Loop-Mediated Isothermal Amplification Assay for Identification of Five Human Plasmodium Species in Malaysia.

Yee-Ling Lau,Jenarun Jelip,Rohela Mahmud,Mun-Yik Fong,Meng-Yee Lai

doi:10.4269/ajtmh.15-0569

Abstract

The lack of rapid, affordable, and accurate diagnostic tests represents the primary hurdle affecting malaria surveillance in resource- and expertise-limited areas. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cheap diagnostic method. Five species-specific LAMP assays were developed based on 18S rRNA gene. Sensitivity and specificity of LAMP results were calculated as compared with microscopic examination and nested polymerase chain reaction. LAMP reactions were highly sensitive with the detection limit of one copy for Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae and 10 copies for Plasmodium knowlesi and Plasmodium ovale. LAMP positively detected all human malaria species in all positive samples (N = 134; sensitivity = 100%) within 35 minutes. All negative samples were not amplified by LAMP (N = 67; specificity = 100%). LAMP successfully detected two samples with very low parasitemia. LAMP may offer a rapid, simple, and reliable test for the diagnosis of malaria in areas where malaria is prevalent.

Highlights

Malaria is a devastating disease causing approximately 584,000 deaths in 2013,1 but diagnosis still remains a challenge.[2]
Several Loop-mediated isothermal amplification (LAMP) methods have been developed for malaria diagnosis based on Plasmodium falciparum histidine-rich protein-2 (PfHRP2),8 18S rRNA,[9] Plasmodium berghei sporozoite protein with MACPF related domain (PbSPECT2),[10] beta tubulin (β-tubulin),[11] Plasmodium falciparum gametocyte specific genes (Pfs)[16] and Pfs[25,12] mitochondrial DNA,[13] and apical membrane antigen 1 (AMA-1).[14]
We report the attainment of higher sensitivity of detection of all five human malaria species through the use of LAMP method developed based on Plasmodium small subunit ribosomal RNA (18S rRNA)

Summary

Introduction

Malaria is a devastating disease causing approximately 584,000 deaths in 2013,1 but diagnosis still remains a challenge.[2]. We report the attainment of higher sensitivity of detection of all five human malaria species through the use of LAMP method developed based on Plasmodium small subunit ribosomal RNA (18S rRNA). Results of microscopy, nested PCR, and LAMP assay for detection of all Plasmodium species from patients and healthy donors samples

Results

Conclusion