Abstract
Visceral leishmaniasis (VL), also known as kala-azar caused by several species of Leishmania, is a life-threatening and disseminated parasite disease and considered one of the most neglected tropical diseases in the Old World. Rapid and accurate diagnosis is needed to provide appropriate treatment to VL patients. Identification of the species that cause VL is useful to develop an epidemiology of leishmaniasis and reveal the biology of each species of Leishmania. Herein, we describe a new multiplex real-time PCR assay to distinguish L. donovani from L. infantum with high sensitivity; both these organisms are major causes of VL. The real-time PCR assay targeting kinetoplast minicircle DNA can identify the infection of Leishmania species with high sensitivity in the DNA extracted from the peripheral blood of patients. Two assays were designed to distinguish the difference in the cysteine protease B gene copies between L. donovani and L. infantum species. The endogenous control assay targeting mammalian ribonuclease P RNA component H1 (RPPH1) was used to confirm whether the PCR reaction progressed precisely and to quantify the clinical sample input amount. Multiplex real-time PCR with these 4 assays successfully detected Leishmania DNA with high sensitivity and distinguished L. donovani from L. infantum with high specificity using DNA samples extracted from cultured parasites or the peripheral blood of patients. Multiplex real-time PCR can contribute to the therapeutic strategy for patients with kala-azar and to the research of Leishmania epidemiology and biology.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Trouiller P, Olliaro P, Torreele E, Orbinski J, Laing R, Ford N. Drug development for neglected diseases: a deficient market and a public-health policy failure. Lancet. 2002;359(9324):2188–94.
Alvar J, Velez ID, Bern C, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE. 2012;7(5):e35671.
Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol. 2002;9(5):951–8.
Chappuis F, Sundar S, Hailu A, et al. Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol. 2007;5(11):873–82.
Takagi H, Itoh M, Islam MZ, et al. Sensitive, specific, and rapid detection of Leishmania donovani DNA by loop-mediated isothermal amplification. Am J Trop Med Hyg. 2009;81(4):578–82.
Antinori S, Calattini S, Longhi E, et al. Clinical use of polymerase chain reaction performed on peripheral 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;44(12):1602–10.
Aviles H, Belli A, Armijos R, Monroy FP, Harris E. PCR detection and identification of Leishmania parasites in clinical specimens in Ecuador: a comparison with classical diagnostic methods. J Parasitol. 1999;85(2):181–7.
Srivastava P, Dayama A, Mehrotra S, Sundar S. Diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg. 2011;105(1):1–6.
Pereira MR, Rocha-Silva F, Graciele-Melo C, Lafuente CR, Magalhaes T, Caligiorne RB. Comparison between conventional and real-time PCR assays for diagnosis of visceral leishmaniasis. Biomed Res Int. 2014;2014:639310.
Dymond JS. Explanatory chapter: quantitative PCR. Methods Enzymol. 2013;529:279–89.
Mohammadiha A, Mohebali M, Haghighi A, et al. Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples. Exp Parasitol. 2013;133(1):89–94.
Hide M, Banuls AL. Species-specific PCR assay for L. infantum/L. donovani discrimination. Acta Trop. 2006;100(3):241–5.
Oshaghi MA, Ravasan NM, Hide M, et al. Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L. infantum/L. donovani discrimination. Exp Parasitol. 2009;122(1):61–5.
Goncalves-de-Albuquerque Sda C, Pessoa ESR, de Morais RC, et al. Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis. J Venomous Anim Toxins Including Trop Dis. 2014;20:16.
de Pita-Pereira D, Cardoso MA, Alves CR, Brazil RP, Britto C. Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay. Acta Trop. 2008;107(1):66–9.
Lachaud L, Marchergui-Hammami S, Chabbert E, Dereure J, Dedet JP, Bastien P. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J Clin Microbiol. 2002;40(1):210–5.
El-Beshbishy HA, Al-Ali KH, El-Badry AA. Molecular characterization of cutaneous leishmaniasis in Al-Madinah Al-Munawarah province, western Saudi Arabia. Int J Infect Dis. 2013;17(5):e334–8.
de Paiva-Cavalcanti M, de Morais RC, Pessoa ESR, et al. Leishmaniases diagnosis: an update on the use of immunological and molecular tools. Cell Biosci. 2015;5:31.
de Ruiter CM, van der Veer C, Leeflang MM, Deborggraeve S, Lucas C, Adams ER. Molecular tools for diagnosis of visceral leishmaniasis: systematic review and meta-analysis of diagnostic test accuracy. J Clin Microbiol. 2014;52(9):3147–55.
Galai Y, Chabchoub N, Ben-Abid M, et al. Diagnosis of mediterranean visceral leishmaniasis by detection of leishmania antibodies and leishmania DNA in oral fluid samples collected using an Oracol device. J Clin Microbiol. 2011;49(9):3150–3.
Bossolasco S, Gaiera G, Olchini D, et al. Real-time PCR assay for clinical management of human immunodeficiency virus-infected patients with visceral leishmaniasis. J Clin Microbiol. 2003;41(11):5080–4.
Wortmann G, Hochberg L, Houng HH, et al. Rapid identification of Leishmania complexes by a real-time PCR assay. Am J Trop Med Hyg. 2005;73(6):999–1004.
Haralambous C, Antoniou M, Pratlong F, Dedet JP, Soteriadou K. Development of a molecular assay specific for the Leishmania donovani complex that discriminates L. donovani/Leishmania infantum zymodemes: a useful tool for typing MON-1. Diagn Microbiol Infect Dis. 2008;60(1):33–42.
Wong SS, Fung KS, Chau S, Poon RW, Wong SC, Yuen KY. Molecular diagnosis in clinical parasitology: when and why? Exp Biol Med (Maywood). 2014;239(11):1443–60.
Hide M, Banuls AL. Polymorphisms of cpb multicopy genes in the Leishmania (Leishmania) donovani complex. Trans R Soc Trop Med Hyg. 2008;102(2):105–6.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer International Publishing
About this chapter
Cite this chapter
Hamasaki, Y. et al. (2016). Applicability of Multiplex Real-Time PCR to Visceral Leishmaniasis. In: Noiri, E., Jha, T. (eds) Kala Azar in South Asia. Springer, Cham. https://doi.org/10.1007/978-3-319-47101-3_15
Download citation
DOI: https://doi.org/10.1007/978-3-319-47101-3_15
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-43611-1
Online ISBN: 978-3-319-47101-3
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)