Diagnostic Accuracy of Loop-mediated Isothermal Amplification Assay as a Field Molecular Tool for Rapid Mass Screening of Old World Leishmania Infections in Sand Flies and In Vitro Culture

Background: We employed a highly sensitive loop-mediated isothermal amplification (LAMP) by targeting 18S rRNA gene to identify the rapid mass screening of Leishmania infections in captured sand flies of southwest Iran and In vitro culture.

Methods: One hundred fifty sand flies were collected from 11 sites adjacent to Iraqi's borders in southern parts of Khuzestan Province by using sticky sheets of paper and CDC miniature light traps during late May 2014 to Nov 2015. Following morphological identification of sand flies species, the DNA of infected samples was extracted and amplified by PCR and LAMP assays by targeting ITS-rDNA and 18S rRNA genes. The PCR amplicons were directly sequenced to conduct the phylogenetic analysis.

Results: Ten (6.6%) Leishmania infections were identified by LAMP assay (detection limit 0.01 parasites DNA) among infected Sergentomyia baghdadis, S. sintoni and Phlebotomus papatasi sand flies that was more sensitive than PCR (n=6.4%; (detection limit 10¹ parasites DNA). LAMP can identify 10¹-10⁶ promastigotes/100 μl RPMI 1640 while PCR recognized 10⁴-10⁶ promastigotes. The majority infection rate of sand flies was confirmed to L. major inferred by phylogenetic analysis.

Conclusion: This is the first exploration characterized the Old World Leishmania infections by LAMP technique in both infected sand flies and In vitro conditions. The LAMP method because of its shorter reaction time, robustness, more sensitivity, lack of requirement of complicated equipment and visual discriminatory of positivity can be appeared a promising tool instead of PCR to identify low Leishmania loads and entomological monitoring of leishmaniasis in resource-limited endemic of the world.