Dynamics of the Composition of Plasmodium Species Contained within Asymptomatic Malaria Infections in the Central Region of Ghana

Background Monitoring changes in the composition of the Plasmodium species circulating within the population over a period can inform appropriate treatment recommendations. This study monitored variations in the prevalence of four common human Plasmodium species carried by children with asymptomatic malaria infections over a two-year period. Methods Two cross-sectional studies were conducted in November 2017 and December 2019. A total of 210 children aged between 4 and 13 years were recruited in 2017, and 164 similarly aged children were recruited in 2019. Approximately 150 μl of finger-pricked blood was used to prepare thick and thin blood smears as well as spot Whatman® #3 filter paper. Genomic DNA was extracted from the dried blood spots and used in PCR to amplify the 18S rRNA gene from four different human Plasmodium parasites. Results Parasite prevalence by microscopy and the prevalence of P. falciparum detected by PCR was relatively similar at the two time points (Pearson chi-square = 0.405, p=0.525, and Pearson chi-square = 0.452, p=0.501, respectively). However, the prevalence of PCR detectable P. malariae increased by 8.5-fold, whilst P. ovale increased from 0 to 9% in the children sampled in 2019 relative to the children sampled in 2017. The only parasite species identified by microscopy in this study was P. falciparum, and no P. vivax was identified by either microscopy or PCR in the study population during the study period. Conclusion There is the need to implement molecular diagnostic tools for malaria parasite surveillance in Ghana. This will enable the identification and treatment of all circulating malaria parasites including P. malariae and P. ovale, whose population is expanding in parts of Ghana including Simiw.


Introduction
All human malaria parasite species are capable of causing symptomatic as well as asymptomatic infections [1]. Symptomatic malaria is predominantly caused by highdensity parasite infections [2], whilst asymptomatic infections are predominantly caused by submicroscopic densities of parasites [3]. Asymptomatic malaria parasite carriage can persist for long periods of time without treatment, during which the parasite can continuously produce gametocytes that fuel then malaria transmission [4]. Asymptomatic infections are prevalent in both low and high malaria-endemic regions and are important reservoirs for sustaining malaria transmission [4]. e gold standard for the diagnosis of symptomatic malaria in endemic areas is microscopy. However, the inability of microscopy to detect low-density infections [5][6][7] does not make it ideal for the detection of Plasmodium parasites contained in asymptomatic infections [8]. A number of studies that have used molecular approaches such as polymerase chain reaction (PCR) to assess human malaria parasite carriage have shown that the prevalence of malaria parasites, especially P. malariae and P. ovale, is greater than previously reported [9,10]. Infections containing P. malariae and P. ovale most often present as low-density asymptomatic infections that require molecular tools for accurate detection [11,12]. e prevalence of the non-falciparum Plasmodium parasites including P. ovale and P. malariae is less common than P. falciparum in sub-Saharan Africa. Although P. falciparum is the leading cause of symptomatic malaria in sub-Saharan Africa, including Ghana [13], P. malariae and P. ovale can cause chronic infections [14] and have contributed significantly to anemia and morbidity in some populations, including individuals living in Gabon [15]. Some studies from the Central, Eastern, and Greater Accra Regions of Ghana have reported asymptomatic P. falciparum prevalence to be as high as 31%, 32%, and 44%, respectively [7,9].
Successful malaria control interventions are expected to reduce the prevalence and density of malaria parasites circulating within a population over a period of time. e effectiveness of malaria control interventions implemented in a country or community can therefore be evaluated by determining changes in the prevalence and density of malaria parasites contained within infected individuals over time. An earlier report from a P. falciparum-endemic country, Tanzania, has suggested that a decrease in the prevalence of P. falciparum can result in an increase in the prevalence of non-falciparum malaria parasites [16]. is suggests that the enhanced control of P. falciparum in Ghana over the years could result in an increase in the prevalence of the non-falciparum malaria parasites due to a decline in the prevalence of P. falciparum. In this study, we sought to determine the changes in the prevalence of four common human malaria parasite species carried by asymptomatic children living in Simiw, a community in the Central Region of Ghana over a two-year period.

Ethics Approval and Consent to Participate.
e Institutional Review Board of the Noguchi Memorial Institute for Medical Research granted ethical clearance for this study (IRB Approval #: 009/15-16). Permission to conduct this study was also obtained from the Komenda Edina Eguafo Abrem (KEEA) Municipal Director of Education. e study was explained to the parents and or guardians of the school children as well as the school children through a series of interactive meetings. Parents/guardians who were willing to enroll their children into the study provided written parental consent prior to the child being recruited into the study. Children aged 12 years and above were made to endorse a child assent form.

Study Type, Site, and Sample Collection.
Two crosssectional studies were conducted over a two-year period. e first was conducted in November 2017, where 210 children aged between 4 and 13 years were enrolled. e second study was conducted in December 2019 where 164 children aged between three and 13 years were recruited. e study was conducted in Simiw, a community in the KEEA District of the Central Region of Ghana ( Figure 1). Recruitment of study participants was done from the Simiw Basic School at both time points. All children aged between 3 and 13 years were illegible to be enrolled into the study.
Approximately 150 μl of finger-pricked blood was obtained from each child. e blood was used to prepare a thick and thin blood film as well spot a strip of Whatman ® #3 filter paper (GE Healthcare, USA). e blood films were processed for microscopy, whilst the blood spots were air-dried and stored in a Ziplock ® bag containing silica gel beads. e samples were then transported to the NMIMR for onward processing and malaria parasite identification.

Malaria Parasite Identification and Quantification by
Microscopy.
e blood films were processed using the standard WHO protocol [17] and read by two trained malaria microscopists. Plasmodium parasite density (PD) was estimated by multiplying the number of parasites identified per 200 WBCs by 40 [5]. A thick smear was considered negative for Plasmodium parasites if no parasite was observed in 200 high-powered fields. Discordant slides in terms of presence or absence of Plasmodium parasites were re-examined by a third malaria microscopist. Discordant results agreeing with the third reading were considered final.

DNA Extraction.
Genomic DNA was extracted from the dried filter paper blood spots (DBS) using the Chelex-saponin extraction method as previously described [18] with very minor modifications. In brief, two 3 mm disks punched from each dried blood spot. e spots were placed in a 1.5 ml microcentrifuge tube and washed with ice cold phosphate buffered saline (pH, 7.2). A 200 μl aliquot of 6% Chelex-100 was then added to each spot and heated at 95°C for 10 minutes. Finally, tube containing the heated mixture was centrifuged at 12, 000 × g for 1 minute, and 120 μl of the supernatant containing the extracted DNA was transferred into a new sterile labeled 0.5 ml microcentrifuge tube. e DNA was stored at −20°C or used immediately.

Malaria Parasite Identification by PCR.
e conserved regions of the 18s rRNA gene of P. falciparum, P. malariae, and P. ovale were amplified from extracted genomic DNA using a previously described PCR protocol [9]. e 18s rRNA gene of P. vivax was amplified using a similar protocol [19]. e 15 μl primary reaction mixture contained 20-40 ng of DNA, 80 nM of the genus specific primer rPLU5 (forward) and rPLU6 (reverse), 1X PCR buffer, 167 nM dNTP mix, 2.5 mM MgCl 2 , and 1 U of OneTaq DNA polymerase.
Four independent nested (secondary) reactions were performed, with each set up containing a similar composition of reagents as the primary reaction; however, the template used was 0.5 μl of the primary reaction product, and the primer set used was one of the four species-specific primers rFAL1/rFAL2 (133 nM), rMAL1/rMAL2 (333 nM), rOVA1/rOVA2 (333 nM), and rViv1/rViv2 (333 nM) (Supplementary Table S1). e primary and nested PCR reaction cycling conditions included an initial denaturation at 95°C for 5 minutes followed by 35 cycles of 94°C for 30 seconds, 55°C (for primary) or 58°C (for nested) for 1 minute, 68°C for 1 minute, and a final extension at 68°C for 5 minutes. e nested PCR products were resolved on a 2% agarose gel prestained with ethidium bromide and subsequently visualized on a Vilber gel documentation system (Vilber, France).
2.6. Data Analysis. IBM SPSS ver 22 was employed in the descriptive statistics such as frequency and also to identify significant difference between parasite prevalence and PCRdetectable P. falciparum, P. malariae, and P. ovale (Fisher's exact test) in the two sites. e results were either described as a whole or categorized into young children aged below 10 years and older children aged 10 years and above. e independent samples T-test was used to identify differences in the mean age and parasite densities. Differences in the prevalence of males as well as the prevalence of the different Plasmodium species detected by PCR were determined using Fisher's exact test. p values less than 0.05 were considered statistically significant.

Results
ere were a total of 374 children aged between 3 and 13 years recruited into this study. Although the age range of the children recruited in 2017 and 2019 were similar, the children recruited in 2019 had a higher mean age than those recruited in 2017 (Table 1). ere was no significant difference in the prevalence of asymptomatic parasite carriers detected by microscopy (25.7% in 2017 and 28.7% in 2019) or the parasite densities contained in the infections (geometric mean 614 in 2017 and 727 in 2019). e proportion of males in the study population recruited in 2017 and 2019 was also similar and ranged between 49.5% in 2017 and 40.2% in 2019 (Table 1).

Microscopy Estimation of Parasite Prevalence and Species.
Overall, the prevalence of asymptomatic malaria infections detected by microscopy in 2017 and 2019 was similar (Fisher's exact test, p � 0.56) ( Table 1). Although the maximum parasite density identified in the samples from 2019 was almost double that of the 2017 samples, the geometric mean parasite densities in the children recruited in 2017 and 2019 samples were similar (Table 1). e only Plasmodium species identified by microscopy in this study was P. falciparum.
When the children were grouped into young children

Polymerase Chain
None of the samples collected in 2017 was identified as positive by the P. ovale species-specific PCR; however, 9.1% (15/164) of the children tested in 2019 had P. ovale. A majority (87%; 13/15) of the P. ovale infections were contained in mixed infections with P. falciparum. e difference in parasite prevalence between children under 10 years old and those 10 years old and above was similar over the study duration, although smaller in 2017 (22.6%) than in 2019 (21.9%) (Figure 3 and Supplementary  Table S2). e children aged 10 years and above had a higher prevalence of all the different parasite species observed in the study than the younger children (<10) (Figure 3 and Supplementary Table S2).

Discussion
e National Malaria Control Program of Ghana has implemented numerous vector control interventions including enhanced distribution of insecticide-treated materials and adulticiding and larviciding of mosquitoes in addition to parasite control interventions including adapting the T3: Test, Treat, Track Initiative. ese interventions have resulted in a reduction in the incidence and prevalence of severe manifestations of malaria and uncomplicated malaria but not asymptomatic malaria [20]. e escalation of global efforts to eliminate malaria [21,22] however requires improved rapid and effective diagnosis of symptomatic as well as asymptomatic infections caused by all human malaria parasites [23,24].
is study determined the prevalence of children harboring P. falciparum in asymptomatic infections to have remained stable over the two-year study period. is supports an earlier report that malaria control in several parts of sub-Saharan Africa, including Ghana, remains a substantial challenge [10]. Behavioral practices of a population, including delayed healthcare seeking behavior [10,25] and the use of untreated bed nets by some groups of people in Ghana [26], have been suggested to account for a lack of a reduction in the prevalence of malaria despite enhanced nationwide malaria control interventions. Additionally, the continued emergence of drug-resistant parasites and insecticide-resistant mosquitoes [13] as well as the presence of fake and substandard drugs on the market could have added to the persistence of parasitaemic individuals. e significant increase in the prevalence of P. malariae over the two-year period is likely a result of P. malariae presenting primarily as asymptomatic infections, which are not treated or symptomatic infections that are treated with alternative antimalarials other than the first-line ACTs. e WHO recommended treatment for P. malariae and P. falciparum is artemisinin-based combination therapy (ACTs), which when taken appropriately should clear both P. falciparum and P. malariae. e WHO recommended treatment for P. ovale or P. vivax is an ACT plus primaquine to clear the hypnozoites [27]. e absence of antimalarials that are effective against the hypnozoites of P. ovale on the Ghanaian market [28] is likely to be a major contributor to the increased prevalence of P. ovale in Simiw. e hypnozoite stage of P. ovale is known to become active after several days to months of dormancy. Infections with P. malariae and P. ovale most often present as asymptomatic infections [29], which are usually not detected and thus remain untreated [30]. A previous study conducted in the Eastern Region of Ghana also identified a high prevalence of children with asymptomatic infections containing both P. ovale and P. malariae [9], suggesting a probable increase in non-falciparum malaria parasites across Ghana and not just in Simiw. e trend of an increase in parasite carriage by older children relative to younger children has also been identified in studies from other parts of Ghana including the Eastern Region [9] and in other parts of Africa including Equatorial Guinea [31] and Uganda [32]. Younger children are suggested to have a lower incidence of asymptomatic malaria parasite carriage but a higher incidence of symptomatic malaria relative to the older children due to their lower levels of immunity compared to the older children [33].
Plasmodium malariae and P. ovale parasites were predominantly identified in mixed species infections containing P. falciparum. Malaria infections containing P. falciparum in addition to a non-falciparum parasite is a common occurrence in many tropical regions where Plasmodium falciparum is endemic [34] including Ghana [20] and Uganda [35].

Conclusion
ere is the need to implement the use of molecular diagnostic tools for malaria parasite surveillance.
is will enable the identification and treatment of all infecting malaria parasites including P. malariae and P. ovale, whose population is expanding in parts of Ghana including Simiw.