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.

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.

Use of a whole blood assay to monitor the immune response to mycobacterial antigens in leprosy patients: a predictor for type 1 reaction onset?

Longitudinal studies are more appropriate than cross-sectional studies for investigating changes in the immune response to Mycobacterium leprae during leprosy, such as occur in type 1 (reversal) reactions. A test for predicting the onset of reactions in leprosy would greatly reduce disability associated with leprosy. Whole blood assays are appropriate for longitudinal studies of the in vitro T-cell response, as they are robust and reproducible, and require only a small volume of blood. Whole blood assays were used to assess the natural variation in the 'normal' T-cell response to mycobacterial antigens in healthy UK donors, and healthy Nepali donors, tested over 6 months. This was compared with variation in T-cell responses measured over 6 months in 22 leprosy patients in Nepal, including eight who developed type 1 reactions during this time. The in vitro T-cell response to M. leprae sonicate, M. tuberculosis PPD, the mitogen PHA, and (in the UK study) recombinant mycobacterial antigens (70 kD and 30/31 kD proteins) was measured by lymphoproliferation and interferon-gamma (IFN gamma) responses, and variation in responses over time in each subject calculated as a coefficient of variation (CV). The baseline high, low or non-responder status of the healthy UK donors remained stable. The magnitude of IFN gamma responses varied by mean CV ranging from 26% (to PPD) to 63% (to Mtb 70 kD); proliferation responses showed less variation, ranging from mean CV of 18% (to PHA) to 47% (to Mtb 70 kD). Response variation was independent of lymphocyte number in culture. Similar variation in lymphoproliferation responses to MLS, PPD and PHA was observed in the group of healthy Nepali subjects, and in Nepali leprosy patients who did not experience reactions during the study. Of the eight leprosy patients who developed type 1 reactions, four (two BT, one BB, one BL) showed significantly increased proliferation to MLS at the time of reaction (74-300% above baseline); four (one BB, two BL, one LL) remained low or non-responders to MLS throughout. An alternative marker of immune response--anti-phenolic glycolipid-1 (PGL-1) antibody titre--was not predictive of reaction onset in these patients. This study demonstrated that whole blood assays provide reproducible in vitro measurements that can be used to monitor changes in T-cell responses to M. leprae antigens; their practical use as a diagnostic marker of type 1 reaction onset is discussed.

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.

Drug resistance in Nepali leprosy patients.

Although multidrug therapy (MDT) was introduced into Nepal in 1983, the MDT coverage only recently exceeded 67%. In view of the large number of patients who were still receiving dapsone monotherapy, it is relevant to investigate the current levels of dapsone and rifampin resistance. The study was undertaken at a leprosy referral hospital near Kathmandu. Over a 5 1/2-year period, 157 leprosy patients with a bacterial index (BI) > or = 2.0 were investigated for drug resistance according to the method of Rees. Among previously untreated cases, 6% of 88 isolates showed low-dose dapsone resistance; among previously treated patients with a presumed relapse, 47% of 34 isolates demonstrated dapsone resistance. In the remaining 35 cases there was no growth in control mice. Rifampin resistance was not confirmed in any case.

Serological monitoring of the response to chemotherapy in leprosy patients.

Sixty-five patients initially seropositive for IgM anti-phenolic glycolipid-I (PGL-I) antibodies were tested for antibody levels to PGL-I, lipoarabinomannan (LAM), and the 35-kDa protein of Mycobacterium leprae at regular intervals for up to 30 months following the commencement of multidrug therapy (MDT). There was a steady decline in IgM anti-PGL-I and anti-35-kDa antibody levels to a mean of 17% and 14%, respectively, of the starting level at 24 months. The development of type 1 and type 2 reactions or the presence of drug-resistant organisms in a small number of patients had no significant influence on the changes in antibody level. The rate of decline was similar in different disease categories, but a higher proportion of lepromatous patients remained seropositive at the end of 2 years of treatment than borderline tuberculoid patients. By contrast, the mean IgG anti-LAM antibody levels remained stable or increased. Again the occurrence of type 1 or type 2 reactions had no significant effect on antibody level over 2 years. Falls in the IgM anti-PGL-I antibody levels mirrored the falls in the bacterial index in individual patients and provide an additional parameter for monitoring the response to chemotherapy.

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.

Serological responses in primary neuritic leprosy.

The serological responses to 2 Mycobacterium leprae specific epitopes and one common mycobacterial antigen were examined in 46 untreated patients with primary neuritic (PN) leprosy. M. leprae specific antibodies to the terminal disaccharide of phenolic glycolipid and/or the ML-04 defined epitope on the 35 kDa protein were detected in 41% of PN patients and 47% responded to one of the 3 antigens. This serological response mirrored that observed in paucibacillary leprosy patients. There was a significant increase in the level of antibody response when more nerve trunks were involved. Changes in antibody levels in seropositive PN patients may prove useful in monitoring the response to chemotherapy.

Operational value of serological measurements in multibacillary leprosy patients: clinical and bacteriological correlates of antibody responses.

The antibody responses of 100 previously untreated multibacillary (MB) leprosy patients to one protein and two carbohydrate antigens were examined: 94% of the patients had Mycobacterium leprae-specific antibodies; 89% directed to the species-specific epitope on phenolic glycolipid (PGL-I), 89% against the specific epitope on the 35-kDa protein, and 94% against one or both of the two. By contrast, 67% of the patients had anti-lipoarabinomannan (LAM) antibodies. There were trends for the seropositivity rate and the antibody level to rise with the increasing extent of the disease and as patients moved to the polar lepromatous end of the spectrum. The bacillary load, as measured by the bacterial index, was moderately correlated with the IgM anti-PGL-I and the anti-35-kDa antibody levels and, to a lesser extent, with the IgG antibodies directed at the common mycobacterial carbohydrate LAM. The sensitivity of the IgM anti-PGL-I antibodies for detecting smear-positive MB disease was 91%; that for the anti-35-kDa antibodies was 92%.

Heterogeneity of serological responses in paucibacillary leprosy--differential responses to protein and carbohydrate antigens and correlation with clinical parameters.

We have examined the serological responses of 154 untreated paucibacillary (PB) leprosy patients to two carbohydrate and one protein antigens of Mycobacterium leprae. There was a heterogeneous response with 20% of PB patients having IgM anti-PGL-I antibodies and a similar proportion with IgG anti-LAM antibodies, while 33% had antibodies to the M. leprae-specific epitope on the 35-kDa protein. There was overlap in the responses such that 43% of the patients were seropositive in one of the two M. leprae-specific assays, while 49% were positive in any assay. There was a gradation in seropositivity with increasing extent of disease for each of the clinical parameters measured. Those with established disability at the time of presentation were more likely to be seropositive in each assay.