Is drug-resistant Mycobacterium leprae a real cause for concern?: First approach to molecular monitoring of multibacillary Colombian patients with and without previous leprosy treatment.

INTRODUCTION: There is no information in Colombia on Mycobacterium leprae primary and secondary drug resistance in regards to the WHO-multidrug therapy regime. On the other hand, public health authorities around the world have issued various recommendations, one of which prompts for the immediate organization of resistance surveillance through simple molecular methods. OBJECTIVE: To determine the prevalence of Mycobacterium leprae drug resistance to rifampicin, ofloxacin and dapsone in untreated and previously treated patients at the Centro Dermatológico Federico Lleras Acosta during the 1985-2004 period. MATERIALS AND METHODS: We conducted a retrospective study which included multibacillary patient biopsies through elective sampling: 381 of them from new patients and 560 from previously treated patients. Using a microtome, we obtained six slides from each skin biopsy preserved in paraffin, and we extracted M. leprae DNA. We amplified three molecular targets through PCR and obtained the patterns of drug resistance to dapsone, rifampicin and ofloxacin by reverse hybridization. Finally, we collected epidemiological, clinical and demographical data for analyses. RESULTS: From 941 samples under study, 4.14% of them were resistant to one or more drugs, and 5.77 and 3.04% had resistant genotypes in new and previously treated patients, respectively. Total resistance for each drug was 0.43% for dapsone, 3.19% for rifampicin and 1.17% for ofloxacin. We found statistically significant differences for rifampicin and for the total population when comparing the results from untreated versus previously treated patients. Two thirds of the resistant samples were resistant to rifampicin alone or combined. CONCLUSIONS: The standard multidrug therapy schemes continue being effective for leprosy cases; however, it is necessary to guarantee adherence and regularity. Surveillance to drug resistance in new and previously treated leprosy cases should be established.

Is drug-resistant Mycobacterium leprae a real cause for concern?: First approach to molecular monitoring of multibacillary Colombian patients with and without previous leprosy treatment / ¿Es la resistencia de Mycobacterium leprae a los medicamentos un verdadero motivo de preocupación?: Primera aproximación a la vigilancia molecular de pacientes colombianos multibacilares con tratamiento previo para lepra y sin él

Introduction: There is no information in Colombia on Mycobacterium leprae primary and secondary drug resistance in regards to the WHO-multidrug therapy regime. On the other hand, public health authorities around the world have issued various recommendations, one of which prompts for the immediate organization of resistance surveillance through simple molecular methods. Objective: To determine the prevalence of Mycobacterium leprae drug resistance to rifampicin, ofloxacin and dapsone in untreated and previously treated patients at the Centro Dermatológico Federico Lleras Acosta during the 1985-2004 period. Materials and methods: We conducted a retrospective study which included multibacillary patient biopsies through elective sampling: 381 of them from new patients and 560 from previously treated patients. Using a microtome, we obtained six slides from each skin biopsy preserved in paraffin, and we extracted M. leprae DNA. We amplified three molecular targets through PCR and obtained the patterns of drug resistance to dapsone, rifampicin and ofloxacin by reverse hybridization. Finally, we collected epidemiological, clinical and demographical data for analyses. Results: From 941 samples under study, 4.14% of them were resistant to one or more drugs, and 5.77 and 3.04% had resistant genotypes in new and previously treated patients, respectively. Total resistance for each drug was 0.43% for dapsone, 3.19% for rifampicin and 1.17% for ofloxacin. We found statistically significant differences for rifampicin and for the total population when comparing the results from untreated versus previously treated patients. Two thirds of the resistant samples were resistant to rifampicin alone or combined. Conclusions: The standard multidrug therapy schemes continue being effective for leprosy cases; however, it is necessary to guarantee adherence and regularity. Surveillance to drug resistance in new and previously treated leprosy cases should be established.

Introducción. Colombia no dispone de información sobre farmacorresistencia primaria y secundaria de Mycobacterium leprae al esquema de terapia múltiple de la Organización Mundial de la Salud (OMS) y las autoridades de salud pública del mundo han emitido varias recomendaciones, entre las cuales está organizar de inmediato la vigilancia a la resistencia empleando métodos moleculares simples. Objetivo. Determinar la prevalencia de la resistencia de M. leprae a rifampicina, ofloxacina y dapsona en pacientes del Centro Dermatológico Federico Lleras Acosta con tratamiento previo y sin él durante el período de 1985 a 2004. Materiales y métodos. Se realizó un estudio retrospectivo. Mediante muestreo electivo se incluyeron biopsias de pacientes multibacilares: 381 de pacientes nuevos y 560 de pacientes previamente tratados. Se obtuvieron con micrótomo seis cortes de cada biopsia de piel incluida en parafina, y se realizó la extracción de ADN de M. leprae. Se llevó a cabo la amplificación de tres blancos moleculares mediante PCR y se obtuvieron los patrones de resistencia a los medicamentos dapsona, rifampicina y ofloxacina por hibridación inversa. Se recolectaron datos epidemiológicos, clínicos y demográficos para llevar a cabo los análisis. Resultados. De las 941 muestras estudiadas, 4,14 % era resistente a uno o más fármacos, y se detectaron 5,77 y 3,04 % con genotipos resistentes en pacientes nuevos y previamente tratados, respectivamente. La resistencia total para cada fármaco fue de 0,43 % a dapsona, 3,19 % a rifampicina y 1,17 % a ofloxacina. Se encontró una diferencia estadísticamente significativa para rifampicina y para la población total al comparar los resultados de los pacientes no tratados con los de los pacientes tratados previamente. Dos tercios de las muestras resistentes lo fueron a rifampicina sola o combinada. Conclusiones. Los esquemas de terapia múltiple estándar siguen siendo efectivos para los casos de lepra; sin embargo, es necesario garantizar el cumplimiento y la regularidad y establecer la vigilancia de la farmacorresistencia en pacientes nuevos y previamente tratados.

[Delay in leprosy diagnosis as a predictor of disability in a cohort of patients in Colombia, 2000-2010]. / Retraso en el diagnóstico de lepra como factor pronóstico de discapacidad en una cohorte de pacientes en Colombia, 2000-2010.

OBJECTIVE: Evaluate predictive factors of disability at time of leprosy diagnosis in a cohort of Colombian patients, from 2000 to 2010. METHODS: Descriptive and analytical observational study of a retrospective cohort of patients admitted with a leprosy diagnosis to the Centro Dermatológico Federico Lleras Acosta in Bogotá, Colombia, from 2000 to 2010. Variables were analyzed descriptively and predictive factors for disability at diagnosis were identified through simple and multifactorial analyses (Cox proportional hazards model); hazard ratios for each factor in the model were calculated. RESULTS: Time between first symptoms and diagnosis in the 333 cohort patients was 2.9 years on average; 32.3% had certain degree of disability, especially for the feet. Delay in diagnosis and disability was greater in men than in women and in patients with multibacillary rather than paucibacillary leprosy. Disability was significantly associated with delays of ≥ 1 year in diagnosis, age ≥ 30 years, initial bacillary index of ≥ 2, multibacillary leprosy, and natives of the Cundinamarca or Santander departments. Protective factors were female sex, having some education, and residence in Boyacá. CONCLUSIONS: Time between first symptoms and diagnosis is the key predictive factor of disability at time of leprosy diagnosis. Strengthening of active searching for infected people and promotion of early diagnosis are recommended.

Retraso en el diagnóstico de lepra como factor pronóstico de discapacidad en una cohorte de pacientes en Colombia, 2000 - 2010 / Delay in leprosy diagnosis as a predictor of disability in a cohort of patients in Colombia, 2000 - 2010

OBJETIVO: Evaluar los factores pronósticos de la presencia de discapacidad al momento del diagnóstico de lepra en una cohorte de pacientes colombianos de 2000 a 2010. MÉTODOS: Estudio analítico y observacional descriptivo de una cohorte retrospectiva de pacientes ingresados con diagnóstico de lepra en el Centro Dermatológico Federico Lleras Acosta, de Bogotá, Colombia, entre 2000 y 2010. Se realizó el análisis descriptivo de las variables y se identificaron factores pronósticos de la presencia de discapacidad al momento del diagnóstico mediante análisis simple y multifactorial (modelo de riesgos proporcionales de Cox); se calcularon las razones de riesgo (hazard ratio) para cada uno de los factores incluidos en el modelo. RESULTADOS: El tiempo entre los primeros síntomas y el diagnóstico en los 333 pacientes de la cohorte fue en promedio 2,9 años; 32,3% de ellos tenían algún grado de discapacidad, especialmente en los pies. Hubo una mayor proporción de retraso en el diagnóstico y discapacidad en hombres que en mujeres y en pacientes con lepra multibacilar que con paucibacilar. La discapacidad se asoció significativamente con demoras ≥ 1 año en el diagnóstico, edad ≥ 30 años, índice baciloscópico inicial ≥ 2, lepra multibacilar y proceder de Cundinamarca o Santander. Los factores protectores fueron ser del sexo femenino, tener algún grado de escolaridad y residir en Boyacá. CONCLUSIONES: El tiempo entre los primeros síntomas y el diagnóstico constituye el factor pronóstico clave de la discapacidad al momento del diagnóstico de lepra. Se recomienda reforzar la búsqueda activa de personas infectadas y promover el diagnóstico precoz.

OBJECTIVE: Evaluate predictive factors of disability at time of leprosy diagnosis in a cohort of Colombian patients, from 2000 to 2010. METHODS: Descriptive and analytical observational study of a retrospective cohort of patients admitted with a leprosy diagnosis to the Centro Dermatológico Federico Lleras Acosta in Bogotá, Colombia, from 2000 to 2010. Variables were analyzed descriptively and predictive factors for disability at diagnosis were identified through simple and multifactorial analyses (Cox proportional hazards model); hazard ratios for each factor in the model were calculated. RESULTS: Time between first symptoms and diagnosis in the 333 cohort patients was 2.9 years on average; 32.3% had certain degree of disability, especially for the feet. Delay in diagnosis and disability was greater in men than in women and in patients with multibacillary rather than paucibacillary leprosy. Disability was significantly associated with delays of ≥ 1 year in diagnosis, age ≥ 30 years, initial bacillary index of ≥ 2, multibacillary leprosy, and natives of the Cundinamarca or Santander departments. Protective factors were female sex, having some education, and residence in Boyacá. CONCLUSIONS: Time between first symptoms and diagnosis is the key predictive factor of disability at time of leprosy diagnosis. Strengthening of active searching for infected people and promotion of early diagnosis are recommended.

Confiabilidad y concordancia de dos escalas de lectura de baciloscopias para clasificación y seguimiento del tratamiento con múltiples medicamentos de los pacientes con lepra / Reliability and agreement of two smear reading scales for classification and monitoring of multidrug therapy in leprosy patients

Introducción. El diagnóstico de la lepra es clínico pero requiere métodos de clasificación para establecer el tratamiento y el pronóstico de los pacientes, acordes con su carga bacilar; ésta se detecta en extendidos de piel y se establece mediante la escala logarítmica de Ridley. En Colombia se emplea una escala semicuantitativa.Objetivo. Establecer la reproducibilidad entre observadores para las dos escalas y establecer el nivel de correlación de la concordancia entre el índice bacilar obtenido con la escala colombiana y con la de Ridley, para evaluar cuánto se pueden intercambiar. Materiales y métodos. Se estandarizó la lectura de las baciloscopias por dos lectores, con posterior evaluación del acuerdo entre observadores en un ensayo ciego. Cada lector cuantificó la carga bacilar de las muestras, usando la escala colombiana y la internacional. El grado de concordancia entre observadores se evaluó con el coeficiente kappa ponderado. El nivel de correlación de la concordancia entre las mediciones del índice bacilar, se estableció con el coeficiente de Lin. Resultados. El coeficiente kappa ponderado entre observadores fue de 0,83, y de 0,85 para las escalas colombiana y la de Ridley, respectivamente. El coeficiente de Lin fue de 0,96 para la evaluación de correlación de la concordancia de los índices bacilares obtenidos con ambas escalas. Conclusiones. La concordancia entre observadores obtenida para ambas escalas fue excelente, al igual que la correlación de la concordancia de los índices bacilares calculados con los dos métodos. Con los puntos de corte establecidos, se obtuvo un nivel de concordancia excelente, lo que garantiza que las escalas son intercambiables a la hora de establecer si la carga bacilar es alta o baja.

Introduction. After the clinical diagnosis of leprosy, classification methods are necessary to define a treatment and prognosis of patients consistent with bacterial load. Bacteria are detected in skin smear, and bacterial load typically is established by the internationally used Ridley’s logarithmic scale, However, in Colombia an alternative semiquantitative scale is used. Objective. The interobserver reproducibility was established for the Ridley and Colombia scales, and the level of correlation-matching was identified between the bacillary indices obtained in order to assess the degree of interchangeability.Materials and methods. Standardization was attained by a reading of the smears by 2 readers with subsequent, blinded evaluation of inter-observer agreement. Each reader quantified the bacterial load of for each sample (n=325) using the Colombian and the Ridley scales. The degree of interobserver agreement was assessed with weighted kappa coefficient. The level of correlation and agreement between the measurements of the bacillary index was established with coefficient of Lin. Results. The interobserver weighted kappa coefficient was 0.83 for the Colombia scale and 0.85 for the Ridley scale. The Lin coefficient was 0.96 for the correlation-matching of bacillary indexes.Conclusions. Interobserver agreement obtained for both scales was excellent as the correlation-matching bacillary indices determined with both methods. With the cut-off points yielded a good level of agreement, ensuring interchangeability between the scales defining the high or low bacterial load.

[Reliability and agreement of two smear reading scales for classification and monitoring of multidrug therapy in leprosy patients]. / Confiabilidad y concordancia de dos escalas de lectura de baciloscopias para clasificación y seguimiento del tratamiento con múltiples medicamentos de los pacientes con lepra.

INTRODUCTION: After the clinical diagnosis of leprosy, classification methods are necessary to define a treatment and prognosis of patients consistent with bacterial load. Bacteria are detected in skin smear, and bacterial load typically is established by the internationally used Ridley's logarithmic scale, However, in Colombia an alternative semiquantitative scale is used. OBJECTIVE: The interobserver reproducibility was established for the Ridley and Colombia scales, and the level of correlation-matching was identified between the bacillary indices obtained in order to assess the degree of interchangeability. MATERIALS AND METHODS: Standardization was attained by a reading of the smears by 2 readers with subsequent, blinded evaluation of inter-observer agreement. Each reader quantified the bacterial load of for each sample (n=325) using the Colombian and the Ridley scales. The degree of interobserver agreement was assessed with weighted kappa coefficient. The level of correlation and agreement between the measurements of the bacillary index was established with coefficient of Lin. RESULTS: The interobserver weighted kappa coefficient was 0.83 for the Colombia scale and 0.85 for the Ridley scale. The Lin coefficient was 0.96 for the correlation-matching of bacillary indexes. CONCLUSIONS: Interobserver agreement obtained for both scales was excellent as the correlation-matching bacillary indices determined with both methods. With the cut-off points yielded a good level of agreement, ensuring interchangeability between the scales defining the high or low bacterial load.

Avian tuberculosis of zoonotic importance at a zoo on the Bogotá Andean plateau (Sabana), Colombia.

Given that exposure to captive wild animals at circuses or zoos can be a source of zoonotic infection, a case and control study was carried out with a collection of exotic fowl at a zoo in Bogotá, Colombia. The presence of Mycobacterium avium-II was directly related to the death of birds kept in the original enclosure, and of 50% of a group of sentinel birds. Failure to detect the organism in a control group of birds outside the enclosure indicated that the infection was limited to the original enclosed area. We demonstrated that M. gordonae-IV was disseminated in all organs from 1 bird with macroscopic granulomatous lesion, a finding which has not been reported previously. We emphasize the importance of establishing handling norms to reduce the risk of zoonotic transmission.

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis.

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.

Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set.

We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by GST. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.

Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance.

Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.

[Phenotypic and genotypic methods for detecting multidrug resistance in Mycobacterium tuberculosis]. / Evaluación de métodos fenotípicos y genotípicos para la detección de farmacorresistencia de Mycobacterium tuberculosis.

BACKGROUND: Expeditious charactization of drug susceptibility in Mycobacterium tuberculosis is difficult and, calls for the design and evaluation of faster, cheaper and more effective new techniques. OBJECTIVE: The aim of the current study was to compare one genotypic and two phenotypic methods for rapid susceptibility detection of M. tuberculosis. MATERIAL AND METHODS: Twenty-one M. tuberculosis strains were evaluated by phenotypic methodologies of oxidation and reduction of Alamar blue and MTT in the presence of streptomycin, isoniazid, rifampin and ethambutol. In all tests, the proportion method was applied as the comparison standard. By means of receiver operative characteristic (ROC) analysis, the performance, predictive values and threshold values for all drugs were determined. In addition, the performance of PCR and reverse line blot hybridization in establishing predictive values for sensitivity and resistance were compared in contingency tables. RESULTS: The susceptibility patterns established by colorimetric techniques were obtained after seven days of incubation. The performances of these tests were excellent for all drugs-the areas under curves were >0.9, 100% of sensitivity (S) and specificity (E) >80%. The genotypic method of RFLP oligotyping detected multidrug resistance with S of 100% and E of 93%. Conclusion. The results indicated that Alamar blue, MTT and RFLP methodologies are rapid and useful tools for characterizing multidrug resistance in M. tuberculosis, particularly for those patients with high risk of developing multidrug resistant tuberculosis.

[Development of a multiantigenic serological test for tuberculosis diagnosis]. / Utilidad de una prueba serológica multiantigénica en el diagnóstico de tuberculosis.

OBJECTIVE: The present work evaluated a multi-antigen printing immunoassay (MAPIA) for the serological diagnosis of tuberculosis. MATERIALS AND METHODS: Sera were obtained from 66 patients with tuberculosis, verified clinically and bacteriologically and from 47 healthy individuals (control group). Sample sera were used for detection of antibodies against 3 enriched mixtures of proteins and 5 unique recombinant antigens. The antigens were presented in a solid matrix. Sensitivity, specificity and predictive values were evaluated and confirmed by a logistic regression analysis. A prevalence value was calculated and used for the selection of the best antigenic combination. RESULTS: The sensitivity and specificity values of individual antigens varied between 5-83% and 9-100%. The enriched mixtures values were more accurate than those obtained with the recombinant antigens. Combinations of several antigens improved the sensitivity values up to the 81% level. In most cases, specificity values of 57% or less were obtained. CONCLUSIONS: These results suggested that the multiantigenic test can be a useful screening tool, to be used in conjunction with the more definitive diagnostic tests.

Evaluación de métodos fenotípicos y genotípicos para la detección de farmacorresistencia de Mycobacterium tuberculosis / Evaluation of phenotypic and genotypic methods for detecting multidrug resistance in Mycobacterium tuberculosis

Introducción. La dificultad en el diagnóstico temprano y oportuno de la susceptibilidad a los medicamentos de Mycobacterium tuberculosis hace necesario el diseño y la evaluación de nuevas técnicas que superen la efectividad, reduzcan el costo y el tiempo que toman los métodos tradicionales. Objetivo. Evaluar y comparar dos metodologías fenotípicas y una genotípica, útiles en la determinación rápida de susceptibilidad de M. tuberculosis, teniendo el método de las proporciones múltiples como referencia. Materiales y métodos. Se incluyeron 21 cepas de M. tuberculosis para la evaluación de las metodologías fenotípicas de oxidación y reducción de Alamar azul (Maba) y el bromuro de 3-(4-5-dimetiletiazol-2-il)-2,5-difenil tetrazolio (MTT o Tema), frente a estreptomicina, isoniacida, rifampicina y etambutol. Mediante análisis ROC (receiver operative characteristic curves) se determinó el desempeño de las pruebas y se determinaron los valores pronósticos y el punto de corte para cada antibiótico. Además, mediante tablas de contingencia se determinó el desempeño y los valores predictivos de la metodología genotípica de PCR e hibridación reversa o rifoligotyping, versión 3, en la determinación de las cepas multirresistentes. Resultados. Los perfiles de susceptibilidad por las pruebas colorimétricas se obtuvieron a los 7 días, con valores de área bajo la curva menores de 0,9, sensibilidad de 100 por ciento y especificidad mayor de 80 por ciento. La metodología de rifoligotyping resultó eficaz en la detección de mutirresistencia, con sensibilidad de 100 por ciento y especificidad de 93 por ciento. Conclusión. Con los resultados obtenidos consideramos las pruebas Maba, Tema y rifoligotyping como alternativas rápidas y eficaces en la detección de farmacorresistencia de M. tuberculosis, aplicables a pacientes con alto riesgo de desarrollar tuberculosis multirresistente

Utilidad de una prueba serológica multiantigénica en el diagnóstico de tuberculosis / Development of a multiantigenic serological test for tuberculosis diagnosis

Introducción. La heterogeneidad en el patrón de reconocimiento antigénico observado en los enfermos infectados con Mycobacterium tuberculosis hace evidente la necesidad de contar con una técnica rápida y accesible para el diagnóstico de la enfermedad. La disponibilidad de pruebas que empleen diferentes antígenos podría ser de utilidad para aumentar la sensibilidad de la detección.Objetivo. Evaluar el uso de una prueba multiantigénica (Mapia) de fácil manejo y evaluación visual en el diagnóstico serológico de la tuberculosis. Metodología. Se estudiaron 66 sueros de pacientes con enfermedad tuberculosa comprobada y 47 sueros de individuos no tuberculosos, y se detectó la presencia de anticuerpos para 8 antígenos: 3 mezclas enriquecidas en antígenos y 5 antígenos recombinantes, incluidos en una matriz sólida cuya lectura se realizó cualitativamente. Los antígenos fueron evaluados por sus características de sensibilidad, especificidad y valores predictivos, confirmados mediante análisis de regresión logística del cual se obtuvo la razón de prevalencia utilizada para la selección de la combinación antigénica mas adecuada. Resultados. Los valores de sensibilidad y especificidad de los antígenos individuales variaron entre 5 por ciento y 83 por ciento y 9 por ciento y 100 por ciento, respectivamente; los valores de las mezclas enriquecidas fueron mejores que los valores presentados por los antígenos recombinantes. La combinación de varios antígenos mejoró notablemente los valores de sensibilidad hasta 81 por ciento, pero en la mayoría de los casos se obtuvieron valores de especificidad menores al 57 por ciento

Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean.

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.

[Respiratory syntomatic prevalence, infection and tuberculosis disease and associated factors: population-based study]. / Prevalencia de sintomáticos respiratorios, de infección y enfermedad tuberculosa y factores asociados: estudio basado en población, Mitú, Vaupés, 2001.

A cross sectional survey on TB epidemiological characteristics was carried out in Mitú (Vaupes, Colombia) with the aim of measuring the prevalence of TB cases, the prevalence of TB suspected cases, the coverage with BCG vaccine and the prevalence of infection with Mycobacterium tuberculosis. One hundred and sixty five (165) households were included in the survey using a randomized cluster sampling design (n=20 clusters) which yielded a sample size of 972 subjects. The prevalence of TB suspect cases was 3.6% (C.I.95% 2.6-4.9%); coverage with BCG vaccine was 94%. Vaccinated people had a lower chance of being a TB suspected case (OR=0.37 C.I.95% 0.15-0.95). TB prevalence was 1.4%. People vaccinated with BCG had a lower chance of having been a TB case (OR=3.3 C.I.95% 1.0-14). These data recommend that 10% of people with respiratory symptoms be screened for in the National Control Program,and that the results be reviewed with surveys based at health centers. The data also reinforce the need for better vaccination coverages with BCG in high endemic areas.

Caracterización clínica y sociodemográfica de casos nuevos de lepra en municipios endémicos y no endémicos de Colombia / Clinical and sociodemographical caracterization of new cases of leprosy in both endemic and non-endemic municipalities in Colombia

OBJETIVO: Describir algunas características sociodemográficas, clínicas y epidemiológicas de los casos nuevos de lepra y sus convivientes de nueve municipios endémicos y uno no endémico para lepra en Colombia. METODOLOGíA: Se realizó un análisis descriptivo, a partir de la información de los casos nuevos de lepra ingresados al Programa de Control y Eliminación de Lepra de los municipios participantes en el estudio de noviembre de 2002 a octubre de 2003 RESULTADOS: El 62,2 por ciento de los casos presentó la forma multibacilar y el 63 por ciento correspondía al género masculino, la mediana de edad fue de 46.5 años. El 55,6 por ciento de los casos nuevos tenía en el momento del diagnóstico discapacidades grado 1 y 2, mayor al reportado a escala nacional (37,5 por ciento) para el año 2003. Se observó una correlación positiva entre el porcentaje de Necesidades Básicas Insatisfechas por municipio y la incidencia de la enfermedad (r=0,69, p=0,0180). CONCLUSIONES: Nuestros hallazgos resaltan la poca respuesta del Programa de Control de Lepra en algunos municipios para realizar la detección activa y oportuna de los enfermos, además de evidenciar la asociación que históricamente esta enfermedad ha tenido con la pobreza.(AU)

OBJECTIVE: To describe some socio-demographical, clinical and epidemiological characteristics of the new cases of leprosy and household reported from 9 endemic and 1 non-endemic towns in Colombia. METHODOLOGY: A descriptive analysis was made from the information of the new leprosy cases included in the leprosy control and elimination program of the participant towns in the study carried out between November 2002 and October 2003. RESULTS: 62.2 % of the cases presented the multibacillary form and 63 % of them were males; the median age was 46.5 years. 55.6 % of the new cases had first or second level impairments at the moment of diagnosis. This percentage is higher than the one reported in 2003 at a nationwide level, 37.5 %. A positive correlation was observed between the Unsatisfied Basic Needs Index (NBI) and the disease incidence by town (r=0.69, p=0.0180). CONCLUSIONS: Our findings highlight the little response of the Leprosy Control Program in some towns to carry out the active and timely detection of the diseased people, besides making evide nt the historical relationship that this disease has had with poverty.(AU)