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OBJECTIVES: The aim of the present work was to standardise a PCR-Restriction Fragment Length Polymorphism analysis (PRA) as a tool to detect the mycobacteriologic composition of lepromas from leprosy patients used in the production of lepromin to improve the quality of the Mitsuda test. DESIGN: PCR-Restriction Fragment Length Polymorphism analysis using hsp65 and rpoB genes were applied to 11 reference strains of mycobacteria, including M. leprae, and the obtained PRA profiles were compared to mycobacteria in clinical specimens. RESULTS: Out of the biopsies studied, 522% had DNA fragment amplified for both genes (hsp65 and rpoB) for M. leprae. However, other Mycobacterium species were observed in samples of lepromatous leprosy patients. Here we discussed the importance of mycobacteria identification in the antigen of Mitsuda production to be used in the evaluation of leprosy. CONCLUSIONS: Our results suggest that the use of the molecular approach for sample selection can contribute to an improvement in the quality of produced lepromin.

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Considerando a complexidade das manifestaçöes clínicas da hanseníase e sua correlaçäo com as alteraçöes imunológicas encontradas dentro do espectro clínico da doença, e, ainda, a importância do tratamento específico no controle do quadro clínico, imunológico e epidemiológico, säo relatados os aspectos de maior relevância, incluindo resultados de investigaçäo imunológica para a melhor compreensäo dos mecanismos fisiopatológicos envolvidos na evoluçäo da hanseníase. Nas consideraçöes sobre terapêutica, säo apresentados esquemas práticos para controle dos surtos reacionais e a eficácia da poliquimioterapia no controle e na baixa freqência de recidivas da doença

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Basándose en el concepto de que "vacunar contra la lepra no es inocular B.C.G., sino comproblar que Mitsuda ha virado", los autores exponen sus experiencias con los 4 siguientes grupos de sujetos. Grupo I: 28 pacientes lepromatosos (LL), Mitsuda negativos. Grupo II: 10 pacientes tuberculoides (LT), Mitsuda positivos. Grupo III: 7 contactos (niños de 7 a 14 años de edad), Mitsuda negativos. Grupo IV: 26 contactos (niños de 6 a 15 años de edad), Mitsuda negativos. A los integrantes de los primeros 3 grupos mencionados se les aplicó el Antígeno Zulia, constituido por una suspensión bacilar de micobacterias aisladas en cultivo a partir de pacientes lepromatosos (0'1 ml. por vía intradérmica), y simultáneamente, o de 1 a 3 meses después, se les sometió a la prueba clásica de Mitsuda. A los contactos reunidos en el Grupo IV y ensayados con fines comparativos, se les aplicó B.C.G., en substitución del Antígeno Zulia. Estos ensayos demostraron que en 24 de los 28 pacientes L.L. (Grupo I), y en 5 de los 7 contactos (Grupo III, todos inicialmente Mitsuda negativos, se presentó un inminente viraje de los valores inmunológicos, fenómeno inducido por la aplicación del Antígeno Zulia y manifesto por la aparición de reacciones positivas a la lepromina clásica. Por otra parte se hizo notorio que de los 26 contactos vacunados con la única dosis de B.C.G., solamente 14 habían virado su Mitsuda a positivo. Los autores suponen que el "virage" mencionado es una expresión de mecanismos defensivos en individuos en los cuales éstos estaban bloqueados (probablemente por desaparisión de la capacidad linfocitaria de producir el factor de migración de macrófagos) y señalan que el Antígeno Zulia se destaca ampliamente por su capacidad de convertir a los sujetos Mitsuda negativos en sujetos Mitsuda positivos.

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O autor fez um estudo comparativo entre o método clássico de Ziehl-Neelsen e o de Faraco, para a coloração do bacilo de Hansen em cortes de parafina. Empregou o seguinte material: 36 casos de lepra pobres em bacilo; 8 lesões lepromatosas ricas em bacilo e lesões de grande número de reaões de Mitsuda. Nos 36 primeiros pacientes, a pesquisa de bacilo foi positiva em 10 no método de Ziehl-Neelsen e em 14 no de Faraco. A tabela 1 apresenta os resultados nos casos lepromatosos e em algumas lesões das reações de Mitsuda, mostrando a positividade muito maior com o processo, admitindo, porém, que seja mais efetivo nas lesões lepromatosas idosas. Nestas existem muitos bacilos não  ácido-resistentes (11), cuja ácido-resistência ‚ restabelecida pelo engorduramento (2, 10). Acha que a ação da substância gordurosa, ou oleosa, no processo de Faraco, seja por mecanismo físico ou f¡sico-qu¡mico. O autor observou, empregando o método clássico de Ziehl-Neelsen, a perda da  ácido-resistência de vários bacilos em material conservado em bloco de parafina há 5 anos (Tab. 2); e inalteração desta propriedade nos cortes do mesmo material, não corados, pegados na lâmina, feitos na ocasião da inclusão. Com o processo de Faraco, porém, não notou alteração da  ácido-resistência, apresentando igual número de bacilos corados tanto os cortes antigos, como os recentes do material conservado em bloco. (Ambos os cortes foram tomados de pontes próximos do bloco).

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1. A study been made of the progress of the disease in relation to the results of the lepromin test, in 160 cases of leprosy (109 neural, 46 lepromatous, and 5 of doubtful classification) first tested six years ago. 2. the prognostic value of the test is best illustrated in cases of the neural type. Of the 109 cases of the neural type lepromin test was positive group, improvement was seen in about 84 per cent of the cases. In the disease becoming worse in only 14 per centin the lepromin-negative and doubtful group improvement was seenonly in 53 per cent of the cases the disease becoming worse in 47 per cent. In only one case has the disease changed from the neural to the lepromatous type, and this occurred in a lepromin-negative case. The degree of positivity of the test also influence the prognossis of the 92 lepromin-positive neural cases 20 had strongly positive 49 moderately positive and 23 weak positive reactions

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1- Of ten patients, with various dermatoses, attending the outpatient clinic at New York Skin and Cancer Hospital, all reacted positively to tuberculin. Of this group 9 showed early and 8 late positive reactions to lepromin. 2- Of four patients with Boeck´s sarcoid, three reacted negatively to tuberculin and to lepromin. The fourth was weakly positive to tuberculin, negative to lepromin on the early reading but did develop a positive Mitsuda reaction. 3- Of 108 tuberculous patients at Seaview Hospital, 70.4 per cent were positive to lepromin on the early reading and 46.2 on the late reading. This high proportion of Fernández positives may be due to the fact that all individuals were tuberculin positives. If so, comparative figures for Mitsuda positives would indicate that the Mitsuda reaction is less affected by consensitization with m. tuberculosis than is the Fernández reaction.

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The experiments which have been reported demonstrate that: 1- It is possible to sensitize presumably non-leprous persons to leprominby intradermal injection of either an oily or an aqueous suspension of Mycobacterium leprae killed by hat. Furthermore, this sensitization ensues in a high percentage of subjects. 2- Sensitization to lepromin can be produced also by intradermal injection of suspensions of Mycobacterium tuberculosis killed by heat. This is in agreement with the opinion which we have expressed (10,11) in discussing positive lepromin reactions in patients with cutaneous tuberculosis and in persons who had been vaccinated with B.C.G. 3- The early lepromin reaction is attributable to previous intradermal injection of purified lepromin protein (L.P.P.), nor by injection of a suspension of E. typhosus killed by heat. 5- As regards the duration of sensitivity to lepromin, a final answer cannot be given. We have observed positive early reactions to the antigen L.P.P. in presumably non-leprous individuals who have received endermal injections of integral lepromin five years previously. 6- As to the practical value of allergic response as an element of protection against later infection, we believe that it is more desirable for an individual exposed to leprosy to have a positive lepromin reaction. Unfortunately it is not possible to provoke this supposedly protective allergy, since apparently it is dependent on an unknown factor which may be constitutional. When that factor is lacking, nothing can be gained by the intradermal injection of the antigen. However, if the factor is present in an individual as evidency by his capacity to reactin an allergic manner to the antigen, we believe that he will always have a high degree of immunity to leprosy, regardless of his previous state os sensitization.

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I. A method of preparing standard lepromin dried and partly defatted leprosy bacilli is described. The bacilli are obtained by extracting the nodules with chloroform, evaporating the chloroform extract, suspending the resudue in ether and centrifugalising the ethereal suspension in cold. The standardisation is done by weight of the bacterial powder, I mg. of the powder being suspended in 10 c.c. of carbol-saline, and 0.1 c.c. of suspension being used for the test 2. This preparation, like the ordinary lepromin prepared directly form the leprous tissue, produces both early and late reactions in the cases of the neural type of leprosy and no reactions, early or late in case of the lepromatous Type. However, with this preparation the early reactions are stronger, and the late reactions considerably weaker than corresponding, reactions pruduced by ordinary lepromin. This is considered to be an advantage

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A study has been made of the variations in the results of the lepromin test in cases the neural type, demonstrated by repeated testing of the same cases in different circumstances There apper to be at least five possible factores which, acting singly or in combination, may influence the result of the test. These different factors are: variations in clinical activity, variations in bacteriological findings, variations in the time year at which the test is done, deterioration of the lepromin on keeping, and the use of different lots of lepromin Our stdy has shown that the two most important factors are the variations in clinical activity and tima of the yaer at which the test is done

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I. A method for abtaining from excised nodules leprosy bacilli free from tissue is described 2. The bacilli were ground for several hours in an agate mortar and were fractionated into a saline-soluble portion and an insoluble residue. From the soluble portion a protein has been obtained by precipitation with trichlor-acetic acid; from the insoluble residue various lipoid fractions been separated 3. The antigenic actions of the following preparations have been tested by intradermal tests in cases of leprosy. (a) Nodular tissue freed from bacilli, (b) lipoids of the nodular tissue, (c) whole bacilli, (d) groud bacilli, (e) saline extract of ground bacilli, (f) faltrate from the saline extract, (g) protein precipitated from the saline extract, (h) residue of bacilli after saline extraction, (i) the different lipoid fractions separated from the residue, and (j) final residue after the extraction of lipoids

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The purpose of the work here reported was to determine whether or not the Mitsuda reaction can be produced by leproma emulsions in which the bacillary elements have been destroyed, at least to the point of loss of acid-fastness, by application of ultra-supersonic waves. Vaccines so prepared, it has been found, retain antigenic capacity, though it is much weakened. They are similar to the standard Mitsuda antigen in that they fail to cause positive reactions in lepromatous cases, while neural-type cases and healthy persons react positively. The quality of the U-vaccine is not affected by heating. In view of the fact that all of the components of the bacilli remain in the U-vaccine, the weakening of the reaction indicates that it depends upon the presence of the formed leprosy bacilli themselves. This is further indicated by the fact that a filtrate of the U-vaccine is incapable of producing positive reactions, though it must contain all of the soluble bacterial substances. On the other hand, it is also a fact that the small numbers of bacilli present in the U-vaccine can, in some healthy persons and maculo-neural cases, cause reactions approximately as strong as those induced by the Mitsuda antigen itself. The fact that few bacilli can cause strong reactions has already been proved with diluted Mitsuda vaccine in cases of "akuter Schub". The same results have been obtained with the U-vaccine containing small numbers of bacilli that are losing their acid-fastness. Why can the U-vaccine cause the Mitsuda reaction, despite the fact that it may contain nothing which can be stained by methylene blue or by the Ziehl-Neelsen method? It is a fact that this preparation is turbid, though of course much less so than the original Mitsuda vaccine. Yhis turbidity is due to a mass of fine bacillary particles that are not acid-fast. These particles still retain the capability of causing the Mitsuda reaction, though of much weakened grade. This agrees with Hayashi's findings with leprosy bacilli treated with hydrochloric acid. The fact that, contrary to our expectations, the filtrate of this vaccine does not cause the same reaction as the unfiltered substance is further evidence of the essential nature of the Mitsuda reaction. Comparing this filtrate and one of the Mitsuda vaccine, the former, in the first several days, caused stronger reactions than the latter in healthy and neural persons,...

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