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      Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples

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          Abstract

          Background

          Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed.

          Methods

          The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively.

          Results

          The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3 %, 58.6 %, 40.5 % and 10.8 % by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97 % for both methods.

          Conclusion

          This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China.

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          Most cited references38

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          Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

          Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. This technology has been developed into commercially available detection kits for a variety of pathogens including bacteria and viruses. The current focus on LAMP methodology is as a diagnostic system to be employed in resource-limited laboratories in developing countries, where many fatal tropical diseases are endemic. The combination of LAMP and novel microfluidic technologies such as Lab-on-a-chip may facilitate the realization of genetic point-of-care testing systems to be used by both developed and developing countries in the near future. This review will describe the historical, current, and future developments of such technologies.
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            LeishVet guidelines for the practical management of canine leishmaniosis

            The LeishVet group has formed recommendations designed primarily to help the veterinary clinician in the management of canine leishmaniosis. The complexity of this zoonotic infection and the wide range of its clinical manifestations, from inapparent infection to severe disease, make the management of canine leishmaniosis challenging. The recommendations were constructed by combining a comprehensive review of evidence-based studies, extensive clinical experience and critical consensus opinion discussions. The guidelines presented here in a short version with graphical topic displays suggest standardized and rational approaches to the diagnosis, treatment, follow-up, control and prevention of canine leishmaniosis. A staging system that divides the disease into four stages is aimed at assisting the clinician in determining the appropriate therapy, forecasting prognosis, and implementing follow-up steps required for the management of the leishmaniosis patient.
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              The current status of zoonotic leishmaniases and approaches to disease control.

              Leishmaniases are a complex of world-wide diseases with a range of clinical and epidemiological features caused by Leishmania spp. of protozoan parasites. Among 15 well-recognised Leishmania species known to infect humans, 13 have zoonotic nature, which include agents of visceral, cutaneous and mucocutaneous forms of the disease in both the Old and New Worlds. Currently, leishmaniases show a wider geographic distribution and increased global incidence of human disease than previously known. Environmental, demographic and human behavioural factors contribute to the changing landscape of leishmaniasis, which includes increasing risk factors for zoonotic cutaneous leishmaniases and new scenarios associated with the zoonotic visceral leishmaniases. The latter consist of the northward spread of Leishmania infantum transmission in Europe and America, the identification of unusual mammal hosts, and the decline of HIV-Leishmania co-infections in southern Europe following the introduction of the highly active antiretroviral therapy. Few advances have been made in the surveillance and control of the zoonotic leishmaniasis, however a number of tools have been developed for the control of the canine reservoir of L. infantum. These include: (i) several canine vaccine candidates, in particular an FML Leishmania enriched fraction showing good clinical protection, has been registered in Brazil for veterinary use; (ii) a number of insecticide-based preparations have been specifically registered for dog protection against sand fly bites. Laboratory and field studies have shown improved efficacy of these preparations for both individual and mass protection.
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                Author and article information

                Contributors
                gaochunhua1976@aliyun.com
                eledd123@126.com
                wang_junyun@yahoo.com
                dsteverding@hotmail.com
                xiawang@hotmail.com
                yangyuetao@aliyun.com
                shifeng18sep@aliyun.com
                Journal
                Parasit Vectors
                Parasit Vectors
                Parasites & Vectors
                BioMed Central (London )
                1756-3305
                15 July 2015
                15 July 2015
                2015
                : 8
                : 370
                Affiliations
                [ ]National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Laboratory of Parasite and Vector Biology, Ministry of Public Health, National Center for International Research on Tropical Diseases, WHO Collaborating Centre for Malaria, Schistosomiasis and Filariasis, Shanghai, 200025 China
                [ ]BioMedical Research Centre, Norwich Medical School, Norwich Research Park, University of East Anglia, Norwich, NR4 7TJ UK
                Article
                991
                10.1186/s13071-015-0991-2
                4501202
                26169060
                90f6866b-3280-4d3e-892f-dacc20208c23
                © Gao et al. 2015

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 21 May 2015
                : 7 July 2015
                Categories
                Research
                Custom metadata
                © The Author(s) 2015

                Parasitology
                loop-mediated isothermal amplification,conjunctional swabs,zoonotic visceral leishmaniasis,asymptomatic canine reservoir host,leishmania infantum

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