Variation in Neutrophil Levels and Artemisinin-Based Combination Therapy Ecacy in West-africa

White blood cells, including abundant neutrophils, constitute the rst line of innate defence against pathogens. Neutrophils are involved in pathogen clearance by phagocytosis. However, their role on malaria parasite clearance and on the ecacy of Artemisinin-based Combination Therapy (ACT) are poorly understood. In a prospective longitudinal in vivo study, conducted from 2010 to 2014 in Mali, Burkina Faso and Guinea, in which 5360 malaria cases were enrolled, neutrophil rates were compared with malaria carriage after treatment with different ACTs. i.e. Artemether - lumefantrine (AL), Artesunate - amodiaquine (ASAQ), Dihydroartemisinin - piperaquine (DHAPQ) or Pyronaridine artesunate (PA). Depending on the level of neutrophils in the blood, study cases were classied as having neutropenia, normal neutrophils level or neutrophilia. A secondary analysis on data of 4845 cases was performed using R ggplot2 statistical package to compare the post treatment neutrophil levels means and standard deviation between different treatment arms at subsequent malaria episodes. threshold was set at 50%. Discordant results were subjected to a third independent reading. Asexual parasites were counted against 200 leukocytes (in accordance with WHO standards [26]). If the number of parasites was less than 10 parasites per 200 leukocytes, the parasite count was extended to 500 leukocytes. Parasitaemia was estimated by reporting the number of parasites per microliter of blood based on the count of 8,000 leukocytes. Non-PCR corrected drug ecacy results were used in this study. Pre-treatment and days 3, 7 and 28 neutrophils status and parasitaemia carriage were used to evaluate drug ecacy. in repeated treatment of of and over


Background
In healthy individuals, polymorphonuclear neutrophils (PMN), which are the most important component of the white blood cells (WBC), play an important role in the host's immunological response against bacterial, fungal and malaria infections because of their capacity to phagocytose pathogens [1,2]. In addition, they contribute to the recruitment of both nonspeci c and speci c immune effector cells [3]. Finally, since WBC and PMN respond to produced cytokines [4,5], they also modulate the balance between humoral and cell-mediated immunity by contributing to the promotion of T helper cells (i.e. TH1 or TH2) response [6]. PMNs are engaged in a complex cross-talk with immune and endothelial cells that bridges innate and adaptive immunity.
In some individuals' neutrophil levels may be lower than normal i.e. neutropenia, which and can be acute, congenital or cyclic. Neutropenia, de ned as an absolute decrease in the number of neutrophils circulating in the blood [7], increases the risk of infection, in particular from pyogenic and enteric bacteria. In addition, the risk of fungal infections increases further when neutropenia is severe and prolonged [8]. Acute neutropenia, the resolvable short-term drop in absolute neutrophil counts due to an infection or lesion, is much more common than congenital or cyclic neutropenia [9]. Neutropenia is known to be quite common in African populations [10,11] and a genetic deletion of the Duffy antigen receptor for chemokines (DARC-null genotype) is likely to be a major determinant for neutropenia [11]. In West Africa, more than 90% of the population is believed to be Duffy antigen negative [12].
In analogy to the way PMN can control mycobacterial infection by phagocytosis of pathogens at the site of infection [13], these cells could act with similar activities against malaria infection. PMN are known to phagocytose infected Red Blood Cell (iRBC) in vivo [14]. It has been shown that neutrophils can phagocytose merozoites and occasionally trophozoites in bone marrow [15]. Neutrophils can also clear pathogens by respiratory burst called Reactive Oxygen Species (ROS). Malaria parasites growth inhibition by ROS occurs during the intra-erythrocytic development stage [16]. Furthermore, it has been demonstrated in vitro that neutrophils from children infected with Plasmodium are better able to inhibit parasite growth than neutrophils from uninfected subjects [17]. Besides the asexual forms of the malaria parasite, neutrophils can phagocyte extracellular gametocytes, while, intra-erythrocytic gametocytes are not very susceptible to neutrophils [18].
Artemisinin-based combination therapy (ACT) is now for almost two decades recommended by the World Health Organization (WHO) as the rst-line treatment of uncomplicated falciparum malaria [19]. However, the e cacy of ACTs is an ongoing concern , due to the emergence of parasite resistance to artemisinins, [20]. Furthermore, the relationship between malaria, ACT treatment and neutrophils is largely understudied. In a previous analysis, using data from 7 randomized trials conducted in 9 countries assessing the e cacy of artesunate-amodiaquine, amodiaquine mono-therapy, artesunate mono-therapy, artemether-lumefantrine, artesunate and sulphadoxine-pyrimethamine, and dihydroartemisinin, it was found that neutropenia was frequently recorded as an adverse event at a rate of 11% [21].
Despite the role neutrophils play in innate immunity, their role in the immune response against malaria infections has been little studied. In particular, there are no extensive studies on the role neutrophils play in parasite clearance [22].
The aim of the present study was to assess the impact of neutrophil levels on the in vivo e cacy of four frequently used artemisinin-based combinations: Artemether lumefantrine (AL), Artesunate amodiaquine (ASAQ), Dihydroartemisinin piperaquine (DHAPQ) and Pyronaridine artesunate (PA).

Study sites and study participants
This is a secondary analysis of the WANECAM study [23], which was a clinical trial involving 5360 volunteers from West-Africa assessing e cacy and safety of several ACTs. The original WANECAM study was a randomised, multicentre, open-label, longitudinal clinical study carried out across seven study sites in three countries: Burkina Faso, Guinea, and Mali (http://www.wanecam.org/fr/clinical-study-protocol/) [23]. Brie y, study cases were enrolled and followed up over 2 years for consecutive malaria episodes between October 24, 2011, and February 1, 2016. Patients were treated either with AL, ASAQ, DHAPQ or PA at recommended doses. Patients, irrespective of their gender (age ≥ 6 months and body weight ≥ 5 kg), were eligible for enrolment in the study, if they had acute uncomplicated microscopically con rmed Plasmodium sp. malaria with a parasite density <200 000 parasites per μL blood, and with fever or a history of fever within 24 hours and Haemoglobin (Hb) concentration > 5 g/dL [23]. Before enrolment, all adult study cases provided informed consent after being informed on the aims, bene ts and constraints of the study in their own language. For children (< 18 years), the informed consent was obtained from parents or legal guardians after the consent taker was satis ed that the child is willing to participate. Ethical review and clearance for this project was provided by the respective ethical review boards.

Neutrophils status
Blood samples were collected from patients from an alcohol-cleaned peripheral vein into sterile venous blood collection tubes with ethylene diamine tetraacetic acid (EDTA). Blood counts, including neutrophils, lymphocytes, WBC and Hb were measured immediately at enrolment using ABX Pentra 60 -HORIBA (Montpellier, France) [24]. Blood count measurements were also performed at day 3, 7 and 28 after treatment.

Drug e cacy
During the rst two days after inclusion, nger-prick blood was taken every 12 hours to make thick and thin smears to determine the presence and number of malaria parasites. Subsequently, study cases were seen on days 3, 7, 14, 21, 28, 35, 42 and on days of recurrent illness for clinical examination and nger-prick for thick and thin smears, which were stained with 10% Giemsa for 15 minutes. Parasites density was determined by double reading of the thick blood smear using an Olympus CX21 microscope (Tokyo, Japan). The discordance threshold was set at 50%. Discordant results were subjected to a third independent reading. Asexual parasites were counted against 200 leukocytes (in accordance with WHO standards [26]). If the number of parasites was less than 10 parasites per 200 leukocytes, the parasite count was extended to 500 leukocytes. Parasitaemia was estimated by reporting the number of parasites per microliter of blood based on the count of 8,000 leukocytes. Non-PCR corrected drug e cacy results were used in this study. Pre-treatment and days 3, 7 and 28 neutrophils status and parasitaemia carriage were used to evaluate drug e cacy.

Statistical analysis
Frequency of patients with neutropenia, normal neutrophils levels and neutrophilia was computed and descriptive statistics were calculated (percentages, median and quartiles). Chi-square and Fisher tests were used to compare proportions. The threshold of 0.05 was used as statistical signi cance. Bar plots were used to assess the changes in gametocyte prevalence during follow-up after the administration of antimalarial drugs. Logistic regression analyses were performed to predict the effect of neutrophils status, treatment arms, lymphocytes levels, Hb levels, patients gender and age categories on the e cacy of the treatment. Using 'meta' R package (version 4.13-0), a forest plot was performed to investigate the association among socio-demographic parameters and treatment used on gametocytes reappearance at Day 3 according to neutrophils variation.

Results
General study characteristics Data of 4845 patients were used for this secondary analysis. The data of 515 participants were not used because neutrophil counts of these cases were not recorded. The study cases were distributed amongst the treatment arms as follows: 1163 were treated with PA, 1154 with DHAPQ, 961 with ASAQ and 1567 with AL (Table 1). With respect to gender balance, 2489 (51.4%) of patients were male and 2356 (48.6%) were female (p = 0.7476). A total of 2334 patients were enrolled in Mali, 1739 in Burkina and 770 in Guinea. Children (n=2981) with age between 5 and 14 years were the most represented (61.5%) in the study ( There was also a signi cant difference in patients with neutropenia in the amount of trophozoites at baseline level. These patients had a signi cantly lower parasite count compared with the other groups (p < 0.0001) (table 1).

ACTs e cacy according to neutrophils count variations
The dynamics of malaria parasite carriage after treatment according to neutrophil status is presented in Table 2. Irrespective of the type of malaria drugs used in this study, patients with neutropenia had a signi cantly higher malaria parasite carriage frequency up to day 28 after treatment compared to cases with normal neutrophils level and subjects with neutrophilia (p < 0.0001). However, patients with neutropenia had at baseline the lowest parasite density compared to the other groups (Table 1). Microscopy revealed that at day 7 only patients in the neutropenia group, and treated with AL, had a positive blood smear (Table 2). Data from day 28 showed that the chance of parasite recurrence is higher in patients with neutropenia regardless of the type of ACT used for treatment in this study ( Table 2). Fig. 1 shows that, irrespective of the treatment arm, the pre-treatment prevalence of gametocytemia was higher in patients with normal neutrophils level compared to neutropenia and neutrophilia groups (p < 0.0001). Three days after antimalarial drug administration, and regardless of the type of ACT used, the data show an increase in the prevalence of gametocytemia in the neutropenic group while gametocyte clearance is observed in patients with normal neutrophil counts and those with neutrophilia (Fig. 1). On day 28, after treatment, only patients with neutropenia and treated with ASAQ, DHAPQ and PA carried gametocytes (Fig. 1). A forest plot (Fig. 2)

Secondary neutropenia after ACTs administration
At the rst malaria episode (Fig. 3 A), neutrophil levels on follow-up days 3, 7 and 28 were signi cantly lower (p < 0.001) compared to pretreatment neutrophil levels irrespective of the antimalarial treatment received by the patients. This decrease in neutrophils levels was more pronounced at day 3 after treatment. Regardless of the ACT used, the overall neutrophil level was 52.70% before treatment and decreased to 33.44 on day 3 after treatment (p < 0.0001). Seven days after treatment, the level of neutrophils increased signi cantly (p < 0.001 regardless of treatment arm), but without reaching the threshold of normality and subsequently decreased again signi cantly (p < 0.001 regardless of treatment arm) from day 7 to day 28 (Fig. 3).
During consecutive malaria episodes (episode 2: Fig. 3 B, episode 3: Fig. 3 C and episode 4: Fig. 3 D), the dynamics of neutrophil levels followed the same trend as in the initial episode (Fig. 3 A). Irrespective of the malaria episodes, three days after drug administration the neutrophil counts were higher in the ASAQ treatment arm (p < 0.0001) (Fig. 3).

Association between neutrophil count variations and reinfection parasitemia after treatment
Logistic regression analysis (Table 3) showed that neither being in the neutrophilia category (OR = 1.57, p = 0.6771) nor having normal neutrophil levels (OR = 0.97, p = 0.9471) was associated with a risk of malaria parasite reappearance at Day 28 after treatment compared to patients with neutropenia.
Other factors were found to be associated with malaria parasite reappearance during post treatment follow-up (Table 3). Compared to AL treatment arm, ASAQ (OR = 0.27, p = 0.0024), DHAPQ (OR = 0.07, p < 0.0001) and PA (OR = 0.13, p < 0.0001) showed a protective effect against malaria parasite reappearance. Each increase of lymphocyte count (OR = 0.93, p = 0.0063) was observed to be associated with a decrease of the probability of post treatment Day 28 parasitaemia. Compared to adults, children under 5 years (OR = 5.64, p = 0.0393) were at risk of malaria parasite reappearance at Day 28 of follow-up.

Discussion
This study showed patients with neutropenia have lower baseline parasitaemia, a higher rate of recurrent parasitaemia and higher rate of gametocyte carriage by day 28 post-artemisinin-based combination therapy compared to patients with normal or high neutrophil levels. These differences were found to be independent of the type ACT used in this study. The baseline malaria parasite density was lower in patients with neutropenia. This nding is in line with the ndings of Olliaro et al who showed that during acute uncomplicated malaria the increase of neutrophils is positively associated with parasitaemia [27]. It has also been reported that in semi-immune travellers neutrophil counts increase with the severity of malaria infection compared to those with uncomplicated malaria [28]. An important nding form this study with regards to transmission potential of malaria is the fact that the prevalence of post treatment gametocytemia was higher in patients with neutropenia regardless of treatment arm. Neutrophils constituting the most important component of the leukocyte population [2] plays an important role in malaria infection due to its phagocytic activity [1]. The role of neutrophils in the phagocytosis of extracellular gametes has been previous described [18], and this could explain the almost total absence of gametocytes in post treatment follow-up in subjects with neutrophilia. This observation could also be due to faster clearance of asexual parasites in patients with neutropenia, which would leave very little time for differentiation into sexual stages [29].
Approximately one third of the patients had neutropenia and these results support previous studies indicating that neutropenia is quite common in African populations [10,11] where more than 90% of the African population are Duffy antigen negative [12] and that the genetic deletion of the Duffy antigen receptor is a major determinant for neutropenia [11].
In addition, the study provides the evidence of secondary neutropenia when uncomplicated malaria is treated with these four ACT regimens. In this study a secondary neutropenia is observed three days after the administration of antimalarial drugs, regardless of the treatment arm. It has been previously described that 70-90% of acute and severe neutropenia has been shown to be attributable to drugs [30]. Several medications including antimalarial drugs are described to be associated with the decrease of neutrophils levels [30,31]. A larger analysis including data from 7 clinical trials carried out in 9 countries comparing the changes in haematologic parameters of ASAQ to AQ mono-therapy, AS mono-therapy, AL, AS + SP and DHA, revealed 11% of post treatment neutropenia [21]. A study conducted in Ghana to compare the e cacy of ASAQ versus AL in children showed that 3, 7, 14 and 28 days post treatment neutrophil counts were signi cantly lower (p < 0.01) compared to day 0 counts in both treatment arms [32].
The present study revealed that of all four drug combinations used in this analysis, AL is the least protective against parasites carriage during 28 days' post treatment follow-up in patients with normal neutrophil levels. Artemether with a half-life of two to three hours is rapidly eliminated from plasma whereas lumefantrine is eliminated more slowly [33]. The combination allows a rapid clearance of parasitaemia but does not prevent new infections. In DHAPQ combination, piperaquine is characterized by a slow absorption and long half-life, which might prevent new Plasmodium infections [34]. While the half-life of amodiaquine is about 5 hours [35,36], the combination AS+AQ presented a protective effect in the multivariate model. Pyronaridine is described to have a mean half-life of 194.8 ± 47.8 hours [37].
Furthermore, the present research showed that compared to AL treatment arm, patients treated with ASAQ, DHAPQ or PA were less likely to carry gametocytes post treatment. A meta-analysis of individual patient data demonstrated that the appearance of gametocytes in patients' blood was lowest after AL treatment and signi cantly higher after DHAPQ (adjusted hazard ratio (AHR), 2.03; 95 % CI, 1.24-3.32; p = 0.005 compared to AL) and ASAQ (AHR, 4.01; 95 % CI, 2.40-6.72; p < 0.001 compared to AL) [38]. A previous study conducted in Bougoula-Hameau, Mali, to investigate the differential infectivity of gametocytes after artemisinin-based combination treatment, revealed that the Day 3 gametocytes prevalence was lower in AL arm compared to ASAQ and ASSP arms [29]. This could be explained by the fact that unlike AL which is rapidly eliminated from the patient's blood, the other combinations used in this study would remain at sub-therapeutic levels for a longer time promoting the production of gametocytes.
A limitation of the present study is the absence of parasitaemia data six or eight hours after treatment. Such data would have permitted the precise measurement of parasite clearance time and the slope half-life of parasite clearance using the WorldWide Antimalarial Resistance Network (WWARN) Parasite Clearance Estimator (PCE) [39] and relate these to patients' neutrophils count variations. In addition, the lack of a Duffy antigen is a limitation to determine the share of benign ethnic neutropenia [11].
A major strength of this study is the participation of several countries to represent West Africa, which makes this study relevant for a large population in Africa. In addition, the large sample size allowed this study to have su cient power to detect even small differences or effects. This study effectively compared the four major anti-malaria drugs in relation to neutropenia and parasite recurrence.
This study suggests that neutropenia could ultimately decrease the e cacy of ACTs in West Africa. The immunological mechanisms involved warrant further investigations.