Polyunsaturated Fatty Acid-Derived Lipid Mediators as Potential Biomarkers for Leprosy Among Individuals with Asymptomatic Mycobacterium leprae Infection.

Intra-household contacts (HCs) of leprosy patients are at increased risk of infection by Mycobacterium leprae and about ∼5-10% will develop active disease. A prognostic tool to identify HCs with the greatest risk of progressing to active disease would enhance early leprosy diagnosis and optimize prophylactic intervention. Previous metabolomics studies suggest that host lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are potential biomarkers for leprosy. In this study, we investigated retrospective sera of leprosy HCs by liquid chromatography-mass spectrometry and enzyme-linked immunoassay to determine whether circulating levels of ω-3 and ω-6 PUFA metabolites were altered in HCs that developed leprosy (HCDL) in comparison to those that did not (HCNDL). Sera were collected from HCs at the time of index case diagnosis and before clinical signs/symptoms of leprosy. Our findings showed that HCDL sera exhibited a distinct metabolic profile in comparison to HCDNL. Specifically, arachidonic acid, leukotriene B4, 11-hydroxyeicosatetraenoic acid, prostaglandin D2, and lipoxin A4 were elevated in HCDL. In contrast, prostaglandin E2 levels were reduced in HCDL. The ω-3 PUFAs, docosahexaenoic acid, eicosapentaenoic acid, and the docosahexaenoic acid-derived resolvin D1 and maresin-1 were also elevated in HCDL individuals compared to HCNDL. Principal component analyses provided further evidence that lipid mediators could serve as an early biomarker for progression to active leprosy. A logistic model identified resolvin D1 and D2, and prostaglandin D2 as having the greatest potential for early detection of HCs that will manifest leprosy.

Reduced vitamin D receptor (VDR) and cathelicidin antimicrobial peptide (CAMP) gene expression contribute to the maintenance of inflammatory immune response in leprosy patients.

Leprosy is an infectious disease influenced by genetic, immunological, and environmental factors. Reduced gene expressions may be associated with the immunological response pattern and leprosy susceptibility. We investigated the direct and indirect effects of Vitamin D Receptor (VDR) and Cathelicidin Antimicrobial Peptide (CAMP) gene expressions on the serum levels of vitamin D, Cathelicidin, and cytokines in newly-diagnosed leprosy patients and post-six-months of multidrug therapy (MDT). Thirty-four leprosy patients were assessed, paucibacillary (PB; n = 14) and multibacillary (MB; n = 20) cases, untreated or having received six months of MDT, 18 healthy controls, and 25 household contacts. VDR and CAMP gene expression levels were strongly correlated to some important cytokines in both, untreated leprosy patients (PB, r = 0.9319; MB, r = 0.9569) and patients who had undergone MDT (PB, r = 0.9667; MB, r = 0.9569). We observed that both gene expressions directly influenced IL-2, IFN-γ, and IL-17F serum levels in leprosy patients compared to the household contacts and healthy individuals. VDR and CAMP gene expressions induced a persistent inflammatory response in PB and MB leprosy patients, even after six months of MDT, to fight the Mycobacterium leprae infection. Due to the persistent inflammatory profile, multidrug therapy is suggested to be maintained for more than six months, especially for MB patients. Vitamin D supplementation is recommended from the onset as a transcription factor to improve VDR and CAMP gene expression in leprosy patients.

Disease Burden and Current Therapeutical Status of Leprosy with Special Emphasis on Phytochemicals.

BACKGROUND: Leprosy (Hansen's disease) is a neglected tropical disease affecting millions of people globally. The combined formulations of dapsone, rifampicin and clofazimine (multidrug therapy, MDT) is only supportive in the early stage of detection, while "reemergence" is a significant problem. Thus, there is still a need to develop newer antileprosy molecules either of natural or semi-synthetic origin. OBJECTIVES: The review intends to present the latest developments in the disease prevalence, available therapeutic interventions and the possibility of identifying new molecules from phytoextracts. METHODS: Literature on the use of plant extracts and their active components to treat leprosy was searched. Selected phytoconstituents were subjected to molecular docking study on both wild and mutant types of the Mycobacterium leprae. Since the M. leprae dihydropteroate synthase (DHPS) is not available in the protein data bank (PDB), it was modelled by the homology model method and validated with the Ramachandran plot along with other bioinformatics approaches. Two mutations were introduced at codons 53 (Thr to Ile) and 55 (Pro to Leu) for docking against twenty-five selected phytoconstituents reported from eight plants that recorded effective anti-leprosy activity. The chemical structure of phytochemicals and the standard dapsone structure were retrieved from the PubChem database and prepared accordingly for docking study with the virtual-screening platform of PyRx-AutoDock 4.1. RESULTS: Based on the docking score (kcal/mol), most of the phytochemicals exhibited a higher docking score than dapsone. Asiaticoside, an active saponin (-11.3, -11.2 and -11.2 kcal/mol), was proved to be the lead phytochemical against both wild and mutant types DHPS. Some other useful phytoconstituents include echinocystic acid (-9.6, -9.5 and -9.5 kcal/mol), neobavaisoflavone (-9.2, -9.0 and -9.0 kcal/mol), boswellic acid (-8.90, -8.90 and -8.90 kcal/mol), asiatic acid (-8.9, -8.8 and -8.9 kcal/mol), corylifol A (-8.8, 8.0, and -8.0), etc. Overall, the computational predictions support the previously reported active phytoextracts of Centella asiatica (L.) Urban, Albizia amara (Roxb.) Boivin, Boswellia serrata Roxb. and Psoralea corylifolia L. to be effective against leprosy. CONCLUSION: A very small percentage of well-known plants have been evaluated scientifically for antileprosy activity. Further in vivo experiments are essential to confirm anti-leprosy properties of such useful phytochemicals.

Post-exposure prophylaxis (PEP) efficacy of rifampin, rifapentine, moxifloxacin, minocycline, and clarithromycin in a susceptible-subclinical model of leprosy.

BACKGROUND: Subclinical infection with Mycobacterium leprae is one potential source of leprosy transmission, and post-exposure prophylaxis (PEP) regimens have been proposed to control this source. Because PEP trials require considerable investment, we applied a sensitive variation of the kinetic mouse footpad (MFP) screening assay to aid in the choice of drugs and regimens for clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: Athymic nude mice were inoculated in the footpad (FP) with 6 x 103 viable M. leprae and treated by gastric gavage with a single dose of Rifampin (SDR), Rifampin + Ofloxacin + Minocycline (SD-ROM), or Rifapentine + Minocycline + Moxifloxacin (SD-PMM) or with the proposed PEP++ regimen of three once-monthly doses of Rifampin + Moxifloxacin (RM), Rifampin + Clarithromycin (RC), Rifapentine + Moxifloxacin (PM), or Rifapentine + Clarithromycin (PC). At various times post-treatment, DNA was purified from the FP, and M. leprae were enumerated by RLEP quantitative PCR. A regression analysis was calculated to determine the expected RLEP value if 99.9% of the bacilli were killed after the administration of each regimen. SDR and SD-ROM induced little growth delay in this highly susceptible murine model of subclinical infection. In contrast, SD-PMM delayed measurable M. leprae growth above the inoculum by 8 months. The four multi-dose regimens delayed bacterial growth for >9months post-treatment cessation. CONCLUSIONS/SIGNIFICANCE: The delay in discernable M. leprae growth post-treatment was an excellent indicator of drug efficacy for both early (3-4 months) and late (8-9 months) drug efficacy. Our data indicates that multi-dose PEP may be required to control infection in highly susceptible individuals with subclinical leprosy to prevent disease and decrease transmission.

Hypovitaminosis D and reduced cathelicidin are strongly correlated during the multidrug therapy against leprosy.

Mycobacterium leprae infection depends on the competence of the host immune defense to induce effective protection against this intracellular pathogen. The present study investigated the serum levels of vitamin D and the antimicrobial peptide cathelicidin, to determine the statistical correlation between them in leprosy patients before and post-six months of multidrug therapy (MDT), household contacts, and healthy individuals. Previous studies associated these molecules with high risks to develop mycobacterial diseases, such as tuberculosis and leprosy. A total of 34 leprosy patients [paucibacillary (n = 14), multibacillary (n = 20)], and 25 household contacts were recruited. Eighteen healthy adults were selected as a control group. Serum concentrations of vitamin D (25(OH)VD3) and cathelicidin were measured using high-performance liquid chromatography (HPLC), and an enzyme-linked immunosorbent assay (ELISA) kit, respectively. There were no significant differences in serum levels of 25(OH)VD3 between all groups, and the overall prevalence rate of vitamin D deficiency was 67.1%. Cathelicidin levels were significantly lower in both untreated and treated patients when compared to controls and household contacts (p < 0.05). Strong correlations between hypovitaminosis D and reduced cathelicidin in untreated (r = 0.86) and post-six months of MDT (r = 0.79) leprosy patients were observed. These results suggest that vitamin D status and cathelicidin levels are strongly correlated during multidrug therapy for leprosy and nutritional supplementation from the beginning of treatment could strengthen the immune response against leprosy.

Evidence for Mycobacterium leprae Drug Resistance in a Large Cohort of Leprous Neuropathy Patients from India.

Resistance to anti-leprosy drugs is on the rise. Several studies have documented resistance to rifampicin, dapsone, and ofloxacin in patients with leprosy. We looked for point mutations within the folP1, rpoB, and gyrA gene regions of the Mycobacterium leprae genome predominantly in the neural form of leprosy. DNA samples from 77 nerve tissue samples were polymerase chain reaction (PCR)-amplified for M leprae DNA and sequenced for drug resistance-determining regions of genes rpoB, folP1, and gyrA. The mean age at presentation and onset was 38.2 ± 13.4 (range 14-71) years and 34.9 ± 12.6 years (range 10-63) years, respectively. The majority had borderline tuberculoid leprosy (53 [68.8%]). Mutations associated with resistance were identified in 6/77 (7.8%) specimens. Mutations seen were those associated with resistance to rifampicin, ofloxacin, and dapsone. All the six patients were drug-naive. The clinical and pathological manifestations in this group did not differ from the drug-sensitive group. This study highlights the occurrence of resistance to the standard multidrug therapy and ofloxacin in leprosy. Among the entire cohort, 1/77 (1.3%) showed resistance to rifampicin, 2/77 (2.6%) to dapsone, and 5/77 (6.4%) to ofloxacin. Six new patients showing infection by mutant strains indicated the emergence of primary resistance. Resistance to ofloxacin could be due to frequent use of quinolones for many bacterial infections. The results of the study indicate the need for development of a robust and strict surveillance system for detecting drug resistance in leprosy in India.

Estudo observacional retrospectivo do perfil epidemiológico e clínico dos menores de 15 anos acometidos por hanseníase diagnosticados em Serviço de Referência no Rio de Janeiro no período de 1987 a 2018 / Retrospective observational study of the epidemiological and clinical profile of children under 15 years of age affected by leprosy diagnosed in a Reference Service in Rio de Janeiro from 1987 to 2018

A hanseníase é uma doença crônica transmissível causada pelo bacilo Mycobacterium leprae, que acomete sobretudo a pele e os nervos periféricos, podendo provocar perda das funções sensitiva e motora, usualmente evoluindo com deformidades e estigma. Apesar de estratégias mundiais e nacionais visando a eliminação da doença, a mesma persiste como relevante problema de saúde pública em diversos países, incluindo o Brasil, que apresenta elevado número absoluto de casos da doença e indicadores de transmissão ativa, traduzidos pelas taxas de detecção em menores de 15 anos. As crianças representam um grupo vulnerável à infecção, principalmente pela maior exposição a indivíduos doentes dentro da família, especialmente quando estes apresentam a classificação operacional multibacilar (MB) da hanseníase. O diagnóstico precoce seguido de tratamento dos casos é de grande importância, tanto para a redução da carga da hanseníase, quanto para a prevenção de incapacidades físicas nos pacientes. Nesse sentido, o exame de contatos é apontado como relevante ferramenta estratégica. Pretendendo avaliar o perfil epidemiológico, a evolução clínica e a magnitude da associação entre a classificação operacional para fins de tratamento e o modo de detecção em crianças diagnosticadas com hanseníase em um serviço de referência no Rio de Janeiro, foi realizado um estudo retrospectivo com dados de 335 pacientes menores de 15 anos diagnosticados com hanseníase e registrados no Ambulatório Souza Araújo, Laboratório de Hanseníase­ IOC ­ FIOCRUZRJ, no período de janeiro de 1987 a dezembro de 2018. Como limitações podemos mencionar dificuldades no levantamento e no recrutamento de todos os contatos, tratar-se estudo retrospectivo e o fato de ser realizado em um serviço de referência. Foram realizadas análises univariadas e bivariadas para verificar as distribuições dos pacientes com relação a todas as variáveis do estudo, e o modelo de regressão logística foi aplicado, considerando como variável dependente a classificação operacional MB, para avaliar a associação entre esta classificação e o modo de detecção (encaminhamento ou exame de contato) nos pacientes estudados. Para esta análise foram incluídos os pacientes diagnosticados através de encaminhamentos e apenas os casos coprevalentes do grupo diagnosticado através do exame de contato. As crianças detectadas através de encaminhamentos apresentaram chance duas vezes maior [OR 2,00 (IC95% 1,02 ­ 3,92)] de serem diagnosticadas com a hanseníase MB, quando comparadas àquelas detectadas através do exame de contato. Nos pacientes do sexo masculino foi observada chance 2,16 vezes maior (IC95% 1,13 ­ 4,14) de apresentarem a classificação operacional MB quando comparados aos do sexo feminino. Nos pacientes com idade entre 10 e 14 anos, a chance para apresentarem hanseníase MB foi 3,57 vezes maior (IC95% 1,84 ­ 6,90) em relação àqueles com idade entre 0 a 9 anos. A presença cicatriz de BCG, indicando imunização recebida após o nascimento, apresentou redução de chance de apresentarem classificação operacional MB [OR 0,45 (IC95% 0,24 ­ 0,84)]. Esses dados confirmam a importância da realização do exame dos contatos de casos novos registrados para tratamento poliquimioterápico como estratégia para o diagnóstico precoce da doença, principalmente em menores de 15 anos.

Leprosy is a chronic transmissible disease caused by the bacillus Mycobacterium leprae, which mainly affects the skin and peripheral nerves, and can cause loss of sensory and motor functions, usually progressing with deformities and stigma. Despite global and national strategies to eliminate the disease, it persists as a relevant public health problem in several countries, including Brazil, which has a high absolute number of cases of the disease and indicators of active transmission, translated by detection rates in children under 15 years. Children represent a vulnerable group to infection, mainly due to the greater exposure to sick individuals within the family, especially when they present the multibacillary operational classification (MB) of leprosy. Early diagnosis followed by treatment of cases is of great importance, both for reducing the burden of leprosy and for preventing physical disabilities. In this sense, the examination of contacts is identified as a relevant strategic tool. In order to assess the epidemiological profile, clinical evolution and the magnitude of the association between the operational classification for treatment purposes and the mode of detection in children diagnosed with leprosy in a reference service in Rio de Janeiro, a retrospective study was conducted with data from 335 patients under 15 years of age diagnosed with leprosy and registered at the Souza Araújo Ambulatory, Leprosy Laboratory - IOC - FIOCRUZ- RJ, from January 1987 to December 2018. As limitations, we can mention difficulties in the survey and recruitment of all contacts, a retrospective study and the fact that it was carried out in a reference service. Univariate and bivariate analyzes were performed to verify the distribution of patients in relation to all study variables, and the logistic regression model was applied, considering the MB operational classification as a dependent variable, to assess the association between this classification and the mode of detection (referral or contact examination) in the studied patients. For this analysis, patients diagnosed through referrals and only co-prevalent cases in the group diagnosed through contact examination were included. Children detected through referrals were twice as likely [RC 2.00 (95% CI 1.02 - 3.92)] to be diagnosed with MB leprosy, when compared to those detected through contact examination. Male patients were 2.16 times more likely (95% CI 1.13 - 4.14) to have the MB classification when compared to females. In patients aged 10 to 14 years, the chance of having a MB classification was 3.57 times greater (95% CI 1.84 - 6.90) compared to those aged 0 to 9 years. The presence of BCG scar, indicating immunization received after birth, presented a reduced chance of having an MB operational classification [OR 0.45 (95% CI 0.24 - 0.84)]. These data confirm the importance of examining the contacts of new cases registered for polychemotherapy treatment as a strategy for the early diagnosis of the disease, especially in children under 15 years of age.

Diet and nutrition: An important risk factor in leprosy.

Leprosy, once considered as poor man's disease may cause severe neurological complications and physical disabilities. Classification of leprosy depends upon the cell mediated and humoral immune responses of the host, from tuberculoid to lepromatous stage. Current therapy to prevent the disease is not only very lengthy but also consists of expensive multiple antibiotics in combination. Treatment and the duration depend on the bacillary loads, from six months in paucibacillary to a year in multibacillary leprosy. Although as per WHO recommendations, these antibiotics are freely available but still out of reach to patients of many rural areas of the world. In this review, we have focused on the nutritional aspect during the multi-drug therapy of leprosy along with the role of nutrition, particularly malnutrition, on susceptibility of Mycobacterium leprae and development of clinical symptoms. We further discussed the diet plan for the patients and how diet plans can affect the immune responses during the disease.

Neutrophil extracellular traps contribute to the pathogenesis of leprosy type 2 reactions.

Up to 50% of patients with the multibacillary form of leprosy are expected to develop acute systemic inflammatory episodes known as type 2 reactions (T2R), thus aggravating their clinical status. Thalidomide rapidly improves T2R symptoms. But, due to its restricted use worldwide, novel alternative therapies are urgently needed. The T2R triggering mechanisms and immune-inflammatory pathways involved in its pathology remain ill defined. In a recent report, we defined the recognition of nucleic acids by TLR9 as a major innate immunity pathway that is activated during T2R. DNA recognition has been described as a major inflammatory pathway in several autoimmune diseases, and neutrophil DNA extracellular traps (NETs) have been shown to be a prime source of endogenous DNA. Considering that neutrophil abundance is a marked characteristic of T2R lesions, the objective of this study was to investigate NETs production in T2R patients based on the hypothesis that the excessive NETs formation would play a major role in T2R pathogenesis. Abundant NETs were found in T2R skin lesions, and increased spontaneous NETs formation was observed in T2R peripheral neutrophils. Both the M. leprae whole-cell sonicate and the CpG-Hlp complex, mimicking a mycobacterial TLR9 ligand, were able to induce NETs production in vitro. Moreover, TLR9 expression was shown to be higher in T2R neutrophils, suggesting that DNA recognition via TLR9 may be one of the pathways triggering this process during T2R. Finally, treatment of T2R patients with thalidomide for 7 consecutive days resulted in a decrease in all of the evaluated in vivo and ex vivo NETosis parameters. Altogether, our findings shed light on the pathogenesis of T2R, which, it is hoped, will contribute to the emergence of novel alternative therapies and the identification of prognostic reactional markers in the near future.

Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae.

The lipolytic protein LipU was conserved in mycobacterium sp. including M. tuberculosis (MTB LipU) and M. leprae (MLP LipU). The MTB LipU was identified in extracellular fraction and was reported to be essential for the survival of mycobacterium. Therefore to address the problem of drug resistance in pathogen, LipU was selected as a drug target and the viability of finding out some FDA approved drugs as LipU inhibitors in both the cases was explored. Three-dimensional (3D) model structures of MTB LipU and MLP LipU were generated and stabilized through molecular dynamics (MD). FDA approved drugs were screened against these proteins. The result showed that the top-scoring compounds for MTB LipU were Diosmin, Acarbose and Ouabain with the Glide XP score of -12.8, -11.9 and -11.7 kcal/mol, respectively, whereas for MLP LipU protein, Digoxin (-9.2 kcal/mol), Indinavir (-8.2 kcal/mol) and Travoprost (-8.2 kcal/mol) showed highest affinity. These drugs remained bound in the active site pocket of MTB LipU and MLP LipU structure and interaction grew stronger after dynamics. RMSD, RMSF and Rg were found to be persistent throughout the simulation period. Hydrogen bonds along with large number of hydrophobic interactions stabilized the complex structures. Binding free energies obtained through Prime/MM-GBSA were found in the significant range from -63.85 kcal/mol to -34.57 kcal/mol for MTB LipU and -71.33 kcal/mol to -23.91 kcal/mol for MLP LipU. The report suggested high probability of these drugs to demolish the LipU activity and could be probable drug candidates to combat TB and leprosy disease.

Leprosy and its reactional episodes: Serum levels and possible roles of omega-3 and omega-6-derived lipid mediators.

The disease leprosy is caused by Mycobacterium leprae. The disease displays a spectrum of clinical manifestations relating to the stage of the infection and the pathogen-specific immune response. The most frequent M. leprae-specific hypersensitivity reactions are erythema nodosum leprosum (ENL) and type-1 (reversal) reaction (T1R). Omega-3 and omega-6 fatty acid-derived lipid mediators are involved in the regulation of these M. leprae-specific inflammatory and immune responses. Studies on lipid mediators showed their presence during different manifestations of leprosy-before and after multidrug therapy (MDT) and during T1R. This review aims to compare the lipid mediators at different stages of the disease. This review also presents new data on the significance of lipid mediators (cysteinyl leukotrienes and leukotriene B4, prostaglandin E2 and D2, lipoxin A4 and resolvin D1) on ENL.

Dapsone Resistance in Leprosy Patients Originally from American Samoa, United States, 2010-2012.

Skin biopsies from US leprosy patients were tested for mutations associated with drug resistance. Dapsone resistance was found in 4 of 6 biopsies from American Samoa patients. No resistance was observed in patients from other origins. The high rate of dapsone resistance in patients from American Samoa warrants further investigation.

Clinical pilot study: Clarithromycin efficacy in multibacillary leprosy therapy.

Background: Rifampicin is one of the important components in the multidrug therapy (MDT)-World Health Organization regimen for leprosy. Clarithromycin is one of the alternative therapies of rifampicin. Methods: This clinical pilot study was to compare the efficacy of 2,000 mg clarithromycin to 600 mg rifampicin in combination with dapsone and clofazimine for 3 months in multibacillary (MB) leprosy patients. They were divided into an MDT-MB regimen group that consists of rifampicin-dapsone-clofazimine and clarithromycin-dapsone-clofazimine (CDC) regimen group, each group consisted of seven patients. Results: The morphological index (MI) was reduced insignificantly after 3 months therapy in MDT-MB group (P = 0.248). While in the CDC group, the MI decrement showed a significant result (P = 0.004). The comparison of MI reduction in MDT-MB and CDC groups showed an insignificant difference (P = 0.130). Skin discoloration was occurred in both groups, whereas mild-nausea was presented in the CDC group, in addition, red-colored urine was developed in the MDT-MB group. Conclusion: We concluded that 2,000 mg clarithromycin is as effective as 600 mg rifampicin in combination with dapsone and clofazimine regimen in MB leprosy patients. Hence, clarithromycin can be considered as an alternative therapy for leprosy patients who resistance and/or allergy to rifampicin.

Antimicrobial resistance in leprosy: results of the first prospective open survey conducted by a WHO surveillance network for the period 2009-15.

OBJECTIVES: Antimicrobial resistance (AMR) is a priority for surveillance in bacterial infections. For leprosy, AMR has not been assessed because Mycobacterium leprae does not grow in vitro. We aim to obtain AMR data using molecular detection of resistance genes and to conduct a prospective open survey of resistance to antileprosy drugs in countries where leprosy is endemic through a WHO surveillance network. METHODS: From 2009 to 2015, multi-bacillary leprosy cases at sentinel sites of 19 countries were studied for resistance to rifampicin, dapsone and ofloxacin by PCR sequencing of the drug-resistance-determining regions of the genes rpoB, folP1 and gyrA. RESULTS: Among 1932 (1143 relapse and 789 new) cases studied, 154 (8.0%) M. leprae strains were found with mutations conferring resistance showing 182 resistance traits (74 for rifampicin, 87 for dapsone and 21 for ofloxacin). Twenty cases showed rifampicin and dapsone resistance, four showed ofloxacin and dapsone resistance, but no cases were resistant to rifampicin and ofloxacin. Rifampicin resistance was observed among relapse (58/1143, 5.1%) and new (16/789, 2.0%) cases in 12 countries. India, Brazil and Colombia reported more than five rifampicin-resistant cases. CONCLUSIONS: This is the first study reporting global data on AMR in leprosy. Rifampicin resistance emerged, stressing the need for expansion of surveillance. This is also a call for vigilance on the global use of antimicrobial agents, because ofloxacin resistance probably developed in relation to the general intake of antibiotics for other infections as it is not part of the multidrug combination used to treat leprosy.

Possible cases of leprosy from the Late Copper Age (3780-3650 cal BC) in Hungary.

At the Abony-Turjányos dulo site, located in Central Hungary, a rescue excavation was carried out. More than 400 features were excavated and dated to the Protoboleráz horizon, at the beginning of the Late Copper Age in the Carpathian Basin, between 3780-3650 cal BC. Besides the domestic and economic units, there were two special areas, with nine-nine pits that differed from the other archaeological features of the site. In the northern pit group seven pits contained human remains belonging to 48 individuals. Some of them were buried carefully, while others were thrown into the pits. The aim of this study is to present the results of the paleopathological and molecular analysis of human remains from this Late Copper Age site. The ratio of neonates to adults was high, 33.3%. Examination of the skeletons revealed a large number of pathological cases, enabling reconstruction of the health profile of the buried individuals. Based on the appearance and frequency of healed ante- and peri mortem trauma, inter-personal (intra-group) violence was characteristic in the Abony Late Copper Age population. However other traces of paleopathology were observed on the bones that appear not to have been caused by warfare or inter-group violence. The remains of one individual demonstrated a rare set of bone lesions that indicate the possible presence of leprosy (Hansen's disease). The most characteristic lesions occurred on the bones of the face, including erosion of the nasal aperture, atrophy of the anterior nasal spine, inflammation of the nasal bone and porosity on both the maxilla and the bones of the lower legs. In a further four cases, leprosy infection is suspected but other infections cannot be excluded. The morphologically diagnosed possible leprosy case significantly modifies our knowledge about the timescale and geographic spread of this specific infectious disease. However, it is not possible to determine the potential connections between the cases of possible leprosy and the special burial circumstances.

Discovery of a potential lead compound for treating leprosy with dapsone resistance mutation in M. leprae folP1.

Dapsone resistance is a serious impediment to the implementation of the present leprosy control strategies. In the recent past, many studies have been undertaken to address the antibiotic activity and binding pattern of dapsone against both native and mutant (Pro55Leu) folP1. Yet, there is no well-developed structural basis for understanding drug action and there is dire need for new antibacterial therapies. In the present study, molecular simulation techniques were employed alongside experimental strategies to address and overcome the mechanism of dapsone resistance. In essence, we report the identification of small molecule compounds to effectively and specifically inhibit the growth of M. leprae through targeting dihydropteroate synthase, encoded by folP1 which is involved in folic acid synthesis. Initially, ADME and toxicity studies were employed to screen the lead compounds, using dapsone as standard drug. Subsequently, molecular docking was employed to understand the binding efficiency of dapsone and its lead compounds against folP1. Further, the activity of the screened lead molecule was studied by means of molecular dynamics simulation techniques. Furthermore, we synthesized 4-(2-fluorophenylsulfonyl)benzenamine, using (2-fluorophenyl)boronic acid and 4-aminobenzenesulfonyl chloride, and the compound structure was confirmed by (1)H NMR and (13)C NMR spectroscopic techniques. Most importantly, the antibacterial activity of the compound was also examined and compared against dapsone. Overall, the result from our analysis suggested that CID21480113 (4-(2-fluorophenylsulfonyl)benzenamine) could be developed into a promising lead compound and could be effective in treating dapsone resistant leprosy cases.

[EXPERIMENTAL STUDY OF SPECIFIC ACTIVITY OF 1.3-DIAZINON-4 COM- POUND PYaTd1 DERIVATIVE IN VIVO].

AIM: Study anti-leprosy activity of.a 1.3-diazinon-4 compound derivative under the labora- tory code PYaTd1 on the model of intra-plantar infection of mice and evaluate the character of its antibacterial effect. MATERIALS AND METHODS: Study of specific activity was carried out in vivo on the experimental model of leprosy, proposed by Shepard C.C., that assumes execution of intraplantar infection of mice with a suspension of mycobacteria, produced from lepromas or autopsy tissue of a non-treated leprosy infected, or from tissues of experimental mice, previously infected with Mycobacterium leprae from non-treated patients. The study was carried out on 120 CBA line-mice infected with M.leprae (VIII passage) from patient M; Dapsone and PYaTdl compound were administered to animals next day after the infection with forage at a dose of 25 mg/kg for 4.5, 6, 9 and 11 months. The mice were split into 3 groups: control (infected.without treatment), com- parison (infected, receiving.dapsone), experimental (infected, receiving PYaTdl). After.the control term the mice were euthanized under chloroform anesthesia. Suspensions for quantification of mycobacteria were prepared from paw pads. Smears were stained by Ziehl-Nilsson. RESULTS: After 4.5 months the intensity of infect reproduction under, the effect of dapsone and PYaTd1 was reduced compared with control by 18 - 25 times. After a 6-mont course - by 50 - 75% and after 9 months - by 85 - 90%. After 11 months in mice that had received PYaTd1, an intensive suppression of microorganism reproduction as observed: the yield in paws was 70 times lower than in control. In the group that had received dapsone, a reduction of the number of mycobacteria by 20 - 25 times was detected, it was significantly less effective than under the conditions of PYaTd1 admnistration. CONCLUSION: A novel 1.3-diazinon- 4 derivative under the code PYaTd1 can actively supress reproduction of-M. leprae, that gives evidence regarding its specific anti-mycobacterial activity and determines perspectives of its further studies.

Evaluation of anti-bacterial activity of Rifapentine, Clarithromycin, Minocycline, Moxifloxacin, Ofloxacin and their combinations in Murine Model of Rifampicin Resistant Leprosy.

Leprosy, a debilitating disease of the skin and peripheral nerves is caused by Mycobacterium leprae (M. leprae) and is treated by multidrug therapy (MDT) comprising of Dapsone, Rifampicin and Clofazimine. Resistance to any of these drugs poses a threat to the current disease control strategies. With the emergence of Rifampicin resistance in leprosy, it is important that alternative drugs need to be tested to develop a treatment strategy to combat drug resistant leprosy. In the current study, we have investigated WHO MDT, Rifapentine, Clarithromycin, Minocycline, Moxifloxacin, Ofloxacin and their combinations in intermittent and daily dose regimens in rifampicin resistant strains of M. leprae through mouse foot pad experiments in order to determine the loss in viability of M. leprae in response to these drugs and their combinations. Our findings suggest that WHO MDT is still the best combination in Rifampicin resistance cases. Combination of Moxifloxacin with Minocycline and Clarithromycin may also be taken up for clinical trials in cases with Rifampicin resistant leprosy. Rifapentine and Moxifloxacin can be effective alternative drugs to replace Rifampicin where required either in daily dose shorter duration regimens or intermittent dose longer regimen to treat resistant strains.

The Complete Genome Sequence of the Emerging Pathogen Mycobacterium haemophilum Explains Its Unique Culture Requirements.

UNLABELLED: Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear. Using a combination of Illumina, PacBio, and Sanger sequencing, the complete genome sequence of M. haemophilum was determined. Guided by this sequence, experiments were performed to define the basis for the unique growth requirements of M. haemophilum. We found that M. haemophilum, unlike many other mycobacteria, is unable to synthesize iron-binding siderophores known as mycobactins or to utilize ferri-mycobactins to support growth. These differences correlate with the absence of genes associated with mycobactin synthesis, secretion, and uptake. In agreement with the ability of heme to promote growth, we identified genes encoding heme uptake machinery. Consistent with its propensity to infect the skin, we show at the whole-genome level the genetic closeness of M. haemophilum with Mycobacterium leprae, an organism which cannot be cultivated in vitro, and we identify genes uniquely shared by these organisms. Finally, we identify means to express foreign genes in M. haemophilum. These data explain the unique culture requirements for this important pathogen, provide a foundation upon which the genome sequence can be exploited to improve diagnostics and therapeutics, and suggest use of M. haemophilum as a tool to elucidate functions of genes shared with M. leprae. IMPORTANCE: Mycobacterium haemophilum is an emerging pathogen with an unknown natural reservoir that exhibits unique requirements for iron supplementation to grow in vitro. Understanding the basis for this iron requirement is important because it is fundamental to isolation of the organism from clinical samples and environmental sources. Defining the molecular basis for M. haemophilium's growth requirements will also shed new light on mycobacterial strategies to acquire iron and can be exploited to define how differences in such strategies influence pathogenesis. Here, through a combination of sequencing and experimental approaches, we explain the basis for the iron requirement. We further demonstrate the genetic closeness of M. haemophilum and Mycobacterium leprae, the causative agent of leprosy which cannot be cultured in vitro, and we demonstrate methods to genetically manipulate M. haemophilum. These findings pave the way for the use of M. haemophilum as a model to elucidate functions of genes shared with M. leprae.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae.

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.