Understanding a Research Article on Leishmania donovani Isolation and Characterisation

Question

Vivien C: "Hi, I am struggling to understand what they did in this research article. Can anyone, in simple terms, summarize what they are doing? Thank you"

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238840

Here's the Abstract we'll be simplifying:

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease.

Answer 1

The abstract of a research paper summarizes the researchers' experiment. You can expect to find the following information from this section: 1. the purpose and significance of the study; 2. the study's overall design or methodology; and 3. major findings and conclusions

1. Here's the purpose and significance of the study in the researchers' own words:

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays.

Here, they're telling us that visceral leishmaniasis (VL) is a disease that's difficult to treat. It's also hard to know when patients with VL are "cured," meaning they are no longer carriers of leishmania parasites. When it comes to parasites, it's important to have a reliable way to test for patients' "confirmation of cure after treatment," because if patients stop treatment before they are cured, they could become sick again, and even spread the disease.

So the purpose of the study is to explore a reliable way to test patients after VL treatment, to make sure that they really are "cured." They believe that testing for L. donovani antigens in urine samples is an accurate, non-invasive way to see if a patient is a carrier of leishmania parasites (the definition of non-invasive is "not requiring the introduction of instruments into the body;" so an example of an "invasive procedure" would be drawing blood); (an antigen is a molecule, structure (ex. protein), or piece of a pathogen, virus, parasite, etc. that triggers our immune system.)

2. Here's the study's methodology in the researchers' own words:

VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification.

First, they tell you about the study's participants: "VL patients from India and Sudan." Then they tell you how the antigens were isolated. A urine sample can have A LOT of different molecular structures and substances in it, so to isolate the L. donovani antigens, the researchers added antibodies to the samples-- anti-L. donovani polyclonal antibodies, which specifically bind with, or "capture" L. donovani antigens.

Once the "captured" antigens were separated from the unneeded parts of the urine sample, the researchers needed a way to unbind the antigens from the added antibodies. "Elution" is essentially "the reverse process of binding" (Urh, M. et al, 2009); so the mentioned "eluted materials" are the added antibodies and the now isolated, "captured" antigens.

The eluted material from Indian samples were then "submitted to mass spectrometry for protein identification." Mass spectrometry is commonly used to identify the molecular components of samples. The identified proteins of the VL samples were added to a data base to see if any were unique to L. donovani, and thus could be used in future assays or tests to distinguish L. donovani from other pathogens or organisms.

The VL samples from Sudan were also submitted to mass spectrometry, but some of the samples were first "analysed by western blot." Western blots are another way to isolate and identify proteins of interest, as well as measure protein size and amount in sample.

3. Results and Conclusions:

"We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease."

All methods yielded L. donovani proteins by mass spectrometry, and 5 of 6 proteins were highly specific to Leishmania. Therefore, the identified proteins can be used to develop a non-invasive and accurate test for VL diagnosis and "confirmation of cure."

*Source Links:

1. (Urh, M. et al, 2009) https://www.sciencedirect.com/topics/immunology-and-microbiology/elution
2. https://microbeonline.com/western-blot-technique-principle-procedures-advantages-and-disadvantages/
3. https://www.who.int/news-room/fact-sheets/detail/leishmaniasis
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903764/
5. https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/massspec/masspec1.htm