Loop-mediated isothermal amplification (LAMP), the novel method developed by Notomi et al , is able to amplify DNA with great efficacy as its detection limit for DNA in the reaction mixture is as low as six copies, within an hour under isothermal conditions (65°C). LAMP is also an easy, convenient and cost-saving method, which only requires simple laboratory apparatus such as water bath or heating blocks to perform the test. Therefore, it has been widely applied as a diagnostic tool for several viral, bacterial, and parasitic diseases [7–9]. Han et al had reported a species-specific LAMP method for the diagnosis of four well-known human malaria Plasmodium species . This study is the first report whereby LAMP assay is used for the diagnosis of the fifth human malaria P. knowlesi infection in human blood samples. In this study, P. knowlesi DNA was successfully amplified within an hour at 65°C by using LAMP primers that target the AMA gene of P. knowlesi.
Previous studies have indicated that LAMP assay has a higher sensitivity compared to nested PCR in detection of parasites, such as Trypanosoma spp. , Babesia spp. , and T. gondii . The capability of LAMP assay to detect a single microfilaria in samples, as demonstrated by Aonuma et al, clearly shows that the method is highly sensitive . In order to evaluate the sensitivity of LAMP assay for the detection of P. knowlesi in human samples, the LAMP assay was compared with conventional nested PCR, which targeted on P. knowlesi SSU gene and microscopic examination. Under microscopy, the early trophozoites of P. knowlesi morphologically resembled those of P. falciparum, while the late and mature trophozoites, schizonts and gametocytes of P. knowlesi in human infections were generally indistinguishable from those of P. malariae. Hence, the lack of distinguishable morphological features of P. knowlesi makes it extremely difficult to identify P. knowlesi infections by microscopy alone and laboratory misdiagnosis of P. knowlesi as P. malariae is inevitable .
In this study, 13 patient samples which were suspected to be either P. knowlesi/P. malariae or P. knowlesi/P. falciparum infections and were unable to be determined solely by microscopic examination, showed positive for P. knowlesi via LAMP assay, while P. knowlesi nested PCR was unable to detect one of these cases (12 positive as P. knowlesi infections among the 13 samples). The parasitaemia level of that particular case was found to be very low (< 0.01%). On the other hand, there were two cases whereby microscopic examination showed it to be P. knowlesi/P. malariae or P. knowlesi/P. falciparum infection but both LAMP and nested PCR showed negative for P. knowlesi. This might be due to misidentification by using microscopic examination as it requires training, skills and experience. These results showed that LAMP method which employs four primers and targets six distinct sequences on the P. knowlesi DNA could be used as a molecular confirmatory test of P. knowlesi infection instead of microscopic examination which is unable to differentiate the species among P. knowlesi, P. malariae and P. falciparum. Furthermore, it is more sensitive than nested PCR and the detection limit of LAMP assays is also lower relative to nested PCR.
Besides sensitivity, the high specificity of LAMP assay was demonstrated by screening genomic DNAs other Plasmodium species (P. falciparum, P. simium, P. cynomolgi, Plasmodium fragile, P. brasilianum, Plasmodium inui and Plasmodium simiovale), and extracted DNA from blood samples of other Plasmodium malaria and healthy donors. Results showed that LAMP assay which was specifically designed for P. knowlesi did not amplify DNAs of other Plasmodium spp. and no cross-reactivity occurred with all the negative controls. The result indicates that by using LAMP assays, P. knowlesi can be efficiently distinguished from other malaria parasites.
The simplicity and high efficiency of LAMP to amplify DNA under isothermal conditions within an hour suggests that LAMP could be a potential alternative for detection of P. knowlesi in samples compared to nested PCR, which requires an expensive PCR machine and is time-consuming. Another advantage of using LAMP assay is due to its turbidity-based detection of the positive reaction. The positive and negative amplifications could be distinguished by observing the turbidity of reactions with the naked eye, without using gel electrophoresis and therefore the result can be analysed within a few minutes . Another way to analyse the LAMP result is by adding a DNA intercalating dye, SYBR green I to the end-products and visualizing it under UV light. The turbidity can be better observed under UV light. Besides that, the amplicon sequence was confirmed by sequence analysis to eliminate false positive and contaminated reactions.
There are however, limitations to this method. For visualization of results under UV light, the product tubes need to be opened for the dye to be added and this might lead to cross-contamination. This limitation can be overcome by practicing sterile pipetting techniques or using LAMP master mix, which contains pre-added fluorescence dye. Another drawback is the size of target DNA. The efficiency of LAMP is affected by the size of target DNA as the strand displacement DNA synthesis step limits the rate or amplification. The size of target DNA has to be lesser than 300 base pair in order to obtain satisfying results . Hence, target DNA, which is larger than 500 bp is not suitable to be used as it will lead to poor amplification.