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      Loop-mediated isothermal amplification (LAMP) assays targeting 18S ribosomal RNA genes for identifying P. vivax and P. ovale species and mitochondrial DNA for detecting the genus Plasmodium

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          Abstract

          Background

          Loop-mediated isothermal amplification (LAMP) has been widely used to diagnose various infectious diseases. Malaria is a globally distributed infectious disease attributed to parasites in the genus Plasmodium. It is known that persons infected with Plasmodium vivax and P. ovale are prone to clinical relapse of symptomatic blood-stage infections. LAMP has not previously been specifically evaluated for its diagnostic performance in detecting P. ovale in an epidemiological study, and no commercial LAMP or rapid diagnostic test (RDT) kits are available for specifically diagnosing infections with P. ovale.

          Methods

          An assay was designed to target a portion of mitochondrial DNA ( mtDNA) among Plasmodium spp., the five human Plasmodium species and two other assays were designed to target the nuclear 18S ribosomal DNA gene ( 18S rDNA) of either P. vivax or P. ovale for differentiating the two species. The sensitivity of the assays was compared to that of nested PCR using defined concentrations of plasmids containing the target sequences and using limiting dilutions prepared from clinical isolates derived from Chinese workers who had become infected in Africa or near the Chinese border with Myanmar.

          Results

          The results showed that 10 2 copies of the mitochondrial target or 10 2 and 10 3 copies of 18S rDNA could be detected from Plasmodium spp., P. vivax and P. ovale, respectively. In 279 clinical samples, the malaria Pan mtDNA LAMP test performed well when compared with a nested PCR assay (95% confidence interval [CI] sensitivity 98.48–100%; specificity 90.75–100%). When diagnosing clinical cases of infection with P. vivax, the 18S rDNA assay demonstrated an even great sensitivity (95.85–100%) and specificity (98.1–100%). The same was true for clinical infections with P. ovale (sensitivity 90.76–99.96%; specificity 98.34–100%). Using plasmid-positive controls, the limits of detection of Malaria Pan, 18S rDNA P. vivax and 18S rDNA P. ovale LAMP were 100-, 100- and tenfold lower than those of PCR, respectively.

          Conclusion

          The novel LAMP assays can greatly aid the rapid, reliable and highly sensitive diagnosis of infections of Plasmodium spp. transmitted among people , including P. vivax and P. ovale, cases of which are most prone to clinical relapse.

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          Supplementary Information

          The online version contains supplementary material available at 10.1186/s13071-021-04764-9.

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          Loop-mediated isothermal amplification of DNA.

          T. Notomi (2000)
          We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
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            Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis.

            Takafumi Tsuboi, Hideyuki Iriko, Risa Watanabe … (2007)
            Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.
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              Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice.

              Sebastian Mikolajczak, Ashley Vaughan, Niwat Kangwanrangsan … (2015)
              Plasmodium vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections likely initiated by activation of dormant liver stage parasites-hypnozoites. The lack of tractable P. vivax animal models constitutes an obstacle in examining P. vivax liver stage infection and drug efficacy. To overcome this obstacle, we have used human liver-chimeric (huHep) FRG KO mice as a model for P. vivax infection. FRG KO huHep mice support P. vivax sporozoite infection, liver stage development, and hypnozoite formation. We show complete P. vivax liver stage development, including maturation into infectious exo-erythrocytic merozoites as well as the formation and persistence of hypnozoites. Prophylaxis or treatment with the antimalarial primaquine can prevent and eliminate liver stage infection, respectively. Thus, P. vivax-infected FRG KO huHep mice are a model to investigate liver stage development and dormancy and may facilitate the discovery of drugs targeting relapsing malaria.
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                Author and article information

                Contributors
                Yaming Huang: 1724501964@qq.com
                Zhaoqing Yang: zhaoqingy92@hotmail.com
                Journal
                Journal ID (nlm-ta): Parasit Vectors
                Journal ID (iso-abbrev): Parasit Vectors
                Title: Parasites & Vectors
                Publisher: BioMed Central (London )
                ISSN (Electronic): 1756-3305
                Publication date (Electronic): 24 May 2021
                Publication date PMC-release: 24 May 2021
                Publication date Collection: 2021
                Volume: 14
                Electronic Location Identifier: 278
                Affiliations
                [1 ]GRID grid.285847.4, ISNI 0000 0000 9588 0960, Laboratory of Pathogen Biology and Immunology, , Kunming Medical University, ; Kunming, 650500 Yunnan People’s Republic of China
                [2 ]GRID grid.285847.4, ISNI 0000 0000 9588 0960, Department of Pathogen Biology and Immunology, , Kunming Medical University, ; Kunming, 650500 Yunnan People’s Republic of China
                [3 ]GRID grid.433871.a, Zhejiang Provincial Center for Disease Control and Prevention, ; No. 3399 BinSheng Road, Binjiang District, Hangzhou, 310051 Zhejiang People’s Republic of China
                [4 ]Shanglin County People’s Hospital, Shanglin, 530500 Guangxi People’s Republic of China
                [5 ]GRID grid.462844.8, ISNI 0000 0001 2308 1657, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses (CIMI), , Sorbonne Université, ; 75013 Paris, France
                [6 ]GRID grid.508984.8, Animal Parasitic Disease Laboratory, , USDA-Agricultural Research Service, ; 10300 Baltimore Avenue, Beltsville, MD 20705 USA
                [7 ]GRID grid.418332.f, Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, ; Nanning, 530021 Guangxi People’s Republic of China
                Article
                Publisher ID: 4764
                DOI: 10.1186/s13071-021-04764-9
                PMC ID: 8147439
                PubMed ID: 34030725
                SO-VID: 341389f9-a2e8-4045-84cb-12ae2f397709
                Copyright © © The Author(s) 2021
                License:

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                Date received : 9 December 2020
                Date accepted : 3 May 2021
                Funding
                Funded by: National Science Foundation of China
                Award ID: 31860604
                Award ID: U1802286
                Award Recipient :
                Funded by: Major science and technology projects of Yunnan Province
                Award ID: 2018ZF0081
                Award Recipient :
                Funded by: International Science and Technology Cooperation-Yunnan International Science and Technology Cooperation Base
                Award ID: 202003AE140004
                Award Recipient :
                Funded by: Yunnan Applied Basic Research Projects-Union Foundation
                Award ID: 2018FE001-190
                Award ID: 2019FE001-015
                Award Recipient :
                Funded by: Guangxi Zhuang Autonomous Region Health Commission of Scientific Research Project
                Award ID: Z20190892
                Award Recipient :
                Funded by: Education Department Fund of Yunnan Province
                Award ID: 2019J1184
                Award Recipient :
                Categories
                Subject: Research
                Custom metadata
                issue-copyright-statement © The Author(s) 2021

                ScienceOpen disciplines: Parasitology
                Keywords: lamp,genus plasmodium spp.,plasmodium vivax,plasmodium ovale
                Data availability:
                ScienceOpen disciplines: Parasitology
                Keywords: lamp, genus plasmodium spp., plasmodium vivax, plasmodium ovale

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