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 (Table 1).
The neutrophil group with the highest frequency was the neutrophilia category in Mali with 905 cases (38.8%) and Burkina Faso with 735 cases (42.3%), respectively. In contrast, there were more patients with neutropenia 340 (44.0%) in Guinea. The median and interquartile range (IQR) of patient’s bodyweight was significantly higher in neutrophilia category 24.5 kg (IQR, 17.6-37.8), compared to patients in neutropenia (19.6 kg, IQR= 14.0-28.8) and normal neutrophils levels (21.8 kg, IQR= 15.8-32.7) (p = 0.0011). Furthermore, the patient’s median temperature was higher in neutrophilia group with 38.2°C (IQR, 37.4-38.8) (table 1). The prevalence of gametocytemia was significantly higher in patients with normal neutrophils level with 133 cases (40.6%) compared to those with neutropenia and neutrophilia (p < 0.0001).
There was also a significant difference in patients with neutropenia in the amount of trophozoites at baseline level. These patients had a significantly lower parasite count compared with the other groups (p < 0.0001) (table 1).
The median WBC count was significantly higher in patients with neutrophilia 8.0 (IQR, 6.3-10.6) compared to subject in neutropenia 6.5 (IQR, 5.0-8.6) and normal neutrophils levels 6.9 (IQR, 5.2-8.7) (p < 0.0001). In contrast, the median lymphocytes count and interquartile range was significantly higher in patients with neutropenia 48.6% (IQR, 42.92-53.7) compared to those with normal or high neutrophil counts with respectively 32.6% (IQR, 27.0-38.2) and 15.5% (IQR, 8.1-20.3) (p < 0.0001) (Table 1).
ACTs efficacy 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 significantly 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) shows that the ACT used (AL: OR = 0.80 [0.26; 2.46], ASAQ: OR = 1.73 [0.85; 3.52], DHAPQ: OR = 0.99 [0.47; 2.06], PA: OR = 0.97 [0.45; 2.06]) was not associated with a risk of post treatment Day 3 gametocytemia. This figure also shows that neither the country of residence (Burkina Faso: OR = 0.88 [0.46; 1.68], Guinea: OR = 0.29 [0.07; 1.19], Mali: OR = 1.51 [0.87; 2.61]), nor age category (under 5 years old children: OR = 0.48 [0.09; 2.53], teenager: OR = 0.56 [0.24; 1.26]) was associated with a risk of gametocytemia 3 days after treatment in patients with neutropenia compared to those without neutropenia.
Secondary neutropenia after ACTs administration
At the first malaria episode (Fig. 3 A), neutrophil levels on follow-up days 3, 7 and 28 were significantly lower (p < 0.001) compared to pre-treatment 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 significantly (p < 0.001 regardless of treatment arm), but without reaching the threshold of normality and subsequently decreased again significantly (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.