High levels of inflammatory cytokines are associated with poor clinical response to steroid treatment and recurrent episodes of type 1 reactions in leprosy.

Levels of leprosy antigen-induced interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) were measured in 96 leprosy patients with type 1 reactions (T1R) before, during and after a standard 12-week course of steroids. Peripheral blood mononuclear cells (PBMC) from leprosy patients with untreated T1R produced significantly more TNF-alpha than leprosy patients without T1R. Median levels of IFN-gamma and TNF-alpha in T1R patients fell during treatment with steroids; however, TNF-alpha levels increased as the steroid dose was reduced. Median IL-10 levels increased throughout the steroid treatment period and were associated strongly with TNF-alpha levels. Patients with high cytokine levels had a poorer recovery of sensory or voluntary muscle nerve function, a higher risk of reactivation of symptoms during steroid treatment, and a higher risk of another episode of T1R within 2 months of completing the steroid regimen. Rapid and effective reversal of the inflammatory process in T1R is critical to prevent permanent nerve damage from T1R and monitoring cytokine levels during treatment may be useful.

Prediction of 'highly skin smear positive' cases among MB leprosy patients using clinical parameters.

Although 'highly skin smear positive' MB leprosy cases are known to be at high risk of relapse after release from treatment, and have been recommended to receive 'prolonged duration' MDT, government field-based control programmes without skin smear facilities have no simple alternative method to detect such cases. This study reports a significant prevalence of 'highly smear positive' cases amongst 2374 new multibacillary cases recently surveyed by skin smears in Nepal, and retrospectively analyses 555 newly detected, previously untreated BL and LL cases to identify clinical and laboratory parameters that may be associated with a 'highly positive skin smear'. While some parameters showed high sensitivity in predicting 'highly positive smear' status, none showed both high sensitivity and high specificity simultaneously.

DNA encoding a single mycobacterial antigen protects against leprosy infection.

The continuing incidence of leprosy infection around the world and the inability of Mycobacterium bovis bacille Calmette-Guérin (BCG) to protect certain populations clearly indicates that an improved vaccine against leprosy is needed. The immuno dominant 35 kDa protein, shared by Mycobacterium leprae and Mycobacterium avium, but not Mycobacterium tuberculosis or BCG, is recognised by >90% of leprosy patients, making it an ideal candidate antigen for a subunit vaccine. Immunization of outbred Swiss Albino mice with a DNA-35 vaccine stimulated specific T cell activation and IFN-gamma production. DNA-35 immunization induced significant levels of protection against M. leprae footpad infection, comparable to that produced by BCG. Therefore, DNA immunization with the 35 kDa antigen is effective against M. leprae infection and genetic immunization with a combination of antigens holds the potential for an improved vaccine against leprosy.

A method for rapid detection of rifampicin-resistant isolates of Mycobacterium leprae.

A genotypic method for predicting rifampicin resistance in Mycobacterium leprae has been developed and rigorously tested on mouse footpad-derived and clinical specimens. A series of immobilized oligonucleotide capture probes can discriminate between wild type and mutant rpoB alleles, and positive controls are available for the most frequent mutation affecting Ser425. Two different non-radioactive detection formats have been tested with comparable success in both an industrialized and a developing country. The standardized procedure could now be used in a prospective study of potential rifampicin resistance among multibacillary patients.

Vaccination with DNA of the Mycobacterium tuberculosis 85B antigen protects mouse foot pad against infection with M. leprae.

A DNA vaccine composed of the gene for the common mycobacterial secreted protein antigen 85B was demonstrated to protect the mouse foot pad against infection with Mycobacterium leprae. The protective effect was demonstrated by a 61%-88% reduction in the bacterial number, a protective effect less than that of BCG. The same DNA vaccine has been shown to protect mice against M. tuberculosis infection, and the importance of testing other candidate tuberculosis vaccines for their potential to protect against leprosy is discussed.

Interferon-gamma responses to candidate leprosy skin-test reagents detect exposure to leprosy in an endemic population.

New tools for the detection of leprosy exposure in a community will be necessary for the eradication of leprosy. Candidate leprosy skin-test antigens derived from the fractionation of the leprosy bacillus into cytoplasmic and cell-wall proteins free of immuno-inhibitory mycobacterial lipoglycans and carbohydrates were used in an overnight blood test to determine whether exposure to leprosy can be detected by the production of the cytokine interferon gamma (IFN-gamma). Strong IFN-gamma responses were detected in leprosy contacts to both skin-test antigens compared with control subjects from the same endemic communities. There was little response in patients with tuberculosis. Responses were greatest in contacts with recent leprosy exposure. The implications of these findings for the application of these reagents in a field trial as skin tests to detect exposure to leprosy are discussed in light of the strong association between overnight IFN-gamma to PPD and the tuberculin skin-test responses previously reported.

Immunoprophylaxis against Mycobacterium leprae infection with subunit vaccines.

We have investigated the effect of subunit vaccines against infection with Mycobacterium leprae, employing DNA plasmids as the vaccine vectors, and the immunodominant 35 kDa protein of M. leprae as the candidate antigen. A DNA vaccine that expresses the M. leprae 35 kDa protein both stimulated interferon-gamma (IFN gamma)-secreting T cells in mice, and demonstrated protection against M. leprae-infection of mice.

Risk factors for erythema nodosum leprosum.

A retrospective study of new borderline lepromatous and lepromatous patients reporting for multidrug therapy (MDT) for leprosy at the Anandaban Leprosy Hospital, Kathmandu, Nepal, over an 8-year period was conducted to determine the prevalence of erythema nodosum leprosum (ENL), the time and frequency of reactions, and clinical and laboratory parameters associated with ENL. An overall prevalence of ENL in this cohort of 19% was found. One third of these reactions occurred in patients before MDT was given, one third in the first 6 months and one third after 6 months of treatment. Nearly 1 in 10 of the ENL reactions occurred in patients who had completed 2 years of MDT; 45% of patients with ENL had more than one episode. Data collected at the patients' first presentation was used to identify four major risk factors. Patients with lepromatous disease, skin infiltration or a bacterial index (BI) of > 4+ were at significantly increased risk. Patients older than 40 were at significantly decreased risk of ENL. There was a linear relationship in the risk of ENL with an increasing BI and an inverse relationship to increasing age. These observations should enable clinicians to recognize patients at first presentation who will be likely to develop ENL.

Rapid method for diagnosis of leprosy by measurements of antibodies to the M. leprae 35-kDa protein: comparison with PGL-I antibodies detected by ELISA and "dipstick" methods.

A new rapid immuno-chromatographic test card for the detection of antibodies to the Mycobacterium leprae 35-kD protein is described. The new assay is compared in the same group of subjects with a direct enzyme ELISA method for 35-kD antibodies and with assays for anti-phenolic glycolipid-I (PGL-I) antibodies using a standard ELISA as well as the recently described "dipstick" method. Good concordance was found between the rapid methods and the corresponding ELISA methods. The detection of untreated paucibacillary leprosy by the 35-kD test card was 59% compared with 27% for the PGL-I dipstick; however, the specificity for the 35-kD test card was 90% compared with 100% for the PGL-I dipstick in an endemic population. The potential application of these new, rapid serologic methods for the diagnosis of leprosy under field conditions is discussed.

Specific serological diagnosis of leprosy with a recombinant Mycobacterium leprae protein purified from a rapidly growing mycobacterial host.

In this report we demonstrate the utility of an monoclonal antibody inhibition enzyme-linked immunosorbent assay based on the Mycobacterium leprae 35-kDa protein, purified from the rapidly growing host Mycobacterium smegmatis, for the serodiagnosis of multibacillary leprosy. The assay proved highly specific (97.5%) and sensitive (90%) and compared favorably with two other established methods routinely utilized for leprosy serodiagnosis.

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein.

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.

Contribution of type 1 reactions to sensory and motor function loss in borderline leprosy patients and the efficacy of treatment with prednisone.

The changes in nerve function tests in 297 new leprosy patients over an average period of 30 months were measured. The impact of type 1 reactions (T1R) on sensory and voluntary muscle function was measured by standard tests. Sensory function was improved in patients with single episodes of cutaneous T1R, but not improved in patients with neural T1R or with multiple episodes of either kind of T1R. Patients over 40 years of age improved less than younger patients, and patients admitted for treatment of T1R improved more than those treated as outpatients. These data point to a need to find better regimens for the treatment of nerve damage in T1R.

Risk factors for type 1 reactions in leprosy.

A cohort of new borderline leprosy patients seen over a 7-year period were examined retrospectively for risk of type 1 reactions (T1R) associated with 12 clinical and laboratory parameters. Logistic regression analysis was used to identify a strong link between facial patches and cutaneous T1R and enlarged ulnar nerves and neural T1R. Anti-phenolic glycolipid-I seropositivity, a positive bacterial index, and disease in more than two body areas were also identified as risk factors for T1R. These data indicate that there are important clinical data which can be used to predict an individual patient's risk of developing T1R.

IgM anti-phenolic glycolipid-I antibody measurements from skin-smear sites: correlation with venous antibody levels and the bacterial index.

Measurements of anti-phenolic glycolipid-I antibodies were made in 200 matched samples of capillary blood from the skin-smear site, venous blood collected on filter paper, and sera. A close correlation among the three samples was observed and a weaker correlation among the antibody levels and the average and skin-smear bacterial index. Capillary blood from the skin-smear site had a consistently higher level of antibodies in each sample than did the sera. The collection of capillary blood from skin-smear sites is a convenient and economical method of obtaining samples for serology and for measuring local antibody levels, and it may be more sensitive than measurements of antibodies in sera.

A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae.

The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum. We have investigated the host response to a 35-kDa protein of M. leprae. Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients. These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli. The M. smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested. Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M. leprae characteristic of this form of the disease. Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen. This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M. leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG. Therefore, the M. leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M. leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease.