Haematological consequences of acute uncomplicated falciparum malaria: a WorldWide Antimalarial Resistance Network pooled analysis of individual patient data

Background Plasmodium falciparum malaria is associated with anaemia-related morbidity, attributable to host, parasite and drug factors. We quantified the haematological response following treatment of uncomplicated P. falciparum malaria to identify the factors associated with malarial anaemia. Methods Individual patient data from eligible antimalarial efficacy studies of uncomplicated P. falciparum malaria, available through the WorldWide Antimalarial Resistance Network data repository prior to August 2015, were pooled using standardised methodology. The haematological response over time was quantified using a multivariable linear mixed effects model with nonlinear terms for time, and the model was then used to estimate the mean haemoglobin at day of nadir and day 7. Multivariable logistic regression quantified risk factors for moderately severe anaemia (haemoglobin < 7 g/dL) at day 0, day 3 and day 7 as well as a fractional fall ≥ 25% at day 3 and day 7. Results A total of 70,226 patients, recruited into 200 studies between 1991 and 2013, were included in the analysis: 50,859 (72.4%) enrolled in Africa, 18,451 (26.3%) in Asia and 916 (1.3%) in South America. The median haemoglobin concentration at presentation was 9.9 g/dL (range 5.0–19.7 g/dL) in Africa, 11.6 g/dL (range 5.0–20.0 g/dL) in Asia and 12.3 g/dL (range 6.9–17.9 g/dL) in South America. Moderately severe anaemia (Hb < 7g/dl) was present in 8.4% (4284/50,859) of patients from Africa, 3.3% (606/18,451) from Asia and 0.1% (1/916) from South America. The nadir haemoglobin occurred on day 2 post treatment with a mean fall from baseline of 0.57 g/dL in Africa and 1.13 g/dL in Asia. Independent risk factors for moderately severe anaemia on day 7, in both Africa and Asia, included moderately severe anaemia at baseline (adjusted odds ratio (AOR) = 16.10 and AOR = 23.00, respectively), young age (age < 1 compared to ≥ 12 years AOR = 12.81 and AOR = 6.79, respectively), high parasitaemia (AOR = 1.78 and AOR = 1.58, respectively) and delayed parasite clearance (AOR = 2.44 and AOR = 2.59, respectively). In Asia, patients treated with an artemisinin-based regimen were at significantly greater risk of moderately severe anaemia on day 7 compared to those treated with a non-artemisinin-based regimen (AOR = 2.06 [95%CI 1.39–3.05], p < 0.001). Conclusions In patients with uncomplicated P. falciparum malaria, the nadir haemoglobin occurs 2 days after starting treatment. Although artemisinin-based treatments increase the rate of parasite clearance, in Asia they are associated with a greater risk of anaemia during recovery. Supplementary Information The online version contains supplementary material available at 10.1186/s12916-022-02265-9.

Page 2 of 19 The WorldWide Antimalarial Resistance Network Falciparum Haematology Study Group BMC Medicine (2022) 20:85 Background Malaria remains a major cause of anaemia in malaria endemic countries, with a complex pathogenesis attributable to red cell destruction and haematopoietic suppression [1] that can be compounded by malnutrition, helminth carriage and inherited blood disorders [2]. Artemisinin-based combination therapy (ACT) is the first-line antimalarial treatment for uncomplicated malaria in almost all endemic countries [3], achieving high cure rates, rapid parasite clearance and reduced ongoing transmission of the parasite [4,5]. However, artemisinin derivatives can suppress reticulocytosis and contribute to haemolysis; their use has been associated with delayed-onset anaemia [6,7]. The haematological recovery and adverse consequences of the artemisinin derivatives, following the treatment of falciparum malaria, may vary between different ACTs [8].
To assess the comparative benefits of different antimalarial treatment regimens, it is critical to quantify the haematological impact attributable to P. falciparum infection and the clinical and demographic factors that underlie this. The aim of this study was to determine the pattern of haematological recovery following uncomplicated falciparum malaria and define the risk factors for moderately severe haematological outcomes at baseline and during early follow-up.

The WWARN repository and study selection
Haemoglobin concentrations are often not reported in antimalarial trial publications, even if these data are collected. Since a review of published literature would not provide sufficiently comprehensive information, the focus of this individual patient data meta-analysis was on studies identified in the WWARN repository. The WWARN repository contains data from 451 antimalarial efficacy studies in which patients were enrolled from locations in 69 countries, with a diverse range of P. falciparum transmission intensities. Data in the repository have been standardised and collated using methodology described previously in the WWARN Clinical Module Data Management and Statistical Analysis Plan [9].
The WWARN repository was searched in August 2015 for all antimalarial efficacy studies of uncomplicated P. falciparum malaria in non-pregnant patients that followed subjects prospectively for a minimum of 28 days and reported haemoglobin concentration (or haematocrit) at least at baseline (day 0). Investigators of the identified studies were invited to participate in this study group and information was made available on the WWARN website [10]. Uncomplicated P. falciparum malaria was defined as microscopy-proven falciparum malaria without features of severe malaria [11]. Patients were excluded if they had severe malaria.

Outcomes of interest
The primary outcome of the analysis was the risk of moderately severe anaemia (Hb < 7 g/dL) on day 7 after initiation of treatment. Secondary outcomes included the mean fall in haemoglobin at day of nadir and day 7, the timing of nadir haemoglobin, risk of moderately severe anaemia at days 0 and 3, and the risk of a large reduction in haemoglobin from baseline, defined as a fractional fall in Hb of ≥ 25% on day 3 or 7.

Statistical methods
All statistical analyses were done using R (Version 3.2.5, The R Foundation for Statistical Computing) or Stata MP 15, based on an a priori statistical plan shared with data contributors [10].
Haematocrit measurements were converted to haemoglobin concentrations using the following formula: Haemoglobin = (Haematocrit − 5.62)/2.60 [12]. The timing of sampling was defined as day 0 if occurring on the day of enrolment / first day of treatment, with sequential numbering thereafter. Data were stratified by region (Africa, Asia and South America). Univariable and multivariable mixed effects logistic regression models were used to model risk of (i) moderately severe anaemia on day 0, 3 or 7 and (ii) large reduction in haemoglobin on day 3 or 7. Study site (sites within countries) was included as a random intercept in these models.
Changes in mean haemoglobin over time were examined, after stratifying by region, using linear mixed effects models. Fractional polynomial terms for time were fitted as random effects for patients to capture the nonlinear associations and random intercepts for patients and study site. All available haemoglobin measurements were included in these analyses. Additional analyses of mean haemoglobin over time within each region were undertaken, stratified by age group (< 5 years and ≥ 5 years).
For all regression models, independent risk factors were identified following the strategy recommended by Collet [13]. Covariates examined included the following: age in years (categorised as < 1 year, 1 to 4 years, 5 to 11 years and ≥ 12 years), sex, fever (temperature > 37.5 °C) on enrolment, baseline parasitaemia (after log transformation), mixed Plasmodium species infection, underweight (defined as weightfor-age Z-score <−2 for children younger than 5 years) [14], high parasitaemia (defined as > 100,000 parasites/μL [15]), presence of gametocytaemia on enrolment, transmission intensity, treatment (artemisinin-based therapy versus non-artemisinin-based therapy) and parasite clearance (early clearance on day 1 or day 2 versus delayed parasite clearance on day 3 or later

Results
A total of 200 P. falciparum clinical trials undertaken between 1991 and 2013 met the inclusion criteria and were available for analysis ( Fig. 1 Table S3).

Haematological status at enrolment
The haematological exclusion criteria differed between studies. The commonest haematological exclusion criterion was a haemoglobin < 5 g/dL (used in 126 studies where 39,940 patients were included), with 2 studies (483 patients) excluding patients with a haemoglobin < 6 g/dL, 9 studies (5964 patients) excluding patients with a haemoglobin < 7 g/dL and 3 studies (566 patients) excluding patients with a haemoglobin < 8 g/dL. In 60 studies, haematological exclusion criteria were not stated; Additional file 1: Table S2. There were 208 patients with a haemoglobin < 5 g/dL at baseline, who were excluded from further analysis, since they met the WHO criteria for severe malaria.
The median haemoglobin at enrolment was 9.9 g/dL (range 5.0-19.7 g/dL) in Africa, 11.6 g/dL (range 5.0-20.0 g/dL) in Asia and 12.3 g/dL (range 6.9-17.9 g/dL) in South America (Table 1). Moderately severe anaemia was defined as haemoglobin concentration < 7 g/dL and was present in 4891 (6.9%) patients at baseline, with a prevalence of 8.4% (4284/50,859) in Africa, 3.3% (606/18,451) in Asia and 0.1% (1/916) in South America. Owing to the limited numbers of patients from South America, all subsequent analyses were restricted to patients from either Africa or Asia and stratified by geographical location.
The mean haemoglobin at enrolment varied with both age and baseline parasitaemia (Additional file 2: Figure  S2). The main risk factors for moderately severe anaemia at baseline in both Africa and Asia were young age and presenting without high parasitaemia (> 100,000/ μL); Additional file 3: Table S4 and S5. In Africa, the risk of moderately severe anaemia was inversely related to parasitaemia, whereas in Asia the risk rose to a peak at 10,000 parasites/μL, and decreased thereafter (Fig. 2).

Haemoglobin profile following the start of treatment
A linear mixed effects model of all haemoglobin concentrations over time showed that following the start of treatment haemoglobin concentration fell rapidly to a nadir on day 2 (Fig. 3). The estimated mean fall in haemoglobin in Africa was 0.57 g/dL at day 2 and 0.03 g/dL at day 7, with corresponding estimates in Asia of 1.13 g/dL and 0.78 g/dL. Haemoglobin concentrations returned to baseline by day 8 in Africa and day 22 in Asia, and thereafter continued to increase, reaching a mean concentration at day 42 of 11.40 g/dL (95%CI 11.28-11.52) in Africa (1.47 g/dL above baseline) and 12.17 g/dL (95%CI 11.98-12.36) in Asia (0.60 g/dL above baseline) (Fig. 3).
Haematological recovery was assumed to have occurred by day 42, and therefore, the observation on this day represented the baseline Hb of this patient population without infection. In African patients, 71.3% of the total fall in Hb from predicted baseline occurred before treatment and 28.3% after treatment. The corresponding percentages in Asia were 34.7% and 65.3% respectively.
Differences in the haemoglobin profiles between Africa and Asia were largely attributable to the variation in the age distributions of the study populations. When Fig. 2 Relationship between predicted probabilities of moderately severe anaemia (haemoglobin < 7 g/dL) on enrolment and continuous covariates. Results are generated from the final multivariable models and are adjusted for mean values of other covariates (age, baseline parasitaemia, sex and fever). The model was restricted to children age > 0.75 years due to instability at the extremes of the data the analyses were stratified by age, the haemoglobin profiles were similar for the two continents (Fig. 3).
Compared with those treated with non-artemisininbased treatments, patients in Asia treated with artemisinin-based therapy were at significantly greater risk of moderately severe anaemia on day 7 (AOR = 2.06 (95%CI 1.39-3.05); p < 0.001), but this was not the case in African patients (AOR = 1.01 (95%CI 0.56-1.82); p = 0.987). In Asia, the difference in risk between artemisinin-and non-artemisinin-based treatments remained when only the 8570 patients enrolled before 2007 (when artemisinin resistance was first described in the Greater Mekong Subregion) were included in the model (AOR = 1.98 (95%CI  Table S7-S8 and Additional file 2: Figure S3). The fractional fall in haemoglobin on day 7 was correlated positively with the baseline haemoglobin (r = 0.47; p < 0.001 adjusted for clustering of study site). A high baseline haemoglobin was associated with a greater risk of a large fractional fall (≥ 25%) on day 7 in Africa (AOR for every 1 g/dL increase in baseline haemoglobin = 1.52 (95%CI 1.40-1.65); p < 0.001) and in Asia (AOR for every 1 g/dL increase in baseline haemoglobin = 1.43 (95%CI 1.35-1.52); p < 0.001) (Additional file 2: Figure S4). Other risk factors for a large fractional fall in haemoglobin on day 7 were similar to the risk factors for moderately severe anaemia on this day (Additional file 3: Table S9).

Assessment of potential bias
Methodological factors potentially contributing to bias are presented in Additional file 1: Table S2. Although many studies were unblinded, haemoglobin measurement is automated, thus minimising the risk of observer bias. Publication bias was unlikely, since haemoglobin  measurements were not a primary outcome in any of the publications and haemoglobin concentrations are unlikely to have influenced the decision to publish. Exclusion due to variable haemoglobin criteria will have caused a small reduction in the apparent proportion of patients with moderately severe anaemia at baseline and may also have artificially reduced the proportion of patients becoming severely anaemic during follow-up. In a sensitivity analysis, exclusion of patients from the 14 studies that had baseline haemoglobin cut-offs greater than 5 g/ dL had minimal impact on the results (Additional file 3: Tables S10 and S11).

Discussion
Our study provides a detailed analysis of haemoglobin concentration kinetics in patients with falciparum malaria, enrolled across geographically diverse regions. The available data, exceeding > 70,000 individual data from patients of all ages, provides unprecedented power to define the factors associated with the acute fall in haemoglobin before and after treatment. Malaria is due to an intraerythrocytic infection which results in a reduction of red blood cells, intra-and extravascular haemolysis, bone marrow suppression and sequestration [178]. The administration of antimalarial drugs inhibits these pathological processes by preventing parasite replication and limiting the duration of dyserythropoiesis. Hence, the haematological manifestations of malaria are a function of the duration and degree of parasitaemia prior to treatment and the speed of therapeutic response to antimalarial treatment. Our analysis demonstrated that in Africa, hence in generally relatively high transmission regions, approximately three quarters of the malaria-attributable fall in haemoglobin occurs before presentation and one quarter after treatment, whilst in Asia, in generally relatively low transmission settings, one third occurs before presentation and two-thirds after treatment. The relative drop in haemoglobin was positively correlated with baseline haemoglobin.
Although the greatest fall in haemoglobin occurred before treatment in Africa, our analysis focused primarily on factors associated with the subsequent fall and recovery which may be more amenable to clinical intervention. Consistent with a recent pooled analysis from Africa [179], our study found that in both Africa and Asia, the nadir haemoglobin occurred within 2 days of starting treatment and haemoglobin generally rose thereafter. Whilst a previous analysis identified that nadir haemoglobin occurred on day 7, this was based on weekly assessments, and thus would have missed the true nadir occurring between weekly observations [1]. In vulnerable populations, such as young children and pregnant women, who are at risk of adverse clinical outcomes, antimalarial clinical trials should implement a routine haemoglobin assessment at day 2 or 3 to ensure early diagnosis of severe anaemia.
The baseline haemoglobin in patients with falciparum malaria varied substantially with age and parasite density at presentation. After controlling for confounding factors, significant site to site variation remained, likely reflecting variations in transmission intensity, host immunity and factors unrelated to malaria. Patients from Asia tended to be older than those enrolled in Africa, but after controlling for age there were minimal differences in haemoglobin between regions, either at baseline or during follow-up. Following treatment, the absolute and proportional reductions in haemoglobin were greater in patients from Asia compared to Africa and were correlated with the higher baseline haemoglobin in Asian patients. Hence, patients presenting with a low haemoglobin concentration were less likely to experience a further fall in their haemoglobin.
The relationship between level of parasitaemia and degree of anaemia is complex [1,179]. In Africa, anaemia at presentation was greatest in patients with low parasitaemias. There are several possible explanations for this. First, in highly endemic parts of Africa, robust immunity develops early, suppressing parasitaemia and symptoms. A substantial proportion of patients presenting with fever and low-level Plasmodium parasitaemia in these regions will have an alternative diagnosis, such as bacterial sepsis, which is also associated with anaemia [180]. Second, immune-mediated suppression of malaria symptoms can result in chronic, untreated parasitaemia that, over time, leads to significant suppression and dysregulation of haematopoiesis. Third, repeated episodes of malaria can result in splenic sequestration, with low-level peripheral parasitaemia, associated splenomegaly and dilutional anaemia [178,181].
In Asia, the risk of anaemia at presentation increased with rising parasitaemia, peaking at 10,000 parasites/μL before decreasing thereafter. As transmission intensity in endemic parts of Asia is generally significantly lower than in Africa, immunity is less robust and a much greater proportion of infections will be symptomatic and of short duration. In this setting, anaemia will be related primarily to acute destruction of both parasitised and unparasitised red cells, the severity of which is correlated with the level of parasitaemia.
Treatment with artemisinin-based therapy in Asia was associated with a twofold higher risk of moderately severe anaemia (but not a large fractional fall in haemoglobin) within 7 days compared with non-artemisinin-based therapy, whereas in Africa, artemisinin-based treatment was not associated with an excess risk of early anaemia. This relationship was not attributable to the presence of artemisinin resistance. We hypothesise that rapid killing of intraerythrocytic parasites by artemisinins in non-immune Asian adults likely leads to more rapid clearance of whole red blood cells from the circulation than that occurring after slower acting drug treatments. In immune African patients, a greater proportion of infected red cells undergo targeted intraerythrocytic parasite removal (pitting) followed by a return to circulation, thus ameliorating the early development of anaemia [6]. Reticulocytosis probably also occurs more rapidly after treatment in immune compared with non-immune individuals [6]. Further studies are warranted to explore the differences in haematological response to treatment with artemisinin derivatives in populations with different levels of immunity. Our analysis is based upon studies conducted prior to 2014. In the last 5 years, artemisinin-resistant parasites have spread across the Greater Mekong Subregion [182], with recent reports confirming their presence in Sub-Saharan Africa [183,184]. Slower parasite clearance times and subsequent emergence of resistance to partner drugs will ultimately lead to treatment failure that will impact the generally prompt haemoglobin recovery that we observed in our analysis. Our study has a number of limitations. The analysis focused on the acute haematological impact of malaria and the early recovery phase and did not address the influence of late treatment failure on subsequent recovery to baseline haemoglobin concentrations. This will be addressed in a subsequent analysis. Our estimates of the pattern of haemoglobin changes during the first few days after diagnosis may have been influenced by selection bias, as only a small subset of patients had multiple haemoglobin measurements during the first 7 days of follow-up. Although we did not employ a traditional systematic review to identify eligible studies, our analysis is the largest meta-analysis to date of patients treated for malaria in both Africa and Asia. This unprecedented data collection ensures robust parameter estimates and minimises the risk of inclusion bias. Furthermore, a systematic review would not preclude bias, since some studies recorded haemoglobin/haematocrit measurements but did not present these data in published manuscripts. Whilst the results of the current study are likely to be generalisable to Africa and Asia, the small number of patients from the Americas prevents the generalisability of our findings to this region. An additional potential cause of bias is the exclusion of patients from the original studies, prior to pooling, according to variable definitions of severe anaemia. Almost two-thirds of studies excluded patients with a haemoglobin < 5 g/dL, with a few studies excluding patients based on higher cut-offs and the remaining 30% having an unknown cut-off. Additional limitations of our study include the use of various methodologies to measure haematocrit or haemoglobin, a lack of a robust conversion factor to adjust haematocrit to haemoglobin in different studies' populations and no reliable data on the following confounding factors that can influence haemoglobin and its recovery: the duration of prior parasitaemia (which has been shown to correlate with anaemia at presentation [178]), host genetic factors associated with anaemia (e.g. sickle cell anaemia, thalassaemia), administration of haematinics (or treatment for anaemia) and hydration status.

Conclusions
In conclusion, the majority of patients with uncomplicated falciparum malaria had a modest fall in haemoglobin following treatment, before subsequent improvement in haemoglobin during recovery. Despite highly effective treatment, some patients remained at significant risk of moderately severe anaemia. Young children had a particularly high risk, likely related to lower immunity and high initial peripheral parasitaemia. The risk of anaemia is exacerbated by prolonged parasitaemia prior to presentation [1] or delayed parasite clearance, both of which are associated with suboptimal treatment regimens particularly in areas where antimalarial drug resistance was emerging [185]. Whilst artemisinin-based treatment generally ensured rapid parasite clearance and high efficacy, in Asia their use was associated with a greater risk of moderately severe anaemia on day 3 and day 7 that could not be accounted for by an underlying rise in artemisinin resistance. Early diagnosis of malaria and treatment with highly effective antimalarials remains critical in minimising anaemia associated with P. falciparum infection.
Additional file 1: Table S1. Describes studies included in the analysis. Table S2. Describes assessment of bias by included study.
Additional file 2: Figure S1. Describes study sites. Figure S2. Describes the relationship between haemoglobin on enrolment and continuous covariates. Figure S3. Describes the relationship between the predicted probability of moderately severe anaemia on day 3 and continuous covariates. Figure S4. Describes the relationship between the predicted probability of a large fractional fall in haemoglobin on day 7 and continuous covariates.
Additional file 3: Table S3. Describes the overview of antimalarial treatments. Table S4. Describes the risk factors for moderately severe anaemia at enrolment (univariable logistic regression). factors for moderately severe anaemia at enrolment (multivariable logistic regression). Table S6. Describes the risk factors for moderately severe anaemia at day 7 (univariable logistic regression). Table S7. Describes the risk factors for moderately severe anaemia at day 3 (univariable logistic regression). Table S8. Describes the risk factors for moderately severe anaemia at day 3 (multivariable logistic regression). Table S9.
Describes the risk factors for a large fractional fall in haemoglobin by day 7. Table S10. Describes the sensitivity analysis for risk factors for moderately severe anaemia at enrolment (multivariable logistic regression). Table S11. Describes the sensitivity analysis for risk factors for moderately severe anaemia at day 7 (multivariable logistic regression).