Abstract
Control of visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani primarily relies on chemotherapy using an increasingly compromised repertoire of antileishmanial compounds. For evaluation of novel drugs, the Syrian golden hamster is considered as a clinically relevant laboratory model. In this study, two molecular parasite detection assays were developed targeting cathepsin-like cysteine protease B (CPB) DNA and 18S rRNA to achieve absolute amastigote quantification in the major target organs liver and spleen. Both quantitative PCR (qPCR) techniques showed excellent agreement with a strong correlation with the conventional microscopic reading of Giemsa-stained tissue smears. Using multiple single tissue pieces and all three detection methods, we confirmed homogeneity of infection in liver and spleen and the robustness of extrapolating whole organ burdens from a small single tissue piece. Comparison of pre- and post-treatment burdens in infected hamsters using the three detection methods consistently revealed a stronger parasite reduction in the spleen compared to the liver, indicating an organ-dependent clearance efficacy for miltefosine. In conclusion, this study in the hamster demonstrated high homogeneity of infection in liver and spleen and advocates the use of molecular detection methods for assessment of low (post-treatment) tissue burdens.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1:307–310
Bourgeois N, Lachaud L, Reynes J, Rouanet I, Mahamat A, Bastien P (2008) Long-term monitoring of visceral leishmaniasis in patients with AIDS: relapse risk factors, value of polymerase chain reaction, and potential impact on secondary prophylaxis. J Acquir Immune Defic Syndr 48:13–19
Bretagne S, Durand R, Olivi M, Garin J-F, 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 Diag Lab Immunol 8:828–831
Brewster S, Aslett M, Barker DC (1998) Kinetoplast DNA minicircle database. Parasitol Today 14:437–438
Dereure J, Duong Thanh H, Lavabre-Bertrand T, Cartron G, Bastides F, Richard-Lenoble D, Dedet JP (2003) Visceral leishmaniasis. Persistence of parasites in lymph nodes after clinical cure. J Infect 47:77–81
Dorlo TP, Balasegaram M, Beijnen JH, de Vries PJ (2012) Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis. J Antimicrob Chemother 67:2576–2597
Engwerda CR, Ato M, Kaye PM (2004) Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis. Trends Parasitol 20:524–530
Gan CS, Chong PK, Pham TK, Wright PC (2007) Technical, experimental, and biological variations in isobaric tags for relative and absolute quantitation (iTRAQ). J Proteome Res 6:821–827
Gangalum PR, de Castro W, Vieira LQ, Dey R, Rivas L, Singh S, Majumdar S, Saha B (2015) Platelet-activating factor receptor contributes to antileishmanial function of miltefosine. J Immunol 194:5961–5967
Gonzalez-Andrade P, Camara M, Ilboudo H, Bucheton B, Jamonneau V, Deborggraeve S (2014) Diagnosis of trypanosomatid infections: targeting the spliced leader RNA. J Mol Diagn 16:400–404
Hendrickx S, Boulet G, Mondelaers A, Dujardin JC, Rijal S, Lachaud L, Cos P, Delputte P, Maes L (2014) Experimental selection of paromomycin and miltefosine resistance in intracellular amastigotes of Leishmania donovani and L. infantum. Parasitol Res 113:1875–1881
Hendrickx S, Mondelaers A, Eberhardt E, Delputte P, Cos P, Maes L (2015) In vivo selection of paromomycin and miltefosine resistance in Leishmania donovani and L. infantum in a Syrian hamster model. Antimicrob Agents Chemother 59:4714–4718
Hide M, Bras-Goncalves R, Banuls AL (2007) Specific cpb copies within the Leishmania donovani complex: evolutionary interpretations and potential clinical implications in humans. Parasitology 134:379–389
Kloehn J, Saunders EC, O’Callaghan S, Dagley MJ, McConville MJ (2015) Characterization of metabolically quiescent Leishmania parasites in murine lesions using heavy water labeling. PLoS Pathog 11:e1004683
Lachaud L, Marchergui-Hammami S, Chabbert E, Dereure J, Dedet JP, Bastien P (2002) Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J Clin Microbiol 40:210–215
Loria-Cervera EN, Andrade-Narvaez FJ (2014) Animal models for the study of leishmaniasis immunology. Rev Inst Med Trop Sao Paulo 56:1–11
Maes E, Valkenborg D, Baggerman G, Willems H, Landuyt B, Schoofs L, Mertens I (2015) Determination of variation parameters as a crucial step in designing TMT-based clinical proteomics experiments. PLoS ONE 10:e0120115
Marschner N, Kotting J, Eibl H, Unger C (1992) Distribution of hexadecylphosphocholine and octadecyl-methyl-glycero-3-phosphocholine in rat tissues during steady-state treatment. Cancer Chemother Pharmacol 31:18–22
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
Moreira ND, Vitoriano-Souza J, Roatt BM, Vieira PMA, Ker HG, de Oliveira Cardoso JM, Giunchetti RC, Carneiro CM, de Lana M, Reis AB (2012) Parasite burden in hamsters infected with two different strains of Leishmania (Leishmania) infantum: “Leishman Donovan units” versus real-time PCR. PLoS One 7:e47907
Mukherjee P, Ghosh AK, Ghose AC (2003) Infection pattern and immune response in the spleen and liver of BALB/c mice intracardially infected with Leishmania donovani amastigotes. Immunol Lett 86:131–138
Mukherjee AK, Gupta G, Adhikari A, Majumder S, Kar Mahapatra S, Bhattacharyya Majumdar S, Majumdar S (2012) Miltefosine triggers a strong proinflammatory cytokine response during visceral leishmaniasis: role of TLR4 and TLR9. Int Immunopharmacol 12:565–572
Murray HW (2001) Tissue granuloma structure-function in experimental visceral leishmaniasis. Int J Exp Pathol 82:249–267
OpenWetWare (2010) PCR inhibitors
Ozturk OG (2012) Using biological variation data for reference change values in clinical laboratories. Biochem Analyt Biochem 1:e106
Purkait B, Kumar A, Nandi N, Sardar AH, Das S, Kumar S, Pandey K, Ravidas V, Kumar M, De T, Singh D, Das P (2012) Mechanism of amphotericin B resistance in clinical isolates of Leishmania donovani. Antimicrob Agents Chemother 56:1031–1041
Requena JM, Soto M, Doria MD, Alonso C (2000) Immune and clinical parameters associated with Leishmania infantum infection in the golden hamster model. Vet Immunol Immunopathol 76:269–281
Ricós C, Perich C, Minchinela J, Álvarez V, Simón M, Biosca C, Doménech M, Fernández P, Jiménez CV, Garcia-Lario JV, Cava F (2009) Application of biological variation—a review. Biochemia Med 19:250–259
Srivastava A, Sweat JM, Azizan A, Vesely B, Kyle DE (2013) Real-time PCR to quantify Leishmania donovani in hamsters. J Parasitol 99:145–150
Stauber LA (1958) Host resistance to the Khartoum strain of Leishmania donovani. Rice Instit Pamphlet 45:80–96
Sundar S (2001) Drug resistance in Indian visceral leishmaniasis. Trop Med Int Health 6:849–854
Tellevik MG, Muller KE, Lokken KR, Nerland AH (2014) Detection of a broad range of Leishmania species and determination of parasite load of infected mouse by real-time PCR targeting the arginine permease gene AAP3. Acta Trop 137:99–104
Tupperwar N, Vineeth V, Rath S, Vaidya T (2008) Development of a real-time polymerase chain reaction assay for the quantification of Leishmania species and the monitoring of systemic distribution of the pathogen. Diagn Microbiol Infect Dis 61:23–30
van den Bogaart E, Schoone GJ, Adams ER, Schallig HDFH (2014) Duplex quantitative reverse-transcriptase PCR for simultaneous assessment of drug activity against Leishmania intracellular amastigotes and their host cells. Int J Parasitol: Drugs Drug Resist 4:14–19
van der Meide W, Guerra J, Schoone G, Farenhorst M, Coelho L, Faber W, Peekel I, Schallig H (2008) Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites. J Clin Microbiol 46:73–78
van Eys GJ, Schoone GJ, Kroon NC, Ebeling SB (1992) Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Mol Biochem Parasitol 51:133–142
Weirather JL, Jeronimo SM, Gautam S, Sundar S, Kang M, Kurtz MA, Haque R, Schriefer A, Talhari S, Carvalho EM, Donelson JE, Wilson ME (2011) Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples. J Clin Microbiol 49:3892–3904
WHO (2010) Control of the leishmaniasis: report of a meeting of the WHO Expert Committee on the Control Leishmaniases. In Word Healt Organ Tech Rep Ser (Geneva: WHO), pp. 1–186
WHO (2015a) Kala-Azar elimination programme: report of a WHO consultation of partners (Geneva, Switzerland)
WHO (2015b) Leishmaniasis fact sheet N°375, WHO, ed
Acknowledgments
The authors acknowledge An Matheeussen and Pim-Bart Feijens for their technical assistance. The work was funded by the Research Fund Flanders (FWO: project G051812N and scholarship 11V4315N), the Agency for Innovation by Science and Technology in Flanders (IWT: project 121474), a research fund of the University of Antwerp (TT-ZAPBOF 33049), and additionally supported by the Drugs for Neglected Diseases initiative (DNDi, Geneva, Switzerland). LMPH is a partner of the “Antwerp Drug Discovery Network” (ADDN, www.addn.be) and the Excellence Centre “Infla-Med” (www.uantwerpen.be/infla-med).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethics statement
The use of laboratory rodents was carried out in strict accordance with all mandatory guidelines (European Union directive 2010/63/EU on the protection of animals used for scientific purposes and the Declaration of Helsinki) and was approved by the ethical committee of the University of Antwerp (UA-ECD 2011–74).
Additional information
Eline Eberhardt, Annelies Mondelaers, Louis Maes and Guy Caljon contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Fig. S1
Linearity and PCR efficiency for L. infantum. Standard curves of the CPB DNA qPCR (top row) and 18S rRNA qPCR assay (bottom row) for liver (left) and spleen (right) with standard deviations on each data point (JPG 102 kb)
Fig. S2
Linearity and PCR efficiency for L. donovani. Standard curves of the CPB DNA qPCR (top row) and 18S rRNA qPCR assay (bottom row) for liver (left) and spleen (right) with standard deviations on each data point (JPG 98 kb)
Rights and permissions
About this article
Cite this article
Eberhardt, E., Mondelaers, A., Hendrickx, S. et al. Molecular detection of infection homogeneity and impact of miltefosine treatment in a Syrian golden hamster model of Leishmania donovani and L. infantum visceral leishmaniasis. Parasitol Res 115, 4061–4070 (2016). https://doi.org/10.1007/s00436-016-5179-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-016-5179-y