Reevaluating malaria severity metrics: insights from PfHRP2-derived biomass estimates in Ghanaian children

Abstract

Background

Malaria remains a major cause of morbidity and mortality in sub-Saharan Africa. Severe Plasmodium falciparum malaria is primarily driven by parasite sequestration in deep vascular tissues. Standard diagnostic tools, such as peripheral parasitaemia determination, do not always reflect the total parasite burden. Plasma Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) level has emerged as a potential biomarker for estimating total parasite biomass, which may better reflect disease severity than peripheral parasitaemia. However, it is still unclear how PfHRP2-estimated parasite biomass varies across different clinical malaria syndromes, how well it predicts severity compared to circulating parasitaemia, and how prior antimalarial treatment influences these measures. Addressing these gaps is critical to improving severity assessment and guiding timely interventions.

Methods

Data from 118 children diagnosed with cerebral malaria (CM, n = 58), severe malaria anaemia (SMA, n = 28), or uncomplicated malaria (UM, n = 32) were collected in five referral hospitals in Accra from 2012 to 2016. Total parasite burden (PTot) was estimated using PfHRP2-based biomass measurement while the circulating parasite burden (PCir) was determined from peripheral parasite density. The sequestered parasite burden (PSeq), which represent the difference between PTot and PCir, was also evaluated. Additionally, the impact of prior antimalarial treatment on parasite burden was assessed.

Results

PTot and PSeq were consistently higher than PCir in severe malaria syndromes. In UM, PTot and PCir were similar, while the median PCir was lower in CM than in UM, suggesting greater sequestration in severe disease. After regaining consciousness, CM patients exhibited decreased PTot and PSeq values compared to their values at initial clinical evaluation. Higher PSeq estimates were associated with coma. Prior antimalarial treatment also reduced PCir but did not significantly change PTot.

Conclusion

PfHRP2-derived total parasite biomass demonstrated a stronger association with severe malaria syndromes than peripheral parasitaemia. Accounting for prior antimalarial treatment is essential, as it may lower circulating parasite counts without affecting total biomass. Incorporating total parasite biomass assessments into clinical evaluation could enhance disease severity classification and inform timely interventions in endemic regions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12936-026-05804-4.

Background

Malaria remains a leading cause of morbidity and mortality among children in sub-Saharan Africa, with Plasmodium falciparum responsible for the most severe manifestations of the disease [1]. Clinically, P. falciparum malaria presents as a spectrum from uncomplicated malaria (UM) to severe or complicated forms, including acute respiratory distress, severe malarial anaemia (SMA) and cerebral malaria (CM). The burden of severe malaria shows striking regional variations across sub-Saharan Africa. CM accounts for nearly 13% of severe paediatric cases in high-burden regions [2], while SMA emerges as an even more pervasive threat, accounting for 23–38% of hospital admissions [3–5], depending on transmission intensity and demographic factors. There is an evolving pattern in severe malaria presentation in sub-Saharan Africa, with geographic and temporal variations in the relative frequency of CM and SMA [6]. Among children under 5 years, severe malaria syndromes such as CM and SMA are prevalent and often linked to malaria parasite sequestration in vital organs. In CM, parasitized red blood cells (RBCs) sequester in the brain microvasculature, leading to vascular congestion, tissue hypoxia, inflammation, and potential organ dysfunction [7, 8]. SMA arises from extensive red blood cell destruction, resulting in severe anaemia characterised by fever and minimal convulsions [9].

Sequestered parasites, which can be assessed through the water‑soluble Histidine‑Rich Protein 2 (HRP2) released by P. falciparum during the rupture of infected RBCs, may not be detected using standard or routine peripheral blood smears. This limitation may lead to a significant underestimation of the total parasite burden in cases of severe malaria. Addressing this limitation requires comprehensive public health strategies for early and accurate assessment of the malaria severity for guiding timely clinical interventions. In sub-Saharan Africa, the majority of severe malaria syndrome patients often present to health facilities only after unsuccessful initial treatment attempts [10]. This is largely due to the complex pathophysiology of P. falciparum, where the parasites sequester in deep tissues to evade immune clearance [11].

Total parasite biomass (PTot), estimated from plasma PfHRP2, provides a more accurate representation of parasite burden by accounting for both circulating and sequestered parasites [12, 13]. PTot correlates strongly with disease severity and mortality compared to peripheral parasitaemia alone, which is commonly used in the clinical assessment of malaria severity [13–15]. However, the extent to which peripheral parasitaemia underestimates total parasite burden across different malaria syndromes in children remains inadequately elucidated.

Additionally, the sequestration index (SI), derived as the ratio of PfHRP2-based total biomass to circulating parasitaemia, enhances our understanding of severe malaria dynamics and host responses [15]. Yet, the role of sequestration in modulating coma depth in CM is not fully understood. Again, despite the widespread availability of effective antimalarials, many patients present at hospitals with increased complications even after receiving initial treatment. This is a critical but understudied phenomenon in malaria management, with little known about how these medications affect total parasite burden at the critical juncture of hospital presentation. This study aimed to firstly evaluate the effectiveness of PfHRP2 as a measure of total parasite biomass in various malaria syndromes (CM, SMA, and UM) compared to conventional circulating parasites (PCir), investigate the influence of parasite sequestration on coma severity in paediatric CM, and finally to evaluate the impact of prior antimalarial treatment on parasite biomass during hospital presentation.

Methods

Study design

This was a prospective cohort study that involved paediatric malaria patients presenting to tertiary and secondary healthcare facilities in the Greater Accra Region of Ghana between May 2012 and August 2016. Children were enrolled prospectively and categorized based on clinical presentation into CM, SMA, or UM following comprehensive clinical and laboratory evaluation. Children with CM, mainly presenting in coma were admitted to the paedriatric wards of the health facilities and closely monitored until they recovered from coma (CMRec). Following the recovery of consciousness defined as Blantyre coma score (BCS) > 2, patients were re-evaluated within 12 h. During this period, blood samples were collected for PfHRP2-derived biomass estimation to assess the parasite load and sequestration dynamics during this critical transitional phase of infection.

Study sites and setting

Participants were recruited from five major hospitals in the Greater Accra Metropolitan Area: Princess Marie Louise Children’s Hospital (PML), La General Hospital (La), Ledzokuku Krowoh Municipal Assembly Hospital (LEKMA), Greater Accra Regional Hospital (Ridge), and Tema General Hospital in the port city of Tema. The selection of these hospitals was based on their status as primary referral centres for paediatric emergencies within the Greater Accra Region of Ghana. All study facilities were conveniently located within an hour’s drive of the laboratory at Noguchi Memorial Institute for Medical Research (NMIMR), where the samples underwent subsequent analysis.

The Greater Accra Region is a coastal urban-periurban zone with perennial malaria transmission and seasonal peaks during the rainy seasons. All five participating hospitals are the major referral hospitals with dedicated paediatric departments, 24-h emergency services, and laboratory capacity including microscopy, basic haematology (complete blood count), and biochemical testing.

Study population and sampling

Children aged 1–12 years presenting with suspected malaria underwent systematic evaluation for enrolment eligibility. Children presenting to the emergency department of any of the hospitals with coma and/or fever underwent screening and diagnosis by paediatricians for SMA and/or CM. Similarly, those presenting to the outpatient department (OPD) of the hospitals were screened for UM. Screening for malaria was performed using both microscopy and HRP2-based rapid diagnostic tests (RDTs). Thick and thin blood smears were prepared for malaria parasite identification and density quantification. Complete blood count with haemoglobin measurement, blood glucose, and blood culture were also performed. Lumbar puncture with cerebrospinal fluid analysis was performed in all suspected CM cases to exclude meningitis/encephalitis. UM cases were defined as microscopically confirmed P. falciparum infection with fever (≥ 37.5 °C) but without signs of severe disease, whereas severe malaria included cases fulfilling WHO criteria such as severe anaemia, coma, or respiratory distress. The treatment history of CM patients prior to their initial clinical evaluation was collected either through a structured questionnaire completed by parents or guardians at the time of admission, or, when available, from referral documents. This questionnaire included critical questions about the type of medication, method of administration, and its source. Medications were classified as ‘prior antimalarial treatment’ if parents reported that an antimalarial drug was administered to the child between onset of symptoms and their admission to the hospital.

Inclusion criteria for CM, SMA and UM

Children aged 1–12 years who presented with suspected malaria underwent comprehensive evaluation, including detailed medical histories and physical examinations conducted by attending nurses and physicians. The inclusion criteria for malaria cases required a history of fever or current fever with an axillary temperature exceeding 37.5 °C, confirmed malaria parasitaemia, and syndrome-specific clinical conditions as detailed below. SMA was defined by haemoglobin (Hb) levels below 5.0 g/dL, while CM was identified primarily through impaired consciousness (BCS ≤ 2), lasting longer than 60 min with no history of prior head trauma, bacteraemia (assessed by blood culture) or other neurological disorders. Cerebrospinal fluid analyses were conducted to exclude alternative diagnoses such as meningitis or encephalitis. UM were characterized by patients who were fully conscious and did not meet WHO’s criteria for severe malaria [16]. All malaria cases received standard medical care according to Ghana Health Service guidelines [17].

Laboratory data, blood collection and processing

Whole blood samples of 3 mL and 2 mL were collected from each participant into EDTA tubes for routine clinical testing and PfHRP2 quantification, respectively. Samples designated for routine clinical analyses, including complete blood count (CBC); which is made up of parameters like haemoglobin (Hb), hematocrit (HCT), platelet, white blood cells (WBC), etc., blood culture, and sickling test, were promptly sent to the hospital laboratory. Samples intended for PfHRP2 quantification were transported to the Immunology Laboratory at NMIMR, where they were centrifuged at 1000 × g for 15 min to separate plasma from cellular components. Both plasma and the corresponding blood cell pellets were subsequently stored at − 30 °C for future analysis.

Ethical considerations

Informed written consent was obtained from the parents or guardians of all participants, with the study receiving approval from the Institutional Review Boards of the Noguchi Memorial Institute for Medical Research (NMIMR) with approval number NMIMR-IRB CPN/030/08–09 and the Ghana Health Service Ethical Committee (GHS-ERC: 11/11/11).

ELISA for HRP2 detection

Plasma concentrations of PfHRP2 were determined by enzyme-linked immunosorbent assay (ELISA) using a commercial Malaria Ag CELISA kit (Cellabs, Sydney, Australia). PfHRP2 was measured following the manufacturer's instructions, with the kit’s positive PfHRP2 protein control (1ug/mL) used to generate the standard curve 20 ng/mL, 10 ng/mL, 5 ng/mL, and 2 ng/mL, all in duplicates. The assay was validated for performance before use in measuring patients' plasma levels of HRP2. Optical density (OD) readings at 450 nm were obtained using a microplate reader and PfHRP2 concentrations in diluted clinical samples were converted to PfHRP2 protein levels using the calibration curve as described [18].

Parasite biomass estimation

Circulating, total (PfHRP2-derived), and sequestered parasite biomass estimates were calculated using formulas derived by Dondorp et al., [14] with adjustments in initial parasite replication rate and elimination constant to reflect parasite kinetics in sub-Saharan populations based on empirical data from African paediatric cohorts [15]. Briefly,

$$\begin{array}{cc}\hfill \text{Total biomass}\left(\text{PTot}\right)& =7.3\times PfHRP2\left(g/L\right)\times \left(1-HCT\right)\times \text{body weight}\left[\mathit{kg}\right]\times {10}^{13},\hfill \\ \hfill \text{Circulating burden}\left(\text{PCir}\right)& =\text{parasites}/ul\times {10}^6\times 0.08\times \text{weight}\left[\mathit{Kg}\right],\hfill \\ \hfill \text{Sequestered burden}\left(\text{PSeq}\right)& =\text{PTot}-\text{PCir}.\hfill \\ \hfill \end{array}$$

Sequestration index (SI) was defined as the ratio of PTot to PCir. Parasite biomass was expressed as parasites/body to account for the participants' variation in size. Positive and negative values for sequestered biomass occur because the model by Dondorp et al., assumes sampling in the mid-erythrocytic cycle, such that total biomass may be over- or underestimated in an individual depending on the maturation stage of parasites at the time of taking blood samples.

Definitions

Total parasite biomass (PTot) refers to the total malaria parasite burden in an individual, estimated from PfHRP2 concentrations, and represents both circulating and sequestered parasites. Circulating parasite burden (PCir) is the estimated total parasites in peripheral blood, derived from peripheral parasitaemia (parasite density) and expressed as parasites/body. Sequestered parasite burden (PSeq) reflects the hidden parasites in deep tissues.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism version 10.5.0 (GraphPad Software Inc.). For PTot and PCir, data were log₁₀ transformed. Data for PSeq was not log₁₀ transformed due to the presence of negative values in the UMs. The distribution of the log-transformed data was further assessed for normality using Shapiro–Wilk tests and Q-Q plots to inform the choice of statistical test. One-way ANOVA with Tukey’s post hoc test was used to compare normal distributions while Kruskal–Wallis test was employed for non-normal distributions, followed by Dunn’s post hoc test to account for multiple pairwise comparisons. For comparisons where variables (PTot and PCir) are measured from the same individuals, a paired t-test was performed. Comparative analyses were performed across PTot, PCir, PSeq, and the impact of antimalarial therapy administered before hospital presentation. Correlation analyses were conducted using Spearman's rank correlation coefficient. A threshold of P < 0.05 was considered indicative of statistical significance.

Results

Patient characteristics

A total of 165 participants were assessed in the study. Complete datasets, including laboratory results (haemoglobin, parasitaemia, PfHRP2 concentration) and anthropometric data (body weight), were available for 118 children (CM = 58, SMA = 28 and UM = 32), forming the analytical cohort for parasite biomass analysis (Fig. 1). Forty-seven patients were excluded at initial clinical presentation due to insufficient plasma volume for PfHRP2 quantification and/or lack of clinical data such as weight and hemoglobin levels. A total of 48 CM patients recovered from coma. Follow up data on 10 CM patients were either not complete (n = 6) or patient died (n = 4) during hospitalization and were not available for further analysis. Within the CM group, 23 patients had received prior antimalarial treatment upon hospital presentation but continued to exhibit the documented symptoms. Information on prior antimalarial treatment was available for only CM patients.

Fig. 1.

Study participants enrolled and analysed

The demographic and clinical characteristics are summarized in Table 1. CM and UM patients were older (P < 0.05) than SMA patients, with a male predominance in CM and SMA cohorts. UM patients exhibited the highest temperatures at hospital presentation. CM patients had significantly lower platelet counts than SMA and UM patients, while UM patients demonstrated highest peripheral parasite densities (47,420/uL). Both CM and SMA patients displayed significantly higher (P < 0.05) white cell counts, PfHRP2 concentrations, PTot, and sequestration indices compared to UM (Table 1).

Table 1.

Study participants’ Demographic/Clinical/Laboratory Characteristics. Values are represented as Baseline Mean ($\pm$ SEM) unless otherwise specified

Characteristics	Cerebral Malaria (n = 58)	Severe Malarial Anaemia (n = 28)	Uncomplicated Malaria (n = 32)
Age (Years)	5.5 ($\pm 0.3)$ a	2.7 ($\pm$ 0.4)b	6.1 ($\pm 0.5)$ a
Sex M/F	35/23	15/13	13/19
Temperature (0C)	38.1 ($\pm 0.1$)a	37.8 ($\pm 0.2$)a	39.2 ($\pm 0.1$)b
Haemoglobin Concentration g/dL	8.56 ($\pm 0.21$)a	4.05 ($\pm 0.13$)b	10.07 ($\pm 0.34$)c
Leucocytes (× 109/L)	10.48 ($\pm 0.66$)a	14.36 ($\pm 1.55$)a	7.7 ($\pm 0.47$)b
Platelets (× 103/uL)	63.8 ($\pm 5.7$) a	145.2 ($\pm 21.3$)b	97.0 ($\pm 12.3$)b
Parasite density/uL, Median (IQR)	760 (100–47860) a	24,480 (990–115780)a,b	47,420 (4640–133180)b
Random Blood Sugar (mmol/L)	7.8 ($\pm 0.4$)a	8.2 ($\pm 0.7$)a	N/A
Blantyre Coma Score (BCS)	1 (47%) 2 (53%)	5 (100%)	5 (100%)
Plasma P. falciparum HRP2 concentration (ng/mL)	2316.0 ($\pm 373.4$)a	1115 ($\pm 187.8$)a	195.6 ($\pm 37.4$)b
HRP2-based Total Parasite Estimation (PTot) (× 108/body), Median (IQR)	9545 (2117–29,700)a	5770 (2537–11,850)a	1425 (558–2700)b
Circulating Parasites (PCir) (× 108/body), Median (IQR)	7.7 (0.5–329)a	215 (6.9–1180)a,b	864 (68.6–1890)b
Sequestration Index (SI)	12,666.0 ($\pm 5812$)a	707.7 ($\pm 267.5$)a	11.4 ($\pm 4.4$)b

NB: In each comparison, there is/are no statistical difference(s) for values with the same letters (a,b,c). Values for Sex and BCS were not compared. Statistical significance was set at P < 0.05. BCS presented as categorical distribution. CM patients were stratified by consciousness level at sampling: BCS 1 or BCS 2. All SMA and UM patients were fully conscious (BCS = 5). N/A: data not collected (RBS routinely not done for UM cases).

Evaluating parasite biomass estimations to predict disease severity in malaria

PTot varied across the different groups, with the highest burden observed in CM (P < 0.0001) (Fig. 2A). Mean levels of PCir were significantly higher (P < 0.05) in UM patients compared to CM (Fig. 2B). Except for UM patients, PTot estimates were consistently and significantly higher (P < 0.0001) than their corresponding PCir values across all study groups (Fig. 2C). After regaining consciousness, CM patients exhibited significantly lower (P < 0.0001) PTot and PCir values compared to their corresponding values at initial clinical evaluation (Fig. 2D).

Fig. 2.

Total and Circulating Parasite Biomass Across Malaria Groups (CM, SMA, UM and CMRec). Comparison of PTot (A) and PCir (B) between groups. (C) Intra-group comparison of PTot and PCir between groups. (D) Time point comparison between acute CM and CMRec

Parasite sequestration patterns in malaria syndromes

Children diagnosed with CM and SMA exhibited a significantly higher (P < 0.05) burden of sequestered parasites compared to those with UM (Fig. 3A). During the recovery phase from coma, CM patients exhibited a marked reduction in parasite sequestration (P < 0.0001) relative to their initial clinical presentation (Fig. 3B). Visual presentations show general reduction in the PTot and PSeq (Supplementary Fig. 2). PCir showed strong inverse correlations with PSeq in both CM (rₛ = − 0.8215, P < 0.0001) and SMA (rₛ = − 0.8915, P < 0.0001) patients (Supplementary Table 1). Additionally, both PCir (r_s = − 0.5600) and PTot (r_s = − 0.5165) showed negative correlations with platelet counts in SMA patients (both P = 0.0472), with white blood cell counts in CM patients showing weak negative correlations (r_s = − 0.2640, P = 0.0472) with PTot (Supplementary Table S1).

Fig. 3.

Sequestration Intensity in Malaria Groups. Comparison of Sequestered Parasite biomass between (A) CM, SMA and UM groups and (B) within the CM group

Association of parasite sequestration with coma score in cerebral malaria

The degree of PSeq at the initial assessment of CM patients, did not show significant difference (P > 0.05) across the various levels of consciousness assessed by the BCS (Fig. 4A). During the coma phase, PSeq accounted for approximately 93% of the total parasite biomass in BCS 1, decreasing to about 86% in BCS 2 (Fig. 4B).

Fig. 4.

Sequestration Intensity in Cerebral Malaria stratified by Blantyre Coma Score (BCS)

Impact of prior antimalarial treatment on parasite biomass in cerebral malaria

CM patients presenting with prior antimalarial treatment showed a relatively slight increase in PTot values compared to the untreated groups, showing a mean ($\pm SEM$) of 11.93 ($\pm 0.17$) and 11.77 ($\pm 0.14)$ respectively (Fig. 5). This trend was different in peripheral parasitaemia estimates (Fig. 5), where PCir was significantly (P = 0.03294) higher in the untreated group [9.63 ($\pm 0.27$)] compared to the treated [8.93 ($\pm 0.27)]$.

Fig. 5.

Impact of Prior Antimalarial Therapy on PTot and PCir in Cerebral Malaria

Discussion

This study explored the dynamics of parasite biomass in relation to malaria severity, and how plasma PfHRP2-derived parasite biomass could be a better marker for assessing malaria severity compared to peripheral parasitaemia. Plasma PfHRP2-derived total parasite biomass (PTot) showed stronger associations with malaria disease severity than microscopy-based circulating parasite burden (PCir) in our study groups. In severe malaria cases (CM and SMA), PTot significantly exceeded PCir, considering the presence of parasite sequestration which are often seen in these severe malaria cases. In contrast, uncomplicated malaria (UM) patients exhibited no significant difference between PTot and PCir, suggesting minimal sequestration in mild diseases of malaria. These findings suggest that peripheral parasitaemia have limited discriminatory value in differentiating malaria syndromes, particularly in conditions characterised by substantial parasite sequestration. Peripheral parasite counts predominantly reflect circulating ring-stage parasites, which are less virulent compared to mature, sequestered forms in the microvasculature of critical organs such as the brain and lungs [19, 20]. PfHRP2 accumulates in plasma from both circulating and sequestered parasites, thereby serving as a robust proxy for assessing total parasite burden [13, 21, 22].

In cases of UM, despite elevated peripheral parasitaemia, PfHRP2 levels consistently remained lower compared to CM patients, also reflecting minimal sequestration [23]. The inverse correlations between PCir and PSeq in severe malaria likely indicates that higher sequestration is associated with fewer detectable malaria parasites in peripheral blood. Sequestration in CM enables the parasites to evade peripheral detection despite concurrently contributing to elevating plasma PfHRP2 levels [14]. PfHRP2-derived biomass varied significantly across different malaria groups, with the highest level in CM, followed by SMA and then UM. Conversely, PCir displayed an inverse pattern, with UM cases showing markedly higher circulating densities compared to CM. The notable decline of PCir in CM patients with improved consciousness despite persistent levels of PTot, likely reflects delayed PfHRP2 clearance or persistent endothelial cytoadherence of dying parasites [24, 25]. This challenges the reliability of peripheral parasitaemia alone to monitor treatment response in CM.

Sequestered parasite burden (PSeq) was analysed across different groups to evaluate the intensity in various malaria syndromes. PSeq increased progressively with disease severity reflecting the pivotal role of microvascular parasite accumulation in severe disease manifestations [26]. There was no statistically significant difference in PSeq between SMA and CM. The study showed a significant reduction in parasite sequestration at recovery (BCS > 2) from coma, accompanied by a relatively decreased proportion of PSeq in BCS2 (85.6%) compared to BCS 1 (92.8%). This pattern suggests a potential association between coma score and the extent of parasite sequestration [23, 27–29].

In Ghana, most cases of severe malaria syndromes are only reported to health facilities after treatment failure, thereby highlighting the need to assess the impact of prior-hospital antimalarial therapy on parasite burden in severe malaria cases. Although previous reports suggest that unsuccessful antimalarial treatment may transiently increase the levels of PfHRP2 [30], PTot in our CM cohort did not differ significantly between patients who self-medicated or received prior antimalarial treatment at a referral facility, compared to the untreated group. In contrast, mean PCir was significantly lower in the treated group. Our sample size for treatment comparisons provided insufficient statistical power to definitely characterize the effect of prior-hospital antimalarial on parasite burden despite the observed elevation in PTot for the treated individuals. Consequently, it is essential to take the patient's treatment history into account when interpreting PfHRP2 measurements. This calls for future studies with serial PfHRP2 measurements to correlate these biomass patterns to better understand the clinical consequences of treatment-altered parasite distribution.

Conclusions

Overall, this study demonstrates that total malaria parasite burden estimated using plasma PfHRP2 levels gives a stronger association with malaria disease severity within clinical syndromes with substantial sequestration than peripheral parasitaemia alone. Higher PfHRP2 concentrations and sequestration indices may be linked to most severe malaria forms, especially cerebral malaria, and are associated with coma.

Recommendations and limitations

Integrating PfHRP2-derived total parasite biomass measurements into routine malaria severity assessments holds the potential to enhance the early identification of severe cases. The study elucidates associations but has not established the predictive validity of PfHRP2-derived biomass estimates for clinical application. It is noteworthy that PfHRP2 levels can persist even after parasite clearance, which may lead to an overestimation of total biomass in recovering patients. Again, sequestered parasites estimation was made indirectly (estimated from PfHRP2 concentrations) rather than direct visualization using histological or imaging techniques. We, therefore, advocate for future research involving a larger, multi-center prospective cohort to validate PfHRP2 thresholds for risk stratification, conduct serial PfHRP2 measurements to characterize clearance kinetics, and evaluate treatment effects.

Abbreviations

PfHRP2

Plasmodium falciparum Histidine-Rich Protein 2

CM

Cerebral Malaria

SMA

Severe Malarial Anaemia

UM

Uncomplicated Malaria

CMRec

Cerebral Malaria Recovered

PTot

Total Parasite Biomass

PCir

Circulating Parasite Burden

PSeq

Sequestered Parasite Burden

SI

Sequestration Index

BCS

Blantyre Coma Score

EDTA

Ethylenediaminetetraacetic Acid

CBC

Complete Blood Count

OPD

Outpatient Department

RBCs

Red Blood Cells

SEM

Standard Error of the Mean

RBS

Random Blood Sugar

OD

Optical Density

ELISA

Enzyme-Linked Immunosorbent Assay

NMIMR

Noguchi Memorial Institute for Medical Research

WHO

World Health Organization

Author contributions

*DO and BG designed the study and the overall research framework. *DO, BG, TA, and DO carried out patient recruitment, clinical assessments, and data acquisition from participating hospitals. *DO, BG, EKB, TA and KAK performed experiments. EAK, AOO, CKDC, and *DO analysed the data. EAK, AOO, TA, EKB, DO, LEA, KAK and *DO drafted the manuscript. *DO, BG and KAK oversaw research activities, data integrity, and manuscript preparation. All authors read and approved the final manuscript.

Funding

No external funding was received for this study. The authors conducted the research using internal resources from their respective institutions.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research (CPN/030/08-09) and the Ghana Health Service Ethical Committee (GHS-ERC: 11/11/11). Informed written consent was obtained from parents or legal guardians after detailed explanations of the study’s purpose, procedures, risks, and benefits. Participation was voluntary, with the option to withdraw at any time without affecting the standard of care.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.