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Using microfluidics and flow cytometry to develop novel diagnostics for Leishmaniasis


   Institute of Infection, Immunology and Inflammation

  Dr T Hammarton, Dr M Jimenez  Applications accepted all year round  Self-Funded PhD Students Only

About the Project

The Leishmaniases are a group of neglected tropical diseases caused by >20 species of Leishmania parasites that are spread by >90 species of phlebotomine sand flies. 1 billion people in Africa, Asia, the Americas and the Mediterranean are at risk, with the poor and malnourished being disproportionately affected. >1 million new symptomatic cases are thought to occur annually (https://www.who.int/news-room/fact-sheets/detail/leishmaniasis) with symptoms ranging from disfiguring skin lesions (cutaneous leishmaniasis, CL), destruction of mucous membranes (mucocutaneous leishmaniasis, MCL) and visceral disease (VL) which is fatal in ~95% cases without treatment. However, it may take weeks to months after being bitten by an infected sand fly for symptoms to manifest, and many infected patients do not seem to develop symptoms [1]. In the absence of a vaccine, early diagnosis and prompt effective treatment are important to reduce the morbidity and mortality associated with Leishmania infection as well as to reduce the transmission and prevalence of leishmaniasis. With environmental and climate changes causing displacement of people from Leishmania-endemic areas and likely to result in migration of sand fly vectors northwards into new, naive areas, there is an urgent need to increase surveillance and enable early diagnosis of Leishmania infections to identify and reduce reservoirs that could sustain infection and transmission in these areas [2,3].

Leishmania promastigote forms are introduced into the skin of mammalian hosts when an infected sand fly bites. There they are taken up by host neutrophils and dermal tissue-resident macrophages, as well as some monocytes and monocyte-derived dendritic cells, where they differentiate to amastigotes and replicate. Apoptotic infected neutrophils may also be subsequently taken up by macrophages, increasing the infected macrophage population [4]. There is a plethora of methods used to diagnose leishmaniasis, ranging from observing clinical symptoms (which can be variable, non-specific and/or atypical if a patient is co-infected with e.g. HIV, malaria or pulmonary tuberculosis) to parasitological methods (the gold standard) involving direct visualisation of the parasite in splenic, bone-marrow or lymph node aspirates or following culturing from the aspirates or blood, as well as immunological and molecular methods [5]. There are disadvantages to all of these methods. Some have low sensitivity and/or specificity or only work for some species of Leishmania or may not be able to distinguish between past and present infection. For others, there are issues of the tests requiring specialised equipment and/or expertise meaning they are costly and time-consuming to perform. Finally, some rely on obtaining aspirates from e.g. bone marrow or spleen, liver biopsies or lesion scrapings, which can be painful or even dangerous for the patient.

Being able to diagnose Leishmania infection rapidly and accurately from a simple blood sample would be highly beneficial and might also allow infected but asymptomatic patients to be identified so that they can be closely monitored and treated promptly if they subsequently develop symptoms. It would also allow large scale screening for infections and cases, aiding epidemiological and surveillance studies. Studies have shown that Leishmania parasites can be cultured or detected microscopically from blood samples, buffy coat preparations and isolated peripheral blood mononuclear cells in patients with CL and VL, but that these methods of detection lack sensitivity [6,7]. Microfluidic sorting of blood or purified blood fractions using small spiral channels may offer a route to enriching for Leishmania-infected cells to increase the sensitivity of diagnosis. Microfluidic inertial-focussing is a simple, rapid, portable and high throughput (>106 cells/min) method that enables separation of cells based on differences in their shape, size and deformability [8,9]. Currently, we are optimising this method to separate different cell cycle stages of Leishmania promastigote parasites. This proposed PhD project will complement this work by investigating whether microfluidic sorting can be adapted to sort Leishmania-infected white blood cells from non-infected cells. Infection of host cells by parasites such as Plasmodium spp. and Trypanosoma cruzi is known to increase host cell stiffness, in part due to molecular changes that the parasite induces, and in part due to the physical presence of the parasite itself [10,11]. While changes to host cell deformability following infection with Leishmania parasites have not been investigated in detail, studies have shown that Leishmania spp. alter the composition of the macrophage plasma membrane, suggesting that changes in stiffness are likely [12,13]. As such, by optimising the dimensions of microfluidic spiral channels, we believe it should be possible to exploit alterations in stiffness to separate Leishmania-infected white blood cells from uninfected cells.

A combination of deformability and imaging flow cytometry and microscopy will be employed to investigate changes in the deformability, size and shape of white blood cells such as neutrophils, monocytes and macrophages upon infection by a range of Leishmania species (e.g. L. mexicana, L. major, L. infantum etc.). This information will shed light on the Leishmania infection process, allowing hypotheses about the molecular changes to the host cell induced by the parasite to be developed and tested using e.g. proteomics or lipidomics technologies, and will enable the optimisation of the dimensions and outlets of microfluidic spiral devices to separate infected white blood cells from non-infected cells. It is hoped this will provide proof of principle for a novel diagnostic method for leishmaniasis.  

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Funding Notes

Self-funded students with ~£10-12K annual bench fees

References

1. Singh O, Hasker E, Sacks D, et al. (2014). Asymptomatic Leishmania infection: a new challenge for Leishmania control, Clin Infect Dis 58: 1424–1429. https://doi.org/10.1093/cid/ciu102
2. Chalghaf B, Chemkhi J, Mayala B et al. (2018). Ecological niche modeling predicting the potential distribution of Leishmania vectors in the Mediterranean basin: impact of climate change. Parasit Vectors 11, 461. https://doi.org/10.1186/s13071-018-3019-x
3. Curtin JM, & Aronson NE (2021). Leishmaniasis in the United States: emerging issues in a region of low endemicity. Microorganisms, 9: 578. https://doi.org/10.3390/microorganisms9030578
4. Chaves MM, Lee SH, Kamenyeva O et al. (2020). The role of dermis resident macrophages and their interaction with neutrophils in the early establishment of Leishmania major infection transmitted by sand fly bite. PLoS Pathog 16: e1008674. https://doi.org/10.1371/journal.ppat.1008674
5. Thakur S, Joshi J & Kaur, S (2020). Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods. J Parasit Dis 44: 253–272. https://doi.org/10.1007/s12639-020-01212-w
6. Nakkash-Chmaisse H, Makki R, Nahhas G et al. (2011). Detection of Leishmania parasites in the blood of patients with isolated cutaneous leishmaniasis. Int J Infect Dis 15: e491-4. https://doi.org/10.1016/j.ijid.2011.03.022
7. Diro E, Yansouni CP, Takele Y et al. (2017). Diagnosis of visceral Leishmaniasis using peripheral blood microscopy in Ethiopia: a prospective Phase-III study of the diagnostic performance of different concentration techniques compared to tissue aspiration. Amer J Trop Med Hyg 96: 190–196. https://doi.org/10.4269/ajtmh.16-0362
8. Jimenez M, Miller B, Bridle HL (2017). Efficient separation of small microparticles at high flowrates using spiral channels: application to waterborne pathogens. Chem Eng Sci 157: 247–54.
9. Guzniczak E, Otto O, Whyte G et al. (2020) Deformability-induced lift force in spiral microchannels for cell separation. Lab Chip 20: 614-625.
10. Hosseini SM & Feng JJ (2012). How malaria parasites reduce the deformability of infected red blood cells. Biophys J 103: 1–10. https://doi.org/10.1016/j.bpj.2012.05.026
11. Mott A, Lenormand G, Costales J et al. (2009). Modulation of host cell mechanics by Trypanosoma cruzi. J Cell Physiol 218: 315-22. https://doi.org/10.1002/jcp.21606
12. Forero M, Marín M, Corrales A et al. (1999). Leishmania amazonensis infection induces changes in the electrophysiological properties of macrophage-like cells. J. Membrane Biol. 170: 173–180. https://doi.org/10.1007/s002329900547
13. Ghosh M, Roy K, Das Mukherjee D et al. (2014). Leishmania donovani infection enhances lateral mobility of macrophage membrane protein which is reversed by liposomal cholesterol. PLoS Negl Trop Dis 8: e3367. https://doi.org/10.1371/journal.pntd.0003367
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