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      Development and Clinical Evaluation of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Brazil

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      Publication date (Electronic):
      Journal: BioMed Research International
      Publisher: Hindawi

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          Abstract

          Visceral leishmaniasis (VL) is considered a major public health concern in Brazil and several regions of the world. A recent advance in the diagnosis of infectious diseases was the development of loop-mediated isothermal amplification (LAMP). The aim of this study was to develop and evaluate a new LAMP assay for detection of K26 antigen-coding gene of L. donovani complex. A total of 219 blood samples of immunocompetent patients, including 114 VL cases and 105 non-VL cases, were analyzed for the diagnosis of VL in the present study. Diagnostic accuracy was calculated against a combination of parasitological and/or serological tests as a reference standard. The results were compared to those of kDNA Leishmania-PCR. The detection limit for the K26-Lamp assay was 1fg L. infantum purified DNA and 100 parasites/mL within 60 min of amplification time with visual detection for turbidity. The assay was specific for L. donovani complex. Sensitivity, specificity, and accuracy were 98.2%, 98.1%, and 98.2%, respectively, for K26-LAMP and 100%, 100%, and 100%, respectively, for kDNA Leishmania-PCR. Excellent agreement was observed between K26-LAMP and kDNA Leishmania-PCR assays (K = 0.96). A highly sensitive and specific LAMP assay targeting K26 antigen-coding gene of L. donovani complex was developed for diagnosis in peripheral blood samples of VL patients.

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          Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects.

          Tsugunori Notomi, Yasuyoshi Mori, Norihiro Tomita … (2015)
          Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3'-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.
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            Case-control and two-gate designs in diagnostic accuracy studies.

            Anne Rutjes, Johannes Reitsma, Jan P. Vandenbroucke … (2005)
            In some diagnostic accuracy studies, the test results of a series of patients with an established diagnosis are compared with those of a control group. Such case-control designs are intuitively appealing, but they have also been criticized for leading to inflated estimates of accuracy. We discuss similarities and differences between diagnostic and etiologic case-control studies, as well as the mechanisms that can lead to variation in estimates of diagnostic accuracy in studies with separate sampling schemes ("gates") for diseased (cases) and nondiseased individuals (controls). Diagnostic accuracy studies are cross-sectional and descriptive in nature. Etiologic case-control studies aim to quantify the effect of potential causal exposures on disease occurrence, which inherently involves a time window between exposure and disease occurrence. Researchers and readers should be aware of spectrum effects in diagnostic case-control studies as a result of the restricted sampling of cases and/or controls, which can lead to changes in estimates of diagnostic accuracy. These spectrum effects may be advantageous in the early investigation of a new diagnostic test, but for an overall evaluation of the clinical performance of a test, case-control studies should closely mimic cross-sectional diagnostic studies. As the accuracy of a test is likely to vary across subgroups of patients, researchers and clinicians might carefully consider the potential for spectrum effects in all designs and analyses, particularly in diagnostic accuracy studies with differential sampling schemes for diseased (cases) and nondiseased individuals (controls).
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              Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity.

              Charles Mary, Francoise Faraut, Laurie Lascombe … (2004)
              A real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to reach a sensitivity of 0.0125 parasites/ml of blood. In order to analyze the incidence of heterogeneity and number of minicircles, we performed comparative PCR by using the Leishmania DNA polymerase gene as a reporter. Assays performed in both promastigote and amastigote stages showed variations among different L. infantum and Leishmania donovani strains and the stability of the minicircle numbers for a particular strain. Analysis of blood samples from a patient who presented with Mediterranean visceral leishmaniasis confirmed the reliability of such an assay for Leishmania quantification in biological samples and allowed an estimation of positivity thresholds of classical tests used for direct diagnosis of the disease; positivity thresholds were in the range of 18 to 42, 0.7 to 42, and 0.12 to 22.5 parasites/ml for microscopic examination, culture, and conventional PCR, respectively. At the time of diagnosis, parasitemia could vary by a wide range (32 to 188,700 parasites/ml, with a median of 837 parasites/ml), while in bone marrow, parasite load was more than 100 parasites per 10(6) nucleated human cells. After successful therapy, parasitemia levels remain lower than 1 parasite/ml. In the immunocompromised host, relapses correlate with an increase in the level of parasitemia, sometimes scanty, justifying the need for assays with high sensitivity. Such sensitivity allows the detection of Leishmania DNA in the blood of 21% of patients with no history of leishmaniasis living in the Marseilles area, where leishmaniasis is endemic. This technique may be useful for epidemiologic and diagnostic purposes, especially for the quantification of parasitemia at low levels during posttherapy follow-up.
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                Author and article information

                Contributors
                Daniel Moreira de Avelar:
                ORCID: https://orcid.org/0000-0001-7907-7913
                Journal
                Journal ID (nlm-ta): Biomed Res Int
                Journal ID (iso-abbrev): Biomed Res Int
                Journal ID (publisher-id): BMRI
                Title: BioMed Research International
                Publisher: Hindawi
                ISSN (Print): 2314-6133
                ISSN (Electronic): 2314-6141
                Publication date Collection: 2019
                Publication date (Electronic): 24 July 2019
                Volume: 2019
                Electronic Location Identifier: 8240784
                Affiliations
                Fundação Oswaldo Cruz, Instituto René Rachou, Pesquisa Clínica e Políticas Públicas em Doenças Infecciosas e Parasitárias, Belo Horizonte, MG, Brazil
                Author notes

                Academic Editor: Mansour El-Matbouli

                Author information
                https://orcid.org/0000-0001-7907-7913
                Article
                DOI: 10.1155/2019/8240784
                PMC ID: 6681617
                PubMed ID: 31428648
                SO-VID: d76f4339-a9fc-4814-9ffd-7f9b5b860c63
                Copyright © Copyright © 2019 Daniel Moreira de Avelar et al.
                License:

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 7 March 2019
                Date revision received : 26 May 2019
                Date accepted : 18 June 2019
                Funding
                Funded by: Ministério da Saúde
                Award ID: TC 278/2013
                Funded by: Research Foundation of the State of Minas Gerais
                Award ID: 310938/2017-1
                Funded by: Conselho Nacional de Desenvolvimento Científico e Tecnológico
                Categories
                Subject: Research Article

                Data availability:

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