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INTRODUCTION: Despite multidrug therapy, leprosy remains a public health issue. The intradermal Bacillus Calmette-Guérin (BCG) vaccine, Mitsuda test (lepromin skin test), and anti-phenolic glycolipid I (PGL-I) serology are widely used in leprosy studies and have shown great epidemiological value. METHODS: This longitudinal study evaluated the relative risks and benefits of these three tools by comparing results observed in household contacts (HHCs) of leprosy patients who developed leprosy with those of HHCs who did not in a population of 2,992 individuals monitored during a 10-year period. RESULTS : Seventy-five (2.5%) new leprosy cases were diagnosed, including 28 (0.9%) co-prevalent cases. Therefore, for the risk-benefit assessment, 47 (1.6%) HHCs were considered as truly diagnosed during follow-up. The comparison between healthy and affected contacts demonstrated that not only did BCG vaccination increase protection, but boosters also increased to 95% relative risk (RR) reduction when results for having two or more scars were compared with having no scars [RR, 0.0459; 95% confidence interval (CI), 0.006-0.338]. Similarly, Mitsuda reactions >7mm in induration presented 7-fold greater protection against disease development compared to reactions of 0-3mm (RR, 0.1446; 95% CI, 0.0566-0.3696). In contrast, anti-PGL-I ELISA seropositivity indicated a 5-fold RR increase for disease outcome (RR, 5.688; 95% CI, 3.2412-9.9824). The combined effect of no BCG scars, Mitsuda reaction of

Hypersensitivity reactions called reversal reaction (RR) and erythema nodosum leprosum (ENL) occur in leprosy. They are characterized by an increase in tumor necrosis factor-alpha (TNF-alpha). Thalidomide is an effective treatment for ENL but not RR. Its effectiveness in ENL is attributed to inhibition of TNF-alpha, and this does not explain its failure to treat RR. We assessed thalidomide's effect on TNF-alpha in RR. Mononuclear cells from RR and non-RR patients and healthy individuals were treated with thalidomide and M.leprae (AFB), a cytosol fraction of M. leprae or Dharmendra lepromin. Thalidomide suppressed TNF-alpha, but when some RR patients' cells were stimulated with AFB, it enhanced TNF-alpha.

This report describes a promising mode of treatment of lepromin-unresponsive, far-advanced, lepromatous (LL) leprosy patients with antileprosy vaccines as an adjunct to multidrug therapy (MDT). The Trial Groups included 50 highly bacilliferous, lepromin-negative, untreated LL patients. They were given MDT for 2 years. Of them, 30 patients were administered a mixed antileprosy vaccine containing killed Mycobacterium leprae of human origin plus M. bovis BCG. The remaining 20 patients were given M. bovis BCG. Depending on the severity of lepromin unresponsiveness, they were given one to six inoculations at 3-month intervals. Another 20 similar LL patients were taken in the Control Group. They were given only MDT for 2 years. From the start of the study, all patients belonging to the Trial and Control Groups were followed every 3 months for clinical, bacteriological and immunological outcomes. Within 2 years all 50 patients of the Trial Groups and 19 of the 20 patients of the Control Group became clinically inactive and bacteriologically negative. However, the clinical cure and the falls of the bacterial and morphological indexes were much faster in those patients receiving the mixed vaccine therapy than in those patients who were given BCG plus MDT or only MDT. The immunological improvements in the patients of the Trial and Control Groups were assessed by: a) lepromin testing at the beginning of the study and at 3-month intervals and also by b) the in vitro leukocyte migration inhibition (LMI) test at both the beginning and end of the study. As the patients were given more and more vaccinations, the incidence of lepromin conversion increased, more so in the patients receiving the mixed vaccine. Thus, 63%, 15% and 5% of the patients became lepromin positive in those patients receiving the mixed vaccine, BCG, and MDT only, respectively. Lamentably, the vaccine-induced lepromin positivity was temporary and faded away within several months. At the beginning of the study, the LMI test against specific M. leprae antigen was negative in all patients of both the Trial and Control Groups. After the end of the chemo-immunotherapy schedule, the LMI test became positive in 50% and 20% of LL patients receiving the mixed vaccine and BCG, respectively. None of the Control Group could show LMI positivity after completion of the MDT schedule. These results show that treatment of LL patients with the mixed vaccine and MDT could quickly reverse the clinical course of the disease, remove immunologic anergy in some patients, and induce a rapid decrease in the bacterial load in them.

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.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.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.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.

Ultraviolet radiation (UVR) modulates cellular immunity in humans and experimental animals and can interfere with immune responses against infectious agents in animal models. We used the lepromin reaction, a cell-mediated immune response to antigens of Mycobacterium leprae, to determine whether UVR affects the cellular immune response to an infectious agent in humans. We selected 29 healthy, lepromin-positive contacts of leprosy patients and determined their minimal erythema dose (MED) of UVR. Immediately afterward, each subject was injected with 0.1 ml of lepromin in two areas of the buttocks: one at the site that had received twice the MED of UVR and the other on the contralateral, unirradiated site. The irradiated site was given twice the MED every 4 d for a total of five treatments. One week after the last irradiation, both lepromin reactions were measured and biopsied. The size of the lepromin-induced granulomas was significantly reduced in the irradiated site, as was the number of lymphocytes. Immunohistochemical analysis showed a depletion in the number of infiltrating cells and a lower percentage of T cells, particularly the CD4+ subpopulation, in granulomas formed in UV-irradiated skin. This study demonstrates that local UV irradiation reduces the granulomatous reaction to lepromin in sensitized individuals. These findings are of clinical relevance because of the fundamental role played by the delayed-type hypersensitivity response in defense against intracellular pathogens and because of potential increases in the amount of UVR in sunlight reaching the earth's surface.

SUMMARY T cell proliferation and interferon-gamma (IFN-γ) production of peripheral blood mononuclear cells (PBMC) from 20 household contacts were tested against the 18- and 65-kD heat shock proteins from Mycobacterium leprae (ML 18 and ML65 respectively) and antigen 85 from Myco. bovis bacille Calmette-Guerin (BCG) (Ag 85) during a 12-months follow-up study. Among the eight contacts that became positive, eight showed positive reactivity against Ag 85, 5/8 against ML65 and 4/8 against ML 18 at the end of the study. Of the 16 contacts who were lepromin-positive either at first or second testing, all responded to Ag 85, 11 to ML 65, but only eight reacted to ML18 antigen. Contacts who were lepromin-positive at first testing developed responses to ML18 only at second testing. In contrast, among the four contacts that remained lepromin-negative during the follow up, three proliferated to Ag 85 either at first or second testing, but only one produced IFN-γ against Ag 85 at the end of the study. These results demonstrated that T cell reactivity and particularly IFN-γ secretion against Ag 85, but not against ML18 and ML65, might be a predominant mechanism in the early stages of acquired protective immunity against Myco. leprae.

A case of infectious leprosy in residential accommodation in the UK prompted a study of the cellular and humoral response to Mycobacterium leprae in two groups of individuals who were in contact with the index case for almost a year. In the younger staff group (mean age 44 years) 23 of 30 individuals had positive Mitsuda skin tests, 25 showed lymphocyte transformation to a soluble sonicate of M leprae and 2 had slightly raised IgM antibody concentrations to the terminal disaccharide of Mleprae phenolic glycolipid-1. In the older group of residents (mean age 83 years) 7 of 36 individuals were skin-test-positive, 25 of 33 were positive by lymphocyte transformation, but none had raised antibody levels. When retested on two further occasions, the same 2 individuals in the younger group still had raised antibody concentrations, 1 of whom had a persistent lepromin skin-test response for over 8 months and showed a pronounced increase in lymphocyte transformation to mycobacterial antigens. The findings suggest that transmission of M leprae may have occurred in these 2 contacts, who were therefore given 6 months' chemoprophylaxis with rifampicin.

The Leprosy Control Program of Antioquia, (post-elimination leprosy state of Colombia), had registered by 1999, 56 lepromatous leprosy patients and their household contacts (HHC). Our interest was to detect Mycobacterium leprae infection in these HHC. Clinical examination, acid-fast bacillary staining (AFB) in nasal secretions, and slit skin samples, IgM anti-PGL-I in serum and Lepromine A (Mitsuda) reactivity were tested. Two hundred forty eight HHC were studied, 49% were male. After clinical examination, two HHC were diagnosed as multi bacillary patients; 13% showed positive IgM anti-PGL-I titers; Mitsuda reaction (or = 4 mm) was positive in 59%; AFB was negative in all samples, except in the two new patients. HHC were classified according to test results. Group 1: two new multi bacillary patients. Group 2: 15 HHC seropositive, Mitsuda-negative. Group 3: 13 HHC seropositive, Mitsuda-positive. Group 4: 130 HHC seronegative, Mitsuda-positive. Group 5: 88 HHC seronegative, Mitsuda-negative. These results are an indication that the transmission of the infection is still happening in a region considered in the post elimination phase. The two new patients represent an infection source for others contacts, and groups 2 and 3 are infected HHC that could develop the disease in future. Follow up of high risk population is necessary to achieve real elimination of leprosy.

A 22-year-old male polar lepromatous leprosy patient who became Mitsuda positive after 36 months of multidrug therapy (MDT) is reported. Lepromatous leprosy (LL) is a state of specific immunosuppression and is invariably irreversible. The finding of Mitsuda positivity in histopathologically proven polar lepromatous leprosy is extremely uncommon, and conversion of lepromin status following MDT has not so far been reported. This case reports confirms the observations made by Waters et al. regarding lepromin conversion in lepromatous patients.

The course of leprosy in patients with HIV infection has been a controversial issue for a long time. It is still a matter of debate whether the HIV status of an individual has any impact on the natural history of leprosy and response to anti-leprosy treatment. We report here three HIV-positive leprosy cases (two BT and one BB) along with their CD4 counts and HIV staging with anti-leprosy therapeutic response. Both BT cases responded well to conventional WHO MDT (PB) for 6 months, whereas the BB case relapsed 3 months after completion of MDT (MB) for one year. However, he became inactive again following a further one-year course of MDT (MB).

The Ridley-Jopling system of classification of the variegated clinical pattern of leprosy is based on the specific cell-mediated immunity observed in the histopathology of skin lesions conforming to a spectrum from TT at one end to LL at the other. In this study a fairly large sample of 90 patients was classified on clinical grounds; the histopathology of the skin lesions was studied blind. There was an overall concordance of 90% between the clinical and histological classifications. In addition, the systemic cell-mediated and humoral immune responses were studied. The in vivo cell-mediated immune response, namely the Mitsuda skin response, mostly conformed to the clinical classification. While the in vitro lymphoproliferative responses to BCG and its sonicate were high, the lymphoproliferative responses to Dharmendra lepromin were surprisingly poor. Humoral responses to 35 kDA protein of M. leprae and PGL-1 were good in most LL, BL patients and tapered off towards TT. IgG antibodies to recombinant ML 65 kDa proteins denoted mycobacterial presence.

A total of 20 bacteriologically positive multibacillary (MB) leprosy patients older than 18 years of age with a bacterial index (BI) of 2+ or greater were given standard World Health Organization multiple drug therapy (MDT-MB) for 12 consecutive months plus four intradermal doses of Mycobacterium w vaccine at 3 monthly intervals (Study group). Twenty age-matched MB patients were given WHO/MDT alone (Control group). The patients of both groups were followed up for 1 year. Improvements in the patients were periodically monitored by clinical (Ramu's score), bacteriological (SSS), histopathological (skin biopsy) and immunological (lepromin conversion) parameters. Study group patients showed more significant improvements in all parameters except for lepromin conversion compared to patients in the Control group. The incidence of type 1 reaction was more in the Study group (30% vs 10%), while the incidence of type 2 reaction was more in the Control group (25% vs 15%). Neuritis associated with reactions was seen more often in the Control group compared to the Study group (20% vs 10%). The addition of Mycobacterium w vaccine as an adjunct to the 1-year WHO/MDT regimen appears to be significantly more beneficial in MB leprosy patients with a high initial BI compared to WHO/MDT given alone. Studies on larger numbers of patients with extended follow up will be in order.

The immune responses of 19 treated lepromatous patients who had remained smear negative for a long period were assessed for specific cell-mediated immunity (CMI), anti-Mycobacterium leprae antibodies and cytokine release in response to challenge with M. leprae soluble antigen (MLSA). All of these patients remained anergic to Mitsuda lepromin. Lymphoproliferation in response to M. leprae antigen was noted in only two patients. Significant reduction in the phenolic glycolipid I (PGL-I) antibody response in treated patients with no difference in the M. leprae 35-kDa antibody response was observed when these responses were compared with those of active lepromatous patients. More treated patients produced interleukin-2 (IL-2) and interferon gamma (IFN-gamma) than did active patients. On the other hand, fewer treated patients produced IL-10 than did active patients. These limited findings suggest that the host immune response makes an attempt toward upregulation of CMI in some treated LL/BL patients.

Thirty sib-pairs were ascertained through unrelated lepromatous probands. They consisted of 22 healthy individuals and 8 leprosy patients. The Mitsuda reactions of all sibs were evaluated both macroscopically and histologically, and high molecular weight genomic DNA was extracted from the white blood cells of all sib-pairs. Three DNA polymorphisms identified by polymerase chain reaction (274C/T, D543N, 1729 + 55del4) were used as chromosome markers at the NRAMP1 locus. Sib-pair comparisons did not disclose any sign of close linkage between the Mitsuda reaction and the genetic markers.

Primary neuritic leprosy (PNL) presents as a peripheral neuropathy with no visible skin patches and skin smears negative for acid fast bacilli. The pathogenesis of PNL is poorly understood. The aim of the study was to document the histological changes in the nerve, apparently normal skin and nasal mucosa in PNL and to study its significance to the pathogenesis of leprosy lesions. The study is based on a cohort of 208 PNL patients registered at the Schieffelin Leprosy Research and Training Centre, Karigiri. All patients had a nerve biopsy, 196 had a skin biopsy and 39 had a nasal mucosal biopsy. The findings reveal that PNL patients exhibit a spectrum of disease histologically in the nerve ranging from lepromatous to tuberculoid leprosy with a significant proportion (46%) manifesting a multibacillary leprosy histology. Findings in the apparently normal skin and nasal mucosa reveal that there are widespread changes due to leprosy in tissues such as the skin and nasal mucosa even when the disease appears clinically confined to a few nerves. PNL may be an early stage in the pathogenesis of the disease before the appearance of skin lesions. The number of nerves enlarged and lepromin status did not give any clue to the nature of underlying disease.

Anergy, or contrarily, Mitsuda-type responses towards 4 chemoautotrophic nocardioform antigens (CAN-Ags) and a control standard lepromin were tested in 73 LL, TT and borderline cases of leprosy. The antigens injected per patient varied from a maximum of 5 to a minimum of 2. Complete anergy to CAN-Ags was seen in 92/92 instances tested on 24 LL cases. The anergy was weakly modified or unmodified in 3 other LL cases which had been vaccinated before. Concurrent studies with the same antigens tested on 33 TT cases showed clear-cut, dose-dependent, Mitsuda-type late responses in 80/81 instances. The CAN bacteria, therefore, despite their origin from different unrelated leprous human, mouse footpad (MFP) and armadillo tissues, appeared to be identical with each other and also probably related to the leprosy bacillus, on the basis of these parameters.

The authors studied the Mitsuda reaction in 37 leprosy patients (18 reactional tuberculoid, 19 reactional borderline cases) and compared the results with clinical findings, histopathology and bacilloscopy. Evaluation of the Mitsuda reaction was carried out on days 30, 60, 90 and 120. Most of the reactional tuberculoid patients showed a Mitsuda reaction of +3 in opposition to the reactional borderline patients who showed only +. Bacilloscopic analysis revealed that in 75% of the reactional tuberculoid cases there were rare or no bacilli; bacilli were present in 95% of the reactional borderline cases. The authors conclude that reactional tuberculoid cases have a greater ability to clear bacilli than reactional borderline cases, and that the Mitsuda reaction is a useful tool for the differentiation between these two types of leprosy.

It is amazing how after years of scientific research and therapeutic progress many simple and basic questions about protective immunity against Mycobacterium leprae remain unanswered. Although the World Health Organization (WHO) has recommended short-term multidrug therapy (WHO/MDT) for the treatment of paucibacillary (PB) leprosy patients, from time to time several workers from different parts of the globe have reported inadequate clinical responses in a few tuberculoid and indeterminate leprosy patients following adequate WHO/MDT despite the fact that they are Mitsuda responsive. A few borderline tuberculoid patients harbor acid-fast bacilli (AFB) in their nerves for many years even though they become clinically inactive following MDT, a fact which has been ignored by many leprosy field workers. Keeping these patients in mind, we have attempted to investigate the cause of the persistence of AFB in PB cases and have looked into the question of why Mitsuda positivity in tuberculoid and indeterminate leprosy patients, as well as in healthy contacts, is not invariably a guarantee for protectivity against the leprosy bacilli. We have: a) analyzed the histological features of lepromin-induced granulomas, b) studied the bacteria-clearing capacity of the macrophages within such granulomas, and c) studied the in vitro leukocyte migration inhibition factor released by the blood leukocytes of these subjects when M. leprae sonicates have been used as an elicitor. The results of these three tests in the three groups of subjects have been compared and led us to conclude that the bacteria-clearing capacity of the macrophages within lepromin-induced granuloma (positive CCB test) may be taken as an indicator of the capability of elimination of leprosy bacilli and protective immunity against the disease. This important macrophage function is not invariably present in all tuberculoid and indeterminate leprosy patients or in all contacts even though they are Mitsuda responsive and are able to show a positive leukocyte migration inhibition (LMI) test. It is likely but not certain that this deficit of the macrophage is genetically predetermined and persists after completion of short-term WHO/MDT. Thus, after discontinuation of treatment slow-growing, persisting M. leprae multiply within macrophages leading to relapse.

Sequential estimates of the levels of circulating immune complexes (CIC), complement catabolic fragment C3d, complement-mediated immune complex solubilization (CMS) and immunoglobulins were made in 24 newly diagnosed with borderline tuberculoid leprosy over a 20 month period after initiation of chemotherapy. Fourteen of these patients had not suffered from reversal reactions either at the time of presentation or during the follow-up. The levels of CIC were evaluated in them from the third to the eleventh month after starting chemotherapy and immunoglobulin G (IgG) levels were evaluated up to eight months. The concentrations of C3d and immunoglobulins A (IgA) and M (IgM) were normal in these patients. The other ten patients had reversal reaction at the time of diagnosis which subsided by the third month after starting treatment. They did not have reversal reactions later. The levels of CIC and IgG were elevated and those of CMS were depressed throughout the study period. Serum C3d level was initially elevated but came down to normal by the third month while IgA and IgM levels were within normal limits. The relevance of these findings to the genesis of reversal reaction is discussed in this communication.

The present report, which describes management of lepromin-negative borderline leprosy patients with low-dose Convit vaccine, is an extension of our earlier study on the treatment of lepromatous leprosy patients with low-dose Convit vaccine as an adjunct to multidrug therapy (MDT). The test Group I, consisting of 50 lepromin-negative, borderline leprosy patients, were given low-dose Convit vaccine plus MDT. The control group II consisted of 25 lepromin-negative, borderline leprosy patients given BCG vaccination plus MDT and 25 lepromin-negative, borderline leprosy patients given killed Mycobacterium leprae (human) vaccine plus MDT. The control group III consisted of 50 lepromin-positive, borderline leprosy patients not given any immunostimulation but given only MDT. Depending upon the lepromin unresponsiveness, the patients were given one to four inoculations of the various antileprosy vaccines and were followed up every 3 months for 2 years for clinical, bacteriological and immunological outcome. All patients belonging to the test and control groups showed clinical cure and bacteriological negativity within 2 years. However, immunologic potentiation, assessed by lepromin testing and the leukocyte migration inhibition test (LMIT), was better in the test patients receiving low-dose Convit vaccine plus MDT than in the control patients receiving BCG vaccine plus MDT or killed M. leprae vaccine plus MDT or MDT alone. But the capacity of clearance bacteria (CCB) test from the lepromin granuloma showed poor bacterial clearance in the test patients. However, there was no relapse during 6 years of follow up. Two mid-borderline (BB) patients had severe reversal reactions with lagophthalmos and wrist drop during immunotherapy despite being given low-dose Convit vaccine.

Phase-II and extended Phase-II studies were conducted in three different sets of the population in Thiruthani Taluk, Chengalpattu District, South India, involving BCG and killed Mycobacterium leprae (KML) combination vaccines to ascertain the acceptability of the vaccines. In the Phase-II study, 997 healthy volunteers were vaccinated on individual randomization with one of the vaccines arms: BCG 0.1 mg + 6 x 10(8) KML, BCG 0.1 mg + 5 x 10(7) KML, BCG 0.1 mg + 5 x 10(6) KML, BCG, 0.1 mg or normal saline. Blood samples were taken and the serum was tested for antibody levels against phenolic glycolipid-I (PGL-I) and the 35-kDa protein of M. leprae. In this study, we observed regional suppurative adenitis in 6% (6 out of 100), 3% (3 out of 100), and 3% (3 out of 100) of the vaccinees in the BCG 0.1 mg + 6 x 10(8) KML, BCG 0.1 mg + 5 x 10(7) KML, and BCG 0.1 mg + 5 x 10(6) KML vaccine arms, respectively, in the 13-70 year age group. Earlier BCG scar status, skin-test reactions to lepromin-A, Rees' MLSA, and serum antibody levels against PGL-I and the 35-kDa protein did not help to identify the group at risk of developing suppurative adenitis. Suppurative adenitis appears to have a different relationship between the age of the subject and the dose of the vaccine. In order to overcome the problem of regional suppurative adenitis and to know the mechanism involved, an extended Phase-II study was conducted in similar groups of the population by reducing the BCG and KML doses, i.e., with BCG 0.05 mg + 6 x 10(8) KML, BCG 0.05 mg + 5 x 10(7) KML, and BCG 0.01 mg + 5 x 10(7) KML. Biopsy specimens were collected from lymph nodes of the suppurative adenitis cases and were subjected for culture and histopathological examination. The observations showed that regional suppurative adenitis could be reduced to 1% in the BCG 0.05 + 6 x 10(8) KML group, 0.5% in the BCG 0.05 + 5 x 10(7) KML group, and 0.5% in the BCG 0.01 + 5 x 10(7) KML group. This phenomenon of suppurative adenitis appears to be related to the total dose of mycobacterial antigens. Suppurative adenitis was seen by weeks 18 and 20 post-vaccination in the latter two lower doses; whereas it was seen by week 8 in the higher dose of the combination vaccines. No case of suppurative adenitis was observed in the BCG 0.1 mg group. Culture and histopathology ruled out the possibilities of progressive BCG infection and superadded infection. Considering the above results, BCG 0.05 mg + 6 x 10(8) KML was acceptable for a large-scale vaccine trial in South India.

M.w vaccine is one of the antileprosy vaccines under test in an ongoing comparative vaccine trial in South India. The objective of the present study was to examine the sensitizing ability, as measured by skin test reactions to Rees' MLSA and lepromin, and reactogenicity of M.w vaccine in the local population. Two doses of M.w, 1 x 10(9) bacilli and 5 x 10(9) bacilli, were used, in two separate studies of 395 and 400 "healthy" individuals aged 1-65 years. In each study, the study subjects received either M.w vaccine or normal saline (control), by random allocation. The results showed that healing of vaccination lesions was uneventful although the healing process was somewhat prolonged with the higher dose. The mean size of lesions was 7.0 mm and 9.5 mm with the low and high doses of the vaccine, respectively. The results also showed that M.w vaccine in a dose of 1 x 10(9) bacilli, failed to induce post-vaccination sensitization as measured by reactions to Rees' MLSA and by Fernandez and Mitsuda reactions to lepromin-A. However, when the dose of the vaccine was increased to 5 x 10(9) bacilli the mean sizes of post-vaccination reactions to Rees' MLSA and lepromin-A (both early and late) were significantly larger in the vaccine group compared to that in the control group. The sensitizing effect attributable to the vaccine was of the order of 1.5 mm to 1.8 mm.

ICRC vaccine is one of the candidate anti-leprosy vaccines under test in a large scale comparative vaccine in trial. The objectives of the present study was to study the sensitization potential, as measured by Rees' MLSA and lepromin, and reactogenicity of this vaccine preparation in the local population. The study included 368 'healthy' individuals aged 1-70 years. Each individual received either ICRC vaccine or normal saline (control) by random allocation. They were also tested with Rees' MLSA and lepromin-A, 12 weeks after vaccination. Reactions to Rees' MLSA were measured after 48 hours and those to lepromin-A after 48 hours and three weeks. Character and size of local response, at the vaccination site, were recorded at 3rd, 8th and 15th week after vaccination. The results of the study showed that healing of vaccination lesion was uneventful, the mean size of the lesion being 10.3 mm. The mean sizes of post-vaccination reactions, to Rees' MLSA and lepromin (both early and late reactions), were significantly higher in the vaccine group compared to that in the normal saline group; the sensitizing effect attributable to the vaccine was of the order of 3.5 mm, 1.7 mm and 2.2 mm respectively. In conclusion, the study has demonstrated that ICRC vaccine was 'safe' and produced significant sensitizing effect as measured by post-vaccination sensitization to Rees' MLSA and lepromin, in the local population.

Thirty-two subjects with suspected leprosy lesions were investigated to assess various modalities of sensibility and sweat function and these were correlated with immunological and histological parameters. It was found that pain and temperature, mediated by small unmyelinated fibres were impaired in the early lesions. Impairment of sweat function was seen only when one of the modalities of sensibility was also affected. Antibodies specific to a protein (35 kDa) antigen and phenolic glycolipid 1 of Mycobacterium leprae were positive in nine and 12 cases respectively, while 15 of the 31 biopsies revealed the presence of mycobacterial antigens in these lesions. The implications of these findings are discussed.

Cell mediated immunity was studied in 50 patients of leprosy and 15 control volunteers, by estimation of peripheral blood lymphocytes (PBL), total rosette forming cells (TRFC) and active rosette forming cell (ARFC) counts in vitro. PBL, TRFC and ARFC counts were slightly but not significantly increased in patients of tuberculoid leprosy (TT) and muculoanaesthetic variant of tuberculoid leprosy as compared to control group. However, gradual decrease in T-cell subsets, occurred in borderline tuberculoid leprosy (BT) as compared to tuberculoid type (TT)-(p0.05). Significant decrease in lymphocytes and T-cell subsets was observed in midborderline leprosy (BB), (p0.01):borderline lepromatous leprosy (BL), (p0.001) and lepromatous subpolar and polar types (LL); (p0.001) as compared to control group. Mycobacterium leprae (M.leprae) were positive in BT-(20%); BB-(72.7%); BL-(83.2%) and LL-(100%). Delayed hypersensitivity reactions (DHR) revealed significantly increased lepromin positivity in TT (83.3%) and BT (80%) which decreased in BB (63.6%) and BL (50%). Lepromin test showed anergic state in LL group (28.5% positivity). Dinitrochlorobenzene (DNCB) skin test showed 100% positivity in TT group and controls while gradual significant decrease was observed from BT (p0.05) to LL scale (p0.001). Leprosy spectrum of Ridley and Jopling scale is directly co-related with inherent cell mediated immune status of the patients which has a significant prognostic role in treatment and long term management.

The authors analysed some immunological criteria in leprosy patients diagnosed as borderline tuberculoid by the presentation of different grades of skin lesions as well as different grades of nerve involvement. Only 50% of the patients presented a single skin lesion and 58% had none or only one affected nerve. Nineteen patients (39.6%) showed a positive lepromin reaction (induration > or = 5 mm). Patients with a positive skin test had a greater number of skin lesions when compared with patients with a negative lepromin test. Fifty-seven percent of the patients were found to be positive using a lymphoproliferation test (LTT) in response to Mycobacterium leprae antigens. Positive LTT results did not correlate with the number of skin lesions, but patients unresponsive to LTT had a lesser extent of nerve involvement. Four out of 18 patients (22%) released high IFN gamma levels in PBMC culture stimulated by M. leprae. (mean U/ml +/- SD = 142 +/- 72). All of these 4 patients presented only one skin lesion, although three of them had more than one affected nerve. Nineteen out of 21 patients (90.5%) showed no anti-PGL-1 antibodies in their serum. The low levels of anti-PGL-1 antibodies among these patients confirmed their tuberculoid background even in those with multiple skin lesions. These findings seem to attribute an important role to IFN gamma in restraining the spreading of the infection in the skin, but IFN gamma may have an opposite effect on the nerves. The potential pathological effects of IFN gamma during the delayed type of hypersensitivity can be related to its ability to synergise with other inflammatory cytokines such as TNF alpha, IL-1 beta, and others.

Sixteen neuritic cases have been seen developing cutaneous lesions. These cutaneous lesions by and large appear within 4 months after the diagnosis of neuritic leprosy. Leprosy pathology in cutaneous lesions has been found ranging between indeterminate and borderline lepromatous group. Development of cutaneous lesions does not seem to be influenced by age, sex or number of nerves or lepromin status. Neither lesions seem to appear in any particular part of the body. Therapy, duration and type i.e. monodrug or multidrug, also does not seem to influence the development of cutaneous lesions in either way. It appears that neuritic cases with either very early (indeterminate) or with advanced multibacillary neural pathology may develop skin lesions. Skin lesion possibly appear following reversal reaction in skin. Cases with newly developed skin lesions well respond to standard therapy. Development of cutaneous lesions by neuritic cases possibly indicates towards the natural history of the disease, conforming to the hypothesis that leprosy is basically neural in inception and that all other forms emerge from it.

In a clinico-pathological study of Indeterminate leprosy, fifty-six cases were chosen based on specified clinical criteria. Their clinical features were noted, the smears for acid fast bacilli (AFB) were prepared from lesions, lepromin inoculation and biopsies were performed from the lesional edges. They were subsequently treated with a modified extended WHO regimen for paucibacillary leprosy. On routine hematoxylin eosin (HE) and Fite-Faraco staining of paraffin embedded sections, histopathological confirmation of Indeterminate leprosy was observed in only 17/56 (31%) of the clinically diagnosed cases whereas the remaining were labelled as non-specific pathology. Histometric analysis of all HE stained sections did not show any characteristic finding which could be considered as characteristic and discriminatory for Indeterminate leprosy. Immunoperoxidase staining for demonstration of mycobacterial antigen by direct staining procedure using conjugated rabbit anti-BCG and indirect three step procedure using primary rabbit anti-BCG and avidin biotin complex, was next performed on the sections exhibiting non-specific pathology. With the direct immunoperoxidase method, antigen was demonstrable in (11/35) 31% of the cases. The more sensitive indirect method could demonstrate the presence of antigen in (21/35) 60% of the cases. This study thus shows that demonstration of mycobacterial antigen by simple and inexpensive immunoperoxidase techniques enhances the histopathologic diagnosis of Indeterminate leprosy.

In this study, we measured in vitro proliferative responses of peripheral blood mononuclear cells from both leprosy patients across the clinical spectrum and also healthy contacts from a leprosy-endemic population to delipidified cell components of Mycobacterium leprae (DCC) and Dharmendra lepromin. Dharmendra lepromin was poor in inducing in vitro T cell proliferation in all the study groups, even though it elicited marked in vivo skin test reaction in tuberculoid leprosy patients and healthy contacts. In contrast, Dharmendra preparation of BCG induced marked T-cell response in tuberculoid as well as bacterial index negative lepromatous patients. DCC induced a significantly higher lymphoproliferative response than Dharmendra lepromin in all study groups. A significant positive correlation was observed between the lymphoproliferative responses to DCC and BCG. The present study, based on a large number of leprosy patients and healthy contacts, clearly demonstrates that DCC, depleted of glycolipids and lipopolysaccharides, is a good antigenic preparation for evaluating T-cell reactivity to M. leprae.

This study is an extension of a previous study on an antileprosy combination vaccine of BCG plus killed Mycobacterium leprae (KML) regarding its sensitization potential and reactogenicity. The study was extended to see if by reducing the dose of BCG in the combination vaccine the incidence of suppurative adenitis could be reduced without a significant reduction in the level of postvaccination skin-test responses. The study included 860 individuals, and three preparations of the combination vaccine [BCG 0.05 mg + 6 x 10(8) KML (I), BCG 0.05 mg + 5 x 10(7) KML (II), BCG 0.01 mg + 5 x 10(7) KML (III)] along with normal saline (i.v.) were used. Each individual received one of these four preparations by random allocation. They were also tested with Rees' M. leprae soluble antigen (MLSA) and lepromin A 12 weeks after vaccination. Reactions to the MLSA were measured after 48 hr; reactions to lepromin A after 48 hr and 3 weeks. The character and size of the local response at the vaccination site were recorded at the third, eighth, and 15th week postvaccination. The results of the study showed that by halving the dose of BCG in the combination vaccine BCG plus 6 x 10(8) KML a) the incidence of suppurative regional adenitis was reduced significantly, b) there was no significant change in the post-vaccination response at 12 weeks as measured by Rees' MLSA and lepromin A, and c) the evolution of the vaccination lesion was somewhat prolonged. This dose was found satisfactory for use in a comparative antileprosy vaccine trial in South India.

A total of 64,570 household and other close contacts of about 2000 leprosy cases were screened for eligibility for entry into a trial of a new leprosy vaccine. The screening procedure included a clinical examination for leprosy and for the presence of BCG and lepromin scars. Ninety-five new cases of leprosy were identified, and the prevalence of BCG and lepromin scars among them was compared with similar data from matched controls selected from among those with no evidence of leprosy. The difference in the prevalence of BCG scars in the two groups was used to estimate the protection against leprosy conferred by BCG vaccination. One or more BCG scars was associated with a protective efficacy of 56% (95% confidence limits 27% to 74%). There was a trend of increasing protection with four or more BCG scars, but this was not statistically significant. There was no evidence that the efficacy of BCG varied with age or according to whether or not the contact lived in the same household as a case. The protective effect was significantly higher among males, and was significantly greater for multibacillary than for paucibacillary leprosy.

In a hospital based study, 362 household contacts of multibacillary leprosy patients were screened for evidence of leprosy and 54 (14.9%) were found to be having leprosy. The remaining 308 apparently healthy contacts were lepromin tested and 109 (35.4%) were observed to be negative to Mitsuda lepromin. M.w vaccine was administered intradermally to 95 of these 109 lepromin negative contacts. Sixty eight of them could be retested for lepromin A reactivity. Fifty six (82.35%) manifested lepromin conversion. The twelve subjects who did not show lepromin conversion, received a second dose of the vaccine, and eleven subsequently became lepromin positive. The overall lepromin conversion rate was thus 98.5% (67 out of 68). Follow-up of these contacts upto a period of 30 months did not demonstrate reversion of lepromin positivity back to negativity status. No untoward effects of vaccination were observed except for local ulceration at the site of vaccine administration.

A study was conducted in 997 individuals in two villages in south India to find the acceptability and sensitizing effect of the antileprosy combination vaccine of BCG plus killed Mycobacterium leprae (KML). Three preparations of the combination, BCG 0.1 mg + 6 x 10(8) KML (I), BCG 0.1 mg + 5 x 10(7) KML (II), and BCG 0.1 mg + 5 x 10(6) KML (III), along with BCG 0.1 mg (IV), and normal saline (V), were used in the study. Each individual received one of the above five preparations by random allocation. They were also tested with Rees' M. leprae soluble skin-test antigen (MLSA) and lepromin-A, both at intake and 12 weeks after vaccination. Reactions to Rees' MLSA were measured after 48 hr; those to lepromin-A after 48 hr and 3 weeks. The character and size of the local response at the vaccination site were recorded at 3, 8, 12, 15, and 27 weeks after vaccination. The mean sizes of postvaccination sensitization to both Rees' MLSA and lepromin-A in the vaccine groups were significantly larger than those in the normal saline group, clearly demonstrating the ability of the vaccines to induce sensitization as measured by responses to the two skin tests. The sensitizing effect was the highest following vaccination with vaccine I. It was not significantly different for vaccines II, III, and IV, although, generally, a dose-response effect was observed. The sensitizing effect attributable to the vaccine was more clearly seen in children than in adults. The above conclusions were the same irrespective of which results were considered, reactions to Rees' MLSA or Fernandez or Mitsuda reactions to lepromin-A. A significant finding of the study was that at intake the Mitsuda reactions provided a measure of sensitizing effect due to vaccine. The healing of vaccination lesions was uneventful. In more than 90% of vaccinated individuals, the lesions had healed by the 12th week in vaccine groups II, III, and IV, and by the 15th week in vaccine group I. The results showed that vaccination with BCG or combination vaccines was equally safe in individuals with or without previous BCG scars. Thirteen persons, aged 10 years or older, developed suppurative lymphadenitis around the 8th week (7 in vaccine group I, 3 each in vaccine groups II and III). However, healing was prompt after drainage in these individuals.

Tumor necrosis factor--alpha (TNF), one of the mediators of septic shock, has a role in the immunopathological complications of several infections. However, its role in leprosy is yet unclear. In this study, serum TNF and IL-1 levels in 64 patients spread over the spectrum of leprosy [lepromatous leprosy (LL), 30; borderline lepromatous, 12; borderline borderline, 8; and borderline tuberculoid-tuberculoid leprosy, 14] were measured at the time of admission. Elevated levels of TNF ranging from 15 to 4500 pg/ml were detected in lepromatous leprosy cases (399 +/- 189) and low levels ranging from 15 to 160 pg/ml were detected in the tuberculoid form of leprosy. Patients undergoing type 1 and type 2 lepra reactions also exhibited high TNF levels of 15-2100 pg/ml. Of the 14 clinically healthy individuals studied, 3 showed TNF levels of 15, 50, and 58 pg/ml. Interleukin 1-beta (IL-1) levels were found to be significantly higher in LL cases (70-5000 pg/ml) (328 +/- 184) in comparison to other groups or normal controls (9 +/- 3). The coefficient of correlation between TNF and IL-1 levels was statistically significant in LL and reaction cases (r = 0.96, P less than 0.001). These patients were followed up as outpatients for a period of 1 year. It was observed that 4 out of 8 patients with TNF levels greater than 100 pg/ml went into lepra reactions between 2 and 6 months after entry into the study, whereas only 5 out of 56 with less than 100 pg/ml went into mild lepra reactions (chi 2 = 9.7, P less than 0.01). Determination of TNF and IL-1 levels thus seems to have a prognostic significance in terms of lepra reaction in patients.

An assessment has been made of 108 neuritic leprosy patients to find out if the number of affected nerves and the clinical presentations of these patients give any indication of the underlying severity (classification) of the disease. Detailed clinical recordings, skin smears, lepromin testing with Dharmendra antigen, and a leukocyte migration inhibition test (LMIT) using sonicated Mycobacterium leprae antigens were done in these patients. Nerve biopsies of available affected nerves were taken in 39 patients. The results show that neuritic leprosy patients also have a spectrum. However, none of the clinical parameters, including the number and distribution of affected nerves, the immune response and the nerve histology, were found to be inter-related. Further, even though all of the patients were skin-smear negative, a significant proportion showed lepromatous histology and nearly two thirds had a moderate-to-heavy bacterial load within the nerves.

Three patients with solitary skin lesions showing the cardinal signs of leprosy were seen and clinically classified among the paucibacillary cases. Initially, they were treated with two drugs (rifampin and dapsone) as recommended by the WHO Expert Committee. On the first visit of their follow-up, they were seen to be histopathologically either in the borderline (BB) or borderline lepromatous (BL) group, and acid-fast bacilli were demonstrated in the sections. Later they were put on three drugs (rifampin, dapsone and clofazimine) as given for multibacillary cases, and therapeutically they also behaved like bacilliferous leprosy. Such cases are rare and the reasons for the occurrence are not clear. Further studies on the subtle relationship between the local host factors and the virulence of the organisms grown from these lesions may offer an explanation. In light of these cases and previous reports of even lepromatous leprosy presenting as a single skin lesion, field workers--including both medical and paramedical workers--should carefully perform and interpret slit-skin smears from clinically diagnosed paucibacillary cases so that such unusual presentations of the disease are treated appropriately and not missed.

A 6-year prospective study of 79 household contacts of leprosy cases was made in order to correlate the development of the disease with their specific T-cell immunity, measured by the Mitsuda test, and levels of anti-Mycobacterium leprae antibodies determined in three consecutive observations with the FLA-ABS test. Overall in the contacts, 71.7% were Mitsuda positive and 93.6% showed seropositivity, without regard to their age, sex, or leprosy type of their index case. Households were divided into lower-risk and higher-risk groups according to either the paucibacillary or multibacillary character of their index case. The lower-risk group consisted of 19 contacts of 2 tuberculoid (TT) and 5 indeterminate cases. The higher-risk group was made up of 60 household contacts of 18 active lepromatous (LL) cases. All but two contacts in the former group had a positive Mitsuda reaction; the most common antibody titer was 1:160, with a tendency to stabilize or decrease over time. In the two Mitsuda-negative contacts, increased antibody levels were observed. In the higher-risk group, 61.6% were Mitsuda positive and showed a humoral profile similar to those Mitsuda positive in the lower-risk group. In most of the Mitsuda-negative LL contacts, the antibody levels remained constant or progressively increased, suggesting a high probability of active subclinical infection. This assumption was partially supported by the finding of a new borderline lepromatous (BL) leprosy case in the Mitsuda-negative LL contact group. Nevertheless, the contribution of the close and extensive contact with a multibacilliferous case as a risk factor was difficult to evaluate because of the small size of the sample studied.

A total of 438 household contacts of leprosy patients and 228 non-contacts were studied for anti-PGL-I antibodies and cell mediated immune response to Mycobacterium leprae using ELISA and lepromin tests. The contacts showed relatively higher positivity to ELISA (29.4%) and lepromin (70.5%) as compared to non-contacts who showed 9.6 and 57 per cent positivity. In contacts as well as non-contacts the seropositivity was not found to be influenced by age and sex. However, seropositivity was higher (39.7%) in the contacts of patients with multibacillary leprosy as compared to contacts (27.7%) of patients with paucibacillary leprosy.

26 Patients of leprosy presenting with hypopigmented lesions were divided on morphological grounds into 3 Sub groups, Group I (9 patients) with well-defined single patch with moderate to complete sensory loss; Group II (8 patients) with single ill-defined lesion having partial sensory loss; and Group III (9 patients) having multiple hypo-pigmented patches with mild to moderate sensory loss. Epidermal atrophy was a conspicuous histological finding in all groups. Only patients in Group I showed epitheloid cells in dermal infiltrate with erosion of epidermis in one case. This group may be labelled as maculoanesthetic leprosy. Patients in Group II and III showed mononuclear cell infiltrate in dermis, around neurovascular bundles and appendages. They were histologically consistent with indeterminate leprosy. Follow-up biopsy after six to eight months of treatment showed healing of the lesion of reduction in the infiltrate in most cases.

870 household contacts of leprosy patients were examined for sub-clinical infection with M. leprae by smear (skin and nasal), lepromin and FLA-ABS tests. 0.6%, 3.3%, 71.5% and 14.4% of the contacts were found to be positive for skin smear, nasal smear, lepromin and FLA-ABS tests respectively. An analysis of the results revealed that 4% of the lepromin positive contacts and 3.6% of the lepromin negative contacts were positive to both FLA-ABS and skin or nasal smear.

While studying circulating immune complexes (CIC) in the sera of lepromatous patients by the polyethylene glycol (PEG) precipitation technique, we found (by light microscopy) abundant acid-fast bacilli (AFB), morphologically similar to those seen in slit skin smear preparations from these patients, precipitated with the PEG precipitated materials. Both solid and non-solid AFB could be readily identified. Ultrastructures of these AFB in the PEG aggregates showed some similarity with those detected in the PEG precipitates prepared from armadillo-derived lepromin under identical conditions. The most striking difference between the AFB in the test sera and that in the armadillo-derived lepromin was the absence of any transverse band in the former. This suggested that the AFB in the patients' circulation were not in division stage. Furthermore, electron-dense material were deposited on the AFB co-precipitated from the patients' sera, which were not found on the AFB co-precipitated from the armadillo-derived lepromin.

Earlier studies of immunologic reconstitution therapy of active lepromatous leprosy patients by repeated infusions of viable allogeneic blood lymphocytes indicated a beneficial effect. In order to avoid cell separation and its associated risks, we attempted to transfer immunity passively into seven seriously ill lepromatous patients by repeated transfusions of fresh blood donated by healthy, but tuberculin and lepromin positive, subjects. The results showed clinical improvement in some cases with the elimination of Mycobacterium leprae, histological reversion and return of immunologic responsiveness.

Transfer factor or whole lymphocytes from donors with delayed hypersensitivity to antigens of Mycobacterium leprae were employed to reconstitute delayed hypersensitivity in nine patients with anergic leprosy. Five received a mean of 4.1 X 108 lymphocytes, and four received transfer factor from equivalent cell numbers. One to six days after transfer, six of nine patients experienced erythematous indurative changes within leprous skin infiltrates that regressed by the 12th day. Simultaneously, erythema nodosum occurred in four patients, and fever and arthralgia in three. Six of seven converted from anergy to a delayed hypersensitivity response to M. leprae antigens. The seventh became skin-test positive at the "local" site of injection only, and the test reverted to negative seven days later. Two of three patients showed an increase in perivascular lymphocytic infiltration in the post-transfer skin-test biopsy site. Delayed hypersensitivity reactions to M. leprae antigens can be produced, with lymp...

In a study of 37 leprosy patients, the oral administration of levamisole failed to provoke an increase in both the Fernandez and Mitsuda reactions to lepromins of human and armadillo origin. We interpret this as evidence against an effective specific immunostimulatory capability of levamisole in leprosy patients under the conditions of the study. Current knowledge of the mechanism of levamisole action supports the concept that the fundamental immunologic defect in lepromatous leprosy may reside in the lymphocyte and not the macrophage, or the respective related functions of these two cell forms.

Eleven cases of borderline-borderline leprosy were subjected to levamisole therapy for 3 months. Levamisole was given 150 mg/day on three consecutive days every fortnight along with dapsone (DDS). Immuno-stimulation was assessed by lepromin test using Lepromin-A supplied by W.H.O. containing 4.0 X 10(7) bacilli per milli litre. It was found that there was statistically significant change in lepromin reaction after levamisole therapy.

A comparative study on the Fernandez and on the Mitsuda reaction was made in 104 normal people employing whole and sonicated lepromin. Both types of lepromin can induce a good early reaction, and there was no significant difference in the results of the Fernandez reaction. The sonicated lepromin, though it contains all of the components of the original preparation, gives weaker late skin reactions. The results of the Mitsuda reaction were statistically significant.