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      Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

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          Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria ( affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who need them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates ( n = 41), buffy coat specimens from R. typhi PCR-positive Lao patients ( n = 42), and diverse negative controls ( n = 47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus ( n = 266). The limit of detection was ∼40 DNA copies/LAMP reaction, with an analytical sensitivity of <10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%]) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies.

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          The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.

          Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
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            ACT: the Artemis Comparison Tool.

            The Artemis Comparison Tool (ACT) allows an interactive visualisation of comparisons between complete genome sequences and associated annotations. The comparison data can be generated with several different programs; BLASTN, TBLASTX or Mummer comparisons between genomic DNA sequences, or orthologue tables generated by reciprocal FASTA comparison between protein sets. It is possible to identify regions of similarity, insertions and rearrangements at any level from the whole genome to base-pair differences. ACT uses Artemis components to display the sequences and so inherits powerful searching and analysis tools. ACT is part of the Artemis distribution and is similarly open source, written in Java and can run on any Java enabled platform, including UNIX, Macintosh and Windows.
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              Rickettsioses as paradigms of new or emerging infectious diseases.

               D Raoult,  V Roux (1997)
              Rickettsioses are caused by species of Rickettsia, a genus comprising organisms characterized by their strictly intracellular location and their association with arthropods. Rickettsia species are difficult to cultivate in vitro and exhibit strong serological cross-reactions with each other. These technical difficulties long prohibited a detailed study of the rickettsiae, and it is only following the recent introduction of novel laboratory methods that progress in this field has been possible. In this review, we discuss the impact that these practical innovations have had on the study of rickettsiae. Prior to 1986, only eight rickettsioses were clinically recognized; however, in the last 10 years, an additional six have been discovered. We describe the different steps that resulted in the description of each new rickettsiosis and discuss the influence of factors as diverse as physicians' curiosity and the adoption of molecular biology-based identification in helping to recognize these new infections. We also assess the pathogenic potential of rickettsial strains that to date have been associated only with arthropods, and we discuss diseases of unknown etiology that may be rickettsioses.

                Author and article information

                Role: Editor
                J Clin Microbiol
                J. Clin. Microbiol
                Journal of Clinical Microbiology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                March 2014
                March 2014
                : 52
                : 3
                : 832-838
                [a ]Lao-Oxford-Mahosot Hospital–Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao People's Democratic Republic
                [b ]Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, England, United Kingdom
                [c ]Mahidol-Oxford Tropical Medicine Research Unit (MORU), Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
                Author notes
                Address correspondence to Daniel H. Paris, parigi@ .

                S.D. and J.C.-V. contributed equally to this work.

                Copyright © 2014 Dittrich et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.

                Chlamydiology and Rickettsiology

                Microbiology & Virology


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