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.
