Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax

Malaria, caused by the parasite Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene (P. falciparum), 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.


Introduction
Malaria, which is caused by the Plasmodium protozoan and transmitted by mosquitoes, is an epidemic that predominantly occurs in tropical and subtropical regions [1]. Malaria infection has mainly been reported in Africa and several other countries, and more than 40% of the world's population remains at risk from various malarial species [2]. According to the World Health Organization (WHO), 409,000 malaria deaths were reported worldwide in 2019 [3]. Plasmodium species that infect humans include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi [4]. P. falciparum accounts for 99.7% of malaria cases in Africa and causes fatal symptoms. P. vivax induces weaker symptoms than P. falciparum but is known to occur worldwide and has a 75% infection rate in the Americas [3]. Malaria infections commonly cause frequent fevers, anemia, and an enlarged spleen; however, treatments differ for each species [5]. Malaria treatments are largely divided into chloroquine and artemisinin antibiotics [6]. P. vivax and P. ovale are treated using chloroquine antibiotics; P. falciparum, P. malariae, and P. knowlesi are treated with artemisinin antibiotics [7]. These five types of human-affecting Plasmodia are responsible for an infectious disease that can be cured if appropriate treatments are employed. However, if P. falciparum is not detected early, it can lead to severe disease and death [8]. Therefore, it is crucial to diagnose malaria promptly during early stages and distinguish among malaria species [9].
For malaria detection, currently available diagnostic methods include blood smears, the application of a rapid diagnostic kit (RDT), and molecular diagnostics. Blood smear, a representative diagnosis sample for malaria, is simultaneously performed with thickand thin-layer smears, followed by Giemsa staining, in order to identify the presence of Plasmodium species under a microscope [10]. However, blood smears are time-consuming and require trained specialists because the staining process takes 45 min [11] and a trained microscopist consumes 15-30 min for microscopic examination of a single blood slide [11]. Furthermore, with thick-layer smears, it is difficult to identify the type of malaria, and with thin-layer smears, it can be challenging to confirm the diagnosis if the infection density is low [12]. RDT results can be confirmed within 20 min, but the sensitivity is lower than that of other diagnostic methods [13]. Polymerase chain reaction (PCR), a molecular diagnostic method, has the highest sensitivity and specificity among malaria diagnosis methods and can identify Plasmodium, as well as distinguish between species [14]. However, PCR takes approximately 3 h and requires expensive equipment with thermal cycling capabilities and skilled professionals [15]. Thus, several isothermal amplification methods for malaria diagnoses have been proposed as molecular diagnostic methods to overcome these limitations, including loop-mediated isothermal amplification (LAMP) [16], lateral flow recombinase polymerase amplification (LF-RPA) [17], and quantitative nucleic acid sequence-based amplification (QT-NASBA) [18]. Among these isothermal amplification methods, LAMP is the most extensively investigated method for malaria diagnosis [19][20][21][22]. LAMP requires four primers, containing six parts of the target gene sequence and two loop primers, that enhance the performance of the loop structure. This primer set reacts with the target gene to form a loop structure, thus robustly amplifying the target gene at 58-65 • C using Bst polymerase, a displacement DNA polymerase.
To date, a variety of LAMP detection methods have been developed, including colorimetric and fluorescence detection, detection using fluorescent probes, and detection using lateral flow devices [23]. Among these, detection methods using fluorescent probes can be adapted to multiplex LAMP assays. Multiplex LAMP methods that use currently available probes include the methylation-specific LAMP (MS-LAMP), assimilating probe-LAMP, fluorescence of loop primer upon self-enriching-LAMP (FLOS-LAMP), detection of amplification by release of quenching (DARQ), quenching of unincorporated amplification signal reporters (QUASR), and molecular beacon LAMP (MB-LAMP) [24][25][26][27][28][29].
In the present study, we developed a multiplex malaria Pan/Pf/Pv/IC LAMP method to detect all Plasmodium species (Pan), P. falciparum (Pf), P. vivax (Pv), and the internal control (IC; human beta-actin), based on a slightly modified DARQ probe method. The limit of detection (LOD) test for the multiplex malaria Pan/Pf/Pv/IC LAMP assay was performed using four Plasmodium species, namely P. falciparum, P. vivax, P. malariae, and P. ovale. In addition, the performance of the multiplex Pan/Pf/Pv/IC LAMP assay was compared with that of the commercial RealStar ® Malaria S&T PCR Kit 1.0.

Clinical Samples and DNA Extraction
Clinical whole blood samples were collected from patients infected with malarial parasites at the Korea University Guro Hospital from June 2000 to July 2017. All clinical samples were diagnosed by microscopic examination of Giemsa-stained blood smears and in-house PCR tests at the Korea University Guro Hospital. In total, 208 whole blood specimens were used in the present study, including 118 positive specimens of four types of malaria (61 P. falciparum, 54 P. vivax, 2 P. malariae, and 1 P. ovale) and 90 negative specimens. DNA extraction from the 208 whole blood samples was performed using the NX-48 Viral NA Kit, with a Nextractor NX-48 system (Genolution, Seoul, Korea), according to the manufacturer's instructions. DNA was stored at −50 • C. All tests were performed in a blinded manner, with the operator unaware of any previous test results.
The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Korea University Guro Hospital (2017GR0769 (8 March 2017), 2020GR0451 (7 October 2020)).

Primer Design
Before use in the LAMP assays, P. falciparum LAMP primer sets were designed for the lactate dehydrogenase (LDH) gene of P. falciparum and assessed for specificity via a BLAST search of sequences in GenBank (National Center for Biotechnology Information [NCBI], Bethesda, MD, USA). P. falciparum LAMP primers, including two outer primers (forward primer F3 and backward primer B3), two inner primers (forward inner primer FIP and backward inner primer BIP), and two loop primers (forward loop primer LF and backward loop primer LB), were designed using the Primer Explorer software (Version 4; Eiken Chemical Co., Tokyo, Japan). To detect all malaria species and P. vivax, we used malaria Pan and P. vivax LAMP primer sets, designed by Mohon et al. and Chen et al., respectively [30,31]. For the IC, we used the previously designed LAMP primer set for the actin beta gene [32]. A multiplex LAMP assay was designed using two types of additional oligonucleotide sequences at the 5 LB primers for probing and complementary oligonucleotide sequences for quenching. For the multiplex LAMP assay, FAM, HEX, Texas Red, and Cy5 fluorescence tagging probes were used for Malaria Pan, P. falciparum, P. vivax, and IC, respectively. All LAMP primers and probes were synthesized by Macrogen Inc. (Seoul, Korea; Table 1).

Multiplex Malaria Pan/Pf/Pv/IC LAMP Assay
The multiplex malaria Pan/Pf/Pv/IC LAMP assay was performed using a Mmiso DNA amplification kit (M monitor, Daegu, South Korea). The LAMP reaction mixture comprised of 12.5 µL of 2x reaction buffer, 0.625 µL of malaria Pan LAMP primer mix, 0.625 µL of P. falciparum LAMP primer mix, 1.25 µL of P. vivax LAMP primer mix, 0.6 µL of IC LAMP primer mix, 720 nM quencher 1 solution, 216 nM quencher 2 solution, 2 µL of enzyme mix, and 5 µL of sample DNA (final reaction volume: 25 µL). The LAMP primer mix was composed of 4 µM of two outer primers (F3 and B3), 32 µM of two inner primers (FIP and BIP), 10 µM of loopF primer, 6 µM of loopB primer, and 4 µM of loopB probe. The LAMP assay was run on a CFX 96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) at 60 • C for 40 min. In addition, a negative control (human serum RNA and distilled water) was used as the baseline. The positive signal was determined by examining whether the signal was steep or gradual, considering the baseline, as the baseline of the LAMP assay was unstable when compared with quantitative PCR (qPCR) or RT-PCR.

Real-Time Reverse Transcription (RT)-PCR
The RealStar ® Malaria Screen & Type PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) was performed with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's protocol. Thermocycling parameters specified in the kit were as follows: inactivation at 95 • C for 2 min, 45 cycles of denaturation at 95 • C for 15 s, annealing with fluorescence detection at 58 • C for 45 s, and extension at 72 • C for 15 s. The RealStar ® Malaria Screen & Type PCR Kit 1.0 was used with two tubes, one tube to diagnose P. falciparum and P. vivax, as well as the other tube to diagnose P. knowlesi, P. malariae, and P. ovale.

Limit of Detection (LOD) Tests of the Multiplex Malaria Pan/Pf/Pv/IC LAMP Assay
The LOD of the multiplex malaria Pan/Pf/Pv/IC LAMP was determined using pTOP Blunt V2 vectors, including the 18S rRNA partial gene sequences of Plasmodium spp., LDH partial gene sequences of P. falciparum, the 16S rRNA gene partial sequences of P. vivax, and beta-actin partial gene sequence of humans. All vectors were constructed by Macrogen, Inc. The vectors were serially diluted 10-fold, from 1.0 × 10 7 copies/µL to 1.0 × 10 1 copies/µL, to determine the LOD of the multiplex malaria Pan/Pf/Pv/IC LAMP assay. In addition, the LOD of malaria Pan/Pf/Pv/IC LAMP was tested with serially diluted clinical samples (10 −1 to 10 −6 ) infected with P. falciparum, P. vivax, P. malariae, and P. ovale. The LOD of the multiplex malaria Pan/Pf/Pv/IC LAMP for clinical malaria samples was compared with that of the RealStar ® Malaria Screen & Type PCR Kit 1.0.

Limit of Detection (LOD) of the Multiplex Malaria Pan/Pf/Pv/IC LAMP Assay
The analytical sensitivity of the multiplex malaria Pan/Pf/Pv/IC LAMP assay was compared with that of the corresponding monoplex LAMP primer sets against synthetic pan, Pf, Pv, and IC vectors, ranging from 10 7 to 10 1 DNA copies/µL ( Figure 2, Table 2). The monoplex malaria Pan, Pf, Pv, and IC LAMP revealed an LOD of 1 × 10 2 , 1 × 10 2 , 1 × 10 2 , and 1 × 10 3 copies/µL, respectively. In the multiplex malaria Pan/Pf/Pv/IC LAMP assay, the LOD results for the four channels were the same as those for the corresponding monoplex LAMP assays; the Ct values of the multiplex malaria Pan/Pf/Pv/IC LAMP assay were delayed, when compared with those of the corresponding monoplex LAMP assay. Furthermore, the LOD of the LAMP assay was compared with that of monoplex LAMP and commercial RealStar ® Malaria Screen & Type PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) using serially diluted P. falciparum, P. vivax, P. malariae, and P. ovale clinical samples (clinical whole blood DNA sample dilution range of 10 −1 to 10 −6 ; Table 3  The monoplex and multiplex malaria Pan/Pf/Pv/IC LAMP assays were performed with synthetic malaria Pan, P. falciparum, P. vivax, or beta-actin vectors, ranging from 10 7 to 10 1 RNA copies/µL. Colors (red, orange, green, blue, purple, pink, black, and olive) indicate vector copy numbers/µL (1.0 × 10 7 to 1.0 × 10 1 copies/µL) and distilled water (DW; negative control). The LOD test was performed in duplicate. IC, internal control; LAMP, loop-mediated isothermal amplification.  To confirm the clinical performance of the multiplex malaria Pan/Pf/Pv/IC LAMP assay, the assay sensitivity and specificity were compared with those of RealStar ® Malaria Screen & Type PCR Kit 1.0 for P. falciparum (n = 61), P. vivax (n = 54), P. malariae (n = 2), P. ovale (n = 1), and healthy patient (n = 90) whole blood specimens (Table 4). For P. falciparum clinical samples (n = 61), the sensitivities for P. falciparum and IC were 100% and 95.08%, respectively, for the RealStar ® Malaria Screen and Type PCR Kit (1.0). The sensitivity of the multiplex malaria Pan/Pf/Pv/IC LAMP assay was 100% in the malaria Pan channel (FAM), 100% in the Pf channel (HEX), and 75.41% in the IC channel (Cy5). The cross-reactivity of the Pv channel of the multiplex malaria Pan/Pf/Pv/IC LAMP assay was 100%, whereas that of the Pv channel of the RealStar ® Malaria Screen & Type PCR Kit 1.0 was 98.36% against P. falciparum clinical samples (n = 61). For P. vivax clinical samples (n = 54), the sensitivities for P. vivax and IC were 100% with the RealStar ® Malaria Screen and Type PCR Kit 1.0; the sensitivities of the multiplex malaria Pan/Pf/Pv/IC LAMP assay were 100% in the malaria Pan channel (FAM), 100% in the Pv channel (HEX), and 81.48% in the IC channel (Cy5). The Pf channels of the two assays showed no cross-reactivity with the P. vivax clinical samples. For the P. malariae (n = 2) and P. ovale clinical samples (n = 1), the sensitivities and specificities of both assays were 100%.

Discussion
The accurate classification of malaria species during the early stage of diagnosis is valuable for the proper treatment of infected patients and prevention of overexposure to antimalarial drugs, as treatment policies for different species tend to vary, such as artemisinin-based combination therapy (ACT) for P. falciparum and chloroquine for nonfalciparum species [7,33]. Currently, microscopy is the gold standard diagnostic method for malaria (including the classification of malaria species); however, an expert with a high level of training and experience is needed for accurate diagnosis, especially for species identification [34,35].
In the present study, we developed multiplex differential diagnostic tools to classify the malaria Pan, P. falciparum, and P. vivax. Developing multiplex LAMP assays lacking non-specific signals is substantially more difficult than developing multiplex qPCR, as one LAMP assay uses six or seven primer/probe mixtures, and combinations of 2-4 LAMP sets can potentially result in the non-specific amplification of several primers. In particular, in the case of LAMP with the DARQ probe method, the probe sequence corresponding to the binding region of the LAMP product needs to be designed with consideration of the low homology with sequences of other primers and LAMP products, given that it induces non-specific signals by probe-primer or probe-LAMP product interaction. Herein, the multiplex malaria Pan/Pf/Pv/IC LAMP assay was designed by combining a newly designed Pf LAMP primer set with Malaria pan and P. vivax LAMP primer sets, designed by Mohon et al. and Chen et al., respectively [30,31]. This combination of four LAMP primer sets (malaria Pan, Pf, Pv, and IC) exhibited no cross-reactivity with each other, as well as 100% specificity for non-infected samples and distilled water. In the LOD test for P. falciparum and P. ovale, the multiplex malaria Pan/Pf/Pv/IC LAMP showed a LOD 10-fold and 100-fold higher than that of the commercial RealStar ® Malaria S&T PCR Kit 1.0, respectively. For P. vivax and P. malariae, the LOD of the LAMP assay was the same as that obtained with the commercial kit. Notably, the multiplex malaria Pan/Pf/Pv/IC LAMP assay showed a slightly higher LOD than the commercial PCR kit. However, after assessing 208 clinical samples, the multiplex malaria Pan/Pf/Pv/IC LAMP assay revealed sensitivity (100%) and specificity (100%) similar to those detected with the commercial RealStar ® Malaria S&T PCR Kit 1.0 (100%).
As the isothermal amplification method does not require an expensive temperature control device to change the temperature for target gene amplification, the isothermal amplification method has the advantage of easy application for on-site diagnosis, which is difficult to implement in general PCR [15,36]. Unfortunately, the multiplex LAMP assay developed using fluorescence is limited, in terms of field applications [37]. Isothermal amplification, or a portable PCR device for detecting multiple fluorescence signals, is required to utilize the multiplex LAMP assay for point-of-care testing (POCT). Most available field isothermal amplifiers have been developed for single or two fluorescence channels [38]. However, Bio Molecular Systems (BMS, Australia) recently developed a 2 kg portable magnetic induction cycler (Mic) PCR machine with four real-time channels available. Thus, along with the Mic PCR Machine, the multiplex malaria Pan/Pf/Pv/IC LAMP assay, developed in the present study, can be used for POCT for diagnosing malaria species.
In the present study, we developed a multiplex malaria Pan/Pf/Pv/IC LAMP assay, which can classify malaria Pan, P. falciparum, and P. vivax within 40 min. On evaluating 118 malaria-infected clinical samples, the multiplex malaria Pan/Pf/Pv/IC LAMP assay revealed a performance similar to the commercial RealStar ® Malaria Screen & Type PCR Kit 1.0, which is based on the qPCR method. Thus, with portable multi-channel isothermal equipment, such as the Mic PCR Machine, the multiplex malaria Pan/Pf/Pv/IC LAMP assay can be applied in resource-limited settings for point-of-care diagnostics.