Adenosine A2a receptor as a potential regulator of Mycobacterium leprae survival mechanisms: new insights into leprosy neural damage

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells­glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host­pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron­glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae­Schwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. nzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR nalysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzymelinked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.

Padronização do uso da monoazida de propídio (PMAxxTM) na detecção de viabilidade do Mycobacterium leprae / Standardization of the use of propidium monoazide (PMAxxTM) in the detection of Mycobacterium leprae viability

Introdução A hanseníase é uma doença infecciosa crônica, causada peloMycobacterium leprae, que se manifesta na pele e pode invadir o sistema nervoso periférico do paciente. O cultivo de seu agente etiológico em meios de cultura artificiaisou celulares ainda é um desafio e obstáculo para estudos relacionados à sua microbiologia. Para avaliar a viabilidade de células bacterianas, utiliza-se corantes fluorescente, como a monoazida de propídeo (PMA). O corante penetra somente nas células que estão com a membrana celular comprometida e reage com fração de hidrocarboneto a fim de resultar em uma modificação permanente do DNA. Objetivo Padronizar a utilização do corante monoazida de propídeo (PMAxx™) em combinação com a técnica de reação em cadeia da polimerase em tempo real (RT qPCR) para detecção da viabilidade do M. leprae. Metodologia Diferentes concentrações de PMAxx™ foram adicionadas a 250µl de suspensão bacilar purificada, proveniente de coxim plantar de camundongos previamente infectados. As amostras foram incubadas no escuro por diferentes tempos. Após a incubação, foram fotoativadas por exposição em lâmpada halógena de 650 W. Foram avaliados os parâmetros de concentração bacilar, tempo de incubação no escuro, tempo de exposição à luz e concentração do PMAxx™. O DNA do bacilo foi extraído utilizando-se um kit comercial e amplificadas por RT qPCR, com uso de primers específicos para as regiões Specific Repetitive Element (RLEP) do DNA de M. leprae Resultados Não houve diferença significativa no valor do ΔCt em nenhuma das concentrações de bacilos, indicando que não foi possível fazer a discriminação entre células vivas e inviáveis. O tempo ideal de incubação no escuro foi de 60 minutos, pois apresentaram uma diferenciação significativa do ΔCtvivo-morto com PMAxxTM e ΔCtmorto com e sem PMAxxTM. Em relação ao tempo de fotoativação, o maior valor de ΔCt observado foi submetido a sete minutos em exposição à luz. A concentração do PMAxxTM que apresentou uma diferenciação de ΔCt maior foi de 25µL. Discussão Os resultados mostram que o PMAxx™ tem uma boa eficácia com outras bactérias, mas ainda apresenta dificuldades em intercalar ao DNA de M. leprae. O uso do corante após análise com RT qPCR/RLEP para o bacilo é um método que ainda necessita de ajustes nos parâmetros como purificação da amostra, tempo de exposição e fotoativação. Esses dados ainda são preliminares e não inviabilizam a perspectiva de novos experimentos a partir dos ajustes nos parâmetros já avaliados.

Introduction Leprosy is a chronic infectious disease, caused by Mycobacterium leprae, which manifests itself in the skin and may invade the peripheral nervous system of the patient. Culturing its etiologic agent in artificial or cell culture media is still a challenge and obstacle for studies related to its microbiology. To assess the viability of bacterial cells, fluorescent dyes such as propidium monoazide (PMA) are used. The dye penetrates only cells with a compromised cell membrane and reacts with a hydrocarbon fraction to result in a permanent modification of the DNA Objective To standardize the use of the dye propidium monoazide (PMAxx™) in combination with the real-time polymerase chain reaction (RT qPCR) technique for detection of M. leprae viability Methodology Different concentrations of PMAxx™ were added to 250µl of purified bacillary suspension from plantar cushion of previously infected mice. The samples were incubated in the dark for different times. After incubation, they were photoactivated by exposure in a 650 W halogen lamp. The parameters of bacillary concentration, incubation time in the dark, light exposure time and concentration of PMAxx™ were evaluated. The bacillus DNA was extracted using a commercial kit and amplified by RT qPCR using specific primers for the Specific Repetitive Element (RLEP) regions of the M. leprae Results There was no significant difference in the ΔCt value at any of the bacilli concentrations, indicating that discrimination between live and non-viable cells was not possible. The optimal incubation time in the dark was 60 minutes, as they showed a significant differentiation of ΔClive-dead with PMAxxTM and ΔCtdead with and without PMAxxTM. Regarding photoactivation time, the highest value of ΔCt observed was subjected to seven minutes in light exposure. The concentration of PMAxxTM that showed a greater differentiation of ΔCt was 25µL Discussion The results show that PMAxx™ has good efficacy with other bacteria, but still presents difficulties in intercalating to M. leprae DNA. The use of the dye after analysis with RT qPCR/RLEP for the bacillus is a method that still needs adjustments in parameters such as sample purification, exposure time and photoactivation. These data are still preliminary and do not preclude the prospect of new experiments based on adjustments in the parameters already evaluated.

Gene expression profile of Mycobacterium leprae contribution in the pathology of leprosy neuropathy

Peripheral neuropathy is the main cause of physical disability in leprosy patients.Importantly, the extension and pattern of peripheral damage has been linked to how the host cell will respond against Mycobacterium leprae (M. leprae) infection, in particular, how the pathogen will establish infection in Schwann cells. Interestingly, viable and dead M. leprae have been linked to neuropathology of leprosy by distinct mechanisms. While viable M. leprae promotes transcriptional modifications that allow the bacteria to survive through the use of the host cell's internal machinery and the subvert of host metabolites, components of the dead bacteria are associated with the generation of a harmful nerve microenvironment. Therefore, understanding the pathognomonic characteristics mediated by viable and dead M. leprae are essential for elucidating leprosy disease and its associated reactional episodes. Moreover, the impact of the viable and dead bacteria in Schwann cells is largely unknown and their gene signature profiling has, as yet, been poorly explored. In this study, we analyzed the early differences in the expression profile of genes involved in peripheral neuropathy, dedifferentiation and plasticity, neural regeneration, and inflammation in human Schwann cells challenged with viable and dead M. leprae. We substantiated our findings by analyzing this genetic profiling in human nerve biopsies of leprosy and non-leprosy patients, with accompanied histopathological analysis. We observed that viable and dead bacteria distinctly modulate Schwann cell genes, with emphasis to viable bacilli upregulating transcripts related to glial cell plasticity, dedifferentiation and anti-inflammatory profile, while dead bacteria affected genes involved in neuropathy and pro-inflammatory response. In addition, dead bacteria also upregulated genes associated with nerve support, which expression profile was similar to those obtained from leprosy nerve biopsies. These findings suggest that early exposure to viable and dead bacteria may provoke Schwann cells to behave differentially, with far-reaching implications for the ongoing neuropathy seen in leprosy patients, where a mixture of active and non-active bacteria are found in the nerve microenvironment.

Classification and general aspects of leprosy

Leprosy is a chronic infectious disease whose etiological agent is Mycobacterium leprae. Recently, Mycobacterium lepromatosis is also implicated as a causative agent and has been identified in different forms of the disesase. Leprosy is a comples disease from a clinical histopathological, and molecular point of view. The wide complex disease from a clinical, histopathological characteristics observed throughout the disease spectrum and reactions render it a challenging disease in clinical and pathological practice. This chapter discusses the main aspects of the disease and its hispathological classification. An important approach to the bacilloscopic examination, which is fundamental for the histopathological classification of the disease, showing its quantitative and qualitative aspects, is discussed. the various photographic panels demonstrate the bacillus' ability to parasitize different types of tissues and cells of the skin and other organs of the human body...

Caracterização de modelo murino de granuloma não infeccioso em comparação à técnica de Shepard de infecção por M. leprae / Characterization of a murine model of non-infecious granuloma compared to the Shepard technique of M. leprae infection

Na hanseníase, o modelo de Shepard de inoculação por Mycobacterium leprae (M. leprae) em patas de camundongos vem sendo utilizado em diversos estudos sobre a resposta imune, avaliação de novas drogas e esquemas terapêuticos, além da dinâmica da infecção precoce e crônica. Para estudar o papel do microambiente granulomatoso na hanseníase, além de modelos animais convencionais ou imunocomprometidos, o desenvolvimento de modelos murinos de granuloma não infeccioso pode adicionar parâmetros patogênicos a serem comparados no desenvolvimento da doença. Preparações de nitrocelulose estão entre as formas de desenvolvimento de granulomas não imunogênicos em experimentação animal. O presente estudo investigou a formação de granulomas não infecciosos induzidos por partículas de nitrocelulose, em comparação a lesões induzidas por M. leprae. Grupos de camundongos nude e BALB/c, foram constituídos e inoculados, conforme a técnica de Shepard, com suspensão de M. leprae (ML), suspensão de nitrocelulose (NT), associação de M. leprae com nitrocelulose (ML/NT) e veículo controle (CTRL). Após 07, 14, 21 e 28 dias, amostras foram coletadas e analisadas histopatologicamente pelas colorações, HematoxilinaEosina e Fite-Faraco. Os grupos experimentais demonstraram a formação de granulomas em ambas os fenótipos murinos. Principalmente nos grupos NT e ML/NT, as lesões foram caracterizadas por infiltrado inflamatório mononuclear, predominantemente macrofágico, com presença de células epitelioides, eventuais macrófagos vacuolizados e ausência de células gigantes multinucleadas. As lesões induzidas exclusivamente por M. leprae pareceram menos exuberantes que àquelas observadas nos demais grupos, indicando que a nitrocelulose intensificou a resposta macrofágica nos espécimes avaliados e sugerindo que esse composto pode ser utilizado não só para o desenvolvimento de granulomas não imunogênicos, mas também na exacerbação da resposta imune em granulomas induzidos por agentes infecciosos, como M. leprae.

In leprosy, the Shepard model of inoculation by Mycobacterium leprae (M. leprae) in mouse footpad has been used in several studies on the immune response, evaluation of new drugs and therapeutic schemes, in addition to the dynamics of early and chronic infection. To study the role of the granulomatous microenvironment in leprosy, in addition to conventional or immunocompromised animal models, the development of murine models of non-infectious granuloma can add pathogenic parameters to be compared in the development of the disease. Nitrocellulose preparations are among the ways of developing non-immunogenic granulomas in animal experimentation. The present study investigated the formation of non-infectious granulomas induced by nitrocellulose particles, in comparison to lesions induced by M. leprae. Groups of athymic nude and BALB/c mice were set up and inoculated, according to Shepard technique, with M. leprae suspension (ML), nitrocellulose suspension (NT), M. leprae association with nitrocellulose (ML/NT) and control vehicle (CTRL). After 07, 14, 21 and 28 days, samples were collected and histopathologically analyzed by Hematoxylin-Eosin and FiteFaraco staining. The experimental groups demonstrated the formation of granulomas in both murine strains. Mainly in the NT and ML/NT groups, the lesions were characterized by mononuclear inflammatory infiltrate, predominantly macrophagic, with the presence of epithelioid cells, eventual vacuolated macrophages and absence of multinucleated giant cells. The lesions induced exclusively by M. leprae seemed less exuberant than those observed in the other groups, indicating that nitrocellulose intensified the macrophage response in the specimens evaluated, and suggesting that this compound can be used not only in the development of non-immunogenic granulomas, but also in exacerbation of the immune response in granulomas induced by infectious agents, such as M. leprae.

Involvement of TNF-Producing CD8+ Effector Memory T Cells with Immunopathogenesis of Erythema Nodosum Leprosum in Leprosy Patients.

Type 2 reaction (T2R) or erythema nodosum leprosum (ENL), a sudden episode of acute inflammation predominantly affecting lepromatous leprosy patients (LL), characterized by a reduced cellular immune response. This possibly indicates a close relationship between the onset of T2R and the altered frequency, and functional activity of T lymphocytes, particularly of memory subsets. This study performed ex vivo and in vitro characterizations of T cell blood subpopulations from LL patients with or without T2R. In addition, the evaluation of activity of these subpopulations was performed by analyzing the frequency of these cells producing IFN-γ, TNF, and IL-10 by flow cytometry. Furthermore, the expression of transcription factors, for the differentiation of T cells, were analyzed by quantitative real-time polymerase chain reaction. Our results showed an increased frequency of CD8+/TNF+ effector memory T cells (TEM) among T2Rs. Moreover, there was evidence of a reduced frequency of CD4 and CD8+ IFN-γ-producing cells in T2R, and a reduced expression of STAT4 and TBX21. Finally, a significant and positive correlation between bacteriological index (BI) of T2R patients and CD4+/TNF+ and CD4+/IFN-γ+ T cells was observed. Thus, negative correlation between BI and the frequency of CD4+/IL-10+ T cells was noted. These results suggest that CD8+/TNF+ TEM are primarily responsible for the transient alteration in the immune response to Mycobacterium leprae in ENL patients. Thus, our study improves our understanding of pathogenic mechanisms and might suggest new therapeutic approaches for leprosy.

Hanseníase neural, aspectos diagnósticos da forma neural pura e mecanismos imunopatogênicos da lesão do nervo na doença. Participação de quimiocinas CCL2 e CXCL10 e metaloproteinases 2 e 9 / Neural leprosy, pure neural leprosy diagnosis and imunopatogenic mechanisms of nerve damage during the disease. Participation of chemokines CCL2 and CXCL10) and metalloproteinases 2 and 9

O diagnóstico da hanseníase neural pura baseia-se em dados clínicos e laboratoriais do paciente, incluindo a histopatologia de espécimes de biópsia de nervo e detecção de DNA de Mycobacterium leprae (M. leprae) pelo PCR. Como o exame histopatológico e a técnica PCR podem não ser suficientes para confirmar o diagnóstico, a imunomarcação de lipoarabinomanana (LAM) e/ou Glicolipídio fenólico 1 (PGL1) - componentes de parede celular de M. leprae foi utilizada na primeira etapa deste estudo, na tentativa de detectar qualquer presença vestigial do M. leprae em amostras de nervo sem bacilos. Além disso, sabe-se que a lesão do nervo na hanseníase pode diretamente ser induzida pelo M. leprae nos estágios iniciais da infecção, no entanto, os mecanismos imunomediados adicionam severidade ao comprometimento da função neural em períodos sintomáticos da doença. Este estudo investigou também a expressão imuno-histoquímica de marcadores envolvidos nos mecanismos de patogenicidade do dano ao nervo na hanseníase. Os imunomarcadores selecionados foram: quimiocinas CXCL10, CCL2, CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, e metaloproteinases 2 e 9. O estudo foi desenvolvido em espécimes de biópsias congeladas de nervo coletados de pacientes com HNP (n=23 / 6 BAAR+ e 17 BAAR - PCR +) e pacientes diagnosticados com outras neuropatias (n=5) utilizados como controle. Todas as amostras foram criosseccionadas e submetidas à imunoperoxidase. Os resultados iniciais demonstraram que as 6 amostras de nervos BAAR+ são LAM+/PGL1+. Já entre as 17 amostras de nervos BAAR-, 8 são LAM+ e/ou PGL1+. Nas 17 amostras de nervos BAAR-PCR+, apenas 7 tiveram resultados LAM+ e/ou PGL1+. A detecção de imunorreatividade para LAM e PGL1 nas amostras de nervo do grupo HNP contribuiu para a maior eficiência diagnóstica na ausência recursos a diagnósticos moleculares...

The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of M. leprae DNA by polymerase chain reaction (PCR). Given that histopathological examination and PCR methods may not be sufficient to confirm diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL1) M. leprae wall components were utilized in the first step of this investigation in an attempt to detect any vestigial presence of M. leprae in AFB- nerve samples. Furthermore, it´s well known that nerve damage in leprosy can be directly induced by Mycobacterium leprae in the early stages of infection; however, immunomediated mechanisms add gravity to the impairment of neural function in symptomatic periods of the disease. Therefore, this study also investigated the immunohistochemical expression of immunomarkers involved in the pathogenic mechanisms of leprosy nerve damage. These markers selected were CXCL10, CCL2 chemokines and CD3, CD4, CD8, CD45RA, CD45RO, CD68, HLA-DR, metalloproteinases 2 and 9 in nerve biopsy specimens collected from leprosy (23) and nonleprosy patients (5) suffering peripheral neuropathy. Twenty-three PNL nerve samples (6 AFB+ and 17 AFB-PCR+) were cryosectioned and submitted to LAM and PGL1 immunohistochemical staining by immunoperoxidase; 5 nonleprosy nerve samples were used as controls. The 6 AFB-positive samples showed LAM/PGL1 immunoreactivity. Among the 17 AFB- samples, only 8 revealed LAM and/or PGL1 immunoreactivity. In 17 AFB-PCR+ patients, just 7 had LAM and/or PGL1-positive nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL1 in the nerve samples would have contributed to enhanced diagnostic efficiency in the absence of molecular diagnostic facilities...

Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Increased expression of regulatory T cells and down-regulatory molecules in lepromatous leprosy.

T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-γ (IFN-γ) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-ß (TGF-ß), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-ß. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.

Administration of M. leprae Hsp65 interferes with the murine lupus progression

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatrory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K409 A recombinant Hsp65 of M. leprae in an animal mode of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F1 mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The teatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K409 A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule, that counteracts the progression of the SLE, and that the point mutated K409 A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process.

Epidemiology and prevention of leprosy: a cohort study in Indonesia

Leprosy is an infectious disease caused by Mycobacterium leprae, a bacterium which primarily affects the nerves and skin. Worldwide 513,798 new patients were detected in 2003. Trend analysis showed that the new case detection rate did not decline between 1985 and 2000. Leprosy diagnosis is aminly based on the presence of skin lesions, loss of sensitivity and thickened nerves. The clinical pattern varies widely between patients due to a different level of celular immunity that an infected person mounts to the infection. For treatment purposes patients are divided into paucibacillary (PB) and multibacillary (MB) patients. Effective treatment against leprosy is available (chapter 1). In the last five decades cohort studies have investigated risk factors for developing leprosy. Most of these studies were conducted among contacts of leprosy patients and a few were population-based. Risk factors for developing leprosy were grouped into three levels: (1) individual level; (2) household and community levels; (3) meso/macro levels (chapter 1). As leprosy case detections rates did not decline in the last 15 years despite effective multi-drug treatment, new approaches and strategies to definitely eliminate leprosy as a public health problem are required. Four research questions were addressed in this thesis: "Which patients transmit leprosy?", "What are risk factors for developing leprosy?", "Can rifampicin, used as chemoprophylaxis, prevent leprosy?" and "How can chemoprophylaxis be most efficiently implemented?". All studies presented in this thesis were preformed in the same study area, five geographically isolated islands in the Flores Sea, Indonesia, and are part of still continuing population-based cohort study. Chaper 2 mainly serves as background information on the leprosy epidemic in the study area. It describes how an enormous load of backlog patients existed on these islands, with only irregular leprosy control before 2000. The overvall new case detection ra...

Population survey to determine risk factors for Mycobacterium leprae transmission and infection

Background: Not every leprosy patient is equally effective in transmitting Mycobacterium leprae. We studied the spatial distribution of infection (using seropositivity as a marker) in the population to identifity which disease characteristics of leprosy patients are important in transmission. Methods: Clinical data and blood samples for anti-M.leprae ELISA were collected during a cross-sectional survey on five Indonesian islands highly endemic for leprosy. A geographic information system (GIS) was used to define contacts of patients. We investigated spatial clustering of patients and seropositive people and used logist regression to determine risk factors for seropositivity. Results: Of the 3986 people examined for leprosy, 3271 gave blood. Seroprevalence varied between islands (1.7-8.7%) and correlated significantly with leprosy prevalence. Five clusters of patients and two clusters of seropositives were detected. In multivariate analysis, seropositivity significantly differed to be the best discriminator of contact groups with higher seroprevalence: contacts of seropositive patients had an adjusted odds ratio (aOR) of 1.75 (95% CI: 0.92-3,31). This increased seroprevalence was strongest for contact groups living _< 75 metres of two seropositive patients (aOR:3.07;95%CI:1.74-5.42). Conclusions: In this highly endemic area for leprosy, not only household contacts of seropositive patients, but also persons living in the vicinity of seropositive patient were more likely to harbour antibodies against M.leprae. Through measuring the serological status of patients and using a broader definition of contacts, higher risk groups can be more specifically identified

Single nucleotide polymorphisms (SNPs) at -238 and -308 positions in the TNFalpha promoter: clinical and bacteriological evaluation in leprosy.

Tumor necrosis factor alpha (TNFalpha) plays a key role in orchestrating the complex events involved in inflammation and immune response. The presence of single nucleotide polymorphisms (SNPs) within the promoter region of the TNFa gene has been associated with a number of diseases. The aim of this study was to investigate the distribution of polymorphisms at positions -238 (G/A) and -308 (G/A) at the TNFalpha promoter, and its association to the outcome of different clinical forms of leprosy. Furthermore, the bacteriological index (BI) was evaluated among genotyped multibacillary (MB) patients in order to investigate the possible influence of each polymorphism on the bacterial load. This study included a total of 631 leprosy patients being 401 MB and 230 paucibacillary (PB), that was further separated according to its ethnicity (Afro- and Euro-Brazilians). The combination of SNPs in haplotypes generated three different arrangements: TNFG-G, TNFG-A and TNFA-G. In spite of the marked differences observed in the frequency of the haplotypes along the ethnic groups, no statistical differences were observed in haplotype frequencies between MB and PB patients. The BI analyses showed a lower bacteriological index among the -308 carriers, while the BI of the -238 carriers was higher. Although no significance has been achieved in this analysis regarding the influence of the polymorphisms to the development of the clinical outcome, it seems that in a different stage (among the MB patients) the polymorphisms could contribute to the degree of severity observed.

Neuropathic pain in leprosy patients.

The introduction of multidrug therapy by the World Health Organization has dramatically reduced the world prevalence of leprosy but the disease is still a public health problem in many countries, with a world prevalence of almost 600,000 cases in 2001. Damage to peripheral nerves is a key component of leprosy and the sensory and motor loss that follows is the basis for many of the classical features of this disease, such as skin wounds, cracks, plantar ulcers, clawed hands, drop foot, and incomplete closure of the eyelids. One of the most remarkable aspects of leprosy to lay persons and health care workers alike is that patients are reputed to feel no pain. However, neuropathic pain is arising as a major problem among leprosy patients. It can be nociceptive due to tissue inflammation, which mostly occurs during episodes of immune activation or neuropathic due to damage or dysfunction of the nervous system. This study, conducted among 358 leprosy patients, reveals a considerable prevalence of neuropathic pain and presents evidence that this common problem should be a high priority of those in charge of leprosy control programs.

Neuropathic pain in leprosy patients

The introduction of multidrug therapy by the World Health Organization has dramatically reduced the world prevalence of leprosy but the disease is still a public health problem in many countries, with a world prevalence of almost 600,000 cases in 2001. Damage to peripheral nerves is a key component of leprosy and the sensory and motor loss that follows is the basis for many of the classical features of this disease, such as skin wounds, cracks, plantar ulcers, clawed hands, drop foot, and incomplete closure of the eyelids. One of the most remarkable aspects of leprosy to lay persons and health care workers alike is that patients are reputed to feel no pain. However, neuropathic pain is arising as a major problem among leprosy patients. It can be nociceptive due to tissue inflammation, which mostly occurs during episodes of immune activation or neuropathic due to damage or dysfunction of the nervous system. This study, conducted among 358 leprosy patients, reveals a considerable prevalence of neuropathic pain and presents evidence that this common problem should be a high priority of those in charge of leprosy control programs.

Genes required for mycobacterial growth defined by high density mutagenesis

Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.