Current progress in the various aspects of the project are reported below:
Case record forms
Following the successful completion of our first clinical grading working in March 2018 led by experts in the field, and attended by local consultant dermatologists and collaborators from Brazil, India and the UK, a case report form (CRF) has now been developed to collect meta-data from patients with cutaneous leishmaniasis in a standardised manner. A second CRF is now finalised for multiple lesion leishmaniasis. In addition, these CRFs provide the basis for database searches.
The cutaneous leishmaniasis CRF is available here (restricted access).
The multiple leishmaniasis CRF is available here (restricted access).
Patient sample collection
Ethical review has been completed in India, Sri Lanka and Brazil and the collection of patient biopsies has now begun.
Tools for molecular pathology
A molecular pathology pipeline is now established through pilot studies and is ready to be implemented. Flow cytometry and whole blood RNAseq is done on PBMCs to look at systemic gene expression. Simultaneously, tissue biopsy samples are collected, and a pan tissue transcriptome profiling is done using Nanostring. This helps not only to understand the gene pathways that are dysregulated but can provide insights into patient diversity. To provide a spatial context to these gene signatures, Nanostring Digital Spatial Profiling is then used to mark regions of interest (ROIs) in the tissue and data is then directly collected from these areas. Host/pathogen gene targets that are predicted by Nanostring, are then validated by qPCR, IHC, and FISH.
We are now beginning to integrate all the ‘omics’ data with the pathological and treatment data for each sample for easy access via LeishPathNet.
A) A molecular pathway pipeline to study disease mechanisms
B) Example of cross patient variability (n=7) for immune cell subset gene signatures clustered as pre (light blue) and post treatment (dark blue). Heat map scale ranges Log2FC -4 (blue) to +4 (yellow). C) Example of a skin biopsy sample with regions of interest (ROIs) digitally marked using DSP. Green shows pan cytokeratin expression in epidermis whereas red shows CD3 expression in dermis. (D-D’) An example of parasite distribution (delta amastin marker; white dots; FISH) in macrophages (CD68 marker; red; IHC) and T cells (CD3 marker; green; IHC). Scale bar is 10 pixels.
WSI Database (Zen Browser)
LeishPathNet provides direct access to the ZenBrowser database (see SLIDES). Currently, the database is being housed on a 20TB server and will in future allow direct upload / download access to network members. Currently the database is populated by exemplar data from a study of L. donovani infection in the mouse. A schematic of the database import process is provided below, which allows matching of WSIs and their associated scanner metadata (contained in a .czi file) with the patient metadata contained in the CRF (converted to a .csv file via Excel):
Our first workshop aimed at introducing leishmaniacs to economists and vice versa, and focused on setting an agenda for future research took place at the The Principal, York on 17th October 2017. The agenda for the meeting can be found at: LeishModel_EconomicsAgendaFINAL (open access)
Workshop Presentations (restricted access)
Current project slide database
The projects below have an existing slide database. Access to the database can be found at: SLIDES
The CRACK-IT project entitled “A multiscale model to minimise animal usage in leishmaniasis drug development” was the winner of the Innovate UK / NC3Rs CRACK-IT Challenges Award in Virtual Infectious Diseases Research (https://crackit.org.uk/challenge-16-virtual-infectious-disease-research).
This project has developed a novel computational model of visceral leishmaniasis coupled to a user-friendly simulation interface. The tool will be used to predict more effective combined therapies for use in patients suffering from leishmaniasis, as well as supporting the identification of new chemo- and immuno-therapeutic targets, and will help to understand how confounding factors such as malnutrition and coinfection may influence drug and vaccine efficacy.
Summary: CRACK-IT-1 was designed to provide tissue transcriptomic data and histopathology on the early response of BALB/c mice to Leishmania donovani infection in the presence or absence of drug treatment.
Staining: Frozen sections. H&E
For further detail: CRACK_IT_1ExpData
Summary: CRACK-IT-1 was designed to provide tissue transcriptomic data and histopathology on the late response of BALB/c mice to Leishmania donovani infection in the presence or absence of drug treatment.
Staining: Frozen sections. H&E
For further detail: CRACK_IT_2ExpData