Summary of clinical and laboratory data from malaria patients
Malarial lung tissues were obtained from 17 patients, 9 cases with PE and 8 cases with non-PE. Six normal lung tissues samples were used as control. No difference in clinical complications of severe malaria (ie. cerebral malaria, acidosis, hypoglycaemia, shock, disseminated intravascular coagulopathy) was observed between the two groups, except for the presence of acute kidney injury (p = 0.03). The demographic data from the severe P. falciparum malaria patients is documented in Table 1. There was a significant difference in the patient haematocrit between non-PE and PE (p = 0.04). No significant difference in age, blood urea nitrogen (BUN), creatinine, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, globulin, total bilirubin, direct bilirubin, WBC counts, and parasitaemia (all p > 0.05) was observed.
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 the PE group (19.22±0.90), compared to non-PE group (12.13±0.09, p = 0.001) (Fig. 2).
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, the number of CD68 positive cells was significantly higher in both the PE group (36.64±4.50/HPF), and the non-PE group (24.41±3.40/HPF), compared to the control group (3.58±0.99/HPF, p < 0.05). In terms of the number of macrophages per alveoli, the number of CD68 positive cells was significantly increased in the PE group (2.09±0.27) when compared to the non-PE group (1.20±0.21, p = 0.025) and the control group (0.89±0.22, p = 0.007).
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 expression of CD40 was prominent in the group of severe P. falciparum malaria with PE (97.88±1.71%) compared to non-PE (72.81±7.17%, p = 0.001) and the control group (19.83±11.81%, p = 0.003) (Fig. 3, Panel B). Macrophages expressing CD40 in the alveolar septal area and within the alveoli show similar trends between PE and non-PE groups, as depicted in Fig. 4A.
Expression CD163
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 and non-PE show an increase in CD163 expression, compared to the control group (p < 0.01). No significant difference in the mean percentage of CD163 positive cells was observed between PE and non-PE groups (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).