• Research priorities: Diagnostic tests
• Country: Brazil
• Project no.: 706.18.57
• Budget: €69,946
• Duration: June 2018 - October 2020
• Status: Completed

Full project title:
Using plasma metabolomics to increase our understanding of metabolic changes in leprosy and associated reactions: A proof-of-concept study.

Project Coordination
Emory University School of Medicine, USA

Partner
Faculdade da Saúde e Ecologia Humana (FASEH), Brazil

Aim: There are many knowledge gaps surrounding leprosy transmission, diagnostics and morbidity management. This research project studied the interaction between the host (humans) and the pathogen (Mycobacterium leprae) by looking for “metabolic signatures” using innovative methods.

Final project summary:
High resolution metabolomics (HRM) has led to better understanding of host-pathogen interactions of many infectious diseases but has rarely been used in studies of metabolism in leprosy. Thousands of small molecules can be detected through HRM and advanced data extraction. Our objectives were to identify unique metabolic signatures related to leprosy presentation and to explore possible links between nutrients, host metabolism and disease manifestations. Between June 2018 and December 2019, adults newly diagnosed with leprosy and healthy controls were recruited from leprosy referral clinics in Minas Gerais, Brazil. Plasma samples were analyzed at the Clinical Biomarkers Laboratory at Emory University, Atlanta GA. Metabolites were detected using an established HRM workflow and characterized by accurate mass m/z and retention time. The mummichog informatics package was used to compare metabolic pathway activity between groups. Additionally, select individual metabolites were quantified and compared. Analyses was controlled for age and sex of study participants. Forty-two individuals with leprosy were enrolled, of which 26 (62%) were multibacillary (MB) and 16 (38%) werepaucibacillary (PB). Persons with leprosy were compared to 25 asymptomatic controls (9 community controls and 16 household contacts). We found statistically significant differences in arachidonic acid and prostaglandin metabolism as well as differences in vitamin D, retinol, carnitine shuttle,  and tryptophan metabolism. Our findings suggest interdependency of these pathways at the intersection of metabolismand immunity and strongly support further investigations of these metabolic signatures that can greatly increase our understanding of the underlying pathophysiology of leprosy. However, the in vivo, real world data that plasma HRM can provide is critical to compliment the invitro mechanistic studies. As described, many of these pathways are highly interdependent and the output provides further avenues of research both within HRM as well as other methods.

In summary, this study described many metabolic differences across healthy controls, MB leprosy and PB leprosy especially in regards to fatty acids, energy metabolism, micronutrients, and tryptophan metabolism. These findings indicate that future studies investigating the intersection of host metabolism and the immune response to M. leprae are warranted. Furthermore, understanding metabolic differencesin persons with leprosy and asymptomatic persons with evidence of prior exposure would enhance our ability to understand the pathophysiology of disease and identify biomarkers that differentiate “active” and “latent” disease. Further prospective well-controlled and well-powered studies using HRM in individuals susceptible to leprosy, may be useful to identify metabolites or metabolic profiles that could eventually be adapted as plasma biomarkers of leprosy and predict progression of this ancient disease. In a disease with anunculturable pathogen and long incubation period, HRM clearly shows a path to better understanding of the disease that will advanceearlier diagnosis, prevention, and treatment.

Impact

High-Resolution Plasma Metabolomics Identifies Alterations in Fatty Acid, Energy, and Micronutrient Metabolism in Adults across the Leprosy Spectrum. Fairley J, Ferreira J, Fraga L, et al. The Journal of infectious diseases. 2023;

ASTMH 2021 annualmeeting: oralpresentation: High resolutionmetabolomics highlightdifferences in lipid andnutritional metabolismacross the leprosyspectrum providingavenue for advances inleprosy host-pathogenresearch; Jessica K.Fairley, José A. Ferreira,Thomas R. Ziegler, DeanP. Jones, Lucia A. Fraga,Sandra Lyon, Jeffrey M.Collins

• Research priorities: Diagnostic tests
• Country: Brazil
• Budget: € 51,652 | Project number: 706.18.39
• Duration: February 2018 - July 2024
• Status: Ongoing

Two variants in the LRRK2 gene were found to be strong candidates to control leprosy susceptibility in a unique family affected by leprosy. By infecting macrophages in vitro with M. leprae, this genetic susceptibility will be further studied.

Functional analysis of candidate variants in the early-onset leprosy phenotype using a novel cellular model

Project Coordination

Pontifical Catholic University of Parana, Brazil (PUCPR)

Partners

• Federal University of Piaui, Brazil (UFPI)
• University of Sao Paulo, Brazil (USP)
• McGill University, Canada

Project summary

Leprosy is an ancient disease and still is a public health problem in South American, African and Asian countries, in which around 200,000 new individuals are affected every year. Over the past decades, intensive research has resulted in strong evidence for the existence of an innate, genetic component controlling susceptibility to leprosy; however, the exact mechanisms involved is not yet fully understood. In previous studies by the investigator, the entire genome of members of a family presenting a rare leprosy case was sequenced: the disease manifested in two twin sisters at the very early age of 22 months. Also, their father and grandmother were affected, the only unaffected member was the mother. Complex computational analysis reveal two variants in a gene named LRRK2 as strong candidates to control leprosy susceptibility in the studied family; interestingly, several previous studies also have shown LRRK2 function in controlling immune response, and previous genetic studies have already associated LRRK2 variants with leprosy. Although the genetic results are promising, functional validation is necessary. In this study, the investigators will obtain and modify cells from the skin of affected and unaffected family members to generate the LRRK2 variants. Next, these cells will be reprogrammed and differentiated into M. leprae cells target, such as macrophages. The macrophages will be infected in vitro with M. leprae and cellular events will be evaluated to inquire how such variants could contribute to leprosy susceptibility. Results may contribute to a better understanding of the molecular basis of the etiology of leprosy.

• Grant: LRI Regular Grant
• Research priorities: Diagnostic tests
• Country: India, USA
• Project no.: 704.16.59
• Budget: € 110,000
• Duration: January 2017 - December 2018
• Status: Completed
• Co-funding partners: Turing Foundation

Full project title:
Biomarkers for early detection of leprosy using comparative transcriptomics

Project coordination
ICMR-National Institute of Research in Tribal Health

Partner
National Hansen's Disease Program (USA)

Aim: This project aimed to better understand how some infected people can resist an infection with the leprosy bacteria while others develop the disease.

Final project summary
Leprosy diagnosis is mainly guided by clinical symptoms (loss of sensation due to nerve damage) and positive smear from lesions, which is usually a late stage until when the transmission continues to other healthy contacts and community members. Microbial pathogens possess specific virulence factors which in combination with other molecules (called Pathogen Associated Molecular Patterns or PAMPs), elicit a characteristic gene-expression pattern (referred as transcriptional profile) that determines a differential outcome (disease or clearance) of an infection in different hosts. The main research quest is to understand “how some infected people can resist M. leprae infection while others progress to disease?”

Laboratory diagnosis of leprosy is challenging due to complex factors related to host genetics. The nine-banded armadillo is the only available animal model to study leprosy progression and pathogenesis with defined duration and dose of M. leprae infection. Interestingly, armadillos also exhibit differential susceptibility to leprosy, with ~20% animals being able to resist experimental inoculation of M. leprae. Thus, in the present study, the research team has used armadillo model for identifying the biomarkers of leprosy progression by comparing the gene expression profiles of leprosy-susceptible and -resistant animals after experimental infection of leprosy bacilli. Peripheral blood mononuclear cells (PBMCs) collected and cryo-preserved at the 4th and 18th month after infection from the leprosy-resistant and -susceptible animals were revived and stimulated with M. leprae antigens and their transcriptome profiles were compared using RNA-Seq & bioinformatics tools. This analysis has identified differentially expressed genes (DEGs). It was noticed that these gene-expression profiles were able to differentiate the animals according to their differential susceptibility to leprosy, including one animal which has an intermediate level of susceptibility and indeed clustered separately between the groups of the resistant and susceptible animals. Bioinformatic analysis and comparison of differentially expressed genes by literature mining was performed. In addition, the gene expression pattern observed at the 4th month time-point had remarkable similarities with that observed at the 18th month time-point, indicating that the gene-expression profiles can potentially predict the disease even during the pre-symptomatic stages of leprosy, and thus can serve as valuable biomarkers for early detection of the disease.

In this study, the researchers have identified a list of candidate pathways and genes (IDO-1, C-X-C motif chemokine ligand CXCL9/CXCL10, CD34 andIL10 etc.) which could be playing important role in leprosy pathogenesis and progression. Particularly, the IDO1 has been associated with immunosuppressive activity and may be linked to the poor cell-mediated immunity observed in lepromatous leprosy cases. The results were also confirmed using quantitative-PCR. Many of these genes have been previously implicated in leprosy pathogenesis (such as C-X-Cmotif chemokine ligand CXCL9 /CXCL10, myelin protein zero MPZ etc.) and have been found to be differentially expressed upon infection in different studies using clinical samples or cell-line based experiments. A significant number of DEGs and pathways are involved in host innate immunity and neurological processes, consistent with leprosy pathogenesis and neuro-predilection of M. leprae. The analysis of the affected pathways has revealed gene networks which are associated with leprosy pathogenesis and disease progression in a susceptible host which can be potentially useful for early detection of leprosy, particularly in form of a point-of-care diagnostics, considering theirrobustness in different clinical specimens.

Impact

Sharma, M., & Singh, P. (2022). Advances in the diagnosis of leprosy. Frontiers in Tropical Diseases, 3, 893653.

Avanzi, C., Lécorché, E., Rakotomalala, F. A., Benjak, A., Rapelanoro Rabenja, F., Ramarozatovo, L. S., ... & Cole, S. T. (2020). Population genomics of Mycobacterium leprae reveals a new genotype in Madagascar and the Comoros. Frontiers in microbiology, 11, 711.

Schaub, R., Avanzi, C., Singh, P., Paniz-Mondolfi, A., Cardona-Castro, N., Legua, P., ... & de Thoisy, B. (2020). Leprosy transmission in Amazonian countries: current status and future trends. Current Tropical Medicine Reports, 7, 79-91.

Sharma, M., & Singh, P. (2022). Repurposing drugs to combat drug resistance in leprosy: A review of opportunities. Combinatorial Chemistry & High Throughput Screening, 25(10), 1578-1586.

Sharma, M., & Singh, P. (2022). Role of tlyA in the biology of uncultivable mycobacteria. Combinatorial Chemistry & High Throughput Screening, 25(10), 1587-1594.

Sharma, M., Gupta, Y., Dwivedi, P., Kempaiah, P., & Singh, P. (2021). Mycobacterium lepromatosis MLPM_5000 is a potential heme chaperone protein HemW and mis-annotation of its orthologues in mycobacteria. Infection, Genetics and Evolution, 94, 105015.

• Grant: LRI Regular Grant
• Research priorities: Diagnostic tests
• Country: Brazil
• Project no.: 703.15.45
• Budget: € 60,269
• Duration: August 2015 - July 2018
• Status: Completed

Full project title:
Evaluation of the qPCR in household contact monitoring

Project coordination
Laboratório de Hanseníase Fundação Oswaldo Cruz/ FIOCRUZ (Brazil)

Partners
Fundação Afredo da Mata (Brazil)
Universidade Federal de Juiz de Fora (Brazil)
Ludwig-Maximilians University (Germany)

Aim: There are no diagnostic tests that could predict whether or not a “contact” will develop the disease. This project investigated the possible use of M.leprae DNA detection as a early detection method in over 1600 household contacts of leprosy patients. 

Final project summary
Leprosy is a disease caused by a bacterium and proper diagnosis can completely cure patients, but clinical signs of the disease are sometimes difficult to detect. The bacteria infect skin and nerves and late diagnosis are generally associated with permanent nerve injuries. It is well known that the group of individuals at greatest risk are the family members and household contacts that live in close proximity to patients.

Since there is a lack of diagnostic tests that could predict whether or not a “contact” will develop the disease, the aim of this research project was to provide a better way of diagnosis. In the past few years, novel technologies to amplify the causative agent, Mycobacterium leprae, of leprosy DNA are available and these tests are becoming more accurate, faster and cheaper. This project has investigated the use of quantitative PCR and PGL-I serology in monitoring household contacts right after the diagnosis of the index case and followed them for at least three years.

The researchers were able to perform examination of a group of more than 2,000 people who were at risk to develop leprosy because they lived in the household of a patient with active disease. Approximately 1,600 individuals were tested for the presence of the bacterial DNA and 955 followed-up. The results so far indicate that qPCR from ear lobes slit skin smear is not recommended to estimate risk of leprosy, but qPCR is very helpful increasing the precision of diagnosis of household contacts when they present suspected lesions, since skin biopsies were performed for confirmation and qPCR was complementary to histopathological analysis.

Impact

Quantitative polymerase chain reaction in paucibacillary leprosy diagnosis: A follow-up study. Barbieri RR, Manta F, Moreira SJ M, et al. PLoS neglected tropical diseases. 2019; 13 (3) : e0007147