Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria

To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings. Plasmodium vivax-specific consensus repeat sequence (CRS)-based and Plasmodium falciparum-specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp (TM) Malaria Detection (Pan/Pf, Pv) kit was investigated using nested PCR confirmed mono- and co-infections of P. vivax and P. falciparum positive (n = 149) and negative (n = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min (limit of detection, LOD 0.7 parasites/mu L) and Pf LAMP assay in 30 min (LOD 2 parasites/mu L). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% (95% CI, 95.96-100% and 89.85-100%, respectively). The Loopamp (TM) Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas Loopamp (TM) Pv showed a sensitivity of 98.36% (95% CI, 91.28-99.71%) and specificity of 100% (95% CI, 87.54-100%). The developed two-tube LAMP assay is highly sensitive (LOD <= 2 parasite/mu L), demonstrating comparable results with the commercial Loopamp (TM) Malaria Detection (Pf/pan) kit, and was superior in detecting the P. vivax co-infection that remained undetected by the Loopamp (TM) Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both P. falciparum and P. vivax malaria.

Automated summary

Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are crucial for strengthened surveillance in low-resource settings. The developed two-tube LAMP assay demonstrated high sensitivity and specificity for detecting P. vivax and P. falciparum infections, with comparable results to commercial kits and superior performance in detecting co-infections.