Development and Assessment of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Iran.
AbstractBackground: 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.
Mohebali M, Edrissian GhH, Shirzadi MR, Akhoundi B, Hajjaran H, Zarei Z, et al. An observational study on the current distribution of visceral leishmaniasis in different geo-graphical zones of Iran and implication to health policy. Travel Med Infect Dis. 2011 Mar;9(2):67-74.
Desjeux P. Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis. 2004 Sep;27(5):305-18.
Mohebali M, Edrissian GH, Nadim A, Hajjaran H, Akhoundi B, Hooshmand B. Application of direct agglutination test (DAT) for the diagnosis and seroepidemiological studies of visceral leishmaniasis in Iran. Iranian J Parasitol 2006;1(1):15-25.
Mohebali M, Hamzavi Y, Edrissian GH, Forouzani A. Seroepidemiological study of visceral leishmaniasis among humans and animal reservoirs in Bushehr province, Islamic Republic of Iran. East Mediterr Health J. 2001 Nov;7(6):912-7.
Alvar J, Canavate C, Gutierrez-Solar B, Jimenez M, Laguna F, Lopez-Velez R, et al. Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clin Microbiol Rev. 1997 Apr;10(2):298-319.
Desjeux P, Alvar J. Leishmania/HIV co-infections: epidemiology in Europe. Ann Trop Med Parasitol. 2003 Oct;97 Suppl 1:3-15.
Rijal S, Boelaert M, Regmi S, Karki BM, Jacquet D, Singh R, et al. Evaluation of a urinary antigen-based latex agglutination test in the diagnosis of kala-azar in eastern Nepal. Trop Med Int Health. 2004 Jun;9(6):724-9.
Salam MA, Khan MG, Mondal D. Urine antigen detection by latex agglutination test for diagnosis and assessment of initial cure of visceral leishmaniasis. Trans R Soc Trop Med Hyg. 2011 May;105(5):269-72.
Singh DP, Goyal RK, Singh RK, Sundar S, Mohapatra TM. In search of an ideal test for diagnosis and prognosis of kala-azar. J Health Popul Nutr. 2010 Jun;28(3):281-5.
Ghatei MA, Hatam, G.R., Hossini, M.H., Sarkari, B. Performance of latex agglutination test (KAtex) in diagnosis of visceral leishmaniasis in Iran. Iranian Journal of Immunology. 2009;6(4):202-7.
Antinori S, Calattini S, Longhi E, Bestetti G, Piolini R, Magni C, et al. Clinical use of polymerase chain reaction performed on per-ipheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in HIV-infected and HIV-uninfected patients: a single-center, 8-year experience in Italy and review of the literature. Clin Infect Dis. 2007 Jun 15;44(12):1602-10.
Cruz I, Canavate C, Rubio JM, Morales MA, Chicharro C, Laguna F, et al. A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus. Trans R Soc Trop Med Hyg. 2002 Apr;96 Suppl 1:S185-9.
Lachaud L, Chabbert E, Dubessay P, Reynes J, Lamothe J, Bastien P. Comparison of various sample preparation methods for PCR diagnosis of visceral leishmaniasis using peripheral blood. J Clin Microbiol. 2001 Feb;39(2):613-7.
Reithinger R, Dujardin JC. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol. 2007 Jan; 45(1):21-5.
Singh RK, Pandey HP, Sundar S. Visceral leishmaniasis (kala-azar): challenges ahead. Indian J Med Res. 2006 Mar;123(3):331-44.
Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol. 2002 Sep;9(5):951-8.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000 Jun 15;28(12):E63.
Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, et al. Loop-mediated isothermal amplification for detection of African trypanosomes. J Clin Microbiol. 2003 Dec;41(12):5517-24.
Maruyama F, Kenzaka T, Yamaguchi N, Tani K, Nasu M. Detection of bacteria carrying the stx2 gene by in situ loop-mediated isothermal amplification. Appl Environ Microbiol. 2003 Aug;69(8):5023-8.
Endo S, Komori T, Ricci G, Sano A, Yokoyama K, Ohori A, et al. Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LA-MP) method. FEMS Microbiol Lett. 2004 May 1;234(1):93-7.
Okafuji T, Yoshida N, Fujino M, Motegi Y, Ihara T, Ota Y, et al. Rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification. J Clin Microbiol. 2005 Apr;43(4):1625-31.
Adams ER, Schoone GJ, Ageed AF, Safi SE, Schallig HD. Development of a reverse transcriptase loop-mediated isothermal amplifi-cation (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples. Am J Trop Med Hyg. 2010 Apr;82(4):591-6.
Takagi H, Itoh M, Islam MZ, Razzaque A, Ekram AR, Hashighuchi Y, et al. Sensitive, specific, and rapid detection of Leishmania donovani DNA by loop-mediated isothermal amplification. Am J Trop Med Hyg. 2009 Oct;81(4):578-82.
Zhang H, Thekisoe OM, Aboge GO, Kyan H, Yamagishi J, Inoue N, et al. Toxoplasma gondii: sensitive and rapid detection of infection by loop-mediated isothermal amplification (LA-MP) method. Exp Parasitol. 2009 May;12-2(1):47-50.
Noyes HA, Reyburn H, Bailey JW, Smith D. A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. J Clin Microbiol. 1998 Oct;36(10):2877-81.
Njiru ZK, Mikosza AS, Matovu E, Enyaru JC, Ouma JO, Kibona SN, et al. African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol. 2008 Apr;38(5):589-99.
Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, et al. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem. 2006 Feb;52(2):303-6.
Plutzer J, Karanis P. Rapid identification of Giardia duodenalis by loop-mediated isothermal amplification (LAMP) from faecal and env-ironmental samples and comparative findings by PCR and real-time PCR methods. Parasitol Res. 2009 Jun;104(6):1527-33.
Bakheit MA, Torra D, Palomino LA, Thekisoe OM, Mbati PA, Ongerth J, et al. Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isoth-ermal DNA amplification and confirm-ation of generated LAMP products by sequencing. Vet Parasitol. 2008 Nov 25;158(1-2):11-22.
Aryan E, Makvandi M, Farajzadeh A, Huygen K, Bifani P, Mousavi SL, et al. A novel and more sensitive loop-mediated isothermal amp-lification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex. Microbiol Res. 2010 Mar 31;165(3):211-20.
Brewster S, Aslett M, Barker DC. Kinetoplast DNA minicircle database. Parasitol Today. 1998 Nov;14(11):437-8.
Rogers WO, Wirth DF. Kinetoplast DNA minicircles: regions of extensive sequence divergence. Proc Natl Acad Sci U S A. 1987 Jan;84(2):565-9.
Khan MG, Bhaskar KR, Salam MA, Akther T, Pluschke G, Mondal D. Diagnostic accuracy of loop-mediated isothermal amplification (LA-MP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors. 2012;5:280.
Copyright (c) Iranian Journal of Parasitology
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.