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

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.

Identificación de especies de Leishmania en pacientes y flebotominos en áreas de transmisión en una región del Perú / Identification of Leishmania species in patients and phlebotomines in transmission areas in a region of Perú

Objetivos. Identificar las especies de Leishmania presentes en las lesiones cutáneas del paciente y en las Lutzomyias que cohabitan en las áreas endémicas de la región La Libertad en el Perú. Materiales y métodos. Se usaron métodos moleculares basados en PCR y RFLP lo cual permitió obtener datos eficientes con poca muestra (pequeños especímenes), debido a su alta sensibilidad y las facilidades de aplicación en el trabajo de campo. Resultados. Los resultados del PCR de pacientes y de vectores, mostraron la presencia de Leishmania (V.) peruviana como principal agente causal de la Leishmaniosis tipo andina, transmitidas por Lutzomyia peruensis. Así mismo, se reveló la presencia de Leishmania (V.) guyanensis en Lutzomyia ayacuchensis. Conclusiones. Se mostró la presencia de L. (V.) peruviana y L. (V.) guyanensis en las áreas andinas en estudio. Hallazgos que exigen realizar una investigación más amplia sobre la distribución geográfica de L. (V.) guyanensis y las características clínicas relacionadas con la infección en áreas endémicas de Leishmaniosis cutánea.

Objectives. To identify the species of Leishmania present in the skin lesions of patients and Lutzomyias living in endemic areas of La Libertad, Peru. Materials and methods. Molecular methods based on PCR and RFLP were used, which allowed to have efficient data with small amounts of samples (small specimens), due to their high sensitivity and ease of application in the field work. Results. The results of PCR of clinical samples of patients and insect vectors showed the presence of Leishmania (V.) peruviana as a major causative agent of andean leishmaniasis transmitted by Lutzomyia peruensis. The presence of Leishmania (V.) guyanensis in Lutzomyia ayacuchensis, was found as well. Conclusions. The presence of L. (V.) peruviana and L. (V.) guyanensis in the Andean areas under study was found. These findings remark the need of a wider research about the geographical distribution of L. (V.) guyanensis and clinical features related to the infection in endemic areas of cutaneous leishmaniasis.