Development Of DNA-Based Assays For Molecular Surveillance And Point-Of-Care Diagnosis Of Non-Falciparum Malaria

Abstract

Background In recent years, the clinical significance of Plasmodium malariae and Plasmodium ovale is increasingly gaining public health attention as the global transmission of falciparum malaria is decreasing. However, the most readily available and cost-effective malaria diagnostic tools, which include microscopy and antigen-based rapid diagnostic tests, lack adequate sensitivity and specificity for accurate surveillance and point-of-care diagnosis of these non-falciparum species. This challenge poses a major setback to global efforts aimed towards malaria control and elimination. In this study, DNA-based assays with improved sensitivity and specificity were developed for species-specific detection of P. malariae and P. ovale. Methods SYBR Green-based real-time quantitative polymerase chain reaction (qPCR) assays were developed for the detection of P. malariae and P. ovale using a new set of primers called cooperative primers. The cooperative primer-based qPCR assays were used in a cross-sectional study to determine the prevalence rates of P. malariae and P. ovale using field samples obtained from three malaria transmission settings in Ghana. Following this, the associations between P. malariae and P. ovale infections and haematological indices were assessed. For point-of-care diagnosis of P. malariae and P. ovale associated malaria, in-house real-time loop-mediated isothermal amplification (RT-LAMP) assays were developed and the diagnostic performance of the RT-LAMP assays were compared to the cooperative primer-based qPCR assays. In addition, label-free DNA-based electrochemical biosensors were developed for ultrasensitive detection of P. malariae and P. ovale.

Results Both the P. malariae and the P. ovale cooperative primer-based qPCR assays had a detection limit of approximately 1.0 parasite/μL, which is at least 10-fold lower than the corresponding conventional primer-based qPCR assays. Using the cooperative primer-based qPCR assays in a cross-sectional study, the prevalence rates of P. malariae and P. ovale infections among the combined study population were 13.3% and 4.8%, respectively. Notably, study participants harbouring mixed infections of P. falciparum with either P. malariae or P. ovale had the greatest risk of developing anaemia. The diagnostic sensitivity and specificity of the in-house RT-LAMP assays were in the range of 86.2% - 97.5% when compared to the cooperative primer-based qPCR assays. Remarkably, both the P. malariae and the P. ovale DNA-based biosensors showed a sensitivity of 100% using purified genomic DNA samples. However, the specificities of the biosensors were 100% and 66.7% for P. malariae and P. ovale, respectively. Conclusion In summary, the results demonstrate that the DNA-based assays described in this study have adequate sensitivity and specificity for the detection of P. malariae and P. ovale in clinical isolates. This study highlights the importance of including detection tools with lower detection limits in the routine surveillance and point-of-care diagnosis of non-falciparum species. The availability of reliable and cost-effective species-specific detection tools for P. malariae and P. ovale will be necessary for comprehensively assessing the effectiveness of malaria interventions and control measures aimed towards global malaria elimination.