Introduction

Asymptomatic parasite carriers are a major challenge to malaria elimination, as they fuel the persistence of malaria globally1. They occur as a result of continuous exposure to malaria infections, leading to the acquisition of partial immunity against complications such as cerebral malaria2. Asymptomatic carriers often harbor low densities of parasitaemias, which often goes undetected by conventional malaria diagnostic tools (microscopy and rapid diagnostic tests, RDT) and untreated, and can persist for more than 18 months3,4. In addition, asymptomatic malaria poses several challenges, such as cognitive impairment, anaemia, and stunting in schoolchildren, as well as a significant cause of absenteeism in school5,6. Most malaria surveillance efforts in Ghana focus on younger children and pregnant women, which may lead to underrepresentation of schoolchildren in nationwide malarial surveillance7,8 hence posing a threat to malaria elimination8,9. A study in sub-Saharan Africa has shown schoolchildren contribute about 62% of asymptomatic malaria10. Recent studies have also indicated that there is a gradual shift in malaria infection prevalence from young children (< 5 years) to school age children who are increasingly being at high risk for malaria11,12.

Ghana has three major ecological zones namely the Sahel savannah zone in the north, the Forest zone in the middle, and the Coastal savannah zone in the south13,14. The Sahel savannah zone has a unimodal rainfall pattern while the forest and coastal savannah zones have bimodal rainfall pattern. Hence malaria transmission is seasonal in the Sahel savannah zone, but peaks twice in the forest and coastal savannah zones. The transmission of these parasites depends on the interplay of factors including climatic conditions, vegetation, distribution of malaria vectors, interventions and ecology15,16.

A large proportion of malaria infections in sub-Saharan Africa is caused by Plasmodium falciparum, contributing to 99.7% of malaria cases17. The non-falciparum species (P. malariae and P. ovale) which are less frequent, have a prevalence between 1% and 17%18,19. These non-falciparum species occur as mixed infections with the dominant species P. falciparum and P. vivax, leading to inaccurate estimation of parasite prevalence20. P. malariae is often known to induce chronic nephrotic syndrome, which can be fatal, but they can also go for extended periods of time without showing any symptoms21,22. Plasmodium ovale has two subspecies P. ovale curtisi and P. ovale wallikeri which differ in morphology, clinical manifestations and genetics23,24. These P. ovale subspecies have been implicated in several diseases including severe anaemia, pulmonary impairment and death when treatment delays25,26. Plasmodium vivax infections are rarely identified in sub-Saharan Africa due to large proportion of Duffy-negative population in central and west Africa.

Effective malaria intervention strategies namely widespread use of long-lasting insecticide nets (LLINs), seasonal malaria chemoprevention (SMC), indoor residual spraying (IRS) and improved case management with artemisinin combination therapy (ACTs), are expected to reduce the burden of the disease in a country over time27. In Ghana, for instance, nation-wide distribution of LLINs and introduction of ACTs as the first-line treatment since 2005 have resulted in a notable decline in malaria cases28. Reduction in malaria prevalence has been reported in areas where indoor residual spraying and insecticide treated nets have been implemented29. Hence, the efficiency of current intervention strategies implemented in the various communities in Ghana can be assessed by investigating the dynamics of parasite carriage over a period. A previous study in Uganda reported an increase in malaria transmission over time due to limited coverage of interventions such as insecticide treated nets (ITNs) and effective medications30. This shows that parasite prevalence could increase in Ghana over time amidst current intervention strategies. Therefore, the spatiotemporal variations of asymptomatic P. falciparum malaria among school children in three ecological zones of Ghana by comparing infection in school children in 2023 to previously published data from 201714.

Results

Demographics of study participants

A total of 1,154 subjects were enrolled in the current 2023 study, while 1,109 participants were involved in our previous study conducted in 201714. The median ages of children in 2023 (7 years, 8–11 years) and in 2017 (9 years, 5–9 years) were significantly different (Mann Whitney U test, p. value = 0.0001). The study population consisted of 54.77% (632/1154) males in 2023 whiles 48.24% (535/1109) males in 2017 (Table 1). There was a significant difference in the gender distribution in 2023 and 2017 (χ2 = 9.6387, df = 1, p. value = 0.002) (Table 1).

Microscopic detection of Plasmodium falciparum carriage

The infection rate by microscopy was 1.21% (14/1154) in 2023 whereas it was 16.05% (178/1109) in 2017 (χ2 = 162.82, df = 1, p < 0.0001) (Table 2). At all the study sites, there was a reduction in infection rates among the schoolchildren between 2023 and 2017. There were no infections in Dwease during the dry season (0%, 0/145), Kpalsogou dry season (0%, 0/147) and Pagaza rainy season (0%, 0/138) in 2023 (Table 3). Seasonally, and during both years, the infection rates were higher during the rainy season than the dry season in all study sites except in Pagaza where infection rates was high in dry season (1.87%, 2/107) than rainy season (0%, 0/138) (Table 3).

Asymptomatic data was collected during the rainy periods in both 2017 and 2023 as this aligns with peak malaria transmission due to increased mosquito breeding and biting activity. Between 2017 and 2023, there was a significant increase in parasite density, from approximately 1,600 parasites/µl (1120–2500 parasites/µl) in 2017 to 2,520 parasites/µl (840–8560 parasites/µl) in 2023 (Mann Whitney U test, p. value = 0.2275) (Table 2). This increase was noted across all study sites, except in Kpalsogou and Pagaza, where parasite densities decreased. In Kpalsogou, no parasites were detected in either the dry or rainy seasons in 2023, whereas in 2017, parasite densities were 2,320 parasites/µl during the dry season and 1,700 parasites/µl during the rainy season. In Pagaza, parasite density reduced from 1,320 parasites/µl (dry season) and 1,580 parasites/µl (rainy season) in 2017 to 1,220 parasites/µl (dry season) and no parasite density during the rainy season in 2023 (Table 3). Across most sites, parasite density was higher during the rainy season compared to the dry season, consistent with seasonal transmission patterns. However, exceptions were observed. In Dwease (2017), parasite density was slightly higher in the dry season (1,480 parasites/µl) than in the rainy season (1,320 parasites/µl). Similarly, in Kpalsogou (2017), higher parasite density was recorded in the dry season (2,320 parasites/µl) than the rainy season (1,700 parasites/µl), and in Pagaza (2023), parasite density was recorded only during the dry season (1,220 parasites/µl), with no parasites detected in the rainy season.

Overall, the gametocyte count detected in 2023 was significantly lower than in 2017 (Mann Whitney U test, p = 0.0007) (Table 2). Across most study sites, an increase in gametocyte count was observed; however, an exception was noted in Anyakpor, where the total number of gametocytes was higher in 2023 (50 gametocytes) compared to 2017 (41 gametocytes) (Table 3). Seasonal trends showed higher gametocyte counts during the rainy season across most sites. Notable exceptions were observed in Kpalsogou (2017), where the dry season had more gametocytes (2) than the rainy season (1), and in Anyakpor (2023), where the dry season also recorded more gametocytes (49) compared to the rainy season (1), contrary to the general trend (Table 3).

Molecular detection (PCR) of Plasmodium falciparum carriage

The parasite infection significantly decreased by 57% between 2017 (39.95%, 443/1109) and 2023 (17.33%, 200/1154) (χ2 = 116.1, df = 1, p < 0.0001), revealing a decrease in infections between the two study periods (Table 2). This trend was consistent across all the ecological zones with the reduction most observed in the Sahel savannah zone, where reduction was about 60% (Table 2). Despite the decline in prevalence, the forest zone recorded the highest malaria prevalence in both 2017 and 2023 (Table 2). A significant reduction in Plasmodium falciparum infection prevalence among schoolchildren was observed across study sites between 2017 and 2023. In Kpalsogou, the infection decreased from 24.71% (21/85) in the 2017 dry season to 13.61% (20/147) in 2023, and from 49.24% (65/132) to 14.09% (21/149) during the rainy season. Similarly, in Pagaza, prevalence declined from 28.16% (29/103) to 12.15% (13/107) in the dry season, and from 42.19% (81/149) to 10.14% (14/138) in the rainy season (Table 3).

Seasonal patterns indicated that infection prevalence was generally higher during the rainy season compared to the dry season across most sites. However, exceptions were recorded in the 2023 data from Anyakpor and Dwease, where the dry season prevalence was significantly higher than in the rainy season. In Anyakpor, the dry season prevalence was 20.27% (30/148) compared to 15.29% (26/170) in the rainy season (χ² = 112.38, df = 1, p < 0.0001). In Dwease, the dry season prevalence was 28.28% (41/145), exceeding the rainy season rate of 23.33% (35/150) (χ² = 90.73, df = 1, p < 0.0001).

The estimation of parasite infection by PCR was significantly higher than microscopy in 2023 (χ2 = 28.9479, df = 1, p < 0.0001), and also in 2017 (χ2 = 157.0568, df = 1, p < 0.0001). Submicroscopic infections were less prevalent in 2023 in all study sites except in Anyakpor where prevalence was 18.92% (28/148) and 12.35% (21/170) during the dry and rainy season of 2023 and 8.62% (10/116) and 4.73% (7/148) during the dry and rainy season of 2017 (Fig. 1A-B). The highest submicroscopic infections were found among children living in Dwease during the dry season (2023 = 28.28%, 41/145 and 2017 = 47.01%, 55/117) (Fig. 1B). Submicroscopic Plasmodium falciparum infections were generally more prevalent during the dry season compared to the rainy season across all study sites. However, an exception was observed in 2017 among children in Pagaza and Kpalsogou, where higher prevalence was recorded during the rainy season. In Pagaza, submicroscopic infections increased from 14.56% (15/103) in the dry season to 24.48% (47/192) in the rainy season. Similarly, in Kpalsogou, prevalence increased from 17.65% (15/85) in the dry season to 31.06% (41/132) during the rainy season. (Fig. 1A-B).

Table 1 Demographics of study participants per ecological zone per Year.
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Table 2 Clinical data of study participants per ecological zone per Year.
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Table 3 Proportion of Plasmodium falciparum detected in the various study sites per season.
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Fig. 1 
figure 1

Prevalence of submicroscopic malaria infections in the four study sites of Ghana during the rainy and dry seasons between 2017 and 2023. (A) Prevalence of the Infections in the study sites during the rainy season between 2017 and 2023. (B) Prevalence of the Infections in the study sites during the dry seasons between 2017 and 2023.

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Discussion

With the shift from malaria control to elimination, much attention has been drawn to asymptomatic malaria infections as they represent an infectious reservoir that contributes significantly to the onward transmission of Plasmodium parasites3. Reports reveal that schoolchildren represent an important infectious reservoir affecting malaria control and elimination strategies10,31. Previous studies in Ghana reported the presence of asymptomatic falciparum and non-falciparum malaria infections as being prevalent in communities in Ghana32,33. However, none of the studies conducted in Ghana have investigated the changing prevalence of P. falciparum in asymptomatic malaria across different seasons and ecological zones of Ghana. The temporal changes in prevalence of parasite carriage is essential for determining the success of an intervention strategy implemented by the National Malaria Elimination Program (NMEP). This study investigated the temporal and spatial variations of asymptomatic malaria in the different ecological zones with different transmission intensities by comparing the prevalence of asymptomatic carriage infections in 2023 and previously published data in 2017 14.

The large proportion of the demography in sub-Saharan Africa is a youthful population with more than 27% being schoolchildren34. The proportion of schoolchildren in the population makes them important reservoirs for malaria because they are more attractive and available to mosquito bites35. However, malaria in schoolchildren has received little attention and have not been given necessary intervention36. Due to the repeated exposure to P. falciparum, schoolchildren develop partial immunity which reduced clinical symptoms and facilitates the establishment of asymptomatic malaria37. These asymptomatic reservoirs are more infectious to mosquitoes than symptomatic38. Studies have reported that schoolchildren are responsible to about 50.4% of malaria transmission to mosquito in Uganda, and 20–50% of transmission from human to mosquito39. In region of sub-Saharan Africa with stable malaria transmission, the prevalence of infections is likely driven by the gradual development of naturally acquired immunity resulting from repeated malaria exposure40,41.

The findings from this study revealed 57% reduction in asymptomatic P. falciparum malaria across all communities between 2017 and 2023. The temporal decrease was consistent across all study sites; with the trend most observed in the Sahel savannah zone (Kpalsogou and Pagaza). The decrease in parasite prevalence may be as a result of effective interventions deployed in Ghana during this period. Gradually, there have been improvement in seasonal malaria chemoprevention (SMC) administration in northern Ghana, moving from two months implementation in 2015 when it started, to 4 months in 202242. There were changes in insecticide use for indoor residual spraying during the period from pyrethroid to neonicotinoids in 2021 which has proved very effective43. These improved interventions together with others such as ACTs for managing malaria cases, IPTp and LLIN continuous distribution might have contributed to the reduction in prevalence of infection, despite interruption by COVID-1944[,45. The success of these interventions at reducing parasite prevalence calls for the implementation of these interventions in addition to other more effective interventions including seasonal malaria chemoprevention (SMC) and intermittent preventive treatment in schoolchildren (IpTsc) to reduce parasite burden across the country. This finding also supports recent reports that there are reduced burden of malaria in many countries in sub-Saharan Africa, including Ghana42,46,47. Parasite carriage was high in rainy seasons at both time points; however, high parasite carriage was observed in Anyakpor and Dwease in 2023. The high parasite carriage in both dry and rainy season is probably due to the availability of mining activities and irrigation systems in these sites which serve as breading sites and create a conducive environment for the female Anopheles mosquito to reproduce48,49. A study conducted in Western Kenyan found that irrigated areas was associated with more than two-fold increase in malaria transmission with residents experiencing persistent low-density parasite infections compared to non-irrigated areas50. Children living in forest zone had the highest parasite carriage at both timepoints. The high parasite carriage in forest zone could be likely due to activities of mining in these areas which support the continuous Anopheles vector breeding thereby enabling parasite transmission51,52. These suggest that there might be parasite transmission throughout the year in the forest zone.

The observed increase in parasite densities during the rainy season is because rains provides a suitable habitat for the Anopheles mosquito to breed and multiply which increases transmission of parasites53 and the same person could be bitten by many infected mosquitoes. The increased number of gametocyte carriers in the dry season identified in 2023, corroborates findings from a recent study conducted in Ghana54. As a survival mechanism, more asexual parasite forms might be triggered to undergo gametocytogenesis during the dry season to enable onward transmission during the rainy season when Anopheles vectors are abundant55. A significant proportion of submicroscopic parasites in 2017 observed in this study has also been reported in other studies56,57. The high prevalence of infections particularly in Dwease (Forest zone) and Anyakpor (Coastal savannah zone) during the dry season may reflect lower parasite densities associated with reduced transmission58. In resource-limited settings, these low-density infections often go undetected by routine microscopy allowing asymptomatic carriers to persist a hidden reservoir of transmission. This challenges malaria control and elimination strategies as undetected infections can sustain transmission within these communities despite significant reduction in infections. Also, the test, treat and track (TTT) program could be rendered ineffective as asymptomatic individual remain undetected, and are not given recommended treatments. Hence, efficient malaria surveillance with more sensitive molecular tools and targeted interventions during the dry seasons may be essential in these communities. While this study provides valuable insights on the temporal and spatial distribution of asymptomatic falciparum malaria, it is worth noting that purposive sampling strategy was used. Hence, the findings may not be generalizable to all schoolchildren in the ecological zones. Nevertheless, this study captures relevant information on the trends of malaria transmission in Ghanaian communities, which were selected to represent the ecological zones.

Conclusion

This study highlights a significant reduction in the prevalence of asymptomatic falciparum malaria among schoolchildren between 2017 and 2023, with sahel savannah zone recording the most reduction and the forest zone recording the least reduction. Temporal patterns reveal high transmission during the rainy season across all ecological zones compared to the dry season. Submicroscopic infections were consistently higher during the dry season across the ecological zones between 2017 and 2023.

We recommend that more effective interventions remain essential to further reduce the disease burden in Ghana, particularly among children living in the forest area. There should be targeted interventions for submicroscopic infections during the dry seasons across all ecological zones. The data from this study can guide implementation of effective intervention strategies such as the seasonal malaria chemoprevention (SMC) and the Intermittent Preventive Treatment in Schoolchildren (IpTsc) that would help in eliminating malaria in Ghana.

Materials and methods

Study design and sites

A cross-sectional survey was carried out among schoolchildren aged 3–12 years living in communities across the three ecological zones of Ghana. Thick and thin blood smears on microscope slides, along with blood blots on filter papers, were collected from each assented participant during both the dry and rainy seasons of 2023 for microscopy and PCR analysis. Malaria parasite prevalence data from 2017 was obtained from a previously published study by Amoah et al.14which employed similar sampling procedures and study design as the current study ensuring comparability. The study sites selected were Kpalsogou (09°24′27′′N 00°51′12′′W) in Kumbungu District and Pagaza (09°24′27′′N 00°51′12′′W) in Tamale District both located in the Sahel savannah zone, Dwease (0 6°37′00′′N 01°13′00′′W) in the Asante Akim Central District represented the forest zone, and Anyakpor (5°47′N 0°38′E) in Dangbe East District situated in the coastal savannah zone (Fig. 2).

Kpalsogou and Pagaza have a unimodal rainfall pattern from May to November, and a long dry season from December to April. The average yearly temperature is about 28 °C, however it can get to as high as 42 °C. The mean annual rainfall is around 1000–1300 mm59. Kpalsogou lies near a dam that is connected to an irrigation system; hence farming may continue all year round. Water from the dam gets to the farms via small channels that spills water all over, creating breeding sites for malaria mosquitoes to breed all year round. Kpalsogou is supported by the indoor residual spraying (IRS) program by the U.S. President’s Malaria Initiative (PMI)60. Kpalsogou and Pagaza are under the seasonal malaria chemoprevention (SMC)44.

Dwease is a town in the Ashanti Akyem District. Farming, informal gold and manganese surface mining are the main sources of income for the locals. The abandoned gold mines serve as breeding sites for mosquitoes. The average yearly rainfall is 1200 mm59 with temperatures ranging from 23 °C to 33 °C. Dwease has a bimodal rainfall pattern, with the major rainfall falling between April to June and the minor one in September and October60.

Anyakpor is also a rural community in the Ada East District of Ghana. There is an irrigated vegetable farming in this area which provides the main employment for the people. These informal irrigation scheme creates breeding sites for mosquitoes year-round, sustaining malaria transmission. The area has temperatures ranging from 23 °C to 33 °C and has a bimodal rainfall pattern, with the major rainfall falling between April to June and the minor one in September and October60. The coastal savanna receives relatively little yearly rainfall, with an average of 800 mm.

Sample size and sampling techniques

The sample size was determined based on the estimated prevalence of malaria parasite infection in the study area, using standard formula. With a confidence level of 95%, a margin of error of 5%, an expected malaria prevalence of 15% based on prevalence on previous surveillance61 a sample size (n) of 196 was calculated using the formula:

.

$$n\,=\,\:\frac{{Z}^{2}\times\:\text{p}(1-\text{p})\:}{{e}^{2}}$$

This study employed a non-probability purposive sampling approach. The study was conducted in the only primary school within the communities to represent the ecological zones (Table 4). Within each ecological zone, enrolled pupils from 3 years to 12 years were eligible for inclusion. The school attendance register was used as a sample frame for selecting eligible study participants.

Table 4 List of schools within the various communities of the ecological zones.
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Details of primary schools involved in the study across three ecological zones of Ghana. Each school represents the only public primary school in its respective community. Sampling was conducted during both the dry and rainy seasons in 2017 and 2023.

Inclusion and exclusion criteria

Afebrile schoolchildren between the ages of 3 years to 12 years were recruited in the study who show no symptoms of malaria thus temperature should be less or equal to 37.5 °C. Schoolchildren with temperature above 37.5 °C with no consent form were excluded from the study. The age range of schoolchildren corresponds to the age range according to the Ghanaian Educational System62,63.

Ethical statement and consideration

Ethical clearance was obtained from the Protocol and Ethical Review Committee of the Ghana Health Service (GHS-ERC: 008/01/24). Written informed consent was obtained from both parents and guardians of the schoolchildren. All experiments and methods were performed in accordance with the institution’s guidelines and regulations. All schoolchildren who tested positive for malaria were referred to the nearest health facility for treatment. The list of all malaria positive schoolchildren was given to the headmaster or headmistress, who coordinated the referral at the nearest health facility.

Blood collection procedures

A finger prick blood sample (about 100 µl) was collected from each schoolchild and 50 µl used to prepare dried blood spot (DBS) on a Whatman™ filter paper. The filter paper was air-dried, individually packaged, and wrapped in zip-lock bags with silica gel to avoid mold growth on samples. Thin and thick smears were prepared on the same slide, stained with 10% Giemsa for 10 min, washed with distilled water and air-dried according standard protocols64. The slides and filter papers were sent to the Noguchi Memorial Institute for Medical Research for microscopy and molecular analysis. All microscopy work was conducted by well-trained microscopists with Global health certification as well as training from the Ghana health service. Discordant were resolved by discussion with a third microscopist. To ensure reliability and minimize bias, microscope slide readings were blinded and independently verified. Plasmodium parasite density and species identification were determined by microscopy of the thick and thin blood smears respectively. If parasites were found against 200 leucocytes, the slide was identified as positive. Asexual parasite density (asexual and gametocyte) was determined as parasites/µl of blood = (total number of Plasmodium parasites counted x 8000 leucocytes/µl)/total number of leucocytes counted.

Fig. 2
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The study sites in the different ecological zones of Ghana. This map was designed for this study by Nutifafa Efui Abusah, Department of medical microbiology, University of Ghana with Photoshop version 25.6.0.

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Genomic DNA extraction

Genomic DNA was extracted from the dried filter paper blood spots using the Chelex extraction as previously described by Amoah et al., 202265. The DBS was punched-out into sterile 1.5 ml microfuge tubes containing 1 ml of 1x phosphate buffered saline (PBS) supplemented with Tween-20. The tubes were incubated overnight at room temperature. The DBS punches were washed in ice-cold PBS and incubated at 4 °C for 30 min and the supernatant discarded. Finally, 50 µl of freshly prepared 20% Chelex-100 in distilled water and 100 µl of distilled water was added to each tube. The tubes were then heated at 95 °C for 10 min. The tubes were centrifuged at 14,000 x g for 8 min, and 120 µl of the supernatant transferred into a new sterile 0.5 ml microtube and stored at −20 °C until used for molecular analysis.

Amplification of Cox 3 gene for Plasmodium falciparum speciation

The mitochondrial cytochrome oxidase III (Cox3) gene of P. falciparum was amplified for the identification of parasite species, as described by Isozumi et al. 201566 with some modifications. Two rounds of amplification (nest 1 and 2) were done where the products of the nested 1 reaction served as a template for the nest 2 reactions.

A total reaction volume of 10 µl was used in nest 1, which contained 1X PCR buffer, 0.25 µM of genus-primers (Table 5), 2 mM of magnesium chloride, 0.20 mM dNTP mix, 0.025 µl of One Taq polymerase and 2 µl of the DNA extracts. The cycling conditions were 94 °C for 30 s (initial denaturation), with 40 cycles of 94 °C for 30 s (denaturation), 63 °C for 1 min (primer annealing), 68 °C for 1 min (elongation), and a final extension of 68 °C for 5 min (extension) and 4 °C for infinity.

The nest 2 involved the use of species-specific primer for P. falciparum. Each amplification was carried out in a 10 µl reaction volume containing 1X PCR buffer, 0.20 µM each of P. falciparum species-specific primers (Table 5), 2.08 mM magnesium chloride, 0.20 mM of dNTP mix, 0.05 µl of One Taq polymerase and 1 µl of 1:00 dilution of nest 1 product. The cycling conditions were 94 °C for 30 s (initial denaturation), with 35 cycles of 94 °C for 30 s (denaturation), 56 °C for 1 min (primer annealing), 68 °C for 1 min (elongation), and a final extension of 68 °C for 5 min and 4 °C for infinity. The products of the nest two reactions were resolved using 2% agarose gel electrophoresis and subsequently viewed with a Vilber UV gel documentation system with an expected band size of 201 bp. For nest 1 and nest 2 reactions, 3D7 genomic DNA and nuclease-free water were used as positive and negative controls, respectively.

Table 5 List of primer sequences for Plasmodium speciation. Primer list for Nested PCR of Plasmodium falciparum sequence and band size of amplicon previously published by Isozumi et al.66
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Calculation for Submicroscopic Infections.

PCR prevalence – Microscopy Prevalence as describe by Okell et al.67.

Data analysis

The microscopy and PCR data were entered in Microsoft Excel 2016 and analyzed using STATA v17.0 and GraphPad Prism 9.1.2 software. The Chi-square test was used to test the statistical significance of differences in the proportion of positive samples. The Mann Whitney U-test and Kruskal-Wallis test was used to compare median ages of participants, parasite density and gametocyte between the two timepoints, seasons and across the study sites. A p-value less than 0.05 was considered statistically significant.