Validation of a Biomeme smartphone Based- DNA Real-Time PCR Assay for Diagnosis of Human Malaria at the Point-of-Care

Abstract

Malaria poses a diagnostic challenge in the majority of developing countries due to financial constraints. In 2017, there were approximately 219 million new cases of malaria, with over 435 000 deaths worldwide. Children under the age of five were the most affected, accounting for 70% of all cases. In some African countries with high transmission rates, a large proportion of the population is infected but asymptomatic, and the patients have developed enough immunity to protect them from malaria disease but not from infection. Cost and ease of diagnostic performance are major considerations in these countries. While microscopy and other modern malaria diagnosis methods are available, the development of an accurate, sensitive, and cost- effective rapid diagnostic tool would go a long way toward alleviating those challenges. This study evaluated the ability of a portable real-time PCR (smartphone- based real-time) assay tool from Biomeme Inc. to provide better diagnostic capability than existing methods. The study was conducted in Western Kenya from April 2016 to February 2018. Patients aged two and above who presented with malaria symptoms, such as an axillary temperature of 37.5°C or a history of fever 48 hours prior to admission to the health facilities, were recruited. The consent of the 315 respondents, including children who had their parents or legal guardians sign for them, was required. The initial parasitemia had to be in the range of 1,000 to 200,000 parasites per μL of blood. Patients with severe anemia and malaria were excluded, as were those with a hemoglobin level of 5 g/d, the presence of other diseases that cause febrile conditions, the presence of any other Plasmodium species other than falciparum, and patients with a history of adverse events with ACT and sulphonamide drugs. The Biomeme sample preparation kit was used to extract DNA, which was then compared to the Chelex method. The concentration, purity, and turbidity of the DNA obtained using the two extraction methods were measured using a NanoDrop spectrophotometer. Amplification of two Plasmodium falciparum gene markers, MSP1 and 18S rRNA, was used to determine the detection limits of the DNA obtained from Biomeme and Chelex. The Student's t-test was used to compare the means of the two methods, and the level of agreement was determined using a Kappa statistic value. The results of parasitemia densities effect on heamoglobin concentration showed that, the higher the parasitemia density the lower the heamoglobin concentration were. The age factor was also an influential indicator given that; the elderly were having comparatively lower heamoglon concentration with corresponding increase of parasitemia as compared to young ones. When compared to the Chelex method, the Biomeme sample preparation kit produced the highest yields and concentrations of DNA. Thus, the Biomeme yielded a score of (33.39mg±8.36), whereas Chelex yielded a score of (7.6mg±2.48). The 18S rRNA amplification detection results on Biomeme and Chelex amplicons were as follows: sensitivity (97.44±0.71 percent, 94.44±0.53 percent), specificity (50±0.71, 60±0.53 percent), and Kappa value (0.473, 0.544) respectively. MSP1 amplifications had the following results: sensitivity (97.370.49 percent, 91.67±0.43 percent), specificity (67±0.49 percent), and Kappa value (0.6401, 0.4755) respectively with p˂0.05. On the other hand, the genetic diversity analysis showed that MSP1 and Biomeme amplicon had higher number of strains as compared the Chelex ones. In addition, 18S rRNA had lesser number of the predictive strains in comparison to MSP1 while using both DNA amplicons from Biomeme and Chelex. The amplicon intensities of Biomeme were clearly higher than those of Chelex. Biomeme real-time PCR had relatively lower CT values as compared ABI 7500 real-time PCR. In this project study, it was incident that the method of DNA extraction, genomic marker and the platform of amplification had a considerable impact on the result. Despite the turbidity ratio, which indicated that it contained some chaoatrophic salts, these findings suggest that Biomeme was a rapid and accurate diagnostic technique for malaria that can be used at the point of care.