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 10 4 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.

Micro-costing from healthcare professional’s perspective and acceptability of cutaneous leishmaniasis diagnostic tools in Morocco: A mixed-methods study

“…In Eastern Mediterranean countries where Cutaneous Leishmaniasis is endemic, the standard laboratory test involves light microscopy examination of a smear prepared by scraping a suspected lesion. This method enables the visual identification of parasites following Giemsa stain colouration [ 7 ]. Ideally, results are obtained in approximately one and a half hours when colouration and microscopy readings are performed directly [ 8 ].…”

“…The CL-RDT received notable acceptance for its user-friendliness and time efficiency compared to traditional microscopy. Micro-costing data revealed that the average unit cost for microscopy is 15 MAD [ 7 – 31 ], whereas 75 MAD [52–131] for CL-RDT. Altogether, the diagnostic cost for microscopy is 115 MAD±4, marginally higher than the 102 MAD±2 for CL-RDT (p = 0,05).…”

Rapid and Highly Sensitive Detection of Leishmania by Combining Recombinase Polymerase Amplification and Solution-Processed Oxide Thin-Film Transistor Technology

“…Although some leishmaniasis types, such as mucocutaneous and visceral leishmaniasis, may cause severe health issues and death, cutaneous leishmaniasis is typically not life threatening and can heal independently in many cases [ 25 , 26 ]. For Leishmania detection, LAMP is a widely used isothermal amplification method and has been reported to show similar results to nested PCR [ 27 ]. Sereno et al have reviewed more isothermal amplification methods in parasites of the Trypanosomatidae family, such as NASBA, LAMP, RPA, and PSR, in which RPA and LAMP rank highly in terms of field applicability [ 28 ].…”