title: Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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. gaochunhua1976@aliyun.com.
authors.forename: Chun-hua
authors.initials: CH
authors.lastname: Gao
authors.affiliation: 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. eledd123@126.com.
authors.forename: Dan
authors.initials: D
authors.lastname: Ding
authors.affiliation: 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. wang_junyun@yahoo.com.
authors.forename: Jun-yun
authors.initials: JY
authors.lastname: Wang
authors.affiliation: BioMedical Research Centre, Norwich Medical School, Norwich Research Park, University of East Anglia, Norwich, NR4 7TJ, UK. dsteverding@hotmail.com.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Steverding
authors.affiliation: BioMedical Research Centre, Norwich Medical School, Norwich Research Park, University of East Anglia, Norwich, NR4 7TJ, UK. xiawang@hotmail.com.
authors.forename: Xia
authors.initials: X
authors.lastname: Wang
authors.affiliation: 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. yangyuetao@aliyun.com.
authors.forename: Yue-tao
authors.initials: YT
authors.lastname: Yang
authors.affiliation: 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. shifeng18sep@aliyun.com.
authors.forename: Feng
authors.initials: F
authors.lastname: Shi
mesh_terms: D000818:Animals; D002681:China; D003228:Conjunctiva / Q000469:parasitology*; D017931:DNA Primers / Q000235:genetics; D004283:Dog Diseases / Q000469:parasitology*; D004285:Dogs; D018314:Leishmania infantum / Q000235:genetics / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000469:parasitology / Q000662:veterinary*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers