Association of intraleukocytic malaria pigment with disease severity in children with plasmodium falciparum Malaria
Shimaponda, Nzooma Munkwangu
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In malaria diagnosis, peripheral parasitaemia is used as an indicator of malaria severity. However, this does not quantify central sequestration, which is important in the pathogenesis of severe malaria. Intraleukocytic malaria pigment or haemozoin, recognizable within the cytoplasm of phagocytic cells by light microscopy, may represent a peripheral marker for parasite biomass. This study evaluated the association of intraleukocytic haemozoin with the severity of Plasmodium falciparum malaria in Zambian children. The study sought to determine peripheral Plasmodium falciparum malaria parasite densities in blood slides of children with parasitaemia, determine malaria severity, assess presence and quantities of pigment-laden leukocytes and evaluate the association of intraleukocytic malaria pigment with malaria severity. A sample size of 204 children divided into 37 severe malaria, 80 uncomplicated malaria and 87 controls were analyzed prospectively for presence of intraleukocytic haemozoin. Previous studies on the association of intraleukocytic malaria pigment with disease severity and other markers of malaria disease severity were reviewed. The study was conducted at the University Teaching Hospital and Chilenje Health Center in Lusaka, Mpongwe District Hospital on the Copperbelt and Mpulungu Health Center in the Northern Province of Zambia from December 2006 to May 2007. This was a hospital based case-control study. Cases were classified as severe or uncomplicated malaria based on the modified criteria put forth by the World Health Organization. Patients whose blood slides were negative for malaria parasites and were without severe disease were enrolled into the control category. Severity of malaria was determined based on haemoglobin concentrations and coma scores. Three blood slides of thick and thin films were prepared at enrollment of each patient prior to therapy. The thick blood smears were dehaemoglobinized and stained with 10% Giemsa at pH 7.2 for 15 minutes. Peripheral parasite density was determined from the thick films and calculations were based on the number of asexual forms/mm3 per 200 leukocytes and later converted to asexual forms per ul. Thin smears were fixed in absolute methanol for one minute and then stained at pH 6.8 using the MayGrunwald-Giemsa stain method; for 15 minutes in Maygrunwald stain, 15 minutes in Giemsa stain and two minutes in Sorensen's phosphate buffer. Differential counts were determined manually by two expert microscopists. Malaria pigment was detected on thin films by counting 500 leucocytes and determining the proportions of pigment-laden monocytes, lymphocytes and neutrophils. The study observes that the rate of severe malaria is significantly higher in children under five years than in children above five years of age due to the less developed immunity at that stage in life. Secondly, that the presence of neutrophilic, lymphocytic and monocytic malaria pigment is strongly associated with severe and not uncomplicated malaria. Thirdly, that there is a significant association between pigment-laden neutrophils, lymphocytes and monocytes with severe anaemia and pigment-laden neutrophils with coma and with coma/severe anaemia. Fourthly, our study concludes that high parasitaemia (> 250,000 asexual forms per ul) is not associated with pigment-laden leukocytes and neither is it associated with severe malaria. Our data shows justification to reject the null hypothesis that the presence of intraleukocytic pigment is not associated with malaria severity. This study validates intra neutrophilic and monocytic malaria pigments as markers for disease severity, and demonstrates that only pigment-laden neutrophils are associated with coma in children with severe malaria. The evidence of this diagnostic indicator may provide a basis to stratify patients for appropriate treatment and medical attention according to severity of disease and we recommend that absolute pigment-laden neutrophil count of > 469189 per microlitre may be considered as an indicator for severe malaria.