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Loop-mediated isothermal amplification (LAMP) is a novel method that rapidly amplifies target DNA with high specificity under isothermal conditions. It has been applied as a diagnostic tool for several infectious diseases, including viral, bacterial, and parasitic diseases. In the present study, we developed a LAMP method for the molecular diagnosis of Plasmodium knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability. We designed three sets of PkLAMP primers for the species-specific β-tubulin gene. The primer sets for PkLAMP specifically amplified the autologous DNA extracts of P. knowlesi, and the sensitivity of the test was 100-fold that of single-PCR assay. These results indicate that our PkLAMP method can be used to efficiently distinguish between P. knowlesi and other malaria parasites. To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR (nPCR) method and microscopic examination were made with blood samples from two experimentally infected monkeys. These studies showed that P. knowlesi infection can be identified much earlier with PkLAMP than with nPCR and microscopy. Moreover, the detection performance of PkLAMP using whole blood as the template was identical to that of PkLAMP when genomic DNA extracts were used. These results suggest that the PkLAMP method is a promising tool for molecular diagnosis of P. knowlesi infection in areas of endemicity.
Naturally acquired human infections with a macaque malaria parasite, Plasmodium knowlesi, have now been referred to as the fifth human malaria (4, 17). In fact, recent studies have shown that naturally occurring P. knowlesi malaria cases are not rare and are widely distributed in Southeast Asia, particularly in forested areas inhabited by the natural macaque host and vectors such as the Anopheles leucophyrus group (4, 5, 16).
Until recently, numerous cases of P. knowlesi infections in humans may have been misdiagnosed as ordinary Plasmodium malariae malaria (4, 5, 16), since the morphological characteristics of the blood stages of P. knowlesi parasites are similar to those of P. malariae, and it can be easily misidentified as P. malariae on microscopic examination (16). Moreover, our recent study showed that some commercial rapid malaria diagnostic tests based on the detection of parasite lactate dehydrogenase enzyme (pLDH) are unable to distinguish between human malaria parasites and P. knowlesi, since certain antibodies to pLDH that were thought to be specific for Plasmodium falciparum and Plasmodium vivax also bind to P. knowlesi (9). Although the development of a PCR diagnostic method has been essential to solving these problems of misdiagnosis, PCR assays are not a simple method of detection and are not a viable option for routine diagnosis.
Loop-mediated isothermal amplification (LAMP) has been developed as a novel method to amplify DNA with high specificity and simplicity (13). It consists simply of incubating a mixture of the target gene, four or six different primers, Bst DNA polymerase, and substrates. The significant advantages of the LAMP method are (i) high amplification efficiency under isothermal conditions (63 to 65°C) and (ii) visual judgment based on the turbidity or fluorescence of the reaction mixture, which is kept in the reaction tube (10, 12). LAMP has thus emerged as a powerful tool to facilitate genetic testing for the rapid diagnosis of several infectious diseases, including viral, bacterial, and parasitic diseases (8, 11). Although the detection performances of LAMP for four human malaria parasites have been assessed in clinical and epidemiological settings, the LAMP method has not yet been evaluated for the diagnosis of P. knowlesi infection (3, 7, 14). In the present study, we developed a LAMP method for diagnosis of P. knowlesi infection (PkLAMP) and evaluated its sensitivity, specificity, and clinical applicability using blood samples obtained from experimentally P. knowlesi-infected monkeys.
The LAMP method requires a set of four specific primers: a forward inner primer (FIP), a backward inner primer (BIP), and two outer primers (F3 and B3), which recognize a total of six distinct nucleotide sequences (B1, B2, B3, F1, F2, and F3) on the target gene (10, 12, 13). Since it has been demonstrated that additional loop primers increase the amplification efficiency, loop primers for each target gene were also synthesized. The specific primers for P. knowlesi were designed against species-specific β-tubulin gene sequences (GenBank accession number AY639984) (Fig. (Fig.1A).1A). For easy confirmation of the amplified sequences, we modified the FIP and BIP by inserting a restriction enzyme (EcoRI) cleavage site between the F1 complementary sequence and F2 and between the B1 complementary sequence and B2, respectively, as shown in Fig. Fig.1B1B.
The PkLAMP reaction was performed as described previously (10, 12, 13). Briefly, the reaction was performed with 25 μl of a mixture containing 1 μl of the extracted DNA template, 40 pmol each of the FIP and BIP, 5 pmol each of the F3 and B3 primers, 20 pmol each of the forward loop primer (FLP) and backward loop primer (BLP), and 1 μl of fluorescent detection reagent (Eiken Chemical Co., Ltd., Tokyo, Japan) with Loopamp DNA amplification kit (Eiken Chemical Co., Ltd., Tokyo, Japan). The PkLAMP reaction was performed as described above with each of the specific primers. In a conventional heat block, the mixture was incubated at 66°C (temperatures of 47 to 72°C were also tested) for 60 min, and the reaction was then terminated by heating the mixture at 80°C for 5 min. For the initial validation study, PkLAMP was confirmed with real-time monitoring of the increase of turbidity using a Loopamp real-time turbidimeter (LA-200; Teramecs, Kyoto, Japan). To confirm the amplified DNA products of each parasite, 1 μg/μl of the product was digested with the EcoRI at 37°C for 1 h. The nontreated and EcoRI-digested LAMP products were subjected to electrophoresis on a 2% agarose gel and then visualized under UV light after staining with ethidium bromide (Sigma). Digested LAMP DNA products were purified after 2% agarose gel electrophoresis and then cloned into a pCRII cloning vector using a TA cloning kit (Invitrogen, Carlsbad, CA). The nucleotide sequences of inserts were determined using a Big Dye Terminator kit (Applied Biosystems Japan, Ltd.) with an automated DNA sequencer (ABI Prism 3100 genetic analyzer; Applied Biosystems Japan, Ltd.). The Genetyx 7 package (Software Development Co., Ltd., Tokyo, Japan) was used to align the determined sequences. For the challenge infections, the amplified products in the reaction tube were directly detected with the naked eye using Loopamp fluorescent detection reagent (Eiken Chemical Co., Ltd.) according to the manufacturer's instructions.
Specificity of the PkLAMP primers was tested using genomic DNAs (gDNAs) of various Plasmodium species in a gel electrophoresis and fluorescent analysis. The gDNAs of P. falciparum, P. vivax, P. malariae, and P. ovale were kindly provided by Takefumi Tsuboi of Ehime University of Japan. Blood samples infected with P. inui, P. simiovale, P. fieldi, P. fragile, P. hylobati, and P. gonderi were obtained from American Type Culture Collection (ATCC), and gDNAs of these parasites were extracted from frozen infected blood with a QIAamp DNA blood mini kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. P. coatneyi- and P. cynomolgi-infected blood samples were obtained from experimentally infected monkeys and were subjected to DNA extraction with the QIAamp DNA blood mini kit. These purified DNA samples were used as templates for the subsequent PkLAMP and single-PCR assays. As a negative control, DNA extracted from normal monkey blood was prepared as described above.
For sensitivity testing, the PkLAMP reaction was tested using 10-fold serial dilutions of plasmid DNA containing the target sequence by cloning from P. knowlesi H strain genomic DNA and compared against results of the single-PCR assay using F3 and B3 primers. PCR amplification was performed in 25 μl of a mixture containing 1 μl of the extracted DNA template, 50 pmol of each primer, 200 μM each deoxynucleoside triphosphate (dNTP), and 1.25 U of Taq Gold DNA polymerase (Applied Biosystems, Foster City, CA) in a PCR buffer (Applied Biosystems). The reaction was performed for 35 cycles under the following conditions: 10 min at 95°C to activate the Taq Gold DNA polymerase, 1 min of denaturation at 94°C, 1 min of annealing at 60°C, 1 min of extension at 72°C, and 10 min of final extension at 72°C in a Gene Amp PCR system 9700 (Applied Biosystems). The PCR products were subjected to agarose gel electrophoresis and then visualized as described above.
PkLAMP was evaluated for fluorescence detection of P. knowlesi target DNA using blood samples obtained from experimentally P. knowlesi-infected monkeys. Two monkeys, J58 (male) and J64 (male), which were 3-year-old Japanese macaques (Macaca fuscata) weighing 4.2 kg and 4.7 kg, respectively, were used in this experiment. Both monkeys were second-generation offspring bred in captivity. The investigators adhered to the Guidelines for the Use of Experimental Animals authorized by the Japanese Association for Laboratory Animal Science. Monkey J58 was inoculated intravenously with 1 × 108 fresh P. knowlesi H strain (ATCC 30158) parasitized red blood cells (PRBCs) obtained from another infected Japanese macaque. Monkey J64 was inoculated intravenously with frozen P. knowlesi Hackeri strain (ATCC 30153)-infected blood obtained from the ATCC. After infection, Giemsa-stained thin blood films were prepared daily from peripheral blood obtained by ear prick, and parasitemia in the infected monkeys was monitored by microscopic examination. Heparinized blood samples for PkLAMP assay were obtained daily from the infected monkeys during the course of infection. The infected blood samples were subjected to DNA extraction with a QIAamp DNA blood mini kit (Qiagen) as described above. The DNA extracts and whole blood samples were frozen at −80°C until use.
We compared the sensitivities of PkLAMP and conventional nested PCR (nPCR) assays using DNA extract of P. knowlesi and whole blood obtained from two infected monkeys during the course of infection. The nPCR assay, based on the Plasmodium DNA sequence of the small-subunit (SSU) rRNA gene, was performed according to a standard protocol as described previously (15). The nest 1 reaction was carried out in a 50-μl reaction mixture containing 2× PCR master mix (AmpliTaq Gold PCR master mix; Applied Biosystems), 250 nM each primer (rPLU1 and rPLU5) (15), and 2 μl of DNA template. The reaction mixture for nest 1 PCR amplification was placed in a thermal cycler (TP600; Takara Bio Inc., Shiga, Japan) at 95°C for 5 min for initial denaturation. This was followed by 40 cycles of 94°C for 30 s, 55°C for 60 s, and 72°C for 120 s for amplification and then 72°C for 10 min for final extension. Nest 2 PCR amplification was performed in a 20-μl reaction mixture containing 2× PCR master mix (Applied Biosystems), 250 nM each primer (Pmk8 and Pmkr9) (16), and 2 μl of the nest 1 PCR products used as DNA templates. the reaction mixture for nest 2 PCR amplification was placed in a thermal cycler (TP600) at 95°C for 5 min for initial denaturation. This was followed by 40 cycles of 94°C for 30 s, 60°C for 60 s, and 72°C for 60 s for amplification and then 72°C for 10 min for final extension. Nest 2 PCR products were electrophoresed separately on a 2% agarose gel and illuminated with UV light.
The specificity of the PkLAMP primers was investigated by using various Plasmodium gDNAs as templates for PkLAMP. As shown in Fig. Fig.2A,2A, a typical ladder pattern was detected in P. knowlesi DNA (lane 1) but not in the DNAs of other Plasmodium species. Moreover, fluorescent detection was also specifically obtained in the reaction tube including gDNA of P. knowlesi, as shown in Fig. Fig.2B.2B. The sizes of the PkLAMP fragments digested by EcoRI were identical to the predicted sizes for the parasite (data not shown). To evaluate the accuracy and robustness of the LAMP method, the PkLAMP reaction was carried out in a water bath at 47 to 72°C separately. Positive ladder patterns were observed at 48 to 71°C and strongly at 56 to 70°C. These findings demonstrated that a set of species-specific primers was highly specific for the detection of the corresponding parasite in PkLAMP. To confirm the nucleotide sequences of the LAMP products, the amplified and digested DNA products were purified from the positive controls and cloned into a vector. The determined sequences of the DNA fragments were completely identical to the reported ones (data not shown) (P. knowlesi, accession no. AY639984).
To examine the sensitivity of PkLAMP, three PkLAMP detection methods were compared with conventional single PCR using two outer primers, F3 and B3, for the detection of P. knowlesi β-tubulin gene. As shown in Fig. Fig.3A,3A, amplification by real-time PkLAMP was obtained in reaction tubes containing from 108 to 102 copies/μl of the DNA template in a 60-min reaction with a turbidity assay. On gel electrophoresis analysis, the amplified products also showed ladder-like patterns from 108 to 102 copies/μl (Fig. (Fig.3B).3B). The amplified products in these positive reaction tubes were also visually detectable using the Loopamp fluorescent detection reagent, as shown in Fig. Fig.3C.3C. In contrast, the limit of detection for PCR using the F3 and B3 primers was 108 to 104 copies/μl (Fig. (Fig.3D).3D). Therefore, it appeared that the sensitivity of the PkLAMP, regardless of the detection method, was 100-fold higher than that of the single-PCR assay.
The course of infection of Macaca monkeys experimentally infected with P. knowlesi was monitored by PkLAMP and nPCR for detecting parasite DNA (Table (Table1).1). Both monkeys infected with P. knowlesi developed a fulminating acute infection, and they finally became lethargic and severely withdrawn just before autopsy. In monkey J58 inoculated with fresh PRBCs of P. knowlesi strain H, the parasites in the peripheral blood were first detected by microscopy on day 1; parasite densities then increased to around 10% within 3 days after infection. P. knowlesi DNA could be detected by PkLAMP as well as nPCR assay on all days during the course of infection (Table (Table1).1). In monkey J64 inoculated with frozen PRBCs of P. knowlesi strain Hackeri, the parasites were first detected by microscopy on day 6; parasite densities then increased sharply to around 58% within 9 days after infection. P. knowlesi DNA could be detected by PkLAMP throughout the course of infection, while the earliest detection of parasite DNA by the nPCR assay was on the third day after infection (Table (Table11).
We also compared the amplification efficiencies of PkLAMP and nPCR using frozen whole blood as a template. As shown in Table Table1,1, PkLAMP could amplify the target from whole blood with an efficiency similar to that with DNA extracts throughout the course of infection. These results clearly indicate that PkLAMP could detect even the target DNA from nonpurified whole blood. In contrast, nPCR assay using whole blood from J58 and J64 could amplify parasite DNA only on day 3 and day 9, respectively, when parasite densities were markedly increased in the blood (Table (Table11).
The diagnosis of malaria at regional clinics in areas of endemicity has been performed mainly by microscopic examination of blood smears because of its ease and rapid application. However, the morphology of the asexual stages of the zoonotic simian Plasmodium parasites substantially resembles that of human parasites, particularly on thick blood films, and laboratory technicians are trained to recognize only the four species of human parasites (16). In fact, numerous human cases of P. knowlesi infection have been misdiagnosed by microscopy as P. malariae due to their morphological similarities (4, 5, 16). The application of DNA amplification to the diagnosis of malaria can solve these problems. Amplification of parasite DNA using a specific PCR has been applied to various Plasmodium species, including four human malarial parasites and P. knowlesi (5, 15, 16). However, despite the excellent specificity and sensitivity of PCR and real-time PCR, these methods require complicated procedures and sophisticated instrumentation such as a thermal cycler, and they are often impractical under conditions requiring field diagnosis. In this regard, the LAMP method has the advantages of simplicity, specificity, and sensitivity compared to other molecular diagnostic methods. Thus, the LAMP method is a promising candidate for wide use in regional clinics and under field conditions.
In the present study, we successfully developed a LAMP method for detecting P. knowlesi infection, using a primer set that targets the β-tubulin genes of parasites. The specificity of the primers was evaluated using nine species of simian malaria parasites and four species of human malaria parasites. The results showed that the primer set for PkLAMP amplified only the autologous DNA samples of P. knowlesi in typical ladder bands. In contrast, no ladder bands were obtained from any other control. These findings indicate that this primer set is specific for P. knowlesi and can be used to examine for P. knowlesi malaria as well as to distinguish between it and other types of malaria. The sensitivity of the test was evaluated, and the results showed that PkLAMP was 100-fold more sensitive than single-PCR assay using the F3 and B3 primers. Moreover, the present study showed that an isothermal reaction time of 1 h was enough to amplify 109 copies of the target DNA in reaction tubes containing from 108 to 102 copies/μl of the DNA template and that results could be easily judged by visual inspection of the turbidity or fluorescence of the reaction mixture (10, 13). These results suggest that the PkLAMP assay is reliable and useful for the diagnosis of P. knowlesi malaria.
To evaluate the feasibility of using in vivo materials, comparisons of PkLAMP and the conventional nested PCR method and microscopic examination were made with blood samples from two infected monkeys. These studies validated PkLAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early and advanced infections of P. knowlesi. Early species identification in the diagnosis of malaria is very important in preventing disease progression. In particular, early identification of P. knowlesi infection is essential, since the unique 24-h asexual replication cycle among human and simian malaria parasites can rapidly result in high levels of parasitemia with a fatal outcome in humans (4, 5). Although nPCR and sequencing have been applied to species identification for malaria diagnosis, a more rapid diagnostic test such as PkLAMP would be a convenient and powerful tool for enabling the delivery of prompt and adequate medical treatment.
The present study also assessed the detection performance of PkLAMP with different DNA template preparations, including frozen whole blood or genomic DNA extracts. The detection efficiency of PkLAMP using whole blood was identical to that of PkLAMP when gDNA extracts were used as the template. However, the detection performance of nPCR using the whole-blood templates was quite poor. It appears that this is due to blood components, such as myoglobin, hem-blood protein complexes, and immunoglobulin G, that inactivate the Taq DNA polymerase used in standard PCR (1). In contrast, such inhibitors do not affect the Bst polymerase used in LAMP (6). According to previous reports, the specificity and sensitivity of detection appear to be unaffected by LAMP processing conditions or sample type, including whole blood, filter paper- or card-processed blood, serum, sputum, and crudely processed tissue samples (8). Furthermore, Poon et al. have reported that P. falciparum DNA was detected by LAMP using a promising simple DNA template method of preparation from heat-treated blood (14). Further improvement of template production methods for PkLAMP will be required to optimize and simplify template preparation.
In conclusion, PkLAMP can be considered an efficient candidate for the molecular diagnosis of P. knowlesi infection in areas of endemicity. Thekisoe et al. reported that LAMP reagents are stable at ambient temperature for up to 2 weeks (16a). In addition, a recent study of the LAMP method showed that it is able to detect both Plasmodium oocysts and sporozoites from an “all-in-one” template using whole mosquito bodies (2). These observations further emphasize the potential usefulness of the LAMP method as a diagnostic and new epidemiological surveillance tool for malaria. Our studies will also provide a powerful method for the diagnosis and monitoring of P. knowlesi infection in the field.
We are grateful to Mayumi Ohshita and Mayu Tanaka at the Center for Tropical Medicine and Parasitology, Dokkyo Medical University, for technical support.
This study was supported in part by the following four grants: (i) a grant from the program for Promotion of Fundamental Studies in Health Sciences (no. 04-09) from the National Institute of Biomedical Innovation, (ii) a grant from the Promotion of Basic Research Activities for Innovative Biosciences program (PROBRAIN), (iii) a grant from the 21st Century COE Program (A-1) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and (iv) a cooperative research grant (2007-19-3) from the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine.
Published ahead of print on 5 May 2010.