Comparison of Nested-PCR and Loop-Mediated Isothermal Amplification for Cutaneous Leishmaniasis Diagnosis

Abstract

Background: Leishmaniasis is a protozoan disease caused by Leishmania genus and its most common form is cutaneous leishmaniasis. The number of reported cases of cutaneous leishmaniasis in Khuzestan, southwest Iran, continues to increase. Therefore, early, accurate diagnosis is crucial for successful therapy. Objectives: The Nested PCR is a molecular method with high sensitivity and specificity in detecting leishmaniasis, but this method requires advanced equipment and skilled labor. Therefore, developing a simple yet accurate technique to diagnose leishmaniasis is essential. This study was designed to evaluate the possibility of replacing Nested PCR with loop-mediated isothermal amplification (LAMP) method for diagnosis of cutaneous leishmaniasis. Methods: We obtained 75 clinical samples from cutaneous leishmaniasis patients whose infection had already been confirmed by microscopic examination. The LAMP assay with pre-added malachite green was performed using a set of four primers targeting conserved sequences of the18S ribosomal RNA gene. The nested PCR method was performed using specific kinetoplast minicircle DNA primers. Cultured promastigotes of Leishmania major (MHOM /IR/75/ER), L. tropica (MHOM/IR/02/Mash10), and the virulent RH strain of Toxoplasma gondii were used as controls. Results: Our results showed that LAMP was positive for 100% of microscopically positive samples, which was similar to Nested PCR. The detection of L. major was improved at 104 parasites/ml using the LAMP method. Conclusions: Our findings suggest that considering the simplicity, specificity, and sensitivity of LAMP, it would be a potentially useful method in the diagnosis of leishmaniasis and detection of its various strains/species. This cost and time-effective method can be used as a suitable alternative for surveillance of leishmaniasis, as well as in epidemiological studies.