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.
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| Issue | Vol 9 No 1 (2014) | |
| Section | Articles | |
| Keywords | ||
| Human Iran LAMP Nested PCR Peripheral blood Visceral leishmaniasis | ||
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