infection was found to be
generally increasing with age (Figure 3D).jmdjournal.org - The Journal of Molecular Diagnostics
qPCR Detection of Nonfalciaprum Species
Figure 3 Prevalence of Plasmodium species among study participants. A: The prevalence of genus Plasmodium determined by microscopy and real-time
quantitative PCR (qPCR) in Ewim and Sogakope. B: The prevalence of Plasmodium falciparum (Pf), Plasmodium malariae (Pm), and Plasmodium ovale (Po)
determined by SYBR Greenebased qPCR assays and microscopy among the combined study population. C: The proportion of Plasmodium species mono and
mixed infections among study participants in the two study sites. D: The prevalence of Plasmodium species positive cases by age group (in years). The
prevalence was determined by expressing the total number of positive cases as a percentage of the total sample size for each of the stratified age groups.
n Z 560 study participants (A); n Z 178 in Ewin (A); n Z 382 in Sogakope (A).Quantification of Parasite Copy Number
The copy numbers of P. falciparum, P. malariae, and
P. ovale were estimated for the positive clinical samples.
Using qPCR, the median parasite loads of P. falciparum
were significantly higher than those for P. malariae
(P < 0.0001) and P. ovale (P < 0.0001). However, there
was no significant difference between P. malariae and P.
ovale parasite loads (P Z 0.16) (Figure 4A). Across the
stratified age groups, the median P. falciparum copy number
among age group of 6 to 10 years was significantly higher
compared with those for the age group of 0 to 5 years
(P Z 0.04) and the age group of 21 to 40 years
(PZ 0.0002) (Figure 4B). On the other hand, differences in
the parasite loads for both P. malariae and P. ovale among
the age groups did not reach statistical significance
(P > 0.05 for both comparisons) (Figure 4B). Finally, the
qPCR CT values were correlated with parasitemia, as
determined by microscopy (Figure 4C). As expected, there
was a significant negative correlation between the qPCR CT
values and the log-transformed parasitemia (r Z e0.36;
P < 0.001), although the association was not strong.The Journal of Molecular Diagnostics - jmdjournal.orgDiscussion
Because of the limited geographic distribution and marginal
contribution of P. malariae and P. ovale subspecies toward
global malaria burden, these nonfalciparum species have not
received much attention.27 In 2018, the estimated non-
falciparum malaria cases in sub-Saharan Africa were <1%
of all malaria cases.28 However, it is generally thought that
the prevalence of these nonfalciparum species has been
largely underestimated because of the lack of reliable
diagnostic tools.29,30 In addition, the few cases of P.
malariae and P. ovale subspecies are usually detected as
low density and mixed infections with the dominant P.
falciparum, which further present an obstacle to routine
diagnostic tools with poor sensitivity and limited specific-
ity.31e33 As such, there is the need for reliable diagnostic
tools to accurately assess the burden of nonfalciparum
species. In this study, cooperative primers were used to
develop qPCR assays with improved detection limits for the
detection of low-density P. malariae and P. ovale infections
in clinical isolates.1399
Ansah et al
Figure 4 Quantification of Plasmodium species copy numbers.
Data have been presented as box plot, where the boxes represent the
interquartile range, the line through the box is the median, and the
whiskers indicate the 1st and the 99th percentiles. The geometric
mean of the parasite load is denoted by the plus symbol (þ), whereas
the individual dots represent outliers. Parasite copy numbers were
compared using U-test. Statistical significance has been represented.
A: The overall parasite load for Plasmodium falciparum, Plasmodium
malariae, and Plasmodium ovale infections. B: Plasmodium species
parasite load by age group (years) stratification. C: Correlation of
parasite load determined by microscopy and real-time quantitative
PCR (qPCR) CT values. n Z 479 P. falciparum infections (A); n Z 104
P. malariae infections (A); nZ 31 P. ovale infections (A). *P < 0.05,
***P < 0.001, and ****P < 0.0001.One of the major obstacles that limits the specificity,
sensitivity, and detection limit of NAATs is the formation
and propagation of non-specific products, such as primers-
dimers, which result in false negatives or false positives.18
Although several technologies have been described to
mitigate this challenge, cooperative primers were the first
technology that was shown to simultaneously inhibit the
formation and the propagation of primers-dimers up to 2.5
million-fold compared with conventional primers.20 This
study describes the first report on the application of coop-
erative primers for the detection of nonfalciparum species.
The data presented herein suggest that the cooperative
primers had relatively higher CT values than their corre-
sponding conventional primers for a given concentration of
target DNA. A possible explanation is that because the
amplification process for the cooperative primer requires an
initial binding of the cooperative sequence before the
binding of the short low melting temperature primer to its
complementary sequence, it is likely that this additional
time may be a lagging phase that accounts for the differ-
ences in the CT values between the cooperative and the1400conventional primers.20 Notwithstanding this observation,
the results show that the cooperative primers have at least
10-fold lower detection limit compared with their corre-
sponding conventional primers. The lower detection limit of
the cooperative primer-based assays may be explained by
the ability of the cooperative primers to limit primer-dimer
formation.20,34
In Ghana, P. falciparum, P. malariae, and P. ovale are
the three Plasmodium species that have been implicated in
clinical malaria.35 The estimated national prevalence rates
of P. falciparum, P. malariae, and P. ovale are 90% to
98%, <10%, and <2%, respectively.35 However, different
studies across various regions in Ghana have reported
varying prevalence rates for the three Plasmodium spe-
cies.36e38 The data presented herein by microscopy show
that the prevalence rates of P. malariae and P. ovale are
comparable to the national prevalence.35 Using qPCR
analysis, the prevalence rates of P. falciparum, P. malariae,
and P. ovale among the study population were 85.5%,
18.5%, and 5.5%, respectively. These prevalence rates of
P. malariae and P. ovale are comparable to those in ajmdjournal.org - The Journal of Molecular Diagnostics
qPCR Detection of Nonfalciaprum Speciesprevious report,36 but are about twofold higher than the
reported national prevalence,35 and higher than rates in
studies conducted elsewhere in the country.37,38 The higher
prevalence reported in this study maybe due to the lower
detection limits of the cooperative primer-based assays,
even though these studies involve different study pop-
ulations. An undetected population harboring nonfalciparum
species is of great concern because these individuals
potentially serve as parasite reservoir for sustained and long-
term transmission of P. malariae and P. ovale subspecies.
In high transmission settings, malaria incidence generally
peaks in the first few years and then declines and levels off
in the later years.39,40 Consistent with these reports, the
results show that P. falciparum prevalence peaks at the age
of 6 to 12 years and subsequently declines with increasing
age. For nonfalciparum species, P. malariae was found to
be most common among participants aged 11 to 20 years,
whereas P. ovale prevalence was similar across the age
groups. Other studies in Ghana,38 Senegal,41 and Kenya33
also observed that children aged <15 years had a rela-
tively higher risk of P. malariae infection than adults.
Another study in Ghana found no P. ovale infection among
participants aged 10 years, but found infection in younger
participants.36 In Indonesia, it was also observed that the
older population, with a median age of 21 years, had higher
P. malariae infections compared with the younger popula-
tion.35,42 These differences in the distribution of non-
falciparum species among different age groups could be due
to underrepresentation of the different age groups and dif-
ferences in malaria transmission intensity across the various
study sites.33,43
The P. malariae and P. ovale infections are usually
detected as coinfections with P. falciparum.6,44e46 In areas
of high transmission, P. falciparum has been reported to
suppress the prevalence and the density of nonfalciparum
species.6,47,48 Consistent with previous reports,33,36,38,44 the
results show that the parasite density of both P. malariae
and P. ovale parasites in all the mixed infection cases was
lower than P. falciparum. Low density of nonfalciparum
species in cases of mixed infection with dominant P. fal-
ciparum is likely to result in misdiagnosis and affect
appropriate treatment recommendations.49 Nevertheless, it
could be argued that because artemisinin combination
therapies are generally recommended for the treatment of
both uncomplicated P. falciparum and nonfalciparum ma-
laria,50 such misdiagnosis would be of less importance for
antimalarial treatment. However, with the recent reports of
the increasing prevalence of nonfalciparum species despite
artemisinin combination therapy treatment, accurate detec-
tion is necessary.36,51e54
In summary, the study shows at least twofold higher
prevalence of P. malariae and P. ovale among study par-
ticipants compared with the national prevalence in Ghana.
This underlines the need for the employment of such
detection tools with lower detection limits to accurately
assess the burden of nonfalciparum species. The turnaroundThe Journal of Molecular Diagnostics - jmdjournal.orgtime of the current cooperative primer-based assays is
comparable to conventional qPCR assays. However, it is
also important to highlight that the cost of cooperative
primers is relatively higher than their corresponding con-
ventional primers. Notwithstanding the cost, the deployment
of such detection tools will be helpful for reliable diagnosis
and accurate surveillance of nonfalciparum species in a
holistic approach toward malaria elimination.
Acknowledgments
We thank the study participants and their parents or
guardians, the Tackling Infections to Benefit Africa (TIBA)-
Ghana team, the Medical Directors and Biomedical Scien-
tists of Ewim Polyclinic and Richard Novati Catholic
Hospital for helping with the sample collection, and the
TIBA-Ghana local Expert Advisory Group.
Author Contributions
F.A., P.K., Y.A., and G.A.A. conceived and designed the
study; F.A., J.S., D.D., N.G.A., S.O.B., B.K.S.D., A.S.,
P.K., and J.D.C. collected data and performed the experi-
ments; F.A. and Y.A. analyzed the data; F.A. drafted the
manuscript; Y.A. and G.A.A. thoroughly edited the manu-
script draft; Y.A., P.K., L.A.E., and G.A.A. supervised the
study; all authors reviewed and approved the manuscript.
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