Iranian Journal of Parasitology 2014. 9(1):50-59.

Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran.
Mehrdad Ghasemian, Mohammad Javad Gharavi, Lame Akhlaghi, Mehdi Mohebali, Ahmad Reza Meamar, Ehsan Aryan, Hormozd Oormazdi

Abstract


 

Background: Parasitological methods for the diagnosis of Visceral leishmania-sis (VL) require invasive procedures, so serological and molecular approaches have been developed but are not generally applicable in the field. We evaluated a loop mediated isothermal amplification (LAMP) assay using blood from VL pa-tients and compared it to nested PCR.

Methods: Forty-seven subjects with clinical features (fever, hepatosplenomegaly and anemia) were confirmed positive for VL by the direct agglutination test (DAT) at titers >3200. Forty DAT negative individuals from non-endemic areas with no clinical signs or symptoms of VL served as controls. A LAMP assay was performed using a set of six primers targeting Leishmania infantum kinetoplast DNA (kDNA) minicircle gene under isothermal (64 °C) conditions. For nested PCR we used primers targeting the kDNA minicircle gene.

Results: The LAMP assay provided a detection limit of 1 parasite in 1 ml of peripheral blood and detected L. infantum DNA in 44 of 47 DAT-confirmed VL cases, with diagnostic sensitivity of 93.6% (95% CI). No L. infantum DNA was amplified in controls, indicating a specificity of 100%. The nested PCR yielded sensitivity of 96% (95% CI) and a specificity of 100% (95% CI).

Conclusion: The LAMP assay gave results similar to those of nested PCR but in a shorter time. The LAMP method is simple; requires no sophisticated equip-ment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye.

Keywords


Human; Iran; LAMP; Nested PCR; Peripheral blood; Visceral leishmaniasis

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