Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia

Background: Plasmodium knowlesi is the most common cause of malaria in Malaysia. However, microscopic diagnosis is inaccurate and rapid diagnostic tests (RDTs) are insufficiently sensitive. PCR is sensitive and specific but not feasible at a district level. Loop-mediated isothermal amplification (LAMP) shows potential with only basic requirements. A commercially available LAMP assay, the Eiken Loopamp (TM) MALARIA Pan Detection kit, is sensitive for Plasmodium falciparum and Plasmodium vivax, but has not previously been evaluated for P. knowlesi. This study aims to determine the sensitivity of this LAMP assay for detecting P. knowlesi infection. Methods: Study participants included 73 uncomplicated malaria patients with PCR species confirmation: 50 P. knowlesi, 20 P. falciparum and 3 P. vivax. Nineteen malaria-negative, non-endemic area controls were also included. The sensitivity of the Eiken Loopamp (TM) MALARIA Pan Detection kit (Pan LAMP) for detecting each Plasmodium species was evaluated. Sensitivity and specificity of the Eiken Loopamp (TM) MALARIA Pf Detection kit (Pf LAMP) for P. falciparum were also determined. The limit of detection for each LAMP assay was evaluated, with results compared to PCR. All P. knowlesi patients were also tested by CareStart (TM) (Pf/VOM) and OptiMAL-IT (TM) (Pan/Pf) RDTs. Results: The sensitivity of the Pan LAMP assay was 100% for P. knowlesi (95% CI 92.9-100), P. falciparum (95% CI 83.2-100), and P. vivax (95% CI 29.2-100). The Pf LAMP was 100% sensitive and specific for P. falciparum detection, with all P. knowlesi samples having a negative reaction. LAMP sensitivity was superior to both RDTs, with only 10 and 28% of P. knowlesi samples testing positive to CareStart (TM) and OptiMAL-IT (TM), respectively. Limit of detection using the Pan LAMP for both P. knowlesi and P. vivax was 2 parasites/mu L, comparable to PCR. For P. falciparum both the Pan LAMP and Pf LAMP demonstrated a limit of detection of 20 parasites/mu L. Conclusions: The Eiken Loopamp (TM) MALARIA Pan Detection kit is sensitive for detection of P. knowlesi in low parasitaemia clinical infections, as well as P. falciparum and P. vivax. However, a P. knowlesi-specific field assay in a simpler format would assist correct species identification and initiation of optimal treatment for all malaria patients.