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      Poor diagnostic performance of a species-specific loop-mediated isothermal amplification (LAMP) platform for malaria

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          Abstract

          Background: The objective of this study was to assess an in-house loop-mediated isothermal amplification (LAMP) platform for malaria parasite detection and identification on species level.

          Methods: LAMP primers specific for the human Plasmodium spp., namely, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, as well as genus-specific primers, were tested against a composite gold standard comprising microscopy from thick and thin blood films, commercial genus-specific Meridian illumigene Malaria LAMP, in-house real-time polymerase chain reaction (PCR), and commercial fast-track diagnostics (FTD) Malaria differentiation PCR.

          Results: Of the 523 blood samples analyzed, the composite gold standard indicated 243 Plasmodium-species-DNA-containing samples (46.5%). Sensitivity and specificity of the analyzed genus- and species-specific LAMP primers were 71.0%–100.0% and 90.8%–100.0%, respectively. The influence of parasitemia was best documented for P. falciparum-specific LAMP with sensitivity values of 35.5% (22/62) for microscopically negative samples containing P. falciparum DNA, 50% (19/38) for parasitemia ≤50/μL, 84% (21/25) for parasitemia ≤500/μL, and 100% (92/92) for parasitemia >500/μL.

          Conclusions: In our hands, performance characteristics of species-specific in-house LAMP for malaria lack reliability required for diagnostic laboratories. The use of the easy-to-apply technique for surveillance purposes may be considered.

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          Most cited references 57

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          Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis.

          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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            Estimating prevalence from the results of a screening test.

             W Rogan,  B Gladen (1977)
            This paper deals with some basis properties of screening tests. Such tests purport to separate people with disease from people without. Minimal criteria for such a process to be a test are discussed. Various ways of judging the goodness of a test are examined. A common use of tests is to estimate prevalence of disease; frequency of positive tests is shown to be a bad estimate, and the necessary adjustmants are given.
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              Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis of Malaria

              Background Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. Methodology and Significant Findings Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. Conclusion This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.
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                Author and article information

                Journal
                1886
                European Journal of Microbiology and Immunology
                EuJMI
                Akadémiai Kiadó
                2062-8633
                December 2018
                : 8
                : 4
                : 112-118
                Affiliations
                [ 1 ]Department of Microbiology and Hospital Hygiene, Tropical Microbiology and Entomology Unit, Bundeswehr Hospital Hamburg , Hamburg, Germany
                [ 2 ]Department of Preventive Medicine, Bundeswehr Medical Academy , Munich, Germany
                [ 3 ] Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock , Rostock, Germany
                [ 4 ]Department of Infectious Diseases and Tropical Medicine, Bundeswehr Hospital Hamburg , Hamburg, Germany
                [ 5 ] Amplex Diagnostics GmbH, Gars-Bahnhof , Germany
                [ 6 ] Swiss Tropical and Public Health Institute , Basel, Switzerland
                [ 7 ]Faculty of Medicine, University Basel , Basel, Switzerland
                [ 8 ]National Reference Centre for Tropical Pathogens, Bernhard Nocht Institute for Tropical Medicine , Hamburg, Germany
                Author notes
                [*]

                Corresponding author: Hagen Frickmann, M.D., Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, Bernhard Nocht Str. 74, 20359 Hamburg, Germany; E-mail: Frickmann@ 123456bni-hamburg.de

                Article
                10.1556/1886.2018.00020
                6348705
                © 2018 The Author(s)

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the original author and source are credited, a link to the CC License is provided, and changes - if any - are indicated.

                Page count
                Pages: 7
                Categories
                Original Research Paper

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