School-aged children as a silent reservoir of Plasmodium falciparum: findings from a cross-sectional survey conducted in Burkina Faso in 2022

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Asymptomatic Plasmodium falciparum infections sustain malaria transmission and challenge elimination efforts. School-aged children may serve as a key reservoir, yet are often overlooked in malaria control programs. Methods. A cross-sectional survey was conducted in July 2022 across Banfora, Orodara, and Gaoua districts (Burkina Faso), enrolling 1,127 children aged 6 months to 10 years. Malaria diagnosis was performed using HRP2-based RDT, expert microscopy, and qPCR. Children with fever (>37.5°C) were excluded from asymptomatic analysis. Results. Prevalence of asymptomatic Plasmodium falciparum infection was 28.0% by RDT and 31.9% by microscopy. Among school-aged children (5–10 years), 66.5% tested positive by microscopy versus 21.6% in children under five. Gametocyte carriage reached 3.9% overall and was twice as high in school-aged children (67 vs. 32 gametocytes/μL). Microscopy outperformed RDT in sensitivity (76.7% vs. 67.7%), specificity (100% vs. 96.2%), and positive predictive value (100% vs. 83.8%) using qPCR as reference. Most infections were due to P. falciparum (98.2%), with mixed infections being rare. Conclusion. School-aged children are a major reservoir for asymptomatic and potentially infectious malaria. Their inclusion in surveillance and control strategies is critical. Expert microscopy remains a reliable field tool to detect asymptomatic carriage in high-burden areas. Asymptomatic P. falciparum infection School-aged children Burkina Faso Figures Figure 1 Background In 2023, an estimated 263 million malaria cases were reported worldwide, with the WHO African Region bearing 94% of the global burden and accounting for 95% of malaria-related deaths [ 1 ]. In Burkina Faso, a country of over 20 million people, school-aged children (5–14 years) represent the largest demographic group (29.1%), while children under five account for 16.2% [ 2 ]. Despite the implementation of the WHO's "High Burden to High Impact" (HBHI) strategy, malaria incidence remains high, with over 10 million cases reported in recent years [ 3 ]. Mortality is still highest among children under five, but a worrying increase in malaria-related deaths has also been documented among school-aged children, who accounted for 27% of all malaria cases and 18% of deaths in 2022 [ 3 ]. Transmission is driven primarily by vectors of the Anopheles gambiae complex [ 4 ]. Malaria presents in two forms: symptomatic (either uncomplicated or severe) and asymptomatic, the latter being defined by the presence of asexual-stage parasites without clinical manifestations [ 1 , 5 , 6 ]. Asymptomatic infections are critical in sustaining transmission, especially due to their prolonged duration and the higher likelihood of gametocyte maturation and infectivity to mosquitoes [ 7 – 11 ]. A national survey conducted between 2013 and 2014 found a 38.2% prevalence of asymptomatic parasitaemia in children under five in one-third of Burkina Faso's health districts using RDT [ 10 ]. Current national strategies include the deployment of long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), seasonal malaria chemoprevention (SMC), artemisinin-based combination therapies (ACTs), and the rollout of RTS,S/AS01 and R21/Matrix-M vaccines among children under five. However, school-aged children, largely excluded from these interventions, may increasingly serve as a persistent parasite reservoir. Chronic infections in this age group are also associated with cognitive impairment and reduced school performance [ 12 ]. This study aims to quantify the burden of asymptomatic Plasmodium infections in both children under five and school-aged youth, and to assess the diagnostic performance of RDT, light microscopy, and RT-PCR under field conditions. The results provide evidence-based refinement of targeted strategies. Materials and Methods Study site, design, and period. This study is a secondary analysis of a pilot observational study conducted following the 2019 mass distribution campaign of insecticide-treated nets. The primary objective of the initial study was to evaluate the effectiveness and cost-effectiveness of dual active ingredient insecticide-treated nets (dual-AI ITNs), compared to standard pyrethroid-only nets, as well as their impact on the malaria burden [ 13 ]. A cross-sectional household survey was conducted in July 2022 in three health districts of southwestern Burkina Faso: Banfora, Orodara, and Gaoua. These districts lie within the tropical zone, where malaria transmission is perennial, with a pronounced seasonal peak between June and October. The survey targeted children aged 6 months to 10 years residing in randomly selected villages within each district, encompassing both children under five and school-aged youth. Sample size andsampling technique. Sample size was calculated using the standard formula for cross-sectional surveys: n = z² ×p(1 − p)/d² , where z = 1.96 for a 95% confidence level, p = expected prevalence (set at 30%), and d = margin of error (5%). This yielded a minimum of 504 children per age group. Accounting for a 13% non-response rate, the adjusted sample size was 570 children per group. Systematic sampling was applied within sectors to identify eligible households. In each health district, 10 villages were selected by random draw. In total, 1,140 children aged 6 months to 10 years were enrolled across Banfora, Orodara, and Gaoua. Blood sampling and malaria screening. Trained nurses measured axillary temperature and collected capillary blood via finger prick using sterile disposable lancets. Samples were used for on-site malaria diagnosis with an HRP2-based RDT (ADX-004-025 HRP2 Pf Ag, ADVY CHEMICAL PVT.LTD, India). Thick and thin blood films were prepared on a single slide, labeled with unique codes. Additionally, 50 µL of blood was spotted on Whatman903™ filter cards (five spots per card). Filter papers were air-dried and stored at room temperature in sealed bags with desiccants. All samples were transported to the CNRFP laboratory for further analysis. Malaria HRP2 antigen detection (RDT). The RDT used in the field was provided by the Permanent Secretariat for Malaria Elimination of Burkina Faso. Tests were conducted according to manufacturer instructions. Five microliters of capillary blood were applied to the test device, followed by four drops (~ 110 µL) of buffer solution. Results were read after 20 minutes and interpreted as positive or negative based on band appearance. Light microscopy. Thin smears were fixed in methanol for 30 seconds and stained with 3% Giemsa for 45 minutes. Slides were independently examined by two Level 1 WHO-certified microscopists from the National Center for Malaria Research and Training (CNRFP). In case of disagreement (species identification, parasite presence/absence, or > 25% difference in density), a third expert reviewed the slide. The final parasite density was calculated as the arithmetic mean of the two closest readings. Parasite quantification was based on thick film counts using the formula: (parasites × 8,000) / leukocytes counted. A slide was considered negative if no parasite was detected after scanning 200 high-power fields. Molecular detection of P. falciparum by qPCR. Each dried blood spot was cut under sterile conditions and transferred to an Eppendorf tube in the Laboratory of the Institute of Parasitology and Tropical Pathology of Strasbourg (IPPTS) in France. Parasite DNA was extracted using the 96-well protocol of Zainabadi et al . [ 14 ] The eluate was quantified by fluorometry (Qubit, Thermo Fisher), adjusted to 20 ng/µL and stored at − 20°C until use. Samples were first screened using a qualitative SYBR Green-based real-time PCR targeting the Plasmodium cytochrome b gene. Positive samples were subsequently diluted 1:10 and subjected to species-specific real-time PCR assays using primers targeting the same cytochrome b region to distinguish P. falciparum , P. vivax , P. malariae , and P. ovale . All reactions were performed on a BioRad CFX96 thermal cycler using 5 µL of template DNA and SYBR Green PCR Master Mix (Solis Biodyne, Estonia). Primer sequences, cycling conditions, and reaction mix compositions have been described previously by Canier et al. [ 15 , 16 ] Ethical considerations. The study protocol was reviewed and approved by the Health Research Ethics Committee of Burkina Faso (CERS), under authorization no. 2022-05-117. Written informed consent was obtained from all participants or their legal guardians prior to enrolment. Consent also covered the future use of collected biological samples for research purposes. Data analysis. Data entry and verification were performed using Microsoft Excel 2021. Statistical analyses were conducted with MedCalc® version 20.218 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org ; 2023). Descriptive statistics were used to summarize socio-demographic characteristics and are presented as frequencies and percentages. Continuous variables were summarized as means or medians, depending on distribution. Diagnostic performance metrics, including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), were computed using 2×2 contingency tables, with qPCR as the reference standard. Agreement between diagnostic methods was assessed using Cohen’s Kappa (κ) coefficient. Statistical significance was set at p < 0.05, with 95% confidence intervals reported for all estimates. Results Socio-demographic and clinical characteristics. A total of 1,127 children were enrolled across the three study sites. Males represented 48.0% of participants, and children under five years of age accounted for 50.4% of the sample. The median axillary body temperature was 36.7°C (95% CI: 36.7). Children presenting with fever at the time of the survey (≥37.5°C) were excluded from the analysis of asymptomatic carriage, representing 5.4% (61/1,127) of the total sample. The baseline characteristics of the study population are summarized in Table 1. Table 1. Baseline characteristics of the study population. Summary of demographic and clinical variables among the 1,127 children enrolled across the three study sites. Variables include sex distribution, age groups (<5 years and 5–10 years), mean age, parasitemia levels (mean and range), and median body temperature. Only children with axillary temperature below 37.5°C were considered eligible for the analysis of asymptomatic malaria carriage. Characteristics N(%) p-value Gender Male 541(48.0) 0.18 Female 586 (52.0) Age (years) <5 568 (50.4) 0.78 5–10 559 (49.6) Mean parasitemia/µL (95%CI) <5 15652 (5872-25432) 5–10 8828 (5461-12195) Parasitemia range (min-max)/µL 16-544000 Mediantemperature (°C) 36.7°C (95%IC : 36.7). Prevalence of asymptomatic Plasmodium infection. Among non-febrile children, the overall prevalence of asymptomatic P. falciparum infection was 28.0% (95% CI: 25.3–30.8) by RDT and 31.9% (95% CI: 29.1–34.8) by expert microscopy. Parasite densities measured by microscopy ranged from 16 to 544,000 parasites/μL. Age-stratified analysis showed a markedly higher burden among school-aged children (5–10 years), with 66.5% (226/340) testing positive and a geometric mean parasite density of 967 parasites/μL (95% CI: 752–1,243), compared to 726 parasites/μL in children under five. Geographic disparities were pronounced. The Gaoua site accounted for 64.7% of all asymptomatic infections detected, while prevalence at Banfora and Orodara remained below 18%. Except in Banfora, school-aged children consistently exhibited a significantly higher prevalence of asymptomatic carriage than younger children (p = 0.0001). No significant sex-related differences were observed across sites (see Supplemental Table 1). Infections were overwhelmingly monoinfections with P. falciparum (98.2%), while mixed infections with P. malariae (1.5%) and P. ovale (0.3%) were rare. Gametocyte carriage was detected in 3.9% (95% CI: 2.9–5.2) of the population, with higher prevalence and density in school-aged children. Geometric mean gametocyte density reached 67 gametocytes/μL (95% CI: 42–108) in older children, compared to 32 gametocytes/μL (95% CI: 19–54) in those under five. Table 2. Prevalence of asymptomatic P. falciparum infection among children under five and school-aged children, based on RDT and expert microscopy. The table presents the number and proportion of positive cases detected by rapid diagnostic tests (RDT) and microscopy, stratified by age group (<5 years and 5–10 years). Overall prevalence rates are also reported for each diagnostic method. Only non-febrile children (temperature <37.5°C) were included in the analysis. Age (years) N RDT Microscopy 1066 Pos, n (%) Overall Pos, n (%) Pos, n (%) Overall Pos, n (%) <5 529 99 (18.7) 299 (28.0) 114 (21.6) 340 (31.9) 5–10 537 200 (37.2) 226(42.1) Supplemental Table 1. Distribution of asymptomatic P. falciparum infections by sex, age group, and study site, based on RDT and microscopy. This table presents site-specific prevalence rates of asymptomatic P. falciparum infections detected by RDT and expert microscopy, stratified by sex (male/female) and age group (<5 years and 5–10 years) in each of the three health districts (Banfora, Gaoua, and Orodara). p-values indicate statistical significance of differences in prevalence between subgroups within each site. Only non-febrile children (temperature <37.5°C) were included in this analysis. Districts sanitaires/Variables RDT Microscopy Prevalence ( %) p-value Prevalence (%) p-value Banfora Male (n %) 174 (49.7) 8.3 0.52 8.3 0.92 Female (n %) 176 (50.3) 7.1 8.6 <5 years (%) 176 (50.3) 6.3 0.12 6.3 0.02 5-10 years (%) 174 (49.7) 9.1 10.6 Total 350 15.4 16.9 Gaoua Male (n %) 158 (45.7) 28.6 0.06 31.5 0.13 Female (n %) 188 (54.3) 28.6 33.2 <5 years (%) 176 (50.9 21.4 0.0001 25.1 0.0001 5-10 years (%) 170 (49.1) 35.8 39.6 Total 346 57.2 64.7 Orodara Male (n %) 176 (47.6) 5.9 0.91 7.3 0.97 Female (n %) 194 (52.4) 6.8 8.1 <5 years (%) 185 (50.0) 0.8 0.0001 1.4 5-10 years (%) 185 (50.0) 11.9 14.1 0.0001 Total 370 12.7 15.4 Diagnostic tool performance for asymptomatic P. falciparum infection detection A random selection of 40% of available samples from each study site yielded 456 dried blood spots for evaluation. Of these, 440 were successfully processed for DNA extraction and analyzed using cytochrome b qPCR. A total of 180 children tested positive by qPCR, with Ct values ranging from 17.0 to 43.5 and estimated parasitemia spanning from 0.02 to 58,662 parasites/μL. Expert microscopy failed to detect 23.3% (42/180) of qPCR–confirmed infections, while RDT missed 32.2% (58/180). False positives were observed only with RDTs (3.8%; 10/260), while microscopy showed perfect specificity (no false positives). False-negative cases were associated with significantly lower parasite densities. For RDT, the median parasitemia among missed infections was 4.43 parasites/μL, compared to 161.7 parasites/μL in true positives (p < 0.001). Similarly, for microscopy, the median parasitemia of false negatives was 1.0 parasite/μL, versus 158.9 parasites/μL in true positives (p < 0.001). These findings underscore the limitations of both field diagnostic tools in detecting low-density asymptomatic infections. Table 3. Detection gaps of asymptomatic P. falciparum infections by microscopy and RDT, using qPCR as the reference standard. Contingency table shows the number of true positives, false negatives, true negatives, and false positives for microscopy and HRP2-based RDT, compared to P. falciparum cytochrome b qPCR. The percentage of infections missed by each diagnostic method is indicated. Only non-febrile children were included. RT-PCR Positive Negative Total % missed Microscopy Positive 138 0 138 23.3 Negative 42 260 302 Total 180 260 440 RDT Positive 122 10 132 32.2 Negative 58 250 308 Total 180 260 440 Diagnostic accuracy of field-based tests compared to qPCR. Using qPCR as the reference standard, HRP2-based RDTs demonstrated a sensitivity of 67.7% (95% CI: 60.4–74.5) and a specificity of 96.2% (95% CI: 93.1–98.1). Expert microscopy showed higher diagnostic performance, with a sensitivity of 76.7% (95% CI: 70.0–82.6) and perfect specificity (100%; 95% CI: 98.6–100). The area under the curve (AUC) was 0.80 for RDT and 0.90 for microscopy, indicating superior overall performance of microscopy. Positive predictive value (PPV) was 100% for microscopy and 83.8% for RDT. Negative predictive values (NPV) were comparable: 91.0% for RDT and 90.3% for microscopy. The kappa coefficient, measuring agreement with qPCR, was moderate for RDT (κ = 0.67) and substantial for microscopy (κ = 0.80). Overall accuracy reached 89.7% for RDT and 92.6% for microscopy (Table 4). Table 4. Comparative diagnostic performance of RDT and expert microscopy for asymptomatic P. falciparum detection, using qPCR as reference. Diagnostic metrics include sensitivity, specificity, predictive values, likelihood ratios, kappa agreement coefficient, and overall accuracy for both RDT and microscopy. Median parasite densities with 95% confidence intervals are reported for true positives and false negatives. RT-PCR served as the gold standard. Only non-febrile children were included in this analysis. Test Characteristic RDT Microscopy Total sample size (N) 440 440 True positive (N, Parasitaemia (µl), median) 122 (161.7 parasites/µL; 95% CI: 96.2–289.2) 138 (158.9 parasites/µL; 95% CI: 94.6–274.0) False negative (N, Parasitaemia (µl), median) 58 (4.4 parasites/µL; 95% CI: 1.0–21.3) 42 (1.0 parasite/µL; 95% CI: 1.0–3.7) True negative 250 260 False positive 10 0 Sensitivity 67.7% (95% CI: 60.4–74.5) 76.7% (95% CI: 70.0–82.6) Specificity 96.2% (95% CI: 93.1–98.1) 100% (95% CI: 98.6–100) AUC 0.8 (95% CI: 0.8–0.9) 0.9 (95% CI: 0.85–0.91) Positive predictive value 83.8% (95% CI: 76.6–90.5) 100% Negativepredictive value 91.0% (95% CI: 89.1–92.6) 90.3% (95% CI: 87.8–92.3) Positive likelihood ratio 17.6 (95% CI: 9.5–32.6) - Negativelikelihood ratio 0.3 (95% CI: 0.3–0.4) 0.23 (95% CI: 0.18–0.30) Kappa coefficient 0.67 (95% CI: 0.6–0.7) 0.80 (95% CI: 0.8–0.9) Accuracy 89.7% (95% CI: 86.4–92.3) 92.6% (95% CI: 89.8–95.0) Discussion In malaria-endemic settings, asymptomatic P. falciparum infections are substantially more prevalent than symptomatic cases, yet they remain largely unaddressed in the design and implementation of control strategies. These silent reservoirs of infection contribute meaningfully to sustained transmission, particularly in high-burden countries such as Burkina Faso. The present study provides robust field evidence on the magnitude of asymptomatic parasitemia among children and offers a comparative assessment of routine diagnostic tools, with implications for surveillance and elimination efforts. The findings indicate a high overall prevalence of asymptomatic P. falciparum infection, with microscopy detecting P. falciparum in 31.9% of non-febrile children. This prevalence is consistent with estimates from the fifth Demographic and Health Survey in Burkina Faso and with previous studies conducted in similar epidemiological contexts [ 2 , 10 ]. Importantly, the data reveal a marked age-dependent gradient, with school-aged children (5–10 years) bearing the greatest burden of asymptomatic carriage (66.5% by microscopy), significantly exceeding that observed in children under five. These results corroborate recent reports of a demographic shift in malaria epidemiology, wherein older children, often excluded from chemoprevention and vaccine initiatives, have emerged as a dominant reservoir [ 17 – 25 ]. The disproportionately high parasite and gametocyte densities in school-aged children underscore their central role in transmission dynamics. This age group also exhibited the highest prevalence of gametocyte carriage (3.9%), with a mean density more than double that observed in younger children. These findings have direct operational implications, as school-aged children are frequently absent from targeted interventions such as seasonal malaria chemoprevention (SMC), indoor residual spraying (IRS), and vaccine programs. Addressing this oversight may be essential to disrupt persistent transmission. Geographical heterogeneity was also observed across the three study sites, with the Gaoua district contributing nearly two-thirds of all asymptomatic cases. Such spatial variation in prevalence is consistent with previous national data [ 10 ] and highlights the need for context-specific interventions that account for local transmission dynamics [ 26 , 27 ]. Furthermore, the overwhelming predominance of P. falciparum monoinfections (98.2%) supports the continued use of P. falciparum -specific diagnostic tools in this setting. However, the detection of P. malariae and P. ovale , albeit rare, raises concerns about under diagnosis in contexts where HRP2-based RDTs are exclusively used [ 28 ]. No significant sex-based differences in infection prevalence were observed consistent with findings reported by Ndong et al. [ 29 ]. However, variability in sex-related patterns across regions has been previously documented, suggesting that gender is unlikely to be a consistent risk factor [ 17 , 23 ]. From a diagnostic perspective, this study provides important comparative data. When benchmarked against qPCR, expert microscopy exhibited superior sensitivity (76.7%) and perfect specificity (100%) compared to HRP2-based RDTs (sensitivity: 67.7%; specificity: 96.2%). Both tools demonstrated high positive predictive values (PPV > 80%), but their negative predictive values were insufficient to reliably exclude infection, especially in cases of low parasitemia. This is of particular concern given that microscopy and RDTs missed 23.3% and 32.2% of PCR-positive infections, respectively, most of which exhibited submicroscopic parasite densities. False-positive results were limited to RDTs (3.8%) and may be attributed to persistent circulating HRP2 antigen, a well-recognized limitation of this diagnostic method in high-transmission areas [ 30 ]. The analysis also showed that false-negative results were strongly associated with lower parasite densities, highlighting the challenge of detecting low-density infections using field-adapted methods. These findings reinforce concerns about the current trend of systematically and exclusively relying on RDTs in household surveys to estimate parasite prevalence in Malaria Indicator Surveys and Demographic and Health Surveys. These undetected cases, although clinically silent, remain epidemiologically significant due to their potential to harbor transmissible gametocytes [ 24 , 25 , 31 ]. A major strength of this study is the simultaneous use of expert microscopy and molecular methods to quantify asymptomatic carriage in a well-characterized pediatric population across multiple epidemiological settings. The random selection of communities and stratified sampling enhanced the representativeness of the findings. However, the study is not without limitations. The absence of detailed socio-demographic and behavioral data (e.g., net usage, prior treatment history, nutritional status) limited the capacity to explore individual-level risk factors for carriage. Additionally, the cross-sectional design precludes temporal inferences on duration or progression of infection. Overall, these findings advocate for a recalibration of malaria control policies in high-transmission settings. School-aged children, who remain largely invisible in routine interventions, represent a substantial and infectious reservoir. Their systematic inclusion in targeted strategies, such as school-based screening and treatment, or potential chemoprevention, is warranted. Moreover, while molecular diagnostics remain the gold standard for detecting low-density infections, expert microscopy remains a viable and operationally feasible alternative in resource-limited settings, particularly for surveillance and focal screening. Conclusion This study provides compelling evidence that P. falciparum is responsible for most asymptomatic malaria infections among children in high-transmission areas of Burkina Faso. While children under five remain vulnerable, school-aged children emerged as the predominant infectious reservoir, characterized by high prevalence of both asexual parasites and gametocyte carriage. Despite their epidemiological significance, this population remains insufficiently targeted by existing malaria control strategies. The results also highlight the operational value of expert microscopy, which demonstrated high diagnostic performance when benchmarked against molecular reference standards. While it lacks the sensitivity of PCR for detecting low-density infections, its specificity and predictive accuracy make it a practical and scalable option for field-based surveillance. These findings call for a reorientation of malaria elimination strategies to explicitly include school-aged children in screening and treatment interventions. Addressing this neglected reservoir, while strengthening diagnostic capacity at the point of care, is critical to reduce silent transmission and sustain progress toward malaria elimination goals. Future efforts must also account for local transmission heterogeneity and ensure that diagnostic approaches are matched to context-specific epidemiological patterns. Declarations Acknowledgements The authors express their sincere gratitude to all study participants and their parents or guardians for their involvement. We also thank the health personnel and community members in the health districts of Banfora, Gaoua, and Orodara for their valuable collaboration and support throughout the study. Ethics approval and consent to participate. The study protocol was approved by the Health Research Ethics Committee of Burkina Faso (CERS), under authorization no. 2022-05-117. Written informed consent was obtained from all parents or legal guardians prior to enrolment, including consent for the dissemination and scientific use of the results. Authors’ contributions. TWC, Gansané A, DM and DS contributed to study design. TWC, SH, Ganou A, KR and DS contributed to field data collection. TWC, MSY and DN conducted laboratory analyses. Gansané A, and DM supervised the study and provided scientific oversight. TWC, Gansané A and DM contributed to manuscript drafting and revisions. All authors reviewed and approved the final manuscript. Competing interests. The authors declare no competing interests. Funding. This study was financially supported by the Centre National de Recherche et de Formation sur le Paludisme (CNRFP). Molecular analyses were supported by the Université de Strasbourg (grant R42PHAV) and the Institut Universitaire de France (IUF) under the Senior Chair program (2024–2029). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials. The datasets generated and/or analyzed during this study are available from the corresponding author upon reasonable request. References WHO. World malaria report. 2024. Burkina Faso. Enquête Démographique et de Santé. 2021. Ministère de la Santé du Burkina Faso. Annuaire Statistique de la Santé , 2023. Soma DD, Zogo BM, Somé A, Tchiekoi BN, Hien DF de S, Pooda HS, et al. Anopheles bionomics, insecticide resistance and malaria transmission in southwest Burkina Faso: A pre-intervention study. PLoS One. 2020;15:e0236920. Lindblade KA, Steinhardt L, Samuels A, Kachur SP, Slutsker L. The silent threat: asymptomatic parasitemia and malaria transmission. Expert Review of Anti-infective Therapy. 2013;11(6):623–39. White NJ. Severe malaria. Malar J. 2022;21:284. Bousema T, Okell L, Felger I, Drakeley C. Asymptomatic malaria infections: detectability, transmissibility and public health relevance. Nat Rev Microbiol. 2014;12:833–40. Das NG, Dhiman S, Talukdar PK, Goswami D, Rabha B, Baruah I, et al. Role of asymptomatic carriers and weather variables in persistent transmission of malaria in an endemic district of Assam, India. Infect Ecol Epidemiol. 2015;5:25442. Barnes KI, White NJ. Population biology and antimalarial resistance: The transmission of antimalarial drug resistance in Plasmodium falciparum. Acta Trop. 2005;94:230–40. Ouédraogo M, Samadoulougou S, Rouamba T, Hien H, Sawadogo JEM, Tinto H, et al. Spatial distribution and determinants of asymptomatic malaria risk among children under 5 years in 24 districts in Burkina Faso. Malaria Journal. 2018;17(1):460. White NJ. The Consequences of Treating Asymptomatic Malaria Parasitemia. Clinical Infectious Diseases. 2017;64(5):654–5. Cohee LM, Nankabirwa JI, Greenwood B, Djimde A, Mathanga DP. Time for malaria control in school-age children. Lancet Child Adolesc Health. 2021;5(8):537–8. Gansané A, Candrinho B, Mbituyumuremyi A, Uhomoibhi P, NFalé S, Mohammed AB, et al. Design and methods for a quasi-experimental pilot study to evaluate the impact of dual active ingredient insecticide-treated nets on malaria burden in five regions in sub-Saharan Africa. Malar J. 2022;21(1):19. Zainabadi K, Nyunt MM, Plowe CV. An improved nucleic acid extraction method from dried blood spots for amplification of Plasmodium falciparum kelch13 for detection of artemisinin resistance. Malar J. 2019;18:192. Canier L, Khim N, Kim S, Eam R, Khean C, Loch K, et al. Malaria PCR Detection in Cambodian Low-Transmission Settings: Dried Blood Spots versus Venous Blood Samples. Am J Trop Med Hyg. 2015;92(3):573–7. Canier L, Khim N, Kim S, Sluydts V, Heng S, Dourng D, et al. An innovative tool for moving malaria PCR detection of parasite reservoir into the field. Malaria Journal. 2013;12(1):405. Kaghou MM, Metoh TN, Fru CT, Berinyui S, Bindamu M, Chi A, et al. High prevalence and risk factors associated with asymptomatic malaria among children in Nkwen village, Northwest Region, Cameroon. Malar J. 2024;23(1):243. Hayuma PM, Wang CW, Liheluka E, Baraka V, Madebe RA, Minja DTR, et al. Prevalence of asymptomatic malaria, submicroscopic parasitaemia and anaemia in Korogwe District, north-eastern Tanzania. Malaria Journal. 2021;20(1):424. Agaba BB, Rugera SP, Mpirirwe R, Atekat M, Okubal S, Masereka K, et al. Asymptomatic malaria infection, associated factors and accuracy of diagnostic tests in a historically high transmission setting in Northern Uganda. Malaria Journal. 2022;21(1):392. Aninagyei E, Adedia D, Larbi G, Acheampong SO, Nyarko M, Abbew GA, et al. Epidemiology and likelihood of asymptomatic malaria among community dwellers in the Fanteakwa south district of Ghana. Parasite Epidemiol Control. 2024;27:e00378. Biruksew A, Demeke A, Birhanu Z, Golassa L, Getnet M, Yewhalaw D. Schoolchildren with asymptomatic malaria are potential hotspot for malaria reservoir in Ethiopia: implications for malaria control and elimination efforts. Malar J. 2023;22(1):311. Mukomena SE, Philipe CM, Désiré MK, Pascal LT, Ali MM, Oscar LN. Asymptomatic Parasitemia in under five, school age children and households self-medication, Lubumbashi, Democratic Republic of Congo. Pan Afr Med J. 2016;24:94. Mensah BA, Myers-Hansen JL, Obeng Amoako E, Opoku M, Abuaku BK, Ghansah A. Prevalence and risk factors associated with asymptomatic malaria among school children: repeated cross-sectional surveys of school children in two ecological zones in Ghana. BMC Public Health. 2021;21(1):1697. Mulamba C, Odufuwa OG, Kweyamba PA, Lazaro LO, Chabo MS, Kamage JJ, et al. Plasmodium falciparum gametocyte burden in a Tanzanian heterogeneous transmission setting. Malaria Journal. 2025;24(1):54. Oduma CO, Ogolla S, Atieli H, Ondigo BN, Lee MC, Githeko AK, et al. Increased investment in gametocytes in asymptomatic Plasmodium falciparum infections in the wet season. BMC Infectious Diseases. 2021;21(1):44. Asmelash D, Agegnehu W, Fenta W, Asmelash Y, Debebe S, Asres A. The Burden of Asymptomatic Malaria Infection in Children in Sub-Saharan Africa: A Systematic Review and Meta-Analysis Exploring Barriers to Elimination and Prevention. J Epidemiol Glob Health. 2025;15(1):17. Kouna LC, Oyegue-Liabagui SL, Voumbo-Matoumona DF, Lekana-Douki JB. Malaria Prevalence in Asymptomatic and Symptomatic Children Living in Rural, Semi-Urban and Urban Areas in Eastern Gabon. Acta Parasit. 2024;69(1):471–82. WHO. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 6 (2014-2015). Geneva: World Health Organization. 2015. Ndong IC, van Reenen M, Boakye DA, Mbacham WF, Grobler AF. Trends in malaria admissions at the Mbakong Health Centre of the North West Region of Cameroon: a retrospective study. Malar J. 2014;13(1):328. Lamsfus Calle C, Schaumburg F, Rieck T, Nkoma Mouima AM, Martinez de Salazar P, Breil S, et al. Slow clearance of histidine-rich protein-2 in Gabonese with uncomplicated malaria. Microbiology Spectrum. 2024;12(10):e00994-24. Opoku Afriyie S, Addison TK, Gebre Y, Mutala AH, Antwi KB, Abbas DA, et al. Accuracy of diagnosis among clinical malaria patients: comparing microscopy, RDT and a highly sensitive quantitative PCR looking at the implications for submicroscopic infections. Malaria Journal. 2023;22(1):76. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Mar, 2026 Read the published version in Malaria Journal → Version 1 posted Editorial decision: Revision requested 15 Feb, 2026 Reviews received at journal 10 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers agreed at journal 01 Feb, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviewers invited by journal 27 Jan, 2026 Editor assigned by journal 18 Jan, 2026 Submission checks completed at journal 16 Jan, 2026 First submitted to journal 14 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8603000","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":582004994,"identity":"573ecd97-76cc-4bd8-81ba-a21fa9261a36","order_by":0,"name":"Casimire Wendlamita Tarama","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYJCCAwwGcLZNAphKKCBCCw+EnZbAwAbSYoBXBwRAtRyGaGHAo4W//fjDQzcK7jHYs599+OlGzfk8fvnuxA8PDBjk+cUOYNUicSbH4HCOQTEDD0+6sXTOsdvFkm28myWADjOcOTsBqxYDhhwGoJYEoMPSGKRz2G4nbjjGuwGkJcHgNg4t/M8fQLTwP2P+nfPvHEjL5h94tQANhGiRSGOTzm07ANKyDa8tEjfeQLXceMZmnduXnDizLXebRYKBBE6/8PenP/6c8yeBgb0/jfl2zje7xH7ms5tv/qiwkeeXxq4FBuob0K3Hq3wUjIJRMApGAX4AAKpkW7WtIUwiAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Casimire","middleName":"Wendlamita","lastName":"Tarama","suffix":""},{"id":582004995,"identity":"de03258b-aff2-437a-8d41-8766ffdfa273","order_by":1,"name":"Harouna Soré","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Harouna","middleName":"","lastName":"Soré","suffix":""},{"id":582004996,"identity":"ba008049-a212-4163-bcb1-1829a8de378c","order_by":2,"name":"Mamadou Samb Yade","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mamadou","middleName":"Samb","lastName":"Yade","suffix":""},{"id":582004997,"identity":"d3b54659-f13c-4b6a-819a-7035b8aa2400","order_by":3,"name":"Denis Niyomwungere","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Denis","middleName":"","lastName":"Niyomwungere","suffix":""},{"id":582004998,"identity":"5d012f2f-ce2f-47c5-8f97-053c8dbe08f8","order_by":4,"name":"Siaka Débé","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Siaka","middleName":"","lastName":"Débé","suffix":""},{"id":582004999,"identity":"04bc87ee-dc65-40f8-8d18-0c165bf62317","order_by":5,"name":"Réné Kinda","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Réné","middleName":"","lastName":"Kinda","suffix":""},{"id":582005000,"identity":"ebed9640-f717-4097-ac69-33aae360fea4","order_by":6,"name":"Adama Ganou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Adama","middleName":"","lastName":"Ganou","suffix":""},{"id":582005001,"identity":"9c2214fb-633c-4a80-8724-fdea3901607f","order_by":7,"name":"Didier Menard","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Didier","middleName":"","lastName":"Menard","suffix":""},{"id":582005002,"identity":"2a9bcc53-531e-4800-9ec6-61ba1d6d5650","order_by":8,"name":"Adama Gansané","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Adama","middleName":"","lastName":"Gansané","suffix":""}],"badges":[],"createdAt":"2026-01-14 14:53:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8603000/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8603000/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12936-026-05867-3","type":"published","date":"2026-03-30T15:58:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101485315,"identity":"2c15de3a-51f7-449f-aa5f-8e7f12f379c8","added_by":"auto","created_at":"2026-01-30 08:59:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eStudy sites, Burkina Faso, 2022-2023\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8603000/v1/28e3289be976cfaa5a65ee4e.png"},{"id":106343948,"identity":"522fec5c-2012-441d-9ed5-c7df2f562502","added_by":"auto","created_at":"2026-04-07 16:11:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1522297,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8603000/v1/c96b96b1-cc05-4847-8e67-59d3a9de1d7d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"School-aged children as a silent reservoir of Plasmodium falciparum: findings from a cross-sectional survey conducted in Burkina Faso in 2022","fulltext":[{"header":"Background","content":"\u003cp\u003eIn 2023, an estimated 263\u0026nbsp;million malaria cases were reported worldwide, with the WHO African Region bearing 94% of the global burden and accounting for 95% of malaria-related deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Burkina Faso, a country of over 20\u0026nbsp;million people, school-aged children (5\u0026ndash;14 years) represent the largest demographic group (29.1%), while children under five account for 16.2% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite the implementation of the WHO's \"High Burden to High Impact\" (HBHI) strategy, malaria incidence remains high, with over 10\u0026nbsp;million cases reported in recent years [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mortality is still highest among children under five, but a worrying increase in malaria-related deaths has also been documented among school-aged children, who accounted for 27% of all malaria cases and 18% of deaths in 2022 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Transmission is driven primarily by vectors of the \u003cem\u003eAnopheles gambiae\u003c/em\u003e complex [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Malaria presents in two forms: symptomatic (either uncomplicated or severe) and asymptomatic, the latter being defined by the presence of asexual-stage parasites without clinical manifestations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Asymptomatic infections are critical in sustaining transmission, especially due to their prolonged duration and the higher likelihood of gametocyte maturation and infectivity to mosquitoes [\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A national survey conducted between 2013 and 2014 found a 38.2% prevalence of asymptomatic parasitaemia in children under five in one-third of Burkina Faso's health districts using RDT [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Current national strategies include the deployment of long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), seasonal malaria chemoprevention (SMC), artemisinin-based combination therapies (ACTs), and the rollout of RTS,S/AS01 and R21/Matrix-M vaccines among children under five. However, school-aged children, largely excluded from these interventions, may increasingly serve as a persistent parasite reservoir. Chronic infections in this age group are also associated with cognitive impairment and reduced school performance [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aims to quantify the burden of asymptomatic \u003cem\u003ePlasmodium\u003c/em\u003e infections in both children under five and school-aged youth, and to assess the diagnostic performance of RDT, light microscopy, and RT-PCR under field conditions. The results provide evidence-based refinement of targeted strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eStudy site, design, and period.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study is a secondary analysis of a pilot observational study conducted following the 2019 mass distribution campaign of insecticide-treated nets. The primary objective of the initial study was to evaluate the effectiveness and cost-effectiveness of dual active ingredient insecticide-treated nets (dual-AI ITNs), compared to standard pyrethroid-only nets, as well as their impact on the malaria burden [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A cross-sectional household survey was conducted in July 2022 in three health districts of southwestern Burkina Faso: Banfora, Orodara, and Gaoua. These districts lie within the tropical zone, where malaria transmission is perennial, with a pronounced seasonal peak between June and October. The survey targeted children aged 6 months to 10 years residing in randomly selected villages within each district, encompassing both children under five and school-aged youth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSample size andsampling technique.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSample size was calculated using the standard formula for cross-sectional surveys: \u003cem\u003en\u0026thinsp;=\u0026thinsp;z\u0026sup2; \u0026times;p(1\u0026thinsp;\u0026minus;\u0026thinsp;p)/d\u0026sup2;\u003c/em\u003e, where \u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.96 for a 95% confidence level, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;expected prevalence (set at 30%), and \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;margin of error (5%). This yielded a minimum of 504 children per age group. Accounting for a 13% non-response rate, the adjusted sample size was 570 children per group. Systematic sampling was applied within sectors to identify eligible households. In each health district, 10 villages were selected by random draw. In total, 1,140 children aged 6 months to 10 years were enrolled across Banfora, Orodara, and Gaoua.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBlood sampling and malaria screening.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTrained nurses measured axillary temperature and collected capillary blood via finger prick using sterile disposable lancets. Samples were used for on-site malaria diagnosis with an HRP2-based RDT (ADX-004-025 HRP2 Pf Ag, ADVY CHEMICAL PVT.LTD, India). Thick and thin blood films were prepared on a single slide, labeled with unique codes. Additionally, 50 \u0026micro;L of blood was spotted on Whatman903\u0026trade; filter cards (five spots per card). Filter papers were air-dried and stored at room temperature in sealed bags with desiccants. All samples were transported to the CNRFP laboratory for further analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMalaria HRP2 antigen detection (RDT).\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe RDT used in the field was provided by the Permanent Secretariat for Malaria Elimination of Burkina Faso. Tests were conducted according to manufacturer instructions. Five microliters of capillary blood were applied to the test device, followed by four drops (~\u0026thinsp;110 \u0026micro;L) of buffer solution. Results were read after 20 minutes and interpreted as positive or negative based on band appearance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLight microscopy.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThin smears were fixed in methanol for 30 seconds and stained with 3% Giemsa for 45 minutes. Slides were independently examined by two Level 1 WHO-certified microscopists from the National Center for Malaria Research and Training (CNRFP). In case of disagreement (species identification, parasite presence/absence, or \u0026gt;\u0026thinsp;25% difference in density), a third expert reviewed the slide. The final parasite density was calculated as the arithmetic mean of the two closest readings. Parasite quantification was based on thick film counts using the formula: (parasites \u0026times; 8,000) / leukocytes counted. A slide was considered negative if no parasite was detected after scanning 200 high-power fields.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular detection of\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003eby qPCR.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEach dried blood spot was cut under sterile conditions and transferred to an Eppendorf tube in the Laboratory of the Institute of Parasitology and Tropical Pathology of Strasbourg (IPPTS) in France. Parasite DNA was extracted using the 96-well protocol of Zainabadi \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] The eluate was quantified by fluorometry (Qubit, Thermo Fisher), adjusted to 20 ng/\u0026micro;L and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use. Samples were first screened using a qualitative SYBR Green-based real-time PCR targeting the \u003cem\u003ePlasmodium\u003c/em\u003e cytochrome b gene. Positive samples were subsequently diluted 1:10 and subjected to species-specific real-time PCR assays using primers targeting the same cytochrome b region to distinguish \u003cem\u003eP. falciparum\u003c/em\u003e, \u003cem\u003eP. vivax\u003c/em\u003e, \u003cem\u003eP. malariae\u003c/em\u003e, and \u003cem\u003eP. ovale\u003c/em\u003e. All reactions were performed on a BioRad CFX96 thermal cycler using 5 \u0026micro;L of template DNA and SYBR Green PCR Master Mix (Solis Biodyne, Estonia). Primer sequences, cycling conditions, and reaction mix compositions have been described previously by Canier et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthical considerations.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e The study protocol was reviewed and approved by the Health Research Ethics Committee of Burkina Faso (CERS), under authorization no. 2022-05-117. Written informed consent was obtained from all participants or their legal guardians prior to enrolment. Consent also covered the future use of collected biological samples for research purposes.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData analysis.\u003c/h2\u003e \u003cp\u003eData entry and verification were performed using Microsoft Excel 2021. Statistical analyses were conducted with MedCalc\u0026reg; version 20.218 (MedCalc Software Ltd, Ostend, Belgium; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.medcalc.org\u003c/span\u003e\u003cspan address=\"https://www.medcalc.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; 2023). Descriptive statistics were used to summarize socio-demographic characteristics and are presented as frequencies and percentages. Continuous variables were summarized as means or medians, depending on distribution. Diagnostic performance metrics, including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), were computed using 2\u0026times;2 contingency tables, with qPCR as the reference standard. Agreement between diagnostic methods was assessed using Cohen\u0026rsquo;s Kappa (κ) coefficient. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, with 95% confidence intervals reported for all estimates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSocio-demographic and clinical characteristics.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 1,127 children were enrolled across the three study sites. Males represented 48.0% of participants, and children under five years of age accounted for 50.4% of the sample. The median axillary body temperature was 36.7\u0026deg;C (95% CI: 36.7). Children presenting with fever at the time of the survey (\u0026ge;37.5\u0026deg;C) were excluded from the analysis of asymptomatic carriage, representing 5.4% (61/1,127) of the total sample. The baseline characteristics of the study population are summarized in \u003cstrong\u003e\u003cem\u003eTable 1.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Baseline characteristics of the study population.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSummary of demographic and clinical variables among the 1,127 children enrolled across the three study sites. Variables include sex distribution, age groups (\u0026lt;5 years and 5\u0026ndash;10 years), mean age, parasitemia levels (mean and range), and median body temperature. Only children with axillary temperature below 37.5\u0026deg;C were considered eligible for the analysis of asymptomatic malaria carriage.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"556\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 252px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 252px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e541(48.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e586 (52.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 252px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u0026lt;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e568 (50.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5\u0026ndash;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e559 (49.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 252px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean parasitemia/\u0026micro;L (95%CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e\u0026lt;5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e15652 (5872-25432)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 99px;\"\u003e\n \u003cp\u003e5\u0026ndash;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e8828 (5461-12195)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 252px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParasitemia range (min-max)/\u0026micro;L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e16-544000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 252px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMediantemperature (\u0026deg;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e36.7\u0026deg;C (95%IC\u0026nbsp;: 36.7).\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePrevalence of asymptomatic \u003cem\u003ePlasmodium\u003c/em\u003e infection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong non-febrile children, the overall prevalence of asymptomatic \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003einfection was 28.0% (95% CI: 25.3\u0026ndash;30.8) by RDT and 31.9% (95% CI: 29.1\u0026ndash;34.8) by expert microscopy. Parasite densities measured by microscopy ranged from 16 to 544,000 parasites/\u0026mu;L. Age-stratified analysis showed a markedly higher burden among school-aged children (5\u0026ndash;10 years), with 66.5% (226/340) testing positive and a geometric mean parasite density of 967 parasites/\u0026mu;L (95% CI: 752\u0026ndash;1,243), compared to 726 parasites/\u0026mu;L in children under five.\u003c/p\u003e\n\u003cp\u003eGeographic disparities were pronounced. The Gaoua site accounted for 64.7% of all asymptomatic infections detected, while prevalence at Banfora and Orodara remained below 18%. Except in Banfora, school-aged children consistently exhibited a significantly higher prevalence of asymptomatic carriage than younger children (p = 0.0001). No significant sex-related differences were observed across sites (see Supplemental Table 1).\u003c/p\u003e\n\u003cp\u003eInfections were overwhelmingly monoinfections with \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003e(98.2%), while mixed infections with \u003cem\u003eP. malariae\u0026nbsp;\u003c/em\u003e(1.5%) and \u003cem\u003eP. ovale\u0026nbsp;\u003c/em\u003e(0.3%) were rare. Gametocyte carriage was detected in 3.9% (95% CI: 2.9\u0026ndash;5.2) of the population, with higher prevalence and density in school-aged children. Geometric mean gametocyte density reached 67 gametocytes/\u0026mu;L (95% CI: 42\u0026ndash;108) in older children, compared to 32 gametocytes/\u0026mu;L (95% CI: 19\u0026ndash;54) in those under five.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Prevalence of asymptomatic \u003cem\u003eP. falciparum\u003c/em\u003e infection among children under five and school-aged children, based on RDT and expert microscopy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe table presents the number and proportion of positive cases detected by rapid diagnostic tests (RDT) and microscopy, stratified by age group (\u0026lt;5 years and 5\u0026ndash;10 years). Overall prevalence rates are also reported for each diagnostic method. Only non-febrile children (temperature \u0026lt;37.5\u0026deg;C) were included in the analysis.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"611\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 222px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRDT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 251px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003ePos, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eOverall Pos, n (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003ePos, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 157px;\"\u003e\n \u003cp\u003eOverall Pos, n (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e529\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e99 (18.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e299 (28.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e114 (21.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 157px;\"\u003e\n \u003cp\u003e340 (31.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u0026ndash;10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e537\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e200 (37.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e226(42.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 1. Distribution of asymptomatic \u003cem\u003eP. falciparum\u003c/em\u003e infections by sex, age group, and study site, based on RDT and microscopy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis table presents site-specific prevalence rates of asymptomatic \u003cem\u003eP. falciparum\u003c/em\u003e infections detected by RDT and expert microscopy, stratified by sex (male/female) and age group (\u0026lt;5 years and 5\u0026ndash;10 years) in each of the three health districts (Banfora, Gaoua, and Orodara). p-values indicate statistical significance of differences in prevalence between subgroups within each site. Only non-febrile children (temperature \u0026lt;37.5\u0026deg;C) were included in this analysis.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"614\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDistricts sanitaires/Variables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRDT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrevalence\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e( %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrevalence\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"33\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"30\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBanfora\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale (n %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e174 (49.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale (n %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e176 (50.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;5 years (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e176 (50.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5-10 years (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e174 (49.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e9.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e10.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 447px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"13\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e15.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e16.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGaoua\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale (n %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e158 (45.7)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e31.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale (n %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e188 (54.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e33.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;5 years (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e176 (50.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e21.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e25.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5-10 years (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e170 (49.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e35.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e39.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 447px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"19\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e57.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e64.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrodara\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMale (n %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e176 (47.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.97\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemale (n %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e194 (52.4)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e6.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;5 years (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e185 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5-10 years (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e185 (50.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e11.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e14.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 447px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"9\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 167px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e12.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e15.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 167px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 3px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eDiagnostic tool performance for asymptomatic \u003cem\u003eP. falciparum\u003c/em\u003e infection detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA random selection of 40% of available samples from each study site yielded 456 dried blood spots for evaluation. Of these, 440 were successfully processed for DNA extraction and analyzed using \u003cem\u003ecytochrome b\u003c/em\u003e qPCR. A total of 180 children tested positive by qPCR, with Ct values ranging from 17.0 to 43.5 and estimated parasitemia spanning from 0.02 to 58,662 parasites/\u0026mu;L.\u003c/p\u003e\n\u003cp\u003eExpert microscopy failed to detect 23.3% (42/180) of qPCR\u0026ndash;confirmed infections, while RDT missed 32.2% (58/180). False positives were observed only with RDTs (3.8%; 10/260), while microscopy showed perfect specificity (no false positives).\u003c/p\u003e\n\u003cp\u003eFalse-negative cases were associated with significantly lower parasite densities. For RDT, the median parasitemia among missed infections was 4.43 parasites/\u0026mu;L, compared to 161.7 parasites/\u0026mu;L in true positives (p \u0026lt; 0.001). Similarly, for microscopy, the median parasitemia of false negatives was 1.0 parasite/\u0026mu;L, versus 158.9 parasites/\u0026mu;L in true positives (p \u0026lt; 0.001). These findings underscore the limitations of both field diagnostic tools in detecting low-density asymptomatic infections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Detection gaps of asymptomatic\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eP. falciparum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;infections by microscopy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand RDT, using qPCR as\u0026nbsp;the reference standard.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContingency table shows the number of true positives, false negatives, true negatives, and false positives for microscopy and HRP2-based RDT, compared to \u003cem\u003eP. falciparum\u003c/em\u003e cytochrome b qPCR. The percentage of infections missed by each diagnostic method is indicated. Only non-febrile children were included.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"600\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRT-PCR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e% missed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e23.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e302\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e440\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRDT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e32.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e308\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e440\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eDiagnostic accuracy of field-based tests compared to qPCR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing qPCR as the reference standard, HRP2-based RDTs demonstrated a sensitivity of 67.7% (95% CI: 60.4\u0026ndash;74.5) and a specificity of 96.2% (95% CI: 93.1\u0026ndash;98.1). Expert microscopy showed higher diagnostic performance, with a sensitivity of 76.7% (95% CI: 70.0\u0026ndash;82.6) and perfect specificity (100%; 95% CI: 98.6\u0026ndash;100).\u003c/p\u003e\n\u003cp\u003eThe area under the curve (AUC) was 0.80 for RDT and 0.90 for microscopy, indicating superior overall performance of microscopy. Positive predictive value (PPV) was 100% for microscopy and 83.8% for RDT. Negative predictive values (NPV) were comparable: 91.0% for RDT and 90.3% for microscopy. The kappa coefficient, measuring agreement with qPCR, was moderate for RDT (\u0026kappa; = 0.67) and substantial for microscopy (\u0026kappa; = 0.80). Overall accuracy reached 89.7% for RDT and 92.6% for microscopy (Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4. Comparative diagnostic performance of RDT and expert microscopy for asymptomatic\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eP. falciparum\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;detection, using qPCR as reference.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiagnostic metrics include sensitivity, specificity, predictive values, likelihood ratios, kappa agreement coefficient, and overall accuracy for both RDT and microscopy. Median parasite densities with 95% confidence intervals are reported for true positives and false negatives. RT-PCR served as the gold standard. Only non-febrile children were included in this analysis.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest Characteristic\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRDT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal sample size (N)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e440\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTrue positive (N, Parasitaemia (\u0026micro;l),\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emedian)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e122 (161.7 parasites/\u0026micro;L; 95% CI: 96.2\u0026ndash;289.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e138 (158.9 parasites/\u0026micro;L; 95% CI: 94.6\u0026ndash;274.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFalse negative (N, Parasitaemia (\u0026micro;l),\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emedian)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e58 (4.4 parasites/\u0026micro;L; 95% CI: 1.0\u0026ndash;21.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e42 (1.0 parasite/\u0026micro;L; 95% CI: 1.0\u0026ndash;3.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTrue negative\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e260\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFalse positive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e67.7% (95% CI: 60.4\u0026ndash;74.5)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e76.7% (95% CI: 70.0\u0026ndash;82.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSpecificity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e96.2% (95% CI: 93.1\u0026ndash;98.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e100% (95% CI: 98.6\u0026ndash;100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAUC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.8 (95% CI: 0.8\u0026ndash;0.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.9 (95% CI: 0.85\u0026ndash;0.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePositive predictive value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e83.8% (95% CI: 76.6\u0026ndash;90.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNegativepredictive value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e91.0% (95% CI: 89.1\u0026ndash;92.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e90.3% (95% CI: 87.8\u0026ndash;92.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePositive likelihood ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e17.6 (95% CI: 9.5\u0026ndash;32.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eNegativelikelihood ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.3 (95% CI: 0.3\u0026ndash;0.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.23 (95% CI: 0.18\u0026ndash;0.30)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eKappa coefficient\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.67 (95% CI: 0.6\u0026ndash;0.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.80 (95% CI: 0.8\u0026ndash;0.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 221px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAccuracy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e89.7% (95% CI: 86.4\u0026ndash;92.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e92.6% (95% CI: 89.8\u0026ndash;95.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn malaria-endemic settings, asymptomatic \u003cem\u003eP. falciparum\u003c/em\u003e infections are substantially more prevalent than symptomatic cases, yet they remain largely unaddressed in the design and implementation of control strategies. These silent reservoirs of infection contribute meaningfully to sustained transmission, particularly in high-burden countries such as Burkina Faso. The present study provides robust field evidence on the magnitude of asymptomatic parasitemia among children and offers a comparative assessment of routine diagnostic tools, with implications for surveillance and elimination efforts.\u003c/p\u003e \u003cp\u003eThe findings indicate a high overall prevalence of asymptomatic \u003cem\u003eP. falciparum\u003c/em\u003e infection, with microscopy detecting \u003cem\u003eP. falciparum\u003c/em\u003e in 31.9% of non-febrile children. This prevalence is consistent with estimates from the fifth Demographic and Health Survey in Burkina Faso and with previous studies conducted in similar epidemiological contexts [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Importantly, the data reveal a marked age-dependent gradient, with school-aged children (5\u0026ndash;10 years) bearing the greatest burden of asymptomatic carriage (66.5% by microscopy), significantly exceeding that observed in children under five. These results corroborate recent reports of a demographic shift in malaria epidemiology, wherein older children, often excluded from chemoprevention and vaccine initiatives, have emerged as a dominant reservoir [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22 CR23 CR24\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe disproportionately high parasite and gametocyte densities in school-aged children underscore their central role in transmission dynamics. This age group also exhibited the highest prevalence of gametocyte carriage (3.9%), with a mean density more than double that observed in younger children. These findings have direct operational implications, as school-aged children are frequently absent from targeted interventions such as seasonal malaria chemoprevention (SMC), indoor residual spraying (IRS), and vaccine programs. Addressing this oversight may be essential to disrupt persistent transmission.\u003c/p\u003e \u003cp\u003eGeographical heterogeneity was also observed across the three study sites, with the Gaoua district contributing nearly two-thirds of all asymptomatic cases. Such spatial variation in prevalence is consistent with previous national data [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and highlights the need for context-specific interventions that account for local transmission dynamics [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, the overwhelming predominance of \u003cem\u003eP. falciparum\u003c/em\u003e monoinfections (98.2%) supports the continued use of \u003cem\u003eP. falciparum\u003c/em\u003e-specific diagnostic tools in this setting. However, the detection of \u003cem\u003eP. malariae\u003c/em\u003e and \u003cem\u003eP. ovale\u003c/em\u003e, albeit rare, raises concerns about under diagnosis in contexts where HRP2-based RDTs are exclusively used [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNo significant sex-based differences in infection prevalence were observed consistent with findings reported by Ndong et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, variability in sex-related patterns across regions has been previously documented, suggesting that gender is unlikely to be a consistent risk factor [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a diagnostic perspective, this study provides important comparative data. When benchmarked against qPCR, expert microscopy exhibited superior sensitivity (76.7%) and perfect specificity (100%) compared to HRP2-based RDTs (sensitivity: 67.7%; specificity: 96.2%). Both tools demonstrated high positive predictive values (PPV\u0026thinsp;\u0026gt;\u0026thinsp;80%), but their negative predictive values were insufficient to reliably exclude infection, especially in cases of low parasitemia. This is of particular concern given that microscopy and RDTs missed 23.3% and 32.2% of PCR-positive infections, respectively, most of which exhibited submicroscopic parasite densities.\u003c/p\u003e \u003cp\u003eFalse-positive results were limited to RDTs (3.8%) and may be attributed to persistent circulating HRP2 antigen, a well-recognized limitation of this diagnostic method in high-transmission areas [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The analysis also showed that false-negative results were strongly associated with lower parasite densities, highlighting the challenge of detecting low-density infections using field-adapted methods. These findings reinforce concerns about the current trend of systematically and exclusively relying on RDTs in household surveys to estimate parasite prevalence in Malaria Indicator Surveys and Demographic and Health Surveys. These undetected cases, although clinically silent, remain epidemiologically significant due to their potential to harbor transmissible gametocytes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA major strength of this study is the simultaneous use of expert microscopy and molecular methods to quantify asymptomatic carriage in a well-characterized pediatric population across multiple epidemiological settings. The random selection of communities and stratified sampling enhanced the representativeness of the findings. However, the study is not without limitations. The absence of detailed socio-demographic and behavioral data (e.g., net usage, prior treatment history, nutritional status) limited the capacity to explore individual-level risk factors for carriage. Additionally, the cross-sectional design precludes temporal inferences on duration or progression of infection.\u003c/p\u003e \u003cp\u003eOverall, these findings advocate for a recalibration of malaria control policies in high-transmission settings. School-aged children, who remain largely invisible in routine interventions, represent a substantial and infectious reservoir. Their systematic inclusion in targeted strategies, such as school-based screening and treatment, or potential chemoprevention, is warranted. Moreover, while molecular diagnostics remain the gold standard for detecting low-density infections, expert microscopy remains a viable and operationally feasible alternative in resource-limited settings, particularly for surveillance and focal screening.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides compelling evidence that \u003cem\u003eP. falciparum\u003c/em\u003e is responsible for most asymptomatic malaria infections among children in high-transmission areas of Burkina Faso. While children under five remain vulnerable, school-aged children emerged as the predominant infectious reservoir, characterized by high prevalence of both asexual parasites and gametocyte carriage. Despite their epidemiological significance, this population remains insufficiently targeted by existing malaria control strategies.\u003c/p\u003e \u003cp\u003eThe results also highlight the operational value of expert microscopy, which demonstrated high diagnostic performance when benchmarked against molecular reference standards. While it lacks the sensitivity of PCR for detecting low-density infections, its specificity and predictive accuracy make it a practical and scalable option for field-based surveillance.\u003c/p\u003e \u003cp\u003eThese findings call for a reorientation of malaria elimination strategies to explicitly include school-aged children in screening and treatment interventions. Addressing this neglected reservoir, while strengthening diagnostic capacity at the point of care, is critical to reduce silent transmission and sustain progress toward malaria elimination goals. Future efforts must also account for local transmission heterogeneity and ensure that diagnostic approaches are matched to context-specific epidemiological patterns.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude to all study participants and their parents or guardians for their involvement. We also thank the health personnel and community members in the health districts of Banfora, Gaoua, and Orodara for their valuable collaboration and support throughout the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Health Research Ethics Committee of Burkina Faso (CERS), under authorization no. 2022-05-117. Written informed consent was obtained from all parents or legal guardians prior to enrolment, including consent for the dissemination and scientific use of the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions.\u0026nbsp;\u003c/strong\u003eTWC, Gansan\u0026eacute; A, DM and DS contributed to study design. TWC, SH, Ganou A, KR and DS contributed to field data collection. TWC, MSY and DN conducted laboratory analyses. Gansan\u0026eacute; A, and DM supervised the study and provided scientific oversight. TWC, Gansan\u0026eacute; A and DM contributed to manuscript drafting and revisions. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThis study was financially supported by the Centre National de Recherche et de Formation sur le Paludisme (CNRFP). Molecular analyses were supported by the Universit\u0026eacute; de Strasbourg (grant R42PHAV) and the Institut Universitaire de France (IUF) under the Senior Chair program (2024\u0026ndash;2029). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials.\u0026nbsp;\u003c/strong\u003eThe datasets generated and/or analyzed during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. World malaria report. 2024. \u003c/li\u003e\n\u003cli\u003eBurkina Faso. Enqu\u0026ecirc;te D\u0026eacute;mographique et de Sant\u0026eacute;. 2021. \u003c/li\u003e\n\u003cli\u003eMinist\u0026egrave;re de la Sant\u0026eacute; du Burkina Faso. Annuaire Statistique de la Sant\u0026eacute; , 2023. \u003c/li\u003e\n\u003cli\u003eSoma DD, Zogo BM, Som\u0026eacute; A, Tchiekoi BN, Hien DF de S, Pooda HS, et al. Anopheles bionomics, insecticide resistance and malaria transmission in southwest Burkina Faso: A pre-intervention study. PLoS One. 2020;15:e0236920. \u003c/li\u003e\n\u003cli\u003eLindblade KA, Steinhardt L, Samuels A, Kachur SP, Slutsker L. The silent threat: asymptomatic parasitemia and malaria transmission. Expert Review of Anti-infective Therapy. 2013;11(6):623\u0026ndash;39. \u003c/li\u003e\n\u003cli\u003eWhite NJ. Severe malaria. Malar J. 2022;21:284. \u003c/li\u003e\n\u003cli\u003eBousema T, Okell L, Felger I, Drakeley C. Asymptomatic malaria infections: detectability, transmissibility and public health relevance. Nat Rev Microbiol. 2014;12:833\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eDas NG, Dhiman S, Talukdar PK, Goswami D, Rabha B, Baruah I, et al. Role of asymptomatic carriers and weather variables in persistent transmission of malaria in an endemic district of Assam, India. Infect Ecol Epidemiol. 2015;5:25442. \u003c/li\u003e\n\u003cli\u003eBarnes KI, White NJ. Population biology and antimalarial resistance: The transmission of antimalarial drug resistance in Plasmodium falciparum. Acta Trop. 2005;94:230\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eOu\u0026eacute;draogo M, Samadoulougou S, Rouamba T, Hien H, Sawadogo JEM, Tinto H, et al. Spatial distribution and determinants of asymptomatic malaria risk among children under 5 years in 24 districts in Burkina Faso. Malaria Journal. 2018;17(1):460. \u003c/li\u003e\n\u003cli\u003eWhite NJ. The Consequences of Treating Asymptomatic Malaria Parasitemia. Clinical Infectious Diseases. 2017;64(5):654\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eCohee LM, Nankabirwa JI, Greenwood B, Djimde A, Mathanga DP. Time for malaria control in school-age children. Lancet Child Adolesc Health. 2021;5(8):537\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eGansan\u0026eacute; A, Candrinho B, Mbituyumuremyi A, Uhomoibhi P, NFal\u0026eacute; S, Mohammed AB, et al. Design and methods for a quasi-experimental pilot study to evaluate the impact of dual active ingredient insecticide-treated nets on malaria burden in five regions in sub-Saharan Africa. Malar J. 2022;21(1):19. \u003c/li\u003e\n\u003cli\u003eZainabadi K, Nyunt MM, Plowe CV. An improved nucleic acid extraction method from dried blood spots for amplification of Plasmodium falciparum kelch13 for detection of artemisinin resistance. Malar J. 2019;18:192. \u003c/li\u003e\n\u003cli\u003eCanier L, Khim N, Kim S, Eam R, Khean C, Loch K, et al. Malaria PCR Detection in Cambodian Low-Transmission Settings: Dried Blood Spots versus Venous Blood Samples. Am J Trop Med Hyg. 2015;92(3):573\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eCanier L, Khim N, Kim S, Sluydts V, Heng S, Dourng D, et al. An innovative tool for moving malaria PCR detection of parasite reservoir into the field. Malaria Journal. 2013;12(1):405. \u003c/li\u003e\n\u003cli\u003eKaghou MM, Metoh TN, Fru CT, Berinyui S, Bindamu M, Chi A, et al. High prevalence and risk factors associated with asymptomatic malaria among children in Nkwen village, Northwest Region, Cameroon. Malar J. 2024;23(1):243. \u003c/li\u003e\n\u003cli\u003eHayuma PM, Wang CW, Liheluka E, Baraka V, Madebe RA, Minja DTR, et al. Prevalence of asymptomatic malaria, submicroscopic parasitaemia and anaemia in Korogwe District, north-eastern Tanzania. Malaria Journal. 2021;20(1):424. \u003c/li\u003e\n\u003cli\u003eAgaba BB, Rugera SP, Mpirirwe R, Atekat M, Okubal S, Masereka K, et al. Asymptomatic malaria infection, associated factors and accuracy of diagnostic tests in a historically high transmission setting in Northern Uganda. Malaria Journal. 2022;21(1):392. \u003c/li\u003e\n\u003cli\u003eAninagyei E, Adedia D, Larbi G, Acheampong SO, Nyarko M, Abbew GA, et al. Epidemiology and likelihood of asymptomatic malaria among community dwellers in the Fanteakwa south district of Ghana. Parasite Epidemiol Control. 2024;27:e00378. \u003c/li\u003e\n\u003cli\u003eBiruksew A, Demeke A, Birhanu Z, Golassa L, Getnet M, Yewhalaw D. Schoolchildren with asymptomatic malaria are potential hotspot for malaria reservoir in Ethiopia: implications for malaria control and elimination efforts. Malar J. 2023;22(1):311. \u003c/li\u003e\n\u003cli\u003eMukomena SE, Philipe CM, D\u0026eacute;sir\u0026eacute; MK, Pascal LT, Ali MM, Oscar LN. Asymptomatic Parasitemia in under five, school age children and households self-medication, Lubumbashi, Democratic Republic of Congo. Pan Afr Med J. 2016;24:94. \u003c/li\u003e\n\u003cli\u003eMensah BA, Myers-Hansen JL, Obeng Amoako E, Opoku M, Abuaku BK, Ghansah A. Prevalence and risk factors associated with asymptomatic malaria among school children: repeated cross-sectional surveys of school children in two ecological zones in Ghana. BMC Public Health. 2021;21(1):1697. \u003c/li\u003e\n\u003cli\u003eMulamba C, Odufuwa OG, Kweyamba PA, Lazaro LO, Chabo MS, Kamage JJ, et al. Plasmodium falciparum gametocyte burden in a Tanzanian heterogeneous transmission setting. Malaria Journal. 2025;24(1):54. \u003c/li\u003e\n\u003cli\u003eOduma CO, Ogolla S, Atieli H, Ondigo BN, Lee MC, Githeko AK, et al. Increased investment in gametocytes in asymptomatic Plasmodium falciparum infections in the wet season. BMC Infectious Diseases. 2021;21(1):44. \u003c/li\u003e\n\u003cli\u003eAsmelash D, Agegnehu W, Fenta W, Asmelash Y, Debebe S, Asres A. The Burden of Asymptomatic Malaria Infection in Children in Sub-Saharan Africa: A Systematic Review and Meta-Analysis Exploring Barriers to Elimination and Prevention. J Epidemiol Glob Health. 2025;15(1):17. \u003c/li\u003e\n\u003cli\u003eKouna LC, Oyegue-Liabagui SL, Voumbo-Matoumona DF, Lekana-Douki JB. Malaria Prevalence in Asymptomatic and Symptomatic Children Living in Rural, Semi-Urban and Urban Areas in Eastern Gabon. Acta Parasit. 2024;69(1):471\u0026ndash;82. \u003c/li\u003e\n\u003cli\u003eWHO. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 6 (2014-2015). Geneva: World Health Organization. 2015. \u003c/li\u003e\n\u003cli\u003eNdong IC, van Reenen M, Boakye DA, Mbacham WF, Grobler AF. Trends in malaria admissions at the Mbakong Health Centre of the North West Region of Cameroon: a retrospective study. Malar J. 2014;13(1):328. \u003c/li\u003e\n\u003cli\u003eLamsfus Calle C, Schaumburg F, Rieck T, Nkoma Mouima AM, Martinez de Salazar P, Breil S, et al. Slow clearance of histidine-rich protein-2 in Gabonese with uncomplicated malaria. Microbiology Spectrum. 2024;12(10):e00994-24. \u003c/li\u003e\n\u003cli\u003eOpoku Afriyie S, Addison TK, Gebre Y, Mutala AH, Antwi KB, Abbas DA, et al. Accuracy of diagnosis among clinical malaria patients: comparing microscopy, RDT and a highly sensitive quantitative PCR looking at the implications for submicroscopic infections. Malaria Journal. 2023;22(1):76. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Asymptomatic P. falciparum infection, School-aged children, Burkina Faso","lastPublishedDoi":"10.21203/rs.3.rs-8603000/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8603000/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBackground. \u003c/strong\u003e\u003c/em\u003eAsymptomatic \u003cem\u003ePlasmodium falciparum\u003c/em\u003e infections sustain malaria transmission and challenge elimination efforts. School-aged children may serve as a key reservoir, yet are often overlooked in malaria control programs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods. \u003c/strong\u003e\u003c/em\u003eA cross-sectional survey was conducted in July 2022 across Banfora, Orodara, and Gaoua districts (Burkina Faso), enrolling 1,127 children aged 6 months to 10 years. Malaria diagnosis was performed using HRP2-based RDT, expert microscopy, and qPCR. Children with fever (\u0026gt;37.5°C) were excluded from asymptomatic analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults. \u003c/strong\u003e\u003c/em\u003ePrevalence of asymptomatic \u003cem\u003ePlasmodium falciparum\u003c/em\u003e infection was 28.0% by RDT and 31.9% by microscopy. Among school-aged children (5–10 years), 66.5% tested positive by microscopy versus 21.6% in children under five. Gametocyte carriage reached 3.9% overall and was twice as high in school-aged children (67 vs. 32 gametocytes/μL). Microscopy outperformed RDT in sensitivity (76.7% vs. 67.7%), specificity (100% vs. 96.2%), and positive predictive value (100% vs. 83.8%) using qPCR as reference. Most infections were due to \u003cem\u003eP. falciparum\u003c/em\u003e (98.2%), with mixed infections being rare.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion. \u003c/strong\u003e\u003c/em\u003eSchool-aged children are a major reservoir for asymptomatic and potentially infectious malaria. Their inclusion in surveillance and control strategies is critical. Expert microscopy remains a reliable field tool to detect asymptomatic carriage in high-burden areas.\u003c/p\u003e","manuscriptTitle":"School-aged children as a silent reservoir of Plasmodium falciparum: findings from a cross-sectional survey conducted in Burkina Faso in 2022","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 08:59:42","doi":"10.21203/rs.3.rs-8603000/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-16T01:39:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-10T07:03:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T19:43:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252122285301304721196162842590275187932","date":"2026-02-02T06:45:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"190833399547135858442763666167681374245","date":"2026-02-02T04:54:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64331192234258160970977348387981559179","date":"2026-01-30T03:50:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58481042654635924768725449809819614394","date":"2026-01-28T18:51:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-28T01:59:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T00:08:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-16T12:19:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2026-01-14T14:33:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca954183-9732-4063-a1ff-30ef1db97f01","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:06:26+00:00","versionOfRecord":{"articleIdentity":"rs-8603000","link":"https://doi.org/10.1186/s12936-026-05867-3","journal":{"identity":"malaria-journal","isVorOnly":false,"title":"Malaria Journal"},"publishedOn":"2026-03-30 15:58:43","publishedOnDateReadable":"March 30th, 2026"},"versionCreatedAt":"2026-01-30 08:59:42","video":"","vorDoi":"10.1186/s12936-026-05867-3","vorDoiUrl":"https://doi.org/10.1186/s12936-026-05867-3","workflowStages":[]},"version":"v1","identity":"rs-8603000","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8603000","identity":"rs-8603000","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}