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In recent years, advances in medical diagnosis and treatment have greatly attracted our attention, whereas some rare diseases, such as leprosy, have not found a place in the medical education curriculum; their existence may even be forgotten. Although the prevalence and incidence rates for leprosy have been significantly reduced as a result of the control strategies of the World Health Organization, new cases still appear. A total of 214,783 new cases were reported from 143 countries during 2016, corresponding to the global new-case detection rate of 2.9 per 100,000 population. Leprosy proves to be a very interesting model due to its immunologic properties. It joins with syphilis, mycosis fungoides, cutaneous tuberculosis, and sarcoidosis as one of the great imitators. The diagnosis of leprosy can be simple and practical, but considering the diagnosis of leprosy in the differential diagnosis is the first requisite again.

Leprosy is a chronic granulomatous disease caused by the bacillus Mycobacterium leprae. It primarily affects the skin and peripheral nerves and is still endemic in various regions of the world. Clinical presentation depends on the patient's immune status at the time of infection and during the course of the disease. Leprosy is associated with disability and marginalization. Diagnosis is clinical and is made when the patient has at least 1 of the following cardinal signs specified by the World Health Organization: hypopigmented or erythematous macules with sensory loss; thickened peripheral nerves; or positive acid-fast skin smear or skin biopsy with loss of adnexa at affected sites. Leprosy is treated with a multidrug combination of rifampicin, clofazimine, and dapsone. Two main regimens are used depending on whether the patient has paucibacillary or multibacillary disease.

Summary Objectives The aim of the present work was to standardise a PCR-Restriction Fragment Length Polymorphism analysis (PRA) as a tool to detect the mycobacteriologic composition of lepromas from leprosy patients used in the production of lepromin to improve the quality of the Mitsuda test. Design PCR-Restriction Fragment Length Polymorphism analysis using hsp65 and rpoB genes were applied to 11 reference strains of mycobacteria, including M. leprae, and the obtained PRA profiles were compared to mycobacteria in clinical specimens. Results Out of the biopsies studied, 52·2% had DNA fragment amplified for both genes (hsp65 and rpoB) for M. leprae. However, other Mycobacterium species were observed in samples of lepromatous leprosy patients. Here we discussed the importance of mycobacteria identification in the antigen of Mitsuda production to be used in the evaluation of leprosy. Conclusions Our results suggest that the use of the molecular approach for sample selection can contribute to an improvement in the quality of produced lepromin.

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.

Immunotherapy with a vaccine consisting of autoclaved Mycobacterium w, was given in addition to standard chemotherapy (multidrug therapy (MDT)) to 93 multibacillary (MB) leprosy patients. One hundred and seven patients with similar types of disease served as controls and received MDT + placebo injections. The study was a double-blind randomised trial. On opening the codes, results obtained were in concordance with those in a single-blind trial which has been extensively reported. Bacteriological clearances were significantly more rapid in vaccinated patients (p < 0.03). Thirty-five LL or BL patients with a high bacterial index (BI) of 6 were completely cleared of acid-fast bacilli (AFB) after eight doses of vaccine. Only 8 patients in the control group became bacteriologically negative in the same time period. They all had BIs < 4. Associated with decreasing BI was accelerated clinical regression of lesions after vaccination and lepromin conversion rates of 100% for BB, 71% for BL and 70% for LL. A significant number of immunised patients showed histological improvement (p < 0.004). Thirty-six showed a complete disappearance of dermal granulomas and a picture of non-specific infiltration. The vaccine did not precipitate neuritis or deformities; episodes were noted in vaccinated patients as were incidences of Type 2 reaction. The overall improvement was reflected by a shorter duration of treatment and faster release of vaccinated patients.

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.

India with its 4 million cases of leprosy, accounts for one-third of the world's population of leprosy patients. One-fourth of them are below 15 years of age. We report a 5-year follow-up study of healthy children who were close contacts of leprosy patients, in order to: 1. detect subclinical infection and observe the development of overt disease by using the Fluorescent Leprosy Antibody Absorption Technique (FLA-ABS) and the lepromin test which assess the humoral and cell-mediated immunity (CMI), respectively; 2. evaluate the efficacy of dapsone as a chemoprophylactic agent in the 'at risk' contacts. Four-hundred-and-fifty-five healthy contacts were studied. Majority of the contacts of multibacillary patients (303) were FLA-ABS positive (75 percent) and lepromin negative (55 percent) showing that although most of them had been infected, the lepromin status was negative (P < 0.01). On the other hand, the majority of the contacts of paucibacillary patients (152) were lepromin positive (57 percent) (P < 0.05). Furthermore, only 61 percent of contacts of paucibacillary patients were FLA-ABS positive as compared to 75 percent of contacts of multibacillary patients demonstrating that the former had been exposed to a lesser quantum of infection (P < 0.05). On the basis of results of FLA-ABS and lepromin tests, these 455 contacts were classified into four groups, viz. Group I comprising children who were FLA-ABS positive and lepromin positive; Group II, who were FLA-ABS positive and lepromin negative; Group III, who were FLA-ABS negative and lepromin positive; and Group IV who were FLA-ABS negative and lepromin negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Cynomolgus monkeys are a useful model for human tuberculosis, but susceptibility to M. leprae is unknown. A cynomolgus model of leprosy could increase understanding of pathogenesis-importantly, neuritis and nerve-damaging reactions. We administered viable Mycobacterium leprae to 24 cynomolgus monkeys by three routes, with a median follow-up period of 6 years (range = 1-19 years) involving biopsies, nasal smears, antiphenolic glycolipid-1 (PGL-1) antibody serology, and lepromin skin testing. Most developed evanescent papules at intradermal M. leprae inoculation sites that, on biopsy, showed a robust cellular immune response akin to a lepromin skin test reaction; many produced PGL-1 antibodies. At necropsy, four monkeys, without cutaneous or gross neurological signs of leprosy but with elevated PGL-1 antibodies, including three with nasal smears (+) for acid fast bacilli (AFB), showed histological features, including AFB, suggestive of leprosy at several sites. Overall, however, cynomolgus monkeys seem minimally susceptible to leprosy after experimental M. leprae administration.

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.

IL-6 production was examined in PBMC cultures from healthy leprosy contacts and from leprosy patients stimulated with the purified mycobacterial 18-, 65-, and 70-kDa heat-shock proteins (hsp) and the secreted fibronectin-binding antigen 85 (Ag85). In lepromin-negative contacts, the 70-kDa hsp was the only antigen capable of eliciting significant IL-6 production. In lepromin-positive contacts, Ag85, the 65- and the 70-kDa hsp induced substantial IL-6 titers. IL-6 levels induced with the 70-kDa antigen were about fourfold higher than with the 65-kDa hsp or with Ag85. The 18-kDa antigen did not induce any IL-6 in these healthy contacts. PBMC from tuberculoid leprosy patients produced even more elevated levels of IL-6, and PBMC from lepromatous leprosy patients produced extremely high levels of IL-6. All antigens were capable of inducing IL-6 in leprosy patients. Highest levels were found in cultures stimulated with the 65-kDa hsp, and lowest levels were in cultures stimulated with the 18-kDa hsp.

Hansen's disease, or leprosy, although a relatively uncommon disease in the United States, continues to be important because of its implications--physical, psychological, and social--for the patient. Prognosis and treatment of the disease are based largely on clinical classification, which ranges from the multibacillary "lepromatous" to the paucibacillary "tuberculoid" forms, depending on the patient's specific immune capabilities. Traditionally, skin testing with lepromins--suspensions of the etiologic agent of Hansen's disease, Mycobacterium leprae--have been used as adjuncts to clinical parameters for classification in endemic areas. However, these have not been systematically studied in the United States. This report describes the results obtained from skin testing 38 volunteers (22 patients and 16 uninfected persons) with standard lepromin preparations. These results support the adjunctive value of lepromins for clinically classifying Hansen's disease in our "hypoendemic" population.

Leprosy patients undergoing phase II trials in two hospitals of New Delhi, India, were HLA typed to see the association of HLA with differential responsiveness to Mycobacterium w vaccine. The vaccine comprises an atypical, nonpathogenic mycobacterium, Mycobacterium w, which has cross-reactive antigens with M. leprae. Multibacillary patients who are lepromin negative are vaccinated at an interval of 3 months. Considerable improvement is evident in the patients in terms of a decline in bacterial indices and histopathological and immunological upgrading. But all the patients do not respond to the vaccine in the same manner; some are slow responders, while others are good responders. HLA-A28 and DQw3 (DQw8 + 9) were found to be associated with slow responsiveness, while DQw1 and DQw7 were found to be associated with a more rapid responsiveness to the M. w vaccine. However, these associations were not significant after P correction for the number of antigens tested for each locus except for HLA-DQw3 (DQw8 and DQw9) and DQw7. DQw7, a new defined split of HLA-DQw3, seems to be associated with the responsiveness to M. w vaccine.

Delayed Type Hypersensitivity (DTH) and protective immunity are thought to be tightly linked. Remarkable similarity exists between their cellular and immune mechanisms. However, their dissociation is also well known. Here we investigate the immunological mechanisms relevant for their dissociation in a group of non-relapsing cured lepromatous leprosy (CLL) patients. In these patients, using lepromin reaction as a model system of DTH we report critical role of tissue chemokine response in synchronous manifestation of these linked phenomena. Results indicate elevation of the threshold of tissue chemokine induction thus dissociating DTH from protective immunity in lepromin -ive CLL patients. We also show that the DTH anergy in these subjects is not an absolute one but depends on the strength of the stimulus. Our data provide insights into the intricate relationship between DTH and immunity and highlight the persistent presence of effector immune mechanisms involving these two pathways in apparently unresponsive lepromatous leprosy patients.

Isoelectric focusing was used to separate the three components of the antigen 85 complex of Mycobacterium bovis BCG. Antibody responses of leprosy patients against each Mycobacterium bovis BCG. Antibody responses of leprosy patients against each component were quantitated by densitometric analysis of immunoblot assays. The 85A component was recognized by 40% (8/20) of the lepromin positive and negative healthy subjects, by 76% (19/25) of the tuberculoid and by 96% (24/25) of the lepromatous leprosy sera. In contrast, the 85B component was not stained by the control sera, nor by the tuberculoid leprosy sera but by 64% (16/25) of the lepromatous leprosy sera. The results suggest that antigen 85B contains one or several epitopes that are specifically recognized by sera of lepromatous leprosy patients only.

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.

T cells bearing gamma/delta antigen receptors comprise a resident population of intraepithelial lymphocytes in organs such as skin, gut, and lungs, where they are strategically located to contribute to the initial defense against infection. An important unsolved question about antigen-driven gamma/delta T cell responses regards the breadth of their T cell receptor (TCR) repertoire, since many specific epithelial compartments in mice display limited diversity. We have examined the diversity of TCR delta gene expression among human gamma/delta T cells from skin lesions induced by intradermal challenge with Mycobacterium leprae. We show that the vast majority of gamma/delta cells from M. leprae lesions use either V delta 1-J delta 1 or V delta 2-J delta 1 gene rearrangements and, within a given region of the lesion, display limited junctional diversity. This contrasts markedly with the extensive diversity of gamma/delta T cells from peripheral blood of these same individuals, as well as skin from normal donors. These results indicate that the gamma/delta response to M. leprae involves the selection of a limited number of clones from among a diverse repertoire, probably in response to specific mycobacterial and/or host antigens.

It has been reported previously that Mycobacterium leprae modulated CD2 on human peripheral blood T lymphocytes and that this modulation was accompanied by a marked reduction in the proliferative response of these cells to mitogens and antigens. In this study, we report that treatment of peripheral blood mononuclear cells from healthy individuals with Dharmendra preparation of M. leprae inhibited their ability to form rosettes with sheep red blood cells. Flow cytometric analysis of Dharmendra lepromin-treated cells showed that, in addition to CD2, CD4 and CD8 were modulated while the surface expression of CD3 was not affected. The specificity of CD2 modulation was confirmed by similar effects of Dharmendra lepromin on thymocytes and lymph node cells from human CD2 transgenic mice. The modulatory effect of Dharmendra lepromin was not observed at lower temperatures. Dharmendra lepromin treatment of activated T cells resulted in reduced binding of monoclonal antibodies to IL-2R and D66 epitope of CD2. The modulatory effects were not observed with Dharmendra preparation of BCG or other preparations of M. leprae. Our results indicate that certain M. leprae factor(s) specifically modulate(s) CD2, CD4, CD8 and IL-2R but not CD3 on T lymphocytes. The suppressive effect of Dharmendra lepromin on the T-cell proliferative response reported earlier may be explained by its modulatory effect on a number of T-cell surface molecules.

Immunotherapy with a vaccine consisting of autoclaved Mycobacterium w was given in addition to chemotherapy in 54 multibacillary, lepromin negative patients belonging to BB, BL and LL types of leprosy. Thirty-seven patients with similar types of diseases received chemotherapy and placebo injections. The 'vaccine' was repeated every 3 months. Bacterial clearance was more rapid in the vaccinated patients. Two lepromatous leprosy patients with initial bacterial index (BI) of 1.8 and 2.8 became bacteriologically negative in 1 year. One LL patient with BI of 6.0 had a BI fall to 0.16 after four doses of the vaccine. None of the LL patients belonging to placebo group during the same time period became bacteriologically negative. Rapid bacterial clearance was accompanied by distinct signs of clinical improvement. One hundred percent of BB, 85.7% of BL patients and 61.5% of LL patients converted to lepromin positivity after four doses of the vaccine. A significant number of vaccinated patients demonstrated an upgrading in skin lesions histopathologically.

Controversies over the vitamin D receptor (VDR) acting as a susceptibility factor in Mycobacterium sp. infections may be the result of incorrect population stratification. The risk of leprosy occurrence conditioned by VDR polymorphism was investigated by stratifying the population of a highly endemic Brazilian region into negative and positive Mitsuda responses. Leprosy patients (102) and a group of healthy nonconsanguineous household contacts (68) were genotyped for the VDR TaqI polymorphism (T/t). TT and Tt genotypes were not considered to be risk factors as their odds ratios (OR) were not different from those presented by the negative Mitsuda response individuals. The combination of the tt genotype and the negative Mitsuda test provided an occurrence rate 13 times higher in leprosy patients than in controls with positive Mitsuda responses. This suggests that there is a higher risk of leprosy development when individuals carry this unfavorable combination, and demonstrates a possible synergistic role of these two variables in leprosy susceptibility via effects on cellular immunity.

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.

Background. Leprosy is a chronic infectious disease caused by Mycobacterium leprae. The Mitsuda reaction is a delayed granulomatous skin reaction elicited by intradermal injection of heat-killed M. leprae. Interestingly, results of the Mitsuda test are positive in the majority of individuals, even in areas not endemic for M. leprae. Like leprosy, the Mitsuda reaction is thought to be genetically controlled, but its mode of inheritance is unknown, although the role of the NRAMP1 gene has previously been reported. Methods. We conducted a segregation analysis of quantitative Mitsuda reactivity in 168 Vietnamese nuclear families ascertained through patients with leprosy. Results. We found strong evidence ( ) for a major gene controlling the Mitsuda reaction independently 9 P ! 10 of leprosy clinical status. Subsequent linkage analysis showed that this major gene was distinct from NRAMP1. Under the major-gene model, ∼12% of individuals are homozygous for the recessive predisposing allele and are predicted to display high levels of Mitsuda reactivity (mean, ∼10 mm, versus 5 mm in other individuals). Conclusion. We provide evidence that the Mitsuda reaction is controlled by a major gene. Our study paves the way for the identification of this gene and should provide novel insight into the mechanisms involved in granuloma formation, especially in M. leprae infection. Leprosy is a chronic infectious disease, caused by Mycobacterium leprae, that mainly affects skin and peripheral nerves [1]. Despite a dramatic decrease in prevalence during the past 15 years, partly due to the efficacy of multidrug therapy [2], the worldwide leprosy incidence is still approaching 500,000 novel cases/year. Whereas most infected individuals do not develop clinical disease, even after sustained exposure to M. leprae, others present a broad spectrum of clinical symptoms. Leprosy ranges from the tuberculoid form, characterized by mature, well-delineated, and well-differentiated

Controversial results have been achieved by attempting to associate the NRAMP1 gene with Mycobacterium leprae susceptibility as well as with the Mitsuda reaction, which represents a specific immune response to M. leprae. This study evaluated this association as well as the interaction of the polymorphism (GT)(n) in the promoter region of the NRAMP1 gene with a specific immune response to M. leprae measured by the intradermal Mitsuda test in leprosy patients and in non-consanguineous household contacts. The study aimed to evaluate the association of this gene polymorphism with resistance or susceptibility to the disease, and/or with clinical forms of the disease, in a population in an endemic area served by the State Reference Center in Sanitary Dermatology and Leprosy, Federal University of Uberlandia, MG, Brazil. Leprosy patients (90) were diagnosed according to Ridley and Jopling criteria and they grouped into multibacillary (MB) and paucibacillary (PB) patients. The control group consisted of 61 non-consanguineous contacts. NRAMP1 promoter genotypes were obtained through amplification by the polymerase chain reaction (PCR) followed by the detection through the low ionic-strength single strand conformational polymorphism (LIS-SSCP) electrophoretic technique. There were no significant differences in the allelic and genotypic frequencies for alleles 2, 3, and 4 in relation to the Mitsuda test among patients and household contacts, nor between those with MB and PB forms. However, individuals with a negative lepromin response associated with genotypes 22 and 23 presented a 7- and 8-fold greater chance of developing leprosy, respectively. Therefore, the NRAMP1 gene promoter polymorphism exhibited an interaction with the lepromin response, suggesting that allele 2 of the NRAMP1 promoter is an independent genetic factor that predisposes cells to enable pathogen survival, probably due to its low efficiency in iron transport. However, establishment of the infection and disease development may be conditioned by other immunological and genetic factors.

A lthough the World Health Organization (WHO) has assumed the authoritative role for the guidelines for the diagnosis and treatment of leprosy, certain endemic countries such as India and Brazil and countries with traditional interest in the disease such as England and the United States have utilized their own programs which have been modified by the WHO. The prevalence of leprosy in endemic areas has been significantly reduced, but unfortunately the incidence continues to be the same. However, the WHO is optimistic about the elimination of leprosy as a public health problem in the near future; such optimism is not shared by many leprologists. The practicing physician especially in the non-endemic areas, such as the USA, should be aware of the current diagnostic and therapeutic approach to leprosy as recommended not only by the National Leprosy Center of the USA and its satellite clinics, but also by the WHO. If the practicing dermatologist wants to treat leprosy and become a ‘‘dermatoleprologist,’’ it is mandatory that he is knowledgeable about its diagnosis, treatment, reactions, the associated neuritis, and the prevention and management of its disabilities. With the potential decline of leprosy, leprosy control will become part of primary health care, which must promote early detection, adequate treatment, prevention, and management of disabilities.

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).

This is a retrospective study of 276 patients consisting of 157 active and 119 reactive patients of borderline leprosy. They were followed up for 10 years after sulphone monotherapy. The presenting symptoms were carefully examined from the records and systematically presented. Frequency of reactions was least in BT cases and most in BL cases. Risk factors of reaction appear to be the type of leprosy, multiplicity of lesions, high BI and, possibly, psychological stress. Biopsy of skin lesions was performed in all cases initially, and at the subsidence of the disease. Histological findings closely correlated with clinical classification. While all the cases showed clinical subsidence, histological subsidence was found in 200 (73%) cases, and the condition was static in 36 cases (13%). Immunological upgrading was seen in 110%, while 4% showed downgrading. Bacteriological status and lepromin reaction of active and reactive cases were compared. All these factors need to be taken into consideration for instituting prompt and proper treatment.

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 eight patients with indeterminate leprosy (HI), at least 4 to 6 years after discharge from multibacillary (MB) or paucibacillary (PB) schemes of anti leprosy multidrug therapy (MDT), were submitted to traditional diagnostic procedures for leprosy and to polymerase chain reaction (PCR) analysis of different clinical samples for detection of Mycobacterium leprae DNA. No significant difference was observed for any of the parameters analyzed between PB or MB schemes of treatment and no indications were found for more efficient outcome of HI using the MB scheme. Remarkably, 18 (54.5%) of the individuals were PCR positive in at least one of the samples: positivity of PCR was highest in blood samples and four individuals were PCR positive in blood and some other sample. Upon comparison of PCR results with clinical and histopathological parameters, no correlation was found between PCR-positivity and eventual relapse. This is the first report on detection of M. leprae DNA in PB patients, more than half a decade after completion of MDT, suggesting that live bacilli are present and circulating much longer than expected, although reinfection of the individuals can not be excluded. Overall, we feel that because of the high sensitivity of the assay, extreme care should be taken about association of PCR results, efficacy of treatment and disease status.

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.

Skin testing with lepromin, which produces a delayed-type hypersensitivity reaction, has been used in the classification of leprosy, and a good correlation has been found between immunological status and the reaction to lepromin. In addition, the prognostic value of the lepromin test has been demonstrated. More recently, skin testing with two soluble antigens of Mycobacterium leprae showed no difference of the mean size of the reaction between household contacts and non-contacts, indicating that these antigens are not useful for the diagnosis of leprosy. This and other evidence points to the need for a better skin test antigen capable of detecting infection of individuals by M. leprae. Whereas serological assays for antibodies against both PGL-1 and the 35 kDa antigen of M. leprae have been found to yield positive results in 90-100% of patients with lepromatous (BL/LL) leprosy, these assays fail to identify 40-60% of patients with tuberculoid (BT/TT) leprosy, because of the presence of only an insignificant level of antibody against components of M. leprae in these patients' serum, although, in many BT patients, antibody signal could be detected in the local lesions. These data indicate that there remains a need for a specific diagnostic test for leprosy.

One of the most urgent needs from leprosy research is a test for infection. The lepromin test is not suitable as a diagnostic test for leprosy, and neither the Rees nor the Convit soluble antigens has appeared sufficiently specific. Because two new antigens, MLSA-LAM and MLCwA, may not fully meet the requirements for specificity, we have embarked upon the preparation of a second generation of skin test antigens. Size-fractionated cryptozoic proteins were prepared from M. leprae by electroelution from preoperative sodium dodecylsulphate-polyacrylamide gel electrophoresis, and individual fractions were probed with polyclonal and monoclonal antibody reagents to identify both known and novel proteins. In addition, immunological responses were assessed in M. leprae-sensitized guinea pigs against both crude subcellular fractions (cytosol, membrane, and soluble cell wall proteins) and the size-fractionated cytosolic proteins. A particularly promising subcellular fraction is the membrane fraction of M. leprae, which contains many proteins unique to the organism. Clinical trials of the M. leprae membrane proteins are now being planned.

We screened 487 household contacts of multibacillary (MB) patients for evidence of disease and their lepromin status. From the 444 results available, 302 (68.02%) were lepromin positive and 142 (31.98%) were lepromin negative on initial testing. The initial lepromin status as assessed in the group of 54 contacts having disease at the outset showed 24 out of 46 (52.2%) to be lepromin positive and 22 of 46 (47.8%) to be lepromin negative. In the same group, among 24 lepromin positives, 22 (91.7%) had paucibacillary (PB) and 2 (8.3%) had multibacillary (MB) disease; among the lepromin negatives, 12 (54.5%) had PB and 10 (45.5%) had MB disease. Out of 72 initially lepromin-negative contacts administered Mycobacterium w vaccine and followed up, the cumulative percentages show that 53 (73.6%) converted to positivity after a single dose, 10 (87.5%) after a second dose and 67 (93.1%) after the third dose. The incidence of new cases with leprosy was 8 out of 231 (3.46%) among lepromin-positive contacts and 5 out of 93 (5.38%) among lepromin-negative contacts administered Mycobacterium w vaccine. Among 231 lepromin-positive contacts, the new cases occurred in those with a 1+ and 2+ lepromin response only, and no case occurred among 51 contacts with a 3+ lepromin response. The incidence among lepromin-positive contacts in this study (3.46%) was similar to the observations in two other studies: 3.2% by Dharmendra, et al. and 6.9% by Chaudhary, et al. However, the incidence among lepromin-negative contacts administered Mycobacterium w vaccine was significantly lower than that observed among lepromin-negative contacts not administered any vaccination in the other two studies (14.1% by Dharmendra, et al. and 29.0% by Chaudhary, et al.). To conclude, although a study of small sample size, the preliminary evaluation indicates that administration of Mycobacterium w vaccine seems to have the potential to reduce the incidence of leprosy among household contacts of leprosy patients. More explicit results about the vaccine will be available from the ongoing field trials in Kanpur Dehat in the near future.

In 14 nine-banded armadillos the Mitsuda response to nude mouse-derived lepromin (lepromin-nu/nu) was compared to that of armadillo-derived lepromin (lepromin-A) by injecting the reagents intradermally into either side of the abdomen of the animal and examining the biopsies from the sites after 12 days. The histopathologic responses to both antigens were found to be similar, whether the animal was Mitsuda-negative (lepromatous) or Mitsuda-positive (tuberculoid). It is pointed out that armadillos are good experimental models for leprosy, and their use can replace humans in experimental studies.

Healthy children contacts of leprosy patients had their humoral and cell-mediated immunological status assessed using the Fluorescent Leprosy Antibody Absorption Technique (FLA-ABS) and the Lepromin test, respectively. Subsequently, they were followed up for 2 1/2 years to study the development of overt disease. Two-hundred children were studied and classified into four groups, viz. Group I comprised of children who were FLA-ABS positive and Lepromin positive; Group II = FLA-ABS positive and Lepromin negative; Group III = FLA-ABS negative and Lepromin positive; Group IV = FLA-ABS negative and Lepromin negative. The good cell-mediated immune (CMI) response in the 107 children in Group I prevented them from developing the disease though they had been infected. Out of the 37 children in Group II, 15 developed the disease. There were no children in Group III. None of the 56 children in Group IV developed the disease, possibly because they had not been significantly infected. All these findings were statistically significant (P less than 0.01). This study highlights the protective role of cell-mediated immunity in leprosy. It also suggests the need to carry out surveillance surveys in the endemic population to identify and follow up those at risk.

The Mitsuda test, which measures the specific immune response against intradermally injected lepromin, has a high prognostic value for susceptibility or resistance to the lepromatous form of leprosy. A sib-pair linkage analysis between the Mitsuda response and the NRAMP1 gene was done among 20 nuclear families with leprosy (totaling 118 sibs) from Ho Chi Minh City, Vietnam. All family subjects were genotyped for several intragenic and flanking NRAMP1 markers, leading to the definition of a fully informative NRAMP1 haplotype. Significant linkage was observed between NRAMP1 and Mitsuda reaction when considered either as a quantitative (P

The reliability of methods of testing nerve function is important, since diagnostic decision making is a direct function of the quality of the test. Three methods of nerve function testing were investigated at the Danish Bangladesh Leprosy Mission (DBLM) in north Bangladesh, and assessed for inter-observer reliability. The three methods were 1) ballpoint pen test (BPT) for sensory function; 2) graded Semmes Weinstein monofilament test (SWM) for sensory function and 3) voluntary muscle testing (VMT) for motor function. The weighted kappa (kappa w) statistic was used to express inter-observer reliability. Using this statistic, 0 represents agreement no better than random, and 1.0 complete agreement. kappa w values of > or = 0.80 are reckoned to be adequate for monitoring and research. Fifty-three patients were tested, a Senior physiotechnician acting as 'gold standard' against whom four other staff physiotechnicians were assessed. All three testing methods were found to have minimal inter-observer variation, with the kappa w for inter-observer agreement using BPT being 0.86, the SWM 0.92, and VMT 0.94. It is concluded that in trained and experienced hands, all three methods are reliable and repeatable to a level allowing confident use of results obtained in monitoring and research.

A vaccine based on autoclaved Mycobacterium w was administered, in addition to standard multidrug therapy (MDT), to 157 bacteriologically positive, lepromin-negative, multibacillary (LL, BL and BB) leprosy patients. The vaccinees were supported by a well-matched control group of 147 patients with similar type of disease who received a placebo injection in addition to MDT. The MDT was given for a minimum period of 2 years and continued until skin-smear negativity, while the vaccine was given at 3-month intervals up to a maximum of 8 doses. The lepromin response evaluated in terms of percentage of subjects converting to positivity status, measurement in millimeters, and duration of lepromin positivity sustained, reflected a statistically significant better outcome in the vaccine group patients (especially LL and BL leprosy) in comparison to those in the placebo group. The data indicate that lepromin-positivity status seems to have an impact on accelerating the bacteriological clearance, as is evident by the statistically significant accelerated decline in the BI of those patients who converted to lepromin positivity as compared to those remaining lepromin negative throughout therapy and post-therapy follow up. To conclude, the addition of the Mycobacterium w vaccine to standard MDT induces a lepromin response of a statistically significant higher magnitude than that observed with MDT alone.

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.