Development and evaluation of loop-mediated isothermal amplification assays for diagnosis of malaria and sickle cell anaemia

Abstract

Malaria and sickle cell anaemia (SCA) are co-endemic and are among the leading cause of child morbidity and mortality in Uganda. Misdiagnosis of malaria and delayed diagnosis of SCA due to low parasite density and lack of diagnostic facilities at the point of care leads to poor case management. This study developed Loop-mediated isothermal amplification (LAMP) assays for diagnosis of malaria and SCA with the aim of improving the diagnosis of both malaria and SCA at the point of care. The LAMP assay primer sets targeting the Plasmodium falciparum plasmoredoxin (Plrx) gene and the SCA rs334 mutation were designed. The LAMP assays were optimized and their diagnostic performance were evaluated in terms of sensitivity and specificity using blood samples from consented participants. The Plrx based LAMP assay for diagnosis of malaria had a limit of detection of 7 parasites/µL, analytical specificity of 100% and a turnaround time of 30 minutes when DNA is available. The Plrx based LAMP assay had a diagnostic sensitivity of 100% and specificity of 95%, with an almost perfect agreement (κ = 0.929) compared to nested Polymerase chain reaction when performed on 127 blood samples. The SCA rs334 mutation based LAMP assay for diagnosis of SCA had analytical sensitivity of 0.2 ng/µL and failed to accurately differentiate all the rs334 mutations and wild type alleles. The SCA LAMP assay had a turnaround time of 60 minutes, diagnostic sensitivity and specificity of 100% and 64.3% respectively. The Plrx LAMP assay and SCA LAMP assay developed in this study are simple, rapid and require less sophisticated dry heat block and are therefore, good options to be applied at the point of care.