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Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran

BACKGROUND: Parasitological methods for the diagnosis of Visceral leishmaniasis (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...

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Autores principales: GHASEMIAN, Mehrdad, GHARAVI, Mohammad Javad, AKHLAGHI, Lame, MOHEBALI, Mehdi, MEAMAR, Ahmad Reza, ARYAN, Ehsan, OORMAZDI, Hormozd
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Tehran University of Medical Sciences 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4289880/
https://www.ncbi.nlm.nih.gov/pubmed/25642260
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author GHASEMIAN, Mehrdad
GHARAVI, Mohammad Javad
AKHLAGHI, Lame
MOHEBALI, Mehdi
MEAMAR, Ahmad Reza
ARYAN, Ehsan
OORMAZDI, Hormozd
author_facet GHASEMIAN, Mehrdad
GHARAVI, Mohammad Javad
AKHLAGHI, Lame
MOHEBALI, Mehdi
MEAMAR, Ahmad Reza
ARYAN, Ehsan
OORMAZDI, Hormozd
author_sort GHASEMIAN, Mehrdad
collection PubMed
description BACKGROUND: Parasitological methods for the diagnosis of Visceral leishmaniasis (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 patients 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 equipment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye.
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spelling pubmed-42898802015-01-30 Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran GHASEMIAN, Mehrdad GHARAVI, Mohammad Javad AKHLAGHI, Lame MOHEBALI, Mehdi MEAMAR, Ahmad Reza ARYAN, Ehsan OORMAZDI, Hormozd Iran J Parasitol Original Article BACKGROUND: Parasitological methods for the diagnosis of Visceral leishmaniasis (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 patients 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 equipment; has a short reaction time; and results, indicated by turbidity of the reaction mixture, are observable with the naked eye. Tehran University of Medical Sciences 2014-03 /pmc/articles/PMC4289880/ /pubmed/25642260 Text en Copyright: © Iranian Journal of Parasitology & Tehran University of Medical Sciences This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.
spellingShingle Original Article
GHASEMIAN, Mehrdad
GHARAVI, Mohammad Javad
AKHLAGHI, Lame
MOHEBALI, Mehdi
MEAMAR, Ahmad Reza
ARYAN, Ehsan
OORMAZDI, Hormozd
Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran
title Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran
title_full Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran
title_fullStr Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran
title_full_unstemmed Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran
title_short Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran
title_sort development and assessment of loop-mediated isothermal amplification (lamp) assay for the diagnosis of human visceral leishmaniasis in iran
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4289880/
https://www.ncbi.nlm.nih.gov/pubmed/25642260
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