# Clarification of factors associated with post-artesunate delayed hemolysis (PADH): analysis of 327 patients with severe imported Plasmodium falciparum malaria in France

Pierre-Louis Conan, Marc Thellier, Eric Kendjo, Sandrine Houzé, Rémonie Seng, Stéphane Jauréguiberry

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Pierre-Louis Conan, Marc Thellier, Eric Kendjo, Sandrine Houzé, Rémonie Seng, et al. Clarification of factors associated with post-artesunate delayed hemolysis (PADH): analysis of 327 patients with severe imported Plasmodium falciparum malaria in France. Travel Medicine and Infectious Disease, 2025, pp.102801-102801. 10.1016/j.tmaid.2025.102801. hal-05059156

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# Clarification of factors associated with post-artesunate delayed hemolysis (PADH): Analysis of 327 patients with severe imported *Plasmodium falciparum* malaria in France☆

Pierre-Louis Conan<sup>a, *, †</sup>, Marc Thellier<sup>b, c, d</sup>, Eric Kendjo<sup>b, c</sup>, Sandrine Houzé<sup>c, e</sup>, Rémonie Seng<sup>f, i</sup>, Stéphane Jauréguiberry<sup>c, f, g, h</sup>

## ABSTRACT

**Background:** Post-Artesunate delayed hemolysis (PADH) occurs in approximately 15 % of treated patients 2–3 weeks after artesunate administration. Identifying risk markers for PADH would help predict which patients are at higher risk.

**Methods:** In this prospective national cohort study conducted in a non-malaria endemic area from 2011 to 2016, a Cox proportional hazards model was used to assess the association between clinical and biological data available at Day 0 and the occurrence of PADH within 30 days of artesunate administration.

**Results:** In the analyzed population (n = 327), 49 PADH events occurred after a median time of 14 days (IQR, 13–17) after artesunate initiation. Higher initial parasitemia was associated with an increased risk of PADH, with a significant interaction found with patient origin. The cumulative probability of PADH event at Day 30 post-artesunate was 65 % (95 % confidence interval [CI], 44–79) for European patients vs. 14 % (95 % CI, 0–26) for those with recent African ancestry [RAA] when the initial parasitemia was >10 %. After adjustment for weight, history of malaria, initial hemoglobin, very severe malaria and residence in an endemic area, compared to recent African ancestry with initial parasitemia <4 %, the adjusted hazard ratio for PADH occurrence was 18.8 (95 % CI, 4–89) for Europeans and 4.77 (95 % CI, 0.8–29.2) for recent African ancestry with initial parasitemia >10 %.

**Conclusions:** This study showed that initial parasitemia and patient origin were the main predictors of developing PADH, with the highest risk observed in Europeans with an initial parasitemia >10 %.

## 1. Introduction

Malaria, with 249 million cases and 608000 deaths in 2022 world-wide, remains a major public health problem [1]. Artesunate, recommended by the WHO since 2010 as a first-line treatment for severe malaria (SM), is considered safer and better tolerated than quinine [2–6]. However, episodes of post-artesunate delayed hemolysis (PADH) occurring between 2 and 3 weeks after administration have been described [7]. The frequency of PADH in non-endemic malaria areas was estimated at 15 % [8–12], and it has been shown that 15 % of patients

had a hemoglobin less than 7 g/dL and up to 50 % of patients required transfusion support [10,13]. The pathophysiology of PADH is linked to a process called *pitting*, whereby parasites are cleared from infected red blood cells in the spleen, resulting in pitted red blood cells [14,15]. PADH is thought to be partly caused by the synchronous and secondary destruction of these pitted cells [16–19]. In addition to pitting, mechanisms involving autoantibodies and drug-anti-drug immune complexes have at times been proposed as contributing factors to the pathophysiology of PADH, but these hypotheses ultimately failed to gain convincing support [7,20,21]. In France, weekly laboratory monitoring

<footnote><sup>a</sup> Service des Maladies Infectieuses et Tropicales, Hôpital d'Instruction des Armées Bégin, Saint-Mandé, France</footnote>

<footnote><sup>b</sup> Sorbonne Université, Institut Pierre Louis d'Epidémiologie et de Santé publique, Inserm, Paris, France</footnote>

<footnote><sup>c</sup> Centre National de Référence du Paludisme, Paris, France</footnote>

<footnote><sup>d</sup> Sorbonne Université, Service de Parasitologie, Hôpital Pittié-Salpétrière, APHP, Paris, France</footnote>

<footnote><sup>e</sup> Université Paris-Cité, IRD, MERIT, Paris, France</footnote>

<footnote><sup>f</sup> Centre de Recherche en Epidémiologie et Santé des Populations (CESP), INSERM, U1018, Paris, France</footnote>

<footnote><sup>g</sup> Université Paris-Saclay, Service des Maladies Infectieuses et Tropicales, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France</footnote>

<footnote><sup>h</sup> Société Française de Médecine des Voyages, France</footnote>

<footnote><sup>i</sup> Service de Sante Publique et d'Epidémiologie, AP-HP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France</footnote>

<footnote>* The preliminary results of this study were presented orally at the 33rd European Congress of Clinical Microbiology & Infectious Diseases in Copenhagen in April 2023. The manuscript is currently being prepared into its full version.</footnote>

<footnote>* Corresponding author. Service de maladies infectieuses et tropicales, Hôpital d'Instruction des Armées Bégin, 69 avenue de Paris, 94160, Saint-Mandé, France. E-mail address: pierre-louis.conan@intradef.gouv.fr (P.-L. Conan).</footnote>
is recommended until the twenty-eighth day after artesunate adminis-
tration [22]. To date, it is difficult to easily predict which patients will
develop PADH. The occurrence of PADH can be predicted by deter-
mining pitted cells concentration, yet generalization is hindered as this
assessment is typically confined to research laboratories [8,15]. Several
studies have shown that some clinical and biological factors were
associated with the occurrence of PADH (young age, patient origin, and
lack of malaria premunition, initial parasitemia, initial hemoglobin) [7,
16,17,23]. However, these single-center studies, often conducted in
endemic regions with limited patient cohorts, yielded conflicting results.
Identifying patients likely to develop PADH later would allow clinicians
to stop the weekly monitoring for all patients to only focus on patients at
risk and therefore reduce the cost of follow-up for patients at the lowest
risk. The aim of our study is to use a national cohort to determine in a
systematic and robust way factors associated with the occurrence of
PADH based on readily available clinical and biological data at the time
of the first artesunate administration (day 0) in patients with *Plasmo-
dium falciparum* imported SM.

## 2. Methods

### 2.1. Data source and study population

We conducted a prospective cohort analysis of all imported *Plasmo-
dium falciparum* SM that received at least one dose of artesunate cases
reported to the National Reference Center for Malaria (NRC-M) between
2011 and 2016, a network which comprises 110 French hospitals across
metropolitan France and overseas territories.

The source is the Malaria National Reference Center (NRC-M) data-
base. National surveys have shown that cases reported by the NRC-M
surveillance system represent 50–55 % of all malaria cases in France
and are representative of imported malaria cases managed in France
(capture-recapture survey) [24,25]. Data are entered prospectively by
healthcare practitioners responsible for patient care using a standard-
ized questionnaire. It includes sections for anonymized patient details,
diagnostic criteria, clinical and epidemiological data, possible sources of
contamination, and the reporting process for healthcare professionals.
The NRC-M database constitutes a multicenter prospective cohort, with
data entry and follow-up dates dependent on patient attendance at
follow-up consultations, the accuracy of practitioner reporting, and data
entry by the NRC-M. Collected variables include socio-demographic
characteristics, details of stays in endemic areas, characteristics of ma-
laria episodes, their management, and outcomes. Results of biological
analyses conducted at Day 0 upon admission and during follow-up are
also documented within the NRC-M database.

Subjects were required to have undergone laboratory testing before
Day 7 following the beginning of artesunate (D7), and to have had at
least one follow-up assessment after D7. Patients with mixed-species
infections were excluded from the analysis.

### 2.2. Case definitions

SM was defined as malaria in individuals with blood smears positive for asexual forms of *Plasmodium falciparum* parasites and at least 1 severity criterion according to the French definition of SM [26]. According to French guidelines, hyperparasitemia is defined as greater than 4 % and applies uniformly to all patients [26]. Criteria for very severe malaria included respiratory distress and/or shock and/or coma and/or hyperlactatemia and/or acidosis [27]. PADH was defined by i) a new decrease of at least 10 % in hemoglobin levels after D7 (anemia), coupled with a simultaneous increase of at least 10 % in lactate dehydrogenase (LDH) levels (hemolysis) between the last available measurement before D7 and a subsequent measurement after D7, as published before [28]. The concept of patient origin encompasses two realities: individuals born in France of African descent and those born in Africa visiting friends and relatives. This group will be referred to as

RAA (Recent African Ancestry). The NRAA (non recent African Ancestry) group comprises patients without recent ancestors born in Africa.

### 2.3. Statistical analysis

We collected age, sex, patient origin, place of birth, weight, presence of immunosuppression, history of malaria, travel details, chemoprophylaxis, clinical history, criteria for severe malaria, the time of the first dose of artesunate, and biological results at day 0 (D0) to day 30 (D30) follow-up data. We analyzed the time period of inclusion and center activity. Quantitative variables were presented as medians (interquartile range) or means (standard deviation–SD). Qualitative variables were expressed as percentages. Differences between analyzed population and excluded subjects were assessed by using the chi2 test or Fisher exact test when appropriate for categorical variables and the Wilcoxon rank test for quantitative variables. Factors associated with PADH occurrence within 30 days post-artesunate were investigated through Kaplan Meier curves and Cox proportional hazards model. Time was defined as the interval between the first artesunate dose and PADH occurrence, or until the last available hemoglobin measurement or post-artesunate D30. Correlations between all pairs of variables will be assessed and visualized using a correlogram. Highly correlated variables (correlation coefficient >0.7) were managed by selecting a single representative variable for inclusion. Variables with *p* < 0.2 in univariate analysis and those identified in the literature were included in multivariate analysis. Proportional hazards assumptions were tested (inspection of the non-parametric Kaplan-Meier curves for the qualitative variables and temporal distribution of Schoenfeld residuals for the quantitative variables). The analyses were performed with SAS software© version 9.

### 2.4. Missing data

The main analysis was conducted initially with complete data and subsequently repeated after performing multiple imputation of missing data using Markov chain Monte Carlo methods [29,30]. Ten imputations were performed to obtain valid inference and reduce sampling variability. Variables with a missing data rate greater than 20 % were excluded from imputation. All 10 datasets were analyzed and combined using Rubin rules [31].

### 2.5. Sensitivity analyses

We performed sensitivity analysis in which any further delayed decrease (regardless of importance) in hemoglobin after D7 with an associated increase in LDH higher than 10 % was considered as a PAHD event. Another sensitivity analysis was performed with exclusion from the analysis of subjects for whom the decrease in hemoglobin observed after D7 was less than 10 %.

### 2.6. Ethical considerations

As per French law on common public-health missions of all French National Reference Centers (https://www.legifrance.gouv.fr/eli/decre t/2016/6/16/AFSP1614128D/jo/texte/fr), no specific patient consent was required. The analysis of biological samples obtained in the medical care context was considered as non-interventional requiring only the non-opposition of the patient during sampling (article L1221-1.1 and L1211-2 of the French Public Health Code). The database was registered in the general register of treatments of Assistance Publique des Hôpitaux de Paris (APHP) under the number 20220520121419. All collected data were anonymized before analysis.
# 3. Results

## 3.1. Population characteristics

A study flowchart outlining the analysis is depicted in Fig. 1. Among the 1630 patients included in the study, at Day 14, Day 21, and Day 28 — the recommended follow-up dates by the HCSP — a hemoglobin measurement was available for only approximately 14 % of the subjects, and an LDH measurement for around 8 % of the subjects. The thick blood smear, which is also recommended at Day 28, was available for 31 % of the subjects (Supplementary Table S1).

Follow-up data were available for 327 patients with SM (analyzed population) (Table 1). In the population of 327 patients, the average number of hemoglobin and LDH data points per patient was 7.6 (SD 3.8) and 4 (SD 2.9), respectively. The majority of analyzed patients were male (60 %), born in Africa and/or African descendant (69 %), with a median age of 43 (28–54) years. Parasitemia between 4 and 10 % was present in 33 % of subjects and greater than 10 % in 25 %. There was no significant difference in initial parasitemia by patient origin (54 % of RAA had an initial parasitemia >4 % versus 72 % of Europeans; *p* = 0.15).

Hyperparasitemia was the sole sign of severity for 11 % of patients. The median length of hospital stay was 7 days (5–8). Two patients died during follow-up (D27 and D30), neither of whom experienced PADH.

Compared to the 1303 patients excluded from the analysis (Supplementary Table S2), patients in the analyzed population had significantly more severity criteria (*p* < 0.001) and a higher proportion of very severe malaria (*p* < 0.001). The length of hospital stay was significantly longer in the analyzed group than in the excluded population (*p* < 0.001).

## 3.2. Post artesunate delayed hemolysis and anemia

Forty-nine of the 327 patients experienced PADH during 7222 person-days of follow-up. The overall incidence rate of PADH was 6.8

(95 % CI, 6.6–7.0) per 1000 person-days, with rates of 3.7 (95 % CI, 3.4–4.0) and 14.6 (95 % CI, 14.2–15.0) per 1000 person-days in RAA and Europeans, respectively. The median time to onset of the PADH event was 14 days (13–17), with occurrences ranging from 8 to 30 days after the first artesunate administration. For these 49 subjects, the median time between artesunate introduction and hemoglobin nadir was 15 (13–20) days. The median decrease in hemoglobin was 2 g/dL (1.5–2.9), with extremes ranging from 1 to 7.7 g/dL. There was no significant difference in hemoglobin decrease between RAA and Europeans (1.8 (1.4–2.5) g/dL versus 2.1 (1.6–3.2) g/dL; *p* = 0.2). However, hemoglobin decrease was significantly greater in patients with initial parasitemia >10 % compared to those with parasitemia between 4 and 10 % and <4 %, both among RAA and Europeans (Supplementary Fig. S1).

## 3.3. Factors associated with PADH occurrence

The cumulative probability of experiencing PADH by D30 post-artesunate was 20.2 % (Fig. 2). Three distinct phases were identified: an initial phase characterized by a sharp increase in PADH probability between D8 and D15 (25 PADH/49), followed by stabilization around Days 17 or 18 (18/49), and finally, a transition to a stable PADH probability after D21 (6 PADH/49).

## 3.4. Univariate analysis for risk factors (Table 1)

Univariate analysis revealed no significant association between the year of diagnosis or the treatment center's activity and PADH occurrence. Factors associated with a higher risk of PAHD included weight (HR 1.02, 95%CI 1.00–1.03, per increase of 1 kg; *p* = 0.027)), and baseline hemoglobin (HR 1.19, 95%CI, 1.05–1.35, per increase of 1 g/dL hemoglobin; *p* = 0.005). Higher parasitemia levels were also significantly associated with increased PADH risk (*p* < 0.001), with a significant interaction found between patient origin and parasitemia (*p* = 0.048). Patient origin-related differences in PADH occurrence were most

<figure><img src="image_3.png" /><figcaption>Fig. 1. Flowchart of the study population selection.</figcaption></figure>
<table><caption>**Table 1**
Factors associated with the occurrence of PADH (univariate analysis).</caption><thead><tr><th>Characteristic</th><th>Available data</th><th>Patients without PADH during follow-up</th><th>Patients with PADH during follow-up</th><th>Hazard ratio (HR)</th><th>95 % IC</th><th>p-value</th></tr></thead><tbody><tr><td colspan="7"><strong>Socio-demographic characteristics</strong></td></tr><tr><td>Age</td><td>323</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt;15 years</td><td></td><td>20 (7 %)</td><td>2 (4 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>15-30 years</td><td></td><td>59 (22 %)</td><td>9 (18 %)</td><td>1.59</td><td>0.34-7.36</td><td>0.6</td></tr><tr><td>31-45 years</td><td></td><td>75 (27 %)</td><td>18 (37 %)</td><td>2.02</td><td>0.47-8.70</td><td>0.3</td></tr><tr><td>46-60 years</td><td></td><td>82 (30 %)</td><td>15 (31 %)</td><td>1.64</td><td>0.37-7.15</td><td>0.5</td></tr><tr><td>&gt;60 years</td><td></td><td>38 (14 %)</td><td>5 (10 %)</td><td>1.14</td><td>0.22-5.86</td><td>0.9</td></tr><tr><td>Weight, kg</td><td>278</td><td>72 [60-83]</td><td>80 [69-88]</td><td>1.02</td><td>1.00-1.03</td><td><strong>0.027</strong></td></tr><tr><td>Sex</td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Female</td><td></td><td>115 (41 %)</td><td>16 (33 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Men</td><td></td><td>163 (59 %)</td><td>33 (67 %)</td><td>1.45</td><td>0.80-2.64</td><td>0.2</td></tr><tr><td>Patient origin</td><td>308</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>RAA</td><td></td><td>194 (74 %)</td><td>18 (38 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>European</td><td></td><td>67 (26 %)</td><td>29 (62 %)</td><td>3.99</td><td>2.21-7.18</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Birth in endemic areas</td><td>302</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td></td><td>151 (58 %)</td><td>14 (33 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>No</td><td></td><td>108 (42 %)</td><td>29 (67 %)</td><td>2.66</td><td>1.4-5.03</td><td><strong>0.003</strong></td></tr><tr><td>Residence in endemic areas</td><td>287</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td></td><td>54 (22 %)</td><td>5 (11 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>No</td><td></td><td>186 (78 %)</td><td>42 (89 %)</td><td>2.23</td><td>0.88-5.63</td><td><strong>0.090</strong></td></tr><tr><td>Immunosuppression</td><td>312</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>241 (91 %)</td><td>46 (98 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>24 (10 %)</td><td>1 (2 %)</td><td>0.21</td><td>0.03-1.50</td><td>0.12</td></tr><tr><td>History of malaria</td><td>280</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>159 (67 %)</td><td>36 (82 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>77 (33 %)</td><td>8 (18 %)</td><td>0.52</td><td>0.24-1.12</td><td><strong>0.095</strong></td></tr><tr><td>Year of inclusion</td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2011-2013</td><td></td><td>74 (27 %)</td><td>17 (35 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>2014-2016</td><td></td><td>204 (73 %)</td><td>32 (65 %)</td><td>0.63</td><td>0.33-1.20</td><td>0.2</td></tr><tr><td>Inclusion Centre*</td><td>322</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Less than 6 SM per year</td><td></td><td>110 (40 %)</td><td>17 (35 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Between 6-11 SM per year</td><td></td><td>47 (17 %)</td><td>4 (8 %)</td><td>0.52</td><td>1.17-1.54</td><td></td></tr><tr><td>More than 12 SM per year</td><td></td><td>116 (43 %)</td><td>28 (57 %)</td><td>1.47</td><td>0.8-2.68</td><td>0.2</td></tr><tr><td colspan="7"><strong>Features of the stay</strong></td></tr><tr><td>Chemoprophylaxis</td><td>314</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>200 (75 %)</td><td>35 (73 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>66 (25 %)</td><td>13 (27 %)</td><td>1.06</td><td>0.56-2.00</td><td>0.9</td></tr><tr><td>Place of stay</td><td>318</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>West Africa</td><td></td><td>175 (65 %)</td><td>33 (67 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Other</td><td></td><td>94 (35 %)</td><td>16 (33 %)</td><td>0.96</td><td>0.53-1.75</td><td>&gt;0.9</td></tr><tr><td colspan="7"><strong>Characteristics of malaria</strong></td></tr><tr><td>Duration of symptom progression, days</td><td>314</td><td>4 [2-6]</td><td>4 [3-5]</td><td>1.00</td><td>0.99-1.00</td><td>0.3</td></tr><tr><td>Initial parasitemia on Day 0, %</td><td>320</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt;4 %</td><td></td><td>129 (47 %)</td><td>4 (9 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>4-10 %</td><td></td><td>90 (33 %)</td><td>17 (35 %)</td><td>5.74</td><td>1.93-17.1</td><td><strong>0.002</strong></td></tr><tr><td>&gt;10 %</td><td></td><td>53 (20 %)</td><td>27 (56 %)</td><td>13.5</td><td>4.72-38.6</td><td><strong>&lt;0.001</strong></td></tr><tr><td>Initial hemoglobin on day 0, g/dL<sup>a</sup></td><td>319</td><td>12 [10.2-13.6]</td><td>13.6 [11.60-14.5]</td><td>1.19</td><td>1.05-1.35</td><td><strong>0.005</strong></td></tr><tr><td>Very severe malaria</td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>149 (54 %)</td><td>18 (37 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>129 (46 %)</td><td>31 (63 %)</td><td>1.67</td><td>0.93-2.98</td><td><strong>0.084</strong></td></tr><tr><td>Number of severity criteria</td><td>324</td><td>2 [1-3]</td><td>3 [2-4]</td><td>1.18</td><td>0.98-1.41</td><td><strong>0.083</strong></td></tr><tr><td colspan="7"><strong>Severity criteria</strong></td></tr><tr><td>Neurologic impairment<sup>c</sup></td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>159 (57 %)</td><td>30 (61 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>119 (43 %)</td><td>19 (39 %)</td><td>0.84</td><td>0.47-1.49</td><td>0.5</td></tr><tr><td>Cardio-Circulatory impairment<sup>d</sup></td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>242 (87 %)</td><td>39 (80 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>36 (13 %)</td><td>10 (20 %)</td><td>1.66</td><td>0.83-3.33</td><td>0.2</td></tr><tr><td>Spontaneous bleeding</td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>273 (98 %)</td><td>48 (98 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>5 (12 %)</td><td>1 (2 %)</td><td>1.84</td><td>0.25-13.4</td><td>0.5</td></tr><tr><td>Respiratory distress<sup>e</sup></td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>268 (96 %)</td><td>47 (96 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>10 (4 %)</td><td>2 (4 %)</td><td>1.02</td><td>0.25-4.22</td><td>&gt;0.9</td></tr><tr><td>Macroscopic hemoglobinuria</td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>248 (89 %)</td><td>45 (92 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>30 (11 %)</td><td>4 (8 %)</td><td>0.92</td><td>0.33-2.55</td><td>0.9</td></tr><tr><td>Hemoglobin &lt;7 g/dL</td><td>324</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>262 (95 %)</td><td>49 (100 %)</td><td>Ref.</td><td>ref.</td><td></td></tr><tr><td>Yes</td><td></td><td>13 (5 %)</td><td>0 (0 %)</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Renal impairment<sup>f</sup></td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>225 (81 %)</td><td>42 (86 %)</td><td>Ref.</td><td>ref.</td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Characteristic</th><th>Available data</th><th>Patients without PADH during follow-up</th><th>Patients with PADH during follow-up</th><th>Hazard ratio (HR)</th><th>95 % IC</th><th>p-value</th></tr></thead><tbody><tr><td>Yes</td><td></td><td>53 (19 %)</td><td>7 (14 %)</td><td>0.71</td><td>0.32–1.59</td><td>0.4</td></tr><tr><td>Hypoglycemia &lt;2.2 mmol/L</td><td>327</td><td>276 (99 %)</td><td>49 (100 %)</td><td>Ref.</td><td></td><td></td></tr><tr><td>No</td><td></td><td>2 (1 %)</td><td>0 (0 %)</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Jaundice or total bilirubin &gt;50 µmol/L</td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>134 (48 %)</td><td>19 (39 %)</td><td>Ref.</td><td></td><td></td></tr><tr><td>Yes</td><td></td><td>144 (52 %)</td><td>30 (61 %)</td><td>1.4</td><td>0.79–2.48</td><td>0.3</td></tr><tr><td>Hyperlactatemia</td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>174 (63 %)</td><td>27 (55 %)</td><td>Ref.</td><td></td><td></td></tr><tr><td>Yes</td><td></td><td>104 (37 %)</td><td>22 (45 %)</td><td>1.19</td><td>0.68–2.1</td><td>0.5</td></tr><tr><td>Acidosis<sup>g</sup></td><td>327</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td></td><td>261 (94 %)</td><td>47 (96 %)</td><td>Ref.</td><td></td><td></td></tr><tr><td>Yes</td><td></td><td>17 (6 %)</td><td>2 (4 %)</td><td>0.61</td><td>0.15–2.5</td><td>0.5</td></tr></tbody></table>

Factors associated with the occurrence of PADH within 30 days post artesunate were investigated by determination of hazard ratios (HR) with 95 % confidence intervals by the Cox proportional hazards model. For quantitative variables, the HR represents the risk increase per unit increment.

Abbreviations: CI = confidence interval, SM = severe malaria.

* Average annual number of severe malaria cases managed by the center. <sup>a</sup> The median initial hemoglobin was 11.9 g/dL [Q1-Q3 10.5–13.5] and 13.6 g/dL [Q1-Q3 11.6–14.5] for non-PADH and PADH groups respectively.

<sup>b</sup> The number in the patient column corresponds to the number of patients with the severity criterion in the initial phase.

<sup>c</sup> Neurologic impairment = At least lethargy, confusion, multiple convulsion, coma Glasgow Scale <11.

<sup>d</sup> Cardio-circulatory impairment = At least shock (systolic blood pressure #80 mmHg), peripheral signs of hypotension. <sup>e</sup> Respiratory distress = At least respiratory frequency 0.32/mn, crackles, pulmonary infiltrates (chest x-ray), PaO2 <60 mmHg, SpO2,91 %.

<sup>f</sup> Renal impairment = Plasma or serum creatinine >265 µmol/L (3 mg/dL) or blood urea >20 mmol/L.

<sup>g</sup> Acidosis = pH < 7.35 or plasma bicarbonate level <15 mmol/L.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Kaplan Meier curve of the cumulative probability of occurrence of PADH during follow-up.</figcaption></figure>

The median time to the onset of the PADH event was 14 days [Q1-Q3 13–17], with 21 out of 49 patients experiencing PADH between Days 13 and 17. Three distinct phases have been identified: an initial phase (Days 8–15) characterized by a sharp increase in the probability of PADH onset (25 PADH/49). The conclusion of this initial phase (Days 16–20), where the probability of PADH onset begins to stabilize, falls around Day 17 or 18 (18 PADH/49). Finally, a transition to a stable probability of PADH onset begins beyond Day 21. These insights shed light on the variations in PADH onset probability among patients over the study period.

pronounced for initial parasitemia >10 % (Fig. 3). The highest risk was observed in Europeans with initial parasitemia >10 % (cumulative probability of PADH by day 30: 65 %, 95 % CI 44–79). Compared to an RAA subject with a parasitemia <4 %, the PADH occurrence risk was 6 times higher (HR 6.24, 95 % CI, 1.1–34; *p* = 0.035) in an RAA subject with parasitemia >10 %, and 36 times higher for a European subject with parasitemia >10 % (HR 36.4, 95 % CI, 8.6–155; *p* < 0.001).

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Kaplan Meier curves of the probability of occurrence of PADH by patient origin and initial parasitemia.</figcaption></figure>

The blue curve represents European patients, and the red curve represents RAA patients. The log-rank test was used to determine if there was a significant difference in the survival curves of the two groups.

The correlogram revealed a strong overlap between birth in endemic areas and residence in endemic areas, leading to the selection of the latter as a more current and relevant factor for analysis (Supplementary Fig. S2).

### 3.5. Multivariate analysis

After adjusting for weight, history of malaria, initial hemoglobin, residence in endemic area and presence of very severe malaria, the risk of PADH remained significantly higher for Europeans with an initial parasitemia >10 % compared to RAA with parasitemia <4 % (HR 18.8, 95 % CI, 4–89; *p* < 0.001) and RAA with a parasitemia >10 % (HR 4.77, 95 % CI, 0.78–29.2; *p* = 0.09) (Fig. 4). Initial hemoglobin and weight were no longer independent predictors after adjustment (HR 1.03, 95 %
<figure><img src="image_6.png" /><figcaption>Fig. 4. Factors associated with the occurrence of PADH (multivariate analysis).</figcaption></figure>

CI, 0.88–1.2; *p* = 0.46 and HR 1.01, 95 % CI, 0.99–1.0; *p* = 0.192 respectively).

## 3.6. Sensitivity analysis

Similar results were obtained after multiple imputation for missing data on D0 characteristics, with the highest risk observed in Europeans with initial parasitemia >10 % compared to RAA with an initial parasitemia less than 4 %. Sensitivity analyses adjusting PADH event criteria (one by removing the minimum limit of 10 % hemoglobin loss, the other by removing from the analysis the subjects who had moderate decrease in hemoglobin less than 10 %) did not change the meaning of the results (Supplementary Figs. S3, S4, S5 and Supplementary Table S3).

## 4. Discussion

In this nationwide prospective multicenter study, we delved into the factors influencing the risk of PADH within 30 days of initiating artesunate treatment in *Plasmodium falciparum* imported SM. Our multivariate analysis spotlighted the pivotal role of the interaction between patient origin and parasitemia in dictating the subsequent PADH occurrence, offering novel insights into this relationship. Particularly, European patients with a baseline parasitemia exceeding 10 % were identified as the most susceptible group to PADH within the initial 30 days of commencing artesunate treatment, with a substantial 65 % cumulative probability of developing PADH by day 30. In fact, these patients should be monitored more frequently on a weekly basis than other patient groups as described before. If financial resources or technical support are scarce other types of patients with a low risk of PADH may not be systematically monitored for hematological problems, due to the lower risk of late hemolysis.

We highlighted a gradient of PADH risk and severity in relation to parasitemia. Indeed, PADH occurrence risk escalates with parasitemia levels between 4 % and 10 %, reaching its peak when parasitemia surpasses 10 %. Furthermore, we found that higher initial parasitemia corresponds to greater hemoglobin loss during PADH, irrespective of patient origin. The association between initial parasitemia and PADH in SM has been suggested since the initial description of hemolytic anemia

in 2011 [7]. Four out of 6 patients who experienced PADH had initial parasitemia greater than 10 %. Further reinforcing this link, a 2012 study comparing PADH cases with non-PADH subjects found significantly higher initial parasitemia in PADH cases (mean initial parasitemia of 16 % in PADH patients versus 11 % in those without PADH, *p* = 0.02) [17]. Another study observed a trend of higher parasitemia in PADH patients, although statistical significance was not achieved (median initial parasitemia 7.1 % (21 patients with PADH) versus median baseline parasitemia 5 % (51 patients without PADH), *p* = 0.1) [16]. These results align with the notion that patients with PADH have a significantly higher number of circulating pitted erythrocytes than those without PADH. As the amount of pitted erythrocytes is partly dependent on the initial parasitemia, individuals with initial hyperparasitemia are at a heightened risk of developing PADH [8].

However, it is to note that initial parasitemia alone does not provide a complete explanation for PADH. Our study is the first to shed light on the interaction between parasitemia and patient origin on the risk of PADH. Notably, we show that Europeans are significantly more predisposed to PADH than RAA patients with similar parasitemia levels. The gradient of PADH risk with increasing level of parasitemia was less marked in RAA. While no previous studies have explicitly explored the link between patient origin and PADH in non-endemic areas, a study including 11 patients found that only 2 of the 11 PADH cases occurred in patients born in endemic areas [21]. Moreover, studies of African children indicate a lower prevalence of PADH, with hemoglobin decreases observed in only 7 % and 11.4 % of cases [23,32,33]. These figures contrast sharply with the situation in Europe, suggesting that the prevalence of PADH is likely lower in endemic regions. The significant interaction between patient origin and initial parasitemia suggests underlying differences in immune responses that could influence PADH risk. Individuals from endemic areas, such as RAA patients, often develop partial immunity (premunition) through repeated malaria exposure. This acquired immunity can modulate the clearance of infected erythrocytes and mitigates the extent of pitting-induced delayed hemolysis. Conversely, individuals from non-endemic areas, such as Europeans, lack this adaptive immunity, relying more heavily on spleen-mediated parasite clearance. This dependence leads to a higher burden of pitted erythrocytes, significantly increasing the risk of PADH.
Premunition has been shown to reduce parasitemia and delay hemolytic events by promoting immune-mediated parasite clearance [34,35]. Additionally, genetic factors such as the sickle cell trait or specific human leukocyte antigen (HLA) polymorphims prevalent in populations from endemic areas may contribute to lower PADH susceptibility [36, 37]. These mechanisms may explain the gradient of PADH risk observed between Europeans and RAA patients with similar parasitemia levels. Future research should explore biomarkers of immunity, including splenic functions and pitting rates, to deepen understanding of the biological underpinnings of this interaction. Notably, the lower inci- dence of PADH in malaria-endemic regions underscores the protective role of premunition acquired through repeated exposure [19,21]. For instance, Ndour et al., demonstrated rapid, pitting-independent *Plas- modium falciparum* clearance in immune Malian children treated with artesunate [36]. These findings highlight the critical role of immunity in mitigating PADH risk and emphasize the importance of contextualizing our results for diverse geographical and immunological settings to reach broader audiences.

While our univariate analysis indicated an association between baseline hemoglobin levels and PADH risk, this association lost signifi- cance in our study after adjusting for patient origin and parasitemia. Similar result was found in sensitivity analyses, as with using multiple imputation of missing data, suggesting that it likely isn't related to data loss. The link between PADH and late once-infected erythrocyte loss has been recognized since 2014 [8]. It seems logical to think that, akin to high initial parasitemia, high initial hemoglobin levels (and thus a large number of red blood cells) could be directly linked to PADH risk. We previously reported an association between PADH risk and baseline hemoglobin levels at day 0 (median baseline hemoglobin in 21 patients with PADH: 13.6 g/dL [11.6–15.4] versus 11.3 g/dL [9.6–13.1] in 51 patients without PADH; *p* < 0.002) [16]. There is complex interaction of several factors influencing initial hemoglobin levels, including splenic filtration, patient origin, malaria immunity and time from treatment to sequestration. Our study, conducted in a non-endemic malaria setting, did not comprehensively account for all factors, which may weaken the theoretical link between hemoglobin and PADH. Nevertheless, we did not observe any significant association between various factors, such as the presence of very severe malaria criteria, the duration of symptoms before treatment, a history of malaria, age, weight, or sex, and the risk of PADH. PADH is a multifactorial phenomenon, and its occurrence cannot be solely attributed to isolated factors, such as hemoglobin levels. Instead, it likely results from a complex interplay of biological processes, including the pitting process, individual immune responses, and genetic predispositions. Future research should focus on understanding these interactions to refine our knowledge of PADH risk factors, especially in endemic areas.

The definition of PADH was crucial, following established criteria: a 10 % or greater decrease in hemoglobin levels alongside a 10 % or more increase in LDH, a hemolysis marker [8,28,32]. LDH measurements were often available, while haptoglobin or reticulocyte measurements were lacking. Sensitivity analyses have further confirmed the reliability of this definition, advocating its adoption in future studies.

Our work has several limitations. First, not all eligible subjects, i.e. those treated with artesunate for SM, underwent biological monitoring. This limitation arises from the voluntary nature of data entry, which depends on the availability and diligence of healthcare providers man- aging the patients. In France, reporting imported malaria cases and requiring information on severity signs, *Plasmodium* species, travel his- tory to malaria-endemic areas, and suspicion of drug resistance is not mandatory. A more comprehensive online questionnaire is available for reporting, covering clinical and biological variables, treatment details, and follow-up data. Its completion varies based on the diligence of the treating practitioners. This variability explains, for example, the signif- icant lack of hemoglobin data before Day 7 (707 data available out of 1630 patients). Although this is a prospective study, the follow-up of such a cohort leads to missing data and can introduce selection bias.

Furthermore, our data were sourced from the M-NRC database. Despite the extensive network of the M-NRC, it's important to note that only an estimated 50 % of imported malaria cases are reported to the M-NRC. However, national surveys have confirmed the representative nature of cases reported through this surveillance system in France (capture-recapture survey) [37–39]. Patients with mixed-species infections or incomplete follow-up may introduce a bias toward more severe cases or those that were better monitored. However, considering that PADH predominantly occurs in patients with severe malaria, this bias is deemed acceptable within the context of an intention-to-diagnose-and-treat framework.

The attrition, as well as conducting our study in non-endemic areas, limits the extrapolation and generalization of our results to all cases of SM. However, the diversity of our recruitment represents the variety of patients treated for SM in France, and the study provides valuable and accurate information for clinicians managing imported malaria cases in Europe. It is noteworthy that multiple imputation did not alter the re- sults, further supporting the robustness of our findings. We acknowledge limitations, including a 30-day follow-up, standard in French biological monitoring. While late-onset PADH cases are theoretically possible, they are exceedingly rare and unlikely to significantly impact our findings.

This section addresses the limitations of our statistical model and analytical approach, while highlighting measures taken to ensure the robustness and validity of our findings.

One limitation is the use of a p-value threshold (*p* < 0.2) for variable selection in the univariate analysis, which could increase the risk of overfitting. To mitigate this, we applied a two-step strategy: variables were first preselected based on statistical significance, then refined by incorporating clinical relevance and addressing confounders. To further ensure robustness, a correlogram (Supplementary Fig. S2) was used to identify and exclude highly correlated variables, such as “birth in endemic area” and “residence in endemic area,” improving the inter- pretability of the final Cox model.

The exclusion of variables with >20 % missing data, such as *Stay duration*, in the multiple imputation process could potentially introduce bias. However, sensitivity analyses comparing results with and without *Stay duration* showed no significant differences, indicating that its exclusion did not meaningfully affect the robustness of our findings. The variables included in the imputation model were carefully selected based on their clinical relevance and associations with PADH to ensure a rigorous process.

Sensitivity analyses also explored variations in PADH criteria, such as alternative thresholds for hemoglobin reduction and LDH elevation, which demonstrated no significant impact on results. However, minor variations in these criteria could influence the identification of border- line PADH cases, underscoring the need for standardized definitions in future studies.

Wide confidence intervals for certain predictors, such as initial par- asitemia >10 %, reflect small subgroup sizes (e.g., European patients with hyperparasitemia). While the overall findings remain robust, these results require cautious interpretation and emphasize the need for larger studies or meta-analyses to refine risk estimates.

Although this study included one of the largest multicenter cohorts of patients treated for SM in non-endemic settings, no formal sample size calculation was conducted. Nonetheless, the sample size (>300 patients) provided adequate power to detect significant associations. Variability in the frequency and timing of laboratory follow-up assessments across centers also posed a limitation. For instance, if hemoglobin dropped on Day 7 but the next test was conducted on Day 14, the timing of PADH onset may have been misclassified. Standardized monitoring protocols are essential for improving the accuracy of PADH incidence and timing estimates in future studies.

Finally, the study addressed potential biases and confounding through several measures. Selection bias was minimized by using the nationally representative M-NRC database, validated through a capture-recapture survey [24,25]. Information bias was mitigated with multiple
imputation for missing data, and confounding was addressed by including clinically relevant variables and excluding collinear variables identified via a correlogram. Despite these efforts, residual confounding cannot be entirely excluded, and results should be interpreted with this limitation in mind.

Our study highlights that European patients with an initial para-
sitemia of more than 10 % are at the greatest risk of developing PADH
and no other factors seems to be of importance to the incidence of this
hematological phenomenon. Although classification scales for immunity
have been used in previous literature, the multifaceted nature of im-
munity makes it difficult to confine it to a single variable [38,39]. The
RAA group contains two distinct populations: individuals born in Africa
who live in France and return to their home countries to visit family, and
patients born in France with at least one parent of African descent. These
two subgroups do not share the same adaptive immunity profiles, which
could further influence their susceptibility to PADH. Notably, among the
194 patients in the RAA group, only 43 were born in France. This dif-
ference in origins suggests that the overall results of our study were not
significantly altered by the presence of French-born individuals, allow-
ing for a clearer analysis of the broader RAA population. Therefore, our
approach has been to analyse several variables, including patient origin,
birth in an endemic area and residence in an endemic area, as these
factors represent distinct facets of immunity. However, a more
comprehensive study of the relationship between the occurrence of
PADH and immunity to malaria deserves to be considered.

In conclusion, our study underscores the importance of PADH as a relatively frequent event after artesunate treatment, emphasizing the need for rigorous weekly biological monitoring for 28 days. This monitoring must be particularly meticulous for European patients with an initial parasitemia of more than 10 %, as they exhibit more pronounced declines in hemoglobin. It should be noted that while PADH warrants attention, it generally manifests as a benign condition and does not compromise the overall risk-benefit profile of artesunate in the treatment of SM.

CRediT authorship contribution statement

Pierre-Louis Conan: Writing – review & editing, Writing – original draft, Validation, Methodology, Data curation, Conceptualization. **Marc Thellier:** Writing – review & editing, Resources, Data curation. **Eric Kendjo:** Writing – review & editing, Resources, Data curation. **Sandrine Houzé:** Writing – review & editing, Resources, Data curation. **Rémonie Seng:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Data curation, Conceptualization. **Stéphane Jauréguiberry:** Writing – review & editing, Validation, Supervision, Methodology, Data curation, Conceptualization.

Grants and fundings

None

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.tmaid.2025.102801.

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