Immunotherapy with Mycobacterium vaccae and peripheral blood flow in long-treated leprosy patients, a randomised, placebo-controlled trial.

OBJECTIVE: to evaluate immunotherapy as a means of improving peripheral blood flow in chronic leprosy patients. DESIGN: this was a double-blind, randomised, placebo-controlled, clinical trial. MATERIALS: heat-killed Mycobacterium vaccae 1mg plus 0.02 microg Tuberculin protein per 0.1 ml dose in borate buffer, with saline as placebo. Those studied were 92 long-treated residents of a leprosy centre in Iran, 10 of their healthy children and 10 staff members. Evaluation employed the Perimed PF2, Laser-Doppler Flowmeter, a platinum skin thermistor, and a thermal sensibility tester. METHODS: single intradermal injections of test or placebo were given to 103 patients 18 months before the blinded evaluation. Fingerpulp blood flux was measured in controlled conditions and vasomotor reflexes and skin sensation to touch, pain and heat were evaluated in 45 and 47 patients in the placebo and M. vaccae groups, respectively, and in 20 healthy control persons. RESULTS: Laser-Doppler flux, skin temperature, vasomotor reflexes and sensation were impaired in leprosy patients. Immunotherapy improved (p < 0.05) Laser-Doppler flux, skin temperature and temperature sensation. CONCLUSIONS: immunotherapy, given 18 months earlier, significantly improved blood flow and temperature sensation, in fully-treated, chronic, leprosy patients. The same principles might be employed in other conditions of reduced peripheral blood flow.

Environmental mycobacteria in northern Malawi: implications for the epidemiology of tuberculosis and leprosy.

More than 36000 individuals living in rural Malawi were skin tested with antigens derived from 12 different species of environmental mycobacteria. Most were simultaneously tested with RT23 tuberculin, and all were followed up for both tuberculosis and leprosy incidence. Skin test results indicated widespread sensitivity to the environmental antigens, in particular to Mycobacterium scrofulaceum, M. intracellulare and one strain of M. fortuitum. Individuals with evidence of exposure to 'fast growers' (i.e. with induration to antigens from fast growers which exceeded their sensitivity to tuberculin), but not those exposed to 'slow growers', were at reduced risk of contracting both tuberculosis and leprosy, compared to individuals whose indurations to the environmental antigen were less than that to tuberculin. This evidence for cross protection from natural exposure to certain environmental mycobacteria may explain geographic distributions of mycobacterial disease and has important implications for the mechanisms and measurement of protection by mycobacterial vaccines.

Vaccination against leprosy at Ben San Leprosy Centre, Ho Chi Minh City, Vietnam.

Three vaccines, BCG alone, BCG + 10(7) killed Mycobacterium vaccae and 10(8) killed M. vaccae alone, were studied in children living in close contact with leprosy. In the year before vaccination, 14/446 (3.1%) children had developed leprosy. Among those who were not vaccinated, 9/74 (12.2%) developed the disease in the first 4 years of the study and 5/65 (7.7%) developed the disease in the second 4 years. In comparison with this, among those vaccinated, 20/343 (5.8%) developed leprosy in the first 4 years and 5/323 (1.5%) developed leprosy in the second 4 years. This represents 52.5% protection in the first 4 years and 80.5% in the second 4 years. There were no significant differences in protection afforded by each of the three vaccines but the success of the killed preparation of M. vaccae is an important finding.

Application of polymerase chain reaction for the detection of Mycobacterium leprae DNA in specimens from treated leprosy patients.

In this study of leprosy patients apparently cured by dapsone monotherapy, the polymerase chain reaction (PCR), one of the most reliable and sensitive DNA-based assays, was used for the specific detection of Mycobacterium leprae DNA. Sputum and slit-skin samples from 44 such patients at Baba Baghi Leprosy Sanatorium in Iran were examined. Primers for a 530-base-pair fragment of the gene encoding the 36-kDa antigen of M. leprae were used for the study. The PCR results were compared with microscopy for acid-fast bacilli. Of the 44 sputum samples, 2 were positive by PCR (4.5%) and of the 44 slit-skin swabs taken from the same patients, 10 were PCR positive (22.7%). Only one patient was PCR positive for both sputum and slit-skin specimens (2.3%). No positive results were found by acid-fast microscopy. In total, 11 of 44 (25%) patients in this study were found to be PCR positive for M. leprae, and it was thought probable that this indicated the presence of live organisms. Particularly interesting was the statistically significant association of positive results from slit-skin swabs with paucibacillary rather than multibacillary leprosy. It is suggested that whereas relapse or immunological reaction in paucibacillary disease may result from surviving organisms, in multibacillary leprosy this may be due to re-infection.

Circulation and sensation at the fingertips of claw hands.

Measurements of skin blood flow (by laser Doppler flowmetry) and temperature were made under environmental conditions promoting peripheral vasodilatation at the fingertips of a disfigured 'clawed' hand in 12 leprosy patients long-resident at Baba Baghi Leprosy Hospital, Tabriz, Iran. Sensory function was assessed by measuring the responses to light touch, pain and temperature of each finger, and peripheral autonomic function was gauged by estimating palmer sweating and by measuring skin vasomotor reflexes in response to inspiratory gasp. In 2 patients all measured fingers had laser Dopper flux (LDFlux) values and skin temperatures lower than the 95% confidence limits for the mean of 20 healthy controls, i.e. were impaired; in 2 patients all fingers had normal values for LDFlux and temperature; and in 8 patients there was a combination of impairment with most fingers normal for these parameters but with the small finger most commonly impaired. There were 10 (67%) fingers with impaired LDFlux and temperature values who had significant sensory impairment, whereas only 5 (18%) of the fingers with normal LDFlux values and temperatures had a similar sensory deficit. Overall, the fingers with the most impaired sensation had significantly (P < 0.05) lower LDFlux and temperature values than those with no sensory deficit. Microcirculatory impairment was not related to disordered skin vasometer reflexes or dysfunction of sweating. We concluded that the relationship between motor (skeletal muscle) nerve paralysis and any subsequent sensory neuropathy and/or microcirculatory impairment is more complex than might be expected from previous understanding of the disease.

Cellular immune response to common mycobacterial antigens in subjects seropositive for Trypanosoma cruzi.

The immune response is impaired in the silent stage of Chagas' disease. We used quadruple skin-testing with new tuberculins in 37 adults who were symptom-free but seropositive for Trypanosoma cruzi and in 37 matched seronegative controls. Whereas 19% of controls responded to common mycobacterial antigens, none of the Chagas' seropositive group responded to them (p < 0.006), demonstrating specificity in their unresponsiveness. The enhanced tuberculin reactivity after BCG vaccination in the control group was suppressed in seropositive subjects (p < 0.002). Selective loss of response to common mycobacterial antigens may have implications for the autoimmune pathology of Chagas' disease, and for susceptibility to tuberculosis, leprosy, and HIV disease.

Identification of the leprosy bacillus and related mycobacteria by analysis of mycocerosate profiles.

Members of the phthiocerol dimycocerosate family of waxes were extracted from Mycobacterium bovis BCG, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans and a skin biopsy from a leprosy patient. The waxes were degraded by alkaline hydrolysis and the mycocerosic acids converted to pentafluorobenzyl ester. Profiles of the esters, recorded using electron-capture gas-chromatography, gave characteristic profiles for the mycocerosates from M. leprae but those from M. bovis, M. tuberculosis and M. kansasii were superficially similar. The mycocerosate profiles from M. marinum and M. ulcerans were similar, but distinct from the others. Selected ion monitoring negative ion-chemical ionisation gas chromatography-mass spectrometry of of the pentafluorobenzyl esters allowed the analysis of mycocerosate isomers not revealed on gas chromatography alone. M. bovis and M. tuberculosis had similar profiles of C29, C30 and C32 mycocerosates; and additional C33 component was also present in M. kansasii. The mycocerosates from M. marinum and M. ulcerans were C27, C29 and C30 and those from M. leprae were distinct in having C29, C30, C32, C33 and C34 components. These methods have excellent potential for use in the detection of mycobacterial disease by direct analysis of infected tissue without prior cultivation of the causative agent.

Slow bacterial infections or autoimmunity?

In this article, Graham Rook and John Stanford propose that a group of idiopathic diseases that are often associated with a degree of autoimmunity and arthritis, including rheumatoid arthritis, inflammatory bowel disease, sarcoidosis and psoriasis, are caused by extremely slow-growing bacteria. They suggest that these diseases are one end of a continuous spectrum caused by related slow-growing genera, which ranges from rheumatoid arthritis, through Takayasu's arteritis and Whipple's disease, to reach the conventional mycobacterioses such as tuberculosis and leprosy.

Improving on BCG.

BCG is the only vaccine for tuberculosis and leprosy known to be effective in at least some places. Unfortunately it tends to be less successful in just those areas of the developing world where a vaccine is most needed. Although molecular biology offers the prospect of alternatives, these still lie in the indefinite future, and the best use has to be made of BCG. A number of preparations are available from different manufacturers, and a vaccine should be selected with good evidence of efficacy, and a low incidence of complications. Selection of the optimal age for administering BCG should be based on factors pertaining in the area where it is to be used. The influence of contact with environmental mycobacteria, the age at which mycobacterial diseases occur, and the logistics of vaccine delivery must be taken into account. The addition of a suspension of killed Mycobacterium vaccae to BCG may increase its efficacy. Skin test data show that recognition of antigens common to all mycobacterial species and thought to be the first step in the protective immune response, is significantly enhanced by the additive. M. vaccae also contains a substance, or substances, "switching off" the tissue destructive aspect of the Koch phenomenon that is part of the immunopathology of tuberculosis. A suspension of killed M. vaccae alone can be used to enhance immune responses of persons unsuitable for BCG vaccination, such as those already tuberculin positive, and those with scars of earlier BCG vaccination.

Mycobacterium vaccae in immunoprophylaxis and immunotherapy of leprosy and tuberculosis.

Both leprosy and tuberculosis present continuing problems in their control, especially in the developing world, despite the availability of drugs effective in producing a bacteriological cure. Improved immunoprophylaxis, and an effective immunotherapy to be used with chemotherapy are urgently required. Intradermal injection of a suspension of killed Mycobacterium vaccae promotes cell-mediated responses to antigens common to all mycobacteria, and switches off the tissue-necrotizing aspects of the Koch phenomenon. These properties led to the use of the suspensions as an improved vaccine, either alone or in combination with BCG. The same properties led to the employment of the suspension in immunotherapy as an adjunct to chemotherapy in the treatment of both leprosy and tuberculosis. The evidence leading to these conclusions is reviewed and discussed.

Immunotherapy with Mycobacterium vaccae as an adjunct to chemotherapy in the treatment of pulmonary tuberculosis.

47 patients with adult-type pulmonary tuberculosis attending the Chest Diseases Hospital in Kuwait were given a single injection of 10(9) irradiation-killed M. vaccae after 1 month of a 9-month course of chemotherapy. The patients were followed-up for 3 more months in double blind comparison with 65 patients given an injection of saline (placebo). The immunotherapeutic injection produced a small local lesion in 44/47 patients, 18 of which ulcerated and produced small scars. Immunotherapy made no measurable difference to the bacteriological, biochemical, haematological, or radiological parameters measured. However it was associated with significantly improved weight gain, reduced size of skin test response to Tuberculin, increased lymphocyte proliferation to common mycobacterial antigens, and increased antibody levels to mycobacterial antigens. The changes in skin test and LTT responses were related and occurred in 29% of patients whose recognition of common mycobacterial antigens returned to normal. The remaining patients did not differ in these respects from those receiving placebo. The proportion of patients whose responses were improved was very similar to that achieved using the same immunotherapeutic agent in a group of treated multibacillary leprosy patients.