Red blood cell homeostasis in children and adults with and without asymptomatic malaria infection in Burkina Faso.

Asymptomatic malaria infections may affect red blood cell (RBC) homeostasis. Reports indicate a role for chronic hemolysis and splenomegaly, however, the underlying processes are incompletely understood. New hematology analysers provide parameters for a more comprehensive analysis of RBC hemostasis. Complete blood counts were analysed in subjects from all age groups (n = 1118) living in a malaria hyperendemic area and cytokines and iron biomarkers were also measured. Subjects were divided into age groups (<2 years, 2-4, 5-14 and ≥15 years old) and clinical categories (smear-negative healthy subjects, asymptomatic malaria and clinical malaria). We found that hemoglobin levels were similar in smear-negative healthy children and asymptomatic malaria children but significantly lower in clinical malaria with a maximum difference of 2.2 g/dl in children <2 years decreasing to 0.1 g/dl in those aged ≥15 years. Delta-He, presenting different hemoglobinization of reticulocytes and RBC, levels were lower in asymptomatic and clinial malaria, indicating a recent effect of malaria on erythropoiesis. Reticulocyte counts and reticulocyte production index (RPI), indicating the erythropoietic capacity of the bone marrow, were higher in young children with malaria compared to smear-negative subjects. A negative correlation between reticulocyte counts and Hb levels was found in asymptomatic malaria (ρ = -0.32, p<0.001) unlike in clinical malaria (ρ = -0.008, p = 0.92). Free-Hb levels, indicating hemolysis, were only higher in clinical malaria. Phagocytozing monocytes, indicating erythophagocytosis, were highest in clinical malaria, followed by asymptomatic malaria and smear-negative subjects. Circulating cytokines and iron biomarkers (hepcidin, ferritin) showed similar patterns. Pro/anti-inflammatory (IL-6/IL-10) ratio was higher in clinical than asymptomatic malaria. Cytokine production capacity of ex-vivo whole blood stimulation with LPS was lower in children with asymptomatic malaria compared to smear-negative healthy children. Bone marrow response can compensate the increased red blood cell loss in asymptomatic malaria, unlike in clinical malaria, possibly because of limited level and length of inflammation. Trial registration: Prospective diagnostic study: ClinicalTrials.gov identifier: NCT02669823. Explorative cross-sectional field study: ClinicalTrials.gov identifier: NCT03176719.

Introduction

Malaria is a major cause of anaemia in sub-Saharan Africa with a multifactorial aetiology, including hemolysis, dyserythropoiesis and inflammation induced functional iron deficiency [1]. The aetiology of severe anaemia in clinical malaria is often described [2, 3] where destruction of erythrocytes is compounded by suppressed erythropoiesis [4-6], possibly due to cytokine imbalance [3, 7].

In asymptomatic malaria, however, where Plasmodium falciparum (Pf) infection does not result in signs of illness and hemoglobin (Hb) levels are less severely affected [8-11]. Hemolysis and splenomegaly, driven by the magnitude of the infecting biomass and chronicity of infection, are recognised as important factors for the development of anaemia in asymptomatic malaria [12]. Furthermore, defining malaria-attributable anaemia is difficult, as many prevalent comorbidities may also lead to dyserythropoiesis and inflammation induced functional iron deficiency. The capacity of the bone marrow to respond to decreasing Hb levels can however be assessed by analysing reticulocyte counts. So far, only one study reported reticulocyte numbers in asymptomatic malaria patients however [10]. In this study, semi-immune children between 5–15 years old with asymptomatic malaria, had lower Hb levels, increased reticulocyte numbers and erythropoietin levels as well as TNF-α levels, compared to healthy non-malaria carriers, indicating dyserythropoiesis. Although Pf is known to induce a strong pro-inflammatory response and thereby the development of anaemia, malaria can also induce tolerance to subsequent infections or immune challenges which may limit the development of anaemia in asymptomatic malaria [13, 14].

New generation hematology analysers provide new parameters, such as the reticulocyte production index (RPI), immature reticulocyte fraction (IRF), Delta-He and monocyte phenotypes [15], that enable a more comprehensive understanding of hematological changes and better insight into the bone marrow response to anaemia [16]. Reference values are available from the Western population [17] but are lacking from Africa.

The primary aim of present study was to get a more comprehensive understanding of the hematological changes in subjects with asymptomatic malaria in the various age groups. We hypothesized that in asymptomatic malaria, a pro-inflammatory status together with low grade hemolysis will induce anaemia while immune tolerance may have a protective effect on the erythropoiesis. For this study, we used a new generation hematology analyser that provide parameters to monitor erythropoiesis and the results were combined with markers that influence erytropoieis such as iron biomarkers, circulating cytokines and ex-vivo cytokine production capacity of stimulated whole blood. The study was carried out in Burkina Faso, where prevalences of asymptomatic malaria of up to 75% having been described [18].

Materials and methods

Study site and design

Studies were conducted from March 2016 till September 2017 at the Clinical Research Unit Nanoro (CRUN) in a hyperendemic area for Pf malaria [19]. Subjects were recruited during two studies, allowing the inclusion of control groups, such as asymptomatic subjects that are malaria smear negative and patients with clinical malaria. In the present analysis, we included patients with clinical malaria from a prospective diagnostic accuracy study enrolling patients of 3 months and older with acute febrile illness, as described elsewhere [20]. The second study was a cross-sectional field study among 1000 randomly selected healthy volunteers aged over 1-year-old from 24 villages of the Nanoro health and demographic surveillance system (HDSS) [21], allowing inclusion of asymptomatic subjects that are malaria smear negative or positive. See also .

Participants of the two studies were stratified into four age groups: three groups of children (>3 months to <2 years, 2–4 years and 5–14 years) and one group of adults (≥15 years). Furthermore, participants were divided according to case definitions into smear-negative healthy subjects, asymptomatic malaria and clinical malaria. Finally, asymptomatic malaria and smear-negative subjects were combined in a group of asymptomatic subjects and compared with the clinical malaria patients.

Laboratory analyses

Hematology analyses were performed using the Sysmex XN-1000 haematology analyser (Sysmex Corporation, Kobe, Japan), which generated standard full blood counts including reticulocyte counts as well as research parameters (S1 Table) [15]. Furthermore, free haemoglobin (Free-Hb) levels were defined as differences between standard Hb measurements (all RBCs are lysed) and optical Hb level (HGB-O) which measures Hb within intact RBCs. Free-Hb levels above the upper reference limit (i.e. typically ≥0.25 g/L in serum or ≥0.13 g/L in plasma) indicate hemolysis, although free-Hb estimates from the Sysmex analyser and values given by Lippi et al [22] may not be directly comparable. The number of phagocyting monocytes (Phago-MONO), as analysed in the hematology analyser, were used to indicate erythrophagocytosis.

Thick and thin blood films for malaria diagnosis were examined according to World Health Organization procedures [23].

Iron biomarkers Ferritin (FERR), hepcidin (HEP) and soluble transferrin receptor (sTfR) were measured in EDTA plasma samples by quantitative sandwich enzyme immunoassay technique. Four circulating cytokines (IL-6, TNF-α, IFN-ϒ and IL-10) were quantified by MAGPIX technology (Luminex Corporation, Austin, Texas, USA) in EDTA plasma, according to manufacturer’s instructions. Ex-vivo cytokine production of cytokines (IL-1β, IL-6, IL-10, TNF-α and IFN-ϒ) was measured from the supernatant of heparinised whole blood after stimulation with lipopolysaccharide (LPS) by standard “sandwich-type” ELISA procedure (ThermoScientificTM PierceTM). Samples from the under-five year’s group (<5 years) were used because of the highest burden of malarial anaemia in this group.

Data management and statistical aspects

Data are reported as median and interquartile range (IQR) unless stated otherwise. Medians were compared using Mann-Whitney U test, and proportional differences were assessed by Pearson chi-squared or Fisher exact test where appropriate. Spearman correlation was used to assess the relationship between continuous variables of interest. STATA 14 (Stata Corp., TX, USA) was used for the statistical analysis. The p values were adjusted for multiple comparison using Benjamin Hochberg procedure. After correcting for multiple testing, the cut-off point of 0.05 was set as limit of significance. Furthermore, a factor analysis taking into account all the biomarkers was performed using the appropriate R software package in order to assess a correlation patterns/clustering trends among the markers. For each marker, a value test (v. test) representing the importance and the correlation direction (defined by: increase = +; decrease = -) of the marker in the corresponding group was computed.

Ethical considerations

Study protocols were approved by the national ethics committee of Burkina Faso (ref 2016-01-006). The protocol of the diagnostic study was furthermore approved by the institutional review board of Institut de Recherche en Sciences de la Santé (ref A03-2016/CEIRES), the ethical committee of the university hospital of Antwerp (ref 15/47/492) and the review board of the Institute of Tropical Medicine Antwerp (ref 1029/15). Written informed consent was obtained from all participants or their parents/legal guardians. Assent was obtained from all participants aged 7 to 20 years according to the local requirement.

Results

Study population and characteristics

A total of 1118 subjects were enrolled: 191 from the diagnostic accuracy study and 927 from the cross-sectional study (). describes the demographic and clinical characteristics. The mean (standard deviation) of age were 1.5 (0.4) years, 3.2 (0.9) years, 9.7 (3.0) years, 41.8 (19.7) years in the age category of <2years, 2–4 years, 5–14 years and ≥15 years respectively. In the cross-sectional study, 444 out of 927 (48%) asymptomatic subjects had microscopic positive malaria parasitemia. The proportion of asymptomatic malaria infections in ascending age categories was 29.9%, 38.5%, 69.6% and 30.6% respectively. Parasite density (PD) was significantly higher (p<0.002) in all ages in clinical malaria compared to asymptomatic malaria. Pf was the most prevalent species (96.2%).

Flow diagram study subjects.

Diagram represents how the participants included in the analysis were selected from the two studies as described in the methodology section. Only participants with documented malaria result were considered.

Red blood cell reference values and iron and inflammation biomarkers

First we used the data of the 483 smear-negative healthy subjects to determine reference values (. RBC, as well as absolute and relative reticulocyte counts remained stable with age. In contrast, Hb concentrations and hematocrit (Hct) values increased steadily across the age categories from a median Hb and Hct value in the children <2 years of 9.6 g/dL (IQR: 8.8–10.7) and 31.9% (28.5–33.6) to 12.2 g/dL (11.5–13.4) and 37.4% (34.8–39.9), respectively in those ≥15 years. Age related changes were also seen for other RBC markers, such as MCV, MCH, MCHC, HYPO-HE, HYPER-HE, micro-RBC (MicroR), macro-RBC (MacroR), RET-He, RBC-He ().

Hemoglobin (Hb), hematocrit (HCT), red blood cells count (RBC), reticulocyte count (RET#) and percentage (RET%) Reticulocyte Production Index (RPI), Immature Reticulocyte Fraction count (IRF#), nucleated RBC count (NRBC#) and Delta-He per clinical group and age category.

Plots display the status of each hematology parameter per age category. In each age category, participants are divided regarding the health status according to the case definitions whereby healthy smear-negative subjects are represented by “No malaria”, smear-positive asymptomatic infections represented by “Asymptomatic malaria” and smear-positive patients with fever are represented by “Clinical malaria”. Whiskers bottom and top limits are 5 th and 95 th percentiles respectively; (): continuous line is used for comparison between clinical status within the same age category and each clinical status was compared with all the other status; (—): dotted line is used for comparison between age category in the “No malaria” group; *: p value with statistically significant difference (p<0.05) between clinical status within the same age category; ▪: p value with statiscally significant difference (p<0.05) between age category in the “No malaria” group; Mann-Whitney U test was used for comparison between groups; yrs: years; #:absolute count; %: percentage count. No malaria Asymptomatic malaria Clinical malaria.

Although the total number of reticulocytes did not change, their different maturation stages [24] were subject to age-related trends. In addition, both the Hb content of reticulocytes (RET-He) and of RBC (RBC-He) increased across the age categories, resulting in a stable Delta-He (RET-He minus RBC-He) (). Age specific changes in leukocyte and platelet parameters are given in S2 Table.

The level of plasma iron biomarkers and inflammatory cytokines changed in the various age groups. Concentrations of sTfR declined with increasing age, with a median (IQR) concentration of 53.8 nmol/L (40.9–69.9) in the children <2 years and 29.5 nmol/L (23.5–35.9) in those ≥15 years. In contrast, plasma ferritin and hepcidin concentrations increased with age from 9.7 (7.8–24.6) ng/ml to 49.6 (22.6–76.4) ng/ml and from 2.0 (0.9–6.2) pg/ml to 11.6 (5.6–21.4) pg/ml, respectively (). Circulating levels of the proinflammatory cytokine TNF-α decreased across the age categories, while the other cytokines remained stable (IL-6 and IL-10) or were mostly undetectable (IFN-ϒ). ( and S3 Table).

Iron biomarkers, soluble Transferrin Receptor (sTfR), ferritin and hepcidin per clinical group and age category.

Plots display the level of iron biomarker per age category. In each age category, participants are divided regarding the health status according to the case definitions whereby healthy smear-negative subjects are represented by “No malaria”, smear-positive asymptomatic infections represented by “Asymptomatic malaria” and smear-positive patients with fever are represented by “Clinical malaria”. Whiskers bottom and top limits are 5 th and 95 th percentiles respectively; (): continuous line is used for comparison between clinical status within the same age category and each clinical status was compared with all the other status; (—): dotted line is used for comparison between age category in the “No malaria” group; *: p value with statistically significant difference (p<0.05) between clinical status within the same age category; ▪: p value with statistically significant difference (p<0.05) between age category in the “No malaria” group; Mann-Whitney U test was used for comparison between groups; yrs: years. No malaria Asymptomatic malaria Clinical malaria.

Different reticulocyte response compensating malarial anaemia in asymptomatic and clinical malaria

Children with clinical malaria had significantly lower Hb levels than both smear-negative subjects and children with asymptomatic malaria. (). Remarkably, we found no significant differences in Hb levels between the asymptomatic children < 15 years, except those 2–4 years, with or without microscopic parasitemia. The difference in Hb level between asymptomatic subjects and those with clinical malaria was most pronounced in the young children with a maximum difference (median) in Hb level of 2.2 g/dl in children <2 years decreasing to 0.1 g/dl in those aged ≥15 years (12.1 g/dl versus 12.2 g/dl). Haematocrit levels and RBC counts followed similar trends. In adults, there was no difference between the groups in any of these parameters.

Both absolute and relative reticulocyte counts, estimators of bone marrow activity, were elevated among clinical and asymptomatic malaria infections compared to smear-negative subjects (). These differences (in particular for the relative count) were only observed in children in the age categories up to 15 years of age. Delta-He is low in acute inflammation but not in chronic inflammation [25]. Delta-he levels are low, both in clinical and asymptomatic malaria in children, indicating a recent effect of malaria on erythropoiesis (). Adequacy of the bone marrow response for the level of anaemia was analyzed using the reticulocyte production index (RPI). RPI was elevated in children (with significant difference in the <2 years category) with asymptomatic malaria, unlike clinical malaria patients, compared to smear-negative healthy subjects (). This was supported by the finding of similar reticulocyte numbers and a similar immature reticulocyte fraction (IRF), an early marker of erythroid regeneration, among asymptomatic malaria and clinical malaria in children despite different Hb levels. Moreover, we observed a significant negative correlation between absolute reticulocyte counts and Hb levels in asymptomatic malaria (ρ = -0.32, p<0.001) while in clinical malaria, a nearly flat correlation line was found (ρ = -0.008, p = 0.92) (). Furthermore, we found that the percentage of immature reticulocytes (HFR and MFR) increase while the opposite is observed for the mature reticulocytes (LFR). This effect is seen in children < 5 years old with no differences between clinical and asymptomatic malaria (). Finally, the number of nucleated RBC (NRBC) numbers, that are only seen in case of extreme erythropoietic activity, were only increased in children with clinical malaria ().

Correlation between hemoglobin (Hb) and reticulocytes absolute count (RET#) in no malaria, asymptomatic malaria and clinical malaria cases.

Correlation between the hemoglobin level and reticulocytes absolute count in three groups classified according to the health status and based to the case definitions whereby healthy smear-negative subjects are represented by “No malaria”, smear-positive asymptomatic infections represented by “Asymptomatic malaria” and smear-positive patients with fever are represented by “Clinical malaria”. Spearman test was used to assess the correlation. The line represents the nonlinear fit regression line between the variables.

Different circulating cytokine levels and ex-vivo cytokine production capacity in young children with or without asymptomatic malaria

Inflammation may interfere with erythrocyte homeostasis in different ways [26]. Levels of circulating pro- (IL-6, TNF-α and IFN- ϒ) and anti-inflammatory cytokines (IL-10) were highest in clinical malaria. Nevertheless, cytokine levels were also elevated in asymptomatic malaria compared to smear-negative subjects (). In both malaria groups, IL-6 levels correlated significantly with parasite density (ρ = 0.49 and ρ = 0.48 respectively, p<0.001) (). The same trend was seen for TNF-α, IFN-γ and IL-10 (data not shown). Apart from lower levels of circulating proinflammation markers because of lower levels of parasitemia (), immune tolerance may also play a role in asymptomatic malaria. The IL-6/IL-10 ratio, indicating pro/anti-inflammatory balance, was significantly lower in asymptomatic children, compared to clinical malaria (). In addition, ex-vivo cytokine production after LPS stimulation was significantly lower for all cytokines, except for IL-6 (p = 0.053), in the asymptomatic malaria infections below the age of 5 years compared to smear-negative healthy subjects ().

A. Circulating cytokines levels (IL-6, IFN-ϒ, IL-10 and TNF-α) per clinical group and age category and Ratio IL-6/IL-10 in malaria infected subjects according to age groups. In each age category, participants are divided regarding the health status according to the case definitions whereby healthy smear-negative subjects are represented by “No malaria”, smear-positive asymptomatic infections represented by “Asymptomatic malaria” and smear-positive patients with fever are represented by “Clinical malaria”. Whiskers bottom and top limits are 5th and 95th percentiles respectively; (): continuous line is used for comparison between clinical status within the same age category and each clinical status was compared with all the other status; (—): dotted line is used for comparison between age category in the “No malaria” group; *: p value with statistically significant difference (p<0.05) between clinical status within the same age category; ▪: p value with statiscally significant difference (p<0.05) between age category in the “No malaria” group; Mann-Whitney U test was used for comparison between groups; yrs: years. No malaria Asymptomatic malaria Clinical malaria. B. Correlation between IL-6 levels and parasite density (PD) in asymptomatic malaria and clinical malaria cases as previously classified. Spearman test was used to assess the correlation. The line represents the nonlinear fit regression line between the variables. C. Ex-vivo cytokines production (IFN-ϒ, TNF-α, IL-1β, IL-6 and IL-10) after whole blood stimulation with Lipopolysaccharide (LPS) in asymptomatic children less than 5 years old, that were microscopically malaria positive or negative. Whiskers bottom and top limits are 5th and 95th percentiles respectively; (): continuous line is used for comparison between clinical status *: p value with statistically significant difference (p<0.05) between clinical groups; Mann-Whitney U test was used for comparison between groups. No malaria Asymptomatic malaria.

Erythrophagocytosis is increased in asymptomatic malaria, especially in young children

Apart from bone marrow suppression, inflammation may lead to anaemia by reducing survival time of erythrocytes by activating macrophages and increase their phagocytozing capacity. We found that the number of phagocytozing monocytes (Phago-MONO) and activated monocytes (RE-MONO) was increased in both asymptomatic and clinical malaria compared to smear-negative healthy subjects in all age categories, the values being highest for clinical malaria. The differences were most striking in children <5 years of age (). We found no relation between phagocytozing monocytes numbers and inflammation, nor Hb levels. In contrast, the number of activated monocytes, that followed the same trend as phagocytizing monocytes () related well to Hb levels ().

A. The proportion of activated monocytes (RE-MONO%) over total number of monocytes and phagocytozing monocytes count (Phago-MONO) per clinical status and age category. In each age category, participants are divided regarding the health status according to the case definitions whereby healthy smear-negative subjects are represented by “No malaria”, smear-positive asymptomatic infections represented by “Asymptomatic malaria” and smear-positive patienst with fever are represented by “Clinical malaria”. Whiskers bottom and top limits are 5 th and 95 th percentiles respectively; (): continuous line is used for comparison between clinical status within the same age category and each clinical status was compared with all the other status; *: p value with statistically significant difference (p<0.05); yrs: years; Mann-Whitney U test was used for comparison between groups. No malaria Asymptomatic malaria Clinical malaria. B. Correlation between Activated Monocytes count (RE-MONO#), haemoglobin (Hb) and ciculating TNF-α levels in “Asymptomatic malaria” and “Clinical malaria” as previously classified. Spearman test was used to assess the correlation. The lines represents the nonlinear fit regression line between the variables for each clinical status and as indicated in the legend (Red for “Clinical malaria” and Black for “Asymptomatic malaria”).

Next to erythrophagocytosis, reduced survival time of erythrocytes can be caused by hemolysis. Mean (standard deviation) free-Hb levels were 2.4 (4.2) g/L in clinical malaria which is significantly higher than 0.8 (3.4) g/L in asymptomatic malaria infections or 0.9 (3.4) g/L in smear-negative healthy subjects (). Remarkably, free-Hb levels were related to level of parasitemia in asymptomatic malaria infections, but not in clinical malaria ().

A. Free-hemoglobin levels (g/L) per clinical status and age category. In each age category, participants are divided regarding the health status according to the case definitions whereby healthy smear-negative subjects are represented by “No malaria”, smear-positive asymptomatic infections represented by “Asymptomatic malaria” and smear-positive patienst with fever are represented by “Clinical malaria”. Whiskers bottom and top limits are 5 th and 95 th percentiles respectively; (): continuous line is used for comparison between clinical status within the same age category and each clinical status was compared with all the other status; *: p value with statistically significant difference (p<0.05); yrs: years; Mann-Whitney U test was used for comparison between groups. No malaria Asymptomatic malaria Clinical malaria. B. The correlation between the free-haemoglobin (Free-Hb) and the parasite density (PD) in “Asymptomatic malaria” and “Clinical malaria” as previously classified. Spearman test was used to assess the correlation. The line represents the nonlinear fit regression line between the variables.

Iron and inflammation is altered in asymptomatic malaria, especially in young children

Inflammation may also influence iron homeostasis. IL-6 is an important driver of hepcidin and ferritin levels [27]. shows the iron biomarkers in the different age categories and health status. In line with the inflammation data, ferritin and hepcidin levels are high in clinical malaria, followed by asymptomatic malaria and smear-negative healthy subjects in all age categories. At the same time, ferritin and hepcidin levels increase with age in the asymptomatic malaria and smear-negative healthy subjects, while sTfR, that was not different according to the health status, decreases. In contrast, there seems to be no clear age-relation for hepcidin and ferritin in patients with clinical malaria.

The biomarkers are clustered according to malaria status

A factor analysis including all the biomarkers indicate a clustering trend of the biomarkers according to the participants health status as displayed on the The extend (increase or decrease) in which each biomarkers is expressed depending on the health status is presented in the S4 Table.

Discussion

The hematological reference values for people living in a malaria hyperendemic area in Burkina Faso indicate that Hb levels and erythrocyte indices increase with age. The concomitant changing iron and inflammation parameters suggest an age-related improvement of iron incorporation. Hb levels did not differ between asymptomatic malaria and smear-negative healthy subjects, while the presence of higher reticulocyte response in asymptomatic malaria indicate that RBC losses are adequately compensated by bone marrow response. Our data further suggest that the effect of malaria on the bone marrow is mostly acute while a limited pro-inflammatory response in asymptomatic malaria may preserve bone marrow response.

Our study had several strenghts, as the inclusion of more than 1100 participants from different age groups allowed us to provide hematological reference ranges for a malaria hyperendemic area. Control groups were included to compare asymptomatic malaria with smear negative asymptomatic subjects and those with clinical malaria. Furthermore, the combined assessment of hematology parameters, iron biomarkers and circulating and ex-vivo cytokines improved our insight in RBC homeostasis.

Nevertheless, a number of limitations need to be noted. First, malaria classification was based on microscopy therefore sub-microscopic malaria infections may have been included in the smear-negative group. Second, we did not screen for other infections like hookworm or hemoglobinopathies and the ex-vivo cytokines production was only available for the under-five years. For ethical reasons, all smear positive asymptomatic infections were given malaria treatment, so we can not provide evidence for the acute or chronic nature of the infection.

Our finding of similar Hb levels among asymptomatic subjects that are smear positive or negative, is in contrast with previous reports [9, 28–32]. The level of malaria endemicity, co-occuring conditions such as iron deficiency, hemoglobinopathies and intestinal parasites, may explain the found differences. Our study, carried out in a high malaria endemic area with 48% of healthy population being smear positive, shows that asymptomatic malaria infections, unlike clinical malaria patients, have an adequate bone marrow response to decreasing Hb levels, using reticulocyte numbers and RPI as indicators. Previous reports suggest that chronic hemolysis and splenomegaly, driven by the magnitude of the infecting biomass and chronicity of infection, underly the development of anaemia in asymptomatic malaria [12]. Our finding of low Delta-He levels, indicating the difference in hemoglobinization between reticulocytes and erythrocytes, suggests a more acute effect of malaria on the bone marrow however [25]. The hemoglobin content of reticulocytes [reported as RET‐He on Sysmex analysers (Sysmex Corporation, Kobe, Japan) and as CHr on ADVIA analysers from Siemens (Siemens Medical Solutions Diagnostics, Erlangen, Germany)] is an indicator for iron incorporation in the hem over the previous 2–4 days [33]. The hemoglobin content of mature red blood cells (RBC‐He on Sysmex analysers and CH on Bayer analysers) reflect iron availability over a much longer period, as erythrocytes have a lifespan of 100–120 days [33]. Hemolysis may reduce lifespan of RBCs in asymptomatic malaria but the difference with reticulocytes will remain significant.

Free-Hb levels, as measured in our study, were only higher in clinical malaria patients compared to smear negative infections, which argues against a significant role of hemolysis in asymptomatic malaria [12]. However, for acurate estimation, the amount of free-Hb that is bound, sequestred and processed should be known. Furthermore, blood was collected from asymptomatic subjects during a field study and we can not exclude artificial hemolysis due to sample processing, which may obscure differences between those that were smear positive or negative.

Erythropoiesis may be suppressed by increased inflammation. We found increased levels of circulating pro-inflammatory cytokines in asymptomatic and clinical malaria, that related to levels of parasite densities, however, considering also the anti-inflammatory cytokine IL-10, we found that the IL-6/IL-10 ratio was significantly lower in asymptomatic children, compared to clinical malaria, as reported before [34, 35]. This difference in ratio was not noticed in adults, possibly explaining the similar Hb levels in adults with or without clinical malaria. In addition to circulating cytokine measurements, we also assessed ex-vivo whole blood cytokine production after LPS stimulation and found dampened responses in asymptomatic malaria infections as reported [35-37], suggesting immune-tolerance in these subjects. Apart from suppression of erythropoiesis, inflammation may also restrict erythroid cell differentiation and proliferation. We indeed found that NRBCs were prominent in clinical malaria, unlike asymptomatic malaria.

In line with the inflammation data, we found that ferritin as well as hepcidin are high in clinical malaria while only mildly elevated in asymptomatic malaria. Inflammation significantly influences iron homeostasis and both the major iron storage protein ferritin and the major iron transport regulating protein hepcidin are well known acute-phase reactants. A recent study indicated that these iron biomarkers are influenced by acute clinical malaria and during convalescence [38]. On the other hand, levels of sTfR were not different between the various clinical status, which is in contrast to findings from Righetti et al [39]. Evaluation of sTfR levels in our study is complicated as inflammation, malaria, age and iron deficiency all play a role. Looking at the different clinical status in the various age categories, we found increasing Hb levels as subjects get older as well as RBC indices (MCV, MCHC, MCH), increasing HEP and FERR levels combined with decreasing pro-inflammatory cytokines and sTfR levels. Taken together, these data suggest an increased availability of iron for erythropoiesis or increased iron incorporation over time. Also new RBC indices suggest similarly, as MacroR, HYPER-He, RBC-He and RET-He increase while MicroR, HYPO-He decrease.

Apart from hemolysis, iron disturbance and suppression of erythropoietic activity, enhanced erythrophagocytosis may also contribute to anaemia [27]. Phagocytic monocytes (Phago-MONO) are detected on Sysmex hematology analysers as research parameter. The presence of phagocytic monocytes was prominent in clinical malaria, followed by asymptomatic malaria and smear-negative healthy subjects, and mostly only in children <5 years. Importantly, the number of phagocytic monocytes did not correlate with Hb levels nor with circulating cytokine levels, as increased phagocytosis of uninfected RBC is thought to be the main contributor of anaemia in malaria [1, 40, 41]. In contrast, the number of activated monocytes did correlate with Hb levels and circulating cytokine levels.

Conclusions

Bone marrow response, using reticulocyte numbers, RPI and hemoglobin levels as indicators, seem to compensate the increased red blood cell loss in asymptomatic malaria infections, unlike in patients with clinical malaria. Low Delta-He levels as were found in asymptomatic and clinial malaria, suggests mostly a more acute effect of malaria. Our data also suggests that limited inflammation in asymptomatic malaria may play a role in the preservation of the bone marrow response to the higher turnover of RBC driven by the magnitude of the infecting biomass and chronicity of infection.

RBC indices (MCV, MCH, MCHC, MicroR, MacroR, HYPO-He, HYPER-He) and hemoglobinization of reticulocytes (RET-he) and RBC (RBC-He) per clinical group and age category.

Plots display the status of each haematology parameter per age category. In each age category, participants are divided regarding the health status according to the case definitions whereby healthy smear-negative subjects are represented by “No malaria”, smear-positive asymptomatic cases represented by “Asymptomatic malaria” and smear-positive patienst with fever are represented by “Clinical malaria”. Whiskers bottom and top limits are 5th and 95th percentiles respectively; (): continuous line is used for comparison between clinical status within the same age category and each clinical status was compared with all the other status; (—): dotted line is used for comparison between age category in the “No malaria” group; *: p value with statistically significant difference (p<0.05) between clinical status within the same age category; ▪: p value with statiscally significant difference (p<0.05) between age category in the “No malaria” group; Mann-Whitney U test was used for the comparison between groups; NS: not significant; yrs: years; %: percentage. No malaria Asymptomatic malaria Clinical malaria.

(TIF)

Click here for additional data file.

High fluorescence reticulocytes (HFR), medium fluorescence reticulocytes (MFR), low fluorescence reticulocytes (LFR) per clinical group and age category.

Plots display the status of each haematology parameter per age category. In each age category, participants are divided regarding the health status according to the case definitions whereby healthy smear-negative subjects are represented by “No malaria”, smear-positive asymptomatic cases represented by “Asymptomatic malaria” and smear-positive patienst with fever are represented by “Clinical malaria”. Whiskers bottom and top limits are 5th and 95th percentiles respectively; (): continuous line is used for comparison between clinical status within the same age category; (—): dotted line is used for comparison between age category in the “No malaria” group; *: p value with statistically significant difference (p<0.05) between clinical status within the same age category; ▪: p value with statiscally significant difference (p<0.05) between age category in the “No malaria” group; Mann-Whitney U test was used for the comparison of median between groups; NS: not significant; yrs: years; %: percentage. No malaria Asymptomatic malaria Clinical malaria.

(TIF)

Click here for additional data file.

Principal component analysis displaying the clustering of the participants based on their health status in the 3 main health categories.

(TIF)

Click here for additional data file.

Supporting methods.

(DOCX)

Click here for additional data file.

(ZIP)

Click here for additional data file.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

27 Jul 2020

PONE-D-20-20127

Red blood cell homeostasis in children and adults with and without asymptomatic malaria infection in Burkina Faso

PLOS ONE

Dear Dr. KABORE,

Thank you for submitting your manuscript for review to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if the authors revise it to address relevant points raised by the reviewers.  According to reviewers, there are some specific areas where further improvements would be of substantial benefit to the readers, including data analysis and results. For example, reviewer #1 suggests additional analysis for biomarkers correlations;  Reviewer # 2 suggests reviewing data analysis to reduce statistical dispersion;  Reviewer #3 pointed out that the individual data underlying the findings are not fully available in the manuscript and no repository have been provided. For your guidance, a copy of the reviewers' comments is included below.

Please submit your revised manuscript by August 30. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. As your study included minors, please state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

3. Thank you for stating the following in the Financial Disclosure section:

"This work was supported by unrestricted grant from SYSMEX Europe GmbH to AV that supported full funding of the presented studies. Furthermore, SYSMEX Europe GmbH provided the analyzers and technical assistance for running the analyzers. The funding source was involved in the study design, but data collection, analysis and interpretation as well as preparation of this report were done independently.

https://www.sysmex-europe.com"

We note that you received funding from a commercial source: SYSMEX Europe GmbH

Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.

Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1) In Table 1, the percentage of each age group should reflect the proportion of each column instead of each row.

2) There are many biomarkers examined here. It will be great to do some in-depth analysis on the correlations of the biomarkers to cluster and group biomarkers, and to draw some results and conclusion at the hyper level.

Reviewer #2: Red blood cell homeostasis in children and adults with and without asymptomatic malaria

infection in Burkina Faso.

Kaboré B. et al.

This paper describes a large set of hematological parameters in a population living in a malaria hyperendemic area. The novelty of the study lies fundamentally in the integration of reticulocyte and bone marrow related data in symptomatic and asymptomatic subpopulations. The hypothesis is that in asymptomatic malaria immune tolerance may have a protective effect on erythropoiesis, which would compensate the anaemia derived from the pro-inflamatory status and hemolysis. The results support a correlation between the bone marrow response, with higher turnover of RBC, and the development of asymptomatic malaria. Differences were found mostly between children showing clinical malaria and asymptomatic or no malaria subjects.

Overall the study appears well designed, the analytical techniques correctly used and the results convincing, however there are some weaknesses which I recommend to address before publication.

Specific comments:

1. Although the statistical analysis performed yields significant differences among the three main groups analyzed (smear-negative, asymptomatic and clinical malaria) for several parameters, some data display a rather large dispersion (figures 2, 3, 5, 6..), particularly in the adult groups in clinical malaria and some asymptomatic malaria sets. This might be explained if these groups included some subjects displaying characteristics not contemplated in the selection criteria, as a concomitant non malaria health issue, or perhaps the inclusion of pregnant women, which has been reported to display differential response to malaria, leading to heterogeneity in the group. The authors may review the characteristics of the individuals included in each study group and test if the statistical dispersion is reduced by the exclusion or regrouping by these criteria.

2. Participants of the asymptomatic group: the subjects were included in this group based on being smear-positive and healthy at the moment of the test. It is stated also (Supporting methods, study design) that a questionnaire was taken at sample collection, but no information is given if the subjects were questioned for febrile episodes in the previous two weeks to ascertain lack of malaria symptoms. It is possible that some of the asymptomatic subjects were actually either developing symptomatic malaria at an early stage or tested after a malaria episode. Related to the comment #1, this would misrepresent both the symptomatic and asymptomatic groups and add uncertainty to the statistics. Ideally the asymptomatic group should correspond with immune-tolerant individuals. If the authors have such information it would be advisable to be mentioned in the text.

3. The group of subjects �15 years is quite broad, it would be convenient to indicate the average age of this group as blood parameters are most likely affected in what is considered elderly people in endemic areas (>40 years) than in young adults (15-40 years)

Minor comments

“NS” in table 1 and figures S1 and S2 is not defined in the text or legends

Quality of figures: axis labels are difficult to read at the resolution provided in the figures containing large number of plots (fig 2, 5, 7, S1)

Reviewer #3: This manuscript by Kaboré et al describes the hematological alterations shown in asymptomatic or clinical malaria and also investigates the bone marrow response to the loss of RBCs depending on the severity of the infection. The study integrates a wide variety of hematological variables, iron biomarkers and cytokines as a comprehensive approach to understanding how the bone marrow compensates for the condition of anaemia. One of the major highlights of the paper is the demonstration that asymptomatic malaria is associated with enhanced erythropoiesis and immune tolerance compared to those with clinical malaria, probably due to a lower imbalance towards the production of pro-inflammatory factors in individuals with asymptomatic malaria.

In recent years, asymptomatic malaria carriers have become a focus of malaria research, as they are considered the silent reservoir of malaria. The factors that protect these individuals from developing clinical symptoms are still unclear, however, this study provides new insights into the underlying processes of subclinical malaria. In addition, the large and significant number of samples analysed from different ages provides solid and consistent results.

It should be pointed out that the individual data underlying the findings are not fully available in the manuscript and no repository have been provided. Although summary statistics are available, the data points behind means or ranges are not given as stated in the PLOS Data policy.

Most of graphs and statements are clear and well-founded. Nevertheless, there are some points that the authors may review to facilitate the readability and interpretation of the results:

Major comments:

1. As the authors note in the discussion, despite the study is focussed on asymptomatic malaria, the diagnostic method used fails to detect those individuals with submicroscopic malaria, known to be the majority of asymptomatic malaria individuals. In addition, there is no information about other pathologies that may cause hematological alterations such as sickle cell disease. For that reason, smear-negative samples should not be considered reference values, as they may include many submicroscopic malaria and hemoglobin S carriers.

2. Some of the statements do not fit exactly with the results shown in the graphs or are too generalistics. Along the results or discussion the authors describe significant differences between infection groups, however they do not indicate the age groups in which this occurs (e.g. line 270, Fig2, one of the under 15 years groups do not show increased reticulocyte numbers; line 272, Fig2, the authors should indicate that Delta-he levels are low only in children; line 274, Fig2, RPI is only elevated in under-2 children; Line 378, Fig2, the authors claim that erythrocyte indices increase with age, however, the RBC numbers are constant along the age groups). For the benefit of the readers, the authors should be more accurate in their description of the results.

3. Lines 403-405: The authors claim that asymptomatic malaria infections, unlike clinical malaria, have an adequate bone marrow response to decreasing Hb levels, using reticulocyte numbers and RPI as indicators, however, reticulocyte numbers show increased numbers in both subclinical and clinical malaria. It is unclear on what differential results between both groups this statement is based.

4. This reviewer does not see a clear reason why the cytokine production after LPS stimulation is not analysed in clinical malaria patients. In line with the rest of data provided where this group is considered. This analysis is important to provide insights into subclinical malaria immune tolerance in comparison to those who develop clinical symptoms.

5. Figures 2, 3, 5, 6 and S1: When the p-value is marked as a line divided into two segments (e.g. Fig2, RBC, <2yrs) it is not clear whether there are significant differences between smear-negative samples and subclinical malaria and between subclinical and clinical malaria or whether there are also significant differences between smear-negative and clinical malaria. Based on my interpretation, I understand that there are no significant differences between smear-negative and clinical malaria, however, at first glance it seems that there are. Could the authors clarify this point? If there are significant differences between these groups, a clear definition in the legends and graphs on how the p-values are shown, should be provided.

Minor comments:

6. Line 60, 404 and 460: What does “adequate” means? The authors should be more concise describing a main conclusion.

7. Some references are missing along the manuscript (e.g. line 88, the authors should provide the reference of the Western population data; line 367, the authors may provide the reference of IL-6 role in iron homeostasis; line 417, the authors should provide the reference describing the significant role of hemolysis in asymptomatic malaria).

8. Line 264: Could de authors indicate if the difference in Hb level was calculated with the mean of the groups?

9. Line 281: To facilitate a better understanding of this section, I would recommend including the acronyms of the mature and immature reticulocytes that are shown in Fig S2.

10. Lines 330-331: The authors should clarify what the value in brackets means.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Comments_PONE-D-20-20217.docx

Click here for additional data file.

29 Aug 2020

Additional analyses were done regarding the comment of Reviewer 1 for indepth analysis on the correlations of the biomarkers. These analyses were upload in a separate files as supporting documents to response to the reviewers comments.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

2 Oct 2020

PONE-D-20-20127R1

Red blood cell homeostasis in children and adults with and without asymptomatic malaria infection in Burkina Faso

PLOS ONE

Dear Dr. KABORE,

Thank you for submitting your manuscript for review to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if it is revised to address a specific topic raised by the reviewer # 1. More specifically, previous suggestions made by the reviewer meant to make the manuscript more informative to a broad audience. Therefore additional analysis and data interpretation should be presented in the final version of the manuscript, even if only in supplementary materials, rather than just in the supporting file to the reviewers. For your guidance, a copy of the reviewers' comments was included below.

Please submit your revised manuscript by October 20. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1) My question of “in-depth analysis on the correlations of the biomarkers to cluster and group biomarkers, and to draw some results and conclusion at the hyper level” on last version meant to make the manuscript more informative to all the audience instead of just to me, a reviewer. Therefore the relevant analysis results and interpretation should be presented in the manuscript, even if only in supplementary materials, rather than just in the supporting file to the reviewers.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

17 Oct 2020

Comments reviewer #1

Reviewer #1: 1) My question of “in-depth analysis on the correlations of the biomarkers to cluster and group biomarkers, and to draw some results and conclusion at the hyper level” on last version meant to make the manuscript more informative to all the audience instead of just to me, a reviewer. Therefore, the relevant analysis results and interpretation should be presented in the manuscript, even if only in supplementary materials, rather than just in the supporting file to the reviewers.

We thank reviewer for suggesion and we adapted our paper accordingly. See materials and method section: page 8; Lines 148-152 and results section: page 20-21; Lines 387-401. The corresponding figure and table are presented in the supporting materials.

Submitted filename: Response to Revierwers.docx

Click here for additional data file.

4 Nov 2020

Red blood cell homeostasis in children and adults with and without asymptomatic malaria infection in Burkina Faso

PONE-D-20-20127R2

Dear Dr. KABORE,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Luzia Helena Carvalho, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

16 Nov 2020

PONE-D-20-20127R2

Red blood cell homeostasis in children and adults with and without asymptomatic malaria infection in Burkina Faso.

Dear Dr. Kaboré:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Luzia Helena Carvalho

Academic Editor

PLOS ONE

Table 1

Clinical and demographic characteristics.

Characteristics	No malaria	Asymptomatic malaria	P value*	Clinical malaria	P value**
	n = 483	n = 444		N = 191
Gender
Female n (%)	266 (55.1)	223 (50.2)		82 (42.9)
Temperature (°C)	36.5 (36.2–37)	36.5 (36.2–36.9)	NS	38.6 (38–39.7)	<0.001
Parasite density (/ul)
less 2yrs	NA	3405 (515–19418)	NA	48773 (6167–96104)
2-4yrs	NA	4418 (754–16995)	NA	43183(7254–100613)	<0.002
5-14yrs	NA	736 (235–2612)	NA	6939 (679–27683)
15yrs and older	NA	195 (95–645)	NA	1523 (254–8364)
Anthropometry
Less 2yrs (number; %)	96 (19.9)	41 (9.2)		77 (40.3)
Z-wa	-1.2(-1.8.-0.4)	-0.8 (-1.6.-0.1)	0.06	NR
Z-ha	-2.2 (-3.1.-1.3)	-1.9 (-2.7.-1.1)	0.4	-1.2 (-2.2.-0.3)	<0.006
Z-wh	-0.3 (-1.1. 0.4)	-0.2 (-0.9. 1.0)	0.3	NR
MUAC	15 (14–15)	15 (14.2–15.5)	0.9	ND
2-4yrs (number; %)	139 (28.8)	87 (19.6)		66 (34.6)
Z-wa	-1.2 (-1.8.-0.5)	-1.3 (-1.9.-0.5)	0.5	NR
Z-ha	-1.8 (-2.6.-1.2)	-2.2(-3.2.-1.2)	0.7	-1.5(-2.6.-0.3)	<0.006
Z-wh	-0.3 (-0.9.0.5)	-0.2 (-0.8. 0.6)	0.2	NR
MUAC	15 (14.9–16)	15.5 (15–16)	0.2	ND
5-14yrs (number; %)	112 (23.2)	256 (57.7)		28 (14.7)
Z-bmi	15.0 (14.1–16.2)	15.2 (14.2–16.5)	0.6	NR
MUAC	17.5 (16–19)	17.9 (16–19)	0.7	ND
15yrs and older (number; %)	136 (28.2)	60 (13.5)		20 (10.5)
bmi	19.8 (18.4–21.9)	19.0 (18.1–21.4)	0.4
MUAC	25.3 (24–27)	25 (23.3–27.9)	0.9	ND

Data are presented as median and interquartile range unless stated otherwise; Z-wa: weight for age Z-score, Z-ha: height for age Z-score Z-wh: weight for height Z-score and Z-bmi: body mass index (kg/m^2) Z-score according to WHO classification; bmi: body mass index (kg/m^2); MUAC: mid-upper arm circumference; Yrs: years; °C: degree Celsius; NS: not significant; NA: not applicable; ND: not done; NR: not reported. Healthy smear-negative subjects are represented by “No malaria.”

*: Comparison between No malaria and Asymptomatic malaria groups

**: Comparison of Clinical malaria group with No malaria and Asymptomatic malaria

Comparison between groups was done by using Mann-Whitney U test

Table 2

Red blood cells indices reference values (median, and 5th-95th percentile) per age category in the smear-negative healthy (no malaria) group.

Parameters	Age category
	Less 2 yrs (n = 96)	2–4 yrs (n = 139)	5–14 yrs (n = 112)	15+ yrs and older (n = 136)
Hb (g/dL)	9.6 (7.5–11.9)	10.8 (8.1–12.4)	11.6 (9.8–13.2)	12.2 (10.1–14.7)
HCT (%)	31.9 (25.4–36.3)	34 (26.7–38.6)	35.3 (30.7–39.8)	37.4 (30.2–44.1)
RBC (106/μl)	4.5 (3.5–5.4)	4.4 (3.5–5.2)	4.4 (3.5–5.5)	4.4 (3.5–5.5)
MCV (fL)	70.2 (56.2–82.3)	78.2 (65.5–87.2)	79.1 (66.9–88.7)	85.9 (73.0–95.4)
MCH (pg)	21.7 (16.7–27.0)	24.7 (20.0–27.9)	26.2 (21.6–29.7)	28.5 (24.0–32.0)
MCHC (g/dL)	30.9 (27.4–33.9)	31.7 (29.0–34.6)	32.9 (30.5–35.4)	32.7 (30.8–35.7)
RDW-SD (fL)	44.2 (31.5–57.7)	43.3 (37.1–55.6)	40.2 (34.9–49.5)	41.2 (36.5–50.1)
RDW-CV (%)	19.1 (14.5–27.4)	15.8 (12.9–22.5)	14.1 (12.4–17.1)	13.2 (12.1–15.5)
HYPO-He (%)	10.9 (0.9–64.9)	1.9 (0.3–25.1)	0.9 (0.1–8.0)	0.3 (0.1–2.3)
HYPER-He (%)	0.2 (0.0–0.5)	0.3 (0.1–0.5)	0.4 (0.2–0.6)	0.6 (0.3–0.8)
MicroR (%)	28.7 (7.1–71.9)	11.2 (3.0–46.8)	8.0 (1.4–36.7)	2.3 (0.5–16.6)
MacroR (%)	2.9 (0.8–4.4)	3.6 (2.0–4.2)	3.7 (2.4–4.3)	3.7 (3.0–4.8)
RET# (104/μl)	5.7 (2.4–14.0)	6.1 (3.2–23.5)	6.4 (3.2–20.8)	5.7 (2.8–12.2)
RET%	1.3 (0.5–3.4)	1.4 (0.7–6.2)	1.5 (0.7–4.9)	1.3 (0.7–2.7)
HFR# (104/μl)	0.2 (0.02–2.2)	0.1 (0.02–3.2)	0.09 (0.01–1.5)	0.07 (0.01–0.7)
HFR%	3.1 (0.6–19.0)	2.4 (0.4–16.6)	1.5 (0.3–9.7)	1.2 (0.4–7.4)
MFR# (104/μl)	0.7 (0.2–2.7)	0.7 (0.2–3.0)	0.6 (0.2–2.6)	0.5 (0.1–1.4)
MFR%	11.9 (6.5–17.1)	10.7 (5.2–17.9)	9.4 (3.5–17.1)	8.5 (4.4–14.0)
LFR# 104/μl	4.7 (2.2–10.0)	5.1 (2.7–18.2)	5.7 (3.1–15.5)	5.1 (2.6–10.1)
LFR%	83.8 (62.1–92.7)	86.4 (67.6–93.9)	89.0 (74.1–96.0)	90.3 (79.5–95.0)
RPI (%)	0.5 (0.2–1.3)	0.7 (0.3–2.6)	0.8 (0.4–2.4)	0.8 (0.4–1.8)
IRF# (104/μl)	0.9 (0.2–5.0)	0.8 (0.2–6.0)	0.6 (0.2–4.1)	0.6 (0.2–2.0)
IRF%	16.2 (7.3–37.9)	13.6 (6.1–32.4)	11.1 (4.0–25.9)	9.8 (5.0–20.5)
RET-He (pg)	24.6 (16.8–32.3)	28.2 (21.0–33.1)	30.2 (23.7–34.2)	31.7 (26.0–35.6)
RBC-He (pg)	21.7 (15.2–27.1)	25.4 (19.4–28.4)	27.0 (21.7–30.2)	29.2 (23.1–32.4)
Delta-He (pg)	3.1 (-0.4–7.6)	3.0 (-0.2–6.3)	3.2 (0.4–5.5)	2.9 (0.0–4.4)

Data are presented as median, and 5th-95th percentiles.

Yrs: years; #: absolute count; %: percentage count.