Simplified closed tube loop mediated isothermal amplification (LAMP) assay for visual diagnosis of Leishmania infection.

In Thailand, asymptomatic leishmaniasis cases have continuously increased, especially among patients with HIV at risk to develop further symptoms of cutaneous and visceral leishmaniasis. Thus, earlier diagnosis using a simple, sensitive and reliable diagnostic tool is needed because populations at risk mostly reside in rural communities where only basic laboratory equipment is available for health care services. In this present study, a closed tube loop mediated isothermal amplification (LAMP) was developed using a piece of parafilm placed between the dye and LAMP reaction mixture to form semi-layer that partially secured SYBR green I from spilling during amplification. No post-amplification preparation was required and accidental spill of the dye during LAMP amplification was prevented. The result could be visually interpreted under visible and UV lights after dye spinning down. The semi-layer modification of a closed tube LAMP showed successful amplification of Leishmania DNA with clear interpretation using both color and fluorescence dyes when observing by the naked eye. The sensitivity and specificity were as high as 94.4 and 96.9%, respectively whereas detection limits were 102 parasites/mL being ten fold more sensitive than other related studies. This user-friendly inexpensive approach is affordable and suitable for empowering leishmaniasis surveillance without the need of expensive devices in all levels of hospitals, including health services, as well as fieldwork, especially in low income countries.