Mycobacterium leprae is able to infect adipocytes, inducing lipolysis and modulating the immune response.

Leprosy is a chronic infectious disease caused by the intracellular bacillus Mycobacterium leprae (M. leprae), which is known to infect skin macrophages and Schwann cells. Although adipose tissue is a recognized site of Mycobacterium tuberculosis infection, its role in the histopathology of leprosy was, until now, unknown. We analyzed the M. leprae capacity to infect and persist inside adipocytes, characterizing the induction of a lipolytic phenotype in adipocytes, as well as the effect of these infected cells on macrophage recruitment. We evaluated 3T3-L1-derived adipocytes, inguinal adipose tissue of SWR/J mice, and subcutaneous adipose tissue biopsies of leprosy patients. M. leprae was able to infect 3T3-L1-derived adipocytes in vitro, presenting a strong lipolytic profile after infection, followed by significant cholesterol efflux. This lipolytic phenotype was replicated in vivo by M. leprae injection into mice inguinal adipose tissue. Furthermore, M. leprae was detected inside crown-like structures in the subcutaneous adipose tissue of multibacillary patients. These data indicate that subcutaneous adipose tissue could be an important site of infection, and probably persistence, for M. leprae, being involved in the modulation of the innate immune control in leprosy via the release of cholesterol, MCP-1, and adiponectin.

Prediction of the occurrence of leprosy reactions based on Bayesian networks.

Introduction: Leprosy reactions (LR) are severe episodes of intense activation of the host inflammatory response of uncertain etiology, today the leading cause of permanent nerve damage in leprosy patients. Several genetic and non-genetic risk factors for LR have been described; however, there are limited attempts to combine this information to estimate the risk of a leprosy patient developing LR. Here we present an artificial intelligence (AI)-based system that can assess LR risk using clinical, demographic, and genetic data. Methods: The study includes four datasets from different regions of Brazil, totalizing 1,450 leprosy patients followed prospectively for at least 2 years to assess the occurrence of LR. Data mining using WEKA software was performed following a two-step protocol to select the variables included in the AI system, based on Bayesian Networks, and developed using the NETICA software. Results: Analysis of the complete database resulted in a system able to estimate LR risk with 82.7% accuracy, 79.3% sensitivity, and 86.2% specificity. When using only databases for which host genetic information associated with LR was included, the performance increased to 87.7% accuracy, 85.7% sensitivity, and 89.4% specificity. Conclusion: We produced an easy-to-use, online, free-access system that identifies leprosy patients at risk of developing LR. Risk assessment of LR for individual patients may detect candidates for close monitoring, with a potentially positive impact on the prevention of permanent disabilities, the quality of life of the patients, and upon leprosy control programs.

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14 C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial-Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy.

Antimicrobial Resistance among Leprosy Patients in Brazil: Real-World Data Based on the National Surveillance Plan.

Brazil ranks second among countries for new cases and first for relapse cases of leprosy worldwide. The Mycobacterium leprae Resistance Surveillance Plan was established. We aimed to present the results of a 2-year follow-up of the National Surveillance Plan in Brazil. A cross-sectional study of leprosy cases was performed to investigate antimicrobial resistance (AMR) in Brazil from October 2018 to September 2020. Molecular screening targeting genes related to dapsone (folP1), rifampin (rpoB), and ofloxacin resistance (gyrA) was performed. During the referral period, 63,520 active leprosy patients were registered in Brazil, and 1,183 fulfilled the inclusion criteria for molecular AMR investigation. In total, only 16 (1.4%) patients had genetic polymorphisms associated with AMR. Of these, 8 (50%) had cases of leprosy relapse, 7 (43.8%) had cases of suspected therapeutic failure with standard treatment, and 1 (6.2%) was a case of new leprosy presentation. M. leprae strains with AMR-associated mutations were found for all three genes screened. Isolates from two patients showed simultaneous resistance to dapsone and rifampin, indicating multidrug resistance (MDR). No significant relationship between clinical variables and the presence of AMR was identified. Our study revealed a low frequency of AMR in Brazil. Isolates were resistant mainly to dapsone, and a very low number of isolates were resistant to rifampin, the main bactericidal agent for leprosy, or presented MDR, reinforcing the importance of the standard World Health Organization multidrug therapy. The greater frequency of AMR among relapsed patients supports the need to constantly monitor this group.

Drug resistance in leprosy: an update following 70 years of chemotherapy

Leprosy is one of the oldest infectious diseases, reported for more than 2000 years. Leprosy elimination goal as a public health problem set by the World Health Organization, aiming for a global prevalence rate < 1 patient in a population of 10,000, was achieved in 2000 mainly thanks to the worldwide use of leprosy drugs starting in the 1980s and their access at no cost for patients since 1995. However, around 200,000 new cases are still reported each year, particularly in India, Brazil, and Indonesia. As with other bacteria of medical interest, antimicrobial resistance is observed in Mycobacterium leprae strains in several parts of the world, despite multidrug therapy being the recommended standard leprosy treatment to avoid resistance selection since 1982. Therefore, identifying and monitoring resistance is necessary. We provide an overview of the historical facts that led to the current drug resistance situation, the antibiotics effective against M. leprae, their mechanisms of action and resistance, and resistance detection methods. We also discuss therapeutic management of the resistant cases, new genes with potential roles in drug resistance and bacterial adaptation, new drugs under investigation, and the risk for resistance selection with the chemoprophylaxis measures.

Antimicrobial resistance among leprosy patients in Brazil: real-world data based on the National Surveillance plan

Brazil ranks second among countries for new cases and first for relapse cases of leprosy worldwide. The Mycobacterium leprae Resistance Surveillance Plan was established. We aimed to present the results of a 2-year follow-up of the National Surveillance Plan in Brazil. A cross-sectional study of leprosy cases was performed to investigate antimicrobial resistance (AMR) in Brazil from October 2018 to September 2020. Molecular screening targeting genes related to dapsone (folP1), rifampin (rpoB), and ofloxacin resistance (gyrA) was performed. During the referral period, 63,520 active leprosy patients were registered in Brazil, and 1,183 fulfilled the inclusion criteria for molecular AMR investigation. In total, only 16 (1.4%) patients had genetic polymorphisms associated with AMR. Of these, 8 (50%) had cases of leprosy relapse, 7 (43.8%) had cases of suspected therapeutic failure with standard treatment, and 1 (6.2%) was a case of new leprosy presentation. M. leprae strains with AMR-associated mutations were found for all three genes screened. Isolates from two patients showed simultaneous resistance to dapsone and rifampin, indicating multidrug resistance (MDR). No significant relation ship between clinical variables and the presence of AMR was identified. Our study revealed a low frequency of AMR in Brazil. Isolates were resistant mainly to dapsone, and a very low number of isolates were resistant to rifampin, the main bactericidal agent for leprosy, or presented MDR, reinforcing the importance of the standard World Health Organization multidrug therapy. The greater frequency of AMR among relapsed patients supports the need to constantly monitor this group

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial­Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy. (AU)

TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells.

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human ß-defensin-2 (hßD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.

Large-Scale Gene Expression Signatures Reveal a Microbicidal Pattern of Activation in Mycobacterium leprae-Infected Monocyte-Derived Macrophages With Low Multiplicity of Infection.

Leprosy is a disease with a clinical spectrum of presentations that is also manifested in diverse histological features. At one pole, lepromatous lesions (L-pole) have phagocytic foamy macrophages heavily parasitized with freely multiplying intracellular Mycobacterium leprae. At the other pole, the presence of epithelioid giant cells and granulomatous formation in tuberculoid lesions (T-pole) lead to the control of M. leprae replication and the containment of its spread. The mechanism that triggers this polarization is unknown, but macrophages are central in this process. Over the past few years, leprosy has been studied using large scale techniques to shed light on the basic pathways that, upon infection, rewire the host cellular metabolism and gene expression. M. leprae is particularly peculiar as it invades Schwann cells in the nerves, reprogramming their gene expression leading to a stem-like cell phenotype. This modulatory behavior exerted by M. leprae is also observed in skin macrophages. Here, we used live M. leprae to infect (10:1 multiplicity of infection) monocyte-derived macrophages (MDMs) for 48 h and analyzed the whole gene expression profile using microarrays. In this model, we observe an intense upregulation of genes consistent with a cellular immune response, with enriched pathways including peptide and protein secretion, leukocyte activation, inflammation, and cellular divalent inorganic cation homeostasis. Among the most differentially expressed genes (DEGs) are CCL5/RANTES and CYP27B1, and several members of the metallothionein and metalloproteinase families. This is consistent with a proinflammatory state that would resemble macrophage rewiring toward granulomatous formation observed at the T-pole. Furthermore, a comparison with a dataset retrieved from the Gene Expression Omnibus of M. leprae-infected Schwann cells (MOI 100:1) showed that the patterns among the DEGs are highly distinct, as the Schwann cells under these conditions had a scavenging and phagocytic gene profile similar to M2-like macrophages, with enriched pathways rearrangements in the cytoskeleton, lipid and cholesterol metabolism and upregulated genes including MVK, MSMO1, and LACC1/FAMIN. In summary, macrophages may have a central role in defining the paradigmatic cellular (T-pole) vs. humoral (L-pole) responses and it is likely that the multiplicity of infection and genetic polymorphisms in key genes are gearing this polarization.

Improving treatment outcomes for leprosy in Pernambuco, Brazil: a qualitative study exploring the experiences and perceptions of retreatment patients and their carers.

BACKGROUND: Brazil has a high leprosy burden and poor treatment outcomes (TOs), manifesting in high relapse rates. Pernambuco, an impoverished Brazilian state suffering notable geographical health inequalities, has 'hyperendemic' leprosy. Although current literature identifies barriers and facilitators influencing leprosy treatment compliance, inadequate investigation exists on other factors influencing TOs, including carers' roles and psycho-dermatological impact. This qualitative study explores experiences and perceptions of leprosy patients and their carers in Pernambuco, Brazil; to identify location-specific factors influencing TOs, and consequently inform future management. METHODS: 27, semi-structured, in-depth interviews were conducted with 14 patients and 13 carers. Participants were recruited using maximum variation and snowball sampling from three clinics in Petrolina, Pernambuco. Transcripts and field notes from both participant groups were separately analysed using conventional thematic and deviant case analysis. The University of Birmingham Internal Research Ethics Committee and Instituto Lauro de Souza Lima provided ethical approval. RESULTS: Two homologous sets of four, primary, interdependent themes influencing leprosy TOs emerged: 'personal factors'; 'external factors'; 'clinical factors'; and 'the healthcare professional (HCP)-patient-carer relationship'. Poor participant knowledge and lack of symptomatic relief caused patients to distrust treatment. However, because participants thought HCP-led interventions were vital for optimal TOs, patients were effectively persuaded to adhere to pharmaceutical treatments. High standard patient and population education facilitated treatment engagement by encouraging evidence-based medicine belief, and dispelling health myths and stigma. Healthcare, on occasions, was perceived as disorganised, particularly in resource-scarce rural areas, and for those with mental health needs. Participants additionally experienced incorrect/delayed diagnoses and poor contact tracing. Leprosy's negative socio-economic impact on employment - together with stigma, dependency and changing relationships - caused altered senses of identity, negatively impacting TOs. Better dialogue between patients, HCPs and carers facilitated individualised patient support. CONCLUSION: This study highlights the importance of: effective evidence-based leprosy education; communication between HCPs, patients and carers; state-funded support; and healthcare resource distribution. These findings, if prioritised on governmental scales, provide the valuable insight needed to inform location-specific management strategies, and consequently improve TOs. Future research should evaluate the effectiveness of these implementations. Failure to address these findings will hinder regional elimination efforts.

A systematic review and meta-analysis of studies on the diagnostic accuracy and screening of tests to detect antimicrobial resistance in leprosy.

Although multidrug therapy is considered an effective treatment for leprosy, antimicrobial resistance is a serious concern. We performed a systematic review of studies on the diagnostic accuracy and screening of tests for antimicrobial resistance in leprosy. This review was registered in PROSPERO (CRD42020177958). In April 2020, we searched for studies in the PubMed, EMBASE, Web of Science, Scopus, Scielo, and LILACS databases. A random effects regression model was used for the meta-analysis. We included 129 studies. Molecular tests for dapsone resistance had a sensitivity of 78.8% (95% confidence interval [CI] = 65.6-87.9) and a specificity of 97.0% (95% CI = 94.0-98.6). Molecular tests for rifampicin resistance had a sensitivity and specificity of 88.7% (95% CI = 80.0-93.9) and 97.3% (95% CI = 94.3-98.8), respectively. Molecular tests for ofloxacin resistance had a sensitivity and specificity of 80.9% (95% CI = 60.1-92.3) and 96.1% (95% CI = 90.2-98.5), respectively. In recent decades, no increase in the resistance proportion was detected. However, the growing number of resistant cases is still a clinical concern.

TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-kB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human b-defensin-2 (hbD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-kB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.(AU)

Multidrug therapy for leprosy can cure patients with lobomycosis in Acre State, Brazil: a proof of therapy study

Lobomycosis, also referred to as lacaziosis, is an endemic cutaneous and subcutaneous fungal disease that mainly affects Amazonian forest dwellers in Brazil. There is no disease control program in place in Brazil, and antifungal therapy failures are common, and the therapy is inaccessible to most patients. We performed a randomized, unblinded clinical trial testing the cure rate of multiple drug therapy (MDT) for leprosy with surgical excision, with or without itraconazole. A control arm consisted of patients who did not adhere to either therapeutic regimens but continued to be followed up. Multiple drug therapy consisted of monthly supervised doses of 600 mg rifampicin, 300 mg clofazimine, and 100 mg dapsone, in addition to daily doses of 50 mg clofazimine and 100 mg dapsone. The patients in the MDT plus itraconazole arm also received itraconazole 100 mg twice daily. We followed up 54 patients from the MDT group and 26 patients from the MDT plus itraconazole group for an average of 4 years and 9 months. The 23 controls were followed up for 6 months on average. The following endpoints were observed: 1) unchanged (no apparent improvement), 2) improved (reduction in lesion size and/or pruritus), and 3) cured (complete remission of the lesions, no viable fungi, and no relapse for 2 years after the end of the drug treatment). The results indicated a significantly greater likelihood of cure associated with the use of multidrug therapy for leprosy with or without itraconazole when compared with the control group. The addition of itraconazole to MDT was not associated with improved outcomes, suggesting that MDT alone is effective(AU).

A systematic review and meta-analysis of studies on the diagnostic accuracy and screening of tests to detect antimicrobial resistance in leprosy

Although multidrug therapy is considered an effective treatment for leprosy, antimicrobial resistance is a serious concern. We performed a systematic review of studies on the diagnostic accuracy and screening of tests for antimicrobial resistance in leprosy. This review was registered in PROSPERO (CRD42020177958). In April 2020, we searched for studies in the PubMed, EMBASE, Web of Science, Scopus, Scielo, and LILACS databases. A random effects regression model was used for the meta-analysis. We included 129 studies. Molecular tests for dapsone resistance had a sensitivity of 78.8% (95% confidence interval [CI] = 65.6−87.9) and a specificity of 97.0% (95% CI = 94.0−98.6). Molecular tests for rifampicin resistance had a sensitivity and specificity of 88.7% (95% CI = 80.0−93.9) and 97.3% (95% CI = 94.3−98.8), respectively. Molecular tests for ofloxacin resistance had a sensitivity and specificity of 80.9% (95% CI = 60.1−92.3) and 96.1% (95% CI = 90.2−98.5), respectively. In recent decades, no increase in the resistance proportion was detected. However, the growing number of resistant cases is still a clinical concern(AU).

Large-scale gene expression signatures reveal a microbicidal pattern of activation in Mycobacterium leprae-infected monocyte-derived macrophages with low multiplicity of infection

Leprosy is a disease with a clinical spectrum of presentations that is also manifested in diverse histological features. At one pole, lepromatous lesions (L-pole) have phagocytic foamy macrophages heavily parasitized with freely multiplying intracellular Mycobacterium leprae. At the other pole, the presence of epithelioid giant cells and granulomatous formation in tuberculoid lesions (T-pole) lead to the control of M. leprae replication and the containment of its spread. The mechanism that triggers this polarization is unknown, but macrophages are central in this process. Over the past few years, leprosy has been studied using large scale techniques to shed light on the basic pathways that, upon infection, rewire the host cellular metabolism and gene expression. M. leprae is particularly peculiar as it invades Schwann cells in the nerves, reprogramming their gene expression leading to a stem-like cell phenotype. This modulatory behavior exerted by M. leprae is also observed in skin macrophages. Here, we used live M. leprae to infect (10:1 multiplicity of infection) monocyte-derived macrophages (MDMs) for 48 h and analyzed the whole gene expression profile using microarrays. In this model, we observe an intense upregulation of genes consistent with a cellular immune response, with enriched pathways including peptide and protein secretion, leukocyte activation, inflammation, and cellular divalent inorganic cation homeostasis. Among the most differentially expressed genes (DEGs) are CCL5/RANTES and CYP27B1, and several members of the metallothionein and metalloproteinase families. This is consistent with a proinflammatory state that would resemble macrophage rewiring toward granulomatous formation observed at the T-pole. Furthermore, a comparison with a dataset retrieved from the Gene Expression Omnibus of M. leprae-infected Schwann cells (MOI 100:1) showed that the patterns among the DEGs are highly distinct, as the Schwann cells under these conditions had a scavenging and phagocytic gene profile similar to M2-like macrophages, with enriched pathways rearrangements in the cytoskeleton, lipid and cholesterol metabolism and upregulated genes including MVK, MSMO1, and LACC1/FAMIN. In summary, macrophages may have a central role in defining the paradigmatic cellular (T-pole) vs. humoral (L-pole) responses and it is likely that the multiplicity of infection and genetic polymorphisms in key genes are gearing this polarization.

Reductive power generated by Mycobacterium leprae through cholesterol oxidation contributes to lipid and ATP synthesis

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

Multidrug Therapy for Leprosy Can Cure Patients with Lobomycosis in Acre State, Brazil: A Proof of Therapy Study.

Lobomycosis, also referred to as lacaziosis, is an endemic cutaneous and subcutaneous fungal disease that mainly affects Amazonian forest dwellers in Brazil. There is no disease control program in place in Brazil, and antifungal therapy failures are common, and the therapy is inaccessible to most patients. We performed a randomized, unblinded clinical trial testing the cure rate of multiple drug therapy (MDT) for leprosy with surgical excision, with or without itraconazole. A control arm consisted of patients who did not adhere to either therapeutic regimens but continued to be followed up. Multiple drug therapy consisted of monthly supervised doses of 600 mg rifampicin, 300 mg clofazimine, and 100 mg dapsone, in addition to daily doses of 50 mg clofazimine and 100 mg dapsone. The patients in the MDT plus itraconazole arm also received itraconazole 100 mg twice daily. We followed up 54 patients from the MDT group and 26 patients from the MDT plus itraconazole group for an average of 4 years and 9 months. The 23 controls were followed up for 6 months on average. The following endpoints were observed: 1) unchanged (no apparent improvement), 2) improved (reduction in lesion size and/or pruritus), and 3) cured (complete remission of the lesions, no viable fungi, and no relapse for 2 years after the end of the drug treatment). The results indicated a significantly greater likelihood of cure associated with the use of multidrug therapy for leprosy with or without itraconazole when compared with the control group. The addition of itraconazole to MDT was not associated with improved outcomes, suggesting that MDT alone is effective.