RESUMEN
We have reported previously that T cells from patients with multi-drug-resistant tuberculosis (MDR-TB) express high levels of interleukin (IL)-17 in response to the MDR strain M (Haarlem family) of Mycobacterium tuberculosis (M. tuberculosis). Herein, we explore the pathways involved in the induction of Th17 cells in MDR-TB patients and healthy tuberculin reactors [purified protein derivative healthy donors (PPD+ HD)] by the M strain and the laboratory strain H37Rv. Our results show that IL-1ß and IL-6 are crucial for the H37Rv and M-induced expansion of IL-17+ interferon (IFN)-γ- and IL-17+ IFN-γ+ in CD4+ T cells from MDR-TB and PPD+ HD. IL-23 plays an ambiguous role in T helper type 1 (Th1) and Th17 profiles: alone, IL-23 is responsible for M. tuberculosis-induced IL-17 and IFN-γ expression in CD4+ T cells from PPD+ HD whereas, together with transforming growth factor (TGF-ß), it promotes IL-17+ IFN-γ- expansion in MDR-TB. In fact, spontaneous and M. tuberculosis-induced TGF-ß secretion is increased in cells from MDR-TB, the M strain being the highest inducer. Interestingly, Toll-like receptor (TLR)-2 signalling mediates the expansion of IL-17+ IFN-γ- cells and the enhancement of latency-associated protein (LAP) expression in CD14+ and CD4+ T cells from MDR-TB, which suggests that the M strain promotes IL-17+ IFN-γ- T cells through a strong TLR-2-dependent TGF-ß production by antigen-presenting cells and CD4+ T cells. Finally, CD4+ T cells from MDR-TB patients infected with MDR Haarlem strains show higher IL-17+ IFN-γ- and lower IL-17+ IFN-γ+ levels than LAM-infected patients. The present findings deepen our understanding of the role of IL-17 in MDR-TB and highlight the influence of the genetic background of the infecting M. tuberculosis strain on the ex-vivo Th17 response.
Asunto(s)
Memoria Inmunológica , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Mycobacterium tuberculosis/inmunología , Células Th17/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Tuberculosis Pulmonar/inmunología , Adulto , Células Cultivadas , Farmacorresistencia Bacteriana Múltiple , Femenino , Humanos , Interferón gamma/metabolismo , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/genética , Transducción de Señal , Especificidad de la Especie , Células Th17/microbiología , Receptor Toll-Like 2/metabolismo , Tuberculosis Resistente a Múltiples Medicamentos/genética , Tuberculosis Pulmonar/microbiología , Adulto JovenRESUMEN
SETTING: Over 150 potentially pathogenic non-tuberculous mycobacteria (NTM) species have been described, posing an onerous challenge for clinical laboratory diagnosis. OBJECTIVE: To evaluate different approaches for the identification of 40 clinically relevant NTM isolates whose species were not reliably identified using our routine diagnostic workflow comprising phenotypic tests and hsp65 polymerase chain reaction restriction analysis. DESIGN: We used 1) sequencing analysis of four conserved gene targets: 16S rRNA, rpoB, hsp65 and sodA; 2) two commercial reverse hybridisation assays; and 3) protein analysis using matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). RESULTS: Combined, but not individual, sequence analysis allowed reliable species identification for 30/40 (75%) isolates, including species previously unknown to be circulating in Argentina. Commercial kits outperformed our routine identification in only 5/35 isolates, and misclassified many more. MALDI-TOF MS accurately identified species in 22/36 (61%) isolates and did not misidentify any. CONCLUSIONS: Commercial kits did not resolve the problem of species of NTM isolates that elude identification. Combined DNA sequence analysis was the approach of choice. MALDI-TOF MS shows promise as a powerful, rapid and accessible tool for the rapid identification of clinically relevant NTM in the diagnostic laboratory, and its accuracy can be maximised by building up a customised NTM spectrum database.
Asunto(s)
Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Micobacterias no Tuberculosas/genética , Micobacterias no Tuberculosas/aislamiento & purificación , Argentina , Proteínas Bacterianas/genética , Técnicas Bacteriológicas , Chaperonina 60/genética , ADN Bacteriano/genética , Genes Bacterianos , Humanos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Superóxido Dismutasa/genéticaRESUMEN
The ability of countries to control and eradicate bovine tuberculosis (TB) has been jeopardised by various epidemiological and ecological features of the disease. The authors have used epidemiological modelling to develop an analytical framework to assess the likely success of a national TB eradication programme in Argentina. Study results suggest that the current control programme is financially feasible in the long term. However, considering that the costs of the TB eradication programme in Argentina are entirely borne by the producer, the initial investment required and the long-term horizon needed to gain revenue may prevent producers from endorsing the programme. Regionalised programmes that allow differential control strategies to be implemented in specific regions may increase the likelihood of success. This methodological approach could be extended to design and evaluate control and eradication programmes for TB and other infectious diseases in other regions of the world.
Asunto(s)
Simulación por Computador , Modelos Biológicos , Tuberculosis Bovina/prevención & control , Animales , Argentina/epidemiología , Bovinos , Análisis por Conglomerados , Análisis Costo-Beneficio , Estudios de Factibilidad , Modelos Lineales , Prevalencia , Tuberculosis Bovina/economía , Tuberculosis Bovina/epidemiologíaRESUMEN
Tuberculosis cases infected by the same Mycobacterium tuberculosis (MTB) strain are considered to be clustered and involved in a transmission chain. Large clusters are assumed to represent active transmission chains in a population. In the present study, we focused on the analysis of large clusters defined by IS6110-restriction fragment length polymorphism (RFLP) typing in the immigrant population in Madrid. We identified 12 large clusters (involving 43% of the isolates) comprising 4-23 representatives. We proposed a gradient of epidemiological certainty for these large clusters. For a cluster to be considered robust and a good indicator of recent transmission, the MTB strain involved should not have been identified in a geographically and epidemiologically unrelated population and the cluster had to be re-confirmed by another highly discriminative molecular marker (MIRU-VNTR). The clusters that we discovered were classified into three categories: high, intermediate and low expected epidemiological value. In the largest cluster in the study (cluster M6; 23 representatives), failures by both criteria were identified: the representative seven-band RFLP pattern was also the most prevalent in the unrelated population (25 cases) and the cluster was fully split by MIRU-15, suggesting a lack of epidemiological value. The RFLP pattern representative of this cluster was also identified in 64 isolates from five countries in the Latin American genotype database, and again proved to be heterogeneous according to the MIRU-15 analysis. Specific analysis of large clusters, combined with the application of criteria for evaluating their robustness, could help identify uninformative clusters and target epidemiological resources towards those clusters with higher expected epidemiological value.
Asunto(s)
Emigrantes e Inmigrantes , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Tuberculosis/epidemiología , Tuberculosis/microbiología , Técnicas de Tipificación Bacteriana , Análisis por Conglomerados , Elementos Transponibles de ADN , ADN Bacteriano/genética , Humanos , Epidemiología Molecular , Tipificación Molecular , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo de Longitud del Fragmento de Restricción , España/epidemiologíaRESUMEN
The advent of Mycobacterium tuberculosis strain genotyping has allowed differentiation between disease relapse and exogenous re-infection. We report here a remarkable case of multiply recurrent tuberculosis in a patient living with HIV. Between 1995 and 2009, a young HIV-infected intravenous drug user, who was reluctant to comply with anti-retroviral treatment, underwent at least five tuberculosis episodes caused by three distinct M. tuberculosis strains sharply differentiated by drug susceptibility profile, genotype and infectious source. Eventually, the patient died during a relapse of tuberculosis due to a notorious multidrug-resistant outbreak-strain, which infected him during a prolonged hospitalization in the epicentre of such outbreak. Whether recurrent tuberculosis is due to a new infection or to reactivation of a previous one is a century-long controversial question. In our patient, both conditions alternated throughout his 15 years of living with HIV. Cases such as this might not be exceptional in certain underprivileged suburban areas of Argentina and should raise concern over three pending issues in tuberculosis control policies, namely secondary preventing therapy, institutional infection control and patient follow-up throughout the health network system.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/complicaciones , Tuberculosis Pulmonar/complicaciones , Adulto , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Argentina , Técnicas de Tipificación Bacteriana , Elementos Transponibles de ADN/genética , ADN Bacteriano/análisis , Farmacorresistencia Bacteriana Múltiple , Resultado Fatal , Genotipo , Humanos , Masculino , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Cooperación del Paciente , Recurrencia , Factores de Riesgo , Factores de Tiempo , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/fisiopatología , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/fisiopatologíaRESUMEN
We describe the first outbreak of multidrug-resistant tuberculosis (MDR-TB) that occurred in Argentina among transvestite sex workers, and actions undertaken for its control. In Buenos Aires city, transmission was documented between 2001 and 2004 by conventional and molecular methods in a hotel where transvestites used to reside and work. The source case was traced back to 1998. Six secondary cases were diagnosed and treated. Thirty-two contacts were investigated. The outbreak strain had formerly caused nosocomial transmission in Rosario, a city 300 km from Buenos Aires. Our findings highlight the difficulties controlling MDR-TB in Argentina.
Asunto(s)
Brotes de Enfermedades , Trabajo Sexual , Travestismo , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Adulto , Argentina/epidemiología , Dermatoglifia del ADN , Genotipo , Humanos , MasculinoRESUMEN
OBJECTIVE: A multicentre evaluation was performed to assess two rapid low-cost methods, MTT (3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) and resazurin assays, for testing the susceptibility of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and streptomycin (SM). METHODS: Thirty coded M. tuberculosis strains were sent to seven laboratories located in Latin America, representing six countries. Each site performed the colorimetric assays, MTT and resazurin, blind for the first-line drugs RMP, INH, EMB and SM. The minimum inhibitory concentration results obtained were compared to the conventional proportion method on Lowenstein-Jensen medium. RESULTS: After establishing the breakpoint concentrations, excellent results were obtained for RMP, INH and EMB, with levels of specificity and sensitivity of between 96% and 99%. CONCLUSION: MTT and resazurin assays are promising, accessible new alternative methods for middle- and low-resource countries that need low-cost methods to perform rapid susceptibility testing of M. tuberculosis to key anti-tuberculosis drugs.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Colorantes , Farmacorresistencia Bacteriana , Humanos , Indicadores y Reactivos , América Latina , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/patogenicidad , Oxazinas , Valores de Referencia , Reproducibilidad de los Resultados , Sales de Tetrazolio , Tiazoles , Tuberculosis Pulmonar/tratamiento farmacológico , XantenosRESUMEN
Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP) and Double-Repetitive-Element-PCR (DRE-PCR). Two of these strains: IH1 (susceptible to isoniazid) and IH2 (resistant to isoniazid) were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/clasificación , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Adulto , Antituberculosos/administración & dosificación , Antituberculosos/uso terapéutico , Técnicas Bacteriológicas , Dermatoglifia del ADN , Quimioterapia Combinada , Contaminación de Equipos , Infecciones por VIH/complicaciones , Humanos , Isoniazida/administración & dosificación , Isoniazida/farmacología , Isoniazida/uso terapéutico , Masculino , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Selección Genética , Tuberculosis Resistente a Múltiples Medicamentos/complicaciones , Tuberculosis Resistente a Múltiples Medicamentos/prevención & control , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiologíaRESUMEN
Multidrug-resistant tuberculosis (TB) is an increasing problem worldwide, however only three cases have been previously described in transplant recipients, especially involving lung and heart transplant. We describe a case of multidrug-resistant TB in an allogenic bone marrow transplant recipient with good response to second-line therapy.
Asunto(s)
Trasplante de Médula Ósea/efectos adversos , Farmacorresistencia Bacteriana Múltiple , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/etiología , Antituberculosos/uso terapéutico , Femenino , Humanos , Persona de Mediana Edad , Mycobacterium tuberculosis/efectos de los fármacosRESUMEN
Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP) and Double-Repetitive-Element-PCR (DRE-PCR). Two of these strains: IH1 (susceptible to isoniazid) and IH2 (resistant to isoniazid) were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.
Se compararon cepas de Mycobacterium tuberculosis utilizando 2 procedimientos de ADN fingerprinting: polimorfismo de los fragmentos de restricción (RFLP) y Double-Repetitive-Element-PCR (DRE-PCR). Dos de las cepas: IH1 (susceptible a isoniazida) e IH2 (resistente a isoniazida) se recuperaron a partir de casos de tuberculosis pulmonar que ocurrieron en dos hermanos convivientes. La primera fue aislada en julio de 1999 y la segunda un año después. IH1 e IH2 mostraron el mismo patrón de bandas por ambos procedimientos. Estos resultados sugieren que la quimioprofilaxis con una sola droga puede ocasionalmente seleccionar mutantes resistentes, las cuales pueden causar enfermedad y ser reconocidas por estos procedimientos. Las cepas IH3, IH4 e IH5 fueron aisladas de 3 pacientes diferentes, y examinadas por probable contaminación cruzada dentro del laboratorio ya que fueron procesadas el mismo día, por el mismo operador y en la misma cabina de seguridad biológica. Nuevamente, las 3 cepas revelaron el mismo patrón de bandas por RFLP y por DRE-PCR, confirmando la sospecha. Los resultados de la DRE-PCR se obtuvieron luego de 8 horas de trabajo, sin necesidad de subcultivos. Esta técnica permitiría la rápida correción de pautas de tratamiento, evitando la exposición innecesaria de personas y bacterias a drogas antimicrobianas.
Asunto(s)
Adulto , Humanos , Masculino , Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/clasificación , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Antituberculosos/administración & dosificación , Antituberculosos/uso terapéutico , Técnicas Bacteriológicas , Dermatoglifia del ADN , Quimioterapia Combinada , Contaminación de Equipos , Infecciones por VIH/complicaciones , Isoniazida/administración & dosificación , Isoniazida/farmacología , Isoniazida/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa/métodos , Selección Genética , Tuberculosis Resistente a Múltiples Medicamentos/complicaciones , Tuberculosis Resistente a Múltiples Medicamentos/prevención & control , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiologíaRESUMEN
Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP) and Double-Repetitive-Element-PCR (DRE-PCR). Two of these strains: IH1 (susceptible to isoniazid) and IH2 (resistant to isoniazid) were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.
RESUMEN
In the last decade, an unprecedented genetic diversity has been disclosed among Mycobacterium tuberculosis strains found worldwide. However, well-conserved genotypes seem to prevail in areas with high incidence of tuberculosis. As this may be related to selective advantages, such as advanced mechanisms to circumvent [M. bovis Bacille Calmette-Guerin (BCG)-induced] host defence mechanisms, we investigated the influence of strain diversity on the course of experimental disease. Twelve M. tuberculosis strains, representing four major genotype families found worldwide today, and the laboratory strain H37Rv were each used to infect BALB/c mice by direct intratracheal injection. Compared with H37Rv, infections with Beijng strains were characterized by extensive pneumonia, early but ephemeral tumour necrosis factor-alpha (TNF-alpha) and inducible isoform of nitric oxide synthetase (iNOS) expression, and significantly higher earlier mortality. Conversely, Canetti strains induced limited pneumonia, sustained TNF-alpha and iNOS expression in lungs, and almost 100% survival. Strains of the Somali and the Haarlem genotype families displayed less homogeneous, intermediate rates of survival. Previous BCG vaccination protected less effectively against infection with Beijing strains than against the H37Rv strain. In conclusion, genetically different M. tuberculosis strains evoked markedly different immunopathological events. Bacteria with the Beijing genotype, highly prevalent in Asia and the former USSR, elicited a non-protective immune response in mice and were the most virulent. Future immunological research, particularly on candidate vaccines, should include a broad spectrum of M. tuberculosis genotypes rather than a few laboratory strains.
Asunto(s)
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Tuberculosis Pulmonar/microbiología , Animales , Vacuna BCG/administración & dosificación , Ensayo de Unidades Formadoras de Colonias , Citocinas/análisis , Susceptibilidad a Enfermedades , Genoma Bacteriano , Genotipo , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa de Tipo II , Polimorfismo de Longitud del Fragmento de Restricción , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/patologíaRESUMEN
Fifteen episodes of Mycobacterium tuberculosis laboratory cross-contamination suspected between 1996 and 2001 at 6 laboratories in Buenos Aires City and suburbs were investigated by IS6110 RFLP. Thirteen episodes were confirmed. Even though BACTEC 460 produced the highest number of confirmed episodes in a single laboratory, the most extended one occurred while employing conventional culture procedures in solid medium. The double repetitive element-polymerase chain reaction (DRE-PCR) was applied to 8 of these episodes and produced concordant results with those of the RFLP. The DRE-PCR appears to be a valuable tool for the prompt identification of false positive cultures. The timely rectification of defects in laboratory protocols can avert false diagnoses of tuberculosis and unnecessary prolonged treatments.
Asunto(s)
Técnicas Bacteriológicas , ADN Bacteriano/análisis , Contaminación de Equipos , Laboratorios de Hospital , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Tuberculosis/diagnóstico , Aerosoles , Argentina , Técnicas Bacteriológicas/instrumentación , Medios de Cultivo , Reacciones Falso Positivas , Humanos , Radiometría/instrumentación , Manejo de Especímenes , Esterilización/métodosRESUMEN
Fifteen episodes of Mycobacterium tuberculosis laboratory cross-contamination suspected between 1996 and 2001 at 6 laboratories in Buenos Aires City and suburbs were investigated by IS6110 RFLP. Thirteen episodes were confirmed. Even though BACTEC 460 produced the highest number of confirmed episodes in a single laboratory, the most extended one occurred while employing conventional culture procedures in solid medium. The double repetitive element-polymerase chain reaction (DRE-PCR) was applied to 8 of these episodes and produced concordant results with those of the RFLP. The DRE-PCR appears to be a valuable tool for the prompt identification of false positive cultures. The timely rectification of defects in laboratory protocols can avert false diagnoses of tuberculosis and unnecessary prolonged treatments.
Asunto(s)
Humanos , Técnicas Bacteriológicas , ADN Bacteriano , Contaminación de Equipos , Laboratorios de Hospital , Mycobacterium tuberculosis , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa/métodos , Tuberculosis , Aerosoles , Argentina , Medios de Cultivo , Esterilización/métodos , Reacciones Falso Positivas , Radiometría , Manejo de Especímenes , Técnicas Bacteriológicas/instrumentaciónRESUMEN
Fifteen episodes of Mycobacterium tuberculosis laboratory cross-contamination suspected between 1996 and 2001 at 6 laboratories in Buenos Aires City and suburbs were investigated by IS6110 RFLP. Thirteen episodes were confirmed. Even though BACTEC 460 produced the highest number of confirmed episodes in a single laboratory, the most extended one occurred while employing conventional culture procedures in solid medium. The double repetitive element-polymerase chain reaction (DRE-PCR) was applied to 8 of these episodes and produced concordant results with those of the RFLP. The DRE-PCR appears to be a valuable tool for the prompt identification of false positive cultures. The timely rectification of defects in laboratory protocols can avert false diagnoses of tuberculosis and unnecessary prolonged treatments.(AU)
Asunto(s)
Humanos , Técnicas Bacteriológicas , ADN Bacteriano/análisis , Contaminación de Equipos , Laboratorios de Hospital , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Tuberculosis/diagnóstico , Aerosoles , Argentina , Técnicas Bacteriológicas/instrumentación , Medios de Cultivo , Reacciones Falso Positivas , Radiometría/instrumentación , Manejo de Especímenes , Esterilización/métodosRESUMEN
The frequency of Mycobacterium bovis detection in milk samples obtained from infected animals was explored in an intensive dairy area in Argentina. To this end, an "in house" polymerase chain reaction (PCR) method was developed using Mycobacterium tuberculosis Complex specific INS1-INS2 primers, and its performance was compared with that of bacteriological methods. The decontamination procedures previous to culture reduced M. bovis viability. The pathogen was identified in milk samples from 1 of 143 infected cows and in none of 43 uninfected ones. Even though PCR sensitivity was found to be 2-20 times higher than that of bacteriology in experimentally inoculated milk samples, all 186 field samples resulted negative by PCR, including the bacteriologically-confirmed one. In spite of the high prevalence of bovine tuberculosis in Argentinian dairy herds, the detection of M. bovis in milk is an unusual finding.
Asunto(s)
Técnicas Bacteriológicas , Leche/microbiología , Mycobacterium bovis/aislamiento & purificación , Tuberculosis Bovina/microbiología , Animales , Argentina/epidemiología , Bovinos , ADN Bacteriano/análisis , Femenino , Mycobacterium bovis/genética , Reacción en Cadena de la Polimerasa , Prevalencia , Sensibilidad y Especificidad , Tuberculosis Bovina/epidemiologíaRESUMEN
The frequency of Mycobacterium bovis detection in milk samples obtained from infected animals was explored in an intensive dairy area in Argentina. To this end, an [quot ]in house[quot ] polymerase chain reaction (PCR) method was developed using Mycobacterium tuberculosis Complex specific INS1-INS2 primers, and its performance was compared with that of bacteriological methods. The decontamination procedures previous to culture reduced M. bovis viability. The pathogen was identified in milk samples from 1 of 143 infected cows and in none of 43 uninfected ones. Even though PCR sensitivity was found to be 2-20 times higher than that of bacteriology in experimentally inoculated milk samples, all 186 field samples resulted negative by PCR, including the bacteriologically-confirmed one. In spite of the high prevalence of bovine tuberculosis in Argentinian dairy herds, the detection of M. bovis in milk is an unusual finding.
RESUMEN
The frequency of Mycobacterium bovis detection in milk samples obtained from infected animals was explored in an intensive dairy area in Argentina. To this end, an [quot ]in house[quot ] polymerase chain reaction (PCR) method was developed using Mycobacterium tuberculosis Complex specific INS1-INS2 primers, and its performance was compared with that of bacteriological methods. The decontamination procedures previous to culture reduced M. bovis viability. The pathogen was identified in milk samples from 1 of 143 infected cows and in none of 43 uninfected ones. Even though PCR sensitivity was found to be 2-20 times higher than that of bacteriology in experimentally inoculated milk samples, all 186 field samples resulted negative by PCR, including the bacteriologically-confirmed one. In spite of the high prevalence of bovine tuberculosis in Argentinian dairy herds, the detection of M. bovis in milk is an unusual finding.
RESUMEN
Fifteen episodes of Mycobacterium tuberculosis laboratory cross-contamination suspected between 1996 and 2001 at 6 laboratories in Buenos Aires City and suburbs were investigated by IS6110 RFLP. Thirteen episodes were confirmed. Even though BACTEC 460 produced the highest number of confirmed episodes in a single laboratory, the most extended one occurred while employing conventional culture procedures in solid medium. The double repetitive element-polymerase chain reaction (DRE-PCR) was applied to 8 of these episodes and produced concordant results with those of the RFLP. The DRE-PCR appears to be a valuable tool for the prompt identification of false positive cultures. The timely rectification of defects in laboratory protocols can avert false diagnoses of tuberculosis and unnecessary prolonged treatments.