Epidemiological, Physiological and Diagnostic Comparison of Plasmodium ovale curtisi and Plasmodium ovale wallikeri

Nowadays, Plasmodium ovale is divided into two non-recombinant sympatric species: Plasmodium ovale wallikeri and Plasmodium ovale curtisi. In this mini review, we summarize the available knowledge on the clinical/biological aspects of P. ovale spp. malaria and current techniques for the diagnosis/characterisation of P. ovale curtisi and P. ovale wallikeri. P. ovale wallikeri infections are characterized by a deeper thrombocytopenia and shorter latency compared to P. ovale curtisi infections, indicating that P. ovale wallikeri is more pathogenic than P. ovale curtisi. Rapid diagnosis for effective management is difficult for P. ovale spp., since specific rapid diagnostic tests are not available and microscopic diagnosis, which is recognized as the gold standard, requires expert microscopists to differentiate P. ovale spp. from other Plasmodium species. Neglect in addressing these issues in the prevalence of P. ovale spp. represents the existing gap in the fight against malaria.


Introduction
Malaria is a tropical and subtropical infectious disease caused by protozoa of the genus Plasmodium and responsible for 409,000 deaths worldwide in the year 2019 [1]. Among human Plasmodium spp., P. falciparum and P. vivax are the most prevalent species in Africa and the rest of the world, respectively [1]. These two species are involved in the majority of cases of disease severity and therefore the most studied, whereas two other non-falciparum species, viz. P. malariae and P. ovale spp., are relatively less pathogenic, coupled with a low prevalence rate [2,3]. A steady rise in infection due to P. ovale spp. has been observed in several regions of sub-Saharan Africa (sSA) [4][5][6]. Recently, a systematic review highlighted the involvement of P. ovale spp. in severe malaria, which is the same as that of P. vivax and thus deserves as much attention as the other species [2]. P. ovale spp., having two sympatric species, Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow), is the last plasmodial species described in 1922 by Stephens, who named it for the oval shape of some infected erythrocytes [7] commonly found in Africa, Southeast Asia and Papua New Guinea [8,9]. Many epidemiological studies conducted till date do not specifically target either of these two species, though a few have reported on the emergence of Poc and Pow and the evolution of their involvement in severe malaria [2,10,11]. In addition, the available specific screening protocols for these two species are almost non-existent in health centre laboratories for routine malaria diagnosis.
In the year 2014, Fuehrer and Noedl published a review presenting the techniques used for the phylogenetic classification of Plasmodium species based on molecular markers and diagnostic techniques of P. ovale spp. [12]. Three years later, Zaw and Lin presented a more extensive review adding geographical distribution, molecular discrimination techniques and clinical differences between the Poc and Pow species [13]. In the previous two reviews, no information on the performance of the rapid diagnostic test (RDT) relevant for P. ovale spp. was mentioned. The present mini review focusses on two important aspects: (i) a brief comparison of Poc and Pow on clinical and biological aspects; (ii) an overview of the currently available techniques for diagnosis and molecular markers for the genetic characterisation of Poc and Pow, summarizing the available knowledge with respect to these two species.

Ecological and Biological Mechanisms behind the Distinction of Poc and Pow
Since the description of P. ovale spp. by Stephens in 1922, the two species were considered a single species, until 2010, when Sutherland described Poc and Pow as two sympatric species endemic in Africa [7,14]. Poc and Pow are now recognized as two non-recombinant species that diverged about two million years ago and that remain distinct due to a strong genetic barrier [15][16][17]. Three principal hypotheses have been formulated by Sutherland to explain the Poc and Pow segregation [14]. Although an ecological and/or seasonal differential distribution serving as a physical boundary between the two species was first suggested, current knowledge shows that these two species are sympatric and share the same geographical locations [13]. A second hypothesis stipulates host specificity, which is being contradicted by studies that have reported Poc and Pow co-infection [18,19]. The most plausible hypothesis till date is of the genetic barrier, i.e., there is an incompatibility between Poc and Pow gametocytes or the sporozoites obtained from cross-fertilisation between them as they are devoid of the ability to infect [14]. This last hypothesis needs to be thoroughly investigated in all aspects to understand the distinction between the two species despite the limitations as no established in vitro culture technique exists for P. ovale spp.

Epidemiology and Geographic Distribution of Poc and Pow
P. ovale spp. infection accounts for less than 1% of all malaria cases worldwide [1]. Current data on Poc and Pow species distribution and prevalence are not precise, in the sense that the use of species-specific primers is not yet systematically adopted in all epidemiological studies of malaria. However, of late, more studies are being conducted on the detection and differential characterisation of these species [20,21]. Poc and Pow have been reported mainly in sSA and Asia [18,19,[22][23][24][25][26][27] and their prevalence ranges between 0.1% and 4% in Africa and is less than 0.1% in Asia with no significant difference between Poc and Pow [19,[25][26][27][28]. Moreover, misidentification of an increase in imported malaria cases in non-endemic areas have been reported from Singapore, United Kingdom, Spain, Italy, France and China among travellers, mainly from Ivory Coast, Cameroon, Nigeria, Central African Republic, the Democratic Republic of the Congo and Guinea [20,[29][30][31][32][33][34]. Poc and Pow prevalence is supposedly much higher than that currently reported, as P. ovale spp. prevalence (7% to 12%) has been reported without species specification in studies conducted in Zambia, Gabon and Cambodia [5,19,26,35]. This emphasizes the need to popularise the use of more efficient diagnostic and characterisation tools to draw up a real distribution map of these species and to understand their epidemiological dynamics and real contribution to the malaria burden.

Rapid Diagnostic Tests
As with any other disease, rapid diagnosis followed by efficient treatment is one of the cornerstones of malaria control and elimination strategies. RDT remains the most affordable, rapid and available diagnostic technique, requiring little field expertise. During the years 2008 to 2019, the efficacy of 332 RDTs from more than 50 manufacturers was evaluated by the World Health Organization (WHO) to ensure the quality of the kits provided [36]. By 2014, the performance of these RDTs was estimated to be around 93% [37] and, to date, almost all of them have conformed to the WHO's criteria, with continuous effort by manufacturers to improve their performance quality [36]. However, none of these RDTs, are specifically targeted against P. ovale species, as most of the currently marketed RDTs target only P. falciparum and/or P. vivax or all human Plasmodium species, making it impossible to formally diagnose P. ovale spp. infection on this basis and thus resulting in misdiagnosis. Moreover, the sensitivity of existing RDTs is low (<50%) for P. ovale spp. because of low parasite density and/or structural variability of the targeted antigen [20,[38][39][40]. To the best of our knowledge, no RDT till date has had the ability to specifically detect P. ovale spp., let alone differentiate between Pow and Poc.
Few studies have reported on RDT diagnostic efficiency in Poc and Pow but the number of cases were limited even in endemic regions. A recent study from France reported 677 P. ovale spp. imported cases (309 Poc and 368 Pow) and suggested a difference in sensitivity of the two species depending on the RDT type used for diagnosis [32]. Aldolase-RDT (47.8%) showed a higher sensitivity than pLDH-RDT (10.6%) for both species. In addition, two different RDT showed different sensitivity to detect Pow, i.e., 50% and 16% compared to 45.5% and 4.2% for Poc for Aldolase-RDT and pLDH-RDT, respectively; however, the difference was not significant for Aldolase-RDT [32]. The higher pLDH-RDT positivity with Pow may be explained by its ability to produce a higher amount of LDH compared to Poc [20]. Regardless of RDT type the sensitivity was density dependent for both species as previously reported [38,40] though pLDH-RDT would be better for Pow prevalence screening.

Microscopy
Microscopy remains the gold standard for malaria diagnosis and WHO recommends RDT and microscopic diagnosis for all suspected malaria cases [41]. Morphologically identical, Poc and Pow are indistinguishable even by expert microscopists and mostly misidentified as P. vivax [42].
Comparative studies of ultrastructural changes in erythrocytes infected with human Plasmodium species including P. ovale spp. have been carried out, providing some species distinguishing elements upon microscopic diagnosis, among which Schüffner's stippling is present in P. vivax and P. ovale spp. but not in P. falciparum and P. malariae [43]. This approach is still used to distinguish P. ovale spp. from P. malariae and useful for Poc and Pow too.

Molecular Diagnostic Assays
Currently, only molecular techniques, in particular polymerase chain reaction (PCR) based assay, can specifically identify and distinguish Poc and Pow. Several genes depending on their level of sequence conservation are targeted to detect and distinguish between Poc and Pow infection. Commonly used genes in PCR assays are18S rRNA gene, tryptophan rich antigen gene, dihydrofolate reductase thymidylate synthase and sporozoite stage protein gene currently [14,20,33,45].

18S rRNA Gene
The 18S rRNA is the most common targeted gene for confirming the P. ovale spp. [46]. There are reports, which show polymorphism in 18S rRNA gene in Poc and Pow leading to PCR failure in few isolates [14]. Several other genes have also been used for subtyping P. ovale spp. which indicates Poc has greater genetic diversity as compared to Pow (Table 1) [33].

Tryptophane Rich Antigen (tra) Gene
Some studies have targeted tra gene in PCR assay for specific discrimination between Poc and Pow [14,33,47] and also for the identification of genotypic variants within these two species [14]. Out of 22 P. ovale spp. samples, two failures were recorded in Kenya using tra gene compared to three with18S rRNA gene [47]. This indicates that tra gene can be a potential PCR target for the simultaneous identification and characterization of variants between ovale species.

Dihydrofolate Reductase Thymidylate Synthase (dhfr) Gene
Using the dhfr gene as a target in PCR diagnosis, about 30 non-synonymous mutations as well as different amino acid repeats were identified in the P. ovale spp. dhfr gene for easy distinction of Poc from Pow than other human Plasmodium species [14,33,48].

Sporozoite Stage Protein Gene
Sporozoite stage protein such as circumsporozoite protein/thrombospondin-related anonymous related protein (cTrp) and circumsporozoite surface protein (csp) genes, conserved genes, have also been reported by Saralamba and colleagues for Pow and Poc identification [45]. Further, these authors highlight distinct repeat regions in the csp protein in Poc (NPPAPQGEG) and Pow (DPPAPVPQG) as potential targets as monoclonal antibodies that can serologically distinguish sporozoites from these species in future studies.

Kelch 13 Gene
Recently, few authors have used the Kelch 13 gene to distinguish the two ovale species but this gene seems to be less discriminant than cTrp, csp and merozoite surface protein 1 (msp1) genes [34].

Other Method
A post quantitative polymerase chain reaction analysis method called high resolution melting (qPCR-HRM) was developed for Poc and Pow genetic identification based on their DNA melting temperature (Tm) [49]. Specific melting peaks have been identified at 71.26 • C and 71.04 • C respectively for Poc and Pow allowing their distinction.
Although PCR molecular identifications are more sensitive and specific than microscopy and RDT they have certain limitations like, the primers used in existing PCR assays are unable to specifically amplify all field isolates due to emerging genetic diversity [46]. In addition, in certain cases, the primers do not match with P. ovale spp. variants (Pow) resulting in cross reactions with other Plasmodium species, mostly with P. vivax [46]. Thus, the diversity of sequence and primers used could significantly influence the outcome of PCR reactions resulting in compromising the quality of diagnostic results and such epidemiological data regarding Pow and Poc can be misleading at times. Table 1. Some current genes used for molecular identification of P. ovale spp.

Biological and Clinical Comparison of Poc and Pow
In addition to their morphological identity, Poc and Pow share similar life cycle, biological and clinical characteristics [30]. Poc and Pow display same parasitaemia, symptoms, glycaemia, leucocyte count, haemoglobin, aspartate aminotransferase, alanine aminotransferase, bilirubin and albumin levels in infected individuals [20,29,31,32,44]. Though a multi-centric study conducted in Europe reported a significant low haemoglobin and creatinine level in Poc infected patients compare to Pow [20]. The potential differences in pathogenicity between Poc and Pow are not yet clearly established. However, some consistent observations seem to be relevant as they are reported by several independent studies ( Table 2). Thrombocytopenia (platelet count < 150 × 10 3 /mm 3 ) is a common complication in malaria infection, which can reach 94% [50]. A significantly deeper thrombocytopenia in patients with Pow malaria has been reported several times, as well as a shorter latency compared to those infected by Poc [20,29,31,32]. In addition, another study reported a higher temperature and a low albumin level in patients with Pow infection but the difference was not significant [31]. Rojo-Marcos and colleagues show a significantly higher frequency of Pow malaria infection in males (72.7%) compared to females (48.6%) especially in Caucasians group in a study including 35 Poc and 44 Pow cases [20]. In contrast, a recent report including eightfold larger sample size (309 Poc and 368 Pow) demonstrated no significant difference for gender, age or ethnicity [32] as earlier reported studies [29]. All these observations suggested that the variant type Pow could be more pathogenic as compared to the classic type Poc, obviously but this needs more future studies to be conducted on this aspect for efficient conclusive results.

Prophylaxis, Treatment and Relapse
Generally, prophylaxis by chemoprevention consists of taking an antimalarial drug (Chloroquine, Proguanil, Atovaquone/proguanil, Mefloquine, Doxycycline, Primaquine) recommended for anyone traveling to a malaria-endemic region [51]. New findings question the effectiveness of chemoprevention in case of P. ovale species. Many failures of malaria chemoprevention have been recorded in travellers returning from endemic countries [20,29,52,53]. Thirty six percent of chemoprevention failure was recorded in P. ovale spp. infections in British travellers compared to 6.4% and 23.7% for P. falciparum and P. vivax respectively [29]. Gallien et al. report a case of chemoprophylaxis failure using atovaquone-proguanil and another study of malaria surveillance from United States of America and Israel showed that 73% of P. ovale spp. malaria case occurred in travellers on effective prophylaxis treatment [52,53].
Another multicentre study conducted in Europe did not find any difference between those who used chemoprevention to those who did not; highlighting the ineffectiveness of chemoprevention in both Poc and Pow infection as none of molecules used can kill liver-stage hypnozoites [20]. Although the pathophysiological mechanism leading to dormant stage formation and relapse in Poc and Pow is not yet clearly established and is supported only by indirect arguments [54]. Nabarro and colleagues analysed the potential relationship between geographical and seasonal relapse in P. ovale spp. [30]. The authors highlighted the existence of a synchronized relapse in Pow with the malaria season in West Africa suggesting that hypnozoites would be programmed to activate at the time when the probability of being transmitted to a mosquito vector is highest [30]. This observed seasonal relationship in Pow does not exist for Poc.

Conclusions
Poc and Pow are two sympatric species that are almost identical biologically, physiologically and clinically. According to the existing data Pow seems to be the more pathogenic than Poc but this evidence remains to be scientifically established. The development of a simple, affordable and efficient tool for Poc and Pow differential diagnosis is a gap to be filled and should be seriously take into account in future investigations to eliminate this constraint in the fight against malaria. To date, molecular techniques remain the most accurate method to distinguish Poc and Pow.