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.


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.
Chiang Mai University, Faculty of Veterinary Medicine, Department of Veterinary Biosciences and Veterinary Public Health, Muang, Chiang Mai, Thailand.
Saruda
S
Tiwananthagorn
Jichi Medical University, Department of Infection and Immunity, Division of Medical Zoology, Tochigi, Japan.
Hirotomo
H
Kato
Chiang Mai University, Faculty of Veterinary Medicine, Department of Veterinary Biosciences and Veterinary Public Health, Muang, Chiang Mai, Thailand.
Ranchana
R
Yeewa
Chiang Mai University, Faculty of Veterinary Medicine, Department of Veterinary Biosciences and Veterinary Public Health, Muang, Chiang Mai, Thailand.
Amontip
A
Muengpan
Naresuan University, Faculty of Medical Science, Department of Microbiology and Parasitology, Phitsanulok, Thailand.
Raxsina
R
Polseela
Phramongkutklao College of Medicine, Department of Parasitology, Bangkok, Thailand.
Saovanee
S
Leelayoova
D000818:Animals; D018105:DNA, Kinetoplast / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D005260:Female; D006801:Humans; D007891:Leishmania / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D008403:Mass Screening; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction; D011576:Psychodidae / Q000469:parasitology*; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D013785:Thailand
D018105:DNA, Kinetoplast; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S