Loop-mediated isothermal amplification originally developed by Notomi et al. is a less expensive assay which has now been utilized for the development of a number of diagnostic methods to detect several African trypanosome species [16–18], as well as malaria, tuberculosis, and filaria [19–23]. Despite VL being one of the most important parasitic diseases, it still lacks a simple, low-cost and sensitive diagnostic method. Therefore the VL endemic regions in South Asia including Bangladesh requires a definitive diagnostic method that can be performed as a rapid test for VL patients living in immense poverty.
In this study, we have validated the LAMP assay developed by Takagi et al., with 75 confirmed VL cases and 101 controls. It has revealed very encouraging results compared to nested PCR assay which is being used widely for the diagnosis of leishmaniasis. In a study conducted by Takagi et al., diagnostic sensitivity of LAMP was evaluated for 10 confirmed VL patients and 08 (80%) were found to be positive for parasite DNA. In another study conducted with 30 confirmed VL patients, a sensitivity of 83% and specificity of 98% were reported . To compare the sensitivity and specificity of LAMP using buffy coat DNA in our study consisting of 75 confirmed VL cases and 101 controls, we found a higher sensitivity of 90.7% and specificity of 100%. A similar range of sensitivity was reported by a number of investigators using either whole blood or buffy coat (PBMC) for PCR assays targeting the ITS or mini-exon regions for DNA detection [25–27].
Although in this study we observed the performance of LAMP to be slightly lower than Ln-PCR (sensitivity 96% and parasite detection rate is 0.1 parasite against 1 parasite by LAMP), yet it achieved better results than shown in two previous studies. The reason for this could be the use of buffy coat instead of whole blood. The specificity of the LAMP assay has been shown to be very high as the assay utilizes four sets of primers targeting six distinct target DNA sequences [16, 28, 29]. In our study, although LAMP has failed marginally to satisfy the WHO sensitivity level of >95% for any acceptable test there are still several advantageous aspects of the LAMP assay that need to be considered.
The very exciting features of LAMP assay include requirement of just a heat block or even a simple water bath instead of a costly thermal cycler, UV gel documentation and transilluminator, which all are essential components of conventional PCR [20, 24, 30, 31]. Moreover, the assay is performed under isothermal conditions at a temperature range of 60°-65°C, circumventing the time length involved in thermal changes of conventional PCR and also prevents or lowers inhibition observed in its later stages [28, 32]. However, when the LAMP reaction is performed in a water bath the chances of contamination tends to increase more and hence it is recommended to wrap the cap of the tube with paraffin prior to performing the LAMP assay. Further, the requirement of electrical power supply to operate the water bath at the resource limited settings can be avoided by the usage of alternative power sources such as battery, exothermal chemicals and solar power [31, 32].
In addition, results of LAMP assay are interpreted by observing turbidity [20, 31, 32] that reduces cost and time of post conventional PCR analysis [16, 24, 28] as well as eliminating the chance of contamination involved in agarose gel electrophoresis . It is also noted that gel electrophoresis involves handling of potent carcinogenic agents such as ethidium bromide which poses a potential threat for those handling it in the laboratory.
Furthermore, the Bst polymerase enzyme used in LAMP assay is active at relatively high temperatures, which minimizes the possibility of non-specific priming  and is more resistant to inhibitors that prevent conventional PCR . Alongside, the isothermal condition of the assay lowers inhibition observed in the later stages of conventional PCR .
Several reports have indicated the usefulness of using heat-treated samples as a template DNA source without compromising the sensitivity, and thus eliminating the need for DNA extraction which reduces both time and cost [16, 19, 28, 31]. Reports from a previous study indicated that LAMP assay for malaria diagnosis using heat treated blood was priced between US$0.4 and US$0.7, that is lower than currently available RDT's . However, in this study we were not able to validate our assay with heat-treated blood samples for the diagnosis of VL. We speculate that, validation of heat treated buffy coat as a DNA source could further minimize the cost for diagnosis if it can be established for leishmaniasis. Further, there is scope to improve the sensitivity of LAMP in the diagnosis of VL if some unexpected problems associated with sample storage and transportation can be minimized. In our case, we collected VL samples from RMCH and stored them there for a certain length of period, this site is about 300 km away from ICDDR, B. Had these steps been avoided, we believe that the sensitivity of LAMP in our study might be slightly increased.