https://orcid.org/0000-0003-3305-0533
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Figures
Abstract
Current interventions targeting malaria control in sub-Saharan Africa (SSA) are focused on Plasmodium falciparum, the most prevalent species infecting humans. Despite renewed efforts for malaria elimination in SSA, little attention has been paid to the neglected parasites P. malariae, P. ovale spp., and P. vivax and the impact of interventions like long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS) with non-pyrethroid insecticides, and/or seasonal malaria chemoprevention (SMC) on these minor Plasmodium spp. To address this research gap, the aim of this observational study was to assess the impact of two sequential interventions, IRS and SMC combined with LLINs, on the Plasmodium spp. reservoir, with particular emphasis on the non-falciparum minor species, in Bongo District, an area characterized by high seasonal transmission in the Northern Sahelian belt of Ghana. Using an interrupted time-series study design, five age-stratified surveys, each of ~2,000 participants, were undertaken at the end of the wet seasons between 2012 and 2022. Across this 10-year study period, infections with P. malariae and P. ovale spp. were detected using a species-specific PCR targeting the 18S rRNA gene, while P. vivax was not detected. In 2015, following IRS, the prevalence of the minor Plasmodium spp. declined in all ages from baseline in 2012, with participants being significantly less likely to be infected with P. malariae (13.7% vs 1.4%) and P. ovale spp. (5.7% vs 0.4%). Despite this decline, in 2017, 32 months after IRS was discontinued and SMC was introduced, the prevalence of P. malariae (2.9%) and P. ovale spp. (4.0%) rebounded, with approximately a 2- and 10-fold increase, respectively. This rebound in the minor species was observed in all age groups, except for the younger children (< 5 years) targeted by SMC. Finally, when we examined this population in 2020 and 2022 after sustained deployment of SMC, the prevalence of P. malariae continued to increase (7.4% and 5.8%), while the prevalence of P. ovale spp. declined (2.6% and 1.3%). Results show that both IRS and SMC were effective not only against P. falciparum but also reduced the prevalence of P. malariae and P. ovale spp. in Bongo District. Going forward, molecular diagnostics will be critical to identify changes in the submicroscopic reservoir of the minor Plasmodium spp. found in adolescents and adults and to achieve malaria elimination in this region of Ghana.
Author summary
To achieve malaria elimination, the use of molecular diagnostics for surveillance of the Plasmodium spp. reservoir in the human population is essential. Such diagnostics are critical to detect hidden reservoirs of submicroscopic minor-species infections with P. malariae and P. ovale spp. Here we examine the impact of two sequential malaria control interventions, indoor residual spraying (IRS) with non-pyrethroid insecticides and seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine plus amodiaquine administered to all children < 5 years, combined with long-lasting insecticidal nets (LLINs) impregnated with pyrethroids to reduce the reservoir of Plasmodium spp. infections where P. falciparum is dominant and the minor species, P. malariae and P. ovale spp., were also found. This interrupted time-series study, undertaken over 10 years (2012–2022) in Bongo District, located in the Northern Sahelian belt of Ghana, identified that IRS and SMC reduced the prevalence of all Plasmodium spp. IRS significantly reduced the prevalence of single- and mixed-Plasmodium spp. infections across all age groups. However, following its discontinuation a rebound was observed for P. falciparum and minor-species infections, especially in adolescents and adults. Although SMC maintained a near-zero prevalence of all Plasmodium spp. infections, its impact was limited to the target age group (< 5 years). Furthermore, this study showed that following the discontinuation of IRS, the older children, adolescents, and adults, who are not currently eligible for SMC in Ghana, continued to harbour Plasmodium spp. infections, including P. malariae and P. ovale spp., thus sustaining the reservoir. These results show that by combining vector control interventions as well as targeting all age groups with chemoprevention, high-burden countries in sub-Saharan Africa, including Ghana, could bring the Plasmodium spp. reservoir closer to elimination more rapidly.
Citation: Rios-Teran CA, Tiedje KE, Bangre O, Deed SL, Argyropoulos DC, Koram KA, et al. (2026) The dynamics of asymptomatic Plasmodium spp. infections following 10 years of malaria control interventions in Northern Sahelian Ghana. PLoS Negl Trop Dis 20(4): e0014174. https://doi.org/10.1371/journal.pntd.0014174
Editor: Henk Schallig, Academic Medical Center: Amsterdam UMC Locatie AMC, NETHERLANDS, KINGDOM OF THE
Received: September 9, 2025; Accepted: March 20, 2026; Published: April 13, 2026
Copyright: © 2026 Rios-Teran et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data associated with this study, including de-identified individual participant data, are available in the manuscript, Supporting information, and on GitHub (https://github.com/UniMelb-Day-Lab/Minor_Plasmodium_Ghana). Redistribution or reuse of the participant metadata provided in the GitHub repository requires proper attribution and prior approval. Researchers interested in further use of these data should contact the Malaria Reservoir Study Team, represented by the corresponding author, Prof. Karen Day (karen.day@unimelb.edu.au), to identify opportunities for collaboration. The species-specific 18S rRNA PCR protocol and primer sequences are available on GitHub (https://github.com/UniMelb-Day-Lab/SpeciesSpecific_18S_rRNA_PCR) [19].
Funding: This research was supported by Fogarty International Center at the National Institutes of Health through the joint NIH-NSF-NIFA Ecology and Evolution of Infectious Diseases award R01-TW009670 to KAK and KPD; and the National Institute of Allergy and Infectious Diseases, National Institutes of Health through the joint NIH-NSF-NIFA Ecology and Evolution of Infectious Diseases award R01-AI149779 to KAK, ARO, and KPD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Since the early 2000s much of sub-Saharan Africa (SSA) has experienced scale-up of malaria control interventions, including long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), and the treatment of clinical malaria with artemisinin-based combination therapies (ACTs) [1]. These interventions have successfully reduced childhood morbidity and mortality due to malaria but very little is known about the impact of these interventions on the reservoir of asymptomatic Plasmodium spp. infections in the human population that sustain transmission to anopheline mosquitoes, especially in high transmission.
To achieve malaria elimination in high-burden countries in SSA the challenge remains to reduce the reservoir of these asymptomatic infections to zero. Thus, it is critical to understand the composition, population size, and age distribution of this Plasmodium spp. reservoir in humans and the parasitological changes that occur following the implementation of the abovementioned control interventions. To date, there have been limited studies of this reservoir using sensitive molecular diagnostics to investigate the prevalence of all Plasmodium spp. infecting humans. Contemporary studies in SSA have largely focused on the dominant species, Plasmodium falciparum, with few investigations of the minor species, P. malariae, P. ovale spp., and P. vivax (e.g., [2–7]). Furthermore, there have only been a limited number of studies from high-transmission settings that have monitored the impact of the abovementioned interventions on the prevalence of P. falciparum in the reservoir of infection (e.g., [8–12]). None have concurrently evaluated the minor-Plasmodium spp. infections with sensitive molecular diagnostics.
Typical of SSA, Ghana has intensified its national malaria control efforts through the implementation of various intervention strategies (i.e., LLINs, IRS, seasonal malaria chemoprevention (SMC), etc.), across the country’s different epidemiological or ecological zones [13–16]. IRS, in combination with LLINs, has been deployed across much of northern Ghana, specifically, in the Upper West Region since 2011, the Northern Region since 2012, and the Upper East Region since 2013 [16–18]. However, its implementation has not been continuous across all districts. The timing and geographic scale-up of IRS in these regions have been associated with positive impacts on malaria control, as evidenced by reductions in the incidence of confirmed clinical cases [17]. In the Upper West Region, where IRS has been implemented alongside SMC for children aged 3–59 months (< 5 years) since 2015, these combined interventions have been associated with sustained declines in clinical malaria incidence [17]. Despite these successes, the impact of these interventions on the reservoir of Plasmodium spp. infection remains unknown. To address this parasitological research gap, the Malaria Reservoir Study (MRS) was established in Bongo District, Upper East Region, to monitor changes in the human parasite population over a 10-year study period, from a 2012 baseline through the implementation of sequential malaria control interventions out to 2022 [12,19,20]. To our knowledge, this observational study represents the first long-term investigation of the reservoir of Plasmodium spp. infection across all ages in West Africa, with the advantage of a baseline collected prior to the implementation of IRS and SMC.
In Bongo District, located in the Northern Sahelian belt of Ghana, no studies have focused exclusively on characterising minor Plasmodium spp. in the asymptomatic reservoir using molecular diagnostics. Previous investigations undertaken by the MRS detected P. malariae and P. ovale spp. in this district using a species-specific PCR targeting the 18S ribosomal RNA gene (18S rRNA) [19], supporting the presence of these minor species and the need for a more comprehensive investigation. Findings from the MRS underscore that minor Plasmodium spp. are often overlooked, as most studies in Ghana have not investigated these species in community settings across all age groups. Much of the existing evidence comes from cross-sectional studies and is limited to particular regions including the Eastern Region, specifically the Kwahu-South and Akwapim South Municipal Districts [3,21]. Additional studies have identified minor Plasmodium spp. in specific age groups, such as asymptomatic school-aged children in the Central and Ashanti Regions [5,22,23], as well as asymptomatic adults in south-central Ghana, from multiple sites in the forest ecological zone [24] and in the Ashanti Region [25]. Consequently, the prevalence of minor Plasmodium spp. in the asymptomatic reservoir remains largely underdetermined in northern Ghana, emphasizing the need for additional epidemiological studies.
Using an interrupted time-series study design, five age-stratified cross-sectional surveys of ~2,000 participants per survey were undertaken at the end of the wet seasons between 2012 and 2022 to evaluate the impact of two sequential interventions, IRS and SMC, combined with widespread LLIN usage. This study aimed to assess how these interventions influenced the prevalence and composition of the asymptomatic Plasmodium spp. reservoir in Bongo District, an area characterized by high seasonal transmission, with particular emphasis on the non-falciparum minor species P. malariae, P. ovale spp., and P. vivax.
Methods
Ethical approval
This study was reviewed and approved by the ethics committees at the Navrongo Health Research Centre, Ghana (NHRC IRB-131), Noguchi Memorial Institute for Medical Research, Ghana (NMIMR-IRB CPN 089/11–12; NMIMR-IRB CPN 066/20–21), and The University of Melbourne, Australia (Project IDs 13433, 31586, 21649). Individual written informed consent was obtained in the local language (i.e., Gurene) from each enrolled participant by signature or thumbprint, accompanied by the signature of an independent witness. For children < 18 years of age, a parent or guardian provided written consent. In addition, all children between the ages of 12 and 17 provided verbal assent.
Study site and population
This observational study was undertaken in Bongo District, located in the Upper East Region within the Northern Sahelian belt of Ghana, proximal to the border with Burkina Faso (Fig 1A). This district is characterized by high seasonal malaria transmission, where there is a short but intense wet season between June and October (i.e., high-transmission season) and a prolonged dry season between November and May (i.e., low-transmission season), typical of endemic regions at the southern edge of the Sahel sub-region [19,26]. In Bongo District, P. falciparum is the dominant species, while P. malariae and P. ovale spp. are the minor species [19]. For this study, participants were enrolled from two broad catchment areas (i.e., Vea/Gowrie and Soe, with a sampling area of ~60 km2) in Bongo District (hereinafter referred to collectively as “Bongo”), which were selected as they were comparable in terms of population size, age distribution, and ethnic composition but are in different agroecological zones [19]. Vea/Gowrie is located in the south of the district and is proximal to the Vea Dam and irrigation area [19,27], while Soe is situated in the north-west near the border with Burkina Faso and not located near any large bodies of water, although smaller dams for watering animals and gardening are scattered across the catchment area (Fig 1A). Details on the study site, study, population, inclusion/exclusion criteria, and data collection procedures have been previously published [12,19] and briefly described in S1 Text.
(A) Study site locations in Bongo District, Ghana, showing the distribution of the compounds (i.e., households) included from the two catchment areas, Vea/Gowrie (purple, lower left) and Soe (green, upper right), which are ~ 20-40 km apart. The location of Bongo District (red square) in the Upper East Region of northern Ghana is provided in the inset map (lower right). As the population in Bongo District resides in rural communities scattered throughout the district, only those compounds geolocated and included in this study are shown on the map. The map was drawn using the R packages ggmap [28], malariaAtlas [29], and ggspatial [30]. The Bongo District, Upper East Region, Ghana basemap was obtained from OpenStreetMap (OSM; https://www.openstreetmap.org/) and is made available under the Open Data Commons Open Database License (ODbL) by the OpenStreetMap Foundation (https://www.openstreetmap.org/copyright). The Ghana inset basemap was obtained from the Malaria Atlas Project (MAP; https://malariaatlas.org) and is made available under the Creative Commons Attribution 3.0 Unported License (CC BY 3.0 Unported License; https://malariaatlas.org/open-access-policy/). (B) Five age-stratified cross-sectional surveys (N = ~2,000 participants per survey) were conducted in Bongo at the end of the wet seasons between 2012 and 2022 (white circles) in October 2012 (Survey 1, pre-IRS), October 2015 (Survey 2, post-IRS), October 2017 (Survey 3, SMC), November 2020 (Survey 4, SMC), and October 2022 (Survey 5, SMC). Three rounds of IRS with non-pyrethroid insecticides were implemented between 2013-2015 as indicated. Starting in 2016, SMC with SPAQ, was administered to all children (< 5 years) during the wet season (i.e., high transmission). Both IRS and SMC were implemented against a background of widespread LLIN (impregnated with pyrethroid insecticides) usage which were distributed across Bongo District in 2012, 2016, and 2021 through universal coverage campaigns. For additional details on these interventions please see Methods. Fig 1B was adapted from [12,31].
Malaria control interventions
Between 2010 and 2012 and again in 2016 and 2021, LLINs impregnated with pyrethroid insecticides (i.e., PermaNet 2.0, Olyset, or DawaPlus 2.0) were mass distributed in Bongo District through universal coverage campaigns (UCC) by the National Malaria Elimination Programme (NMEP)/Ghana Health Service (GHS) [12,16–18,32]. For those years when no UCC were undertaken, LLINs were provided through routine services (e.g., antenatal clinics, school distributions, etc.) to maintain coverage [12,17].
Starting in 2013, three rounds of IRS with non-pyrethroid insecticides (i.e., organophosphate pirimiphos-methyl formulations) was implemented across the Upper East Region, including Bongo District, with the support of the AngloGold Ashanti Malaria Control Programme (AGAMal) and funding from the Global Fund [12,17,18,33]. AGAMal provided considerable support for the IRS procurement, implementation, and entomological evaluations; however, funds were not explicitly included to concurrently monitor the impact of IRS on malarial disease or the Plasmodium spp. reservoir [34]. The three rounds of IRS were implemented across Bongo District between 2013 and 2015: Round 1: October 2013 – January 2014, (Vectoguard 40WP), Round 2: May - July 2014 (Actellic 50EC), and finally Round 3: December 2014 - February 2015 (Actellic 300CS). Additional details on the IRS intervention and its impact on local entomological parameters have been previously described and published [12]
Following the discontinuation of this short-term IRS in early 2015, SMC was rolled out across the Upper East Region starting in 2016 by Ghana’s NMEP with funding from the Global Fund [17]. SMC involves the monthly administration (i.e., ~ 28–30 days apart) of a full treatment course of an antimalarial to all children between 3–59 months (i.e., < 5 years; SMC-eligible age group), regardless of infection status, for up to four months of the year during the wet or high-transmission season [35]. Like other countries of the Sahel sub-region of Africa where SMC was first deployed, the current drug of choice for SMC in Ghana is sulfadoxine-pyrimethamine plus amodiaquine (SPAQ) [36].
Study design and sample collection
Using an interrupted time-series study design, five age-stratified cross-sectional surveys of ~2,000 participants per survey were undertaken at the end of the wet season between 2012 and 2022 to evaluate the impact of IRS and SMC in combination with LLINs on the prevalence and composition of the Plasmodium spp. reservoir, specifically the non-falciparum minor species P. malariae, P. ovale spp., and P. vivax (Fig 1B). These ~2,000 participants of all ages (1–98 years) represent ~15% of the total population that reside in the Vea/Gowrie and Soe catchment areas [19]. The five cross-sectional surveys were undertaken at the end of the wet season, when parasite prevalence is highest and when the impact of malaria control interventions would be most evident. The surveys can be broken down into four phases (1) October 2012 (Survey 1) prior to IRS and SMC (i.e., baseline); (2) October 2015 (Survey 2) seven months after the final round of IRS; (3) October 2017 (Survey 3) 32 months after IRS was discontinued, but during the deployment of SMC to the younger children (< 5 years); and finally (4) November 2020 (Survey 4) and October 2022 (Survey 5) more than five years after IRS was withdrawn but following the continued deployment of SMC for five and seven years, respectively (Fig 1B).
During each survey, all participants completed a structured questionnaire to collect information on their demographics and socioeconomic characteristics, malaria prevention activities, recent clinical symptoms, blood pressure, body weight, axillary temperature, etc. To monitor for intervention use, information was collected from all participants on IRS coverage, LLIN usage the previous night, and antimalarial treatment in the previous two-weeks (i.e., participants that reported they were sick, sought treatment, and were provided with an antimalarial treatment) [12,19]. In addition, blood samples were collected from all participants for thick/thin blood films, rapid diagnostic tests (RDT; First Response or CareStart P. falciparum antigen, histidine-rich protein 2 (HRP-2)), haemoglobin concentration, and as dried blood spots (DBS) on filter paper (3MM Whatman, Madison, UK) for laboratory-based molecular analyses. All participants who had a positive RDT and were febrile (axillary temperature ≥ 37.5°C) were considered to be symptomatic for malaria and were provided with the appropriate care by clinical personnel at the local health centres via the Ministry of Health (MOH)/GHS, following the current NMEP antimalarial treatment policy [37,38].
This study enrolled 3,239 asymptomatic participants, across the five cross-sectional surveys, spanning all ages (median age: 12 years [IQR = 5–28; range = 1–98]) (Table 1). From these participants, 2,552 (78.8%; median age: 13 years [IQR = 7–32; range = 1–98]) were surveyed in more than one of the five cross-sectional surveys (range of surveys: 2–5) resulting in 9,006 DBS samples (92.9%) from the total 9,693 DBS samples collected and analysed using the laboratory-based molecular diagnostics. For the 687 (21.2%) participants that were only surveyed once in the five surveys considered here, 303 (44.1%) were from children newly enrolled in the final 2022 survey to maintain the age-stratified study design.
Molecular estimation of Plasmodium spp. prevalence
Genomic DNA (gDNA) was extracted from all asymptomatic participant DBS samples collected during each of the five surveys using the QIAamp DNA Mini Kit (QIAGEN, USA) as previously described [19]. All samples were then tested for Plasmodium spp. infections (i.e., P. falciparum, P. malariae, P. ovale spp., and P. vivax) using a nested PCR targeting the 18S ribosomal RNA gene (18S rRNA) [39–41] with previously published primers and protocols, available on GitHub (https://github.com/UniMelb-Day-Lab/SpeciesSpecific_18S_rRNA_PCR) [19]. Using this laboratory-based molecular approach, we were able to identify all participants with single- or mixed-Plasmodium spp. infections (i.e., P. falciparum, P. malariae, P. ovale spp., and/or P. vivax). Since P. ovale was first identified by Stephens in 1922, it has been considered a single species. However, molecular studies have now revealed two genetically distinct forms, commonly referred to as P. ovale curtisi and P. ovale wallikeri [42]. Although the formal taxonomic classification of these species remains under debate [43–46] this study follows the prevailing convention in the literature by referring to them as P. ovale curtisi and P. ovale wallikeri, while collectively describing them as P. ovale spp.
Statistical analysis
For these analyses, participants were categorized into defined age groups, sex, and catchment areas. Continuous variables are presented as medians with interquartile ranges (IQRs), and categorical variables are presented as counts and percentages, where appropriate. Prevalence estimates for each Plasmodium spp. (including single- and mixed-species infections) are presented with 95% confidence intervals (CIs). For these analyses, a single-species infection was defined as an infection with only P. falciparum, P. malariae, P. ovale spp., or P. vivax, while a mixed-species infection was defined as an infection with P. falciparum, P. malariae, P. ovale spp., and/or P. vivax. Associations between the various interventions or study time points (i.e., 2012, 2015, 2017, 2020, and 2022) and the prevalence of each Plasmodium spp. (including single- and mixed-species infections) were estimated using multivariable logistic regression. Confounding variables identified a priori (age groups, sex, catchment area, LLIN usage, and antimalarial treatment in the previous two weeks) were adjusted for in the multivariable regression models. Effect modification by age group, identified a priori, was investigated using an interaction term between this variable and study time points. Using this analysis, we encountered situations where there were no Plasmodium spp. infections (including single- and mixed-species). To address this issue, we employed Firth’s Bias-Reduced logistic regression. Since the majority of the study participants were re-assessed during each of the five cross-sectional surveys the cluster sandwich variance estimator was used to deal with the correlation of these repeated measurements. All statistical analyses were performed in R version 4.4.1 (2024-06-14) and RStudio version 2024.04.2 Build 764 [47] using base R, tidyverse [48], epiR [49], stats [47], gtsummary [50], nnet, clubSandwich [51], and logistf [52].
Results
The study population characteristics for each of the five cross-sectional surveys or study time points (i.e., 2012, 2015, 2017, 2020, and 2022) are provided in Table 1. The study was designed to look at the asymptomatic reservoir in an age-stratified sample of the Bongo population in the two catchment areas (Vea/Gowrie and Soe). No significant differences between the proportion of participants surveyed in each of the catchment areas were observed. Analyses, unless otherwise stated, combined both catchment areas. Likewise, there was a similar proportion of female and male participants across the five surveys. As there was no difference in infection prevalence between the older age groups (i.e., 21–39 and ≥ 40 years), we combined these age groups and described them collectively as the adult population (≥ 21 years).
Species-specific prevalence and species composition of infections
All DBS samples collected during each of the five study time points (N = 9,693) were processed, including gDNA extraction and species-specific 18S rRNA PCR for Plasmodium spp. detection and identification. Among these samples analysed, 55.1% (N = 5,336) were positive for P. falciparum, 6.2% (N = 598) for P. malariae, and 2.8% (N = 267) for P. ovale spp., including both single- and mixed-Plasmodium spp. infections (Table 2). As expected, no samples were PCR-positive for P. vivax. Across all study time points between 2012 and 2022, single-P. falciparum infections were the most common, accounting for 76.2% to 95.4% of PCR-positive samples (Table 2). In contrast, single minor-species infections were rare, with P. malariae and P. ovale spp. each accounting for less than 2.1% and 0.8% of the PCR-positive samples, respectively (Table 2). Instead, most minor-species infections occurred as mixed infections with P. falciparum, the dominant species. Across the study time points mixed-P. falciparum/P. malariae infections ranged from 2.4% to 15.4%, while mixed-P. falciparum/P. ovale spp. infections ranged from 0.9% to 4.6% of the PCR-positive samples (Table 2). Triple-species infections, although observed, were uncommon throughout the study. Given the paucity of single minor-species infections, both single- and mixed-species infections were grouped together for P. falciparum, P. malariae, and P. ovale spp. in subsequent analyses (S1 Table).
Impact of the IRS and SMC interventions on Plasmodium spp. infections
When we investigated the impact of adding IRS with non-pyrethroid insecticides to LLINs impregnated with pyrethroid insecticides to reduce the size of the Plasmodium spp. reservoir, we found that the prevalence of infection in Bongo, including all species, declined from 73.8% at baseline in 2012 (pre-IRS) to 39.6% in 2015 (Table 2, S1 Table) following the final round of IRS, which decreased transmission intensity of P. falciparum by > 90%, as measured by the entomological inoculation rate [12]. Overall, IRS reduced the prevalence of all Plasmodium spp., while P. falciparum remained the major species present (Fig 2A, Table 2, S1 Table). This decline in prevalence was short lived and in 2017, 32 months after IRS was discontinued, but during the deployment of SMC to all children < 5 years, Plasmodium spp. prevalence rebounded to 64.1% (Table 2). Finally in 2020 and 2022, five and seven years after IRS was withdrawn, but with sustained deployment of SMC, Plasmodium spp. prevalence, although lower than the 2017 rebound, remained high, with 51.0% and 51.8% of the population infected in 2020 and 2022, respectively (Table 2). By monitoring this parasite reservoir temporally, we found that although the prevalence of P. falciparum rebounded following the discontinuation of IRS, the minor-species infections, P. malariae and P. ovale spp., did not follow the same pattern as that observed for P. falciparum, both in the population (Fig 2A, S1 Table) and for each age group (Fig 3A, S1 Table).
(A) Prevalence of P. falciparum (green), P. malariae (blue), and P. ovale spp. (red) infections (including single- and mixed-species infections) during each study time point (S1 Table). (B) Forest plots of the adjusted odds ratios for the association between the study time points for P. malariae (blue, left panel) and P. ovale spp. (red, right panel) infection prevalence compared to October 2012 (i.e., baseline pre-IRS reference). Error bars (vertical: prevalence and horizontal: aOR) in the panels represent the upper and lower limits of the 95% confidence intervals (95% CI). For additional details see S2 Table. For the exact timing of the IRS and SMC interventions please see Fig 1.
(A) Prevalence of P. falciparum (green), P. malariae (blue), and P. ovale spp. (red) infections (including single- and mixed-species infections) by age group during each study time point (S1 Table). Forest plots representing the adjusted odds ratios for the associations between the study time points for (B) P. malariae (blue) and (C) P. ovale spp. (red) infection prevalence in each age group (years) compared to October 2012 (i.e., baseline pre-IRS reference). Error bars (vertical: prevalence and horizontal: aOR) in the panels represent the upper and lower limits of the 95% confidence intervals (95% CI). For additional details see S3 and S4 Tables. For the exact timing of the IRS and SMC interventions please see Fig 1.
Impact of the IRS and SMC on minor-Plasmodium spp. infections
Prior to implementation of the IRS and SMC interventions, Table 2 shows P. falciparum as the dominant species with a substantial hidden reservoir of both P. malariae and P. ovale spp. with an overall prevalence of 13.7% and 5.7%, respectively, in single- and mixed-species infections in all ages (Table 2, S1 Table). Following the addition of IRS to LLINs, prevalence dropped for both P. malariae (13.7% to 1.4%) and P. ovale spp. (5.7% to 0.4%) between 2012 and 2015 (Fig 2A, S1 Table). In fact, in 2015, seven months after the final round of IRS, participants were 92% (aOR = 0.08 [95% CI: 0.05-0.11], p-value < 0.001) and 94% (aOR = 0.06 [95% CI: 0.03-0.13], p-value < 0.001) less likely to have a P. malariae and P. ovale spp. infection, respectively, compared to the 2012 baseline (Fig 2B, S2 Table).
Despite this decline, in 2017, 32 months after the discontinuation of IRS, but during SMC, the prevalence of P. malariae (2.9%) and P. ovale spp. (4.0%) rebounded (Fig 2A, S1 Table). In the adjusted model, the odds of infection for the minor species increased relative to 2015, with approximately a 2-fold increase for P. malariae (aOR = 0.14 [95% CI: 0.11-0.19], p-value < 0.001) and a 10-fold increase for P. ovale spp. (aOR = 0.62 [95% CI: 0.46-0.84] p-value = 0.002) (Fig 2B, S2 Table). It is important to point out that although prevalence rebounded in 2017 for the minor species, prevalence did not fully recover, with the population still being significantly less likely to have a P. malariae (aOR = 0.14 [95% CI: 0.11-0.19], p-value < 0.001) or P. ovale spp. (aOR = 0.62 [95% CI: 0.46-0.84], p-value = 0.002) infection, compared to the 2012 baseline (Fig 2B, S2 Table). In this study we found that although IRS significantly impacted the minor species, they did not rebound at the same rate once IRS was withdrawn. In fact, the prevalence of P. malariae did not recover to the same extent compared to P. ovale spp. in 2017 (Fig 2A, S1 and S3 Tables), possibly due to the ability of P. ovale spp. to relapse.
When we investigated the Bongo population (i.e., in all ages) in 2020, five years post-IRS, but during the continued deployment of SMC, the prevalence of P. malariae continued to recover (7.4%), while the prevalence of P. ovale spp. actually declined (2.6%) after rebounding in 2017 (Fig 2A, S1 Table). According to the adjusted models, although the odds of a P. malariae and P. ovale spp. infection in 2020 were still significantly lower than 2012, the odds of a P. malariae infection (aOR = 0.42 [95% CI: 0.34-0.53] p-value < 0.001) continued to increase over time compared to 2017, while for P. ovale spp. it declined (aOR = 0.40 [95% CI: 0.28-0.57], p-value < 0.001) (Fig 2B, S2 Table).
Finally, when we looked at the population in 2022, more than seven years after IRS was discontinued, but with sustained SMC, both P. malariae prevalence (5.8%) and odds of infection (aOR = 0.35 [95% CI: 0.27-0.44], p-value < 0.001) decreased slightly compared to baseline in 2012 (Fig 2B, S2 Table). For P. ovale spp., prevalence continued to decline (1.3%) in 2022 along with the odds of infection (aOR = 0.20 [95% CI: 0.13-0.31], p-value < 0.001) (Fig 2B, S2 Table). Overall, although the prevalence of the minor-species infections rebounded in the population following the discontinuation of IRS, albeit delayed for P. malariae, neither minor species fully recovered relative to the 2012 baseline (i.e., prior to the sequential IRS and SMC interventions).
Age-specific changes in the minor-Plasmodium spp. infections following IRS and SMC
This study was designed to evaluate the impact of IRS and SMC interventions on the prevalence of Plasmodium spp. infection by age. This is particularly important given that SMC is a chemoprevention strategy targeted at children < 5 years starting in 2016, once IRS was discontinued. For all species, prevalence of infection during each study time point remained highest in the older children (5–10 years) and adolescents (11–20 years) (Fig 3A, S1 Table). In 2015, following IRS, prevalence declined, with participants in each age group being ≥ 76% and ≥ 89% less likely to have a P. malariae and P. ovale spp. infection, respectively, compared to 2012 (p-values < 0.001) (Figs 3B and 3C, S3 and S4 Tables). Although the prevalence of the minor-species infections declined in all age groups following IRS, the greatest reductions were observed among the younger (< 5 years) and older (5–10 years) children surveyed. In fact, in 2015, seven months after the final round of IRS, younger and older children were ≥ 97% and 96% less likely to have a P. malariae and P. ovale spp. infection, respectively, compared to the 2012 baseline (p-values < 0.001) (Figs 3B and 3C, S3 and S4 Tables).
In 2017, 32 months after the discontinuation of IRS, minor species prevalence in the younger children (< 5 years) targeted by SMC, remained significantly lower for both P. malariae (aOR = 0.04 [95% CI: 0.00 – 0.14], p-value < 0.001) and P. ovale spp. (aOR = 0.12 [95% CI: 0.01 – 0.58], p-value = 0.007) compared to 2012 (Figs 3B and 3C, S3 and S4 Tables). Meanwhile for the older children (5–10 years), adolescents (11–20 years), and adults (≥ 21 years) that were all ineligible for SMC, the prevalence of minor-species infections rebounded, although once again P. malariae did not recover as quickly compared to P. ovale spp. (Fig 3A, S1 Table). In fact, further analysis of effect modification by age showed that while the odds of a P. ovale spp. infection for the older age groups (i.e., 5–10, 11–20, and ≥ 21 years) rebounded and was comparable to that observed in 2012 (Fig 3C, S4 Table), this was not the case for P. malariae (Fig 3B, S3 Table). In fact, the odds of a P. malariae infection for the older age groups remained significantly lower in 2017 compared to 2012, despite the discontinuation of IRS (Fig 3B, S3 Table).
During 2020 and 2022, the minor species were rarely detected in the younger children (< 5 years) eligible for SMC, with no such infections identified in 2020 and only one P. malariae infection detected in 2022 (Fig 3A, S1 Table). In both study time points, the majority of P. malariae and P. ovale spp. infections were found in older children (5–10 years) and adolescents (11–20 years) (Fig 3A, S1 Table). Although the prevalence of P. ovale spp. declined for the older age groups (i.e., 5–10, 11–20, and ≥ 21 years) starting in 2020, the prevalence of P. malariae increased (Fig 3A, S1 Table). When we examined this trend in more detail, we found that this delayed recovery of P. malariae was most noticeable for the adolescents (11–20 years) (Fig 3A, S1 Table) where the odds of infection in 2020 (aOR = 1.30 [95% CI: 0.92-1.86], p-value = 0.14) was comparable to baseline in 2012 (Fig 3B, S3 Table). In fact, by 2022, P. malariae prevalence had fully recovered for the adolescent (11–20 years) and adult (≥ 21 years) age groups (Figs 3A and 3B, S2 and S3 Tables). This however was not the case of the younger (< 5 years) and older children (5–10 years). In contrast to this delayed rebound for P. malariae, the odds of P. ovale spp. infections in 2022 continued to decline across all age groups from the rebound observed in 2017 (Fig 3C, S4 Table).
Impact of the IRS and SMC on single- and mixed-Plasmodium spp. infections
Data on the proportions of single- and mixed-species infections during each study time point were further analysed to see how these changes following the sequential IRS and SMC interventions (S1 Fig, Table 2). Even after these interventions were deployed, P. falciparum remained the dominant species in Bongo. Across the five study time points between 2012 and 2022, the P. falciparum proportion, as a single-species infection, was greater than 0.75 (S1A Fig, Table 2). On the other hand, mixed-P. falciparum infections declined in 2015, seven months after the final round of IRS, compared to the baseline in 2012 (S1A Fig, Table 2). From 2017 to 2022, as transmission intensity recovered following the discontinuation of IRS, the proportion of mixed-P. falciparum infections steadily increased from 0.03 in 2015 to 0.12 in 2022 (S1A Fig, Table 2).
When we examined the proportion of single- vs. mixed-species infection patterns based on age, we observed that mixed P. malariae and P. ovale spp. infections occurred mostly in the older children (5–10 years) and adolescents (11–20 years) during each study time point including the 2012 baseline (i.e., prior to the sequential IRS and SMC interventions) (S1D, S1E, and S1F Figs). In contrast, the single-P. malariae and P. ovale spp. infections were found mainly in the adults (≥ 21 years) (S1E and S1F Figs). This is consistent with the hypothesis that as immunity to P. falciparum increases, the prevalence of minor species increases such that the peak prevalence of the minor species and dominant species do not coincide [53].
This pattern holds even with intervention. In particular, we draw attention to increases in single-species infections with P. malariae and P. ovale spp., especially after IRS in adolescents (11–20 years) and adults (≥ 21 years) and to a lesser extent to older children (5–10 years) (S1E and S1F Figs). A notable increase in proportion of P. malariae occurs in 2015 when the prevalence of P. falciparum is the lowest.
Discussion
This observational study is the first to assess the impact of two sequential interventions, IRS and SMC in combination with widespread LLIN usage, on the minor Plasmodium spp. in the reservoir of asymptomatic infection in Ghana and indeed West Africa. Employing species-specific molecular diagnostics, we observed a high prevalence of submicroscopic P. malariae at 13.7% and P. ovale spp. of 5.4% occurring largely as mixed infections with P. falciparum at baseline in 2012. The peak prevalences of these minor species occurred in older children (5–10 years) and adolescents (11–20 years) with detectable infections in both younger children (< 5 years) and adults (≥ 21 years). Given the implementation of malaria control interventions across districts in northern Ghana, although often variable as influenced by shifts in funding and NMEP priorities, such high prevalences of submicroscopic minor-Plasmodium spp. infections were more common than we might have expected.
From the baseline in 2012 (pre-IRS) we were able to assess the efficacy of sequential IRS and SMC interventions. This observational study undertaken over a 10-year study period (2012–2022) demonstrates the effectiveness of the short-term IRS against all Plasmodium spp., even reducing the prevalence of minor Plasmodium spp. to near zero. Upon discontinuation of IRS, age-specific analyses showed a rebound in the prevalence of P. falciparum in 2017 (i.e., 32 months post-IRS), to levels higher than the 2012 baseline in adolescents and adults [20]. A similar age-specific shift, characterized by higher prevalence of minor-Plasmodium spp. infections in adolescents and adults, was observed in 2020 after the initial rebound in 2017 following the withdrawal of IRS and five consecutive years of SMC. These age-shifts in prevalence of all Plasmodium spp. are likely due to loss of acquired immunity in all ages during the short-term IRS and SMC targeting children < 5 years. The older children showed a similar pattern, although with a relatively smaller increase in prevalence for both P. falciparum and minor Plasmodium spp. This can be explained by children who aged out of the < 5 years age group during the study; having received SMC previously, they may have reduced immunity and thus be more susceptible to infection. Additionally, these data could indicate “leakage” of SMC to older children outside the SMC-eligible age range, suggesting administration to children not formally targeted by the NMEP [54,55]. These upward age-specific shifts in prevalence make clear the importance of molecular surveillance to detect low-density asymptomatic infections in all ages. This is not the typical practice in Ghana nor other countries implementing these interventions in West Africa. Instead, the common practice is to undertake surveillance by focusing on children aged 2–10 years, as seen in the Malaria Atlas Project [1].
The reduction in the prevalence of all the Plasmodium spp. in the younger children proves that SMC, in combination with LLINs, is effective at protecting this target age group. These results indicate that the benefits of SMC could be expanded by increasing the ages that are eligible, specifically older children and adolescents. They have been shown in this study and others to be vulnerable and act as reservoirs for asymptomatic infections not only P. falciparum but also minor Plasmodium spp. [5,22,23]. Moreover, non-falciparum infections are mainly found at low-densities and are asymptomatic [56]. By expanding the SMC-eligible age range, the reservoir of Plasmodium spp. would be better controlled with targeted chemoprevention.
The WHO recommends the combination SPAQ for SMC in areas with highly seasonal P. falciparum transmission, as it provides protection for ~28 days, is well tolerated, relatively inexpensive, and has demonstrated appropriate efficacy in Africa [57]. Recently, there have been reports of moderate to high prevalence of mutations in the P. ovale spp. dihydrofolate reductase gene (dhfr), associated with resistance to pyrimethamine, in isolates from Central and East Africa [58], as well as travellers returning from Africa [59] In this study, although we did not test for antimalarial resistance markers in minor Plasmodium spp., we found no evidence of emerging resistance to SPAQ after seven consecutive years of SMC implementation. This is supported by the sustained reduction in infection prevalence within the SMC target age group. Among the 1,014 SMC-eligible children (< 5 years) examined between 2017 and 2022, up to seven years after the introduction of SMC, only two P. malariae infections (2017 and 2022) and one P. ovale spp. infection (2017) were detected. Although we did not observe emergence of P. malariae in this age group, a need for monitoring drug resistance mutations in the non-falciparum species in this area remains.
Looking at age-specific changes in the proportions of each species (S1 Fig), complex changes are seen in response to the sequential IRS and SMC interventions. A clear pattern to emerge was that the proportion of single-P. malariae and P. ovale spp. infections increased after the IRS intervention (2015), especially among the adolescents and adults, and also with age after the withdrawal of IRS (i.e., from 2017 onwards). This speaks to a potential interaction with the dominant species P. falciparum where P. falciparum density decreases with age and also after the IRS intervention [12,19]. If there is a carrying capacity for total Plasmodium spp. parasite numbers, as proposed by Bruce et al. [53], a decline in P. falciparum numbers would allow for an increase in density by the minor Plasmodium spp.
More importantly, the implications of these shifts for the transmission potential of the minor species remain uncertain. Previous research has shown that asymptomatic P. falciparum infections are major contributors to onward transmission and make up a substantial portion of the human infectious reservoir [60–64]. However, whether this is the case for P. malariae and/or P. ovale spp. infections remains unclear and warrants further investigation. To better understand their contribution to transmission, it will be important to assess gametocytemia across all age groups and, where feasible, to conduct mosquito membrane feeding assays. These approaches would provide critical insights into the contribution of these minor species to the infectious reservoir and how this reservoir is impacted by malaria control interventions.
The highly sensitive, species-specific 18S rRNA PCR [39–41] revealed that the vast majority of minor-Plasmodium spp. infections in Bongo were submicroscopic and only identifiable through PCR. This makes the PCR-based approach more suitable for monitoring the reservoir than microscopy, which detected virtually no minor-species infections. Despite the greater sensitivity of quantitative real-time PCR for molecular epidemiological surveillance of minor-species infections [6,65,66], broad implementation by National Malaria Programmes in moderate- to high-transmission settings remains challenging due to increased costs (e.g., specialized equipment, reagents). A detailed metagenomic analysis in the same Bongo study population further demonstrated the enhanced sensitivity of PCR compared to microscopy for detecting infections in asymptomatic carriers [67]. Notably, when larger blood volumes were used, the study showed that the prevalence of minor-species infections increased 1.93-fold for P. malariae and 2.50-fold for P. ovale spp. [67]. Combined, these findings demonstrate that microscopy-based surveillance underestimates the reservoir, highlighting the need for more sensitive detection methods.
In summary, we have uncovered a substantial “hidden reservoir” of submicroscopic P. malariae and P. ovale spp. infections in all ages in Bongo District, in the Northern Sahelian belt of Ghana. The prevalence of these minor Plasmodium spp. declined following the sequential IRS and SMC interventions implemented to target P. falciparum, which remained the dominant species throughout the study. From our data, we make the case that the reservoir of infection should be monitored by molecular surveillance targeting P. falciparum and minor Plasmodium spp., as high-burden SSA countries like Ghana advance towards malaria elimination.
Supporting information
S1 Text. Supporting information study design overview.
https://doi.org/10.1371/journal.pntd.0014174.s001
(DOCX)
S1 Table. Parasitological characteristics of the Plasmodium spp. infections (including single- and mixed-species infections) during each study time point.
https://doi.org/10.1371/journal.pntd.0014174.s002
(DOCX)
S2 Table. Association between the study time points and P. malariae and P. ovale spp. infection prevalence using the species-specific 18S rRNA PCR.
https://doi.org/10.1371/journal.pntd.0014174.s003
(DOCX)
S3 Table. Stratum-specific estimates for the association between age groups and P. malariae prevalence during each study time point.
https://doi.org/10.1371/journal.pntd.0014174.s004
(DOCX)
S4 Table. Stratum-specific estimates for the association between age and P. ovale spp. prevalence during each study time point.
https://doi.org/10.1371/journal.pntd.0014174.s005
(DOCX)
S1 Fig. Proportion of single- and mixed-Plasmodium spp. infections identified using the species-specific 18S rRNA PCR from October 2012 (Survey 1, pre-IRS), October 2015 (Survey 2, post-IRS), October 2017 (Survey 3, SMC), November 2020 (Survey 4, SMC), to October 2022 (Survey 5, SMC).
Proportion of single- and mixed-species infections (A-C) in the population and (D-F) by age group for P. falciparum (green), P. malariae (blue), and P. ovale spp. (red) during each study time point. For each Plasmodium spp. darker shades represent those isolates with single-species infections, while the lighter shades denote those isolates with mixed-species infection (including double- and triple-species infections). Blank or white spaces indicate no infections detected by the species-specific 18S rRNA PCR. For the exact timing of the IRS and SMC interventions please see Fig 1.
https://doi.org/10.1371/journal.pntd.0014174.s006
(DOCX)
Acknowledgments
We thank the participants, communities, traditional leadership, and the Ghana Health Service in Bongo District, Ghana for their willingness to participate in this study. We would like to thank the field teams in Bongo for their technical assistance in the field, as well as the laboratory personnel at the Navrongo Health Research Centre for their expertise and for undertaking the sample collections and parasitological assessments. We thank Prof. Mercedes Pascual (Department of Biology and Department of Environmental Studies, New York University, New York, US) for valuable input on this work and for her role as a multi-principal investigator in securing the funding that supported this research (see Funding for additional details).
A research collaboration was developed with scientists from Ghana based at the Navrongo Health Research Centre and the Noguchi Memorial Institute for Medical Research. All collaborators are included as co-authors and the relevant results from the research has been shared with participants, key stakeholders, traditional leadership, and the local community (i.e., Paramount Chief of Bongo District, divisional Chiefs, Queen Mothers, and community members), the Bongo District and Upper East Regional Health Directorates, as well as Ghana National Malaria Elimination Programme. Before this research was undertaken, informed consent was sought and obtained from the key stakeholders, traditional leadership, and the local community in Bongo District. In addition, members of the local community were trained as field workers and were directly involved in liaising with the community and in the collection of the study data. The contribution of these individuals to this research is described in the Acknowledgements. This research addresses a priority concern regarding malaria control and the impact of interventions. These concerns are relevant for both the local community in Bongo District, the Bongo District and Upper East Regional Health Directorates, as well as for the National Malaria Elimination Programme in Ghana.
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