Genotypic Analysis of Genes Associated with Independent Resistance and Cross-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Clinical Isolates.

Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n = 57 ETH-resistant MDR-TB isolates; n = 3 ETH-susceptible MDR-TB isolates; and n = 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB.

Population structure among mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Colombia.

BACKGROUND: Phylogeographic composition of M. tuberculosis populations reveals associations between lineages and human populations that might have implications for the development of strategies to control the disease. In Latin America, lineage 4 or the Euro-American, is predominant with considerable variations among and within countries. In Colombia, although few studies from specific localities have revealed differences in M. tuberculosis populations, there are still areas of the country where this information is lacking, as is a comparison of Colombian isolates with those from the rest of the world. PRINCIPAL FINDINGS: A total of 414 M. tuberculosis isolates from adult pulmonary tuberculosis cases from three Colombian states were studied. Isolates were genotyped using IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and 24-locus Mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTRs). SIT42 (LAM9) and SIT62 (H1) represented 53.3% of isolates, followed by 8.21% SIT50 (H3), 5.07% SIT53 (T1), and 3.14% SIT727 (H1). Composite spoligotyping and 24-locus MIRU- VNTR minimum spanning tree analysis suggest a recent expansion of SIT42 and SIT62 evolved originally from SIT53 (T1). The proportion of Haarlem sublineage (44.3%) was significantly higher than that in neighboring countries. Associations were found between M. tuberculosis MDR and SIT45 (H1), as well as HIV-positive serology with SIT727 (H1) and SIT53 (T1). CONCLUSIONS: This study showed the population structure of M. tuberculosis in several regions from Colombia with a dominance of the LAM and Haarlem sublineages, particularly in two major urban settings (Medellín and Cali). Dominant spoligotypes were LAM9 (SIT 42) and Haarlem (SIT62). The proportion of the Haarlem sublineage was higher in Colombia compared to that in neighboring countries, suggesting particular conditions of co-evolution with the corresponding human population that favor the success of this sublineage.

Características clínicas, epidemiológicas y microbiológicas de una cohorte de pacientes con tuberculosis pulmonar en Cali, Colombia / Clinical, epidemiological and microbiological characteristics of a cohort of pulmonary tuberculosis patients in Cali, Colombia

Introducción. El tratamiento acortado estrictamente supervisado hace parte de una estrategia multifactorial adoptada por la Organización Mundial de la Salud para controlar la tuberculosis y en Colombia alcanza una cobertura de 70%. Objetivo. Establecer las características clínicas, epidemiológicas y microbiológicas y el desenlace de la terapia antituberculosa, en una cohorte de pacientes nuevos con diagnóstico de tuberculosis pulmonar con baciloscopia positiva en Cali, Colombia.Materiales y métodos. Se llevó a cabo un estudio descriptivo, anidado en un ensayo clínico de multicéntrico, en el que se incluyeron 106 pacientes con tuberculosis pulmonar con baciloscopia positiva entre abril de 2005 y junio de 2006, a través de instituciones de salud de la red pública de Cali que administraron el tratamiento antituberculoso. Se practicó baciloscopia seriada, cultivo para micobacterias, prueba de sensibilidad a medicamentos antituberculosos de primera línea, radiografía de tórax postero-anterior y lateral y prueba ELISA para VIH. Se recolectó información clínica y epidemiológica, y se hizo seguimiento por 30 meses, haciendo uso de incentivos de transporte y alimentación.Resultados. La mayoría de los pacientes fueron hombres jóvenes, con diagnóstico hecho más de nueve semanas después del inicio de los síntomas y con baciloscopia muy positiva (2+ o 3+). La resistencia a cualquier medicamento fue de 7,5% y la resistencia inicial a los medicamentos de primera línea fue de 1,9%. La incidencia de efectos secundarios asociados al tratamiento fue de 8,5%. La infección concomitante con VIH fue de 5,7%. El 86,8% de los pacientes completó la terapia con diagnóstico de curación.

Introduction. The World Health Organization recommended strategy for global tuberculosis control is a short-course, clinically administered treatment, This approach has approximately 70% coverage in Colombia. Objective. The clinical, epidemiological and microbiological characteristics along with drug therapy outcomes were described in newly diagnosed, pulmonary tuberculosis patients. Materials and methods. This was a descriptive study, conducted as part of a multicenter clinical trial of tuberculosis treatment. A cohort of 106 patients with pulmonary tuberculosis were recruited from several public health facilities in Cali between April 2005 and June 2006. Sputum smear microscopy, culture, drug susceptibility tests to first-line anti-tuberculosis drugs, chest X- ray and HIV-ELISA were performed. Clinical and epidemiological information was collected for each participant. Treatment was administered by the local tuberculosis health facility. Food and transportation incentives were provided during a 30 month follow-up period.Results. The majority of patients were young males with a diagnostic delay longer than 9 weeks and a high sputum smear grade (2+ or 3+). The initial drug resistance was 7.5% for single drug treatment and 1.9% for multidrug treatments. The incidence of adverse events associated with treatment was 8.5%. HIV co-infection was present in 5.7% of the cases. Eighty-six percent of the patients completed the treatment and were considered cured. The radiographic presentation varied within a broad range and differed from the classic progression to cavity formation. Conclusion. Delay in tuberculosis diagnosis was identified as a risk factor for treatment compliance failure. The study population had similar baseline epidemiologic characteristics to those described in other cohort studies.

[Clinical, epidemiological and microbiological characteristics of a cohort of pulmonary tuberculosis patients in Cali, Colombia]. / Características clínicas, epidemiológicas y microbiológicas de una cohorte de pacientes con tuberculosis pulmonar en Cali, Colombia.

INTRODUCTION: The World Health Organization recommended strategy for global tuberculosis control is a short-course, clinically administered treatment, This approach has approximately 70% coverage in Colombia. OBJECTIVE: The clinical, epidemiological and microbiological characteristics along with drug therapy outcomes were described in newly diagnosed, pulmonary tuberculosis patients. MATERIALS AND METHODS: This was a descriptive study, conducted as part of a multicenter clinical trial of tuberculosis treatment. A cohort of 106 patients with pulmonary tuberculosis were recruited from several public health facilities in Cali between April 2005 and June 2006. Sputum smear microscopy, culture, drug susceptibility tests to first-line anti-tuberculosis drugs, chest X- ray and HIV-ELISA were performed. Clinical and epidemiological information was collected for each participant. Treatment was administered by the local tuberculosis health facility. Food and transportation incentives were provided during a 30 month follow-up period. RESULTS: The majority of patients were young males with a diagnostic delay longer than 9 weeks and a high sputum smear grade (2+ or 3+). The initial drug resistance was 7.5% for single drug treatment and 1.9% for multidrug treatments. The incidence of adverse events associated with treatment was 8.5%. HIV co-infection was present in 5.7% of the cases. Eighty-six percent of the patients completed the treatment and were considered cured. The radiographic presentation varied within a broad range and differed from the classic progression to cavity formation. CONCLUSION: Delay in tuberculosis diagnosis was identified as a risk factor for treatment compliance failure. The study population had similar baseline epidemiologic characteristics to those described in other cohort studies.

[Seroprevalence of Leptospira infection in habitants of peripheral neighborhoods in Cali, Colombia]. / Seroprevalencia de infección por Leptospira en habitantes de barrios periféricos de Cali.

INTRODUCTION: Leptospirosis is a zoonotic disease with varying modes of transmission and clinical expression that affects humans inhabiting urban and rural areas worldwide. In Colombia, little is known regarding leptospirosis in urban settings. OBJECTIVES: To establish the seroprevalence of leptospiral infection in humans, to assess the serological reactivity and to identify factors associated with seropositivity. MATERIALS AND METHODS: We conducted an infection prevalence study in 259 inhabitants of peripheral neighborhoods in the city of Cali, Colombia, stratified by sex and age. Sociodemographic information, time residing in the same neighborhood, occupation, exposure to potentially infectious sources like water or animals, and serum used in a Microagglutination test against 19 Leptospira serovars were obtained. RESULTS: The population was composed mainly of students (37.1%), housewives (32%) and workers in non-formal activities (24.6%). The prevalence of anti-Leptospira antibodies was 23.3% (C195% 18.3-28.3), with high frequency in people older than 57 years of age and significantly higher in men than women (p=0.045). There was an association between the presence of antibodies and contact with animals (p=0.038). Reactivity was observed against 16 of the 19 serovars evaluated, but the antibody titers were low. CONCLUSIONS: The high infection seroprevalence found in this study suggests frequent contact with Leptospira in putatively high risk neighborhoods of Cali. Contact with animals was the most significant factor associated with the presence of anti-Leptospira antibodies.

Seroprevalencia de infección por Leptospira en habitantes de barrios periféricos de Cali / Seroprevalence of Leptospira infection in habitants of peripheral neighborhoods in Cali, Colombia

Introducción. La leptospirosis es una enfermedad zoonótica urbana y rural que afecta a los humanos en todos los continentes, aunque su transmisión y presentación clínica puede variar. En Colombia hay poca información acerca de la importancia de la leptospirosis en entornos urbanos. Objetivos. Establecer la seroprevalencia de infección por Leptospira en humanos, identificar la reactividad serológica y explorar factores asociados con la seropositividad. Materiales y métodos. Se realizó un estudio transversal en 259 habitantes de barrios periféricos de Cali, estratificado por sexo y edad. Se obtuvo información general de cada persona, tiempo de residencia en el barrio, ocupación, exposición a fuentes contaminadas como agua o animales, y suero para la prueba de microaglutinación con 19 serovares de Leptospira. Resultados. La población evaluada correspondió principalmente a estudiantes (37,1 por ciento), amas de casa (32 por ciento) y trabajadores en oficios varios (24,6 por ciento). Se encontraron anticuerpos anti- Leptospira en 23,3 por ciento (IC95 por ciento 18,3 a 28,3) de las personas, siendo más frecuente en mayores de 57 años y significativamente mayor en hombres que en mujeres ( p = 0,045). Se encontró asociación entre la seropositividad y el contacto con animales ( p = 0,038) . De los 19 serovares evaluados, se detectó reactividad frente a 16, pero los títulos de anticuerpos fueron bajos. Conclusiones. La alta prevalencia de infección encontrada en el estudio sugiere la transmisión frecuente de leptospiras en barrios de Cali considerados de alto riesgo. Se destaca el contacto con animales como un factor asociado con la presencia de anticuerpos contra leptospiras.

[Exposure to Leptospira in stray dogs in the city of Cali]. / Evidencia de exposición a Leptospira en perros callejeros de Cali.

In Colombia, little information is available concerning the epidemiology of leptospirosis in urban environments. Furthermore, the role of dogs in the transmission cycle of leptospirosis in the urban setting is unclear. To explore the potential role of canines in the transmission of leptospirosis in Cali, a serological study was conducted with 197 serum samples collected from stray dogs during 2001 and 2003. Serum specimens were screened with the Microscopic Agglutination Test (MAT) and 7 serovars--Icterohaemorrhagiae, Canicola, Gryppotyphosa, Hardjo strain Hardjobovis, Pomona, Hardjo strain Hardjoprajitno and Bratislava. All serovars were provided by the Instituto Colombiano Agropecuario (ICA), Tuluá, Colombia. The MAT was considered positive when 50% or more leptospiras were agglutinated with one or more serovars in a serum dilution of 1:100. At least one serovar showed evidence of infection in 41.1% of the dogs. The most prevalent serovar was Icterohaemorrhagiae, found in 55.6% of the seropositive dogs. 48.1% were co-agglutinations. No reactions against the serovars Pomona, Hardjo strain Hardjoprajitno and Bratislava were observed. These findings suggested that stray dogs are potential reservoirs of Leptospira in Cali and underscored the need to study the epidemiology of this disease in Colombia.

Evidencia de exposición a Leptospira / Exposure to Leptospira in stray dogs in the city of Cali

En Cali, y en Colombia en general, se desconoce la epidemiología de la leptospirosis en ambientes urbanos. Además, el papel del perro en el ciclo de transmisión en dichos ambientes no es claro. Para explorar esta situación, realizamos un estudio serológico en Cali a 197 sueros de perros callejeros durante el 2001 y el 2003, utilizando la prueba de microaglutinación (MAT). En la prueba se incluyeron 7 serovares: Icterohaemorrhagiae, Canicola, Gryppotyphosa, Hardjo cepa Hardjobovis, Pomona, Hardjo cepa Hardjoprajitno y Bratislava aportados por el Instituto Colombiano Agropecuario (ICA) de Tuluá. La prueba se interpretó como positiva por la presencia de una aglutinación ³50 por ciento de las leptospiras, con uno o más serovares, en una dilución del suero ³1:100. Encontramos evidencia de infección en el 41,1 por ciento de los perros con, al menos, uno de los serovares. La mayor reactividad fue a Icterohaemorrhagiae con 55,6 por ciento del total de los seropositivos. Se presentaron 48,1 por ciento de coaglutinaciones. No se registraron reacciones contra los serovares Pomona, Hardjo cepa Hardjoprajitno y Bratislava. Estos hallazgos sugieren que el perro callejero es un posible reservorio de leptospira en Cali y resaltan la necesidad de estudiar la epidemiología de la enfermedad en esta ciudad

Photographic and luminometric detection of luciferase reporter phages for drug susceptibility testing of clinical Mycobacterium tuberculosis isolates.

Luciferase reporter phages (LRPs) have proven to be efficient tools for drug susceptibility testing of Mycobacterium tuberculosis. Luminometric detection of LRP activity offers higher sensitivity and quantitative results, while a Polaroid film detection method offers a "low-tech" inexpensive alternative that is called the Bronx box. In this work we evaluated, improved, and compared the performance of the luminometer and the Bronx box formats for drug susceptibility testing with LRPs by using 51 clinical isolates of M. tuberculosis, with the agar proportion method (PM) serving as reference. The sensitivity in detecting resistance to isoniazid and rifampin, antibiotics that define multidrug resistance (MDR), was 100% for both methods. The turnaround time for results was reduced from 3 weeks for PM to 54 or 94 h for luminometry or the Bronx box, respectively. These results support the utility of LRPs as a screening test for the surveillance of MDR tuberculosis.