Leishmania pp 279-288 | Cite as

Quantification of Parasite Loads by Automated Microscopic Image Analysis

  • Carolina Borsoi MoraesEmail author
  • Laura Maria Alcântara
Part of the Methods in Molecular Biology book series (MIMB, volume 1971)


High content analysis enables automated, robust, and unbiased evaluation of in vitro Leishmania infection. Here, we describe a protocol based on the infection of THP-1 macrophages with Leishmania promastigotes and the quantification of parasite load by high content analysis. The technique is capable of detecting and quantifying intracellular amastigotes, providing a multiparametric readout of the total number of cells, ratio of infected cells, total number of parasites, and number of parasites per infected cells. The technique can be used to quantitate infection of any Leishmania species in virtually all types of permissive host cells and can be applied to quantification of drug activity and studies of the Leishmania intracellular life cycle stage.

Key words

High content screening High content analysis Phenotypic assays Intracellular amastigotes Automated image analysis Leishmania intracellular parasites In vitro infection Leishmania-infected THP-1 macrophages 



We would like to thank Lucio Freitas-Junior for technical discussions. This work was supported by the European Union’s Seventh Framework Programme under grant agreement no. 603240 (New Medicines for Trypanosomatidic Infections—NMTrypI), and the Drugs for Neglected Diseases Initiative. For this project, DNDi received financial support from the following donors: Department for International Development (DFID), UK; Directorate-General for International Cooperation (DGIS), The Netherlands; and Swiss Agency for Development and Cooperation (SDC), Switzerland. The donors had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


  1. 1.
    Mosser DM, Edelson PJ (1985) The mouse macrophage receptor for C3bi (CR3) is a major mechanism in the phagocytosis of Leishmania promastigotes. J Immunol 135:2785–2789PubMedGoogle Scholar
  2. 2.
    Mosser DM, Edelson PJ (1987) The third component of complement (C3) is responsible for the intracellular survival of Leishmania major. Nature 327:329–331CrossRefGoogle Scholar
  3. 3.
    Sampaio WM et al (2007) In vitro binding and survival assays of Leishmania parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi. BMC Vet Res 3:11. Scholar
  4. 4.
    Sundar S, Rai M (2002) Laboratory diagnosis of visceral leishmaniasis. Clin Diagn Lab Immunol 9:951–958PubMedPubMedCentralGoogle Scholar
  5. 5.
    Siqueira-Neto JL et al (2012) An image-based high content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages. PLoS Negl Trop Dis 6:e1671. Scholar
  6. 6.
    De Rycker M et al (2013) Comparison of a high-throughput high content intracellular Leishmania donovani assay with an axenic amastigote assay. Antimicrob Agents Chemother 57:2913–2922Google Scholar
  7. 7.
    Maes L et al (2016) In vitro ‘time-to-kill’ assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and Leishmania infantum. J Antimicrob Chemother 72:428–430. Scholar
  8. 8.
    Espada CR et al (2017) Susceptibility to miltefosine in Brazilian clinical isolates of Leishmania (Viannia) braziliensis. Am J Trop Med Hyg 96:656–659PubMedPubMedCentralGoogle Scholar
  9. 9.
    Seifert K, Escobar P, Croft SL (2010) In vitro activity of anti-leishmanial drugs against Leishmania donovani is host cell dependent. J Antimicrob Chemother 65:508–511CrossRefGoogle Scholar
  10. 10.
    Zulfiqar B, Shelper TB, Avery VM (2017) Leishmaniasis drug discovery: recent progress and challenges in assay development. Drug Discov Today 22:1516–1531CrossRefGoogle Scholar
  11. 11.
    Field MC et al (2017) Anti-trypanosomatid drug discovery: an ongoing challenge and a continuing need. Nat Rev Microbiol 15:217–231CrossRefGoogle Scholar
  12. 12.
    Nühs A et al (2015) Development and validation of a novel Leishmania donovani screening cascade for high-throughput screening using a novel axenic assay with high predictivity of leishmanicidal intracellular activity. PLoS Negl Trop Dis 9:e0004094. Scholar
  13. 13.
    De Muylder G et al (2011) A screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit. PLoS Negl Trop Dis 5:e1253. Scholar
  14. 14.
    Dagley MJ, Saunders EC, Simpson KJ, McConville MJ (2015) High content assay for measuring intracellular growth of Leishmania in human macrophages. Assay Drug Dev Technol 13:389–401CrossRefGoogle Scholar
  15. 15.
    Tsuchiya S et al (1980) Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 26:171–176CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Carolina Borsoi Moraes
    • 1
    Email author
  • Laura Maria Alcântara
    • 1
  1. 1.Department of Microbiology, Institute of Biomedical SciencesUniversity of São PauloSão PauloBrazil

Personalised recommendations