Critical Reviews in Microbiology
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Plasmodium malariae, current knowledge and
future research opportunities on a neglected
malaria parasite species
Eniyou C. Oriero, Lucas Amenga-Etego, Deus S. Ishengoma & Alfred
Amambua-Ngwa
To cite this article: Eniyou C. Oriero, Lucas Amenga-Etego, Deus S. Ishengoma & Alfred
Amambua-Ngwa (2021) Plasmodium￿malariae, current knowledge and future research
opportunities on a neglected malaria parasite species, Critical Reviews in Microbiology, 47:1,
44-56, DOI: 10.1080/1040841X.2020.1838440
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Published online: 28 Jan 2021.
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CRITICAL REVIEWS IN MICROBIOLOGY
2021, VOL. 47, NO. 1, 44–56
https://doi.org/10.1080/1040841X.2020.1838440
REVIEW ARTICLE
Plasmodium malariae, current knowledge and future research opportunities
on a neglected malaria parasite species
Eniyou C. Orieroa, Lucas Amenga-Etegob , Deus S. Ishengomac and Alfred Amambua-Ngwaa
aDisease Control and Elimination Theme, Medical Research Council Unit The Gambia at LSHTM, Fajara, The Gambia; bDepartment of
Biochemistry, Cell and Molecular Biology, University of Ghana, Legon, Ghana; cTanga Research Centre, National Institute for Medical
Research, Tanga, Tanzania
ABSTRACT ARTICLE HISTORY
Plasmodium malariae is often reported as a benign malaria parasite. There are limited data on its Received 10 January 2020
biology and disease burden in sub-Saharan Africa (sSA) possibly due to the unavailability of spe- Revised 19 September 2020
cific and affordable tools for routine diagnosis and large epidemiology studies. In addition, P. Accepted 23 September 2020
malariae occurs at low parasite densities and in co-infections with other species, predominately Published online 25 January
P. falciparum. The paucity of data on P. malariae infections limits the capacity to accurately deter- 2021
mine its contribution to malaria and the effect of control interventions against P. falciparum on KEYWORDS
its prevalence. Here, we summarise the current knowledge on P. malariae epidemiology in sSA - Plasmodium malariae;
overall prevalence ranging from 0-32%, as detected by different diagnostic methods; seropreva- malaria epidemiology; non-
lence ranging from 0–56% in three countries (Mozambique, Benin and Zimbabwe), and explore falciparum; elimination
the future application of next-generation sequencing technologies as a tool for enriching P.
malariae genomic epidemiology. This will provide insights into important adaptive mechanisms
of this neglected non-falciparum species, including antimalarial drug resistance, local and
regional parasite transmission patterns and genomic signatures of selection. Improved diagnosis
and genomic surveillance of non-falciparum malaria parasites in Africa would be helpful in evalu-
ating progress towards elimination of all human Plasmodium species.
Introduction species, namely P. falciparum and P. vivax. Nevertheless,
Between 2000 and 2015, the estimated number of mal- in sSA, most of the population lack the Duffy antigen,
aria cases and malaria deaths worldwide had declined which is required for red blood cell invasion by P. vivax;
by 37% and 60%, respectively, with an estimated 6.2 therefore P. vivax is the least prevalent species, while
million deaths prevented (WHO 2015a). Within the other minor non-falciparum malaria species can be
same period, 17 countries were declared malaria-free or found - P. malariae, P. ovale wallikeri and P. ovale curtisi
had zero indigenous cases (WHO 2007). Nevertheless, (WHO 2007). P. ovale species are only distinguished
malaria elimination, particularly in sub-Saharan Africa where possible in this review due to insufficient data
(sSA), continue to face unique challenges discussed on classification from earlier studies.
extensively elsewhere (Tatem et al. 2010; Rabinovich Amongst these minor species, P. malariae is the
et al. 2017; Shahandeh and Basseri 2019). Indeed, the most common (WHO 2016). However, the true burden
global decline of the malaria burden has currently of these non-falciparum species and the sub popula-
stalled and though there were an estimated 23 million tions most at risk in sSA is not known given the
fewer malaria cases in 2018 than in 2010, no significant absence of routine clinical diagnosis and systematic col-
progress has been achieved since 2014 (WHO 2019). lection of data on these species. It is possible that the
With the current trend, the Global Technical Strategy decline of P. falciparum prevalence in sSA may provide
for Malaria 2016–2030 may not attain the global target a favourable ecological niche for P. malariae expansion.
of reducing malaria by at least 90% (compared with This was recently observed in Tanzania (Yman et al.
2015) of malaria cases and deaths (WHO 2016). Global 2019), where P. malariae has become more common in
targets and interventions to achieve them have been areas of low P. falciparum prevalence after successful
conceived mainly to tackle the two dominant malaria interventions. This phenomenon could become more
CONTACT Alfred Amambua-Ngwa angwa@mrc.gm Medical Research Council Unit The Gambia at LSHTM, Atlantic road, Fajara, P.O.Box 273, Banjul,
The Gambia

 2021 Informa UK Limited, trading as Taylor & Francis Group
CRITICAL REVIEWS IN MICROBIOLOGY 45
common, with the species gaining more public health genomic epidemiological data and improving the
importance. Such an occurrence would mirror findings understanding of P. malariae parasite evolutionary biol-
on P. knowlesi, a zoonotic malaria species in Malaysia, ogy, transmission patterns and signatures of selection.
which is now an important cause of human malaria fol-
lowing the significant decline of P. falciparum and P. Historical or evolutionary background and
vivax to pre-elimination levels (Cooper et al. 2019; life cycle
Fornace et al. 2019).
In areas of seasonal transmission such as The The life history of malaria as a parasitic disease followed
Gambia, P. malariae has been found to be responsible a series of discoveries in the 19th century - the associ-
for about half of the malaria episodes observed outside ation of a previously described black pigment in
the transmission season (Greenwood et al. 1987). This is internal organs of individuals that died of malaria, sepa-
possibly due to its suppression by P. falciparum during rating malaria from other fevers, isolation of the active
peak malaria transmission periods (Mueller et al. 2007) antimalarial component – quinine, and description of
or by other ecological and entomological factors the malaria parasite by Laveran with differential stain-
(Ousmane et al. 2012); observed in Nigeria and Mali, ing to identify blood stage parasites (Coatney et al.
respectively. Comparable data on temporal and spatial 1971; Cox 2010).
variations in P. malariae epidemiology across most sSA The origin and evolutionary history of human
is scarce, particularly with reference to changes and dif- Plasmodium species remain controversial. Comparison
ferences in antimalarial treatment policies. The few of 18S ribosomal DNA gene sequences suggest that the
studies and reports available suggest that P. malariae is divergence of P. falciparum from its only known close
more prevalent in children, mostly occurring in co- relative P. reichenowi, a parasite of chimpanzees, ran
infections with high P. falciparum parasite densities and parallel to the divergence of hominids from chimpan-
seasonal alternation in prevalence with P. falciparum zees, approximately 5–11 million years ago, while the
(Molineaux et al. 1980; Trape et al. 1994; Browne et al. other three human malaria parasite species (P. vivax, P.
2000). The implications of this dynamics are poorly malariae, P. ovale) were lateral transfers from other pri-
understood in terms of sustained transmission, acquisi- mates to humans, probably first encountering the
tion of antimalarial drug resistance and malaria evolving hominids when Homo erectus spread out from
elimination. Africa across Asia approximately one and two million
More accurate information on the P. malariae epi- years ago (Sallares 2002; Sallares et al. 2004). However,
demiology and burden is confounded by the frequent more recent analysis using single genome amplification
occurrence of co-infections with P. falciparum, limiting (SGA) of specimens from a larger collection of apes,
the knowledge on mono infections by this parasite spe- indicate P. falciparum to be of gorilla origin and all
cies (McKenzie and Bossert 1999). Within-host competi- known human strains may have resulted from a single
tion for mixed P. falciparum strains has been reported cross-species transmission event (Liu et al. 2010).
to play a key role in the evolution of drug resistant mal- Older phylogenetic analysis showed the three non-
aria, and may thus impact on management efforts falciparum species of human malaria parasite to fall
(Bushman et al. 2016). It is possible that within-host within a single clade that includes all other mammalian
competition may also impact on the growth, develop- malaria parasites, apart from P. falciparum and P. reiche-
ment, survival and fitness of P. malariae in mixed-spe- nowi. Within this clade, all the primate malaria parasites
cies infections, as P. falciparum multiplies at a faster belonged to one of the lineages represented by P.
rate than P. malariae. With recent advances in next-gen- malariae, P. ovale, or P. vivax, which appear to have
eration sequencing (NGS) technologies, it is now prac- diverged over 100 million years ago, long before the
tically possible to carry out deep sequencing to field emergence of the lines leading to the distinct mamma-
samples and use bioinformatic approaches to perform lian orders of today (Carter and Mendis 2002). More
discriminatory analysis of mixed parasite genomes to recent whole genome sequencing data using a compre-
gain a deeper understanding of the prevalence of these hensive amino acid alignment, however, showed strong
minor species. With the deployment of portable NGS contrasting evidence of P. malariae forming an out-
devices such as the Oxford nanopore platform, detec- group both to rodent-infective species and to a pri-
tion of non-falciparum co-infecting species stands to mate-infective clade that includes P. vivax (Rutledge
improve in sSA. This review summarises current know- et al. 2017a).
ledge on P. malariae in sSA, highlights the knowledge Morphological characteristic features of P. malariae
gaps and explores NGS approaches for generating are the jet-black granular pigment formed in the ring
46 E. C. ORIERO ET AL.
Figure 1. Life cycle of the malaria parasite highlighting differences between P. malariae and other Plasmodium species. Modified
from “Life cycle of the malaria parasite” in Epidemiology of Infectious Diseases. Available at: http://ocw.jhsph.edu. Copyright #
Johns Hopkins Bloomberg School of Public Health. Creative Commons BY-NC-SA.
stages as the parasite grows and stretches across the 1998; Hedelius et al. 2011). Infections with P. malariae
host cell into the band form, without changing the size are considered relatively benign, mostly with low para-
of the host cell (Collins and Jeffery 2007). The life cycle site densities, but chronic infections may cause serious
and presentation of P. malariae is similar to other health problems which may be fatal (Mueller et al.
human Plasmodium species, with the exception of (a) 2007). These clinical manifestations have been seen in
the longer asexual blood stage developmental cycle Africa and Asia, where P. malariae is most prevalent.
also called quartan life cycle (72 h) characterized by a Notable severe clinical outcomes of P. malariae infec-
similar periodicity of chill and fever patterns (paroxysm), tion include chronic nephrotic syndrome, possibly due
(b) the wide range in the duration of the prepatent to immune complex deposition in the glomeruli (Ehrich
period in mosquito transmitted P. malariae (16 to and Eke 2007; Hedelius et al. 2011), and anaemia
59 days), (c) its affinity for older red blood cells (d) the (Langford et al. 2015). In Papua, Indonesia, P. malariae
absence of the dormant hypnozoite stage that is char- mono-infection represented 2.6% of all malaria cases
acteristic of parasite species that cause relapse post reported at Mitra Masyarakat Hospital. These patients
treatment of blood stages and (e) the fewer number of tended to have a lower mean haemoglobin concentra-
merozoites produced in the erythrocytic cycle (Figure tion than patients with P. falciparum, P. vivax and mixed
species infections. Overall, 0.3% of patients with P.
1). A combination of all these factors may be respon-
malariae malaria died within the study period, April
sible for development of immunity to P. malariae infec-
2004 to December 2013 (Langford et al. 2015). In
tions by the human host (Collins and Jeffery 2007).
Dielmo (Senegal), a longitudinal study between 1990
and 2010 found a relatively high prevalence of P. malar-
Disease presentation and case management iae and P. ovale among asymptomatic individuals until
Pathogenesis and morbidity 2004 (Roucher et al. 2014). Following the scale-up of
interventions, the prevalence of all malaria declined,
P. malariae causes chronic infections that may last for with P. malariae and P. ovale together associated with
decades and may recur long time after the initial expos- 5.9% of the malaria burden. More information on infec-
ure. As no dormant liver stages (hypnozoites) have tion and associated clinical manifestation may emerge
been reported for this species, the mechanisms for from recent experimental human blood-stage model
these recurrent infections remain unclear (Vinetz et al. for P. malariae infection (John et al. 2019). With two
CRITICAL REVIEWS IN MICROBIOLOGY 47
volunteers, the model resulted in a safe and reprodu- Diagnosis and prevalence
cible course of infection that was well tolerated, with
Accurate diagnostic tools are needed for precise report-
no renal impairment or recurrent infection three
ing of incidence and prevalence of malaria parasite
months post-treatment (John et al. 2019). In addition, infections. Three approaches are commonly applied,
the experimental infection could be transmitted to the namely thick and thin blood smear microscopy, rapid
vector. The availability of such model would allow diagnostic tests (RDTs) detecting circulating malaria
investigating the parasite biology, and the develop- antigens, and molecular methods detecting parasite’s
ment of P. malariae-specific exposure and infection bio- nucleic acids, for example, PCR. For P. falciparum,
markers. The need for these biomarkers and the poor microscopy is less sensitive than PCR and this probably
performance of current pan-genus lactate dehydrogen- applies to the other Plasmodium species (Daniels et al.
ase enzyme (pLDH) were evident as they remained 2017; Lo et al. 2017; Roman et al. 2018; Schindler et al.
negative despite the presence of symptoms consistent 2019a). P. malariae and P. ovale are not routinely
with malaria 72 h prior to testing (John et al. 2019). detected in health facilities although available RDTs can
also identify non-falciparum infections with limited sen-
sitivity (Gunasekera et al. 2018), reviewed extensively
P. malariae in pregnancy
elsewhere (Abba et al. 2014; Yerlikaya et al. 2018).
Unlike P. falciparum infections, P. malariae infections in Highly sensitive molecular diagnostic assays, which are
population groups such as pregnant women have mainly used in medical research settings have been
much less of a clear clinical outcome. There is much shown to be the most reliable method for detection of
less information on the burden of P. malariae and P. P. malariae and other non-falciparum species
ovale during pregnancy, whereas consequences of P. (Hayashida et al. 2017; Srisutham et al. 2017). The limit
vivax on the outcome of pregnancy have been of detection of most molecular tests for non-falciparum
described in Asia and Latin America (Nosten et al. 1999; species as well as their sensitivity and specificity have
ter Kuile and Rogerson 2008). Most available data in not been clearly defined. However, a new in-house
sub-Saharan Africa (sSA) is on P. falciparum, showing molecular test based on droplet digital PCR (ddPCR)
that pregnant women are at higher risk of malaria, par- and another based on loop-mediated isothermal ampli-
ticularly primigravidae in areas of moderate to intense fication (LAMP) technology, showed excellent sensitivity
transmission, with severe impact on pregnancy out- and specificity for detection of P. malariae and P. ovale
comes (Brabin 1983; De Beaudrap et al. 2013, 2016; (Kollenda et al. 2018; Srisutham et al. 2017). Validated
Walker et al. 2017). While about 1.4% of pregnant sensitive diagnostic approaches applied in quantitative
women were found to harbour non-falciparum infec- real-time PCR commercial kits such as, RealStarV
R
tions across four West African countries (Burkina Faso, Malaria PCR Kits, Altona Diagnostics, Germany;
VR
The Gambia, Ghana and Mali), there was no effect on Genesig Plasmodium species kits, Primerdesign
TM Ltd,
UK; and artus Malaria RG PCR Kit CE, Qiagen, Germany,
pregnancy or outcome (Williams et al. 2016). However,
can be further developed into assays such as high-reso-
another study in Benin reported an association
lution melt (HRM) and LAMP for high-throughput field-
between non-falciparum infections (9.2% P. malariae,
based screening of non-falciparum parasite species
5.7% P. ovale) and adverse pregnancy outcomes such
(Steenkeste et al. 2009; Hayashida et al. 2017; Kassaza
as premature delivery (Doritchamou et al. 2018). In this et al. 2018). Alternatively, serology markers to deter-
study, the prevalence of P. malariae in peripheral blood mine exposure to P. malariae have been explored in
(1%) at delivery was much lower compared to the three different studies, identifying 67.5% of P. malariae-
prevalence in placental blood (5.5%), suggesting a specific antibodies to a recombinant merozoite surface
potential affinity of P. malariae for the placenta protein (MSP-1) antigen among healthy blood donors
(Doritchamou et al. 2018). The mechanisms that under- in Benin; 0–3% as P. malariae single species responses
lie P. malariae gravitation towards placental blood and 8–29% as multiple species responses to a recom-
remain unclear, and the role of chondroitin sulphate binant P. malariae MSP-119 antigen in Zimbabwe; and
moities in this pathology needs to be investigated. P. 46
 56% P. malariae seropositivity to a recombinant
falciparum–infected erythrocytes bind to chondroitin MSP-119 antigen in a baseline survey to evaluate the
sulphate A (CSA) in the placenta, leading to life-threat- impact of control measures on the burden of multiple
ening malaria in pregnant women with severe effects diseases in two districts in Mozambique (Doderer-Lang
on their foetuses and newborns (Singh et al. 2008; et al. 2014; Amanfo et al. 2016; Plucinski et al. 2018).
Khunrae and Higgins 2010). Though species-specific recombinant proteins were
48 E. C. ORIERO ET AL.
used, cross-reactivity between species was seldom rates have been reported for P. malariae in some sSA
reported, and this could be a limitation to the settings (Mombo-Ngoma et al. 2012; Groger et al.
responses and predicted prevalence observed. The 2018). A systematic review summarising data on the
unknown half-life of MSP-1 specific antibodies in P. efficacy and safety of artemisinin combination therapy
malariae infections can also be another drawback of (ACT) for the treatment of non-falciparum malaria was
serological tests for this parasite species. published in 2014 (Visser et al. 2014). The authors iden-
Reports on prevalence of P. malariae in sSA are tified 986 records, out of which only 40 (P. vivax
 35; P.
patchy, showing a wide range (0-32%) in different pop- ovale, P. malariae and P. knowlesi
 5) clinical trials were
ulations, using different detection techniques found eligible for inclusion and only three of these trials
(Borrmann et al. 2002; Baltzell et al. 2013; Noland et al. were done in sub-Saharan Africa. One of the trials done
2014; Sitali et al. 2015; Doctor et al. 2016; Roh et al. in sSA was a systematic assessment of artemether-
2016; Lo et al., 2017; Oboh et al. 2018; Asua et al. 2017; lumefantrine administered for P. malariae, P. ovale and
Daniels et al. 2017; Roman et al. 2018; Schindler et al. mixed-species malaria in Gabon and reported a cure
2019b; Williams et al. 2016) probably because P. malar- rate of 100% with favourable tolerability profile
iae and other non-falciparum parasite species are not (Mombo-Ngoma et al. 2012). However, recent findings
considered to be a significant public health challenge. from a prospective clinical trial assessing species-spe-
Several factors such as the low number of merozoites cific efficacy of artemether-lumefantrine for the treat-
produced per erythrocytic cycle, the extended 72-h ment of non-falciparum malaria parasite species in
asexual blood stage developmental cycle, the prefer- Gabon reported PCR-corrected adequate clinical and
ence to develop in older erythrocytes, possible seques- parasitological response (ACPR) rates of 95.5% for P. fal-
tration or suppression in mixed infections with P. ciparum, 100% for P. malariae, 100% for P. ovale curtisi,
falciparum are thought to account for the poor detec- and 90.9% for P. ovale wallikeri (Groger et al. 2018), sug-
tion and underestimation of P. malariae (Collins and gesting that identification and treatment of different
Jeffery 2007; Mueller et al. 2007). Future approaches parasite species might be necessary.
can explore molecular characterization and develop- Critical challenges to treatment of P. malariae infec-
ment of non-falciparum-specific serological markers, tions in clinical practice is due to lack of appropriate
which would allow for large surveys and monitoring diagnosis tools as most infections occur at densities
the effects of antimalarial treatments and other inter- below detection threshold of the commonly used diag-
ventions on exposure and transmission of these species nostic tools, RDT and microscopy (Mayxay et al. 2004).
in different endemic settings. Nevertheless, the Good Practice Statement of the WHO
reports that “if the malaria species is not known with cer-
tainty, treat as for uncomplicated P. falciparum malaria”
Treatment (WHO 2015b). This however falls short of addressing
Chemotherapy remains one of the major tools used in recommendations for prophylactic treatment such as
malaria control, intervention and elimination strategies. drug combinations used in seasonal malaria chemopre-
The current and historical antimalarial drug regimens vention in some African settings. The efficacy of these
for treatment of P. malariae infections are summarised against P. malariae especially in mixed infection needs
briefly. Overall, P. malariae seems susceptible to quino- to be evaluated. The elimination half-life of artemisinins
lines and the World Health Organisation (WHO) recom- range from 0.4
 2.6 h, while that of sulphadoxine-pyri-
mends treatment of malaria due to P. malariae infection methamine (SP) range from 4
 8 days, mefloquine
with chloroquine (WHO 2015b). Studies on the efficacy from 14
 21 days and half-life of quinolines range from
of various quinoline treatment of P. malariae in Asia 4 days 
 2months (Li and Hickman 2015). Both the
and sSA are rare (Myint et al. 2004; Visser et al. 2014). short half-life of artemisinins and prolonged ones for
Two other studies reported efficacious treatment with partner drugs may have sub-therapeutic implications
chloroquine and pyronaridine in Madagascar and for P. malariae, with respect to its longer life cycle
Cameroon respectively (Barnadas et al. 2007; Ringwald (Rutledge et al. 2017b).
et al. 1997). Beyond these are case reports of treatment
of imported P. malariae from Africa with quinine and Phenotypes and parasite culture
chloroquine (Hong et al. 2012; Brouwer et al. 2013).
Antimalarial resistant phenotypes
Treatment of asymptomatic P. malariae with artesunate
has also been shown to be efficacious, with 100% cure Drug resistance in P. malariae is not widely assessed,
rates by day 7 (Borrmann et al. 2002). High ACT cure though there has been a single reported case of
CRITICAL REVIEWS IN MICROBIOLOGY 49
chloroquine resistance; a prospective 28-day in vivo Johns 1912). More recently, short-term in vitro culture
assessment of the efficacy of chloroquine for treatment of P. malariae erythrocytic stages has been achieved for
of P. malariae reported two cases of persistent parasit- 6 days using RPMI 1640 medium supplemented with
aemia at day 8 and one recurrent parasitemia at day 28, glutamine, hypoxanthine, and 20% human serum
in spite of effective concomitant drug concentration in (Lingnau et al. 1994). Exoerythrocytic stages of P. malar-
the blood (Maguire et al. 2002). Recurrence and recru- iae have also been cultured in vitro using hepatocytes
descence of P. malariae infections has been seen after obtained from liver biopsies of non-human primates
treatment with several antimalarial drugs (chloroquine/ inoculated with sporozoites from mosquito salivary
hydroxychloroquine, atovaquone/proguanil, quinine) glands (Millet et al. 1988).
and broad-spectrum antibiotics. Recurrence has been Varying in vitro culture conditions for other
seen sometimes even decades after the last possible Plasmodium species can be used to generate more
exposure to malaria infections (Kugasia et al. 2014; Teo information on biological processes that enable parasite
et al. 2015; Visser et al. 2016; Grande et al. 2019). adaptation and survival in the different hosts, as
Persistent occurrence of P. malariae infections after observed in a recent in vitro phenomenon reported for
treatment with ACTs in Ghana, Uganda, and in an P. falciparum (Awandare et al. 2018). Altering the para-
imported case, calls for increased vigilance (Dinko et al. site culture conditions by gentle shaking resulted in a
2013; Betson et al. 2014; Rutledge et al. 2017b). The lon- spontaneous and progressive switch of invasion pheno-
ger erythrocytic cycle of P. malariae raises the possibil- types. In line with current in vitro optimization techni-
ity of longer exposure in the blood to antimalarial ques, some of the modifications are being explored for
drugs, including ACTs and possibly selecting drug the culture of P. vivax such as use of different types of
resistant strains. As P. falciparum incidence declines and culture media and supplement combinations, parasite
a favourable ecological niche could be available, such source and target cell sources etc., as reviewed by
selected strains of P. malariae may potentially become Bermu
dez and colleagues (Bermu
dez et al. 2018).
more common and more relevant to public health. New These, including techniques for successful culture of dif-
molecular detection and genotyping assays for P. ferent parasite stages of simian, rodent and avian mal-
malariae are needed to assess any dormant liver-stage aria parasites (Trigg 1985; Schuster 2002) can be
forms of P. malariae and ascertain whether P. malariae considered for in vitro culture of P. malariae.
infections observed after treatment are recrudescent or
new infections. Comparing single nucleotide polymor- Molecular and genomic epidemiology
phisms (SNPs) from whole genome sequence data in
one of the recently reported recurrent cases, the Genome size and base composition
authors reported that the initial infection was poly- The current P. malariae reference genome as in other
clonal and the persistent isolate was a single clone pre- plasmodia is organized into 14 chromosomes and has a
sent at low density in the initial infection (Rutledge total size of 33.6 megabase (Mb). The reference genome
et al. 2017b). Thus, NGS techniques can be used to was constructed from clinical isolates sequenced on
identify markers for molecular genotyping that could Pacific BioSciences long-read sequencer (Rutledge et al.
discriminate between recrudescent and new P. malariae 2017a). The genomes of mammalian Plasmodium spe-
infections, enabling early identification and warning of cies are very similar, however, differences in nucleotide
potential cases of suspected antimalarial resistance bias can be observed. For example, P. vivax and P. fal-
across populations. ciparum have average GC content of approx. 42.3% and
19.4%, respectively (Carlton et al. 2008). Like P. falcip-
arum, the nuclear genome of P. malariae is AT-rich (GC
In vitro culture and invasion
content of approx. 25%) but unevenly distributed
The continuous in vitro culture of malaria parasites, between protein coding and non-coding regions
which was standardised in the 1970s (Trager 1971; (Ansari et al. 2016). Likewise, P. malariae has two extra-
Haynes et al. 1976; Trager and Jensen 1976), has been nuclear DNA; the mitochondrial genome with a size of
instrumental in studying the most deadly species, P. fal- 5.9 kb, containing 12 genes and the apicoplast genome
ciparum. A single study reported successful in vitro cul- with a size of 34.3 kb, containing 55 genes (www.plas-
ture of P. malariae together with P. falciparum and P. modb.org). From rodent Plasmodium species, the mito-
vivax, but further experiments were interrupted to focus chondrial genome codes for only three proteins
on factors affecting Plasmodia in vitro culture, and only (cytochrome b and two subunits of cytochrome oxi-
P. falciparum and P. vivax was taken forward (Bass and dase) as well as two fragmented rRNAs (Otto et al.
50 E. C. ORIERO ET AL.
2014). The circular plastid genome (apicoplast) has a Genetic diversity and population structure
similar origin to the chloroplast of green plants,
The population structure of different malaria parasite
although non photosynthetic. Key functions and meta- species may be driven by geographic isolation, human
bolic processes of the apicoplast such as biosyntheses migration, malaria transmission intensity, and selective
of fatty acids, isoprenoids, iron-sulphur cluster and pressure from drug and vector interventions (Conway
haem are reviewed elsewhere for P. falciparum (Lim and 2007). Unlike P. falciparum, SNP and microsatellite
McFadden 2010). The completed P. malariae reference markers for determining P. malariae structure are few,
genome will facilitate comparative genomics studies with only a limited number of studies done in sSA.
with other Plasmodia and apicomplexan parasites as Using four microsatellites and two minisatellites, early
well as population genetics of the species. studies showed higher diversity in African P. malariae
populations than in those from South America and Asia
Antigenic variation (Bruce et al. 2007). In Malawi, up to 73.2% P. malariae
infections were multiclonal, with multiple genotypes
The alteration of parasite-encoded antigens (variant sur- detected per infection by microsatellite typing.
face antigens) exposed on the surface of infected red However, structure analysis did not reveal significant
blood cells through recombination and expression differentiation or clustering between symptomatic and
switching is well known for parasitic protozoans asymptomatic or between the different study villages
(Kirkman and Deitsch 2012; Recker et al. 2011). In (Bruce et al. 2007, 2011).
Plasmodium species, this occurs in hypervariable multi- The genetic diversity of P. malariae has also been
gene families implicated in virulence and evasion of recently explored with DNA sequencing approaches.
antibody-mediated host immunity by blood stages Sequences of small sub-Unit RNA (SSU rRNA) of P.
(Witmer et al. 2012). These multigene families are often malariae extracted from just four isolates from
located near the telomeres of which the largest family Bangladesh identified three haplotypes, indicating a
is the Plasmodium interspersed repeat – pir genes, a dis- high diversity of P. malariae in that region (Fuehrer
tinctive feature of many Plasmodium species. et al. 2014). Sequences of circumsporozoite surface pro-
Approximately 40% of P. malariae genome is sub-telo- tein (csp) gene from P. malariae in asymptomatic and
meric and contains only a restricted subset of pir gene symptomatic patients from Western Kenya also
repertoires compared to P. ovale and P. vivax (Rutledge revealed high genetic diversity but no clear differenti-
et al. 2017a). The pir gene products are predicted to be ation between geographically separated populations
exported to the infected erythrocyte surface, and lack (Lo et al. 2017). Genetic variations in three antigenic
typical signal peptide sequences and PEXEL/HT erythro- proteins of P. malariae - thrombospondin-related
cyte trafficking motifs (Ansari et al. 2016). About 50% of anonymous protein (TRAP), apical membrane antigen 1
(AMA1), and 6-cysteine protein (P48/45) were assessed
pir genes in P. malariae are pseudogenes (compared to
by analysing diversity of samples collected in Asia at
25% in P. ovale curtisi and 9% in P. vivax), suggesting an
nucleotide and protein levels. This study reported high
even smaller functional repertoire (Rutledge et al.
genetic diversity in P. malariae trap and ama1 com-
2017a). Two other sub-telomeric large gene families
pared to p48/45 (Srisutham et al. 2018). These studies
(fam-l and fam-m), which codes for proteins that are
suggest high diversity in this malaria parasite species
likely exported from the parasite to the infected red and frequent recombination and gene flow between
blood cell surface are observed in P. malariae. Structural populations. With improved sequencing technologies
studies of these proteins, predict a high confidence requiring much less genomic DNA and reduced cost,
overlap with the RH5 protein of P. falciparum, despite more P. malariae samples within and across endemic
having only 10% sequence similarity (Rutledge et al. populations can now be sequenced. This will elucidate
2017a). So far, RH5 is the only known P. falciparum pro- the complexity of infection, dynamics with other spe-
tein that is essential for erythrocyte invasion, through cies and the structure of P. malariae populations.
binding to basigin on the erythrocyte surface (Crosnier Knowledge of the parasite’s population structure will
et al. 2011). Further characterisation of P. malariae sub- further the understanding of its transmission dynamics,
telomeric gene families could provide better under- help the design of interventions strategies, and better
standing of the parasite’s contribution to malaria trans- understanding of distribution of candidate drug resist-
mission in endemic areas and shed more light on the ance markers. With the completion of the P. malariae
reoccurrence of infection after long periods of non- genome, targeted amplicon deep sequencing (TADS) is
exposure without dormant liver stages. now possible towards deeper insights into the genetic
CRITICAL REVIEWS IN MICROBIOLOGY 51
diversity and structure of this parasite species. multigene families were reported from whole-genome
Furthermore, the design of a unique SNP barcode sequence data, which distinguishes different parasite
should provide additional benefits for tracking parasites species (Carlton and Steven 2017). A more accurate
and gene flow across different transmission sources characterisation of the parasite species within the
and sinks. Plasmodium phylogeny is now emerging (Rutledge
et al. 2017a). The P. malariae genome appears to be
indistinguishable from sequences of P. brasilianum, a
Polymorphism in orthologues of antimalarial drug
Plasmodium species found in New World primates
resistance genes
(Rutledge et al. 2017a; Talundzic et al. 2017). This sug-
Drugs have been one of the strongest selection forces gest that P. malariae in humans may be due to a very
on the genome of P. falciparum and this is probably recent zoonotic transmission event and it is possible
true also for other malaria parasite species including P. that zoonotic cycles in current populations may be
malariae. The genetic diversity of P. malariae ortho- occurring. Population genomics studies in sympatric
logues of markers of drug resistance in P. falciparum population with primate hosts of P. brasilianum will
have been assessed in a limited number of populations. help validate this hypothesis. These genomes could be
The genetic diversity of sulfadoxine-pyrimethamine (SP) mined for markers of hosts preference and cell invasion
resistance genes (i.e., dhfr and dhps) were assessed in P. that would shed light on any possible mechanisms of P.
malariae samples collected from sSA and Asia, in areas malariae zoonosis (Carlton and Steven 2017). At the
with different history of SP usage (Tanomsing et al. moment, genomic epidemiology studies of P. malariae
2007; Khim et al. 2012; Tanomsing et al. 2014). remains complicated by usually low parasites densities
Although the African isolates were classified as mostly and predominance of mixed infection with other
wild type in these studies, new non-synonymous muta- human species. Therefore, enrichment approaches
tions were observed in polymorphic sites of the dhfr need to be developed such as species-specific primers
gene (K55E, S58R, S59A, F168S, N194S, D207G, and for selective whole genome amplification (sWGA) or
T221A), leading to the description of six new DHFR hybrid selection methods as previously applied for P.
resistant alleles (Khim et al. 2012). From recent P. malar- falciparum and P. vivax (Oyola et al. 2016; Cowell
iae whole genome data, four synonymous and seven et al. 2017).
non-synonymous SNPs were reported in some drug
resistance genes (sSNPs – multidrug resistance protein
Research gaps and future perspectives
1, P-type ATPase 4 and Bifunctional dihydrofolate
reductase- thymidylate; nsSNPs – multidrug resistance P. malariae is the most common co-infecting species in
protein 2, ABC transporter C family member 2 and P. falciparum and P. vivax endemic populations in sSA,
Bifunctional dihydrofolate reductase- thymidylate) yet it’s biology and epidemiology vis-a-vis interventions
(Rutledge et al. 2017b). Considering non species-spe- and malaria elimination has not received adequate sci-
cific antimalarial treatment history and the use of qui- entific attention. It is closely related to P. brasilianum, a
nolines as partner drugs in current ACTs, orthologues of species found in New World monkeys and also reported
major markers of quinoline resistance (Pfcrt and Pfmdr1) in humans in South America (Lalremruata et al. 2015).
may be under selection. Studies to further evaluate Both species result in morbidity and are therefore minor
these and candidates of resistance to artemisinin deriv- contributors to malaria; but like P. knowlesi could
atives in P. malariae would be informative. In vitro test- become more significant as major human malaria para-
ing and whole-genome sequencing could further site species are eliminated. There are several knowledge
enable, as demonstrated for P. falciparum, the identifi- gaps on the biology and epidemiology of P. malariae in
cation of novel markers by association (Miotto endemic settings that need to be addressed, including:
et al. 2013).
 The burden of P. malariae: Better estimates of P.
malariae infection incidence, prevalence and associ-
Post next-generation sequencing (NGS) genomic
ated malaria would require better diagnostic tools.
epidemiology of P. malariae
Sub-microscopic malaria infections of any parasite
The completion of a reference genome for P. malariae species constitute a reservoir of infection that con-
paves the way for new genomic studies aimed at tributes to transmission and poses a significant
understanding it’s biology and diversity in natural pop- challenge to achieving elimination goals. Therefore,
ulations (Auburn and Barry 2017). Clear differences in efforts should be made to encourage routine
52 E. C. ORIERO ET AL.
diagnosis and reporting of specific non-falciparum biology. Extended to P. malariae across sSA and other
species in endemic settings, especially in sSA. endemic regions, these studies would offer new
 Drug efficacy: Drugs remain a major tool for malaria insights into its genomic epidemiology and population
intervention. While P. malariae is considered to be genetics, impact of interventions such as antimalarial
mostly susceptible to current antimalarial drugs, drugs (or vaccines) on local and regional parasite vari-
assessment of in-vivo efficacy of current and candi- ation. Next-generation sequencing approaches have
date drugs are needed. Though no markers of shown promise as useful tools for monitoring P. falcip-
resistance are known, the dynamics of orthologues arum populations and should be applied to minor spe-
of P. falciparum markers across endemic popula- cies such as P. malariae and P. ovale as we push
tions will serve as surrogate data for monitoring towards sustainable malaria elimination.
resistance in P. malariae. Efficacy studies and more
knowledge of the repertoire and effect of existing Acknowledgments
and new polymorphisms on drug sensitivity will be
beneficial towards drug-based malaria elimin- The authors acknowledge facilitators of the DELGEME
ation approaches. Summer Course, especially Dr. Amed Ouattara, for support in
 ensuring timely preparation of the manuscript as well asP. malariae and P. brasilianum genomes are highly Profs Karine Le Roch and Umberto D’Alessandro for review-
similar yet it is not known if P. malariae is endemic ing the manuscript.
in non-human primates. Studies are required to
identify any existence of a zoonotic reservoir which
will potentially threaten future elimination efforts of Disclosure statement
this species. The authors have no conflicts of interest to declare.
 Long-term in vitro culture of P. falciparum has pro-
vided a lot of information on the biology, pheno- Funding
types and genotypes of the parasite species. So far,
in vitro culture of all stages of P. malariae remains This work was supported through the DELTAS Africa
Initiative [DELGEME grant 107740/Z/15/Z]. The DELTAS Africa
a challenge.
 Initiative is an independent funding scheme of the AfricanProper mechanisms to study the very important Academy of Sciences (AAS)’s Alliance for Accelerating
biological process of erythrocyte invasion for P. Excellence in Science in Africa (AESA) and supported by the
malariae and other non-falciparum species need to New Partnership for Africa’s Development Planning and
be developed. Key proteins in the different stages Coordinating Agency (NEPAD Agency) with funding from the
of the parasite life cycle, metabolic processes and Wellcome Trust [DELGEME grant 107740/Z/15/Z] and the UK
government. The views expressed in this publication are
erythrocyte invasion could be targeted for vaccine, those of the author(s) and not necessarily those of AAS,
drug development and diagnosis. NEPAD Agency, Wellcome Trust or the UK government.
 P. malariae is not known to have dormant hypno-
zoite stages but can resurface after long periods
away from malaria endemic regions. Understanding
ORCID
the mechanisms and processes involved in this
phenomenon can help identify specific targets for Lucas Amenga-Etego http://orcid.org/0000-0003-
new intervention tools. 4468-0506
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