Clinical, histopathological and bacteriological study of 52 referral MB cases relapsing after MDT.

Fifty-two BB-LL relapse cases referred to our centre during 1997-2003 were investigated in detail. Twenty-four cases had been treated with extended MB-MDT [until smear negativity (NON-FDT)]. The remaining 28 cases (54%) had received one of the fixed duration regimens (FDT), of whom 11 had 24 months and 6 had 12 months of WHO MB-MDT. Eleven cases had received rifampicin/ofloxacin (RO) treatment. Follow-up slit skin smear reports were available for 41 cases, all but three cases had been smear negative at some point after release from treatment. None of the cases showed any clinical or bacteriological evidence of upgrading, i.e. LL to BT where as downgrading BB to BL occurred in five cases. The duration between cessation of treatment and reappearance of lesions (DCTR) varied from 2 to 15 years. The mean DCTR was longest (9.4 years) for the NON-FDT and 24 months MB-MDT cases. The mean DCTR was significantly lower in the 12 months MB-MDT and RO treated cases (6.8 and 6.2 years, respectively). Four of RO treated cases and four cases with multiple episodes of reaction had DCTR less than 5 years. Inadequate treatment/poor killing of Mycobacterium leprae results in early onset relapse, whereas 'persisting' or 'drug resistant mutants' contribute to late onset relapse.

Alterations in neurofilament protein(s) in human leprous nerves: morphology, immunohistochemistry and Western immunoblot correlative study.

Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.

Alterations in neurofilament protein(s) in human leprous nerves: morphology, immunohistochemistry and Western immunoblot correlative study

Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.

Viability and drug susceptibility testing of M. leprae using mouse footpad in 37 relapse cases of leprosy.

Mycobacteria leprae isolates obtained from 37 referral relapse cases of leprosy (37 skin and 10 nerve biopsy samples) received during the years 1994-2001, were tested for viability and drug sensitivity in the mouse footpad. A significant M. leprae yield in the footpads of control mice was obtained, with 32/47 (68%) isolates (from 26 cases) thus confirming viability. Of the 28 isolates successfully drug tested, 6 (21%) were resistant to one or more drugs. All except one, were multidrug treated cases (5/24 = 21%). One of the isolates was resistant to all three drugs, i.e., dapsone (di-aminodiphenyl sulphone, DDS), rifampin (RFP), and clofazimine (CLF). Two were resistant to two drugs, i.e., DDS and RFP, and each of the others were mono resistant to DDS, RFP, or CLF. Notably, one of the isolates that showed combined resistance to DDS and RFP was derived from a borderline tuberculoid case. Also, in one case skin and nerve showed that discordance viz: M. leprae derived from skin were resistant to RFP, while those derived from nerve tested sensitive to all three drugs, indicating tissue related difference.

Elevada incidencia y Mycobacterium Leprae visible en lesiones recurrentes en pacientes de lepra tuberculoide Post-MDT

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Clinical and electrophysiological evaluation of nerve function impairment following cessation of multidrug therapy in leprosy.

Seventeen multibacillary (MB) and 15 paucibacillary (PB) cases of leprosy who had had regular and adequate multidrug therapy (MDT) were examined clinically and electrophysiologically at periodic intervals for 1 year following cessation of MDT. All the major nerves were assessed for nerve function impairment (NFI). Overall, two MB (13.3%) and three PB (20%) cases showed signs of deterioration clinically and/or electrophysiologically. The nerve conduction (NC) follow-up studies revealed no significant improvement in the sensory conduction in both the MB and PB groups of nerves, whilst motor conduction showed a significant improvement at the first 6-monthly follow-up among the MB group of nerves. At the study onset, sensory impairment (MB = 62%, PB = 25%) predominated over motor in terms of both severity and frequency. The lower extremity was more frequently and severely affected than the upper in both groups of patients. As an individual test, NC measurement proved to be more sensitive in detecting NFI, but the combination of physical palpatation for nerve thickening and graded nylon test (GNT) was closely comparable to measurement of nerve conduction.

Sciatic nerve of normal and T200x5R Swiss white mice fails to support multiplication of intraneurally injected M. leprae.

In a preliminary study we have shown that freshly harvested Mycobacterium leprae, when injected into the sciatic nerve in normal and immunosuppressed (TR) mice, induce massive but localized epithelioid and macrophage granuloma, respectively, in 3-4 weeks. In order to determine the fate of M. leprae injected intraneurally into normal and TR mice, in the present study we measured sequentially the viability, fold increase and clearance, if any, using semi-quantitative methods. The average M. leprae yield per nerve assessed at regular intervals, beginning at 24 hr and including 72 hr, 1 week, 2, 3, 4, 12, 24 and 48 weeks, showed neither a significant increase nor a decrease in either the normal or the TR mice. The viability of M. leprae, assessed using the standard mouse foot pad method, showed a significant decrease as compared to baseline growth effective at 24 hr and remained static until approximately 4 weeks. A further decline and total loss of viability was noted by 12 months. The results show that injection of M. leprae via the intraneural route in both normal and TR mice failed to sustain the viability and failed to support the multiplication of the organisms.

In vitro studies on extracellular matrix production by M.leprae infected murine neurofibroblasts.

Fibroblasts and a host of macrophage secretory products have been implicated in a number of diseases where excess extracellular matrix (ECM) deposition is the main pathological feature. Fibrosis characterized by excessive deposition of collagen also contributes to the irreversible nerve damage observed in leprosy. Since M. leprae are seen within neurofibroblasts (Nf) in the advanced stages of the disease and macrophages form a common infiltrating cellular constituent of leprous nerves at all stages, secretion of ECM proteins by Nf was studied, in vitro following infection with M. leprae and in the presence of macrophage secretory products. These studies were compared in cells derived from two strains of mice, Swiss White (SW) and C57BL/6, as they differ in their response to M. leprae infection and parallel those observed in lepromatous and tuberculoid patients, respectively. On infection with M. leprae, Nfs showed a decrease in secretion of collagen type IV in SW and type I in C57Bl/6 strain. Macrophages caused a further decrease in the secretion of collagen types affected by M. leprae infection per se, while the other collagen types, viz. I and III in SW strain and III and IV in C57Bl/s strain, were unaffected. This study indicates that neural collagenization in nerves in advanced leprosy may be of Nf origin. However, unlike other diseases with excess collagen deposition, ECM proteins produced by Nfs in response to nerve damage may not be of prime importance in the progression of leprous neuropathy and occur as a general response to loss of cellular content in leprous nerves.

Differential in vitro modulation of Schwann cell proliferation by Mycobacterium leprae and macrophages in the murine strains, Swiss white and C57Bl/6.

The special susceptibility of Schwann cells (SCs) to parasitization by M. leprae and of macrophages to M. leprae-induced defects implicates them in leprous nerve pathogenesis. SC proliferation is an important prerequisite for peripheral nerve regeneration and is regulated by a number of secretory factors. Several of these factors are secreted by SCs themselves as well as by the macrophages which are recruited at the site of lesion to assist in regeneration. SC proliferation, as indicated by 3H-thymidine incorporation, was therefore studied in response to M. leprae infection and in the presence of macrophages in order to determine the role of SC in leprous neuropathy. Cells derived from two strains of mice, Swiss White (SW) and C57Bl/6 were used, as macrophages from these strains have been shown to differ in their response to M. leprae; such differences are similar to those observed in macrophages from lepromatous and tuberculoid leprosy patients, respectively. Infection with M. leprae for a duration of 9 days resulted in reduced proliferation of SCs from SW strain, while SCs from C57Bl/6 remained unaffected. However, in the presence of macrophages, SCs from both strains not only showed enhanced proliferation, but SW SCs also overcame the M. leprae-induced suppression of their proliferation. Altered SC proliferation, therefore, can be implicated as a factor in leprous nerve pathogenesis. The strain variation observed in the response of SCs indicate different nerve damage mechanisms in lepromatous and tuberculoid patients.

Schwann cell extracellular matrix protein production is modulated by Mycobacterium leprae and macrophage secretory products.

Extracellular matrix (ECM) protein deposition is an important feature of leprous nerves, where Schwann cells (SCs) and macrophages are the main hosts for Mycobacterium leprae. Since, SCs are involved in the synthesis of ECM proteins and its production is regulated by macrophage secretory factors, the present study aimed to determine in vitro, the effect of M. leprae infection and macrophage secretory products on secretion of ECM proteins by SCs in two strains of mice, Swiss White (SW) and C57BL/6, that are known to differ in their nerve pathology and macrophage functions in response to infection. Following six days of M. leprae infection, SCs from SW mice responded with increased secretion of 14C-leucine radiolabelled proteins and a concomitant increase in laminin and collagens type I, III and IV, as determined by enzyme-linked immunosorbent assay. In contrast infected C57BL/6 SCs responded with decreased secretion of total proteins and fibronectin. Exposure of SCs to macrophage conditioned medium resulted in decreased ECM protein secretion in both strains of mice. This decrease was a function of protein breakdown by macrophage derived proteases and also active regulation by macrophage secreted cytokines. A similar effect of M. leprae and macrophage secretory products on SC metabolism in leprous nerves would have major ramifications on damage and repair activities. In addition ECM proteins would also influence the composition of the infiltrating cell population in lepromatous and tuberculoid nerves.

Higher incidence of viable Mycobacterium leprae within the nerve as compared to skin among multibacillary leprosy patients released from multidrug therapy.

As identified by a significant growth in the footpads of immunosuppressed mice, the incidence of viable bacteria in a group of 26 multibacillary (BL-LL) patients released from multidrug (MDT) treatment was found to be two times more in the nerves (46%) as compared to skin (23%). Evidently there was a positive correlation between the overall bacterial load and the incidence of viable organisms. Bacterial growth was also observed in two out of five cases where neither the skin nor the nerve homogenate had shown any presence of acid-fast bacilli. Histopathology of biopsies, skin as well as nerve, including those having viable bacteria did not show any features of active disease.