Induction of lepromin positivity following immuno-chemotherapy with Mycobacterium w vaccine and multidrug therapy and its impact on bacteriological clearance in multibacillary leprosy: report on a hospital-based clinical trial with the candidate antileprosy vaccine

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.     

Impact of combined Mycobacterium w vaccine and 1 year of MDT on multibacillary leprosy patients

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.     

Disabilities in multibacillary leprosy following multidrug therapy with and without immunotherapy with Mycobacterium w antileprosy vaccine

A vaccine based on autoclaved Mycobacterium w was administered, in addition to standard multidrug therapy (MDT), to 157 bacteriologically positive, lepromin-negative, multibacillary leprosy patients 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/placebo was given at 3-month intervals up to a maximum of 8 doses in the initial 2 years. The overall incidence of type 1 and type 2 reactions and neuritis during treatment and follow up was nearly equal in the patients in the vaccine and placebo groups; the differences were not statistically significant. The occurrence of disabilities, such as anesthesia, trophic ulcers, claw hand and grade 3 deformities, were not different statistically in the vaccine and placebo groups, an observation valid both for deformities present at induction and for those which developed during the course of therapy and surveillance. A statistically significant difference was observed in the recovery of newly developed trophic ulcers; recovery was quicker in the vaccine group. The recovery rate for motor deformities was marginally higher in the vaccine group, although not significant (p = 0.068) statistically. There was a statistically significant reduction in the incidence of grade 3 deformities following MDT with and without immunotherapy. To conclude, the addition of vaccine to MDT did not precipitate neuritis or deformities over and above that encountered with MDT alone, although it did accelerate bacteriological clearance, histopathological upgrading, conversion to lepromin positivity, and clinical improvement.     

Combined 12-month WHO/MDT MB regimen and Mycobacterium w. vaccine in multibacillary leprosy: a follow-up of 136 patients

A total of 136 patients with BI > or = 2 having been followed up for at least 2 years or more were included in the analyses. Seventy-seven out of 136 patients had completed three years follow up. All patients were given WHO/MDT MB regimen for 12 months and additionally 4 doses of Mycobacterium w. vaccine at 3-month intervals. The age of the patients varied from 6 to 77 years (mean 34 +/- 11.3 years) and they had the disease varying from 3 months to 7 years (mean = 1.9 +/- 1.4 years). The mean of the BI before starting treatment was 3.6 +/- 1.3. At the end of 2 years follow-up, a total of 54 patients out of the 136 (39.7%) had become smear-negative. A larger proportion of patients, 39/46 (84.8%) with BI of < or = 3 had become smear-negative, whereas, only 10/32 (31.3%) patients with BI between 3.1 to 4 and 5/58 (8.6%) highly bacillated patients having initial BI > 4 had become smear-negative at the end of 2 years. Out of the 77 patients who were available for follow up at 3 years, 30/33 (90.9%) patients with BI of < or = 3, 15/20 (75%) patients with BI between 3.1 to 4 and 13/24 (54.2%) patients having initial BI > 4, respectively, had attained smear negativity. Reactions occurred more frequently after 6 months of therapy and over a period of time their frequency gradually decreased, however, they continued to occur even two years after RFT. During the course of MDT and thereafter in follow up 4.6% and 1.3% of the patients developed new deformities or an increase in the existing grade of deformities, respectively. Three relapses (2 in LL and 1 in BL) occurred in patients having initial BI of > 4. One patient relapsed in the second year and the other two relapsed in the third year of follow up and were successfully treated with reintroduction of the same MDT MB regimen. Local ulceration healing with scar formation and regional lymphadenopathy were the only local reactions to the vaccine seen in 47/136 (34.5%) patients. All the patients showed histopathological improvement in the form of a gradual reduction of granuloma fraction. Although the results of this limited period follow up are satisfactory, a long-term follow-up in larger number of patients will settle the issue of safety and efficacy of shortened MDT MB regimen and the place of immunotherapy with M. w. vaccine in multibacillary patients.     

Transmission of leprosy: a study of skin and nasal secretions of household contacts of leprosy patients using PCR

It is generally held that dissemination of Mycobacterium leprae is from nasal mucosa and not through the skin of infected patients. In this study, we evaluated M. leprae in the unbroken skin and nasal secretions of multibacillary (MB) leprosy patients and their contacts. Specimens were examined by direct microscopy and polymerase chain reaction (PCR) for M. leprae DNA. Results showed that 60% of untreated MB leprosy patients examined histologically had acid-fast bacilli in the keratin layer. By PCR studies it was found that 80% of the patients had M. leprae DNA in skin washings and 60% had M. leprae DNA on swabs obtained from the nasal mucosa. Ninety-three contacts of the untreated MB cases were also tested for exposure to M. leprae by analyzing skin washings and nasal secretions by PCR. PCR analysis showed significant skin (17% positive) and nasal muscosal (4%) exposure in contacts before instituting treatment of the index cases. After 2 months of treating the index cases, all contacts tested were negative for M. leprae DNA. These data suggested that both skin and nasal epithelia of untreated MB leprosy patients contribute to the shedding of M. leprae into the environment and contacts of untreated MB cases are at risk for contact with M. leprae through both the nasal mucosa and exposed surfaces of their skin.     

Antibodies to a synthetic analog of phenolic glycolipid-I of Mycobacterium leprae in healthy household contacts of patients with leprosy

Fifty-four household contacts of lepromatous patients, 39 household contacts of tuberculoid patients, and 99 control persons were examined with an enzyme-linked immunosorbent assay for their antibody responses to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae using a synthetic analog (PGL-ISA) with the same terminal sugar epitope, namely, O-(3, 6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(alpha-L-rhamnopyranosyl )-(1----9)-oxynonanoyl-BSA. This study was conducted in the Gurage area of Ethiopia in 15 households with a leprosy patient and 15 matched control households. Household contacts with more than 1 year of exposure to a lepromatous patient had antibodies to PGL-ISA significantly more often (19 of 34 persons) than did household contacts with less than 1 year of exposure to a lepromatous patient (4 of 20 persons), household contacts of tuberculoid patients (8 of 39 persons), and persons without exposure to leprosy in the household (33 of 99 persons). No significant association was found between the prevalence of antibodies to PGL-ISA in the household contacts and disease activity in the lepromatous index patients at the time of examination; nor was there a significant association between antibody responses and age or sex of the contacts. The increased prevalence of antibodies to M. leprae antigen in healthy persons with more than 1 year of contact with a lepromatous patient provides further evidence that subclinical infection in leprosy is common, and is related to the type of leprosy in the index patient. The fact that antibodies to PGL-ISA were detected in one third of the persons without household exposure to leprosy emphasizes the necessity to always include comparable controls from the same endemic area in studies of leprosy contacts.     

Mycobacterium leprae-induced interferon-gamma production by household contacts of leprosy patients: association with the development of active disease

Identification of individuals at risk for developing leprosy and their early diagnosis are central to effective disease control. Lack of immunologic response to Mycobacterium leprae among persons exposed to the infectious agent may be predictive of susceptibility. M. leprae-induced interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells was used as a measure of immune responsiveness. Household contacts of multibacillary patients likely to be at risk of developing active disease were identified, and a preliminary analysis after 2 years of follow-up is presented. A persistent in vitro negative response to M. leprae was present in 34.6% of the contacts, and a decrease in IFN-gamma production was noted in 52.5%. Five contacts (6.41%) developed leprosy during follow-up and, as predicted, belonged to the group of individuals who were negative or showed reduced levels of IFN-gamma in response to the antigen.     

Prospective immunological follow-up in household contacts of Mexican leprosy patients

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.     

Histologic responses in sixty multibacillary leprosy patients inoculated with autoclaved Mycobacterium leprae and live BCG

Sixty lepromatous or borderline lepromatous patients were submitted to immunotherapy with a mixture of autoclaved Mycobacterium leprae and BCG. The histopathologic findings in skin biopsy specimens taken before and after immunotherapy were evaluated independently by six histopathologists in a workshop setting. Their pooled observations on diagnosis and classification were analyzed to assess the histopathologic changes following various periods of immunotherapy. Expressing the results as the average value of five to six independent observations, there were changes in classification of reversal or upgrading toward the tuberculoid end of the leprosy spectrum in 90.5% of the patients initially classified as lepromatous (LL), and in 83.3% of those initially classified as borderline lepromatous (BL). The histopathologic findings amply support the clinical, bacteriologic and immunological changes following immunotherapy from LL or BL, to BL, mid-borderline (BB) or even borderline tuberculoid (BT) leprosy.     

Effect of treatment on PCR positivity in multibacillary leprosy patients treated with conventional and newer drugs ofloxacin and minocycline

In order to develop objective criteria to monitor trends of therapeutic responses positivity of PCR signals and ATP assay methods has been compared in multibacillary (MB) leprosy patients. Biopsies from lesions of 95 BL/LL patients before and after one year of treatment with a new drug regimen comprising of conventional and newer drugs ofloxacin and minocycline have been studied. These biopsies were processed for bacillary ATP assay and PCR positivity for a 36 kDa gene target by earlier published methods. In the untreated patients bacillary ATP levels were detectable in all specimens and ranged from 0.02 to more than 36 pg/millions organisms. After one year of treatment ATP levels were not detectable in any of the 57 biopsies specimens available for analysis. However, PCR signals were detectable in 3 out of 57 biopsies. In two specimens signals were very weak detectable only by hybridization. It may be concluded that DNA based PCR assay may be useful in monitoring the trends of therapeutic responses in MB patients under treatment.     

The response to chemotherapy of serum Mycobacterium leprae-specific antigen in multibacillary leprosy patients

We have examined the Mycobacterium leprae phenolic glycolipid-I (PG-I) antigen levels in the sera of 45 multibacillary leprosy patients commencing chemotherapy. The PG-I antigen levels correlated with the bacterial and morphological indices, but not with the serum IgM anti-PG-I antibody levels. Antigen levels were significantly higher in patients with diffuse skin infiltration, but did not vary significantly with other parameters reflecting the duration and extent of untreated disease. The PG-I antigen levels in 27 patients examined serially decreased consistently over the first year of multidrug therapy.     

Preliminary study of cellular immunity to Mycobacterium leprae protein in contacts and leprosy patients.

Because of the good results obtained in the mononuclear cell (T lymphocyte) proliferative response in tuberculoid leprosy patients and family contacts and healthy Mitsuda-positive volunteers using Mycobacterium leprae soluble extract, we prepared different protein fractions from the soluble extract. We used the T-cell Western blot technique with separation by electrophoresis in SDS-polyacrylamide gels and transfer onto nitrocellulose membranes. Each unstained blot was converted into 18 fractions of antigen-bearing particles and tested with peripheral blood mononuclear cells from 21 individuals including Mitsuda-positive contacts, vaccinated lepromatous leprosy (LL) patients, borderline tuberculoid (BT) patients, and unvaccinated lepromatous patients. The stimulation index (SI) of the contacts was higher to the different fractions in comparison with the leprosy patients. They showed four peaks of stimulation to fractions 66-55, 45-29, 22-18, and 14 kDa. The second highest responders were BT patients, followed by vaccinated LL patients. The unvaccinated patients did not respond significantly to any of the fractions (SI less than 1).     

Seroepidemiological study on 724 household contacts of leprosy patients in French Polynesia using disaccharide-octyl-BSA as antigen

A seroepidemiological surveillance of a contact population was started in 1984 in French Polynesia. The ELISA test was used to measure IgM anti-ND-O-BSA in the sera. Specific antibody levels were higher in healthy Polynesians than in normal individuals living in a nonendemic country. The positive threshold of the reaction was fixed according to this background activity in healthy Polynesians. Under these conditions, 100% of the multibacillary patients were detected as seropositive as compared to 5% of the paucibacillary group. In the population of 724 household contacts tested and observed for 2 years: 93 (12.8%) were seropositive, with 8 (1.1%) showing activity equivalent to multibacillary patients (1 of these 8 individuals developed a lepromatous form of leprosy); 631 (87%) were seronegative and 3 developed a paucibacillary form of the disease (2 BT, 1 I) without any antibody increase. Among those four contacts who developed leprosy, three were related to a multibacillary index case. These data suggest that this test may be useful for the prediction of multibacillary leprosy. A long-term surveillance of this high-risk population will be able to evaluate the diagnostic and prognostic value of the serological assay.     

Typhoid fever in Santiago, Chile: a study of household contacts of pediatric patients

We obtained clinical, epidemiological, and laboratory data (including three stool cultures) from 155 (96%) of 161 household contacts of 24 patients less than 16 years old with culture-confirmed typhoid fever; these 24 patients represented approximately 40% of such patients seen in three hospitals in Santiago during a 12-week period. A chronic typhoid carrier was identified in only one household, with concurrent or secondary cases seen in two other households. When index cases were matched with household members nearest in age, no specific risk factors for illness could be identified. There was evidence of generalized exposure to enteric pathogens within these households, with nine persons from seven different households culture-positive for non-typhoidal Salmonella, and nine, from eight different households, culture-positive for Shigella; transmission of these pathogens within households did not appear to be common since no household had more than one family member with the same serotype or species of either pathogen.     

Lymphocyte transformation test in healthy contacts of patients with leprosy. I. Influence of exposure to leprosy within a household.

Fifty-three household contacts of lepromatous patients, 37 household contacts of tuberculoid patients, and 91 control persons were examined with the lymphocyte transformation test (LTT) for their responses to whole and sonicated antigen preparations from M. leprae, to BCG, M. avium, M. gordonae, and phytohemagglutinin (PHA). The study was carried out in the Gurage area of Ethiopia in 15 households with a leprosy patient and 15 matched control households. Household contacts of lepromatous patients showed significantly greater LTT responses to antigens from M. leprae than the controls, whereas household contacts of tuberculoid patients did not respond differently from controls. Household contacts of lepromatous patients had significantly greater responses to M. leprae antigens when the index patients were "active," i.e., highly bacilliferous, than when they were "inactive," i.e., having a low bacillary load. The degree of sensitization, as indicated by the LTT response, in different exposure groups paralleled the degree of probable infectivity of the index patient. A preparation of antigen from whole M. leprae proved to be more sensitive and more specific in the LTT than did a sonicated preparation. A significant degree of cross-reactivity was found among the various mycobacteria in their LTT responses.     

Serum IgA1 and IgM antibodies against Mycobacterium leprae-derived phenolic glycolipid-I: a comparative study in leprosy patients and their contacts

In order to evaluate the potentials of IgA1 versus IgM as well as of native phenolic glycolipid-I (PGL-I) versus PGL-I-disaccharide coupled to bovine serum albumin (D-BSA) as antigens in the serodiagnosis of leprosy, anti-D-BSA IgA1 and anti-PGL-I IgM were investigated and compared to anti-PGL-I IgA1 in sera from patients and contacts. Anti-D-BSA and anti-PGL-I IgA1 significantly correlate in patients and contacts. The higher IgA1 positivity rates obtained with D-BSA as compared to PGL-I may suggest D-BSA as the favorable antigenic material. In patients but not in contacts anti-PGL-I IgM and IgA1 correlate, IgM predominating over IgA1. In all three antibody systems, the mean values as well as the positivity rates increased from the tuberculoid toward the lepromatous disease pole. Also, the levels of all three antibodies significantly increased with the bacterial index (BI). However, anti-D-BSA (PGL-I) IgA1 appears to be preferable to IgM with respect to sensitivity, i.e., detection of disease activity, in paucibacillary or BI-negative patients. A number of contacts were detected as seropositive with anti-D-BSA and/or anti-PGL-I IgA1 but not with anti-PGL-I IgM. This suggests that IgA1 is a better tool than IgM for the detection of leprosy in its subclinical stage.     

Pattern of bacillary clearance in multibacillary leprosy patients with multidrug therapy

The bacteriological index (BI) of the skin smears is traditionally one of the important parameters of assessment of severity and of progress of leprosy under multidrug therapy. The present study reports on BI clearance among 578 multibacillary treated leprosy patients and the factors that influence this clearance. The patients were treated till smear negativity or for 2 years fixed duration and their skin smears periodically examined every 6 to 12 months till negativity (and even afterwards). We confirm that bacterial clearance is a slow process. The time taken for each log-unit decline in BI is between 13.6 to 24 months probably depending on initial BI level. The rate of smear negativity appears to be dependent on immune competence of the patients as reflected by a rapid BI decline in borderline BT-BB patients vis-à-vis BL-LL lepromatous patients both in the low and high BI group. Patients who had several episodes of ENL, took significantly longer time (63.7 months versus 53.5 months, p<0.0001) to become smear negative than those without ENL.     

IgE in leprosy; effect of a Mycobacterium leprae-BCG vaccine

Since some previous studies have reported elevated total serum IgE levels in leprosy, that may be associated with the existence of a state of generalized T cell anergy, we undertook a carefully controlled study of this immunoglobulin in such patients before and after treatment with a Mycobacterium leprae-BCG vaccine. We found, firstly, that lepromatous leprosy patients suffering active disease had only a moderate elevation of IgE levels that was not statistically significant when compared to appropriate controls. In addition, multiple injections of the vaccine did not cause alterations in the concentration of this immunoglobulin. We were, therefore, unable to confirm the possible existence of a generalized immunodeficiency in lepromatous leprosy that could cause hyper-production of IgE.