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      New Developments in PCR-Based Diagnostics for Bacterial Pathogens Causing Gastrointestinal Infections—A Narrative Mini-Review on Challenges in the Tropics

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          Abstract

          The application of modern PCR approaches for the diagnosis of bacterial gastrointestinal pathogens is on the rise due to their rapidly available results combined with high sensitivity. While multiple studies describe the ongoing implementation of this technique for routine diagnostic purposes in laboratories in Western industrialized countries, reports on successful and also sustainable respective approaches in resource-poor tropical settings are still scarce. In order to shed light on potential reasons for this marked discrepancy, this narrative review summarizes identified challenges for the application of diagnostic PCR targeting bacterial gastrointestinal pathogens from stool samples in the tropics. The identified and discussed issues comprise the lack of generally accepted definitions for (1) minimum standards regarding sample acquisition, storage and transport time for diagnostic PCR analyses in the tropics, (2) nucleic acid extraction standards allowing an optimum detection of all types of pathogens which may be responsible for gastroenteritis in the tropics, (3) validation standards to ensure comparable quality of applied diagnostic assays, and (4) cut-offs for a reliable discrimination of infection and mere colonization in areas where semi-immunity due to repeated exposition associated with poor hygiene conditions has to be expected. Further implementation research is needed to solve those issues.

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          Most cited references141

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          The Measurement of Observer Agreement for Categorical Data

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            Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data.

            Quantification of mRNAs using real-time polymerase chain reaction (PCR) by monitoring the product formation with the fluorescent dye SYBR Green I is being extensively used in neurosciences, developmental biology, and medical diagnostics. Most PCR data analysis procedures assume that the PCR efficiency for the amplicon of interest is constant or even, in the case of the comparative C(t) method, equal to 2. The latter method already leads to a 4-fold error when the PCR efficiencies vary over just a 0.04 range. PCR efficiencies of amplicons are usually calculated from standard curves based on either known RNA inputs or on dilution series of a reference cDNA sample. In this paper we show that the first approach can lead to PCR efficiencies that vary over a 0.2 range, whereas the second approach may be off by 0.26. Therefore, we propose linear regression on the Log(fluorescence) per cycle number data as an assumption-free method to calculate starting concentrations of mRNAs and PCR efficiencies for each sample. A computer program to perform this calculation is available on request (e-mail: bioinfo@amc.uva.nl; subject: LinRegPCR).
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              Inhibition and facilitation of nucleic acid amplification.

              I. WILSON (1997)
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Trop Med Infect Dis
                Trop Med Infect Dis
                tropicalmed
                Tropical Medicine and Infectious Disease
                MDPI
                2414-6366
                02 June 2021
                June 2021
                : 6
                : 2
                : 96
                Affiliations
                [1 ]Institute for Infection Control and Infectious Diseases, University Medical Center Göttingen, 37075 Göttingen, Germany; ulrike.loderstaedt1@ 123456med.uni-goettingen.de
                [2 ]Department of Microbiology and Hospital Hygiene, Bundeswehr Central Hospital Koblenz, Andernacher Str. 100, 56070 Koblenz, Germany; ralfmatthiashagen@ 123456bundeswehr.org
                [3 ]Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, 18057 Rostock, Germany; Hahn.andreas@ 123456me.com
                [4 ]Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, 20359 Hamburg, Germany
                Author notes
                Author information
                https://orcid.org/0000-0002-5157-8369
                https://orcid.org/0000-0002-8967-9528
                Article
                tropicalmed-06-00096
                10.3390/tropicalmed6020096
                8293448
                34199650
                70d11b3f-66a3-4cc6-ab68-24228591b2c1
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 06 May 2021
                : 31 May 2021
                Categories
                Review

                pcr,gastroenteritis,tropics,pre-analytics,interpretation,validation,diagnostics,infection,colonization,nucleic acid extraction

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