Angiogenic and Angiostatic Factors in the Saliva of Malaria Patients (2)

Abstract

Background: The mortality associated with severe malaria remains high even after appropriate treatment with effective anti-malarial drugs. Malaria death is associated with deregulation of host immune responses to inflammatory factors such as C-X-C motif chemokine 10 (CXCL10) and host angiogenic factors such as angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2). Current diagnosis of malaria relies on microscopic detection of the parasites in the blood film. This approach is invasive, increases accidental infections and uncomfortable for some patients. The aim of this study was to investigate biomarkers -CXCL10, Ang-1 and Ang-2 levels in the saliva of malaria patients and compare with plasma levels with regard to their potential use as biomarkers in malaria. This may be useful for further development of highly efficient non-invasive malaria detection methods using saliva. Methods: This was a case control study involving 213 participants: 119 malaria patients and 94 healthy controls, aged 1 -16 years for children and > 16 years for adults. The study was conducted in the Shai-Osudoku District Hospital in Dodowa, Southeastern part of Ghana in the Greater Accra Region. Four millilitres (4ml) of blood and 1.5ml of saliva were collected from each participant into cell preparation tube (CPT) and OMNIgene-Oral tube respectively. The plasma and saliva levels of CXCL10, Ang-1 and Ang-2 of the study participants were measured using enzymelinked immunoassay (ELISA) technique. Complete blood count (CBC) was measured with a Haematology auto analyzer. Plasmodium Lactate Dehydrogenase/Histidine- Rich Protein-2 (pLDH/HRP-2) Antigen Combo Card rapid diagnostic test (RDT) was performed to determine the presence of P. falciparum HRP-2 in participant’s blood Thick and thin blood film slides were prepared, stained with Giemsa solution and examine for the presence of P. falciparum under the microscope. Data were presented as mean ± standard error or median and interquartile range (IQR) where appropriate. Spearman’s rank test was used to find the correlation between plasma and saliva biomarkers in malaria patients. A p-value < 0.05 was considered significant. Results: Of the 213 participants, 119 malaria patients, 44 (37.0%) were male and 75 (63.0%) were female and 94 non- malaria controls, 27 (29%) were male and 67 (71%) were females with ages ranging from 1-78 years, with a median ages of 29 (IQR 22- 35) and 23 (IQR 17-31) years respectively between non-malaria and malaria participants (Median age, p<0.001).There was decreased plasma levels of Ang-1 (p<0.009) and increased plasma levels of CXCL10 (p<0.001) and Ang-2 (p<0.001) in individuals with malaria compared to those without malaria. Similar trends were observed in the saliva samples from study participants. Saliva biomarkers CXCL10, Ang-1 and Ang-2 levels correlated significantly with plasma levels of malaria patients. Finally, Ang-2 was informative when combined with CXCL10 to predict the risk of malaria and could be useful in clinical decision-making. Conclusions: These results provide insight into the use of saliva as a non-invasive diagnostic method and demonstrate that Ang-2 combined with CXCL10 is a promising predictive biomarker in malaria.