Deciphering the proteomic profile of Mycobacterium leprae cell envelope.

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Anti-neutrophil cytoplasmic antibodies in leprosy.

INTRODUCTION: Anti-Neutrophil Cytoplasmic Antibodies (ANCA) are auto-antibodies directed to intracellular components of neutrophils and used to be considered as present almost exclusively in granulomatous vasculitis. Recently, these auto-antibodies have been found in other autoimmune disorders as well as infectious diseases. MATERIALS AND METHODS: We studied patients with leprosy confirmed by bacilloscopy and/or skin biopsy, in reaction phase from the Ambulatório de Hanseníase do Hospital Universitário Professor Edgar Santos. ANCA and Antinuclear antibodies (ANA) were determined by indirect immunofluorescence using commercially available kits. RESULTS: Twenty patients were enrolled in our study, nine males and 11 females. The mean age was 36.9+/-18.2 years. ANCA were present only in one patient, with a perinuclear staining pattern (p-ANCA), and no patient tested positive for ANA. DISCUSSION: Although other studies have shown the presence of ANCA in leprosy, the low frequency of these antibodies in leprosy sera demonstrated in the present study illustrates the high specificity of ANCA for the diagnosis of Wegener granulomatosis.

Fnac study of histoid nodule: an early tool for diagnosis.

Histoid lesion, a variety of lepromatous leprosy, is due to alteration in the growth pattern of Mycobacterium leprae, possibly due to loss of immunity in localized areas. The distinction is based on cellular morphology by demonstrating pallisading arrangement of multi-layered spindle-shaped histocytes. Cytodiagnosis by fine needle aspiration cytology is therefore an early tool to recognize the histoid variety, differentiating it from a conventional LL module, as it is a simple and less traumatic procedure.

Choosing the decolourizer and its strength to stain Mycobacterium leprae. Does it actually matter?

Leprosy bacilli are more easily decolourized during staining than tuberculosis bacilli, so a weaker concentration of decolourizer is usually recommended. In Indonesia, the same 'strong' decolourizer is used for identifying both organisms. In a study to compare the results using different concentrations of different decolourizers, no difference could be found in the bacterial index (BI). It is suggested that the same staining technique can be used for tuberculosis and leprosy.

Hansen's disease (leprosy). Diagnosis by aspiration biopsy of lymph nodes.

A 61-year-old male native of Mexico presented with generalized enlargement of lymph nodes. Fine needle aspiration (FNA) biopsy established lepromatous leprosy as the cause of the lymphadenopathy. The cytologic findings included abundant, frequently multinucleated histiocytes (globus cells), the cytoplasm of which showed multiple vacuoles; cytoplasmic membrane-bound vacuoles were seen free in the background. The vacuoles contained large numbers of acid-fast bacilli. Globus cells, while characteristic, are not specific for Mycobacterium leprae infection and are seen in certain atypical mycobacterioses in immunodeficient patients. This appears to be the first report of lymphadenopathy due to lepromatous leprosy in which the diagnosis was made by FNA biopsy. The immunologic spectrum of leprosy is correlated with clinical and pathologic findings, and the need to remember infectious processes in evaluating lymphadenopathy and the value of reserving air-dried and alcohol-fixed smears for special stains are emphasized.

Morphology of M. leprae (?) in VS3E medium--a preliminary communication.

In a previous attempted culture of M. leprae in VS2M medium non-acid fast organisms were seen initially and acid fast organisms appeared later. A drop of a sonicated suspension from a subculture of this was inoculated in VS3E medium. The inoculum consisted mostly of acid fast granules. The culture yielded pure growth of acid fast organisms. Morphology typical of M. leprae could be seen only after 60th day of culture.

Growth of Mycobacterium leprae in a redox system: III. Evidence of growth at low temperature (psychrophilia) and, further refinement of growth medium.

Considerable growth enhancement, largely as non-acid fast, slender and long rods has been seen when incubated at 10 degrees C. Concentration of some of the media constituents have been reduced that has improved the quantum of growth. A remarkable proneness to physical disintegration of the grown bacilli has been seen and its significance discussed. Also, the possible immunogenic advantage of non-AF M. leprae has been discussed. The question of identification is still not solved, and work is in progress.

Observations on attempted leprosy cultures in two media.

Suspensions of skin tissue material collected from lepromatous leprosy patients and material from mouse foot-pad harvests were inoculated into two media, viz., a biphasic medium and a minimal basal medium. The cultures were incubated at 37 degrees C and 15 degrees C. Small oval (or round) cells appeared in these cultures around the tenth day along with a few cystic structures; and they increased in number later, reaching the maximum around six-seven weeks. The above cells appeared acid-fast in some cultures and some of them appeared to split into pairs of acid-fast bacilli. The cells were most often seen in the biphasic medium at 37 degrees C. The identity of these structures is not known at this stage.

[The modelling of chronic infection (experimental leprosy) against a background of macrophagal immunodeficiency]. / Modelirovanie khronicheskoi infektsii (éksperimental'noi lepry) na fone makrofagal'nogo immunodefitsita.

The dynamics of mycobacterial multiplication was followed in mice with intraplantar leprosy infection and preinduced macrophage insufficiency. The characteristics of Shepard's model appeared to be similar to those of the method proposed by the authors including the susceptibility to the main antileprosy drugs. Peritoneal macrophages were cytochemically studied in the process of development of mononuclear phagocyte deficiency and experimental leprosy. It was concluded that the method proposed preserving all the merits of Shepard's model should allow one to shorten significantly the duration of testing potential drugs for their antileprosy activity.

[Reasons why Mycobacterium leprae cells do not multiply under the cell-free condition].

Our previous paper reported that the intracellular ATP content in cells of M. leprae consistently increased in the medium containing adenosine after 4-6 weeks of cultivation and decreased thereafter. The reason why ATP generation ceased 4-6 weeks after cultivation is not clear, but it was determined that the termination in ATP generation was not a result of deterioration in the culture medium during cultivation because a renewal trial of the old culture medium by freshly prepared culture medium had no effect further maintenance or progressive increase in ATP generation. From the results obtained in a renewal trial of the culture medium, I would like to speculate that the reason why M. leprae cells do not multiply in vitro might be due to the characteristic property of the cell wall of M. leprae, i.e., fragility.

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.

Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. METHODOLOGY/PRINCIPLE FINDINGS: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. CONCLUSIONS: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

Current status of leprosy: epidemiology, basic science and clinical perspectives.

Leprosy has affected humans for millennia and remains an important health problem worldwide, as evidenced by nearly 250 000 new cases detected every year. It is a chronic infectious disorder, caused by Mycobacterium leprae, that primarily affects the skin and peripheral nerves. Recent advances in basic science have improved our knowledge of the disease. Variation in the cellular immune response is the basis of a range of clinical manifestations. The introduction of multidrug therapy has significantly contributed to a decrease in the prevalence of the disease. However, leprosy control activities, including monitoring and prevention programs, must be maintained.