Efectividad de la formación de técnicos de laboratorio para la baciloscopia cutánea y nasal en una institución para la investigación de la lepra en India / No disponible

Durante 2 días, un total de 155 técnicos de laboratorio empleados en distintos laboratorios públicos del gobierno de Tamil Nadu, India, con una experiencia entre 3-25 años, se formaron en la técnica de toma de muestras cutáneas y nasales en el Centro de Enseñanza e Investigación de Chengalpattu entre 2013 y 2014. El objetivo de la formación era centrar su atención en las técnicas de frotis cutáneo y nasal. La formación consistió en demostraciones directas in vivo y formación práctica, evaluación y clasificación de las muestras. Se llevó a cabo una evaluación pre y post formación de cada uno de los participantes. La efectividad de la formación se analizó y había una significativa evidencia (P = 0.004) de que la formación mejor. el conocimiento de los participantes. De promedio, el nivel de conocimientos se incrementó en 10 puntos

A total number of 155 Laboratory Technicians working for the Government of Tamil Nadu, India having an experience of 3 to 25 years in various Public Health Laboratories of the state were deputed to undergo 2 days’ orientation training programme on skin smear and nasal smear techniques at the Central Leprosy Teaching and Research Institute, Chengalpattu in 2013–2014. The aim of the orientation training was to focus their attention on quality skin smear and nasal smear techniques reported by Laboratory Technicians working in various public health laboratories of the state. The training was conducted through live hands-on demonstration, practical performance of trainees and module reading. Pre- and post assessment was carried out for every Laboratory Technician trainee. The effectiveness of this training was analysed and showed that there was strong evidence (P .0.004) that the teaching intervention improves the knowledge of the trainees. On average the level of knowledge improved by approximately 10 points

Downregulation of PHEX in multibacillary leprosy patients: observational cross-sectional study.

BACKGROUND: Peripheral nerve injury and bone lesions, well known leprosy complications, lead to deformities and incapacities. The phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX) encodes a homonymous protein (PHEX) implicated in bone metabolism. PHEX/PHEX alterations may result in bone and cartilage lesions. PHEX expression is downregulated by intracellular Mycobacterium leprae (M. leprae) in cultures of human Schwann cells and osteoblasts. M. leprae in vivo effect on PHEX/PHEX is not known. METHODS: Cross-sectional observational study of 36 leprosy patients (22 lepromatous and 14 borderline-tuberculoid) and 20 healthy volunteers (HV). The following tests were performed: PHEX flow cytometric analysis on blood mononuclear cells, cytokine production in culture supernatant, 25-hydroxyvitamin D (OHvitD) serum levels and (99m)Tc-MDP three-phase bone scintigraphy, radiography of upper and lower extremities and blood and urine biochemistry. RESULTS: Significantly lower PHEX expression levels were observed in lepromatous patients than in the other groups (χ(2) = 16.554, p < 0.001 for lymphocytes and χ(2) = 13.933, p = 0.001 for monocytes). Low levels of 25-(OHvitD) were observed in HV (median = 23.0 ng/mL) and BT patients (median = 27.5 ng/mL) and normal serum levels were found in LL patients (median = 38.6 ng/mL). Inflammatory cytokines, such as TNF, a PHEX transcription repressor, were lower after stimulation with M. leprae in peripheral blood mononuclear cells from lepromatous in comparison to BT patients and HV (χ(2) = 10.820, p < 0.001). CONCLUSION: Downregulation of PHEX may constitute an important early component of bone loss and joint damage in leprosy. The present results suggest a direct effect produced by M. leprae on the osteoarticular system that may use this mechanism.

In vitro and skin lesion cytokine profile in Brazilian patients with borderline tuberculoid and borderline lepromatous leprosy.

OBJECTIVE: We investigated the in vitro and skin lesions production of cytokines in non-treated borderline tuberculoid (BT) and borderline lepromatous (BL) patients. PATIENTS AND METHODS: Seven untreated, non-reactional BT patients and 12 untreated, non-reactional BL patients were studied. Levels of the cytokines IFN-gamma, IL-10, TGF-beta1 and TNF-alpha were measured in supernantant of peripheral blood mononuclear cells (PBMC) cultures, stimulated with specific M. leprae antigen (sonicated and whole). The cytokines iNOS, IL-10 and TGF-beta1 were detected by immunohistochemistry in skin biopsies. RESULTS: BT patients produced higher levels of IFN-gamma than BL patients; iNOS expression in skin lesions was also higher in BT patients. TGF-beta1 was detected in more cells in BL patients; IL-10 expression was similar in both groups. There was a negative correlation between iNOS and TGF-beta1 expression in skin biopsies, positive correlation between TGF-beta1 in skin lesions and bacillary index, as well as positive correlation between iNOS detected in skin biopsies and PBMC IFN-gamma production. CONCLUSIONS: The BT patients had a mainly a Th1-profile of cytokines in their skin lesions and BL patients had a Th2 profile.

Alteration of the cortisol-cortisone shuttle in leprosy type 1 reactions in leprosy patients in Hyderabad, India.

Regulation of inflammation in leprosy may be influenced by local concentrations of active cortisol and inactive cortisone, whose concentrations are regulated by enzymes in the cortisol-cortisone shuttle. We investigated the cortisol-cortisone shuttle enzymes in the skin of leprosy patients with type 1 reactions (T1R), which are characterised by skin and nerve inflammation. Gene expression of the shuttle enzymes were quantified in skin biopsies from 15 leprosy patients with new T1R before and during prednisolone treatment and compared with levels in skin biopsies from 10 borderline leprosy patients without reactions. Gene expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2, which converts cortisol to cortisone, is down-regulated in skin from T1R lesions. However expression levels of 11beta-HSD type 1, which converts cortisone to cortisol, were similar in skin with and without reactions and did not change during anti-leprosy drug treatment. Prednisolone treatment of patients with reactions is associated with an upregulation of 11beta-HSD2 expression in skin. The down regulation of 11beta-HSD2 at the beginning of a reaction may be caused by pro-inflammatory cytokines in the leprosy reactional lesion and may be a local attempt to down-regulate inflammation. However in leprosy reactions this local response is insufficient and exogenous steroids are required to control inflammation.

S-100 as a useful auxiliary diagnostic aid in tuberculoid leprosy.

BACKGROUND: The diagnosis of tuberculoid leprosy is often difficult on hematoxylin and eosin (H&E) due to the absence of demonstrable nerve destruction. This study evaluates the utility of S-100 staining in identifying nerve fragmentation and differentiation of tuberculoid leprosy from other cutaneous granulomatous diseases. METHODS: Fifty cases of leprosy including 38 borderline tuberculoid (BT), two tuberculoid (TT), and 10 indeterminate leprosy (IL) were studied. Eleven controls of non-lepromatous cutaneous granulomatous lesions were included. S-100 was used for identifying the following dermal nerve patterns: infiltrated (A), fragmented (B), absent (C), and intact (D) nerves. RESULTS: On H&E, only 18/38 (47.4%) BT cases and 1/2 (50%) TT cases revealed neural inflammation. On S-100 staining of BT cases, 28/38 (73.7%) showed pattern B followed by patterns C and A in 8/38 (21.1%) and 2/38 (5.3%) cases, respectively. Both the TT cases showed pattern B. Only intact nerves (D) were seen in all the control cases. S-100 identified nerve damage in 4/10 (40%) IL cases. The patterns A, B, and C had sensitivity, specificity, and positive and negative predictive values of 100% in diagnosing tuberculoid (BT + TT) leprosy. CONCLUSIONS: S-100 is superior to H&E in identifying nerve fragmentation (p < 0.01). It also aids the differential diagnosis of tuberculoid leprosy.

Overexpression of vascular endothelial growth factor and its endothelial cell receptor KDR in type 1 leprosy reaction.

The sites of expression of vascular endothelial growth factor (VEGF) and of KDR, its endothelial cell receptor, were investigated in leprosy reaction Type 1, or reversal reaction (RR), by immunohistochemistry and in situ hybridization. In comparison with nonreactional leprosy, overexpression of both VEGF and KDR was seen in granuloma cells, especially epithelioid and foreign body-type giant cells, the epithelium and the vascular endothelium of RR specimens. In granuloma cells, hybridization for VEGF was stronger than immunostaining, a finding that may reflect the rapid turnover of VEGF in an immunologically dynamic situation such as RR. In the epidermis, double immunohistochemistry revealed VEGF overexpression in CDla-positive dendritic cells. The VEGF may not only be relevant for hyperpermeability and mononuclear cell differentiation (the key morphologic features in the acute, clinically evident phase of RR), but it could also be implicated in RR onset, when dendritic cells are activated in response to antigen stimulation.

Expression of inducible nitric oxide synthase and nitrotyrosine in borderline leprosy lesions.

BACKGROUND: In the response to T-helper cell (Th1)-type cytokines and interactions with pathogens, high levels of nitric oxide (NO) are produced by activated macrophages expressing the inducible NO synthase (iNOS). The role and importance of reactive nitrogen intermediates (RNIs) such as NO and peroxynitrite in the host response to diseases caused by intracellular pathogens such as Mycobacterium leprae and M. tuberculosis is unclear. OBJECTIVES: The aim of this study was to investigate the presence of local production of NO and peroxynitrite in borderline leprosy by using antibodies against iNOS and the product of peroxynitrite, nitrotyrosine (NT). METHODS: We detected the presence of iNOS and NT in skin biopsies from borderline leprosy patients, with and without reversal reaction (RR), by immunohistochemistry (n = 26). RESULTS: In general, the granulomas from borderline leprosy lesions with and without RR showed high and specific expression of iNOS and NT. Moreover, strong immunoreactivity to iNOS and NT was observed in granulomas surrounding and infiltrating dermal nerves. The expression of iNOS and NT was also strong in keratinocytes, fibroblasts and endothelial cells in close relation to the granulomatous reaction. In contrast, normal human skin showed no expression of iNOS and NT in these cells. CONCLUSIONS: We conclude that iNOS and NT are expressed in granulomas from borderline leprosy patients with and without RR and propose that RNIs might be involved in the nerve damage following RR in leprosy.

Multibacillary leprosy: lesions with macrophages positive for S100 protein and dendritic cells positive for Factor 13a.

In the defense against Mycobacterium leprae, macrophages play an essential part in the mechanism of bacterial lysis but require the presence of cytokines such as interleukin 2 and gamma interferon from lymphocytes in order to effectively kill the organisms in any number. While there have been many studies of the lymphocytes in lesions of leprosy, less attention has been given to the immunohistochemical characterization of the macrophage populations. In this study, the cutaneous lesions of 69 patients with leprosy (42 lepromatous, 5 mid-borderline, and 22 tuberculoid) were evaluated by immunohistochemistry for the expression of S100 protein, CD1a, CD68, muramidase, HLA-DR, and Factor 13a. The macrophages from lesions of polar, subpolar, and borderline lepromatous leprosy patients expressed S100 protein intensely and constantly. In contrast, the lesions of polar and subpolar tuberculoid leprosy had very few cells that were immunoreactive for S100 protein ('S100+') in the granulomas in the dermis. The macrophages in all lesions were reactive for CD68 and muramidase. In paraffin sections, macrophages of lepromatous lesions failed to stain for HLA-DR, whereas in tuberculoid lesions, they were strongly positive for HLA-DR. Three patients with histoid leprosy (relapse lesions) had lesions that were strongly positive for Factor 13a and were negative for S100 protein ('S100-'). Given the possible chemotactic and migration inhibition effects of the calcium-binding proteins of the S100 family, these data suggest a possibly important role for S100 protein in the accumulation of macrophages in lepromatous leprosy, and also reveal infection of Factor 13a + dermal dendritic cells in histoid leprosy.

Semen biochemistry of leprosy patients.

Studies have been made on the semen of three categories (borderline, borderline tuberculoid and lepromatous) of leprosy patients to evaluate the seminal biochemical constituents viz. fructose, glycerylphosphorylcholine and acid phosphatase besides the physical properties viz. volume, pH, liquefaction time, sperm density and sperm motility. In all categories of leprosy patients, seminal pH, liquefaction time and sperm density underwent significant decline. The decline in the seminal volume and sperm motility was significant only in borderline leprosy. It was observed that seminal glycerylphosphorylcholine (GPC) concentration and acid phosphatase activity declined in all categories of leprosy patients but GPC showed a significant decline only in borderline tuberculoid and acid phosphatase declined significantly only in borderline and lepromatous leprosy.

Detection of transforming growth factor-beta 1 in dermal lesions of different clinical forms of leprosy.

Immunohistochemical studies were performed to determine the presence and distribution of polypeptide transforming growth factor (TGF)-beta 1, a cytokine with macrophage-suppressing activity, in skin biopsies from 41 patients with different clinical forms of leprosy. We used an anti-TGF-beta 1 polyclonal antibody and the avidinbiotin-peroxidase (ABC complex) method. The results demonstrated that the lesions of the lepromatous and borderline lepromatous forms presented intense cytoplasm staining for TGF-beta 1 in the cells of the dermal infiltrate. A reaction of moderate intensity was observed in the cells of granulomas from borderline borderline cases, whereas no detectable immunoreaction was observed in granuloma cells from the tuberculoid and borderline tuberculoid forms. Considering that in the lepromatous leprosy form Mycobacterium leprae multiplies in the cytoplasm of macrophages and the lesions are diffuse and consist of poorly differentiated young macrophages, we believe that these alternations may be explained at least in part by the presence of TGF-beta 1 in the dermal infiltrate. Production of the cytokine may be induced by the presence of the bacillus itself and of its constituents, causing a mechanism of parasite evasion. Similarly, the absence of TGF-beta 1 in tuberculoid leprosy, which progresses with a specific immune response to M. leprae, may explain the intense differentiation of macrophage cells with the formation of well defined epithelioid granulomas capable of eliminating most of the bacilli.

Lipid composition of the stratum corneum of the sole in patients with leprosy.

Several reports support the view that changes of composition of the stratum corneum (SC) lipids may be the cause of impaired barrier function which, in turn, gives rise to xerosis and ichthyotic skin in leprosy. Many reports about abnormalities of serum lipids and cutaneous manifestations, such as xerosis and ichthyotic changes in leprosy, led us to the idea that the composition of SC lipids in patients with leprosy may be different from that in normal subjects. However, the many studies done in the past do not sufficiently account for this. To investigate the composition of SC lipids in patients with leprosy, thin-layer chromatography (TLC) was undertaken. Extraction of the SC lipids with a methanolchloroform-H2O mixture (4:2:1.6, v/v/v, Bligh-Dyer solvent) was carried out after shaving of the SC from the sole. TLC was performed and the composition of lipids was quantitated by photodensitometry. Our study revealed that the composition of SC lipids in the anesthetic lesions of leprosy patients was higher in cholesterol sulfate and triglycerides and lower in sphingolipids and cholesterol esters than that of normal subjects.

Calcium metabolism and its regulating hormones in patients with leprosy.

Calcium metabolism was studied in 47 patients with borderline or lepromatous leprosy. Total and ionized calcium, phosphorus, creatinine, total alkaline phosphatase, parathyroid hormone (PTH), 25-hydroxy vitamin D [25(OH)D], and 1,25-dihydroxy vitamin D [1,25(OH)2D] were measured in serum; calcium and total hydroxyproline were determined in urine. Total subperiosteal diameter and medullar cavity diameter were measured on an X-ray of the hand of all patients. Average values were within normal ranges for all of the biochemical determinations. Total serum calcium was moderately below the normal range in eight patients but ionized calcium levels were within the normal ranges in all of the patients. Four patients, all of them with lepromatous leprosy, had levels of 1,25(OH)2D higher than normal but none of them was hypercalcemic and PTH levels were within normal range. Although all values were within the normal ranges, lepromatous leprosy patients had lower total calcium, higher alkaline phosphatase, and higher urinary hydroxyproline than borderline leprosy patients (9.1 +/- 0.4 vs 9.4 +/- 0.3 mg%, p < 0.001; 10.3 +/- 2.9 vs 7.4 +/- 2.3 King-Armstrong units, p < 0.02 and 27.2 +/- 12 vs 19.4 +/- 5.6 mg/24 hr, p < 0.02, respectively). No differences were found between patients and controls in the average micrometric measurements of the second metacarpal bone but significant osteopenia was found in 19% of the patients. The main finding of the present study in a representative sample of leprosy patients is that the average total serum calcium was in the lowest limit of the normal range, but the ionized serum calcium was in the middle of the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of recombinant interferon gamma administration on lesional monocytes/macrophages in lepromatous leprosy patients.

Hydrogen peroxide (H2O2) and superoxide anion (O2-) were estimated in lesional cells from 10 lepromatous leprosy patients injected intralesionally with recombinant interferon-gamma (rIFN-gamma). Clinically similar contralateral lesions injected with excipient served as controls. Lesional esterase-positive cells (suggestive of monocytes/macrophages) from rIFN-gamma-injected sites of many subjects showed net increments in the H2O2 and O2 levels compared to controls. When these cells were exposed to Mycobacterium leprae in vitro, there was a down-regulation of O2- in 4 of 5 subjects. Such inhibition was not observed in rIFN-gamma-injected sites. From the present studies it was not possible to determine whether the above effects of rIFN-gamma were primarily on lesional mature macrophages or on newly migrated young monocytes. Erythema and induration were observed at the cytokine-injected site but not at the control site between 24 and 72 hr. A monthly slit-skin smear examination of the former site showed a bacterial index (BI) reduction compared to the controls in 4 of 10 patients, this reduction occurring as early as 4 to 8 weeks. Histopathology of the biopsies of 6 of 10 subjects between 9 and 10 months showed a further BI decrease attributable to rIFN-gamma and not to the subsequently instituted chemotherapy.

Investigations into the haemolytic effects of dapsone therapy in leprosy patients.

Investigations into the haemolytic effects of dapsone therapy were carried out in forty four leprosy patients admitted to the Sacred Heart Leprosy Centre, Kumbakonam. They received weight based dapsone dosages varying from 1.3-3.3 mg/kg body weight. Blood levels and urinary Dapsone/creatinine ratio were assessed at 1 day, 7 days and 30 days of Dapsone treatment. At the same points of time, haematological observations were also carried out. Serum bilirubin as well as blood mathaemoglobin were also examined. The findings showed a reduction in Hb levels at 30 days observation in a good proportion of cases on 100 mg. In one case (child) weighing 15 kg and receiving 50 mg dapsone increased mathaemoglobin was observed. It is suggested that dapsone dosage be regulated to body weight and preferably not to exceed 1.5 mg/kg body weight.

Polissacárides séricos nos diferentes tipos e fases da lepra

Utilizando a técnica de Seymour Cohen, os autores fizeram a dosagem de polissacárides em 17 soros normais, 11 soros de portadores de lepra tuberculóide e 60 soros de lepromatosos entre os quais 23 soros de pacientes em diferentes fases de reação leprótica. Tecem ligeiros comentários acêrca da técnica empregada e das possíveis origens dos polissacárides circulantes. Concluem que os polissacárides séricos nos doentes de lepra apresentam-se ligeiramente aumentados em relação às pessoas normais, sendo pequena a diferença entre casos tuberculóides e lepromatosos. Entretanto, na reação leprótica, verifica-se aumento proporcional … intensidade do quadro sindrômico.