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We compared the performance of loop-mediated isothermal amplification (LAMP) with that of a multiplex PCR method for differential detection of human Taenia parasites in fecal specimens from taeniasis patients. The LAMP method, with no false positives, showed a higher sensitivity (88.4%) than the multiplex PCR (37.2%). Thus, it is expected that the LAMP method has a high value for molecular diagnosis of taeniasis.
Differential detection of Taenia species (Taenia saginata, T. asiatica, and T. solium) is a key point for control and prevention of taeniasis/cysticercosis in areas where Taenia disease is endemic. The identification of the carriers of T. solium tapeworms is most important for the prevention of cysticercosis, the most severe Taenia disease. Diagnosis of taeniasis by stool examination to detect taeniid eggs, the most common method, lacks both sensitivity and specificity because the eggs of Taenia species are morphologically indistinguishable. Moreover, Taenia species identification relying on comparative morphology of proglottids also lacks specificity.
The coproantigen detection method has proved to be a useful application in epidemiological surveys (1, 5), but this method is genus specific, not species specific. Recently, a T. solium-specific coproantigen enzyme-linked immunosorbent assay (ELISA) has been developed and shown to be potentially useful for mass screening (4). However, this test fails to identify T. saginata and T. asiatica taeniasis patients. Therefore, molecular tools are more reliable for differential identifications of taeniid parasites. Several PCR technique-based detection methods for Taenia species in fecal samples, including the multiplex PCR method with mitochondrial DNA (18), the PCR-restriction fragment length polymorphism method with mitochondrial DNA (15, 16), and the nested-PCR method with the Tso31 gene encoding the T. solium oncosphere-specific protein (10), have been reported. We have recently developed a loop-mediated isothermal amplification (LAMP) method targeting cytochrome c oxidase subunit 1 (cox1) and cathepsin L-like cysteine peptidase (clp) genes for differential detection of Taenia species (13). This method utilizes a Bst DNA polymerase with strand replacement activity and four primers that recognize six sequences on the target DNA under isothermal conditions. This method has proved to be simple and highly sensitive and specific for differential detection of Taenia species by using DNA prepared from proglottids, cysticerci, and fecal samples of taeniasis patients (12) without using sophisticated and expensive equipment. In the present study, we evaluated its sensitivity and specificity with fecal specimens from taeniasis patients by comparison of the results obtained by the LAMP method with those obtained by the multiplex PCR method.
Thirty-six fecal samples were collected in China from 26 T. saginata taeniasis patients, 5 T. asiatica taeniasis patients, and 5 T. solium taeniasis patients, and 7 fecal samples from Indonesia were obtained from T. saginata taeniasis patients after ethical approvals were received from the local health bureaus in both countries. The fecal samples were collected from patients prior to treatment with antiparasitic drugs to expel the worm, and both fecal samples and recovered parasites were stored in 70% ethanol for further analysis. The expelled tapeworm from each patient was identified by multiplex PCR (18). In addition, taeniid egg-negative fecal samples (n = 11) from persons without a history of tapeworm expulsion were used as negative controls. Copro-DNAs were extracted by using the QIAamp DNA stool Mini kit (Qiagen, Hilden, Germany) as described previously (13), and the extracted DNA was stored at −20°C until use. Moreover, to confirm the specificity of the LAMP method, DNAs prepared from parasite tissues of Ascaris lumbricoides, Enterobius vermicularis, Hymenolepis nana, and hookworms by using the DNeasy tissue kit (Qiagen) were used. Multiplex PCR and LAMP reactions were carried out as described previously (13, 18). In the multiplex PCR, the diagnostic DNA fragments with sizes of 827, 269, 720, and 984 bp were produced in T. saginata, T. asiatica, American/African genotype T. solium, and Asian genotype T. solium, respectively. To verify the specificity of LAMP amplification, the sequences of LAMP amplicons were determined. Briefly, the LAMP products were digested with HinfI (37°C) or ApoI (50°C) enzymes (New England Biolabs, Beverly, MA) and purified by using the NucleoSpin ExTract kit (Macherey-Nagel, Düren, Germany). The digested products were cloned into the pGEM-T vector (Promega, Madison, WI), after filling in sticky ends to provide blunt-ended DNA and the addition of adenine to the ends by Taq DNA polymerase. The ligation mixtures were used to transform Escherichia coli DH5α, and each colony was analyzed by PCR using vector primers. The PCR products were sequenced as described previously (13). McNemar's test was applied to compare the sensitivities of LAMP and multiplex PCR.
Figure Figure11 shows the results of LAMP with cox1 primer sets and multiplex PCR, and Table Table11 shows results for all methods with all samples. The LAMP products appeared as a ladder-like pattern on the agarose gel due to their characteristic stem-loop structure. The LAMP with cox1 primer sets could differentially detect target DNA in 37 out of 43 (86.0%) samples, which had T. saginata (n = 30), T. asiatica (n = 4), and T. solium (n = 3). The LAMP with clp primer sets differentially detected target DNA in 13 (30.2%) samples, which had T. saginata (n = 11), T. asiatica (n = 1), and T. solium (n = 1). No amplification from negative control fecal samples was observed by LAMP with cox1 and clp primer sets or by multiplex PCR (data not shown). All samples positive by LAMP with clp primer sets were positive by LAMP with cox1 primer sets except one sample. The differences between detection rates for the cox1 gene and the clp gene may be responsible for the number of copies of each target gene within samples, since a large amount of mitochondrial DNA exists in a cell, and the amount of mitochondrial DNA is one criterion for selection as a target DNA for detection. Although the cox1 gene was targeted, multiplex PCR could differentially identify Taenia parasites in only 16 samples, 1 sample after the first PCR and 15 samples after the second PCR. The samples negative by the multiplex PCR method were also negative by PCR using one individual primer set specific to each parasite cox1 gene (data not shown). The LAMP products from the multiplex PCR-negative samples were confirmed to be specific for Taenia parasites by sequencing (data no shown), which indicated the high sensitivity of the LAMP methods. Taq DNA polymerase used in PCR is often inactivated by inhibitors present in biological samples, which sometimes cause problems related to sensitivity and reproducibility. Due to the tolerance of the Bst DNA polymerase to inhibitors, in contrast to the Taq DNA polymerase, LAMP appears as a gold standard method to detect pathogens in fecal samples (2, 3, 7-9, 17, 19). The fact that LAMP is less affected by inhibitory substances in biological materials (6) is of great advantage when using fecal specimens that are known to contain a large amount of inhibitors.
Distribution of other intestinal helminthic parasites in areas where taeniasis is endemic raises the possibility of a mixed infection with Taenia and other helminthic parasites. Thus, the specificity of the LAMP method was assessed with DNAs extracted from parasites such as Hymenolepis nana, Enterobius vermicularis, Ascaris lumbricoides, and hookworms. All DNAs from these parasites were negative by LAMP with cox1 and clp primer sets (data not shown). LAMP has been shown to be a highly specific method because the amplification occurs when the four primers specifically recognize the six regions within the target sequence, a unique feature of LAMP (11, 14).
Other interesting advantages of LAMP are as follows: (i) it is possible to distinguish a positive LAMP reaction from a negative LAMP reaction by visual endpoint judgment of the turbidity caused by magnesium pyrophosphate as a by-product of the reaction, and (ii) the LAMP method, performed within 90 min, is faster than the multiplex PCR, which takes at least 4 h for completion of the two-round PCR, because LAMP is carried out under isothermal conditions by using a simple incubator, such as a water bath or a block heater.
In this study, we demonstrated that the LAMP method for identification of taeniid DNA in fecal specimens is more sensitive, easier, and faster than the multiplex PCR method. In addition to these findings, the simplicity and cost-effectiveness of LAMP may make it easily applicable for a clinical practice as well as an epidemiological survey in areas of disease endemicity, especially developing countries. Therefore, the LAMP method as a molecular diagnosis tool will provide the successful control and prevention of taeniasis/cysticercosis in areas of endemicity.
This work was supported by the special fund for the leadership in science and technology in Asia from the Ministry of Education, Culture, Sports, Science and Technology, Japan, by the International Collaboration Research Fund from the Japan Society for the Promotion of Science (JSPS) (grant no. 17256002), by the JSPS Asia/Africa Science Platform Fund (support to A.I.), by the JSPS Japan-China Medical Exchange Program, and by the Japan-China Medical Association Fund (support to M.N.).
Published ahead of print on 14 July 2010.
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