Amplification of DNA of Mycobacterium tuberculosis from peripheral blood of patients with pulmonary tuberculosis

Schluger, Neil W.; Condos, Rany; Lewis, Stuart; Rom, William N.

doi:10.1016/s0140-6736(94)92999-8

Cited by 70 publications

(43 citation statements)

References 8 publications

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“…ii) There is scanty information on the biology of mycobacteremia (Calmette, 1923;Reimer, 1994), and development of an efficient tool to detect mycobacterial DNA would open avenues for further investigation in this field. iii) In addition to our findings with tuberculous lymphadenitis, studies from diverse research groups suggest that a blood-based PCR assay has potential for detection of mycobacterial DNA in blood of patients with pulmonary tuberculosis (especially in AIDS patients) (Aguado et al, 1996;De Francesco et al, 1996;Schluger et al, 1994), other forms of extrapulmonary tuberculosis (Tzoanopoulos et al, 2001), and M. avium subspecies paratuberculosis DNA in patients with Crohn's disease (Naser et al, 2004). iv) Blood presents a number of challenges for successful PCR amplification, making it a model for overcoming potential pitfalls presented by biological specimens from other sources.…”

Section: Discussionsupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

“…The hypothesis is that patients with active infection harbor M. tuberculosis DNA in peripheral scavenging cells (De Francesco et al, 1996;Schluger et al, 1994). This strategy has been useful for diagnosis of mycobacteremia in HIV-positive patients with M. tuberculosis or M. avium infection, with sensitivities comparable to blood cultures (De Francesco et al, 1996;Kulski and Pryce, 1996;Schluger et al, 1994). In our hands, detection of mycobacterial DNA present in PBMCs of 48 non-HIV infected patients with a clinical diagnosis of extrapulmonary disease (tuberculous lymphadenitis) provided an improved sensitivity (65%) and 100% specificity, when compared with microbiological methods such as direct smear and culture (42 and 27% sensitivity, respectively).…”

Section: Introductionmentioning

confidence: 99%

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Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens

Restrepo

Gómez

Shipley

et al. 2006

Journal of Microbiological Methods

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A major challenge for tuberculosis control is mycobacterial detection in paucibacillary disease, particularly in pediatric, extrapulmonary and smear-negative pulmonary infections. We developed a simple and efficient DNA extraction and real-time quantitative PCR (qPCR) protocol for mycobacterial detection and quantification in paucibacillary specimens. The method was refined using an in vitro model mimicking blood specimens which are characterized by the presence of numerous qPCR inhibitors. Mycobacterial DNA detection in blood is of interest given the high sensitivity we previously reported using conventional PCR in blood of patients with tuberculosis lymphadenitis. Mechanical lysis of mycobacteria in the presence of an organic solvent provided the highest sensitivity. Mycobacterial DNA amplification was compromised when the human:bacterial genome ratio was at least 190:1. Separation of the specimen into bacterial-and host-rich fractions prior to DNA extraction improved mycobacterial DNA detection by 30%. Preliminary testing of our protocol in smear-negative, culture-positive specimens (gastric and lymph node aspirates, pleural and cerebrospinal fluid, blood) confirmed the applicability of our technique to a range of paucibacillary specimens for the detection, quantification and speciation (M. tuberculosis versus M. avium) of mycobacteria, several weeks before culture results were available. Our protocol provides a novel efficient and simple strategy to improve the performance of qPCR in paucibacillary specimens, including those with excess human DNA background. This tool is useful to study the pathophysiology of early pulmonary or occult tuberculosis, and for more rapid and accurate diagnosis in difficult to diagnose infections.

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Section: Discussionsupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens

Restrepo

Gómez

Shipley

et al. 2006

Journal of Microbiological Methods

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“…Este tipo de pruebas están basadas en la presencia del microorganismo en sangre (bacteriemia) o, quizá más importante, la presencia de fragmentos de ADN de la micobacteria que está parcialmente digerida por macrófagos que la han fagocitado en el sitio de la infección y, luego, han entrado a la circulación (41)(42)(43). Este evento parece ser común en pacientes con sida e infectados con M. tuberculosis o M. avium, pero también se ha reportado en pacientes sin un inmunocompromiso importante (41)(42)(43)(44)(45)(46)(47)(48). La detección de ADN de la micobacteria en sangre periférica, podría complementar aquellas estrategias que dependen de la respuesta inmune del paciente.…”

Section: ¿Cuál Es El Método De Elección?unclassified

Nuevas herramientas para la detección de la tuberculosis latente.

Restrepo¹

2004

biomedica

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La tuberculosis es una causa importante de muerte en el mundo, principalmente en países en vía de desarrollo. Se estima que cada año hay 9 millones de casos nuevos y 1,5-3 millones de muertes (1,2). Esta infección es causada por Mycobacterium tuberculosis, una bacteria ácido- La tuberculosis es un problema serio de salud pública en el mundo, especialmente en países en vía de desarrollo, inclusive Colombia. En nuestro país estamos enfocados en el manejo clínico de los pacientes con tuberculosis activa, pero no hay campañas efectivas que identifiquen y provean terapia a los individuos con las formas latentes de la infección y con alto riesgo de progresar hacia la enfermedad. En esta revisión se plantea la importancia de realizar dichas campañas para evitar la diseminación de la infección en la comunidad. Esto incluye la búsqueda activa y el tratamiento profiláctico de los contactos de casos recientes, así como la de individuos con tuberculosis latente con alto riesgo de desarrollar la enfermedad. En ausencia de una prueba de oro para detectar la tuberculosis latente, la prueba cutánea de la tuberculina se ha utilizado por más de 100 años para dicho fin, a pesar de sus limitaciones de sensibilidad y especificidad. En esta revisión se evalúan las ventajas y desventajas de una nueva generación de inmunoensayos que incluye la prueba comercial Quantiferon y el desarrollo experimental del ELISPOT. Ambas se basan en la detección de IFNγ secretado por linfocitos de sangre periférica cuando se incuban con antígenos específicos del bacilo tuberculoso. Finalmente, se plantea la importancia de desarrollar pruebas moleculares enfocadas en detectar el ADN de la micobacteria como posible complemento a los inmunoensayos descritos.

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“…Several methods have been published for processing sputa and blood samples for PCR diagnosis of Mycobacterium tuberculosis (1)(2)(3)(4)(5) involving several steps and reagents and are tedious and also increase the chances of cross contamination and infection of the laboratory personnel. As PCR does not require intact DNA for amplification such elaborate and expensive procedures are not required.…”

mentioning

confidence: 99%

A simple method for inhibition free PCR amplification of target DNA directly from clinical specimens

Senthilkumar

Saravanakumar

Kumarasamy

et al. 1997

Indian J Clin Biochem

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KEY WORDS: PCR, M. tuberculosis.Preparation of the clinical specimens for PCR amplification has become an important aspect of nucleic acid probe based diagnosis and the final sensitivity of the diagnostic method is influenced by the sample processing method as PCR requirestarget DNA free of any potential inhibitors. The number of organisms in the clinical specimens like sputum is highly variable and it depends on various factors like the stage of infection, site of infection, the age of the patient. Handling highly infectious material like TB samples preferably should be easy (involve minimum number of steps), simple, economical (particularly for developing countries where the sample size is enormous) and yield at the same time PCR amplifiable target DNA.Several methods have been published for processing sputa and blood samples for PCR diagnosis of Mycobacterium tuberculosis (1-5) involving several steps and reagents and are tedious and also increase the chances of cross contamination and infection of the laboratory personnel. As PCR does not require intact DNA for amplification such elaborate and expensive procedures are not required. We report here a very simple, universal method for preparing the target DNA for PCR amplification from mycobacteria present in various clinical specimens and the method can easily be adopted for any sample.

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Amplification of DNA of Mycobacterium tuberculosis from peripheral blood of patients with pulmonary tuberculosis

Cited by 70 publications

References 8 publications

Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens

Selective enrichment and detection of mycobacterial DNA in paucibacillary specimens

Nuevas herramientas para la detección de la tuberculosis latente.

A simple method for inhibition free PCR amplification of target DNA directly from clinical specimens

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