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      Detection of Antibiotic Resistance in Leprosy Using GenoType LepraeDR, a Novel Ready-To-Use Molecular Test

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          Abstract

          Background

          Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and to second-line drugs (fluoroquinolones) was described worldwide. Since Mycobacterium leprae is not growing in vitro, phenotypic susceptibility testing requires a one year experiment in the mouse model and this is rarely performed. Genetics on antibiotic resistance provide the basis for molecular tests able to detect for antibiotic resistance in leprosy.

          Methodology/Principal Findings

          A reverse hybridization DNA strip test was developed as the GenoType LepraeDR test. It includes DNA probes for the wild-type sequence of regions of rpoB, gyrA and folP genes and probes for the prevalent mutations involved in acquired resistance to rifampin, fluoroquinolones and dapsone, respectively. The performances of the GenoType LepraeDR test were evaluated by comparing its results on 120 M. leprae strains, previously studied for resistance by the reference drug in vivo susceptibility method in the mouse footpad and for mutations in the gene regions described above by PCR-sequencing. The results of the test were 100% concordant with those of PCR sequencing and the mouse footpad test for the resistant strains: 16 strains resistant to rifampin, 22 to dapsone and 4 to ofloxacin with mutations (numbering system of the M. leprae genome) in rpoB (10 S456L, 1 S456F, 1 S456M + L458V, 1 H451Y, 1 G432S + H451D, 1 T433I + D441Y and 1 Q438V), in folP1 (8 P55L, 3 P55R, 7 T53I, 3 T53A, 1 T53V) and gyrA (4 A91V), respectively. Concordance was 98.3% for the susceptible strains, two strains showing a mutation at the codon 447 that in fact was not conferring resistance as shown by the in vivo method.

          Conclusions/Significance

          The GenoType LepraeDR test is a commercially available test that accurately detects for antibiotic resistance in leprosy cases. The test is easy to perform and could be implemented in endemic countries.

          Author Summary

          Although leprosy is a curable disease using a combination of antibiotics for one year, the transmission is still active with 230,000 new cases in 2010. Drug resistance has been described and may prevent eradication of the disease. The infectious agent causing leprosy, Mycobacterium leprae, is not growing in vitro and antibiotic susceptibility testing is possible only in the mouse footpad model that requires a one year experiment. Consequently this testing is rarely done and antibiotic resistance rates in leprosy are unknown. This is the reason why we endeavored to set a new diagnosis test that detects for antibiotic resistance in M. leprae. The test is based on the method of a DNA strip test with a multiplex PCR followed by reverse hybridization. It was developed as an easy-to-use test and it will be available in endemic countries, where these kinds of strip tests are already used for detection of drug resistance in tuberculosis. The results of the new test, Genotype LepraeDR, performed on 120 M. leprae strains were concordant with those of the standard PCR sequencing and mouse footpad susceptibility testing.

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          Most cited references29

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          American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: treatment of tuberculosis.

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            Leprosy now: epidemiology, progress, challenges, and research gaps.

            Leprosy continues to be a challenge to health worldwide, with about 250,000 new cases being detected every year. Despite widespread implementation of effective multidrug therapy, leprosy has not been eliminated. A third of newly diagnosed patients have nerve damage and might develop disabilities, although the proportion varies according to several factors, including level of self-care. Women who develop leprosy continue to be especially disadvantaged, with rates of late diagnosis and disability remaining high in this subgroup. Leprosy was not a specified disease in the Millennium Development Goals, but improvements in the other areas they cover, such as education and levels of poverty, will help leprosy patients and services. We review data and make recommendations for research on diagnosis, treatment, and prevention, such as further use of molecular analysis of the Mycobacterium leprae genome, implementation of BCG vaccination, and administration of chemoprophylaxis to household contacts. We also suggest development of tools for early diagnosis and detection of infection and nerve damage, and formulation of strategies to manage the chronic complications of leprosy, such as immune-mediated reactions and neuropathy. Copyright © 2011 Elsevier Ltd. All rights reserved.
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              THE EXPERIMENTAL DISEASE THAT FOLLOWS THE INJECTION OF HUMAN LEPROSY BACILLI INTO FOOT-PADS OF MICE

              When leprosy bacilli from human patients are inoculated into the foot-pads of CFW mice, a microscopic granuloma containing acid-fast bacilli develops in a characteristic manner. This has been seen in 22 of 22 instances with leprosy bacilli from nasal washings, in 12 of 16 instances with leprosy bacilli from skin biopsies, and in none of 16 cases where the nasal washings were not observed to contain leprosy bacilli. Quantitative studies revealed a relationship between the number of bacilli inoculated and the time required for the appearance of the lesions. The incubation period was usually 1 to 2 months when the dose was 105.5 to 106.0 bacilli and about 6 months when the dose was about 103 organisms. After the development of the lesion, the number of bacilli harvested was usually in the range 104.5 to 106.0, regardless of the number inoculated. When the inoculum has contained 102.0 to 103.5 acid-fast bacilli, and harvests were reasonably prompt, there were regular increases of 50- to 1000-fold. Passage to new groups of mice has been successful 11 of 12 times. Most of these were second passages. One strain has been maintained in 3 passages with a total increase in acid-fast bacilli of 4 x 104-fold. Another strain has been through 4 passages with a total increase of about 4 x 106-fold. Cultures on bacteriological media favorable for the growth of most known mycobacterial species have not shown growth of mycobacteria.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                July 2012
                31 July 2012
                : 6
                : 7
                : e1739
                Affiliations
                [1 ]Université Paris Diderot, EA3964, Paris, France
                [2 ]Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Paris, France
                [3 ]AP-HP, groupe Hospitalier Saint Louis-Lariboisière, Bactériologie, France
                [4 ]Université Pierre et Marie Curie-Paris6, EA1541, ER5, Paris, France
                [5 ]HAIN Lifescience, Nehren, Germany
                [6 ]Leprosy Research Center, National Institute of Infectious Diseases, Higashimurayama-shi, Tokyo, Japan
                [7 ]AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Bactériologie-Hygiène, Paris, France
                Fondation Raoul Follereau, France
                Author notes

                Author Liana Tejmar-Kolar works for the Hain Lifescience company. The other authors have declared that no competing interests exist.

                Conceived and designed the experiments: EC LTK VJ. Performed the experiments: ACN LTK MM. Analyzed the data: EC VJ. Contributed reagents/materials/analysis tools: LTK MM ACN. Wrote the paper: EC VJ.

                Article
                PNTD-D-12-00324
                10.1371/journal.pntd.0001739
                3409109
                22860144
                9290ee5c-f1ef-48d3-863e-b9e2095595e2
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 7 March 2012
                : 5 June 2012
                Page count
                Pages: 7
                Funding
                Funding support for the study on mouse footpad susceptibility testing (animal facilities, animal keeper and technician) was provided by repeated annual grants from the Association Française Raoul Follereau. Development of the DNA strip test work was supported by a specific grant (Magralepre 2007) from Ordre de Malte-France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine
                Diagnostic Medicine
                Infectious Diseases
                Neglected Tropical Diseases
                Leprosy
                Bacterial Diseases
                Skin Infections

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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