Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

Abstract

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors.OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1).METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand.FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Molecular identification of two newly identified human pathogens causing leishmaniasis using PCR-based methods on the 3′ untranslated region of the heat shock protein 70 (type I) gene

“…martiniquensis from ‘ L . siamensis ’ (MHOM/TH/2010/TR) but the size of PCR products could not differentiate parasites in other subgenera [ 28 ]. For the ITS1-PCR method, it is not suitable for the discrimination of ‘ L .…”

“…orientalis ) and L . martiniquensis infection as it generates the same size of products [ 1 , 28 , 47 ].…”

“…L . orientalis ), but the size of the PCR products could not differentiate these parasites from other subgenera [ 28 ]. In 2012, Requena and colleagues analyzed the 3′untranslated region (3′UTR) of Leishmania HSP70-type I ( HSP70-I ) genes from 24 strains representing eleven Leishmania species following PCR amplification [ 29 ].…”

Analytical Performance of a Loop-Mediated Isothermal Amplification Assay for Leishmania DNA Detection in Sandflies and Direct Smears of Patients with Cutaneous Leishmaniasis

“…23,40 Our results differed from those reported by Adams et al 10 with a LoD of 1 × 10 2 parasites/mL for L. donovani and Sriworarat et al 14 with a LoD of 1 × 10 3 parasites/mL for Leishmania siamensis in whole blood samples. In addition, a report by Tiwananthagorn et al 58 concluded that a PCR-kDNA assay was more sensitive than PCR-ITS and LAMP with the 18S rRNA gene. 42 Therefore, a comprehensive study to evaluate these parameters in a vast set of Leishmania species causing CL in the New World is needed.…”

Loop-mediated isothermal amplification (LAMP): An advanced molecular point-of-care technique for the detection of Leishmania infection

“…A similar study also experimentally validated the potential field applicability of the MG-LAMP for detection of L . martiniquensis DNA in sand flies in Thailand; however, no Leishmania DNA was detected by LAMP and PCR when applied to 380 field-caught flies, probably due to low infection rate in the study area [79]. Additionally, the performance of the MG-LAMP was further validated using purified DNA from sand fly pools and was found to be of potential use for the entomological surveillance of CL in Colombia with 100% sensitivity and 96.8% specificity [80].…”