Summary of clinical and laboratory data from malaria patients
The demographic data from severe P. falciparum malaria patients is documented in Table 1. Significant differences were observed in levels of haematocrit (p = 0.032) and creatinine (p = 0.023) between non-PE and PE groups. No significant difference in age, blood urea nitrogen (BUN), alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, globulin, total bilirubin, direct bilirubin, WBC counts, parasitaemia and days of fever (all p > 0.05) was observed. Clinical adult respiratory distress syndrome (ARDS) and acute kidney injury were two complications that showed significant difference between non-PE and PE groups (Additional file 1: Table S1). The causes of death were attributed mainly to cerebral malaria based on clinical manifestations and histopathological findings. Usually patients died of multiple complications of P. falciparum malaria. No difference in other clinical complications of severe malaria (cerebral malaria, jaundice/hepatic dysfunction, severe anaemia, severe metabolic acidosis, hypoglycaemia, shock and disseminated intravascular coagulopathy) was observed between the two groups (Additional file 1: Table S1).
Histopathological changes in the lungs of P. falciparum malaria patients
Common histopathological changes in the lungs of severe P. falciparum malaria included the presence of septal congestion and alveolar haemorrhage (Fig. 1A), alveolar oedema (Fig. 1B), hyaline membrane formation (Fig. 1C), PRBC sequestration in pulmonary capillaries (Fig. 1D), malarial pigment and an increase in the number of macrophages in alveolar spaces and septal area (interstitial area) (Fig. 1E) (all p < 0.05, compared to control lung, Fig. 1F). Mixed inflammatory cells are occasionally seen within the alveoli. Comparing non-PE and PE groups, significant difference in histological findings were noted in the presence of alveolar haemorrhage, accumulation of malarial pigment and the number of lung macrophages (all p < 0.05). The calculated lung injury score (based on histopathological criteria) was significantly higher in both non-PE and PE group, compared to control (p = 0.001, p = 0.002, respectively). Moreover, lung injury score was significantly higher in PE group (19.22±0.90), compared to non-PE group (12.13±0.09, p = 0.001) (Fig. 2). Other non-lung histopathological findings are listed in Table 2. Acute tubular necrosis was more prevalent in PE group compared to non- PE group (p = 0.038).
Immunohistochemistry study of M1/M2
Expression of CD68
CD68 expressing cells were detected as fine brown color in the cytoplasm of lung macrophages in both the septal area and within the alveoli (Fig 3, Panel A). The number of macrophage positive cells was significantly increased in PE group (49.23±5.16/HPF) compared to the non-PE group (31.22±3.81/HPF, p = 0.016) and the normal control group (6.40±1.39/HPF, p = 0.001). In septal areas, no significant difference in CD68 positive cells was noted between PE (36.64±4.50/HPF), and non-PE groups (24.41±3.40/HPF, p = 0.118). However, both malaria groups showed significantly higher CD68 positive cells compared to compared to the control group (3.58±0.99/HPF, p < 0.05).
Expression of CD40
CD40 was used to detect activated M1 subtype lung macrophages. Positive cells expressed a fine brown color on the cell membrane of macrophages. The relative expression of CD40 was prominent in the group of severe P. falciparum malaria with PE (63.44±1.98%) compared to non-PE (53.22±3.85%, p = 0.018) and the control group (43.59±2.61%, p < 0.01) (Fig. 3, Panel B, Fig. 4A). Macrophages expressing CD40 in the alveolar septal area between PE and non-PE groups showed similar trends as in the lung tissues, as depicted in Fig. 4A.
CD163 was used as a marker for the M2 subtype. Positive cells express a fine brown color in the cytoplasm of macrophages. Lung macrophages in severe P. falciparum malaria patients with PE showed a relative decrease CD163 expression, compared to the control group (p < 0.024). No significant difference in the mean percentage of CD163 positive cells was observed between PE and non-PE groups (p = 0.075), and between non-PE and control groups (p = 0.493). In addition, no significant difference in CD163 expression in the septal area was noted among three groups (p > 0.05) (Fig. 3, Panel C, Fig. 4B).
Correlations between M1/M2 surface markers and histopathological changes
There was a significant positive correlation between the ratio of M1/M2 and alveolar haemorrhage (rs = 0.775, p < 0.001), alveolar oedema (rs = 0.672, p = 0.003), presence of malarial pigments (rs = 0.605, p = 0.010), lung macrophages (rs = 0.630, p = 0.007), WBC infiltration (rs = 0.611, p = 0.009) and acute lung injury score (rs = 0.713, p = 0.001) (Fig. 5A-F).