PCR-restriction fragment length polymorphism analysis as a tool for Mycobacterium species identification in lepromas for lepromin production.

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.

Estudio seroepidemiológico de lepra. Provincia Camagüey. Año 2001-2002

Se realizó un estudio de intervención para determinar el comportamiento clínico epidemiológico de la infección por el Mycobacteriumn leprae durante los años 2001-2002 en la provincia de Camagüey. Para esto se realizaron 13.434 pruebas serológicas en población de riesgo de los municipios con una prevalencia de la enfermedad superior a 1 x 10.000, aplicándose una encuesta a los casos con serología positiva. Las variables analizadas fueron: edad, sexo, raza, escolaridad, ocupación, lugar de procedencia, causa del pesquisaje, examen dermato-neurológico, resultado de la baciloscopia, lepromina, tratamiento recibido y seguimiento serológico por un año. Se detectó baja positividad serológica en población de riesgo, predominando las edades entre 25-34 años, el sexo femenino, la raza blanca, nivel de escolaridad medio, los vinculados laboralmente y la fuente de infección ignorada. LOs municipios Con mayor riesgo de transmisión fueron Sierra de Cubitas, Camagüey, y Santa Cruz Sur. NO presentaron lesiones Cutáneas la mayoría de los estudiados, así como leprominas positivas y baciloscopias negativas. Se detectaron personas enfermas y el 50 por ciento de los tratados negativizaron la prueba serológica después de la intervención (AU)

Hanseníase: aspectos clínicos, imunológicos e terapêuticos / Leprosy: clinical, immunological and therapeutical aspects

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

Diagnostic value of gene probes and its correlation with clinical profile of leprosy in children.

Clinico-bacteriological profile of 73 leprosy patients below 16 years of age was studied. Majority of the patients were males and fell in 11-16 years age group (p < 0.05). Skin lesions were present in all cases on both exposed as well as unexposed areas and their number increased with advancing age. Cutaneous sensations were affected in most of the patients while nerve thickening was observed in 41. As age increased, the disease moved from the tuberculoid end of spectrum towards the lepromatous end (p < 0.05) and the positivity of the skin smears increased (p < 0.05). Majority of the paucibacillary cases were lepromin positive while most multibacillary cases were lepromin negative (p < 0.01). Two M. leprae specific gene probes were applied in 42 cases to assess their diagnostic value. Eighty one per cent cases were picked up by the probes indicating presence of active bacilli. These included all lepromin positive cases, all smear positive cases, and most of smear negative cases (p < 0.05). Seven children with inconclusive histology were also positive. Drug treatment and inadequate size of biopsy sample could explain the negative probe results in 19% cases. This study highlights the immense potential of gene probes in diagnosing leprosy in children.

Sobre algunas características antigenicas de micobacterias aisladas en cultivo a partir de material lepromatoso: viraje de Mitsuda en pacientes L.L. y contactos

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.

Valor do método de Faraco para coloração do bacilo de Hansen em cortes / Value Faraco method of staining the bacillus of Hansen in cuts

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).

Prognostic value of the lepromin test

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

The lepromin test in tuberculous persons in a non-endemic area

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.

Sensitization to lepromin in presumably non-leprous individuals

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.

Studies of the lepromin test, A bacillaryu antigen standardised by weight

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

Studies of the lepromin test. Variations in the results of the Mitsuda Test observed in cases of leprosy of the neuro-macular type

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