Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis

Abstract

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.

Comparative Diagnosis of Malaria Infections by Microscopy, Nested PCR, and LAMP in Northern Thailand

“…To confirm the specificity of LAMP primers under our laboratory conditions, DNA of Plasmodium ( P. falciparum , P. vivax , and P. berghei ) and DNA from other protozoan parasites ( Trypanosoma cruzi , Trypanosoma brucei gambiense , Cryptosporidium parvum , Toxoplasma gondii ) were subjected to LAMP with Plasmodium genus primer set. The LAMP assay in this study was conducted as reported previously 21 Statistical methods. The results of all three methods, microscopy, nPCR, and LAMP were analyzed with a statistical calculation program called S-plus 6 Software for Windows (Insightful Corp., Seattle, WA).…”

“…All 130 samples collected in Thailand were tested by using five primer sets reported by Han and others, 21 whereby one set amplifies Plasmodium genus DNA and the four species specific for each of four human infecting Plasmodium species. To confirm the specificity of LAMP primers under our laboratory conditions, DNA of Plasmodium ( P. falciparum , P. vivax , and P. berghei ) and DNA from other protozoan parasites ( Trypanosoma cruzi , Trypanosoma brucei gambiense , Cryptosporidium parvum , Toxoplasma gondii ) were subjected to LAMP with Plasmodium genus primer set.…”

“…Species-specific LAMP assay for P. falciparum and LAMP assays for all four species that infect humans have already been developed. 21,23 Although this new technique has not yet been established for routine diagnosis in the field, the first studies on Plasmodium LAMP highly favor this method because LAMP is almost as specific and sensitive as the nested polymerase chain reaction (nPCR) method for Plasmodium -DNA detection in blood. Precipitation of magnesium pyrophosphate as a by-product of the LAMP reaction causes turbidity, which can either be detected visually or by a realtime turbidimeter.…”

“…Because there is a correlation between the threshold time and the initial concentration of DNA, the LAMP method in combination with a real-time turbidimeter can be used as a quantitative method. 21 Recently, LAMP was applied for the identification of Plasmodium -carrying mosquitoes using rodent malaria parasite ( Plasmodium berghei ) and for filarial parasites detection in the mosquito vectors, suggesting LAMP as more reliable and useful for routine diagnosis of vector mosquitoes in regions where vector-borne diseases such as malaria are endemic. 25,26 This work further validates the application of LAMP assay as a useful tool for malaria diagnosis in human blood samples from Northern Thailand and compares the findings with those obtained by microscopic and nPCR diagnostic methods.…”

Paper‐Origami‐Based Multiplexed Malaria Diagnostics from Whole Blood

“…We now integrate all the required steps into a single device to detect Plasmodium falciparum , Plasmodium vivax ,19 and Plasmodium pan directly from a finger‐prick volume of whole blood within an operator‐blinded study (Figure 1). …”