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.

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.

Use of a whole blood assay to evaluate in vitro T cell responses to new leprosy skin test antigens in leprosy patients and healthy subjects.

Development of an immunological tool to detect infection with Mycobacterium leprae would greatly benefit leprosy control programmes, as demonstrated by the contribution of the tuberculin test to tuberculosis control. In a new approach to develop a 'tuberculin-like' reagent for use in leprosy, two new fractions of M. leprae depleted of cross-reactive and immunomodulatory lipids- MLSA-LAM (cytosol-derived) and MLCwA (cell wall-derived)-have been produced in a form suitable for use as skin test reagents. T cell responses (interferon-gamma (IFN-gamma) and lymphoproliferation) to these two new fractions were evaluated in a leprosy-endemic area of Nepal using a simple in vitro whole blood test. The two fractions were shown to be highly potent T cell antigens in subjects exposed to M. leprae-paucibacillary leprosy patients and household contacts. Responses to the fractions decreased towards the lepromatous pole of leprosy. Endemic control subjects also showed high responses to the fractions, indicating high exposure to M. leprae, or cross-reactive mycobacterial antigens, in this Nepali population. The new fractions, depleted of lipids and lipoarabinomannan (LAM) gave enhanced responses compared with a standard M. leprae sonicate. The cell wall fraction appeared a more potent antigen than the cytosol fraction, which may be due to the predominance of the 65-kD GroEL antigen in the cell wall. The whole blood assay proved a robust field tool and a useful way of evaluating such reagents prior to clinical trials.

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.

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.

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.

Development of a whole blood assay to measure T cell responses to leprosy: a new tool for immuno-epidemiological field studies of leprosy immunity.

A whole blood assay is described to measure T cell mediated immune responses to leprosy and provide an alternative to the conventional lymphocyte transformation test. Optimal conditions were defined for the whole blood assay, and interferon-gamma measurement was found to be a more sensitive way of measuring responses than tritiated thymidine incorporation. The assay was shown to be useful for investigating responses to a range of leprosy antigens. A whole blood assay has the advantages of being quick, simple and requiring only a small volume of blood, making it more appropriate as an immuno-epidemiological field test in leprosy endemic areas.

Cold fingers in leprosy.

Under conditions of maximal thermoregulatory peripheral dilatation, most healthy subjects (both Indian and European) showed raised blood flow in the fingertips (measured by laser Doppler flowmetry) where the skin temperature is only slightly lower than the core body temperature. Most borderline lepromatous (BL) leprosy patients had much colder fingers and the blood flow was slow: borderline tuberculoid (BT) patients had skin temperatures similar to those seen in healthy subjects, but their fingertip blood flow was reduced relative to that in control subjects. The occurrence of cold fingers and slow blood flow was clearly associated with evidence of sensory impairment to light touch, pressure and temperature. Slower fingertip blood flow was strongly associated with impairment of vasomotor control in this anatomical region, suggesting that both may be a consequence of leprosy peripheral neuropathy, at least in patients with early leprosy, but it is likely that leprosy arteriopathy may contribute to the lowered peripheral perfusion in advanced cases. It is suggested that the simple clinical sign of cold fingers may be of value in the preliminary assessment of patients presenting at any leprosy control clinic in the tropics.

Impairment of fingertip vasomotor reflexes in leprosy patients and apparently healthy contacts.

Fingertip blood-flow velocity and its control by vasomotor reflexes were studied in leprosy patients and in healthy controls with a laser Doppler flowmeter. In newly registered patients, the flow was significantly lower than in the healthy controls, and even lower values were recorded in the long-standing patients with lower limb ulcers and/or deformity. The newly registered patients showed substantially impaired vasomotor reflex responses in the fingertips to cold challenge of the opposite hand or deep inspiratory gasp. Low blood flow and impairment of vasomotor reflexes were more prominent in those leprosy patients who showed clinical evidence of neuropathy and/or histological evidence of reaction in a punch biopsy of leprosy skin lesions. This aspect of dysautonomia to cold challenge was particularly prominent in apparently healthy, fully treated ex-patients. There was an unexpectedly high prevalence of impairment of vasomotor reflexes in newly registered and apparently healthy, adequately treated leprosy patients. The method is very sensitive, and it remains to be established whether the lesions it detects are nonprogressive residues, or previous nerve damage, or an indication of on-going nerve damage. A minority of leprosy contacts showed impairment of vasomotor reflexes. Those with two or more affected fingers were more likely to have had a higher level of exposure to Mycobacterium leprae than those with one or no affected fingers. The cause of this unexpected impairment of fingertip vasomotor reflexes in a minority of leprosy control workers has not yet been determined.