Abstract
Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT > 3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7 %) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk.
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References
Alvar J, Cañavate C, Gutierrez-Solar B, Jiménez M, Laguna F, López-Vélez R, Molina R, Moreno J (1997) Leishmania and human immunodeficiency virus coinfection: the first 10 years. Clin Microbiol Rev 10:298–319
Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL (2006) Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol 44:1435–1439
Bossolasco S, Gaiera G, Olchini D, Gulletta M, Martello L, Bestetti A, Bossi L, Germagnoli L, Lazzarin A, Uberti-Foppa C, Cinque P (2003) Real-time PCR assay for clinical management of human immunodeficiency virus-infected patients with visceral leishmaniasis. J Clin Microbiol 41:5080–5084
Bretagne S, Durand R, Olivi M, Garin JF, Sulahian A, Rivollet D, Vidaud M, Deniau M (2001) Real-time PCR as a new tool for quantifying Leishmania infantum in liver in infected mice. Clin Diagn Lab Immunol 8:828–831
Brewster S, Aslett M, Barker DC (1998) Kinetoplast DNA mini-circle database. Parasitol Today 14:437–438
Castilho TM, Shaw JJ, Floeter-Winter LM (2003) New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species. J Clin Microbiol 41:540–546
Cupolillo E, Grimaldi Junior G, Momen H, Beverley SM (1995) Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol 73:145–155
Desjeux P, Alvar J (2003) Leishmania/HIV co-infections: epidemiology in Europe. Ann Trop Med Parasitol 97:3–15
Fraga TL, Brustoloni YM, Lima RB, Dorval MEC, Oshiro ET, Oliveira J, Oliveira ALL, Pirmez C (2010) Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children. Mem Inst Oswaldo Cruz 105(3):310–313
Ghasemian M, Maraghi S, Samarbafzadeh AR, Jelowdar A, Kalantari M (2011) The PCR-based detection and identification of the parasites causing human cutaneous leishmaniasis in the Iranian city of Ahvaz. Ann Trop Med Parasitol 105(3):209–215
Harith A, Salappendel RJ, Reiter I, Knapen F, Korte P, Huigen E, Kolk RHG (1989) Application of a direct agglutination test for detection of specific anti-Leishmania antibodies in the canine reservoir. J Clin Microbiol 27:2252–2257
Khademvatan S, Neisi N, Maraghi S, Saki J (2011) Diagnosis and identification of Leishmania spp. From Giemsa-stained slides, by real-time PCR and melting curve analysis in south- west of Iran. Ann Trop Med Parasitol 105:559–565
Khosravi S, Hejazi SH, Hashemzadeh M, Eslami G, Darani HY (2012) 0 Molecular diagnosis of old world leishmaniasis: real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp. J Vector Borne Dis 49:15–18
Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J (2006) The real time polymerase chain reaction. Mol Aspects Med 27(2–3):95–125
Marfurt J, Niederwieser I, Makia ND, Beck HP, Felger I (2003) Diagnostic genotyping of Old and New World Leishmania species by PCR RFLP. Diagn Microbiol Infect Dis 46:115–124
Mary C, Faraut F, Lascombe L, Dumon H (2004) Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol 42:5249–5255
Mohebali M (2013) Visceral leishmaniasis in Iran: Review of the Epidemiological and Clinical Features. Iran J Parasitol 8(3):348–358
Mohebali M, Hajjaran H, Hamzavi Y, Mobedi I, Arshi S, Zarei Z, Akhoundi B, Naeini K, Manouchehri AR, Fakhar M (2005) Epidemiological aspects of canine visceral leishmaniosis in the Islamic Republic of Iran. Vet Parasitol 129:243–252
Mohebali M, Edrissian GhH, Nadim A, Hajjaran H, Akhoundi B, Hooshman B, Zarei Z, Arshi Sh, Mirsamadi N, Manochehri Naeimi K, Mamishi S, Sanati AA, Moshfe AA, Charedar S, Fakhar M (2006) Application of direct agglutination test (DAT) for the diagnosis and seroepidemiological studies of visceral leishmaniasis in Iran. Iranian J Parasitol 1:15–25
Mohebali M, Edrissian GH, Shirzadi MR, Akhoundi B, Hajjaran H, Zarei Z, Molaei S, Sharifi I, Mamishi S, Mahmoudvand H, Torabi V, Moshfe A, Malmasi A, Motazedian MH, Fakhar M (2011) An observational study on the current distribution of visceral leishmaniasis in different geographical zones of Iran and implication to health policy. Travel Med Infect Dis 9(2):67–74
Nicolas L, Prina E, Lang T, Milon G (2002) Real-time PCR for detection and quantitation of Leishmania in mouse tissues. J Clin Microbiol 40:1666–1669
Noyes HA, Reyburn H, Bailey JW, Smith D (1998) 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 36:2877–2881
Perandin F, Manca N, Calderaro A, Piccolo G, Galati L, Ricci L, Medici M, Arcangeletti CM, Snounou G, Dettori G, Chezzi C (2004) Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol 42:1214–1219
Prina E, Roux E, Mattei D, Milon G (2007) Leishmania DNA is rapidly degraded following parasite death: analysis by microscopy and real-time PCR. Microbes Infect 9(11):1307–1315
Reithinger R, Dujardin JC (2007) Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol 45(1):21–25
Soliman RH, Othman AA (2009) Evaluation of DNA melting curve analysis real-time PCR for detection and differentiation of cryptosporidium species. Parasitol United J 2:47–54
Sundar S, Rai M (2002) Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol 9(5):951–958
Victoir K, De Doncker S, Cabrera L, Alvarez E, Arevalo J, Llanos-Cuentas A, Le Ray D, Dujardin JC (2003) Direct identification of Leishmania species in biopsies from patients with American tegumentary leishmaniasis. Trans R Soc Trop Med Hyg 97:80–87
Vitale F, Reale S, Vitale M, Petrotta E, Torina A, Caracappa S (2004) TaqMan-Based detection of Leishmania infantum DNA using canine samples. Ann N Y Acad Sci 1026:139–143
Wortmann G, Hochberg L, Houng HH, Sweeney C, Zapor M, Aronson N, Weina P, Ockenhouse CF (2005) Rapid identification of Leishmania complexes by a real-time PCR assay. Am J Trop Med Hyg 73(6):999–1004
Acknowledgments
This research was financially supported by Tehran University of Medical Sciences (Project No: p-993). We express our thanks to Z. Zarei and S. Charedar for laboratory help and valuable help with collection of blood samples.
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Ghasemian, M., Gharavi, M.J., Akhlaghi, L. et al. SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples. J Parasit Dis 40, 81–87 (2016). https://doi.org/10.1007/s12639-014-0452-4
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DOI: https://doi.org/10.1007/s12639-014-0452-4