In a point of care setting for the diagnosis of malaria, DNA extraction using conventional methods are time-consuming and complicated. Therefore, in this study we aim to utilize a simple nucleic acid extraction method to directly extract DNA from blood. This would in turn reduce the time, cost and equipment needed to perform DNA extraction. This method is then coupled with LAMP assay for rapid diagnosis of malaria. We obtained 77 malaria samples, of which 36 werePlasmodium (P.) knowlesi, 10 P. vivax, 10 P. falciparum, 1 P. malariae and 20 healthy blood samples from district hospitals from Selangor, Kelantan, Negeri Sembilan, Pahang, and Perak, from 2019 to 2021. All malaria samples tested by LAMP or nested PCR were collected prior to antimalarial treatment. All malaria samples collected were confirmed by microscope at the hospital and cross-checked by Medical Laboratory Technician at the District Health Office. The samples were confirmed by microscopic examination and nested PCR as described by Snounou et al[1] and Imwong et al[2]. The LAMP assay and primers were adapted from Lau et al[3]. The extraction method and buffers were adapted from Zou et al[4] with minor modifications. Blood samples of 60 μL, and 240 μL of lysis buffer [800 mM guanidine hydrochloride, 50 mM Tris (pH 8), 0.5% Triton™ X-100, 1% Tween-20, 40 μg/mL Proteinase K] was used. The tube consisting of blood and lysis buffer mixture was constantly inverted for until homogenous. A 6 mm diameter Whatman grade 1 qualitative filter paper was inserted into the tube was mixed constantly for 1 minute. The filter paper was then removed from the blood lysis mixture and washed in 1 mL of washing buffer [10 mM Tris (pH 8.0), 0.1% Tween-20]. The filter paper was then mixed constantly in the washing buffer for 1 minute. Following that, the filter paper was removed and dipped 5 times into the PCR tube consisting of the LAMP reaction before removing the filter paper. The LAMP assay was incubated in a Loopamp Real time turbidimeter LA 500 (Eiken Chemical Co., Ltd., Japan) at 65 ℃ for 60 minutes and inactivated at 80 ℃ for 2 minutes. Limit of detection of the method was performed by usingP. knowlesi strain A1H1 obtained from the Department of Parasitology, Faculty of Medicine, Universiti Malaya. P. knowlesi strain A1H1 culture blood was ten-fold serially diluted to parasitemia of 1%, 0.1%, 0.01%, 0.001% and 0.000 1% respectively with healthy blood, and was tested with the above method in triplicates. The clinical sensitivity and specificity of LAMP assay was determined using microscopy as the reference standard methods. Sensitivity was calculated as (number of true positives)/(number of true positives+number of false negatives), and specificity was calculated as (number of true negatives)/(number of true negatives+number of false positives). The DNA extraction method coupled with LAMP has a detection limit of 0.001% parasitemia (5 parasites/μL of blood) (Figure 1). A highly trained technician using microscopy can reliably detect as few as 50 parasites/μL of blood, while the published limit of detection of laboratory PCR methods is 0.5 to 5 parasites/μL[5]. When conducting the limit of detection, all samples with parasitemia higher than 0.001% was able to amplify successfully, whereas there was a 1/3 positive amplification at 0.000 1% parasitemia. Clinical sensitivity of LAMP was compared with the results from conventional nested PCR and microscopy. A total of 77 samples were diagnosed using both nested PCR and microscopy. Within the 77 samples diagnosed using both nested PCR and microscopy, there wereP. knowlesi (n=36), P. falciparum (n=10), P. vivax (n=10), P. malariae (n=1) and 20 negative Plasmodium samples. Among 36 P. knowlesi, 3 were not detected using the alternative DNA extraction method coupled with LAMP. This method has successfully amplified allP. falciparum (n=10), P. vivax (n=10) and P. malariae (n=1) positive samples. The 20 healthy blood samples did not show any amplification. With this, the DNA extraction method coupled with LAMP showed 94.7% sensitivity and 100% specificity when compared with microscopy.
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