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

Particle and Fibre Toxicology

Feng Shi²  Yue-tao Yang²  Xia Wang¹  Dietmar Steverding¹  Jun-yun Wang²  Dan Ding²  Chun-hua Gao² 

[1] BioMedical Research Centre, Norwich Medical School, Norwich Research Park, University of East Anglia, Norwich NR4 7TJ, UK;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

关键词: Leishmania infantum;    Asymptomatic canine reservoir host;    Zoonotic visceral leishmaniasis;    Conjunctional swabs;    Loop-mediated isothermal amplification;   

Others  :  1222228
DOI  :  10.1186/s13071-015-0991-2

 received in 2015-05-21, accepted in 2015-07-07,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Gao et al.

【 图 表 】

Fig. 1.

Figure 4.

Fig. 3.

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