Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance.

The aim of this work was to study the diagnostic accuracy of pyrosequencing to detect resistance to fluoroquinolones, kanamycin, amikacin, capreomycin, and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains. One hundred four clinical isolates previously characterized by BACTEC 460TB/MGIT 960 were included. Specific mutations were targeted in gyrA, rrs, eis promoter, and embB. When there was a discordant result between BACTEC and pyrosequencing, Genotype MTBDRsl (Hain Lifescience, Nehren, Germany) was performed. Sensitivity and specificity of pyrosequencing were 70.6% and 100%, respectively, for fluoroquinolones; 93.3% and 81.7%, respectively, for kanamycin; 94.1% and 95.9%, respectively, for amikacin; 90.0% and 100%, respectively, for capreomycin; and 64.8% and 87.8%, respectively, for EMB. This study shows that pyrosequencing may be a useful tool for making early decisions regarding second-line drugs and EMB resistance. However, for a correct management of patients with suspected extensively drug-resistant tuberculosis, susceptibility results obtained by molecular methods should be confirmed by a phenotypic method.

Evaluation of an array-based method for human papillomavirus detection and genotyping in comparison with conventional methods used in cervical cancer screening.

Cervical cancer is the second-most prevalent cancer in young women around the world. Infection with human papillomavirus (HPV), especially high-risk HPV types (HR-HPV), is necessary for the development of this cancer. HPV-DNA detection is increasingly being used in cervical cancer screening programs, together with the Papanicolau smear test. We evaluated the usefulness of introducing this new array-based HPV genotyping method (i.e., Clinical Arrays Papillomavirus Humano) in the cervical cancer screening algorithm in our center. The results obtained using this method were compared to those obtained by the hybrid capture II high-risk HPV DNA test (HC-II) and Papanicolau in a selected group of 408 women. The array-based assay was performed in women that were HC-II positive or presented cytological alterations. Among 246 array-positive patients, 123 (50%) presented infection with >or=2 types, and HR-HPV types were detected in 206 (83.7%), mainly HPV-16 (24.0%). Up to 132 (33.2%) specimens were classified as ASCUS (for atypical squamous cells of undetermined significance), and only 48 (36.4%) of them were HPV-DNA positive by either assay; however, 78.7% of these cases were caused by HR-HPV types. The agreement between both HPV-DNA detection techniques was fairly good (n = 367). Screening with Papanicolau smear and HC-II tests, followed by HPV detection and genotyping, provided an optimal identification of women at risk for the development of cervical cancer. Furthermore, with the identification of specific genotypes, either in single or multiple infections, a better prediction of disease progression was achieved. The array method also made allowed us to determine the possible contribution of the available vaccines in our setting.

Evaluation of an automated nucleic acid extractor for hepatitis C virus load quantification.

The increasing use of molecular methods strongly motivates clinical laboratories to introduce automated nucleic acid extractors. We compared the easyMAG (bioMérieux) with a manual extraction method for hepatitis C virus (HCV) load quantification (RealTime HCV; Abbott). Both methods were comparable, and, therefore, the easyMAG is suitable to be implemented in our laboratory for the management of HCV-infected patients.

Utilidad de la biología molecular en el diagnóstico microbiológico de las infecciones por micobacterias / Utility of molecular biology in the microbiological diagnosis of mycobacterial infections

Las micobacterias constituyen un grupo de bacterias de gran interés en medicina, ya que, junto a especies telúricas y oportunistas, se hallan 2 especies (Mycobacterium tuberculosis y Mycobacterium leprae) de gran importancia en salud pública. A pesar de los esfuerzos realizados para su control, la tuberculosis (TB) sigue siendo en la actualidad uno de los problemas sanitarios de más trascendencia mundial. En los últimos años, la micobacteriología ha experimentado importantes avances tecnológicos. A pesar de ello, el diagnóstico temprano de la infección por micobacterias y, especialmente de la TB, sigue recayendo en el examen microscópico de las muestras teñidas de manera adecuada. En la actualidad, éste sigue siendo el procedimiento más simple, de mejor coste-efectividad y rapidez para proporcionar al clínico una orientación diagnóstica preliminar. El control efectivo de la TB se basa en la detección rápida de M. tuberculosis, seguido por la inmediata implementación del tratamiento antituberculoso adecuado. La emergencia de cepas resistentes a los fármacos antituberculosos agudiza la necesidad de disponer de métodos rápidos de detección de M. tuberculosis y de resistencias. La disponibilidad de métodos de epidemiología molecular de fácil implementación y estandarización, que nos permitan identificar casos relacionados, es fundamental para identificar brotes epidémicos que ayuden a controlar la propagación de la TB. Aun reconociendo los evidentes progresos realizados en el diagnóstico molecular de las infecciones micobacterianas, las técnicas disponibles son todavía insuficientes. En esta revisión, describimos el estado actual de las principales técnicas moleculares para la detección directa de micobacterias en muestras clínicas, para su identificación, detección de resistencias a los principales fármacos antituberculosos y de epidemiología molecular. En cada caso, destacamos las ventajas y las limitaciones de ellas. En un próximo futuro la micobacteriología clínica evolucionará, con bastante probabilidad, hacia la universalización de las técnicas genéticas aplicadas al diagnóstico directo y la detección de resistencias. La epidemiología molecular de la TB se realizará, en sus diferentes aplicaciones, con técnicas más rápidas y automatizadas que las actuales

Species within the Mycobacterium genus are of major medical interest, since, together with environmental and opportunistic species, there are two species (Mycobacterium tuberculosis and Mycobacterium leprae) that remain an important public health challenge. Despite efforts to control tuberculosis (TB), this disease remains one of the most prominent health problems worldwide. In the last few years, mycobacteriology has experienced major technological advances. Nevertheless, the early diagnosis of mycobacterial infection and, especially of TB, is still based on microscopic examination of properly stained samples. At present, this procedure is still the simplest, fastest and most cost-effective method for preliminary diagnostic guidance. Effective control of TB is based on rapid detection of M. tuberculosis, followed by immediate implementation of the appropriate antituberculosis therapy. Because of the emergence of multidrug resistant strains, the development of rapid diagnostic methods, both for identification of M. tuberculosis and susceptibility testing, has become a pressing need. The availability of molecular epidemiology methods that are easy to implement and standardized and that would allow identification of related cases is of key importance to identify epidemic outbreaks and control the spread of TB. Despite the evident progress in the molecular diagnosis of mycobacterial infections, the available techniques are still inadequate. In this review, we describe the state of the art of the main molecular techniques for direct detection of mycobacteria in clinical samples, their identification, detection of resistance to the most important antituberculosis agents, and molecular epidemiology. In each case, we describe the advantages and limitations of current techniques. In the near future, clinical mycobacteriology will probably evolve to the universal use of genetic techniques for direct diagnosis and detection of resistance. The molecular epidemiology of TB will be performed, in its various applications, by faster and more automated techniques than those currently available (AU)

Utilidad de la biología molecular en el diagnóstico microbiológico de las infecciones por micobacterias / Utility of molecular biology in the microbiological diagnosis of mycobacterial infections

Las micobacterias constituyen un grupo de bacterias de gran interés en medicina, ya que, junto a especies telúricas y oportunistas, se hallan 2 especies (Mycobacterium tuberculosis y Mycobacterium leprae) de gran importancia en salud pública. A pesar de los esfuerzos realizados para su control, la tuberculosis (TB) sigue siendo en la actualidad uno de los problemas sanitarios de más trascendencia mundial. En los últimos años, la micobacteriología ha experimentado importantes avances tecnológicos. A pesar de ello, el diagnóstico temprano de la infección por micobacterias y, especialmente de la TB, sigue recayendo en el examen microscópico de las muestras teñidas de manera adecuada. En la actualidad, éste sigue siendo el procedimiento más simple, de mejor coste-efectividad y rapidez para proporcionar al clínico una orientación diagnóstica preliminar. El control efectivo de la TB se basa en la detección rápida de M. tuberculosis, seguido por la inmediata implementación del tratamiento antituberculoso adecuado.(AU)

La emergencia de cepas resistentes a los fármacos antituberculosos agudiza la necesidad de disponer de métodos rápidos de detección de M. tuberculosis y de resistencias. La disponibilidad de métodos de epidemiología molecular de fácil implementación y estandarización, que nos permitan identificar casos relacionados, es fundamental para identificar brotes epidémicos que ayuden a controlar la propagación de la TB. Aun reconociendo los evidentes progresos realizados en el diagnóstico molecular de las infecciones micobacterianas, las técnicas disponibles son todavía insuficientes. En esta revisión, describimos el estado actual de las principales técnicas moleculares para la detección directa de micobacterias en muestras clínicas, para su identificación, detección de resistencias a los principales fármacos antituberculosos y de epidemiología molecular. En cada caso, destacamos las ventajas y las limitaciones de ellas. En un próximo futuro la micobacteriología clínica evolucionará, con bastante probabilidad, hacia la universalización de las técnicas genéticas aplicadas al diagnóstico directo y la detección de resistencias. La epidemiología molecular de la TB se realizará, en sus diferentes aplicaciones, con técnicas más rápidas y automatizadas que las actuales(AU)

Species within the Mycobacterium genus are of major medical interest, since, together with environmental and opportunistic species, there are two species (Mycobacterium tuberculosis and Mycobacterium leprae) that remain an important public health challenge. Despite efforts to control tuberculosis (TB), this disease remains one of the most prominent health problems worldwide. In the last few years, mycobacteriology has experienced major technological advances. Nevertheless, the early diagnosis of mycobacterial infection and, especially of TB, is still based on microscopic examination of properly stained samples. At present, this procedure is still the simplest, fastest and most cost-effective method for preliminary diagnostic guidance. Effective control of TB is based on rapid detection of M. tuberculosis, followed by immediate implementation of the appropriate antituberculosis therapy. Because of the emergence of multidrug resistant strains, the development of rapid diagnostic methods, both for identification of M. tuberculosis and susceptibility testing, has become a pressing need. The availability of molecular epidemiology methods that are easy to implement and standardized and that would allow identification of related cases is of key importance to identify epidemic outbreaks and control the spread of TB. Despite the evident progress in the molecular diagnosis of mycobacterial infections, the available techniques are still inadequate(AU)

In this review, we describe the state of the art of the main molecular techniques for direct detection of mycobacteria in clinical samples, their identification, detection of resistance to the most important antituberculosis agents, and molecular epidemiology. In each case, we describe the advantages and limitations of current techniques. In the near future, clinical mycobacteriology will probably evolve to the universal use of genetic techniques for direct diagnosis and detection of resistance. The molecular epidemiology of TB will be performed, in its various applications, by faster and more automated techniques than those currently available(AU)

[Utility of molecular biology in the microbiological diagnosis of mycobacterial infections]. / Utilidad de la biología molecular en el diagnóstico microbiológico de las infecciones por micobacterias.

Species within the Mycobacterium genus are of major medical interest, since, together with environmental and opportunistic species, there are two species (Mycobacterium tuberculosis and Mycobacterium leprae) that remain an important public health challenge. Despite efforts to control tuberculosis (TB), this disease remains one of the most prominent health problems worldwide. In the last few years, mycobacteriology has experienced major technological advances. Nevertheless, the early diagnosis of mycobacterial infection and, especially of TB, is still based on microscopic examination of properly stained samples. At present, this procedure is still the simplest, fastest and most cost-effective method for preliminary diagnostic guidance. Effective control of TB is based on rapid detection of M. tuberculosis, followed by immediate implementation of the appropriate antituberculosis therapy. Because of the emergence of multidrug resistant strains, the development of rapid diagnostic methods, both for identification of M. tuberculosis and susceptibility testing, has become a pressing need. The availability of molecular epidemiology methods that are easy to implement and standardized and that would allow identification of related cases is of key importance to identify epidemic outbreaks and control the spread of TB. Despite the evident progress in the molecular diagnosis of mycobacterial infections, the available techniques are still inadequate. In this review, we describe the state of the art of the main molecular techniques for direct detection of mycobacteria in clinical samples, their identification, detection of resistance to the most important antituberculosis agents, and molecular epidemiology. In each case, we describe the advantages and limitations of current techniques. In the near future, clinical mycobacteriology will probably evolve to the universal use of genetic techniques for direct diagnosis and detection of resistance. The molecular epidemiology of TB will be performed, in its various applications, by faster and more automated techniques than those currently available.