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

  • John Lukoye Kedogo
  • Fredrick Eyase
  • Bulimo Wallace
  • George Asudi
  • Kimani Francis
  • Damaris Matoke Muhia
  • Eva Aluvaala
Keywords: Biomeme, Malaria-diagnosis, Smartphone-based, Plasmodium Falciparum, Point-of-Care, Resource-Strained.

Abstract

Malaria presents a diagnostic challenge in most developing countries due to their financial status. In the year 2017, there were approximately 219 million new cases of malaria with more than 435 000 deaths globally. Children(70%) under five years were the most affected. In some African countries where transmission rates were high, a large population was infected but remained asymptomatic. Such patients had developed sufficient immunity to protect them from malarial illness but not from the infection. In these countries, cost and ease of performances are major considerations. Whereas microscopy and other contemporary methods for malaria diagnosis are available, development of an accurate, sensitive and cost-effective rapid diagnostic tool would go a long way in alleviating those challenges.

Objectives : This study tested a portable real time PCR (smartphone-based real-time) assay method tool from Biomeme Inc. in order to assess its ability in providing better diagnostic capability compared to existing methods.

Materials and Methodology: From April 2016 to February 2018 a study was carried out in Western Kenya. Nyando River basin which covers an area of 3517 km2. In five administrative wards; Ahero, Kabura, Kabonyo, East Kano and Awasi. Two and above years old patients who presented with malaria symptoms including axillary temperature of 37.50C or history of fever 48 hours prior to the mentioned ward's health facilities were recruitment. Consent of the 315 respondents including children whose parents or legal guardians signing for them was mandatory. Urine test for the presence of chloroquine, quinine or sulphonamides to confirm they had not taken anti -malaria drugs within the previous month was done. The initial parasitemia had to be between 1,000 to <200,000 parasites per μL of blood. Exempted were patients showing signs of severe anemia and malaria; those with a hemoglobin level of <5 g/d; presence of other diseases that causes febrile conditions; presence of any other Plasmodium species other than falciparum; and patients with a history of adverse events against ACT and sulphonamide drugs.

DNA was extracted using the Biomeme sample preparation kit and compared to that of the Chelex method. A NanoDrop spectrophotometer was used to measure the concentration, purity and turbidity of the DNA obtained using the two extraction methods. Limits of detection of the DNA obtained from Biomeme and Chelex were determined by amplification of two Plasmodium falciparum gene markers, MSP1 and 18S rRNA. Student’s t-test was used to compare the means of the two methods and a Kappa statistic value used to determine the level of agreement. Viral screening was performed before the actual blood collection.

During all surveys, nurses collected ~500μl of blood onto slides as thick and thin smears for microscopy onto filter paper for polymerase chain reaction (PCR) and into a HemoCue for immediate testing of hemoglobin levels (densities). Filter papers were dried at ambient temperature in the field, shipped to KEMRI and stored in plastic bags at -20°C containing silicate as desiccant. The samples were spun (350g, 10 min) and the pellets containing packed red blood cells (RBC), and white blood cells (WBC) were frozen with an equal volume of cryopreservation solution (0.9% NaCl, 4.2% sorbitol and 28% glycerol) and transported in liquid nitrogen container to KEMRI.

Results: The Biomeme sample preparation kit recorded the highest yields and concentrations compared to the Chelex method. Biomeme DNA quantification method gave scores as yield of (33.39mg±8.36) while Chelex had yield of (7.6mg±2.48). The 18S rRNA amplification detection results on Biomeme and Chelex amplicons respectively were as follows; sensitivity (97.44±0.71%, 94.44±0.53%), specificity (50±0.71, 60±0.53%) with Kappa value (0.473, 0.544) while for MSP1, the respective results were as; sensitivity (97.37±0.49%, 91.67±0.43%), specificity (67±0.49%, 60±0.43%) and Kappa value (0.6401, 0.4755) p=0.05.

Evaluating the limits of detection, molecular marker in both gels was of 100 base pairs lane 1 and 2. That was representing positive control (3D7) and negative control (free DNAse water) respectively. It was evident that the amplicons intensities of Biomeme were much higher than those of Chelex. The gene fragment sizes were 200 bp on both gels, while serial dilution did not affect the intensities significantly across each gel using the 18S RNA as the gene marker.

Conclusion: These results suggest that, Biomeme was a rapid and accurate diagnostic technique for malaria that can be used at point of care despite the turbidity ratio which showed it had some chaoatrophic salts. Concentrations and the yields of the DNA depend on the platform method used for the extraction.

Recommendations: Biomeme could be an alternative method for resource-strained environments. It is portable and highly connected via web portal system for the transfer of data from one point to another. DNA extracted for molecular biological research work should be pure, free from contamination and toxicity leading to minimal DNA fragmentation.

Keywords: Biomeme, Malaria-diagnosis, Smartphone-based, Plasmodium Falciparum, Point-of-Care, Resource-Strained.

Published
2020-12-07
Section
Articles

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eISSN: 1022-9272