Serologic follow-up of IgG responses against recombinant mycobacterial proteins ML0405, ML2331 and LID-1 in a leprosy hyperendemic area in Venezuela

Leprosy is a slowly evolving disease that occurs mainly in adults. In this study, the Mamaría Village, state of Portuguesa was selected because it had one of the highest prevalence rates (13.25%) of leprosy cases in 1997. Between 1998-2004, 20.2% of the 89 cases registered in this village were less than 15 years old and 61.8% were males. Pau-cibacillary (PB) lesions were the predominant clinical forms identified, although also multibacillary (MB) forms were found. Additionally, 76% of the patients were bacteriologically negative. At the time of diagnosis, 75% of the patients presented with grade 0 disabilities, 23% with grade 1 and 2% with grade 2. Serum samples were collected from 18 PB and 15 MB patients, in addition to 14 family contacts, at the beginning and end of treatment. All the groups were re-evaluated during a three-year period (2008-2011). The proteins used for evaluation were ML0405, ML2331 and LID-1. These mycobacterial proteins were highly specific for Mycobacterium leprae and the IgG responses decreased in both MB and PB patients during multidrug treatment. Our results suggest that these antigens could be used as markers for successful treatment of non-reactional lepromatous patients.

Cojedes: a leprosy hyperendemic state.

BACKGROUND: Leprosy is a chronic infectious disease produced by Mycobacterium leprae. In 1997 Venezuela reached the goal of elimination of leprosy as a public health problem (according to the World Health Organization a prevalence rate of ≤ 1/10,000 inhabitants), but five states still had prevalence rates over that goal. For this study we selected Cojedes State, where prevalence rates remain over the elimination goal. OBJECTIVE: Evaluate the real leprosy situation in high-prevalence areas of Cojedes State. MATERIALS AND METHODS: Seven communities of Cojedes State were selected because they had the highest historic prevalence, as well as the highest prevalence in the year to be studied (1997). RESULTS: A rank correlation using Spearman's test comparing historical prevalence rates (1946-1996) and detection rates (1998-2004) gave a statistically significant P < 0.05 value. Diagnosed leprosy cases were as follows: age: 3.2% under 15 years old; sex: male/female rates between 60% and 91.66% males. The highest number of cases were paucibacillary forms: indeterminate leprosy (33.07%) and borderline tuberculoid leprosy (32.28%); tuberculoid leprosy (7.00%); and multibacillary cases (lepromatous leprosy, LL) were only 2.36%. Bacteriologically, 18.52 patients were M. leprae positive. At the moment of diagnosis, 96.6% showed no disabilities, 3.4% showed grade I disabilities, and there were no grade II or III disabilities. CONCLUSION: This study confirms that several communities in Cojedes State have extremely high leprosy rates.

Serologic follow-up of IgG responses against recombinant mycobacterial proteins ML0405, ML2331 and LID-1 in a leprosy hyperendemic area in Venezuela.

Leprosy is a slowly evolving disease that occurs mainly in adults. In this study, the Mamaría Village, state of Portuguesa was selected because it had one of the highest prevalence rates (13.25%) of leprosy cases in 1997. Between 1998-2004, 20.2% of the 89 cases registered in this village were less than 15 years old and 61.8% were males. Paucibacillary (PB) lesions were the predominant clinical forms identified, although also multibacillary (MB) forms were found. Additionally, 76% of the patients were bacteriologically negative. At the time of diagnosis, 75% of the patients presented with grade 0 disabilities, 23% with grade 1 and 2% with grade 2. Serum samples were collected from 18 PB and 15 MB patients, in addition to 14 family contacts, at the beginning and end of treatment. All the groups were re-evaluated during a three-year period (2008-2011). The proteins used for evaluation were ML0405, ML2331 and LID-1. These mycobacterial proteins were highly specific for Mycobacterium leprae and the IgG responses decreased in both MB and PB patients during multidrug treatment. Our results suggest that these antigens could be used as markers for successful treatment of non-reactional lepromatous patients.

Reactividad serológica y celular frente a proteínas micobacterianas en la enfermedad de Hansen / No disponible

Se diseñó un estudio para evaluar la reactividad inmunológica frente a diferentes preparaciones proteicas micobacterianas utilizando pruebas serológicas y de inmunidad celular. Para el estudio fueron incluidos pacientes con manifestaciones clínicas de lepra predominantemente de la forma multibacilar. El 58% correspondía a la forma clínica de Lepra Lepromatosa (LL) n=81, el 29% a la forma Borderline Lepromatosa (BL) n=41 y 10% a Borderline Bordeline (BB) n=14. Sólo el 3% fueron pacientes Borderline Tuberculoide (BT): 74% masculino y 26% femenino. El fenómeno reaccional más frecuente fue de tipo eritema nodoso leproso (ENL). Las proteínas micobacterianas ensayadas fueron: antígenos proteicos crudos totales de Mycobacterium leprae (MISA), Mycobacterium bovis (MbSA y MbSA de excreción), antígeno proteico de excreción parcialmente purificado con una movilidad relativa de 30 kDa (MI30) y proteínas recombinantes de Mycobacterium (Mt70, Mb 65, MI 36, 28, 18 y 10 KDa) encontrándose que las proteínas recombinantes (MI 10KDa, MI36 kDa) a mayor carga bacilar presentaban una mayor reactividad serológica estadísticamente significativa (p=0.0051 y 0.050 respectivamente). La proteína de 30 kDa) a mayor carga bacilar presentaban una mayor reactividad serológica estadísticamente significativa (p=0.0051 y 0.0050 respectivamente). La proteína de 30 kDa fue predominantemente reconocida por anticuerpos de los pacientes multibacilares. Los resultados demuestran que el promedio de los valores de anticuerpos en pacientes no reaccionales fueron superiores en presencia de proteínas completas (MbSA y MbSA de exreción) en comparación con el grupo de pacientes que presentaron fenómenos reaccionales (p=0.000567 y 0.000061 respectivamente). Este mismo comportamiento se observó frente a las proteínas micobacterianas individuales (30kDA, 10kDa y 36 kDa). La respuesta proliferativa de los linfocitos T en los pacientes multibacilares reaccionales y no reaccionales frente a las proteínas micobacterianas (MISA, ML 10kDa, MbSA, MbSA de excreción) fue negativa en ambos grupos (AU)

The study was designed for evaluating immunological reactivity to various mycobacterial protein preparations using serological and cell-mediated immunological test in patients with clinical leprosy signs, predominantly, with the multibacillary forms. Al patients wer adults with ages between 20 and 39 years. Fifty eight (n=81) percent corresponded to Lepromatous Leprsy (LL), 29% (n=41) to Borderline Lepromatuous Leprosy (BL) and 10% (n=14) to Borderline Borderline Leprosy (BB); only 3% were Bordeline Tuberculoid (BT) patients: 74% males and 26% females. The most frequent reactional phenomenon was of the Erythema Nodosum (ENL) type. The mycobacterial proteins tested were: total crude Mycobacterium leprae antigens (MISA); Mycobacterium bovis (MbSA and excretion MbSA); partially purified excretion protein antigen, with a 30KDa relative movility (Ml30); and recombinant M. leprae protein (Mt70, Mb 65, Ml36, 28, 18 and 10kDa). Two of the recombinant proteints (M110 and Ml36 kDA) presented a statiseally significant higher serological reactivity, directly related with a larger bacillary load (p=0.0051 and 0.050 respectively). The 30kDa protein was predominantly recognized by antibodies from multibacillary patients. Results show that mean antibody values were higher in non reactional patients when tested against complete proteins (MbSA and ex MbSA) when compared with the group of patients who presented reactional phenomena (p=0.000567 and 0.000061, respectively). Comparing reactional with non reactional patients, it was seen that mean antibody values against complete proteins (MbSA and ex MbSA) were higher in non reactional individual (p=0.000567 and 0.000061, respectively). This same behavior occurred towards individual mycobacyterial proteins (30, 10 and 36 kDa). The T lymphocyte prolyphetative response in reactional and non reactional patients towards mycobacterial proteins (MISA, Ml 10 kDA, MbSA, ex MbSA) was negative (AU)

Molecular drug susceptibility testing and genotyping of Mycobacterium leprae strains from South America.

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

Molecular drug susceptibility testing and genotyping of Mycobacterium leprae strains from South America

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

Reactividad serológica y celular frente a proteínas micobacterianas en la enfermedad de Hansen / Serological and cellular reactivity to mycobacterial proteins in Hansen´s disease

Se diseñó un estudio para evaluar la reactividad inmunológica frente a diferentes preparaciones proteicas micobacterianas utilizando pruebas serológicas y de inmunidad celular. Para el estudio fueron incluídos pacientes con manifestaciones clínicas de lepra predominantemente de la forma multibacilar. Todos los pacientes fueron adultos con edad comprendida entre 20 y 39 años. El 58 por ciento correspondía a la forma clínica de Lepra Lepromatosa (LL) n= 81, el 29 por ciento a la forma Borderline Lepromatosa (BL) n=41 y 10 por ciento a Borderline Borderline (BB) n=14. Solo el 3 por ciento fueron pacientes Borderline Tuberculoide (BT): 74 por ciento masculino y 26 por ciento femenino. El fenómeno reaccional más frecuente fue del tipo eritema nodoso leproso (ENL). Las proteínas micobacterianas ensayadas fueron: antígenos proteicos crudos totales de Mycobacterium leprae (MlSA), Mycobacterium bovis (MbSA y MbSA de excreción), antígeno proteico de excreción parcialmente purificado con una movilidad relativa de 30 kDa (Ml 30) y proteínas recombinantes de Mycobacterium (Mt70, Mb 65, Ml 36, 28, 18 y 10 kDa) encontrandose que las proteínas recombinantes (Ml10 kDa, Ml 36 kDa) a mayor carga bacilar presentaban una mayor reactividad serológica estadísticamente significativa (p= 0,0051 y 0,050 respectivamente). La proteína de 30 kDa fue predominantemente reconocida por anticuerpos de los pacientes multibacilares. Los resultados demuestran que el promedio de los valores de anticuerpos en pacientes no reaccionales fueron superiores en presencia de proteínas completas (MbSA y MbSA de exc) en comparación con el grupo de pacientes que presentaron fenómenos reaccionales (p=0,000567 y 0,000061 respectivamente) Este mismo comportamiento se observó frente a las proteínas micobacterianas individuales (30 kDa, 10 kDa y 36 kDa). La respuesta proliferativa de los linfocitos T en los pacientes multibacilares reaccionales y no reaccionales frente a las proteínas micobacterianas...

The study was designed for evaluating immunological reactivity to various mycobacterial protein preparations using serological and cell-mediated immunological tests in patients with clinical leprosy signs, predominantly, with the multibacillary forms. All patients were adults with ages between 20 and 30 years. Fifty eight (n= 81) percent corresponded to Lepromatous Leprosy (LL), 29 percent (n= 41) to Borderline Lepromatous Leprosy (BL) and 10 percent (n=41) to Borderline Borderline Leprosy (BB); only 3 percent were Borderline Tuberculoid (BT) patients: 74 percent males and 26 percent females. The most frequent reactional phenomenon was of the Erythema Nodosum (ENL) type. The mycobacterial proteins tested were: total crude Mycobacterium leprae antigens (MISA); Mycobacterium bovis (MbSA and excretion MbSA); partially purified excretion protein antigen, with a 30kDa relative movility (Ml30); and recombinant M. leprae proteins (Mt70, Mb 65, Ml 36, 28, 18 and 10 kDa). Two of the recombinant proteins (Ml10 and Ml 36 kDa) presented a statiscally significant higher serological reactivity, directly related with a larger bacillary load (p= 0.0051 and 0.050 respectively). The 30 kDa protein was predominantly recognized by antibodies from multibacillary patients. Results show that mean antibody values were higher in non reactional patients when tested against complete proteins (MbSA and ex MbSA) when compared with the group of patients who presented reactional phenomena (p= 0.000567 and 0.000061, respectively). Comparing reactional with non reactional patients, it was seen that mean antibody values against complete proteins (MbSA and ex MbSA) were higher in non reactional individuals (p= 0.000567 and 0.000061, respectively). This same behavior occurred towards individual mycobacterial proteins (30, 10 and 36 kDa). The T lymphocyte prolypherative response in reactional and non reactional patients towards mycobacterial proteins (MlSA, Ml 10 kDa, MbSA...

[Serological and cellular reactivity to mycobacterial proteins in Hansen's disease]. / Reactividad serológica y celular frente a proteínas micobacterianas en la enfermedad de Hansen.

The study was designed for evaluating immunological reactivity to various mycobacterial protein preparations using serological and cell-mediated immunological tests in patients with clinical leprosy signs, predominantly, with the multibacillary forms. All patients were adults with ages between 20 and 30 years. Fifty eight (n = 81) percent corresponded to Lepromatous Leprosy (LL), 29% (n = 41) to Borderline Lepromatous Leprosy (BL) and 10% (n = 41) to Borderline Borderline Leprosy (BB); only 3% were Borderline Tuberculoid (BT) patients: 74% males and 26% females. The most frequent reactional phenomenon was of the Erythema Nodosum (ENL) type. The mycobacterial proteins tested were: total crude Mycobacterium leprae antigens (MISA); Mycobacterium bovis (MbSA and excretion MbSA); partially purified excretion protein antigen, with a 30 kDa relative movility (Ml30); and recombinant M. leprae proteins (Mt70, Mb 65, Ml 36, 28, 18 and 10 kDa). Two of the recombinant proteins (Ml10 and Ml 36 kDa) presented a statiscally significant higher serological reactivity, directly related with a larger bacillary load (p = 0.0051 and 0.050 respectively). The 30 kDa protein was predominantly recognized by antibodies from multibacillary patients. Results show that mean antibody values were higher in non reactional patients when tested against complete proteins (MbSA and ex MbSA) when compared with the group of patients who presented reactional phenomena (p = 0.000567 and 0.000061, respectively). Comparing reactional with non reactional patients, it was seen that mean antibody values against complete proteins (MbSA and ex MbSA) were higher in non reactional individuals (p = 0.000567 and 0.000061, respectively). This same behavior occurred towards individual mycobacterial proteins (30, 10 and 36 kDa). The T lymphocyte prolypherative response in reactional and non reactional patients towards mycobacterial proteins (MlSA, Ml 10 kDa, MbSA, ex MbSA) was negative.

Respuesta inmunitaria de la enfermedad de Hansen: revisión / Immune response of Hansen´s disease: review

La enfermedad de Hansen presenta un amplio espectro de manifestaciones clínicas e histopatológicas, las cuales son un reflejo de la naturaleza de la respuesta inmunológica del individuo ante diversos componentes del Mycobacterium leprae. El sistema inmunológico, integrado por la existencia de una inmunidad innata y adaptativa, ofrece protección frente a infecciones de diversas etiologías, entre ellas las bacterianas. Por supuesto las bacterias han logrado desarrollar múltiples estrategias para evadir las defensas del hospedador, que se basan en mecanismos algunos muy complejos y otros más simples, pero con una sola finalidad de “resistir” el ataque del hospedador y lograr sobrevivir. Estudios realizados en la enfermedad de Hansen han podido determinar los diferentes componentes que puedan estar actuando tanto en la inmunidad innata cómo en la adquirida. En este trabajo se trata de resumir algunos estudios recientes en la enfermedad de Hansen con mayor énfasis en el área de inmunología, sin dejar de considerar que toda enfermedad debe estar muy ligada a otros aspectos tan igual de importantes cómo son los sociales, ambientales y otros de muy difícil resolución en un laboratorio.

Hansen’s disease presents a wide spectrum of clinical and histopathological manifestations that reflect the nature of the immunological response of the host towards diverse Mycobacterium leprae components. The immunological system, composed by both innate and adaptive immunology, offers protection towards infections of various etiologies, among them bacterial. Bacteria, of course, have developed multiple strategies for evading host defenses, based on either very complex or simple mechanisms, but with a single purpose: to “resist” host attacks and to be able to survive. We have tried to summarize some recent studies in Hansen’s disease, with more emphasis in the inmunology area. We think that in the future, all illnesses should also be very strongly related to other important aspects such as the social, environmental and economic, and whose development is not solved in a laboratory.

[Immune response of Hansen's disease. Review]. / Respuesta inmunitaria de la enfermedad de Hansen. Revisión.

Hansen's disease presents a wide spectrum of clinical and histopathological manifestations that reflect the nature of the immunological response of the host towards diverse Mycobacterium leprae components. The immunological system, composed by both innate and adaptive immunology, offers protection towards infections of various etiologies, among them bacterial. Bacteria, of course, have developed multiple strategies for evading host defenses, based on either very complex or simple mechanisms, but with a single purpose: to "resist" host attacks and to be able to survive. We have tried to summarize some recent studies in Hansen's disease, with more emphasis in the inmunology area. We think that in the future, all illnesses should also be very strongly related to other important aspects such as the social, environmental and economic, and whose development is not solved in a laboratory.

Ciertos aspectos inmunológicos en los estados reaccionales en la enfermedad de Hansen: revisión / Some immunological aspects in the reaccional states of Hansen disease: review

El problema clínico crucial en la lepra son los episodios de inflamación aguda que conllevan a daño de los nervios. Aun cuando el Mycobacterium leprae ha sido eliminado mediante el uso de antibióticos, la muerte de la bacteria no es una solución completa al daño ocasionado en los nervios. Dos de los fenómenos inmunopatológicos más frecuentes son: Reacciones tipo I conocidas como reacciones de reversión (RR) y las Reacciones tipo II, como eritema nodoso leproso (ENL). Estas reacciones tipo II se han definido como: una complicación inmunológica en los pacientes multibacilares. Ambos tipos de reacciones están acompañadas por el incremento de citocinas pro-inflamatorias principalmente TNF-a, IL-1, IL-2, IL-4, IL-6, IL-8, INF-g, IL-10, IL-12, entre otras. En un análisis retrospectivo realizado en una población de 150 pacientes del Servicio Central de Dermatología del Instituto de Biomedicina sometidos a terapia multidroga (PQT) y PQT + inmunoterapia, ambos grupos presentaron fenómenos reaccionales tipo II, sólo el grupo que adicionalmente recibió inmunoterapia presentó además reacciones del tipo I. El grupo de pacientes que presentaron ENL estuvo asociado con presencia de gran cantidad de bacilos en las lesiones

[Increase in nitric oxide concentrations in serum and mononuclear cell cultures from patients with Type II reaction state of Hansen's disease]. / Incremento de óxido nítrico en el suero y sobrenadantes de cultivo de células mononucleares de pacientes con la enfermedad de Hansen en estado reaccional tipo II.

The word "reaction" is used in leprosy to describe signs and symptoms of acute inflammation. Type II reactions, including erythema nodosum leprosum (ENL) occur in the multibacillary forms of Hansen's disease. Nitric oxide (NO) could play a role in the response of the host, where a high NO production would be involved in acute inflammatory processes. In this paper we evaluate NO production in serum and in the supernatants of mononuclear cell cultures (MNCC), measured indirectly by Griess' method. The results obtained in serum showed that 52% of patients with ENL (15/29) had a production over 30 microM, distributed as follows: 8/15 had a mean concentration of 36.38 +/- ?5.71 microM; 1/15, 70.5 microM and 6/15 had a mean concentration greater than 100 microM (205.97 +/- 5 microM). Forty eight percent presented nitrite and nitrate levels lower than 30 microM (18.93 +/- 6.15). Only supernatants of mononuclear cell cultures from ENL patients collected at 120 hours of incubation presented NO production levels higher than 10 microM +/- 6.53, as compared with the supernatants from the stable polar forms of the disease (lepromatous leprosy and tuberculoid leprosy), where values were 2.52 microM +/- 1.18 and 2.69 microM +/- 1.07, respectively. These preliminary results show a different metabolic activity in the group of patients with Type II reaction state (ENL).

Incremento de óxido nítrico en el suero y sobrenadantes de cultivo de células mononucleares de pacientes con la enfermedad de Hansen en estado reaccional tipo II / Increase in nitric oxide concentrations in serum and mononuclear cell cultures from patients with type II rection state of Hansen`s disease

El término "reacción" es usado en lepra para describir síntomas y signos de inflamación aguda. En la forma multibacilar de la enfermedad se producen reacciones tipo II, es decir, eritema nodoso lepromatoso (ENL). El óxido nítrico (ON) podría jugar un papel en la respuesta de huésped, donde la producción elevada de ON estaría involucrada en cuadros inflamatorios agudos. En este trabajo se evaluá la producción de ON en suero y en sobrenadantes de cultivos de células mononucleares (CMN). El ON fue medido indirectamente por el método de Griess. En suero, el 52 por ciento de los pacientes con ENL (15/29) presentó niveles de nitritos/nitratos mayores de 30 µM; así 8/15 presentaron una concentración de 36,38 ± 5,71 µM; 1/15 de 70,5 µM y 6/15 mayor de 100 µM (205,97 ± 5 µM). En concordancia con estos resultados, se encontró que sólo los sobrenadantes de cultivos de células mononucleares de los pacientes con ENL colectados a las 120 horas de incubación presentaron niveles significativamente elevados de nitritos/nitratos (10 µM ± 6,53), en comparación con los sobrenadantes de los polos estables de la enfermedad, lepra lepromatosa y lepra tuberculoide, cuyos valores fueron 2,52 µM ± 1,18 y 2,69 µM ± 1,07, respectivamente. Los resultados muestran niveles relativamente encrementados de nitritos/nitratros en el grupo de pacientes con estado reaccional tipo II (ENL), lo cual sugiere la participación de la iNOS en este grupo

Serologic response to mycobacterial proteins in Hansens patients during multidrug treatment

Humoral immune responses were studied in 24 leprosy patients treated with multidrug therapy (MDT) and 16 contacts. The patients were monitored for 2 to 3 years with repeated determination of IgG antibody levels directed to different mycobacterial proteins (Mycobacterium tuberculosis, Mt70; M. bovis, Mb65; M. leprae, Ml36, 28, 18, 10 kDa, and the complete protein M. leprae extract, MLSA). All recombinant antigens were used at 5 micrograms/ml concentration and the complete soluble M. leprae extract at 2 micrograms/ml. The results shown in this study reveal a clear decline in IgG antibodies directed toward mycobacterial proteins in the 12 multibacillary (MB) patients when they were submitted to MDT. Initially we found strong reactivity toward complete cytosolic protein and M. leprae membrane protein. The most reactive recombinant proteins in MB patients were Ml10, Ml36, Mt70 kDa and, finally, Ml18 kDa when compared to the paucibacillary (PB) group. After treatment was completed all lepromatous and borderline lepromatous patients showed low or undetectable levels as compared with their initial values before starting treatment.