Mensah et al. BMC Public Health (2021) 21:1697 https://doi.org/10.1186/s12889-021-11714-8 RESEARCH Open Access Prevalence and risk factors associated with asymptomatic malaria among school children: repeated cross-sectional surveys of school children in two ecological zones in Ghana B. A. Mensah1, J. L. Myers-Hansen1,2, E. Obeng Amoako1, M. Opoku1,2, B. K. Abuaku1† and A. Ghansah1,2*† Abstract Background: Asymptomatic Plasmodium infections significantly drive malaria transmission and impact control and elimination strategies, but are largely uncharacterized. We investigated the prevalence and risk factors of asymptomatic malaria infections to inform malaria control strategies in Ghana. Method: Five cross-sectional surveys were conducted at the end of the peak transmission season (August– September) on 4892 school children aged between 6 and 14 years in two distinct ecological settings in Ghana between 2013 and 2017. The study sites were Begoro (forest ecology) and Cape Coast (coastal ecology). The children were screened for malaria parasites by microscopic examination of Giemsa-stained thin and thick blood films. Hemoglobin levels were measured using the Hemocue HB analyzer. In addition, height was measured and the height-for-age z-scores estimated from the reference population defined by WHO to determine children who were stunted. Proportions of categorical and means of continuous variables were compared using Chi-square test and Student’s t-test respectively, and multivariable logistic regression was done to assess risk factors associated with asymptomatic infections. Results: The overall prevalence of asymptomatic malaria in the school children was higher in Begoro compared to Cape Coast (27% (95% CI: 17, 24%) vs. 24% (95% CI: 17, 24%), p value = 0.04). The study recorded three species of Plasmodium (Plasmodia falciparum, malariae, and ovale) in both sites. Plasmodium falciparum was the predominant species, accounting for about 85% of infections in both study sites. The asymptomatic school children were more likely to be anaemic (OR = 2.01, p value< 0.001) and stunted in growth (OR = 1.46, p value< 0.001). Males carried more asymptomatic infection than females (OR = 1.18, p value = 0.015). School children aged 12–14 years had more asymptomatic infections than those aged 6–8 years (OR = 1.28, p value = 0.005). * Correspondence: Aghansah@noguchi.ug.edu.gh †B. K. Abuaku and A. Ghansah contributed equally to this work. 1Department of Epidemiology, College of Health Sciences, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana 2Department of Parasitology, College of Health Sciences, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mensah et al. BMC Public Health (2021) 21:1697 Page 2 of 9 Conclusion: There is a considerable burden of asymptomatic malaria in the two regions of Ghana, which is associated with males, older children, anaemia, and stunted growth in children, and may have implications for malaria control and elimination strategies in Ghana. Keywords: Asymptomatic infection, Plasmodium, Risk factors, Malaria burden, Ghana Background are more infectious than symptomatics [9, 17, 18] as Malaria is still one of the leading global public health they contribute to infectiousness for longer periods of concerns. Although there was a decline in malaria mor- time when they are not treated [19]. Aside from it being bidity and mortality worldwide in 2018, sub-Saharan Af- a reservoir for malaria transmission, asymptomatic car- rica saw an increase of about one million cases that riage causes several other challenges including but not same year [1]. Economic data from WHO malaria report limited to anaemia, stunting, and cognitive impairment shows that, in 2018, the WHO African Region spend in school children [20, 21]. nearly 3 quarter of the US$ 2.7 billion invested in mal- Like most malaria control programs, control efforts in aria on case management and prevention [1] and cost up Ghana gravitate towards a system, which identifies, to 1.3% of GDP in Africa [2]. In addition, malaria is a treats, and reports people with malaria presenting at major cause of absenteeism among school children in health care facilities giving little attention to asymptom- endemic countries [3–5]. atic cases in the community. However, through seasonal Ghana is a malaria-endemic country and falls within malaria chemoprevention and intermittent preventive the top 15 highest-burden countries [1]. In 2018, Gha- treatments, a subset of asymptomatic infections is na’s disease burden contributed to about 3% of the glo- treated but not tracked. For successful malaria elimin- bal malaria cases recorded [1] and 33% of all the ation, however, the full complement of the burden of in- national Out-Patient Department (OPD) cases recorded fection, including the asymptomatic reservoir should be in 2017 [6]. Pregnant women and children under 5 years the focus. Thus, the asymptomatic carrier must be ac- are the most affected by the disease because they have tively characterized [22]. low immunity and malaria can cause anaemia in both To better understand the asymptomatic burden in groups resulting in debilitating outcomes like cerebral Ghana, we leveraged repeated school-based cross- malaria and death [1]. sectional surveys on the impact of parasite diversity on Plasmodium falciparum infections may result in se- the evolution of drug resistance. We characterized the vere, uncomplicated or asymptomatic malaria. There is a asymptomatic burden and its associated risk factors in wealth of studies and data on uncomplicated and severe two sites in Ghana with varying malaria endemicities. malaria because they are the drivers of malaria-related We show that the asymptomatic burden is significant in morbidity and mortality respectively. Comparatively, both sites and are influenced by risk factors such as age, asymptomatic malaria infection is understudied and re- anaemia, and stunted growth in children. main a challenge to malaria control due to its effect on transmission dynamics [7]. Methods Asymptomatic malaria, is defined as the presence of Study design, setting, population and ethics approval parasites in people and an absence of malaria-related Repeated cross-sectional surveys were conducted in symptoms such as temperature > 37.5 °C. Asymptomatic Cape Coast and Begoro for five consecutive years malaria occurs as a result of continuous exposure to (2013–2017). Cape Coast is in the coastal savanna eco- malaria infections, leading to the acquisition of partial logical zone of Ghana where malaria transmission is low immunity against complications such as cerebral malaria to moderate and perennial. Begoro is situated in the for- and the accumulation of the “reservoir of infection” [8]. est ecological zone of Ghana, where malaria transmis- P. falciparum infection has been shown to persist sion is high and perennial. The detailed description of asymptomatically in semi-immune individuals for more these study sites has been described elsewhere [23]. The than 18 months especially in older children [7] making study participants included children aged between 6 and them important reservoirs for sustaining malaria trans- 14 years attending selected schools in the catchment mission in regions of low and high malaria endemicity areas of the study, whose parents/guardians gave in- [9–15]. The asymptomatic reservoir contributes to gam- formed consent to be part of the study and who also etocyte carriage (the stage of the life-cycle of the para- assented if aged 12 to 14. Three schools were randomly sites that cause mosquito transmission) to drive and selected in each ecological zone and the children aged maintain transmission by the local mosquito vectors [7, between 6 years and 14 years screened for malaria infec- 16]. Some findings suggest that asymptomatic carriers tion using microscopy. Children who were on malaria Mensah et al. BMC Public Health (2021) 21:1697 Page 3 of 9 medication within 2 weeks prior to recruitment into the Stunting in children is defined as height-for-age z- study and tested positive were excluded. The study was score (HAZ) below negative two standard deviations conducted following the latest Declaration of Helsinki from the median of the reference population, using and Good Clinical Practice (GCP). The protocol was ap- the WHO Child Growth Standards proved by the ethics review board of Noguchi Memorial Institute for Medical Research, University of Ghana. Statistical analysis The variables hemoglobin and height-for-age z-scores Sample size and power were categorized into binary variables. Height-for-age z- The study primarily compared the prevalence of asymp- scores were categorized as “stunted” (z-score less than tomatic malaria in the two study sites. A minimum sam- or equal to − 2 SD) and “not stunted” (z-score greater ple size of 1933 per study site provides 80% power to than − 2 SD), haemoglobin was categorized as anaemic determine as low as a 4% difference in asymptomatic (HB < 11 g/dl) and “non-anaemic” (HB ≥ 11 g/dl). All malaria between study sites at a 95% confidence level statistical analyses were performed using the statistical and 5% precision. software STATA version 12. Continuous data were sum- marized into means and standard deviations (SD) (age, weight, height, parasite density, haemoglobin, and axil- Sampling method lary temperature) and categorical data into proportions The list of all schools in the catchment areas of this for descriptive analysis. Parasite density which is not study was obtained from the municipal education office normally distributed was log-transformed and the geo- and three schools randomly selected for each study site. metric mean calculated. Means were compared between All the school children aged 6 to 14 years from the se- study sites using Student’s t-test/Mann Whitney rank lected schools, whose parents gave informed consent test, while proportions were compared using Pearson’s and assented if aged 12 to 14 were screened. Chi-square tests/Fisher exact test. Trends of asymptom- atic malaria over the study period were compared using Sample collection and diagnostic methods the Cochrane Armitage test of trends. Univariate ana- Blood smears were prepared for each participant in the lysis to determine potential risk factors of asymptomatic study. Two slides per participant were obtained: one malaria was performed using the Student’s t-test/Mann with a thick smear and the other with both thick and Whitney rank test for continuous variables and Pearson thin smears. The thick smear slide was stained rapidly Chi-square test/Fisher exact test for categorical variables. (10% Giemsa for 10–15 min) for initial screening, while The association between asymptomatic malaria and an- the thick and thin smear slides were retained for subse- aemia, stunting, gender, age, and study site was explored quent quantification of the parasites if the participant using multivariable logistic regression and the odds ratio tested positive. The parasite density was estimated, by and confidence intervals recorded. The significance level counting the number of asexual parasites against 200 was set at p < 0.05 for all tests. WBC with a hand tally counter. Parasite density was cal- culated as follows: Parasite density (per μl) = (number of Results parasites counted ÷ number of leukocytes counted) × Baseline characteristics of study participants and the 8000. Haemoglobin levels were determined for all pa- prevalence of asymptomatic malaria tients using a portable HemoCue® Hb 301 Analyzer A total of 4892 school children aged between 6 and 14 (HemoCue® AB, Ängelholm, Sweden). The body weight, years, were screened; 2394 from Begoro and 2498 from height, and axillary temperature were also measured. Cape Coast over the five-year study period. Of these, Children who tested positive for malaria and had fever 52.9% were females, 13.9% were anaemic, 11.1% had were treated with artesunate amodiaquine as recom- stunted growth, mean age was 10.3 years, and the geo- mended by the national malaria control program metric mean of parasite density was 289.4. Children in (NMPC). the Cape Coast municipal catchment area were more an- aemic compared to those recruited from Begoro (17% vs Definition of terms 10% respectively, p value< 0.001). Also, the proportion of children from Cape Coast who were stunted was higher Asymptomatic malaria: the presence of parasites in than observed in Begoro (14.5% vs 7.6% respectively, p peripheral thick blood smears, an axillary value< 0.001). With the exception of 2014, the preva- temperature < 37.5 °C, and an absence of malaria- lence of asymptomatic malaria was always higher in related symptoms [7]. Begoro compared to Cape Coast. The overall prevalence Anaemia: Anaemia in children was defined as having of asymptomatic malaria in Begoro was higher than in haemoglobin (Hb) < 11 g/dL Cape Coast (27% vs 24%, p value = 0.04 Table 1), though Mensah et al. BMC Public Health (2021) 21:1697 Page 4 of 9 Table 1 Baseline characteristics of study participants by ecological zones Characteristics Total Ecological zone Forest (FZ) Coastal (CSZ) p value Prevalence of malaria n/N (%) 2013 273/1037 (26.3) 127/478 (26.6) 146/559 (26.1) 0.869 2014 161/781 (20.6) 62/382 (16.2) 99/399 (24.8) 0.003 2015 261/935 (27.9) 142/452 (31.42) 119/483 (24.6) 0.021 2016 241/1027 (23.5) 127/525 (24.2) 114/502 (22.47) 0.576 2017 300/1112 (27.0) 178/557 (32.0) 122/555 (22.0) < 0.001 Total 1236/4892 (25.3) 636/2394 (26.6) 600/2498 (24.0) 0.040 Mean age in years (SD) 10.3 (2.4) 10.3 (2.3) 10.3 (2.5) 0.5258 Age group n (%) in years 0.016 Aged 6 to 8 years 1247 (25.5) 602 (25.2) 645 (25.9) Aged 9 to 11 years 1862 (38.1) 959 (40.1) 903 (36.2) Aged 12 to 14 years 1779 (36.4) 833 (34.8) 946 (37.9) Gender n (%) 0.148 Female 2589 (52.9) 1292 (53.9) 1296 (51.9) Male 2303 (47.1) 1103 (46.1) 1202 (48.1) Anaemia n/N (%) < 0.001 Normal 4160/4831 (86.1) 2139/2390 (89.5) 2021/2441 (82.8) Anaemic 671/4831 (13.9) 251/2390 (10.5) 420/2441 (17.2) Stunted growth < 0.001 Normal 4349 (88.9) 2213 (92.4) 2136 (85.5) Stunted 545 (11.1) 182 (7.6) 363 (14.5) Geometric mean parasitaemia/μL 289.4 289.9 295.0 0.9331 Parasitaemia range (13.79, 95,400) (13.8, 65,910.5) (8.0, 95,400) there was no significant trend over the five-year period non-anaemic children (38% vs. 23%, p value< 0.001). of the study. Three out of the five human Plasmodium Prevalence of asymptomatic malaria was higher in chil- species were found in both study sites (Fig. 1). These dren who were stunted (34%) compared to those who were P. falciparum (PF, 82 and 85.6% in Begoro and were not (34% vs. 24%, p value< 0.001). Children living Cape Coast respectively), P. malariae (PM, 14 and 10% in the Begoro catchment area were more likely to be in Begoro and Cape Coast respectively), P. ovale (PO, < asymptotic carriers than those living in Cape Coast 1% in both Begoro and Cape Coast). Cape Coast had 3% (26.6% vs. 24%, p value< 0.04). mixed infections of PF, PM, and 0.3% of PF, PO, while, Begoro had 2% of PF, PM but no mixed infection of PF, Multivariable analysis of potential risk factors of PO. (Fig. 2 below). asymptomatic malaria A logistic regression model was used to explore the as- Determinants of asymptomatic malaria in school children sociations between the potential risk factors and asymp- Table 2 shows the determinants of asymptomatic mal- tomatic malaria. After adjusting for confounders (age, aria in school-aged children in the two study sites in stunted growth, anaemia and study site), being male in- Ghana. All five risk factors considered: gender, age, an- creased the odds of asymptomatic malaria by 18% (OR = aemia, study site, and stunting were associated with 1.18, 95% CI = 1.03, 1.35, p value = 0.015). The odds of asymptomatic malaria. Asymptomatic carriage was asymptomatic malaria in the 12–14 years age group was higher in males compared to females (28% vs. 23% p 1.28 times higher than that in the 6–8 years age group value = 0.001). In comparison with the other age group- (OR = 1.28, 95%CI = 1.08, 1.53, p value = 0.005). Anaemic ings, the older children (aged between 12 and 14 years) children were twice more likely to carry asymptomatic were more asymptomatically infected (27% vs. 25 and infections (OR = 2.01, 95%CI = 1.77, 2.51, p value< 0.001) 23%, p value = 0.019). The prevalence of asymptomatic than the non-anaemic children and stunted growth in- malaria was higher in anaemic children compared to the creased the odds of carrying asymptomatic parasites by Mensah et al. BMC Public Health (2021) 21:1697 Page 5 of 9 Fig. 1 Map of Ghana showing the location of the two study sites considered in the study. Begorois considered a forest ecological zone where malaria is hyperendemic and has a high transmission intensity. Cape Coast, is considered a coastal savanna zone where malaria is also hyperendemic, but it has a low to moderate transmission intensity Fig. 2 Proportion of Plasmodium sp. in two ecological zones in Ghana. PF = Plasmodium falciparum, PM = Plasmodium malariae, PO = Plasmodium ovale, PF, PM = Plasmodium falciparum and malariae mixed infection, and PF, PO = Plasmodium falciparum and ovale mix infection Mensah et al. BMC Public Health (2021) 21:1697 Page 6 of 9 Table 2 Factors associated with asymptomatic malaria Our repeated cross-sectional surveys estimated the Parameter Asymptomatic malaria p prevalence of the asymptomatic infections in catchment No Yes value areas of two ecological zones in Ghana and also identi- N % N % fied associated risk factors of asymptomatic malaria. The Gender 0.001 study revealed a high asymptomatic burden (~ 20%) in the two catchment areas studied, where most of the in- Male 1669 72.5 633 27.5 fections were P. falciparum. A few infections were (P. Female 1986 76.7 603 23.3 malariae, P. ovale, with mixed infection P. falciparum + Age group (years) 0.019 P. malariae and P. falciparum + P. ovale). As expected, 6 to 8 962 77.2 284 22.8 asymptomatic malaria was higher in Begoro, within the 9 to 11 1396 75.0 466 25.0 forest ecological niche compared to Cape Coast in the 12 to 14 1293 72.7 485 27.3 coastal region of Ghana and was associated with age, gender, anaemia and stunted growth in school children. Anaemia < 0.001 The outcomes of this study highlight the need to expand Non anaemic (≥11) 3193 76.8 965 23.2 control interventions and educational campaigns for Anaemic (< 11) 414 61.7 257 38.3 asymptomatic malaria in Ghana with particular focus on Stunted growth < 0.001 males and older children. Normal 3295 75.8 1053 24.2 The variation in asymptomatic carriage in these differ- Stunted 361 66.2 184 33.8 ent ecological zones is consistent with transmission in- tensity in these two sites. The increased odds of Study site asymptomatic malaria in the forest ecological zone may Begoro 1758 73.4 636 26.6 0.04 be due to more favorable climatic conditions such as Cape Coast 1898 76.0 600 24.0 temperature, abundant rainfall and the vegetative cover, resulting in an abundance of breeding sites for the mos- 46% (OR = 1.46, 95%CI = 1.19, 1.80, p value< 0.001). In quito vector in these areas. These variations observed in addition, the study showed an increased odd of 26% transmission between communities reflect the import- asymptomatic carriage in Begoro (forest ecological zone) ance of micro-ecological factors and transmission in the compared to Cape Coast (in the coastal savanna zone) areas studied [24]. The bulk of malaria cases in sub- after adjusting for age, sex, anaemia and stunted growth Saharan Africa are caused by Plasmodium falciparum, (OR = 1.26, 95% CI = 1.10, 1.44, p value = 0.001) with other Plasmodium species having a low but under- (Table 3). estimated prevalence [1, 25]. The distribution of Plasmo- dium species in this study is consistent with other Discussion studies conducted in Ghana. P. falciparum is the most With the clarion call for malaria elimination, attention common malaria-causing species in Ghana, followed by has been drawn to the asymptomatic reservoir of infec- P. malariae and P. ovale [25–27]. tion as it significantly contributes to Plasmodium trans- The prevalence of asymptomatic infections among mission. Thus, recent epidemiological studies in most children was more predominant in males than females. malaria-endemic countries are not only focusing on This disparity may come as a result of gender roles with symptomatic malaria for disease morbidity and mortality regards to division of labour, hormonal or host genetic rates but also the asymptomatic infection rate to fully factors [28, 29], and behavioral factors like leisure activ- define the disease burden during malaria control and ities and sleeping patterns [30]. Males tend to stay late elimination interventions. outdoors and without proper protective clothing and Table 3 Some risk factors associated with asymptomatic malaria Parameter Asymptomatic malaria Exposed group Unadjusted OR p value Adjusted OR p value (95% CI) (95% CI) Gender Males 1.24 (1.10, 1.42) 0.001 1.18 (1.03, 1.35) 0.015 Age group (years) 9 to 11 1.13 (0.96, 1.34) 0.154 1.11 (0.93, 1.32) 0.2235 12 to 14 1.27 (1.07, 1.50) 0.005 1.28 (1.08, 1.53) 0.005 Anaemia Anaemic 2.05 (1.73, 2.44) < 0.0001 2.01 (1.77, 2.51) < 0.001 Stunted growth Stunted 1.59 (1.32, 1.93) < 0.0001 1.46 (1.19, 1.80) < 0.001 Study site Begoro 1.14 (1.01, 1.30) 0.04 1.26 (1.10, 1.44) 0.001 Mensah et al. BMC Public Health (2021) 21:1697 Page 7 of 9 they also have the tendency to sleep outdoors or not Our study revealed that stunting was associated sleep under a mosquito net. It has been suggested that with asymptomatic malaria though a similar study males usually have lower immune response than females conducted in another forest region of Ghana did not [31]. The immunological differences between males and show an association between stunting and asymptom- females, linked to circulating steroid hormones such as atic malaria [40]. The inconsistency in the results testosterone, oestradiol, and progesterone may explain may be due to smaller sample size in the other study. the increased parasitism in males [31]. Not only do host Evidence of an association between malaria risk and hormones influence infection responses, but parasites in anthropometric indicators such as stunting remains their hosts can also generate and modify hormone con- inconclusive [41]. While some studies have reported centrations [32]. Some of these gender differences that that stunting is associated with a higher risk of affect disease outcomes includes access to immunization, asymptomatic malaria as reported in this study [42, nutritional status, access to, and use of preventive and 43], others have suggested a protective effect [20, 44– curative health care, including differences in the speed 47]. For instance, Mitangala and colleagues in 2013 with which males and females get treatment outside the showed that severely stunted children were at a lower home [30]. Understanding how gendered patterns of be- risk of high-level malaria [46]. Some studies also sug- havior influence exposure to mosquitoes can therefore gest no association between anthropometric indicators assist in developing more-effective recommendations for and asymptomatic malaria risk [40, 48, 49]. How mal- preventing malaria infection especially in males. nutrition influences malaria morbidity and mortality In this study, children aged between 12 and 14 is debatable. Some studies suggest that malnourished years were 21% more asymptomatic than children children seem to be more susceptible to malaria para- aged between 6 and 8 years. This was consistent with site carriage because of decreased immune system findings from other studies conducted in Ghana functioning [50]. This complex relationship may be [33–35]. Age has been shown to be associated with influenced by confounders like socio-economic factors malaria in general and studies have reported its as- such as poverty, level of literacy of parent/guardian, sociation with asymptomatic malaria carriage [34]. common diet of community etc. [51]. An increase in age is positively associated with pro- This study had a few limitations. The study might have tective immunity to malaria in endemic settings under-reported the prevalence of asymptomatic malaria where older children and adults have acquired par- because microscopic detection of parasites as used in the tial immunity and are more likely to carry asymp- study has low sensitivity compared to polymerase chain tomatic infections [34, 36]. reaction (PCR), although it is the gold standard for diag- Our study recorded a doubled risk of anaemia in chil- nosing malaria in endemic countries. Evidence of under- dren who had asymptomatic infections. Malaria- reporting of malaria cases by microscopy has been associated anaemia has been well documented, however, shown [52, 53] compared to PCR. Microscopy reported few studies have looked at the impact of asymptomatic a false negative diagnosis of 19.4% [52]. Also, the study malaria on anaemia in healthy children in schools. In did not consider household factors and the use of in- general, malaria contributes to the loss of iron from secticide treated bed nets, which are also risk factors of lysed cells through excretion, impairment of intestinal malaria. absorption of ingested iron, the release of storage iron from hepatocytes, and recycling of iron that is derived from phagocytosis of senescent or parasitized erythro- Conclusion cytes by macrophages [37]. However, asymptomatic The prevalence of asymptomatic malaria was high in lower-density parasitemia may also contribute to an- both study sites in Ghana. Asymptomatic malaria among aemia, particularly if the parasitemia persists for pro- the school children was age-dependent with a higher risk longed periods due to lack of, or ineffective treatment in male children. A significant association was indicated [38, 39]. In addition, school children living in the Cape between asymptomatic malaria and the risk of anaemia coast catchment area were more anaemic than their and stunting. Thus, malaria control programs should counterparts living in Begoro. Although our study was also focus on asymptomatic malaria as a means to moni- not designed to obtain information on diet, the anec- tor the impact of control interventions and to reduce dotal evidence indicates that the vegetation of Begoro al- malaria morbidity and mortality. lows for an abundant propagation of iron rich green leafy vegetables as staple for the community including Abbreviations our study catchment area in comparison with Cape GCP: Good Clinical Practice; HAZ: Height-for-Age Z scores; OPD: Out-Patient Department; NMPC: National Malaria Control Program; PF: Plasmodium Coast where the vegetation is mostly mangrove with falciparum; PM: Plasmodium malariae; PO: Plasmodium ovale; WHO: World salty patches. Health Organization Mensah et al. BMC Public Health (2021) 21:1697 Page 8 of 9 Acknowledgments 10. Lennon SE, Miranda A, Henao J, Vallejo AF, Perez J, Alvarez A, et al. Malaria The authors will like to thanks the study participants, parents/guardians, staff elimination challenges in Mesoamerica: evidence of submicroscopic malaria and management of the school that participated in the study from Begoro reservoirs in Guatemala. Malar J. 2016;15(1):441. https://doi.org/10.1186/s12 and Cape Coast. 936-016-1500-6. 11. Lin JT, Saunders DL, Meshnick SR. The role of submicroscopic parasitemia in Authors’ contributions malaria transmission: what is the evidence? Trends Parasitol. 2014;30(4):183– BAM implemented the study and wrote the first draft of the manuscript. AG 90. https://doi.org/10.1016/j.pt.2014.02.004. and BKA conceptualized the idea, were involved in the design, 12. Stresman GH, Baidjoe AY, Stevenson J, Grignard L, Odongo W, Owaga C, implementation and writing of the manuscript. JLMH, MO, EOA, were et al. Focal screening to identify the subpatent parasite reservoir in an area involved in data collection and review of the manuscript. The author(s) read of low and heterogeneous transmission in the Kenya highlands. J Infect Dis. and approved the final manuscript. 2015;212(11):1768–77. https://doi.org/10.1093/infdis/jiv302. 13. Sturrock HJ, Hsiang MS, Cohen JM, Smith DL, Greenhouse B, Bousema T, Funding et al. Targeting asymptomatic malaria infections: active surveillance in The study was funded by the National Institute of Health grant number control and elimination. PLoS Med. 2013;10(6):e1001467. https://doi.org/1 R01A1099527. 0.1371/journal.pmed.1001467. 14. Tadesse FG, Slater HC, Chali W, Teelen K, Lanke K, Belachew M, et al. The Availability of data and materials relative contribution of symptomatic and asymptomatic plasmodium vivax The dataset used and/or analysed during the current study is available from and plasmodium falciparum infections to the infectious reservoir in a low- the corresponding author on reasonable request. endemic setting in Ethiopia. Clin Infect Dis. 2018;66(12):1883–91. https://doi. org/10.1093/cid/cix1123. Declarations 15. Zoghi S, Mehrizi AA, Raeisi A, Haghdoost AA, Turki H, Safari R, et al. Survey for asymptomatic malaria cases in low transmission settings of Iran under Ethics approval and consent to participate elimination programme. Malar J. 2012;11(1):126. https://doi.org/10.1186/14 The protocol was approved by the ethics review board of Noguchi Memorial 75-2875-11-126. Institute for Medical Research, University of Ghana (IRB approval no. 056/12– 16. Alves FP, Gil LHS, Marrelli MT, Ribolla PE, Camargo EP, Da Silva LHP. 13). Written informed consent was obtained from the parents or guardians Asymptomatic carriers of plasmodium spp. as infection source for of all children before enrollment. Additionally, children aged 12 years to 14 malaria vector mosquitoes in the Brazilian Amazon. J Med Entomol. years gave their assent before enrollment. The study was conducted 2005;42(5):777–9. https://doi.org/10.1603/0022-2585(2005)042[0777:A following the latest Declaration of Helsinki and Good Clinical Practice (GCP). COPSA]2.0.CO;2. 17. Ouedraogo AL, Goncalves BP, Gneme A, Wenger EA, Guelbeogo MW, Consent for publication Ouedraogo A, et al. Dynamics of the human infectious reservoir for malaria Not applicable. determined by mosquito feeding assays and ultrasensitive malaria diagnosis in Burkina Faso. J Infect Dis. 2016;213(1):90–9. https://doi.org/10.1093/infdis/ Competing interests jiv370. Authors declare no conflicting interest. 18. Schneider P, Bousema JT, Gouagna LC, Otieno S, van de Vegte-Bolmer M, Omar SA, et al. Submicroscopic plasmodium falciparum gametocyte Received: 11 January 2021 Accepted: 29 August 2021 densities frequently result in mosquito infection. Am J Trop Med Hyg. 2007; 76(3):470–4 Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/17360869. 19. Rovira-Vallbona E, Contreras-Mancilla JJ, Ramirez R, Guzmán-Guzmán M, References Carrasco-Escobar G, Llanos-Cuentas A, et al. Predominance of asymptomatic 1. WHO. World malaria report 2019. 2019a .Retrieved from https://www.who. and sub-microscopic infections characterizes the plasmodium gametocyte int/publications-detail/world-malaria-report-2019. reservoir in the Peruvian Amazon. PLoS Negl Trop Dis. 2017;11(7):e0005674. 2. United Nations Children’s Fund. UNICEF report: improving child nutrition: https://doi.org/10.1371/journal.pntd.0005674. the achievable imperative for global progress. 2013. Retrieved from. 20. Goyal S. Protein energy malnutrition and cerebral malaria. J Trop Pediatr. 3. Brooker SJ, Clarke S, Fernando D, Gitonga CW, Nankabirwa J, Schellenberg 1991;37(3):143–4. D, Greenwood B. Malaria in middle childhood and adolescence. Disease 21. Thuma PE, Van Dijk J, Bucala R, Debebe Z, Nekhai S, Kuddo T, et al. Distinct Control Priorities, (Volume 8): Child and Adolescent Health and clinical and immunologic profiles in severe malarial anemia and cerebral Development, 1982. 2017. malaria in Zambia. J Infect Dis. 2011;203(2):211–9. https://doi.org/10.1093/ 4. Kimbi HK, Awah NW, Ndamukong KJ, Mbuh JV. Malaria infection and its infdis/jiq041. consequences in school children. East Afr Med J. 2005;82(2):92–7. https:// 22. Kastenmuller K, Espinosa DA, Trager L, Stoyanov C, Salazar AM, Pokalwar S, doi.org/10.4314/eamj.v82i2.9261. et al. Full-length plasmodium falciparum circumsporozoite protein 5. Nankabirwa J, Brooker SJ, Clarke SE, Fernando D, Gitonga CW, Schellenberg administered with long-chain poly(I.C) or the toll-like receptor 4 agonist D, et al. Malaria in school-age children in Africa: an increasingly important glucopyranosyl lipid adjuvant-stable emulsion elicits potent antibody and challenge. Tropical Med Int Health. 2014;19(11):1294–309. https://doi.org/1 CD4+ T cell immunity and protection in mice. Infect Immun. 2013;81(3): 0.1111/tmi.12374. 789–800. https://doi.org/10.1128/IAI.01108-12. 6. NMCP. National malaria program annual report 2017. 2017. Retrieved from 23. Mensah BA, Aydemir O, Myers-Hansen JL, Opoku M, Hathaway NJ, Marsh http://www.ccmghana.net/index.php/strategic-plans-reports%3Fdownloa PW, et al. Antimalarial drug resistance profiling of plasmodium falciparum d%3D200:nmcp-2017. infections in Ghana using molecular inversion probes and next generation 7. Laishram DD, Sutton PL, Nanda N, Sharma VL, Sobti RC, Carlton JM, et al. sequencing. Antimicrob Agents Chemother. 2020;64(4). https://doi.org/1 The complexities of malaria disease manifestations with a focus on 0.1128/AAC.01423-19. asymptomatic malaria. Malar J. 2012;11(1):29. https://doi.org/10.1186/1475-2 24. Dery DB, Brown C, Asante KP, Adams M, Dosoo D, Amenga-Etego S, et al. 875-11-29. Patterns and seasonality of malaria transmission in the forest-savannah 8. Bousema JT, Gouagna LC, Drakeley CJ, Meutstege AM, Okech BA, Akim IN, transitional zones of Ghana. Malar J. 2010;9(1):314. https://doi.org/10.1186/14 et al. Plasmodium falciparum gametocyte carriage in asymptomatic children 75-2875-9-314. in western Kenya. Malar J. 2004;3(1):18. https://doi.org/10.1186/1475-2 25. Amoah LE, Donu D, Abuaku B, Ahorlu C, Arhinful D, Afari E, et al. Probing 875-3-18. the composition of plasmodium species contained in malaria infections in 9. Gouagna LC, Ferguson HM, Okech BA, Killeen GF, Kabiru EW, Beier JC, et al. the eastern region of Ghana. BMC Public Health. 2019;19(1):1–11. https://doi. Plasmodium falciparum malaria disease manifestations in humans and org/10.1186/s12889-019-7989-1. transmission to Anopheles gambiae: a field study in Western Kenya. 26. Owusu ED, Brown CA, Grobusch MP, Mens P. Prevalence of plasmodium Parasitology. 2004;128(Pt 3):235–43. https://doi.org/10.1017/s0031182003 falciparum and non-P. falciparum infections in a highland district in Ghana, 00444x. and the influence of HIV and sickle cell disease. Malar J. 2017;16(1):1–8. Mensah et al. BMC Public Health (2021) 21:1697 Page 9 of 9 27. Williams O, Meek S. Malaria: country profiles. London: Department of children. Am J Clin Nutr. 1998;68(3):734–41. https://doi.org/10.1093/ajcn/ international development; 2011. 68.3.734. 28. Pathak S, Rege M, Gogtay NJ, Aigal U, Sharma SK, Valecha N, et al. 46. Mitangala P, D’Alessandro U, Donnen P, Hennart P, Porignon D, Balaluka GB, Age-dependent sex bias in clinical malarial disease in hypoendemic et al. Infection palustre et état nutritionnel: résultats d’une cohorte d’enfants regions. PLoS One. 2012;7(4):e35592. https://doi.org/10.1371/journal. âgés de 6 à 59 mois au Kivu en République démocratique du Congo. Rev pone.0035592. Epidemiol Sante Publique. 2013a;61(2):111–20. https://doi.org/10.1016/j. 29. van Eijk AM, Sutton PL, Ramanathapuram L, Sullivan SA, Kanagaraj D, Priya respe.2012.06.404. GSL, et al. The burden of submicroscopic and asymptomatic malaria in India 47. Mzumara B, Bwembya P, Halwiindi H, Mugode R, Banda J. Factors revealed from epidemiology studies at three varied transmission sites in associated with stunting among children below five years of age in Zambia: India. Sci Rep. 2019;9(1):17095. https://doi.org/10.1038/s41598-019-53386-w. evidence from the 2014 Zambia demographic and health survey. BMC 30. Stukel TA, Glynn RJ, Fisher ES, Sharp SM, Lu-Yao G, Wennberg JE. Nutrition. 2018;4(1):51. https://doi.org/10.1186/s40795-018-0260-9. Standardized rates of recurrent outcomes. Stat Med. 1994;13(17):1781–91. 48. Awuah RB, Asante PY, Sakyi L, Biney AAE, Kushitor MK, Agyei F, et al. Factors https://doi.org/10.1002/sim.4780131709. associated with treatment-seeking for malaria in urban poor communities in 31. Klein SL. Hormonal and immunological mechanisms mediating sex Accra, Ghana. Malar J. 2018a;17(1):168. https://doi.org/10.1186/s12936-01 differences in parasite infection. Parasite Immunol. 2004;26(6–7):247–64. 8-2311-8. https://doi.org/10.1111/j.0141-9838.2004.00710.x. 49. Sumbele IUN, Bopda OSM, Kimbi HK, Ning TR, Nkuo-Akenji T. Nutritional 32. Klein PW, Easterbrook JD, Lalime EN, Klein SL. Estrogen and progesterone status of children in a malaria meso endemic area: cross sectional study on affect responses to malaria infection in female C57BL/6 mice. Gend Med. prevalence, intensity, predictors, influence on malaria parasitaemia and 2008;5(4):423–33. https://doi.org/10.1016/j.genm.2008.10.001. anaemia severity. BMC Public Health. 2015b;15(1):1099. https://doi.org/10.11 33. Danquah I, Ziniel P, Eggelte TA, Ehrhardt S, Mockenhaupt FP. Influence of 86/s12889-015-2462-2. haemoglobins S and C on predominantly asymptomatic Plasmodium 50. Bowman NM, Congdon S, Mvalo T, Patel JC, Escamilla V, Emch M, et al. infections in northern Ghana. Trans R Soc Trop Med Hyg. 2010;104(11):713– Comparative population structure of plasmodium falciparum 9. https://doi.org/10.1016/j.trstmh.2010.08.001. circumsporozoite protein NANP repeat lengths in Lilongwe, Malawi. Sci Rep. 34. Kanwugu ON, Helegbe GK, Aryee PA, Abdul-Karim A, Anaba F, Ziblim Z, 2013;3(1):1990. https://doi.org/10.1038/srep01990. et al. Prevalence of asymptomatic malaria among children in the Tamale 51. Deen J, Walraven G, Von Seidlein L. Increased risk for malaria in chronically Metropolis: how does the PfHRP2 CareStart™ RDT perform against malnourished children under 5 years of age in rural Gambia. J Trop Pediatr. microscopy? J Trop Med. 2019;2019:6457628–7. https://doi.org/10.1155/201 2002;48(2):78–83. https://doi.org/10.1093/tropej/48.2.78. 9/6457628. 52. Berzosa P, de Lucio A, Romay-Barja M, Herrador Z, González V, García L, 35. Owusu ED, Buabeng V, Dadzie S, Brown CA, Grobusch MP, Mens PJM j. et al. Comparison of three diagnostic methods (microscopy, RDT, and PCR) Characteristics of asymptomatic Plasmodium spp. parasitaemia in Kwahu- for the detection of malaria parasites in representative samples from Mpraeso, a malaria endemic mountainous district in Ghana, West Africa. Equatorial Guinea. Malar J. 2018;17(1):1–12. https://doi.org/10.1186/s12936- Malar J. 2016;15(1):38. 018-2481-4. 36. Zaw MT, Thant M, Hlaing TM, Aung NZ, Thu M, Phumchuea K, et al. 53. Mfuh KO, Achonduh-Atijegbe OA, Bekindaka ON, Esemu LF, Mbakop CD, Asymptomatic and sub-microscopic malaria infection in Kayah State, eastern Gandhi K, et al. A comparison of thick-film microscopy, rapid diagnostic test, Myanmar. Malar J. 2017;16(1):138. and polymerase chain reaction for accurate diagnosis of plasmodium 37. Verhoef H. Asymptomatic malaria in the etiology of iron deficiency anemia: falciparum malaria. Malar J. 2019;18(1):1–8. https://doi.org/10.1186/s12936-01 a malariologist’s viewpoint. Am J Clin Nutr. 2010;92(6):1285–6. https://doi. 9-2711-4. org/10.3945/ajcn.110.006700. 38. Luxemburger C, Nosten F, Kyle DE, Kiricharoen L, Chongsuphajaisiddhi T, Publisher’s Note White NJ. Clinical features cannot predict a diagnosis of malaria or Springer Nature remains neutral with regard to jurisdictional claims in differentiate the infecting species in children living in an area of low published maps and institutional affiliations. transmission. Trans R Soc Trop Med Hyg. 1998;92(1):45–9. https://doi.org/1 0.1016/s0035-9203(98)90950-6. 39. Sumbele IU, Kimbi HK, Ndamukong-Nyanga JL, Nweboh M, Anchang-Kimbi JK, Lum E, et al. Malarial anaemia and anaemia severity in apparently healthy primary school children in urban and rural settings in the Mount Cameroon area: cross sectional survey. PLoS One. 2015a;10(4):e0123549. https://doi.org/10.1371/journal.pone.0123549. 40. Crookston BT, Alder SC, Boakye I, Merrill RM, Amuasi JH, Porucznik CA, et al. Exploring the relationship between chronic undernutrition and asymptomatic malaria in Ghanaian children. Malar J. 2010a;9(1). https://doi. org/10.1186/1475-2875-9-39 http://europepmc.org/abstract/MED/20122258 http://www.biomedcentral.com/content/pdf/1475-2875-9-39.pdf. 41. Das D, Grais RF, Okiro EA, Stepniewska K, Mansoor R, van der Kam S, et al. Complex interactions between malaria and malnutrition: a systematic literature review. BMC Med. 2018;16(1):186. https://doi.org/10.1186/s12916- 018-1177-5. 42. Akiyama T, Pongvongsa T, Phrommala S, Taniguchi T, Inamine Y, Takeuchi R, et al. Asymptomatic malaria, growth status, and anaemia among children in Lao People’s Democratic Republic: a cross-sectional study. Malar J. 2016; 15(1):499. https://doi.org/10.1186/s12936-016-1548-3. 43. Gari T, Loha E, Deressa W, Solomon T, Lindtjørn B. Malaria increased the risk of stunting and wasting among young children in Ethiopia: results of a cohort study. PLoS One. 2018;13(1):e0190983. https://doi.org/10.1371/journa l.pone.0190983. 44. Ferreira E d A, Alexandre MA, Salinas JL, de Siqueira AM, Benzecry SG, de Lacerda MVG, et al. Association between anthropometry-based nutritional status and malaria: a systematic review of observational studies. Malar J. 2015;14(1):346. https://doi.org/10.1186/s12936-015-0870-5. 45. Genton B, Al-Yaman F, Ginny M, Taraika J, Alpers MP. Relation of anthropometry to malaria morbidity and immunity in Papua New Guinean