Microfasciculation: a morphological pattern in leprosy nerve damage.

AIMS: To study Microfasciculation, a perineurial response found in neuropathies, emphasizing its frequency, detailed morphological characteristics and biological significance in pure neural leprosy (PNL), post-treatment leprosy neuropathy (PTLN) and non-leprosy neuropathies (NLN). METHODS AND RESULTS: Morphological characteristics of microfascicles were examined via histological staining methods, immunohistochemical expression of neural markers and transmission electronmicroscopy. The detection of microfasciculation in 18 nerve biopsy specimens [12 PNL, six PTLN but not in the NLN group, was associated strongly with perineurial damage and the presence of a multibacillary inflammatory process in the nerves, particularly in the perineurium. Immunoreactivity to anti-S100 protein, anti-neurofilament, anti-nerve growth receptor and anti-myelin basic protein immunoreactivity was found within microfascicles. Ultrastructural examination of three biopsies showed that fibroblast-perineurial cells were devoid of basement membrane despite perineurial-like NGFr immunoreactivity. Morphological evidence demonstrated that multipotent pericytes from inflammation-activated microvessels could be the origin of fibroblast-perineurial cells. CONCLUSIONS: A microfasciculation pattern was found in 10% of leprosy-affected nerves. The microfascicles were composed predominantly of unmyelinated fibres and denervated Schwann cells (SCs) surrounded by fibroblast-perineurial cells. This pattern was found more frequently in leprosy nerves with acid-fast bacilli (AFB) and perineurial damage while undergoing an inflammatory process. Further experimental studies are necessary to elucidate microfascicle formation.

Pure neural leprosy: steroids prevent neuropathy progression.

Multidrug therapy (MDT), with rifampicin, dapsone, and clofazimine, treats leprosy infection but is insufficient in arresting or preventing the nerve damage that causes impairments and disabilities. This case-series study evaluates the benefits of the combined use of steroids and MDT in preventing nerve damage in patients with pure neural leprosy (PNL). In addition to MDT, 24 patients (88% male aged 20-79 years, median=41) received a daily morning dose of 60 mg prednisone (PDN) that was gradually reduced by 10 mg during each of the following 5 months. PNL was clinically diagnosed and confirmed by nerve histopathology or PCR. A low prevalence (8.3%) of reaction was observed after release from treatment. However, most of the clinical parameters showed significant improvement; and a reduction of nerve conduction block was observed in 42% of the patients. The administration of full-dose PDN improved the clinical and electrophysiological condition of the PNL patients, contributing to the prevention of further neurological damage.

Role of PGL-I antibody detection in the diagnosis of pure neural leprosy.

Pure neural leprosy (PNL) is difficult to diagnose because skin lesions and acid-fast bacilli (AFB) in slit smears are absent. At present, the gold standard for PNL diagnosis is the histopathological examination of a peripheral nerve biopsy. Even so, detection of bacteria is difficult and histological findings may be non-specific. Furthermore, nerve biopsy is an invasive procedure that is only possible in specialized centres. Therefore, there is a need for additional diagnostic methods that may help to confirm the clinical diagnosis of PNL. In the present study, an additional laboratory test, the ELISA for anti-phenolic glycolipid I (PGL-I) IgM antibodies, was performed on 103 individuals with clinical and neurophysiological signs of peripheral neuropathy, of which 67 were diagnosed as PNL patients and 36 remained as 'not diagnosed as PNL', as well as on a control group of 34 patients with other neurological diseases. An antibody response was present in 14/67 (21%) of the patients diagnosed as PNL as compared with 3/34 (9%) of controls. Anti-PGL-I positivity was observed in 5/8 (63%) of the AFB positive cases. Patients whose diagnosis was confirmed solely by Mycobacterium leprae PCR on the nerve sample had 4/25 (16%) seropositivity. In addition, anti-PGL-I antibodies were detected in 9/40 (23%) of the PNL patients who were PCR negative for M. leprae DNA. Moreover, two patients who showed clinical and eletrophysiological manifestations suggestive of PNL were diagnosed with the help of their positive test results in the anti-PGL-I ELISA. In conclusion, detection of antibodies against PGL-I in patients with peripheral neuropathy is useful as an additional laboratory test to help PNL diagnosis.

Criteria for diagnosis of pure neural leprosy.

The clinical diagnosis of pure neural leprosy (PNL) remains a public health care problem mainly because skin lesions-the cardinal features of leprosy-are always absent.Moreover, the identification of the leprosy bacillus is not easily achieved even when a nerve biopsy can be performed. In an attempt to reach a reliable PNL diagnosis in patients referred to our Leprosy Outpatient Clinic, this study employed a variety of criteria. The nerve biopsies performed on the 67 individuals whose clinical, neurological, and electrophysiological examination findings strongly suggested peripheral neuropathy were submitted to M. leprae identification via a polymerase chain reaction (PCR). Mononeuropathy multiplex was the most frequent clinical and electrophysiological pattern of nerve dysfunction, while sensory impairment occurred in 89% of all cases and motor dysfunction in 81%. Axonal neuropathy was the predominant electrophysiological finding, while the histopathological nerve study showed epithelioid granuloma in 14% of the patients, acid fast bacilli in 16%, and nonspecific inflammatory infiltrate and/or fibrosis in 39%. PCR for M. leprae was positive in 47% of the nerve biopsy samples (n=23). PCR, in conjunction with clinical and neurological examination results, can be a powerful tool in attempting to identify and confirm a PNL diagnosis.

Transforming growth factor-ß1 may be a key mediator of the fibrogenic properties of neural cells in leprosy.

Fibrosis is the main cause of irreversible nerve damage in leprosy. Phenotypic changes in Mycobacterium leprae (ML)-infected Schwann cells (SCs) have been suggested to mediate this process. We found that SC line cultures stimulated with ML upregulated transforming growth factor-ß1 (TGF-ß1), and that TGF-ß1 or ML induced increased numbers of α-smooth muscle actin (α-SMA)-positive cells with characteristic stress fibers. Mycobacterium leprae and TGF-ß1 also induced increased type I collagen and fibronectin mRNA and secretion and augmented mRNA levels of SOX9 and ZEB1, which are involved in the epithelial-mesenchymal transition. These effects could be inhibited by the TGF-ß1 type I receptor (ALK5) inhibitor, SB-431542. In nerve biopsies from leprosy-infected patients with varying grades of fibrosis (n = 11), type I and III collagen and fibronectin were found in the endoneurium and perineurium, α-SMA-positive cells filled the fibrotic perineurium but not the endoneurium, and CD34-positive fibroblasts predominated in the endoneurium. Results of transcriptional studies of 3 leprosy nerves and 5 controls were consistent with these data, but α-SMA and other mRNA levels were not different from those in the control samples. Our findings suggest that TGF-ß1 may orchestrate events, including reprogramming of the SC phenotype, leading to transdifferentiation, connective tissue cell expansion, and fibrogenesis in the evolution of leprosy nerve lesions during some evolutionary stages.

Progression of leprosy neuropathy: a case series study.

A need still exists to determine the clinical and neurophysiological characteristics of leprosy neuropathy at distinct times of the disease by different methods that measure the various nerve fiber functions. A prospective clinical study was performed with 10 paucibacillary (PB) and 12 multibacillary (MB) patients evaluated at diagnosis and one year after cessation of multidrug therapy (MDT). Peripheral nerve function was assessed clinically and by means of the sympathetic skin response, skin vasomotor reflex, and nerve conduction study (NCS). At diagnosis, 73% of the total 22 patients had nerve function impairment (NFI). Autonomic function (χ(2)= 5.5, P= 0.019) and NCS (χ(2)= 7.765, P= 0.01) were significantly more altered in MB than PB patients. At final evaluation, NFI of the MB patients had worsened, especially among the six who had leprosy reaction. As the NFI of PB patients showed improvement, a significant difference between the two groups (χ(2)= 12.320, P= 0.001) was observed. A high prevalence of neuropathy was observed in newly diagnosed patients. Associating different tests with a thorough clinical neurological evaluation increases detection rates.

Expression of metalloproteinases (MMP-2, MMP-9, and TACE) and TNF-alpha in the nerves of leprosy patients.

Matrix metalloproteinases (MMPs) and tumor necrosis factor alpha (TNF-alpha) play important and related roles in the pathogenesis of nerve injury. MMP-dependent and TNF-alpha-dependent processes of neurodegeneration, such as blood-nerve breakdown and immune cell recruitment, are characteristic of leprosy nerve damage. Our work has contributed to the understanding of the role of cytokines in the process, but the role of MMPs in the pathogenesis of neuritic leprosy has not been investigated. This study analyzed the changes in mRNA expression and immunodistribution of MMP-2, MMP-9, TNF-alpha-converting enzyme (TACE), TNF-alpha in nerves of 27 pure neuritic leprosy (PNL) patients, both acid-fast bacilli positive (AFB(+)) and acid-fast bacilli negative (AFB(-)), and 8 non-leprosy patients with control peripheral neuropathic conditions. MMP-2, MMP-9, and TNF-alpha mRNA expression was significantly induced in the AFB(-) relative to the AFB(+) neuritic leprosy group and nonlepritic controls; TACE levels were also elevated in the AFB(-) group, but this change was not statistically significant. Immunoreactive profiles for TNF-alpha and MMPs demonstrated strong reactivity of myelinated axons, infiltrating macrophages, Schwann cells, endothelial cells, and perineurial cells in neuritic leprosy biopsies. This study provides the evidence of the involvement of MMPs in the pathogenesis of PNL neuropathy.

Evaluation of real-time and conventional PCR targeting complex 85 genes for detection of Mycobacterium leprae DNA in skin biopsy samples from patients diagnosed with leprosy.

In spite of the decrease in the number of registered leprosy patients, the number of new cases diagnosed each year (400,000) has remained essentially unchanged. Leprosy diagnosis is difficult due to the low sensitivity of current methodologies to identify new cases. In this study, conventional and TaqMan real-time PCR assays for detection of Mycobacterium leprae DNA were compared to current classification based on clinical, bacteriological, and histological evaluation. M. leprae DNA was extracted from frozen skin biopsy specimens from 69 leprosy patients enrolled in the study and was amplified using specific primers for either the antigen 85B-coding gene or the 85A-C intergenic region by using conventional and real-time PCR. The detection rate was 100% among multibacillary (MB) patients and ranged from 62.5% to 79.2% among paucibacillary (PB) patients according to the assay used. The TaqMan system for 85B gene amplification showed the highest sensitivity, although conventional PCR using the 85A-C gene as a target was also efficient. The cycle threshold (C(T)) values obtained using the TaqMan system were able to statistically (P < 0.0001) differentiate MB (mean C(T), 28.06; standard deviation [SD], 4.51) from PB (mean C(T), 33.06; SD, 2.24) patients. Also, there was a correlation between C(T) values and the bacteriological index for MB patients (Pearson's r, -0.444; P = 0.008). Within the PB patients' group, we tested normal skin from six patients exhibiting the pure neuritic form of leprosy (PNL). Five out of six PNL patients were positive for the presence of M. leprae DNA, even in the absence of skin lesions. In conclusion, the TaqMan real-time PCR developed here seems to be a useful tool for rapidly detecting and quantifying M. leprae DNA in clinical specimens in which bacilli were undetectable by conventional histological staining.

Pure neural leprosy: steroids prevent neuropathy progression

Multidrug therapy (MDT), with rifampicin, dapsone, and clofazimine, treats leprosy infection but is insufficient in arresting or preventing the nerve damage that causes impairments and disabilities. This case-series study evaluates the benefits of the combined use of steroids and MDT in preventing nerve damage in patients with pure neural leprosy (PNL). In addition to MDT, 24 patients (88 percent male aged 20-79 years, median=41) received a daily morning dose of 60 mg prednisone (PDN) that was gradually reduced by 10 mg during each of the following 5 months. PNL was clinically diagnosed and confirmed by nerve histopathology or PCR. A low prevalence (8.3 percent) of reaction was observed after release from treatment. However, most of the clinical parameters showed significant improvement; and a reduction of nerve conduction block was observed in 42 percent of the patients. The administration of full-dose PDN improved the clinical and electrophysiological condition of the PNL patients, contributing to the prevention of further neurological damage.

A poliquimioterapia (PQT), com rifampicina, dapsona, e clofazimina, trata a infecção na hanseníase, mas é insuficiente para interromper ou prevenir o comprometimento neurológico que causa as incapacidades e desabilidades, nesta enfermidade. Este estudo de série de casos avalia o benefício do uso combinado de prednisona e PQT na prevenção do dano neurológico em pacientes com a forma neural pura da hanseníase (FNP). Além do PQT, 24 pacientes (88 por cento homens, com idade variando entre 20-79, mediana=41) receberam uma dose diária de 60 mg prednisona que foi reduzida gradualmente na dose de 10 mg durante cada um dos 5 meses subseqüentes. FNP foi diagnosticada clinicamente e confirmada através do estudo histopatológico ou PCR. Baixa prevalência de reação (8,3 por cento) foi observada apenas após o final do tratamento. A maioria dos parâmetros clínicos mostrou melhora significativa e redução do bloqueio de condução foi observada em 42 por cento dos pacientes. A administração de doses altas de prednisona melhora a evolução clínica e eletrofisiológica de pacientes com a FNP de hanseníase, contribuindo na prevenção de novos comprometimentos neurológicos.

Criteria for diagnosis of pure neural leprosy

The clinical diagnosis of pure neural leprosy (PNL) remains a public health care problem mainly because skin lesions-the cardinal features of leprosy-are always absent.Moreover, the identification of the leprosy bacillus is not easily achieved even when a nerve biopsy can be performed. In an attempt to reach a reliable PNL diagnosis in patients referred to our Leprosy Outpatient Clinic, this study employed a variety of criteria. The nerve biopsies performed on the 67 individuals whose clinical, neurological, and electrophysiological examination findings strongly suggested peripheral neuropathy were submitted to M. leprae identification via a polymerase chain reaction (PCR). Mononeuropathy multiplex was the most frequent clinical and electrophysiological pattern of nerve dysfunction, while sensory impairment occurred in 89% of all cases and motor dysfunction in 81%. Axonal neuropathy was the predominant electrophysiological finding, while the histopathological nerve study showed epithelioid granuloma in 14% of the patients, acid fast bacilli in 16%, and nonspecific inflammatory infiltrate and/or fibrosis in 39%. PCR for M. leprae was positive in 47% of the nerve biopsy samples (n=23). PCR, in conjunction with clinical and neurological examination results, can be a powerful tool in attempting to identify and confirm a PNL diagnosis.