BCG immunotherapy as an adjunct to chemotherapy in BL-lL patients--its effect on clinical regression, reaction severity, nerve function, lepromin conversion, bacterial/antigen clearance and 'persister' M. leprae.

BACKGROUND AND OBJECTIVE: Multidrug therapy in leprosy has failed to eliminate the problem of persister bacilli. Clearance of bacterial antigens is extremely slow which could predispose to continued nerve damage even after release from treatment. In the present study the immunomodulatory efficacy of BCG vaccine administered post-MDT in BL-LL leprosy patients was investigated in depth with a view to determining if augmenting chemotherapy with immunotherapy would help in faster clearance of M. leprae/antigens, bring down the level of persisters and minimise the occurrence/severity of reaction and nerve damage. METHODS: This is a placebo-controlled study in treated BL-LL patients. The patients are matched with respect to age, sex, bacteriological index and history of reaction, stratified and allocated to the two groups. One group (Gr A) received two doses of BCG-MOSCOW (3-33 x 10(5) cells) and the other (Gr B) normal saline (0.85%), injected intra-dermally at 3 month intervals. The Primary outcomes assessed at the end of 6 months were bacterial/antigen clearance, lepromin conversion, granuloma clearance and the occurrence of persisters. The secondary outcomes were clinical regression, occurrence and severity of reaction and changes in nerve functions. MATERIAL: A total of 107 BL-LL patients comprised of 49 in Gr A and 58 in Gr B; of which 36 and 42 respectively completed the study as per protocol, and are included in the final analysis. FINDINGS: The study findings show that both the primary and the secondary out comes were comparable in the two groups. Two doses of BCG administered post-MDT (Gr A) did not significantly alter the level of persisters or help in hastening the bacterial/antigen clearance, clinical regression of lesions and granuloma clearance. Lepromin conversion rates were also comparable. While the frequency of lepra reaction/neuritis following the intervention was comparable, the severity of reactions was significantly higher in Gr A. On the positive side neural functions assessed by nerve conduction studies showed that deterioration of motor nerve conduction was significantly lower in the BCG arm. Since all patients developing moderate to severe reactions, immediately received a course of corticosteroids, it is possible that timely use of it might have helped.

Perceptions, health seeking behaviour and access to diagnosis and treatment initiation among previously undetected leprosy cases in rural Maharashtra, India.

OBJECTIVES: To study sociodemographic profiles, perceptions about leprosy and health seeking patterns among adult leprosy patients and parents of children with leprosy detected through a prevalence survey conducted earlier, in rural areas of Panvel tehsil in Maharashtra. METHODS: The study was cross-sectional and used mixed (qualitative and quantitative) methods. Of the 97 confirmed rural leprosy cases who had been detected through the initial prevalence survey, 58 newly detected adult leprosy cases and parents of 22 children detected with leprosy were interviewed with a semistructured interview schedule between May 2008 and March 2009. FINDINGS: The study revealed that most of the leprosy patients belonged to the poor socioeconomic strata. Nearly 58% of the adult patients reported that they had been detected through the survey within 3 months of noticing their symptom(s) for the first time. Despite having been diagnosed and receiving treatment, only 48% of adult cases knew their condition as leprosy, reflecting their poor knowledge of the disease and lack of communication between providers and patients. The symptom 'patch on the skin' seems to have percolated in the community. Despite approaching the private or public sector for help in the first instance, many patients and children remained undiagnosed and untreated for leprosy. CONCLUSION: Active surveys for leprosy case detection should substitute the self-reporting approach until IEC measures are sufficiently effective to achieve a significant impact on transmission. Nevertheless both approaches will need the presence of staff with active diagnostic skills and optimal drug availability at PHCs.

The effect of corticosteroids usage on bacterial killing, clearance and nerve damage in leprosy; part 3--Study of two comparable groups of 100 multibacillary (MB) patients each, treated with MDT + steroids vs. MDT alone, assessed at 6 months post-release from 12 months MDT.

OBJECTIVE: To investigate effects of therapeutic usage of corticosteroids on M. leprae killing and clearance, on clearance of granuloma and on nerve damage in multibacillary (MB) leprosy patients. DESIGN: From a cohort of 400 untreated MB patients, a comparable group of 100 each receiving MDT + steroids (group A) vs MDT alone (group B) were assessed at 18 months as compared to month zero with respect to clinical and granuloma regression, M. leprae killing and clearance, and nerve functions. Analysis was performed using SPSS version 10.0. The significance of association was tested using Chi square and Fisher's exact tests. RESULTS: Regression of lesions assessed clinically and by histopathology was seen in 52% and 53% patients in group A and 46% and 63% in B respectively (P not significant). Clearance of bacteria assessed by bacteriological index (BI) in slit skin smears (SSS) and extent and intensity of antigen using anti-BCG staining were also comparable in the two groups. Multiplication of M. leprae in the mouse foot pad (MFP) indicating the presence of viable bacilli was seen in 14% and 16% of SSS positive BL-LLs patients in groups A and B respectively (P not significant). The occurrence of viable M. leprae was higher among patients with repeat reaction (19%) than single (11%). Using clinical tests (nerve palpation, monofilament and voluntary muscle testing), the proportion of sensory and motor nerves showing improvement or deterioration were similar in the two groups. However using nerve conduction studies, the overall proportion of nerves showing deterioration (22%) was significantly higher than improvement (9%) (P < 0.001). CONCLUSIONS: Treatment with MDT + corticosteroids does not adversely affect the clearance of granuloma, M. leprae and/or its antigens and M. leprae killing. However the continued presence of viable bacteria in > 14% of BL-LLs patients indicate that 12 months of MDT may be insufficient for complete bacterial killing. In both groups nerve conduction studies indicated that deterioration of nerves was high suggesting, MDT + corticosteroids was not very efficacious in the prevention or reversal of nerve damage. A better immuno-modulatory drug or a modified corticosteroid regime is needed.

Effect of corticosteroid usage combined with multidrug therapy on nerve damage assessed using nerve conduction studies: a prospective cohort study of 365 untreated multibacillary leprosy patients.

The purpose of this study is to determine the extent of nerve involvement and to study the effect of corticosteroids combined with multidrug therapy on nerve damage in leprosy patients using sensory and motor nerve conduction studies. A cohort of 365 untreated multibacillary leprosy patients were prospectively studied using sensory and motor nerve conduction studies on upper and lower limb nerves. They were subgrouped as those to be treated with 12-week regimen of corticosteroids for reaction and/or neuritis or silent neuropathy of <6 months duration along with 12-month multidrug therapy (group A), and those with no reaction were treated with multidrug therapy only (group B). Analysis was performed using SPSS version 10.0. Significance of association was tested using chi(2) test. At registration, abnormality by nerve conduction studies was seen in 92% of patients and majority (65%) showing involvement of more than five sensory and motor nerves. Sensory nerve abnormalities were higher (52%) than motor (37%) (P < 0.001). Affection of sensory and motor nerves was higher in group A (P < 0.001). Notably, 40% nerves in group B also showed impairment at 0 month. This implies that almost all patients showed abnormal nerve conduction studies at onset regardless of reaction, proving nerve damage is more widespread than envisaged. At 18 months, overall percentile deterioration (23%) of nerves was higher than improvement (9%) (P < 0.001) indicating that corticosteroids combined with multidrug therapy failed to significantly improve the nerve status. Sensory nerve (57%) affection was significantly higher than motor (46%) (P < 0.001). Moreover, percentile deterioration of sensory nerves was higher in group A (P < 0.001) implying corticosteroids is not very efficacious in the prevention or reversal of nerve damage. Electrophysiological tests provide valuable information for detecting nerve function impairment and evaluating appropriate therapeutic regimens.

Detection of previously undetected leprosy cases in a defined rural and urban area of Maharashtra, Western India.

INTRODUCTION: Leprosy has been a major public health problem in India for centuries. In India, between 2001 and 2005, the prevalence of leprosy was reduced by 80%. This sharp decline in the prevalence of leprosy alongside the cessation of active surveillance for detection of leprosy cases has raised a sense of alarm in the scientific community. MATERIALS AND METHODS: This is a total population survey aiming to estimate the prevalence of undetected active cases of leprosy in the community in defined rural (Panvel Taluka, Raigad District) and urban (M-East Ward, Mumbai) areas by health workers from Kushth Nivaran Samiti (Panvel) and Lok Seva Sangam (Mumbai). Those provisionally diagnosed with leprosy were subjected to an independent verification using clinical, bacteriological and histopathological investigations at the Foundation for Medical Research. FINDINGS: A population of 196,694 and 600,247 was covered in defined rural and urban areas respectively. In the rural area on examining 178,646 individuals, 120 provisionally diagnosed leprosy cases were detected, of which 65 were paucibacillary (PB) and 55 were multibacillary (MB) based on the WHO operational classification used by health workers at field level. In the urban area, of the 512,434 individuals who were examined, 134 provisionally diagnosed leprosy cases were detected with 92 PB and 42 MB cases. Among the clinically confirmed cases, 35.6% (32/90) and 34.9% (36/109) in rural and urban areas respectively were children. CONCLUSIONS: There are large numbers of undetected leprosy cases in the community with a high proportion of MB patients and children among them. This indicates active transmission pointing to the need for a paradigm shift in leprosy care services and control programme.

Sensitivity and specificity of nerve palpation, monofilament testing and voluntary muscle testing in detecting peripheral nerve abnormality, using nerve conduction studies as gold standard; a study in 357 patients.

OBJECTIVE: To determine sensitivity and specificity of clinical tools viz. nerve palpation (NP), monofilament (MF), and voluntary muscle testing (VMT), for assessing peripheral nerve function impairment (NFI) in leprosy, using nerve conduction studies (NCS) as gold standard. STUDY POPULATION AND METHODS: 357 untreated multibacillary (MB) leprosy patients were assessed using above tests. The nerves assessed were left and right ulnar, median, radial cutaneous, sural, common peroneal and posterior tibial. The concordance between the clinical and NCS tests was done for each nerve. The sensitivity and specificity of clinical tests for detecting nerve impairment was determined, using NCS as gold standard. Analysis was performed using SPSS version 10.0. RESULTS: The sensitivity of NP ranged between 71% to 88% for all nerves, except the median (43%) and sural (59%) nerves. Specificity was > 60% for all, but low for ulnar (34%) and common peroneal (40%) nerves. The specificity of MF testing was > 80% and of VMT assessment was >90% for all nerves. The sensitivity of MF testing ranged between 35-44%, while of VMT assessment was very low i.e. 4-5%, the maximum was for the ulnar nerve (25%). Detection sensitivity of MF testing and VMT assessment improved two fold when combined with NP and was closely comparable to NCS test findings. CONCLUSIONS: Both MF testing and VMT assessment showed good specificity, but moderate to low sensitivity. NP was less specific but more sensitive than MF testing and VMT assessment. Combining NP with MF testing and VMT assessment gives a two fold improvement in the sensitivity for assessing nerve damage and could therefore serve as the most useful clinical tools for diagnosis of leprosy and detecting nerve damage at field level.

Hypophosphorylation of NF-H and NF-M subunits of neurofilaments and the associated decrease in KSPXK kinase activity in the sciatic nerves of swiss white mice inoculated in the foot pad with mycobacterium leprae.

OBJECTIVE: To study the phosphorylation state of neurofilament (NF) proteins and activity of KSPXK kinase in the sciatic nerves of Swiss white (S/W) mice inoculated in the hind foot pads with M. leprae. DESIGN: Test group includes S/W mice inoculated in the foot pads with freshly harvested human derived (viable) M. leprae. Control groups were constituted by (1) Age matched un-inoculated mice, (2) Mice similarly inoculated with M. smegmatis and (3) heat killed M. leprae. Phosphorylation state of NF was studied using Western blot analysis and phosphor-specific NF antibody (SMI 31; Sternberger Monoclonals, Inc.). The KSPXK kinase activity was assayed by using KSPXK fusion protein in a radiometric method using gamma(22)P ATP. RESULTS: Several fold increase in M. leprae numbers was seen in viable M. leprae group while M. smegmatis failed to show any fold increase in the foot pads of S/W mice. Western immunoblot analysis of cytoskeletal preparation from sciatic nerves of uninoculated mice and mice inoculated with M. smegmatis showed immunoreactivity to SMI 31 antibody and protein bands corresponding to both NF-H and NF-M at all the time points from 4-20 months post inoculation. In case of viable M. leprae; SMI 31 reactive protein bands were seen at 4 months but not at any of the later intervals, i.e., between 6-20 months. With heat killed M. leprae transient loss of immunoreactivity to SMI 31 was seen. Decrease in KSPXK kinase activity was recorded in sets inoculated with viable and heat killed M. leprae, and corroborated with loss of immunoreactivity seen in WBs reacted with SMI 31 antibody. CONCLUSIONS: Alterations in the sciatic nerve NF cytoskeleton was seen following inoculation in the hind foot pad with both viable and heat killed M. leprae. The hypophosphorylation of NF observed in this study corroborates with the earlier observations in human leprous nerves.

The effect of corticosteroid usage on the bacterial killing, clearance and nerve damage in leprosy: a prospective cohort study: part 1--study design and baseline findings of 400 untreated multibacillary patients.

OBJECTIVE: To investigate possible adverse effects of therapeutic usage of corticosteroids on the killing and clearance of M. leprae and the clearance of granuloma, in patients with multibacillary (MB) leprosy. DESIGN: A cohort of 400 untreated MB patients were sub-grouped into those to be treated with corticosteroids (prednisolone 40 mg daily tapered to 5 mg over 12 weeks) along with MB-MDT for reaction and/or neuritis or silent neuropathy (SN) of <6 months duration (group A), and those with no reaction and to be treated with MDT only (group B). Clinical, bacteriological, histopathological and neurological test findings at fixed time points were compared. Analysis was performed using SPSS version 10.0. The significance of association was tested using Chi-square test. In the current report, we describe the study design and baseline findings of 400 untreated MB patients, with special emphasis on differences between patients in groups A and B. RESULTS: At baseline, applying Ridley-Jopling classification, 39% patients were BT, 20% BB, 24% BL, 12% sub-polar LL and 5% pure neural (PN). Overall, 60% patients were slit skin smear (SSS) negative and 33% presented with disability either grades 1 or 2. Overall 140/400 (35%) patients presented with reaction and/or neuritis and 11/400 (3%) presented with SN of <6 months duration. Comparing groups A and B, the percentage of patients presenting with DG2 was significantly higher in group A (43%). By clinical tests, monofilaments (MF) and voluntary muscle testing (VMT), the percentage of patients and nerves showing functional impairment was also significantly higher in group A. However, in the more sensitive nerve conduction velocity (NCV) test, the percentage of patients that showed nerve abnormalities was closely comparable; 94% and 91% in groups A and B respectively while number of affected nerves was higher in group A. CONCLUSION: At baseline, as recorded by NCV, peripheral nerve function abnormality was observed in almost all the MB patients regardless of reaction; but among those presenting with reaction or neuritis, the nerve damage was more severe and extensive.

Light and ultrastructural study of sciatic nerve lesions induced using intraneural injection of viable Mycobacterium leprae in normal and immunosuppressed Swiss white mice.

Freshly harvested M. leprae were microinjected into the sciatic nerves of nonimmunosuppressed (non-TR) and immunosuppressed (TR) mice using the technique described by Wisniewski and Bloom. The lesions thus induced, on bypassing the blood-nerve barrier, were biopsied at regular intervals beginning 24 hr and followed up to one year. The fate of M. leprae and the ensuing inflammation and nerve damage were studied using light and electron microscopy. The lesions in both non-TR and TR mice at 24 hr showed an influx of polymorphonuclear leukocytes and an increase in mast cells. The influx and peaking of lymphocytes were delayed by two weeks and 6 weeks, respectively, in TR mice, but the density of lymphocytes at the peak intervals was comparable in both. The plasma cells denoting the humoral response were seen in both, but there was a delay of 3 weeks in non-TR mice. The lesions in non-TR mice showed differentiation of macrophages into epithelioid cells and the formation of giant cells depicting borderline tuberculoid leprosy (BT), Whereas in TR mice, the macrophages showed foamy cytoplasmic changes depicting borderline lepromatous leprosy (BL). Other significant observations common to both non-TR and TR mice were: a) The lesions remained highly localized and showed signs of regression at the 6th and the 12th month intervals. b) The characteristic segmental demyelination and some attempt at remyelination were seen at the site. c) The influx of lymphocytes concorded well with demyelination. d) Bacteria were only seen in the macrophages and never in the Schwann cells or endothelial cells. e) Bacteria persisted in the macrophages, but appeared progressively degenerate at the 6th and 12th post-inoculation months, suggesting loss of viability. The study shows that there was a very effective containment of the infection and that the Schwann cells were resistant to M. leprae infection in the neural milieu. Nerve damage and Schwann cell bacillation do not go hand-in-hand.

Light and ultrastructural study of sciatic nerve lesions induced using intraneural injection of viable mycobacterium leprae in normal and immunosuppressed swiss white mice

Freshly harvested M. leprae were microinjected into the sciatic nerves of nonimmunosuppressed (non-TR) and immunosuppressed (TR) mice using the technique described by Wisniewski and Bloom. The lesions thus induced, on bypassing the blood-nerve barrier, were biopsied at regular intervals beginning 24 hr and followed up to one year. The fate of M. leprae and the ensuing inflammation and nerve damage were studied using light and electron microscopy. The lesions in both non-TR and TR mice at 24 hr showed an influx of polymorphonuclear leukocytes and an increase in mast cells. The influx and peaking of lymphocytes were delayed by two weeks and 6 weeks, respectively, in TR mice, but the density of lymphocytes at the peak intervals was comparable in both. The plasma cells denoting the humoral response were seen in both, but there was a delay of 3 weeks in non-TR mice. The lesions in non-TR mice showed differentiation of macrophages into epithelioid cells and the formation of giant cells depicting borderline tuberculoid leprosy (BT), Whereas in TR mice, the macrophages showed foamy cytoplasmic changes depicting borderline lepromatous leprosy (BL). Other significant observations common to both non-TR and TR mice were: a) The lesions remained highly localized and showed signs of regression at the 6th and the 12th month intervals. b) The characteristic segmental demyelination and some attempt at remyelination were seen at the site. c) The influx of lymphocytes concorded well with demyelination. d) Bacteria were only seen in the macrophages and never in the Schwann cells or endothelial cells. e) Bacteria persisted in the macrophages, but appeared progressively degenerate at the 6th and 12th post-inoculation months, suggesting loss of viability. The study shows that there was a very effective containment of the infection and that the Schwann cells were resistant to M. leprae infection in the neural milieu. Nerve damage and Schwann cell bacillation do not go hand-in-hand.