Preparation of a working seed lot of BCG and quality control by PCR genotyping.

The bacillus Calmette-Guérin (BCG) was obtained in 1920 after successive passages leading to the attenuation of a Mycobacterium bovis strain. For the following 40 years, BCG had been replicated, resulting in substrains with genotypic and phenotypic differences. Several genomic studies have compared two BCG strains, M. bovis and Mycobacterium tuberculosis, and observed that deleted regions in the different strains could be related to differences in antigenic properties. In this work, a working seed lot was obtained from a lyophilized secondary seed lot from the BCG Pasteur strain 1173 P2 and genetically characterized. The genome was analyzed by PCR directed to five regions (RD1, RD2, RD14, RD15, DU2), using the seed lot and different available strains as templates. No genetic differences were found in the fragments studied as compared to the Pasteur strain. A total of 20 passages were carried out and no differences were found in the size of the fragments amplified by PCR. In conclusion, this method allows to control a working seed lot genotypically and to assess the stability of the BCG genome.

Preparation of a working seed lot of BCG and quality control by PCR genotyping / Preparación de un lote semilla de trabajo de BCG y control de calidad por genotipificación por PCR

The bacillus Calmette-Guérin (BCG) was obtained in 1920 after successive passages leading to the attenuation of a Mycobacterium bovis strain. For the following 40 years, BCG had been replicated, resulting in substrains with genotypic and phenotypic differences. Several genomic studies have compared two BCG strains, M. bovis and Mycobacterium tuberculosis, and observed that deleted regions in the different strains could be related to differences in antigenic properties. In this work, a working seed lot was obtained from a lyophilized secondary seed lot from the BCG Pasteur strain 1173 P2 and genetically characterized. The genome was analyzed by PCR directed to five regions (RD1, RD2, RD14, RD15, DU2), using the seed lot and different available strains as templates. No genetic differences were found in the fragments studied as compared to the Pasteur strain. A total of 20 passages were carried out and no differences were found in the size of the fragments amplified by PCR. In conclusion, this method allows to control a working seed lot genotypically and to assess the stability of the BCG genome.

El bacilo de Calmette-Guérin (BCG) se obtuvo en 1920, después de sucesivos pasajes que llevaron a la atenuación de una cepa de Mycobacterium bovis. A lo largo de los 40 años subsiguientes la cepa BCG fue replicada y surgieron subcepas con diferencias fenotípicas y genotípicas. Se realizaron varios estudios de comparación genómica de diferentes cepas de BCG, M. bovis y Mycobacterium tuberculosis, y se observó que las deleciones de regiones en las diferentes cepas podrían estar relacionadas con diferencias en las propiedades antigénicas. En este trabajo se describe la preparación y caracterización genética de un lote semilla de trabajo obtenido a partir de un lote semilla secundaria liofilizado de la cepa BCG Pasteur 1173 P2. Se analizaron por PCR cinco regiones (RD1, RD2, RD14, RD15, DU2) en el lote semilla de trabajo utilizando como control las diferentes cepas disponibles. No se hallaron diferencias genéticas en los fragmentos estudiados al comparar el lote semilla de trabajo con la cepa BCG Pasteur 1173 P2. Asimismo, se efectuaron hasta 20 pasajes y no se encontraron diferencias en el tamaño de los fragmentos amplificados por PCR. En conclusión, se ha puesto a punto un método que permite controlar el genotipo de un lote semilla de trabajo y evaluar la estabilidad del genoma del BCG.

[Clinical utility of a commercial ligase chain-reaction kit for the diagnosis of pulmonary and extrapulmonary tuberculosis in the adult]. / Utilidad clínica de un equipo comercial de reacción en cadena de ligasa para el diagnóstico de la tuberculosis pulmonar y extrapulmonar del adulto.

Microscopy with the Ziehl-Neelsen (ZN) stain is frequently negative in paucibacillary tuberculosis (TB) so that the treatment must be started and continued until the culture results confirm or not the disease. LCx Mycobacterium tuberculosis Assay (Abbott, Lab.) uses the ligase chain reaction for direct amplification of DNA and rapid detection of M. tuberculosis Complex (MTB) in clinical specimens. We evaluated the usefulness of the LCx assay on 1,203 clinical samples: 737 respiratory and 466 extrapulmonary specimens. The LCx results were compared with those obtained by ZN and cultures on solid media and Mycobacteria Growth Indicator Tube (MGIT, Becton Dickinson, Argentina). Since detection and identification of MTB are simultaneously made by the LCx assay, a total of 145 out of 183 patients (79.2%) had a confirmed TB diagnosis in two working days. Positive culture results were predicted in 122 out of 160 cases (76.3%) by LCx and in 70 (43.8%) by ZN as well. The sensitivity (S) and specificity (ES) of LCx assay in ZN positive cases were 93.4% and 100.0% while in ZN negative cases they were 68.0% and 98.6%. The overall S and ES were 79.2% and 98.7%, respectively. We conclude that the LCx assay is a rapid and sensitive technique, which can be a helpful diagnostic tool mainly for paucibacillary TB in reference laboratories.

Utilidad clínica de un equipo comercial de reacción en cadena de ligasa para el diagnóstico de la tuberculosis pulmonar y extrapulmonar del adulto / [Clinical utility of a commercial ligase chain-reaction kit for the diagnosis of pulmonary and extrapulmonary tuberculosis in the adult]

Microscopy with the Ziehl-Neelsen (ZN) stain is frequently negative in paucibacillary tuberculosis (TB) so that the treatment must be started and continued until the culture results confirm or not the disease. LCx Mycobacterium tuberculosis Assay (Abbott, Lab.) uses the ligase chain reaction for direct amplification of DNA and rapid detection of M. tuberculosis Complex (MTB) in clinical specimens. We evaluated the usefulness of the LCx assay on 1,203 clinical samples: 737 respiratory and 466 extrapulmonary specimens. The LCx results were compared with those obtained by ZN and cultures on solid media and Mycobacteria Growth Indicator Tube (MGIT, Becton Dickinson, Argentina). Since detection and identification of MTB are simultaneously made by the LCx assay, a total of 145 out of 183 patients (79.2

) had a confirmed TB diagnosis in two working days. Positive culture results were predicted in 122 out of 160 cases (76.3

) by LCx and in 70 (43.8

) by ZN as well. The sensitivity (S) and specificity (ES) of LCx assay in ZN positive cases were 93.4

while in ZN negative cases they were 68.0

. The overall S and ES were 79.2

, respectively. We conclude that the LCx assay is a rapid and sensitive technique, which can be a helpful diagnostic tool mainly for paucibacillary TB in reference laboratories.

Utilidad clínica de un equipo comercial de reacción en cadena de ligasa para el diagnóstico de la tuberculosis pulmonar y extrapulmonar del adulto. / [Clinical utility of a commercial ligase chain-reaction kit for the diagnosis of pulmonary and extrapulmonary tuberculosis in the adult]

Microscopy with the Ziehl-Neelsen (ZN) stain is frequently negative in paucibacillary tuberculosis (TB) so that the treatment must be started and continued until the culture results confirm or not the disease. LCx Mycobacterium tuberculosis Assay (Abbott, Lab.) uses the ligase chain reaction for direct amplification of DNA and rapid detection of M. tuberculosis Complex (MTB) in clinical specimens. We evaluated the usefulness of the LCx assay on 1,203 clinical samples: 737 respiratory and 466 extrapulmonary specimens. The LCx results were compared with those obtained by ZN and cultures on solid media and Mycobacteria Growth Indicator Tube (MGIT, Becton Dickinson, Argentina). Since detection and identification of MTB are simultaneously made by the LCx assay, a total of 145 out of 183 patients (79.2

) had a confirmed TB diagnosis in two working days. Positive culture results were predicted in 122 out of 160 cases (76.3

) by LCx and in 70 (43.8

) by ZN as well. The sensitivity (S) and specificity (ES) of LCx assay in ZN positive cases were 93.4

and 100.0

while in ZN negative cases they were 68.0

and 98.6

. The overall S and ES were 79.2

and 98.7

, respectively. We conclude that the LCx assay is a rapid and sensitive technique, which can be a helpful diagnostic tool mainly for paucibacillary TB in reference laboratories.

Diagnóstico rápido y sensibilidad de Mycobacterium tuberculosis a antibióticos por el sistema MGIT / Rapid diagnosis and susceptibility of Mycobacterium tuberculosis to antibiotic using MGIT system

Son necesarias nuevas tecnologías que permitan obtener rápidamente el diagnóstico y la sensibilidad a los fármacos de Mycobacterium tuberculosis (MTB) para asegurar un tratamiento temprano y adecuado a la tuberculosis (TB), especialmente cuando ésta es causada por MTB resistente a isoniacida (H) y rifampicina (RMP) simultáneamente (TMBR). Un total de 218 materiales pulmonares obtenidos de 132 pacientes HIV(+) fueron procesados e inoculados en medio de cultivo líquido del sistema fluorescente "Mycobacterial Growth Indicator Tube" (MGIT, Becton Dickinson, USA) y en medios sólidos a base de huevos: Lowenstein-Jensen (L-J) y Stonebrick (SB). Posteriormente se determinó la concentración inhibitoria mínima (CIM) en MGIT de 120 aislamientos a H, RMP, estreptomicina (SM), ácido para-amino-salicílico (PAS) y etambutol (EMB). EL tiempo promedio de detección de crecimiento de MTB a partir de la muestra fue diagnosticado sólo por MGIT. Los resultados de sensibilidad obtenidos por CIM en MGIT se obtuvieron en un promedio de 5 días (3-10 días)y correlacionaron con los obtenidos por el clásico método de las proporciones en L-J (índice de correlación: 0,9974). La discordancia entre aislamientos resistentes a H y SM detectados por uno y otro método fue de 4,7 por ciento (2/42). El MGIT resultó un sistema seguro, confiable y rápido, especialmente para detectar resistencias micobacterianas, que podría ser empleado en laboratorios clínicos para diagnóstico y seguimiento de pacientes con TB(AU)

Diagnóstico rápido y sensibilidad de Mycobacterium tuberculosis a antibióticos por el sistema MGIT / Rapid diagnosis and susceptibility of Mycobacterium tuberculosis to antibiotic using MGIT system

Son necesarias nuevas tecnologías que permitan obtener rápidamente el diagnóstico y la sensibilidad a los fármacos de Mycobacterium tuberculosis (MTB) para asegurar un tratamiento temprano y adecuado a la tuberculosis (TB), especialmente cuando ésta es causada por MTB resistente a isoniacida (H) y rifampicina (RMP) simultáneamente (TMBR). Un total de 218 materiales pulmonares obtenidos de 132 pacientes HIV(+) fueron procesados e inoculados en medio de cultivo líquido del sistema fluorescente "Mycobacterial Growth Indicator Tube" (MGIT, Becton Dickinson, USA) y en medios sólidos a base de huevos: Lowenstein-Jensen (L-J) y Stonebrick (SB). Posteriormente se determinó la concentración inhibitoria mínima (CIM) en MGIT de 120 aislamientos a H, RMP, estreptomicina (SM), ácido para-amino-salicílico (PAS) y etambutol (EMB). EL tiempo promedio de detección de crecimiento de MTB a partir de la muestra fue diagnosticado sólo por MGIT. Los resultados de sensibilidad obtenidos por CIM en MGIT se obtuvieron en un promedio de 5 días (3-10 días)y correlacionaron con los obtenidos por el clásico método de las proporciones en L-J (índice de correlación: 0,9974). La discordancia entre aislamientos resistentes a H y SM detectados por uno y otro método fue de 4,7 por ciento (2/42). El MGIT resultó un sistema seguro, confiable y rápido, especialmente para detectar resistencias micobacterianas, que podría ser empleado en laboratorios clínicos para diagnóstico y seguimiento de pacientes con TB

[Rapid diagnosis and susceptibility of Mycobacterium tuberculosis to antibiotics using MGIT system]. / Diagnóstico rápido y sensibilidad de Mycobacterium tuberculosis a antibióticos por el sistema MGIT.

New technologies for rapid detection and drug susceptibility testing of mycobacteria are needed in clinical laboratories in order to attempt a rapid diagnosis and effective treatment of tuberculosis (TB) specially of cases resistant to isoniazid (H) and rifampin (RMP) (MDR-TB). A total of 218 pulmonary specimens from 132 HIV coinfected patients were processed and inoculated into the Mycobacterial Growth Indicator Tube system (MGIT, Becton Dickinson, MD) and on Lowenstein-Jensen (L-J) and Stonebrink (SB) solid media. The average time for recovering Mycobacterium tuberculosis (MTB) from MGIT was 18.3 days and 31.0 days from solid media. Of the patients 14.4% (19/132) were only diagnosed by MGIT. In another experiment susceptibility tests by the classical proportion method (PM) in L-J medium with H, RMP, streptomycin (SM), para-aminosalycilic-acid and ethambutol (EMB) were carried out on 120 isolates. The results were later compared with those obtained by determining the minimal inhibitory concentration (MIC) for each drug in MGIT against the above mentioned isolates. MIC results from MGIT method were available in an average of 5 days (3-10) and they correlated (correlation index 0.9974) with those of drug susceptibility obtained by the PM. A 4.7% (2/42) of disagreement among detecting isolates resistant to H and SM was found between PM and MGIT. Our results showed MGIT as a useful, safe and timesaving culture system, specially for detecting TB resistance. It might be used in clinical laboratories to improve the proper management of TB patients.

[Research concerning the spontaneous course of 135 cases of Menière's disease. Critical study of operative indications (author's transl)]. / Recherches concernant l'évolution spontanée de 135 cas de maladie de Ménière. Etude critique des indications opératoires.

135 cases of Ménière's disease were observed for five and a half years. Out of 1120 Ent patients, one suffered from Ménière's disease. Out of 1 000 audiogram, 8 involved cases of Ménière's disease. Of 9 cases of non-traumatic vertigo where a vetsibular origin was suspected, I was Ménière's disease. The morbidity was 75 per million. Hearing was stable in 62% of cases, worsened in 15%, improved in 15% whilst in 8% there was total deafness before the onset of vertigo. Vertigo disappeared in 94% of cases. Bilateral Ménière's disease is associated with a particularly unfavourable course with regard to general condition, since the disease would appear to be the consequence of a more deep-seated organic disorder. These figures are exactly the same as those found after vestibular neurectomy. In 98% of cases surgery is useless and hence dangerous.