• 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 annual meeting - oral presentation: 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

• Grant: LRI Regular Grant
• Budget round: 2018
• Research priorities: Diagnostic tests
• Country: Brazil
• Project no.: 706.18.39
• Budget: € 51,652
• Duration: February 2018 - July 2024
• Status: Completed

Project Coordination
Pontifical Catholic University of Parana, Brazil (PUCPR)

Partners
Federal University of Piaui, Brazil 
University of Sao Paulo, Brazil 
McGill University, Canada

Aim: 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.

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

Final project summary:
Leprosy is an infectious disease caused by Mycobacterium leprae, but exposure to the bacteria alone does not explain why some people develop the disease while others remain unaffected. Scientists have long known that the body's immune response plays a critical role in determining susceptibility to leprosy, yet the genetic factors underlying this response are not fully understood.

A unique case in northeastern Brazil provided researchers with an extraordinary opportunity to explore the genetic basis of leprosy. The study focused on a family from Teresina, Piauí, including a pair of identical twin girls who were diagnosed with leprosy at just 22 months of age. This is highly unusual, as leprosy typically has a long incubation period and is rarely diagnosed in very young children. The occurrence of the disease in identical twins at such an early age suggested that inherited genetic factors could play a significant role in their susceptibility.

To investigate this possibility, researchers sequenced the genomes of family members and identified important variations in the LRRK2 gene, a gene that has previously been linked to leprosy susceptibility in different populations around the world. The LRRK2 protein is involved in regulating inflammation and immune responses, helping the body respond to infection. Additional evidence suggested that a variation in another immune-related gene, NOD2, might also contribute to disease development and influence how the immune system responds to leprosy infection.

The study aimed to better understand how these genetic variations affect human immune cells and contribute to the development of leprosy.

To achieve this, researchers used advanced cellular and genetic technologies. They generated induced pluripotent stem cells (iPSCs), which can be reprogrammed to develop into different cell types, and applied gene-editing techniques to study the effects of specific genetic variants. In parallel, they compared cells obtained from different family members based on their natural genetic makeup. The team also examined immune cells called macrophages from individuals with leprosy as well as from healthy individuals who carried the same genetic variants identified in the family.

The research revealed that variations in the LRRK2 gene, and potentially in NOD2, significantly influence how immune cells behave when exposed to biological challenges. The findings confirmed observations previously made in laboratory mouse models and demonstrated that similar mechanisms are active in human cells.

Several important cellular functions were affected by these genetic changes. Researchers observed alterations in:

• Apoptosis (programmed cell death), an essential process that helps the body eliminate infected or damaged cells.
• Production of reactive oxygen species (ROS), molecules that play an important role in destroying pathogens.
• Expression of LRRK2 and NOD2 genes, which are involved in coordinating immune responses.
• Mitochondrial metabolism, affecting the function of mitochondria, the energy-producing structures within cells that also play a critical role in immunity.

Together, these findings provide new insights into how genetic variations may influence the body's ability to respond to Mycobacterium leprae infection and help explain why certain individuals are more vulnerable to developing leprosy.

An additional innovation emerging from the project was the identification of urinary stem cells (USCs) as a promising new model for leprosy research. These cells can be collected non-invasively and may offer an accessible platform for future studies involving gene editing, cell reprogramming, and investigations of disease mechanisms. The development of this model has the potential to accelerate research into leprosy and related immune disorders.

Beyond its scientific discoveries, the project helped strengthen research capacity in Brazil. The work fostered new collaborations and expanded expertise in cell and molecular biology at the Pontifical Catholic University of Paraná (PUCPR), creating opportunities for future research into the genetic and immunological mechanisms of infectious diseases.

This study highlights the important role of genetics in determining susceptibility to leprosy and provides new evidence that variations in the LRRK2 gene can significantly influence immune cell function. By deepening our understanding of how genetic factors shape the body's response to infection, the research contributes to a growing body of knowledge that may ultimately support earlier diagnosis, improved risk assessment, and the development of more personalized approaches to leprosy prevention and treatment.

The findings also demonstrate how cutting-edge technologies such as stem cell modelling, genome sequencing, and gene editing can help unravel the complex interactions between human genetics and infectious diseases, opening new pathways for scientific discovery and innovation in leprosy research.

Impact

Dallmann-Sauer, M., Xu, Y. Z., da Costa, A. L. F., Tao, S., Gomes, T. A., Prata, R. B. D. S., ... & Schurr, E. (2023). Allele-dependent interaction of LRRK2 and NOD2 in leprosy. PLoS Pathogens, 19(3), e1011260.

• 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
• Budget round: 2015
• Research priorities: Diagnostic tests
• Country: Brazil
• Project no.: 703.15.45
• Budget: € 60,269
• Duration: August 2015 - April 2019
• Status: Completed

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. 

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

Final project summary:
Early diagnosis remains one of the greatest challenges in leprosy control. Because there is no single highly accurate diagnostic test for leprosy, diagnosis depends largely on clinical examination. As a result, many patients are only diagnosed after the disease has progressed and symptoms such as nerve impairment have become apparent. This contributes to ongoing transmission and a persistently stable number of new cases.

Household contacts of people affected by leprosy are known to have the highest risk of developing the disease. However, there are currently no reliable markers to predict which contacts are most likely to progress to leprosy. This study investigated whether two laboratory tests—quantitative polymerase chain reaction (qPCR) and anti-PGL-I serology—could help identify contacts at increased risk and improve early diagnosis.

The study followed household contacts attending a reference centre in Rio de Janeiro, Brazil, between 2011 and 2018. In total, 2,437 household contacts were examined. During the first contact surveillance visit, 54 individuals (2.2%) were diagnosed with leprosy through clinical examination. Among the remaining contacts, 25 people presented with skin lesions that were difficult to diagnose. These individuals underwent further investigation using skin biopsy, histopathology, and qPCR testing. Laboratory investigations confirmed leprosy in eight individuals, while the remaining seventeen were diagnosed with other dermatological conditions.

To evaluate the ability of qPCR and anti-PGL-I serology to predict disease progression, a cohort of 955 apparently healthy household contacts recruited between 2011 and 2015 was followed for a minimum of three years and a maximum of seven years. Samples collected included slit-skin smears from the ear lobes for qPCR analysis and blood samples for anti-PGL-I testing.

During follow-up, only five of the 955 contacts (0.5%) developed leprosy. Although a positive qPCR result from ear-lobe samples was associated with an increased relative risk of developing the disease, the test showed low sensitivity. Only one of the five contacts who later developed leprosy had tested positive by qPCR at enrolment. These findings suggest that qPCR performed on ear-lobe slit-skin smears is not suitable as a predictive marker for disease progression among household contacts.

The study also assessed the prevalence of Mycobacterium leprae DNA and anti-PGL-I antibodies among household contacts. Nine percent of contacts tested positive by qPCR, while 12% tested positive for anti-PGL-I antibodies. However, only a small proportion tested positive with both methods. It was not possible to assess the predictive value of anti-PGL-I serology, as none of the contacts who later developed leprosy had tested positive at enrolment.

Although qPCR on ear-lobe samples was not useful for predicting future disease, the study demonstrated an important role for qPCR in supporting diagnosis. Among contacts with suspicious skin lesions, qPCR on skin biopsy samples proved valuable as an additional diagnostic tool alongside histopathological examination. Half of the contacts whose lesions were ultimately confirmed as leprosy tested positive by qPCR, and a positive qPCR result in skin tissue was strongly associated with a diagnosis of leprosy.

The introduction of qPCR into routine practice at the FIOCRUZ clinic also appeared to improve the identification of existing but previously unconfirmed cases among household contacts. Between 2011 and 2018, laboratory testing helped confirm eight additional leprosy cases that might otherwise have remained undiagnosed. This increased the detection of co-prevalent cases—contacts who already had leprosy at the time of screening—and was accompanied by a reduction in the proportion of new cases detected during later follow-up.

The study also identified age as an important factor associated with progression to leprosy. Older household contacts, particularly those over 60 years of age, were found to have a higher risk of developing the disease during follow-up, suggesting that this group may benefit from closer surveillance.

Overall, the findings indicate that routine qPCR testing of ear-lobe slit-skin smears should not be used to predict leprosy risk among household contacts. However, qPCR can be a valuable diagnostic aid when contacts present with suspicious skin lesions, particularly when used alongside histopathological examination of skin biopsy samples. The results also highlight the importance of continued contact surveillance, with particular attention to older household contacts who may be at increased risk of developing leprosy.

Impact

Barbieri, R. R., Manta, F. S., Moreira, S. J., Sales, A. M., Nery, J. A., Nascimento, L. P., ... & Moraes, M. O. (2019). Quantitative polymerase chain reaction in paucibacillary leprosy diagnosis: A follow-up study. PLoS neglected tropical diseases, 13(3), e0007147.

Manta, F. S., Barbieri, R. R., Moreira, S. J., Santos, P. T., Nery, J. A., Duppre, N. C., ... & Moraes, M. O. (2019). Quantitative PCR for leprosy diagnosis and monitoring in household contacts: A follow-up study, 2011–2018. Scientific reports, 9(1), 16675.