Sequential monitoring of leprosy patients with serum antibody levels to phenolic glycolipid-I, a synthetic analog of phenolic glycolipid-I, and mycobacterial lipoarabinomannan

Sequential serum samples from leprosy patients at various stages of antibacterial treatment were tested by an ELISA for antibodies to phenolic glycolipid I (PGL-I), a synthetic PGL-I analog (ND-BSA), and lipoarabinomannan (LAM) from Mycobacterium tuberculosis to determine if these antibodies could be useful in monitoring response to therapy. Among patients with positive initial anti-PGL-I IgM, a significant decrease in this antibody was seen over time (p less than 0.01), whether assayed by PGL-I or ND-BSA. The two antigens showed good agreement in the detection of decrease in anti-PGL-I IgM. The greatest decrease was seen in patients with a high initial anti-PGL-I IgM and a high bacterial index (BI). Patients with a declining BI were seen to have generally declining antibody levels to PGL-I and to LAM; in those patients with a fluctuating BI, antibody levels were less predictable. We conclude that antibodies to PGL-I and LAM can be useful in following response to therapy in leprosy patients and that either the native PGL-I or ND-BSA can serve as antigen for the ELISA.     

Antibodies to phenolic glycolipid-1 and to whole Mycobacterium leprae in leprosy patients: evolution during therapy

Sera from 92 patients were tested by the ELISA method for the presence of IgM antibodies to phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae, and of both IgM and IgG antibodies to the whole M. leprae bacillus. All untreated lepromatous patients exhibited high antibody levels in all three assays. A sharp decline of IgM antibodies to PGL-1 and whole M. leprae was observed during the first two years of therapy, while IgG antibodies to whole M. leprae showed a progressive decrease only over a number of years. Low titers of IgM antibodies to PGL-1 and IgG antibodies to whole M. leprae could be detected in about 50% and 75% of patients, respectively, after more than ten years of treatment, with only 15% showing persisting IgM antibodies to the whole bacillus. Antibody levels as measured by the three assays used were correlated with the bacterial index in patients treated for less than four years. In patients treated longer than four years, only IgM antibodies, whether directed to PGL-1 or to whole M. leprae, remained correlated to the bacillary load. Tuberculoid patients exhibited a different antibody pattern, showing a lower frequency (and lower levels) of antibodies of PGL-1 and of IgG antibodies to whole M. leprae than lepromatous patients, and no detectable IgM antibodies to the whole bacillus. IgG antibodies to whole M. leprae were more frequently noted than antibodies to PGL-1, the latter declining more rapidly during therapy.     

Comparison of synthetic antigens for detecting antibodies to phenolic glycolipid I in patients with leprosy and their household contacts

Three synthetic antigens related to the natural antigen phenolic glycolipid I (PGL-I) were compared for their efficacy in detecting leprosy when used as antigens in an enzyme-linked immunosorbent assay (ELISA) for IgM antibody to PGL-I. Absorbance values for ELISAs using the three antigens correlated well (.79 less than r less than .99) and had a high rate of agreement (89.5% less than a less than 98.4%). Of three subjects (household contacts of patients with leprosy) who later developed the disease, one with lepromatous and one with indeterminate leprosy were seropositive by ELISAs using the three antigens before the clinical onset of disease; one who developed borderline tuberculoid leprosy was seronegative. The predictive value of a positive result for the test was very low (less than 2.4%) and the predictive value for a negative result was high (greater than 99.9%) because of the low prevalence of leprosy in French Polynesia (1.78 per 1000). The high sensitivity, specificity, and efficiency of the tests using the three antigens confirmed their great value for serodiagnosis of leprosy, especially the multibacillary form; the ELISA using NTP seems to be more specific and sensitive for detecting the paucibacillary form.     

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.     

T-cell responses of leprosy patients and healthy contacts toward separated protein antigens of Mycobacterium leprae.

Sonicated extracts of Mycobacterium leprae were separated by two-dimensional gel electrophoresis and electroeluted into 400 distinct soluble fractions. These fractions were probed with T lymphocytes from leprosy patients of different disease types, healthy contacts, and unexposed healthy individuals. Proliferative responses were visualized using three-dimensional stimulation profiles. T cells from many patients and contacts responded to a multitude of antigen fractions of different molecular masses and isoelectric points. T cells from unexposed individuals gave significant responses to lysates or whole organisms of M. leprae, but no or only marginal responses to separated antigen fractions. T cells of polar tuberculoid (TT) and the majority of polar lepromatous (LL) leprosy patients responded only to separated antigen fractions but not to lysates or whole organisms of M. leprae. The remaining LL patients were totally unresponsive and even failed to respond to separated M. leprae fractions. Thus, in some leprosy patients unresponsiveness to M. leprae seems to be caused by distinct components and can be broken by using separated antigen fractions; whereas in others, anergy remains. T cells of borderline tuberculoid (BT) patients, who were under chemotherapy, responded to separated antigen fractions as well as to lysates of M. leprae organisms. In contrast, BT patients who were untreated failed to react with any of the M. leprae preparations. Similarly, T cells of the majority of LL patients responding to separated fractions were under chemotherapy; whereas T cells from untreated LL patients gave no or only marginal responses to any of the M. leprae antigen preparations. These findings suggest some linkage between the degree of T-cell responsiveness and antileprosy drug treatment.     

Serologic responses to Mycobacterium leprae-specific phenolic glycolipid-I antigen in sooty mangabey monkeys with experimental leprosy

Four pairs of sooty mangabey monkeys (Cercocebus atys) were inoculated with serial, 10-fold dilutions of Mycobacterium leprae. The highest-dose pair received 4.8 X 10(10) M. leprae. Serum samples were obtained and clinical signs of leprosy were recorded at intervals of 35 months. Longitudinal serum samples were assayed by an ELISA method for the presence of IgG and IgM antibodies to the M. Leprae-specific phenolic glycolipid-I (PGL-I) antigen. In general, the onset of disease symptoms paralleled the number of M. leprae inoculated, but the ultimate course of disease depended upon individual animal susceptibility. Both IgG and IgM anti-PGL-I isotypes were observed in variable levels and patterns, related to the disease stage, among the eight mangabeys. The data suggest that high IgG and low IgM anti-PGL-I levels correlated with less severe disease; whereas initial high IgM titers and/or rising or sustained high IgM titers, especially together with low IgG anti-PGL-I titers, preceded or corresponded to periods of progressive leprosy. The results show that IgG and IgM anti-PGL-I antibodies can be present in significant titers among mangabeys early after infection with M. leprae. It appears likely that the relative levels of these anti-PGL-I isotypes may be correlated with the susceptibility of individual animals to the development of lepromatous leprosy.     

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).     

IgM antibodies to native phenolic glycolipid-I in contacts of leprosy patients in Venezuela: epidemiological observations and a prospective study of the risk of leprosy

In a randomized, double-blind vaccine trial in Venezuela, about 29,000 contacts of leprosy patients have been vaccinated with either a mixture of heat-killed Mycobacterium leprae and BCG or BCG alone, and are being re-surveyed annually to detect new cases of leprosy. All contacts had a serum sample collected at the time of entry into the trial, and 13,020 of these sera have been analyzed for antibodies to phenolic glycolipid-I (PGL-I). Antibody levels have been related to various characteristics of the contacts and to their risk of developing leprosy in the following 4 years. A strong association was found between PGL-I antibody level and the risk of developing leprosy, in spite of possible modification of the incidence rate induced by vaccination. Antibody levels were higher in females than in males, and declined progressively with age. Household contacts had higher levels than did non-household contacts, and levels were higher in individuals from the state in Venezuela which has the highest incidence of the disease. No substantial differences were found in antibody levels between contacts of multibacillary and paucibacillary patients, which may in part reflect the influence of treatment, and there was no clear association with the presence of BCG or lepromin scars or with skin-test responses to PPD and leprosy soluble antigen. The assay of antibodies to PGL-I seems unlikely to provide a sensitive or specific test for infection with M. leprae, and measuring PGL-I antibody levels as a screening procedure to identify those at high risk of developing leprosy is unlikely to be particularly useful in most leprosy control programs. Such assays may be useful for the epidemiological monitoring of changes in the intensity of infection with M. leprae in a community and for the study of carefully defined groups of contacts during some phases of control programs.     

Recognition of phenolic glycolipid-I (Mycobacterium leprae) and sulfolipid-I (M. tuberculosis) by serum from Mexican patients with leprosy or tuberculosis.

SETTING: Differential diagnosis of leprosy and tuberculosis in regions where both illnesses are endemic is a prerequisite for proper identification and treatment. OBJECTIVE: To evaluate the recognition of phenolic glycolipid-I (PGL-I) of Mycobacterium leprae and sulfolipid-I (SL-I) of M. tuberculosis by serum from patients with leprosy (LL) or pulmonary tuberculosis (PTB). DESIGN: Purified PGL-I and SL-I were used as antigens in an ELISA test set up to assess recognition of these lipids by serum from 43 LL patients, 44 PTB patients and 38 healthy individuals. RESULTS: Leprosy patients gave higher IgM than IgG responses to PGL-I and had comparable IgM and IgG responses to SL-I. A similar situation was observed with PTB serum. Some healthy individuals were found to contain significant levels of antibodies to both lipids. CONCLUSION: There is no specific recognition of either of the two lipid antigens tested by serum from both leprosy and tuberculosis patients; this rules out the possibility of using PGL-I and SL-I as tools for the differential diagnosis of these two mycobacterial diseases.     

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.     

Inhibition of phenolic glycolipid-I synthesis in extracellular Mycobacterium leprae as an indicator of antimicrobial activity

The effects of 22 antimicrobial agents on the incorporation of [U14C] palmitic acid ([U14C] PA) into the unique phenolic glycolipid-I (PGL-I) antigen of Mycobacterium leprae were studied. Nude-mouse-propagated M. leprae were incubated in a modified Dubos medium in the presence of antimicrobial agents for 4 days. [U14C] PA was then added and incubation was continued for 8 days. The antileprosy agents dapsone, rifampin, and clofazimine (2 micrograms/ml each) caused a significant reduction in [U14C] PA incorporation into PGL-I. Among other agents, the most active were erythromycin, chloramphenicol, and cerulenin. Low concentrations of ethionamide, tetracycline, and minocycline stimulated label incorporation. This system may prove useful in the evaluation of antileprosy agents.     

Use of anti-M. leprae phenolic glycolipid-I antibody detection for early diagnosis and prognosis of leprosy

Untreated patients suffering from tuberculoid, lepromatous and indeterminate leprosy, their domiciliary contacts, and healthy controls, all living in Guadeloupe, West Indies, were tested by an ELISA for detecting IgM antibodies to the terminal disaccharide of the phenolic glycolipid-I antigen of Mycobacterium leprae. On most subjects, a Mitsuda test was also performed. A large majority of the tuberculoid patients and healthy subjects were Mitsuda positive. The seropositivity rate reached 44% among tuberculoid patients, and 6% among healthy subjects, with low antibody levels. Lepromatous patients were all Mitsuda negative and seropositive, with antibody production varying from low levels, as seen in tuberculoid patients, to much higher levels. Indeterminate leprosy patients included 62% Mitsuda-positive subjects and 54% seropositive subjects with a large dispersion of antibody levels. Comparing the results of the Mitsuda test to those of the ELISA by factorial analysis allowed us to define several subgroups among this population: some (25%) showed a "lepromatous-like" immune status (Mitsuda negative, seropositive); others (54%) exhibited "tuberculoid-like" profiles (Mitsuda positive without antibodies or with low antibody levels). "Lepromatous-like" cases were significantly older than "tuberculoid-like" patients. A group of subjects (17%) was Mitsuda negative and seronegative, thus displaying a true "indeterminate" immune profile, which had not been seen in other forms of the disease and had been observed in only 2 out of 51 healthy controls. A large majority of contacts was Mitsuda positive, with 33% of them being seropositive, indicating that the prevalence of M. leprae infection greatly exceeds that of overt leprosy in this population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to phenolic glycolipid-I during long-term therapy: serial measurements in individual patients

Levels of IgM antibody to phenolic glycolipid-I (PGL-I) were measured in serum specimens collected over the initial 5 or more years of therapy from 11 leprosy patients. All three patients with paucibacillary disease had undetectable levels of antibody throughout their treatment. The eight patients with multibacillary disease had initially elevated levels which fell quite rapidly with treatment, reaching levels of 10% to 30% of their initial pre-treatment level after 5 years of therapy. The single patient with prolonged therapeutic noncompliance had an increase in antibody level, although clinical or bacteriologic relapse was not documented. These results in individual patients demonstrate that IgM antibody to PGL-I declines rapidly and consistently with treatment in multibacillary patients.     

Management of household contacts of leprosy patients.

We describe an approach to the management of household contacts of leprosy patients and the rationale on which it is based. Initially, all household contacts should be interviewed and examined for symptoms and signs consistent with leprosy and appropriate diagnostic measures taken. Contacts of untreated lepromatous and dimorphous (borderline) leprosy patients are at relatively high risk of disease and should be examined annually for at least 5 years. Dapsone prophylaxis has been shown to prevent secondary cases in contacts up to 25 years old and should be used in these and possibly in older persons. Insufficient data exist to support a recommendation for the use of BCG at present.     

Quantitation of the phenolic glycolipid of Mycobacterium leprae and relevance to glycolipid antigenemia in leprosy

Chemical and immunologic procedures have been developed for quantitation, in the body fluids of patients with leprosy, of phenolic glycolipid I, the major specific antigen of the leprosy bacillus. Serum samples were extracted with CHCl3/CH3OH and fractionated on columns of silicic acid. Thin-layer chromatography with a sensitivity of about 500 ng allowed detection of the glycolipid in untreated lepromatous and borderline patients, and high-pressure liquid chromatography gave a quantitation of 0.8-3.7 micrograms/ml of serum from four patients. An ELISA-inhibition assay with polyclonal antibodies to glycolipid corroborated these figures. Dot-ELISA on nitrocellulose with polyclonal and monoclonal IgG antibodies allowed for much greater sensitivity (500 pg) and semiquantitative evaluation. Small quantities of glycolipid were present in the urine of patients with lepromatous leprosy. In sera obtained from patients undergoing chemotherapy, the amount of glycolipid declined sooner than did titer of antibody. This experimental approach is applicable to diagnosis of leprosy, bacillary quantification, and standardization of skin-test reagents and vaccines.     

IgM serum antibodies to phenolic glycolipid-I and clinical leprosy: two years' observation in a community with hyperendemic leprosy

A village population with hyperendemic leprosy in Papua New Guinea was repeatedly examined for clinical leprosy and for serum IgM antibodies to phenolic glycolipid-I (APGL-I) over 2 years between 1984 and 1986. In 1984, serum APGL-I was elevated in 15% of the subjects without clinical leprosy, and the prevalence of seropositivity was not significantly different in subjects from households with or without leprosy. In 1986, the prevalence of elevated serum APGL-I in leprosy-free subjects had risen to 23%. The incidence of seroconversion from APGL-I negative to APGL-I positive was 9.5% per year (95/1000 person years) in 253 subjects tested in 1984 and 1986. During the same period, 27 of 40 (67%) leprosy-free subjects reverted from positive to negative. The positive seroconversion rate in the community was higher than the incidence of clinical leprosy (11.2/1000 person years) over the same period. However, elevated serum APGL-I was not associated with clinical disease and failed to predict the development of disease over 2 years. The significance of persistent seropositivity found in 14 (5%) leprosy-free subjects is uncertain.     

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.