Assessment of Saliva and Urine Performance for Antimalarial Drug Resistance Molecular Markers Study

Abstract

<i>Background</i>: The malaria diagnostic tools developed to date require blood to be taken. However, certain groups in the population are reluctant to take blood samples because of their cultural habits (blood taboo), or because of the fear associated with the trauma of the injection, especially when the sample is taken repeatedly. Saliva and urine, which are not very invasive to collect, have not been widely used for malaria diagnosis. The aim of this study is to assess the performance of saliva and urine in detecting molecular markers of <i>Plasmodium falciparum </i>resistance to antimalarial drugs. <i>Methodology</i>: Blood, urine and saliva samples were collected in three different localities from 94 patients over 2 years of age with microscopically confirmed <i>Plasmodium falciparum </i>uncomplicated malaria. <i>P. falciparum </i>genomic DNA (Deoxyribonucleic acid) was then extracted and amplified using primers specific for the <i>Pfcrt (Plasmodium falciparum Chloroquine Resistance Transporter), Pfdhfr (Plasmodium falciparum dihydrofolate reductase) </i>and <i>PfK13 propeller (Plasmodium falciparum Kelch13 propeller) </i>genes. The amplification products were processed by electrophoresis and analyzed against blood, saliva and urine samples. A multivariate statistical analysis in R programming environment was performed aiming to assess the performance of blood, saliva and urine samples in detecting molecular markers of <i>P. falciparum </i>resistance. <i>Results</i>: Agarose gel electrophoresis of the amplification products of each gene detected the <i>Pfcrt </i>genes at 80.85% (76/94), <i>Pfdhfr </i>at 95.74% (90/94) and <i>PfK13 Propeller </i>at 98.93% (93/94) in blood. In saliva, gene detection levels were 50% (47/94), 69.14% (65/94) and 4.26% (4/94) respectively for the <I>K13</I> propeller, <i>Pfdhfr</i> and <i>Pfcrt</i> genes. Unlike the <i>Pfcrt </i>gene, which was not detected, 45.74% (43/94) and 38.30% (36/94) of <i>PfK13</i> Propeller and <i>pfdhfr</i> genes respectively were detected in urine. Taking blood as the reference biological sample, statistical analysis showed that unlike urine, saliva exhibited a detection performance for molecular markers of antimalarial drug resistance (<i>pfcrt,</i> <i>pfdhfr</i>, <i>pfK13</i> propeller) close to that of blood (p < 0.05). The performance of saliva and urine was also assessed on the basis of the detection of the molecular markers <i>pfdhfr</i>, <i>pfcrt </i>and <i>pfK13 </i>using ROC (<i>receiver operational characteristic</i>) analysis. The data revealed a high sensitivity of saliva compared with urine in the detection of the <i>pfdhfr</i>, <i>pfcrt </i>and <i>pfK13 propeller </i>genes. <i>Conclusion</i>: The levels of detection of molecular markers of antimalarial drug resistance studied in saliva are close to those in blood. Saliva is a high-performance biological product that could potentially be used as an alternative non-invasive sample for the study of molecular markers of Plasmodium falciparum resistance to antimalarial drugs.