Heterogeneous fitness landscape cues, pknG low expression, and phthiocerol dimycocerosate low production of Mycobacterium tuberculosis ATCC25618 rpoB S450L in enriched broth.

Multidrug-resistant tuberculosis (isoniazid/rifampin[RIF]-resistant TB) ravages developing countries. Fitness is critical in clinical outcomes. Previous studies on RIF-resistant TB (RR-TB) showed competitive fitness gains and losses, with rpoB-S450L as the most isolated/fit mutation. This study measured virulence/resistance genes, phthiocerol dimycocerosate (PDIM) levels and their relationship with rpoB S450L ATCC25618 RR-TB strain fitness. After obtaining 10 different RR-TB GenoType MTBDRplus 2.0-genotyped isolates (with nontyped, S441, H445 and S450 positions), only one S450L isolate (R9, rpoB-S450L ATCC 25618, RR 1 µg/mL) was observed, with H445Y being the most common. A competitive fitness in vitro assay with wild-type (wt) ATCC 25618: R9 1:1 in 50 mL Middlebrook 7H9/OADC was performed, and generation time (G) in vitro and relative fitness were obtained. mRNA and PDIM were extracted on log and stationary phases. Fitness decreased in rpoB S450L and H445Y strains, with heterogeneous fitness cues in three biological replicas of rpoB-S450L: one high and two low fitness replicas. S450L strain had significant pknG increase. Compared with S450L, wt-rpoB showed increased polyketide synthase ppsA expression and high PDIM peak measured by HPLC-MS in log phase compared to S450L. This contrasts with previously increased PDIM in other RR-TB isolates.

Characterization of clinical isolates of Mycobacterium tuberculosis from indigenous peoples of Colombia / Caracterización de aislamientos clínicos de Mycobacterium tuberculosis de pueblos indígenas de Colombia

INTRODUCTION: Tuberculosis continues to be a public health priority. Indigenous peoples are vulnerable groups with cultural determinants that increase the risk of the disease. OBJECTIVE: To determine molecular epidemiology and phenotypical features and of Mycobacterium tuberculosis isolates from indigenous people in Colombia during the period from 2009 to 2014. MATERIALS AND METHODS: We conducted an analytical observational study; we analyzed 234 isolates to determine their patterns of sensitivity to antituberculosis drugs and their molecular structures by spoligotyping. RESULTS: The isolates came from 41 indigenous groups, predominantly the Wayúu (13.10%) and Emberá Chamí (11.35%). We found 102 spoligotypes distributed among seven genetic families (37.2% LAM, 15.8% Haarlem, 8.1% T, 3.4% U, 2.6% S, 2.1% X, and 0.9%, Beijing). The association analysis showed that the non-clustered isolates were related to prior treatment, relapse, orphan spoligotypes, and the Beijing family. The H family presented an association with the Arhuaco and Camëntsá indigenous groups, the U family was associated with the Wounaan group, and the T family was associated with the Motilón Barí group. CONCLUSIONS: This is the first national study on M. tuberculosis characterization in indigenous groups. The study evidenced that diagnosis in indigenous people is late. We described 53% of orphan patterns that could be typical of the Colombian indigenous population. The high percentage of grouping by spoligotyping (62%) could indicate cases of active transmission, a situation that should be corroborated using a second genotyping marker. A new Beijing spoligotype (Beijing-like SIT 406) was identified in Colombia.

Introducción. La tuberculosis es prioridad de salud pública. Los pueblos indígenas son vulnerables debido a los factores culturales determinantes que aumentan el riesgo de tuberculosis. Objetivo. Determinar la epidemiologia molecular y las características fenotípicas de los aislamientos de Mycobacterium tuberculosis de pueblos indígenas de Colombia entre 2009 y 2014. Materiales y métodos. Se hizo un estudio observacional analítico; se analizaron 234 aislamientos para determinar la sensibilidad a los fármacos antituberculosos y la estructura molecular usando spoligotyping. La información epidemiológica se recolectó utilizando el formato único de vigilancia de micobacterias. Resultados. Los aislamientos provenían de 41 grupos indígenas, principalmente los wayúu (13,10 %) y emberá chamí (11,35 %). Se encontraron 102 genotipos distribuidos en siete familias genéticas (37,2 %, LAM; 15,8 %, Haarlem; 8,1 %, T; 3,4 %, U; 2,6 %, S; 2,1 %, X, y 0,9%, Beijing). El análisis de asociación mostró que los aislamientos no agrupados se asociaron con el tratamiento previo, las recaídas, los genotipos huérfanos y la familia Beijing. La familia H presentó una asociación con los grupos indígenas arhuaco y camëntsá, la familia U se asoció con el grupo wounaan y la familia T con el grupo motilón barí. Conclusiones. Este es el primer estudio nacional de caracterización de M. tuberculosis en grupos indígenas. Se evidenció que el diagnóstico en indígenas es tardío, y que 53 % de los patrones huérfanos podrían ser típicos de la población indígena colombiana. El alto porcentaje de agrupamiento por spoligotyping (62%) podría indicar casos de transmisión activa, una situación que debe ser corroborada usando un segundo marcador de genotipificación. Se identificó un nuevo genotipo (Beijing-like SIT 406) en Colombia.

Tuberculosis associated with tumor necrosis factor-α antagonists, case description and analysis of reported cases in Colombia.

Tumor necrosis factor-α (TNF-α) is an important fundamental cytokine during the immune response against cancer and infections such as tuberculosis. This molecule also plays a key pathogenic role in complex and difficult-to-treat diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and ulcerative colitis. The treatment of these diseases frequently needs TNF-α antagonists, which has been related to an increased risk of developing tuberculosis, mycoses, and other severe infections.We report the case of a 68-year-old man with Crohn's disease, who developed  disseminated tuberculosis due to anti-TNF-α immunosuppressive therapy. The diagnosis was based on the histopathological findings and molecular biology assays.We discuss the clinical presentation and workup of this case, and we present a comparative analysis of tuberculosis cases associated with anti-TNF-α reported in Colombia during the last 10 years emphasizing on the diagnosis and treatment of latent tuberculosis.

Tuberculosis asociada a antagonistas del factor de necrosis tumoral alfa, presentación de un caso y análisis de los casos reportados en Colombia / Tuberculosis associated with tumor necrosis factor-α antagonists, case description and analysis of reported cases in Colombia

Resumen El factor de necrosis tumoral alfa (FNTα) es una citocina fundamental en la reacción inmunitaria frente al cáncer y a infecciones tales como la tuberculosis. Esta molécula también desempeña un papel fundamental en la patogenia de enfermedades complejas y de difícil tratamiento, como la artritis reumatoidea, la espondilitis anquilosante, la enfermedad de Crohn, la psoriasis y la colitis ulcerativa, condiciones que suelen requerir el uso de medicamentos que antagonizan la función del factor de necrosis tumoral alfa, el cual se ha relacionado con un incremento del riesgo de desarrollar tuberculosis, micosis y otras infecciones graves. Se reporta el caso de un hombre de 68 años de edad con diagnóstico de enfermedad de Crohn, a quien se le administró tratamiento con antagonistas del FNTα, debido a lo cual desarrolló tuberculosis diseminada. El diagnóstico se hizo con base en los hallazgos histológicos y mediante pruebas de biología molecular. Se discuten la presentación clínica y el manejo del caso, y se hace un análisis comparativo de los casos de tuberculosis asociados al tratamiento con antagonistas del FNTα reportados en Colombia durante los últimos diez años, con especial énfasisen la detección y el tratamiento de la tuberculosis latente.

Abstract Tumor necrosis factor-α (TNF-α) is an important fundamental cytokine during the immune response against cancer and infections such as tuberculosis. This molecule also plays a key pathogenic role in complex and difficult-to-treat diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and ulcerative colitis. The treatment of these diseases frequently needs TNF-αantagonists, which has been related to an increased risk of developing tuberculosis, mycoses, and other severe infections. We report the case of a 68-year-old man with Crohn's disease, who developed disseminated tuberculosis due to anti-TNF-α immunosuppressive therapy. The diagnosis was based on the histopathological findings and molecular biology assays. We discuss the clinical presentation and workup of this case, and we present a comparative analysis of tuberculosis cases associated with anti-TNF-α reported in Colombia during the last 10 years emphasizing on the diagnosis and treatment of latent tuberculosis.

Microcalcificaciones cerebrales en un recién nacido con tuberculosis congénita / Cerebral microcalcifications in a newborn with congenital tuberculosis

La tuberculosis es un problema grave de salud pública a nivel mundial. La Organización Mundial de la Salud estimaba que en el 2012 se habían presentado 8,6 millones de casos nuevos y 1,3 millones de muertes a causa de la enfermedad. En Colombia, la incidencia en 2011 fue de 24 casos por 100.000 habitantes. No hay información sobre la tuberculosis en las mujeres gestantes y la infección congénita se considera una enfermedad rara, de difícil diagnóstico, que genera alta mortalidad y puede confundirse con la adquirida después del nacimiento. La tuberculosis se ha relacionado con la infección por el virus de la inmunodeficiencia humana en madres y neonatos. Por otra parte, los casos de sífilis congénita han aumentado en el mundo y, en Colombia, la prevalencia es de 2,5 casos por 1.000 nacimientos, en tanto que, en el Instituto Materno Infantil-Hospital La Victoria, la frecuencia es de un caso por 57 nacimientos. Se presenta el caso de un recién nacido en tratamiento para sífilis congénita que presentó microcalcificaciones detectadas en una ecografía transfontanelar, lo que alertó sobre la existencia de otro agente infeccioso. La prueba de PCR fue negativa para citomegalovirus, así como los títulos de IgM para toxoplasma, rubéola y herpes I y II. Dado el antecedente de un tratamiento incompleto para tuberculosis en la mujer gestante, se sospechó la presencia de infección por el bacilo de la tuberculosis. No se encontraron bacilos ácido-alcohol resistentes en tres muestras de jugo gástrico, y la prueba de PCR-IS 6110 fue positiva en líquido cefalorraquídeo y en orina, pero no en sangre. El recién nacido recibió tratamiento con penicilina cristalina durante 10 días, así como con isoniacida, rifampicina, pirazinamida y estreptomicina. Actualmente se le hace seguimiento clínico.

Tuberculosis is a serious public health problem worldwide. In 2012, the World Health Organization estimated 8.6 million new cases and 1.3 million deaths due to the disease. In 2011, the incidence in Colombia was 24 cases per 100,000 inhabitants. There is little information about tuberculosis in pregnant women, and congenital infection is considered a rare disease that is difficult to diagnose, leads to high mortality, and may be confused with tuberculosis acquired after birth. In addition, it has been associated with HIV infection in mothers and infants. Moreover, there is increasing incidence of congenital syphilis in the world. In Colombia, the prevalence is 2.5 cases per 1,000 births and its frequency in the Instituto Materno Infantil-Hospital La Victoria is one case per 57 births. We report the case of a newborn under treatment for congenital syphilis and in whom microcalcifications were found in a transfontanelar ultrasound. This finding warned about the existence of another infectious agent. PCR was negative for cytomegalovirus, and IgM titers for toxoplasma, rubella and herpes I and II were also negative. After learning about a history of incomplete treatment for tuberculosis in the mother, we suspected the presence of an infection by the tubercle bacillus in the newborn. No acid-fast bacilli were demonstrated in three gastric juice samples. The IS 6110 PCR assay was found positive in cerebrospinal fluid and urine, but not in blood. The newborn was treated with crystalline penicillin for 10 days along with isoniazid, rifampicin, pyrazinamide and streptomycin. The patient is currently under clinical monitoring.

Mal de Pott en un indígena colombiano / Pott´s disease in a Colombian indigenous man

Cada año mueren alrededor de dos millones de personas a causa de la tuberculosis y se estima que un tercio de la población mundial está infectada con el bacilo que la causa, pero solo entre 5 y 10 % desarrolla la enfermedad. El riesgo de que la enfermedad progrese al estado activo depende de factores endógenos y exógenos. Las comunidades indígenas son un grupo con un alto riesgo de infectarse y enfermar de tuberculosis; además de factores como el aislamiento geográfico, el abandono social y cultural y la desnutrición, se han identificado en ellos polimorfismos genéticos que los hacen más propensos a la infección. La tuberculosis vertebral es la forma más destructiva de la enfermedad y representa cerca de la mitad de los casos de tuberculosis esquelética. Se presenta el caso de un paciente indígena colombiano con tuberculosis vertebral y resultado negativo para HIV. El diagnóstico se basó en los hallazgos clínicos y en los estudios de imaginología, y se confirmó mediante la prueba molecular rápida Genotype MTBDR plus ® y de la reacción en cadena de la polimerasa PCR IS6110; el cultivo fue negativo a las 16 semanas de incubación. Se discuten brevemente la patogénesis, el diagnóstico y el tratamiento, y se comentan algunos aspectos relacionados con la situación de la tuberculosis en las comunidades indígenas colombianas.

Approximately 2 million people die each year from tuberculosis. One third of the world´s population is estimated to be infected with the tuberculosis bacillus, although only 5-10% will develop the disease in their lifetime. The disease progression risk depends on endogenous and exogenous factors. Indigenous communities are a high-risk group for infection and development of tuberculosis. In addition to factors such as geographical isolation, social and cultural neglect and malnutrition, susceptibility to genetic polymorphisms has been identified in them. Spinal tuberculosis is the most destructive form of the disease, which represents approximately half of all cases of skeletal tuberculosis. The case of an HIV negative, indigenous Colombian man is presented. His diagnosis was done based on clinical and image findings, and it was confirmed with the rapid molecular assay Genotype MTBDRplus ® and IS6110 PCR.The culture in solid media was negative after 16 weeks. We briefly discuss the pathogenesis, diagnosis and treatment. Finally, we comment on some aspects of the situation of tuberculosis among indigenous Colombian communities.

[Phenotypic and genotypic diagnosis of bone and miliary tuberculosis in an HIV+ patient in Bogotá, Colombia]. / Diagnóstico genotípico y fenotípico de tuberculosis ósea y miliar en un paciente positivo para HIV en Bogotá, Colombia.

Tuberculosis is the single most frequent cause of death by an infectious agent worldwide. Diagnosis of extra-pulmonary tuberculosis is not always possible through conventional methods, due to the long time required for cultures and the paucibacillary nature of samples; hence the need of rapid molecular methods. HIV infection increases the risk of tuberculosis, and HIV/tuberculosis coinfection is associated with higher mortality. We describe the case of a 56-year old mestizo male patient suspected of having tuberculosis who consulted the San Ignacio Hospital in Bogotá with a two-month history of a painful ulcerated lesion over the distal third area of the right forearm and in whom HIV coinfection was confirmed. Bone and pulmonary histological examination evidenced multiple granulomas, giant cells and fibrosis. Cultures and IS6110-PCR from lung and bone tissues were positive for Mycobacterium tuberculosis complex. Mycobacterium tuberculosis isolates were sensitive to first line drugs.

Diagnóstico genotípico y fenotípico de tuberculosis ósea y miliar en un paciente positivo para HIV en Bogotá, Colombia / Phenotypic and genotypic diagnosis of bone and miliary tuberculosis in an HIV+ patient in Bogotá, Colombia

La tuberculosis se considera la causa más frecuente de muerte producida por un solo agente infeccioso. El diagnóstico de la tuberculosis extrapulmonar no siempre es posible mediante los métodos convencionales debido al lento crecimiento del bacilo y a la naturaleza paucibacilar de las muestras, por lo que es necesario recurrir a las técnicas moleculares. El riesgo de tuberculosis, así como la mortalidad, aumenta en los pacientes con infección por HIV, en quienes el compromiso extrapulmonar es más frecuente. Se describe el caso de un hombre mestizo de 56 años de edad con sospecha de padecer tuberculosis, que asistió a consulta en el Hospital San Ignacio de Bogotá y relató haber tenido dolor en una lesión ulcerada localizada en el tercio distal del antebrazo derecho durante los dos meses anteriores y en quien se confirmó la infección por HIV. El examen histológico de los tejidos óseo y pulmonar demostró la presencia de granulomas múltiples, células gigantes y fibrosis. Tanto los cultivos como la reacción en cadena de la polimerasa en la secuencia de inserción 6110 ( insertion sequence , IS6110) fueron positivos. Los aislamientos de Mycobacterium tuberculosis recuperados fueron sensibles a los medicamentos antituberculosos de primera línea.

Tuberculosis is the single most frequent cause of death by an infectious agent worldwide. Diagnosis of extra-pulmonary tuberculosis is not always possible through conventional methods, due to the long time required for cultures and the paucibacillary nature of samples; hence the need of rapid molecular methods. HIV infection increases the risk of tuberculosis, and HIV/tuberculosis coinfection is associated with higher mortality. We describe the case of a 56-year old mestizo male patient suspected of having tuberculosis who consulted the San Ignacio Hospital in Bogotá with a two-month history of a painful ulcerated lesion over the distal third area of the right forearm and in whom HIV coinfection was confirmed. Bone and pulmonary histological examination evidenced multiple granulomas, giant cells and fibrosis. Cultures and IS6110-PCR from lung and bone tissues were positive for Mycobacterium tuberculosis complex. Mycobacterium tuberculosis isolates were sensitive to first line drugs.

[Pott's disease in a Colombian indigenous man]. / Mal de Pott en un indígena colombiano.

Approximately 2 million people die each year from tuberculosis. One third of the world´s population is estimated to be infected with the tuberculosis bacillus, although only 5-10% will develop the disease in their lifetime. The disease progression risk depends on endogenous and exogenous factors. Indigenous communities are a high-risk group for infection and development of tuberculosis. In addition to factors such as geographical isolation, social and cultural neglect and malnutrition, susceptibility to genetic polymorphisms has been identified in them. Spinal tuberculosis is the most destructive form of the disease, which represents approximately half of all cases of skeletal tuberculosis. The case of an HIV negative, indigenous Colombian man is presented. His diagnosis was done based on clinical and image findings, and it was confirmed with the rapid molecular assay Genotype MTBDRplus® and IS6110 PCR.The culture in solid media was negative after 16 weeks. We briefly discuss the pathogenesis, diagnosis and treatment. Finally, we comment on some aspects of the situation of tuberculosis among indigenous Colombian communities.

A first insight on the population structure of Mycobacterium tuberculosis complex as studied by spoligotyping and MIRU-VNTRs in Bogotá, Colombia.

With an incidence of 25.6/100,000 in 2008, tuberculosis (TB) remains an important public health problem in Colombia. In this study, a total of 152 Mycobacterium tuberculosis complex strains isolated in Bogotá, Colombia between years 1995 and 2007 were genotyped by spoligotyping and 12-loci MIRU-VNTRs. The various spoligotyping-based genotypic lineages in our sample were: Latin American & Mediterranean (LAM) n=75, 49.34%; Haarlem, n=38, 25.0%; ill-defined T group, n=21, 13.82%; S family, n=5, 3.29%; X clade, n=2, 1.32%; Beijing, n=1, 0.65%, while strains with unknown signatures (n=10) represented 6.58% of isolates. Using spoligotyping as a first molecular marker and MIRU-VNTRs as second marker, we obtained 102 single patterns and 14 clustered patterns (n=52 strains from 49 patients, 2-8 strains per cluster). The MIRU-VNTRs patterns corresponded to 50 MITs for 109 strains and 43 orphan patterns. The most frequent patterns were MIT190 (n=12), MIT45 (n=10), and MIT25 (n=9). The Hunter & Gaston discriminatory index (HGDI) of both methodologies used together showed a value of 0.992. In our setting, the HGDI of five loci subset (MIRU10, 16, 23, 26 and 40) contributed most to the discriminatory power of 12-loci format used (HGDI=0.977). The lineage distribution of M. tuberculosis showed that more than 3/4 of strains in Bogotá are commonly found in Latin America, Caribbean, and Europe. This observation might reflect the shared post-Columbus history of Colombia and its Latin-American neighbors as well as strains brought in by 20th century immigrants from Europe. We also demonstrate the usefulness of MIRU-VNTR to detect suspected links among patients and polyclonal infections.

Genome sequence of the Mycobacterium colombiense type strain, CECT 3035.

We report the first whole-genome sequence of the Mycobacterium colombiense type strain, CECT 3035, which was initially isolated from Colombian HIV-positive patients and causes respiratory and disseminated infections. Preliminary comparative analyses indicate that the M. colombiense lineage has experienced a substantial genome expansion, possibly contributing to its distinct pathogenic capacity.

First case of multidrug-resistant tuberculosis caused by a rare "Beijing-like" genotype of Mycobacterium tuberculosis in Bogotá, Colombia.

This report describes a first case due to a genetically distinct and relatively rare "Beijing-like" strain of Mycobacterium tuberculosis isolated from a 15 years old female patient who died shortly after the initiation of antituberculous therapy with second-line drugs. Positive cultures obtained from lung, kidney and adrenal glands upon autopsy were identified as Mycobacterium tuberculosis complex characterized by an identical 15-banded IS6110-RFLP pattern, and were found to be resistant to all the 4 first-line antituberculous drugs tested (rifampin, isoniazid, ethambutol and streptomycin). Spoligotyping followed by comparison with the SITVIT2 database revealed that the isolate belonged to a rare pattern identified as Spoligotype International Type SIT190, which represents only 1.7% of all the Beijing strains worldwide. We present data on its worldwide distribution and present an evolutionary scenario based on available MIRU typing data.

[Molecular epidemiology of tuberculosis in Bogotá in clinical isolates obtained over an 11-year period]. / Epidemiología Molecular de la Tuberculosis en Bogotá en Aislados Clínicos obtenidos durante 11 Años.

OBJECTIVE: Characterising clinical Mycobacterium tuberculosis isolates obtained from 1995 to 2006 in Bogotá , Colombia , using standardised IS6110-based RFLP typing for determining phylogenetic relationships. Calculating cases due to recent infection (grouped cases) cf endogenous reactivation (single patterns). METHODS: This retrospective study characterised 137 clinical Mycobacterium tuberculosis isolates obtained in Bogotá from 1995 to 2006. Study variables consisted of gender, age, HIV status, homelessness, Ziehl Neelsen smear result and date of culture. All isolates were freshly identified by phenotypic methods, confirmed by PRA and evaluated for susceptibility to antimicrobial agents according to the proportional method. Mycobacterium tuberculosis cultures were typed by standardised IS6110-based RFLP. RESULTS: All isolates were confirmed as being M. tuberculosis by phenotypic and genotypic methods. 9,4 % monoresistance and 2,9 % MDR (rifampicin- and isoniazid-resistant) were found. 129 M . tuberculosis isolates were genotyped; 96 (74 %) of them presented unique DNA fingerprints, whilst 35 (26 %) were grouped into 17 clusters consisting of two to four isolates. Direct epidemiological links between patients could not be established in most cases. Only HIV status was a significant predictor of clustering amongst the variables being studied (p<0.05). CONCLUSION: The results of our study revealed a high proportion of unique DNA fingerprints, suggesting high genetic variability between M. tuberculosis strains in Bogotá , Colombia , meaning that most cases of TB in this study were attributed to endogenous reactivation.

Epidemiología Molecular de la Tuberculosis en Bogotá en Aislados Clínicos obtenidos durante 11 Años / Molecular epidemiology of tuberculosis in Bogotá in clinical isolates obtained over an 11-year period

Objetivo: Tipificar molecularmente aislados clínicos de Mycobacterium tuberculosis obtenidas en Bogotá entre los años 1995 a 2006, mediante la técnica RFLP-IS6110 para establecer las relaciones filogenéticas existentes entre ellos y determinar casos debidos a infección reciente (casos agrupados) Vs reactivaciones endógenas (patrones únicos). Métodos: Se realizó un estudio retrospectivo, en el que se incluyeron 137 aislados clínicos pertenecientes al complejo Mycobacterium tuberculosis, obtenidos en Bogotá entre los años 1995 a 2006. Las variables estudiadas para cada paciente fueron: sexo, edad, confección con el Virus de la Inmunodeficiencia Humana , habitante en situación de calle, resultado de la baciloscopia, fecha del cultivo. Los aislados fueron identificados fenotípicamente y confirmados genotípicamente mediante el método molecular PRA y evaluados para sensibilidad a fármacos antituberculosos de primera línea utilizando el método simplificado de las proporciones múltiples. La genotipificación se realizó empleando el método de referencia RFLP-IS6110 (Polimorfismo en la Longitud de los Fragmentos de restricción). Resultados: Todos los aislados fueron confirmados como pertenecientes al complejo M. tuberculosis, mostrando la identificación fenotípica una concordancia del 100 por ciento con la identificación genotípica. La monorresistencia encontrada fue de 9,4 por ciento, y la MDR (resistencia a Rifampicina e Isoniazida) fue 2,9 por ciento. La genotipificación se realizó a 129 aislados, de los cuales 96 (74 por ciento) mostraron diferentes patrones RFLP-IS6110 y 35 aislados (26 por ciento) estuvieron agrupados en 17 clusters conformados por 2 a 4 aislados. La relación epidemiológica entre los pacientes no pudo ser establecida en la mayoría de los casos. De las variables estudiadas solamente el estado de coinfección con VIH fue un predictor significativo para el agrupamiento (p<0.05). Conclusión: Los resultados de nuestro estudio muestran un alto porcentaje de genotipos con patrones RFLP-IS6110 únicos, lo que sugiere gran variabilidad genética entre los aislados de M. tuberculosis circulantes en Bogotá, indicando que la mayoría de los casos de tuberculosis en el estudio pueden ser atribuibles a reactivaciones endógenas.

Objective: Characterising clinical Mycobacterium tuberculosis isolates obtained from 1995 to 2006 in Bogotá , Colombia , using standardised IS6110-based RFLP typing for determining phylogenetic relationships. Calculating cases due to recent infection (grouped cases) cf endogenous reactivation (single patterns). Methods: This retrospective study characterised 137 clinical Mycobacterium tuberculosis isolates obtained in Bogotß from 1995 to 2006. Study variables consisted of gender, age, HIV status, homelessness, Ziehl Neelsen smear result and date of culture. All isolates were freshly identified by phenotypic methods, confirmed by PRA and evaluated for susceptibility to antimicrobial agents according to the proportional method. Mycobacterium tuberculosis cultures were typed by standardised IS6110-based RFLP. Results: All isolates were confirmed as being M. tuberculosis by phenotypic and genotypic methods. 9,4 percent monoresistance and 2,9 percent MDR (rifampicin- and isoniazid-resistant) were found. 129 M . tuberculosis isolates were genotyped; 96 (74 percent) of them presented unique DNA fingerprints, whilst 35 (26 percent) were grouped into 17 clusters consisting of two to four isolates. Direct epidemiological links between patients could not be established in most cases. Only HIV status was a significant predictor of clustering amongst the variables being studied (p<0.05). Conclusion: The results of our study revealed a high proportion of unique DNA fingerprints, suggesting high genetic variability between M. tuberculosis strains in Bogotß , Colombia , meaning that most cases of TB in this study were attributed to endogenous reactivation.

Molecular features of Mycobacterium avium human isolates carrying a single copy of IS1245 and IS1311 per genome.

Human clinical isolates of the Mycobacterium avium complex, from hospitals in Bogotá, were studied using a wide range of molecular tests including PCR restriction-enzyme analysis (PRA) of the hsp65 gene. Up to 21 of the isolates were identified as M. avium PRA variant III (Mav III), a variant obtained only from isolates on the American continent. In contrast to previous reports, restriction fragment length polymorphism analysis using IS1245 and IS1311 showed a single copy for each insertion sequence (IS) in the majority (19/21) of the Colombian Mav III isolates under study. In order to analyse whether the ISs were inserted in a relevant genomic region, experimental conditions were established to determine the insertion loci of each single copy of both ISs in the genome. Analysis of genomic insertion loci indicated that both IS1245 and IS1311 were present in areas containing putatively truncated integrases and/or transposases, which may have an influence on the mobility of the inserted IS. In addition, a conserved genomic region was identified for the insertion of IS1311; this region could be part of the IS1311 itself.

[Mycobacteria-HIV/AIDS association in patients attending a teaching-hospital in Bogotá, Colombia]. / Asociación Micobacterias-VIH/SIDA en Pacientes Atendidos en un Hospital Universitario en Bogotá, Colombia.

OBJECTIVE: Evaluating the frequency of mycobacterium infection in an HIV-positive population and its influence on medium-term survival, along with clinical and epidemiological factors associated with co-infection. METHODS: Several clinical specimens were studied for mycobacteria in a sample of 92 HIV+ patients at the San Juan de Dios teaching-hospital in Bogota, Colombia, during 1996. Factors associated with infection were measured using a prevalence ratio (PR), CI=95%, and logistic regression was used in the multivariable models. The likelihood of survival for three months was measured using Kaplan-Meir curves and factors associated with survival were assessed using Rate ratios and Cox's model. RESULTS: Eight percent of the patients had tuberculosis and 6% of them were found to be infected with atypical mycobacterium. Mycobacterium avium complex (MAC) was the most frequent species, followed by M. fortuitum and M. chelonae. Mixed infections with M. tuberculosis and MAC were diagnosed in one patient and two different species of atypical mycobacterium were isolated in other two cases. Patients suffering from tuberculosis and stages III or IV HIV infection had a 16% survival rate. CONCLUSIONS: Tuberculosis-HIV/AIDS frequency and atypical Mycobacterium-HIV/AIDS' association were very similar. The most frequently isolated atypical mycobacterium specie in this study was MAC. Survival rate was lower for patients infected by M. tuberculosis and even lower when a multi-resistant strain was involved. The most important clinical factor associated with M. tuberculosis was the presence of fever and loss of weight with mycobacterial infection. Blood provided the best samples for isolating mycobacteria.

Asociación Micobacterias-VIH/SIDA en Pacientes Atendidos en un Hospital Universitario en Bogotá, Colombia / Mycobacteria-HIV/AIDS association in patients attending a teaching-hospital in Bogotá, Colombia

Objetivo El objetivo del presente estudio fue evaluar la frecuencia de infecciones micobacterianas en pacientes infectados por el VIH y su repercusión en la sobreviva de los pacientes a mediano plazo, además los factores clínicos y epidemiológicos asociados a la coinfección. Métodos La frecuencia de infección micobacteriana se determinó en una cohorte de 92 pacientes VIH positivos en el Hospital San Juan de Dios en Bogotá (Colombia), en 1996, mediante el empleo de métodos bacteriológicos estandarizados a partir de diferentes muestras clínicas. Los factores asociados con la enfermedad fueron medidos utilizando las razones de prevalencia RP IC= 95 por ciento y la regresión logística como método multivariado. La probabilidad de sobrevivir a tres meses fue evaluada usando las curvas de Kaplan Meir y se midieron los factores asociados con la sobrevida usando razones de tasas de incidencia y el modelo de Cox Resultados El 8 por ciento de los pacientes tenían tuberculosis y el 8,6 por ciento estaba infectado con micobacterias atípicas, siendo el complejo Mycobacterium avium (CMA), el más frecuente, seguido por M. fortuitum y M. chelonae. En un paciente se encontró M. tuberculosis y CMA y en otros dos se encontraron dos especies diferentes de micobacterias atípicas. Pacientes con tuberculosis, en estadios III y IV de enfermedad VIH tenían una sobreviva del 16 por ciento. Conclusiones La frecuencia de la asociación Tuberculosis/VIH-SIDA y Micobacteriosis/VIH-SIDA fue similar. La micobacteria atípica aislada con mayor frecuencia fue CMA. La sobrevida disminuyó en los pacientes con tuberculosis siendo menor si la cepa era multirresistente. El hallazgo clínico mas frecuentemente asociado a la tuberculosis fue la fiebre y la pérdida de peso se asoció a la presencia de micobacteriosis. La sangre fue la mejor muestra para el aislamiento de micobacterias.

Objective Evaluating the frequency of mycobacterium infection in an HIV-positive population and its influence on medium-term survival, along with clinical and epidemiological factors associated with co-infection. Methods Several clinical specimens were studied for mycobacteria in a sample of 92 HIV+ patients at the San Juan de Dios teaching-hospital in Bogota, Colombia, during 1996. Factors associated with infection were measured using a prevalence ratio (PR), CI=95 percent, and logistic regression was used in the multivariable models. The likelihood of survival for three months was measured using Kaplan-Meir curves and factors associated with survival were assessed using Rate ratios and Cox's model. Results Eight percent of the patients had tuberculosis and 6 percent of them were found to be infected with atypical mycobacterium. Mycobacterium avium complex (MAC) was the most frequent species, followed by M. fortuitum and M. chelonae. Mixed infections with M. tuberculosis and MAC were diagnosed in one patient and two different species of atypical mycobacterium were isolated in other two cases. Patients suffering from tuberculosis and stages III or IV HIV infection had a 16 percent survival rate. Conclusions Tuberculosis-HIV/AIDS frequency and atypical Mycobacterium-HIV/AIDS' association were very similar. The most frequently isolated atypical mycobacterium specie in this study was MAC. Survival rate was lower for patients infected by M. tuberculosis and even lower when a multi-resistant strain was involved. The most important clinical factor associated with M. tuberculosis was the presence of fever and loss of weight with mycobacterial infection. Blood provided the best samples for isolating mycobacteria.

Mycobacterium colombiense sp. nov., a novel member of the Mycobacterium avium complex and description of MAC-X as a new ITS genetic variant.

Forty-five mycobacterial strains isolated from 23 Colombian HIV-positive patients were identified as members of the Mycobacterium avium complex (MAC) and were characterized using different molecular approaches. Seven of the isolates showed characteristic features that allowed them to be differentiated from other members of the complex. The isolates had a novel 16S-23S rRNA internal transcribed spacer (ITS 1) gene sequence which is described as a new sequevar, MAC-X. All of the seven novel isolates gave a positive result with the MAC-specific AccuProbe (Gen-Probe), but tested negative for Mycobacterium avium and Mycobacterium intracellulare species-specific probes (64 and 100 % of the isolates, respectively). The novel isolates could be differentiated phenotypically from other members of the MAC on the basis of the production of urease and by a consistent mycolic acid pattern. The novel isolates shared some characteristics with M. avium, such as the avium variant I (av-I) pattern of the hsp65 gene as determined by PCR restriction analysis and a positive PCR result for the mig (macrophage-induced) gene. However, the novel isolates showed a unique 16S rRNA gene sequence. DNA-DNA relatedness values, from 24 to 44 %, confirmed the distinction of the novel isolates from other members of the MAC at the genetic level and their status as members of a separate species. The novel isolates are proposed as representatives of a novel species, Mycobacterium colombiense sp. nov., that is closely related to M. avium within the MAC. The type strain is 10B(T) (=CIP 108962(T)=CECT 3035(T)).

Evaluacion Clinica, imaginologia e inmunologia del absceso hepatico / Clinical, imaginological and immunological diagnostic of liver abcess

Introducción: Entamoeba histolytica es un parásito de distribución mundial. Se estima que el parásito infecta alrededor de 500 millones de personas anualmente y que de ellos 110.000 mueren por complicaciones causadas por. E. histolytica. Las personas infectadas por el parásito se pueden dividir en dos poblaciones diferentes de acuerdo a sus manifestaciones clínicas. En la primera son asintomáticos y está conformada por un 90 por ciento y en la segunda son los sintomáticos representada con un 10 por ciento que manifiestan la enfermedad principalmente como disentería amebiana y como amebiasis extra-intestinal. Objetivo: establecer la capacidad discriminatoria de la clínica, de laboratorio e imagenología en la etiología del absceso hepático. Metodología: estudio prospectivo, descriptivo, abierto, de evaluación clínica y de tecnologías diagnósticas para diferenciar etiología. Resultados: se evaluaron 61 pacientes (durante 46 meses), y se excluyeron 12. Se encontraron 29 casos de AHA (59 por ciento), 16 casos de AHNA (33 por ciento) y 4 mixtos (8 por ciento). Promedio de edad para AHA fue 36 y 45 para AHNA, la relación hombre.-mujer fue 24/5 y 11/5 respectivamente. No se diferencia la etiología por cuadro clínico, examen físico, imagenología o laboratorios (CH, VSG, función hepática) excepto por la mayor prolongación del PT en caso de AHNA. En caso de AHA las pruebas de ELISA fueron positivas en 100 por ciento, las de 10 en 93 por ciento y en el Inmunoblot el orden de frecuencia de las bandas fue: 38 kDa (93 por ciento), 42 kDa (90 por ciento), 80 kDa (86 por ciento), 116 kDa (76 por ciento), 50 kDa (24 por ciento), 97 kDa (17 por ciento), 22.5 kDa (14 por ciento), 45 kDa (10 por ciento), 31 kDa (3 por ciento). Las pruebas de ELISA e ID fueron positivas en todos los pacientes con absceso hepático mixto y en el Inmunoblot el orden de frecuencia de las bandas fue: 80 y 38 kDa (100 por ciento), 42 kDa (75 por ciento), 11 kDa (50 por ciento), 50 y 22.5 kDa (25 por ciento)...