Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker

Abstract

Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3′,5,5′-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.

Method for Malaria Diagnosis Based on Extractions of Samples Using Non-Invasive Techniques: An Opportunity for the Nursing Clinical Practice

“…In relation to malaria diagnosis by qPCR, the presence of Plasmodium DNA in other samples besides blood, such as urine and saliva, was recently demonstrated [16][17][18][19]. The recent demonstration of foreign parasitic DNA presence in hair and epidermal keratinocytes of Leishmania-infected animals opened a new suitable scientific field to study the physiology of these epithelial cells during several diseases, acting as a very specialized tissue to sequestrate and eliminate foreign genetic material [20][21][22]36,37].…”

“…Nowadays, in rural areas of developing countries, the accurate diagnosis based on non-invasive samples is very necessary and demanded, especially in cases where sampling must be repeated [15]. Despite blood is the most used sample for the molecular diagnosis, the presence of Plasmodium DNA has been also described in urine and saliva [16][17][18][19]. This fact shows that nowadays there is a great interest in the research of noninvasive procedures for the diagnostic of malaria, using body secretions and the mucosal surface for the detection of parasitic DNA, without harming the patients.…”

Droplet microfluidics: from proof-of-concept to real-world utility?

“…62 In an attempt to simplify the amplification process, alternative isothermal amplification strategies have recently been explored. Such strategies include loop-mediated isothermal amplification (LAMP), 66,67 rolling circle amplification (RCA), 68 recombinase polymerase amplification (RPA) 69 and exponential amplification reaction (EXPAR). 70 Regardless of the specifics of each method, all operate at moderate temperatures (between room temperature and 60 1C) and do not require thermal cycling.…”

Current Trends of Nanobiosensors for Point-of-Care Diagnostics

“…Nearly half of world's population lives in malaria-endemic regions, and more than half a million deaths resulting from malaria and its complications are reported each year [88], making it a significant global health problem [89]. Significant achievements have been realized in malarial therapeutic development.…”

“…However, eradication of malarial infection especially in low income areas has not achieved much success due to lack of early-stage diagnostic tools. Diagnosis of malaria involves identification and quantification of target metabolites (biomarkers) in biological fluids, mainly blood, urine, and saliva [88, 90]. A variety of biomarkers for malaria exist.…”