Evaluation of MGIT 960 and the colorimetric-based method for tuberculosis drug susceptibility testing.

SETTING: Dr Cetrángolo Hospital, Buenos Aires Province, Argentina. OBJECTIVE: Evaluation of the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system and the colorimetric-based method (CMM) for first- and second-line drug susceptibility testing (FL-DST, SL-DST) against Mycobacterium tuberculosis. DESIGN: FL-DST was studied using SIRE MGIT 960. Minimal inhibitory concentrations (MICs) for isoniazid (INH), streptomycin, rifampicin (RMP), ethambutol (EMB) and levofloxacin (LVX) were also determined by CMM using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). MICs for amikacin (AMK), kanamycin (KM), capreomycin (CPM), ethionamide (ETH), cycloserine, ofloxacin (OFX), linezolide (LZ) and moxifloxacin (MFX) were determined on 94 multidrug-resistant M. tuberculosis isolates by MGIT 960 and CMM. Statistical methods were applied to define drug-susceptible and drug-resistant isolates on the basis of the comparison between results obtained by gold standards. RESULTS: A total of 1626 clinical isolates were studied. Critical drug concentrations could be defined in less than 10 days for both CMM and MGIT 960. CMM was cheaper but more laborious than MGIT 960. The highest performances of both methods were achieved for AMK, RMP, OFX, LZ and MFX, followed by INH, ETH, KM, CPM and LVX (tested only by CMM). CONCLUSIONS: Both methods could be implemented as rapid diagnostic tools to detect drug-resistant isolates in clinical practice.

Tuberculosis transmission of predominant genotypes of Mycobacterium tuberculosis in northern suburbs of Buenos Aires city region.

In 2003, the incidence of tuberculosis in Argentina showed an increase compared to 2002. The severe national crisis at the end of the 90s has probably strongly contributed to this situation. The goal of this work was to estimate the extent of the spread of the most predominant Mycobacterium tuberculosis strains and to assess the spread of predominant M. tuberculosis clusters as determined by spoligotyping and IS6110 RFLP. The study involved 590 pulmonary, smear-positive TB cases receiving medical attention at health centers and hospitals in Northern Buenos Aires (NBA) suburbs, from October 2001 to December 2002. From a total of 208 clinical isolates belonging to 6 major clusters, 63 (30.2%) isolates had identical spoligotyping and IS6110 RFLP pattern. Only 22.2% were shown to have epidemiological connections with another member of their respective cluster. In these major clusters, 30.2% of the 208 TB cases studied by both molecular techniques and contact tracing could be convincingly attributable to a recently acquired infection. This knowledge may be useful to assess the clonal distribution of predominant M. tuberculosis clusters in Argentina, which may make an impact on TB control strategies.

Tuberculosis transmission of predominant genotypes of Mycobacterium tuberculosis in Northern suburbs of Buenos Aires city region / Transmisión de la tuberculosis por genotipos predominantes de Mycobacterium tuberculosis en la región Gran Buenos Aires Norte

In 2003, the incidence of tuberculosis in Argentina showed an increase compared to 2002. The severe national crisis at the end of the 90s has probably strongly contributed to this situation. The goal of this work was to estimate the extent of the spread of the most predominant Mycobacterium tuberculosis strains and to assess the spread of predominant M. tuberculosis clusters as determined by spoligotyping and IS6110 RFLP. The study involved 590 pulmonary, smear-positive TB cases receiving medical attention at health centers and hospitals in Northern Buenos Aires (NBA) suburbs, from October 2001 to December 2002. From a total of 208 clinical isolates belonging to 6 major clusters, 63 (30.2%) isolates had identical spoligotyping and IS6110 RFLP pattern. Only 22.2% were shown to have epidemiological connections with another member of their respective cluster. In these major clusters, 30.2% of the 208 TB cases studied by both molecular techniques and contact tracing could be convincingly attributable to a recently acquired infection. This knowledge may be useful to assess the clonal distribution of predominant M. tuberculosis clusters in Argentina, which may make an impact on TB control strategies.

La incidencia de la tuberculosis en Argentina mostró en 2003 un incremento en comparación con 2002. La grave crisis nacional a fines de los 90 ha probablemente contribuido en gran medida a esta situación. El objetivo del presente trabajo fue determinar la diversidad genética de aislamientos de Mycobacterium tuberculosis y el grado de dispersión de algunas cepas mayoritarias genéticamente relacionadas. El estudio involucró 590 aislamientos clínicos provenientes de muestras respiratorias con examen directo positivo, de pacientes atendidos en los hospitales y centros de salud que conforman la región Gran Buenos Aires Norte (NBA), de octubre de 2001 a diciembre de 2002. De 208 aislamientos que se encontraron en los 6 mayores clusters, 63 (30,2%) tenían patrones idénticos de spoligotyping y de IS6110 RFLP. En el 22,2% de los casos fue posible verificar la conexión epidemiológica con otro miembro del respectivo cluster. Concluimos que el 30,2% de estos agrupamientos principales pueden ser atribuidos a una infección reciente. Estos resultados pueden ser útiles para determinar la distribución clonal de los grupos predominantes de M. tuberculosis en Argentina, lo que puede impactar en las estrategias de control de la tuberculosis.

[Evaluation of a colorimetric micromethod for determining the minimal inhibitory concentration of antibiotics against Mycobacterium tuberculosis]. / Evaluación de un micrométodo colorimétrico para determinar la concentración inhibitoria mínima de drogas antituberculosas frente a Mycobacterium tuberculosis.

Multidrug-resistant tuberculosis (MDR) caused by strains resistant to both isoniazid and rifampin is now considered a serious sanitary problem worldwide. New technical tools for the early detection of these strains are urgently needed to avoid their spread within the community. We have evaluated a microplate colorimetric-based method to determine the minimal inhibitory concentration (MIC) of first-line antituberculosis drugs by using 3-(4, 5 dimethylthiazolyl 1-2 yl)-2,5 diphenyl tetrazolium bromide as a bacterial growth indicator (MTT) (M-MTT). A total of 603 clinical isolates, 507 from respiratory cases (84.1%) and 96 from non-respiratory cases (15.9%) were processed. The proportion method on a Löwenstein-Jensen medium (PM) with isoniazid (INH), 0.20 microg/ml; streptomycin (SM), 4.00 microg/ml; ethambutol (EMB), 2.00 microg/ml and rifampin (RMP), 40.00 microg/ml, was used as the gold standard. The drugs and the concentration range tested were: INH, 1.00-0.03 microg/ml; SM, 8.00-0.25 microg/ml; EMB, 32.00-1.00 microg/ml and RMP, 2.00-0.06 microg/ml. MIC results were obtained on an average of 8 days (range: 7-12). The cut-off values for each drug, calculated by the ROC curve method, were: INH, 0.25 microg/ml, RMP, 0.50 microg/ml, SM, 4.00 microg/ml and EMB, 4.00 microg/ml. Sensitivity and specificity for RMP were 100 %, while for INH, they were 97.8% and 99.5% respectively. The results obtained suggested that M-MTT is a low cost and easy to set up method that could be applied to MDR clinical diagnosis in developing countries.

Evaluación de un micrométodo colorimétrico para determinar la concentración inhibitoria mínima de drogas antituberculosas frente a Mycobacterium tuberculosis / Evaluation of a colorimetric micromethod for determining the minimal inhibitory concentration of antibiotics against Mycobacterium tuberculosis

La tuberculosis multidrogorresistente (MDR), originada por aislamientos de cepas simultáneamente resistentes a isoniacida y rifampicina, es reconocida en la actualidad como un problema sanitario mundial. Nuevas técnicas que permitan detectar en forma temprana cepas MDR son necesarias para evitar su dispersión en la comunidad. En este trabajo hemos evaluado el empleo de un micrométodo colorimétrico para determinar las concentraciones inhibitorias mínimas (CIM) de las drogas de primera línea frente a dichas cepas, usando el indicador: bromuro de 3-(4,5 dimetiltiazol-2-yl)-2,5 difeniltetrazolio (M-MTT). Junto a la cepa de referencia H37Rv se procesaron 603 aislamientos clínicos, 507 provenientes de casos pulmonares (84,1%) y 96 de extrapulmonares (15,9%). Como estándar de referencia se utilizó el método de proporciones en medio de Löwenstein-Jensen (MP), con isoniacida (INH), 0,20 µg/ml; estreptomicina (SM), 4,00 µg/ml; etambutol (EMB), 2,00 µg/ml y rifampicina (RMP), 40,00 µg/ml. Los intervalos de concentraciones de las drogas empleadas en el M-MTT fueron: INH, 1,00-0,03 µg/ml; SM, 8,00-0,25 µg/ml; EMB, 32,00-1,00 µg/ml y RMP: 2,00-0,06 µg/ml. El resultado de la CIM por el M-MTT fue obtenido en un tiempo promedio de 8 días (rango total: 7 a 12 días). Los puntos de corte para cada una de las drogas, calculados mediante el análisis de la curva ROC, fueron: INH, 0,25 µg/ml; RMP, 0,50 µg/ml; SM, 4,00 µg/ml y EMB, 4,00 µg/ml. Los valores de sensibilidad y especificidad fueron 100% en el caso de RMP; y 97,8% y 99,5%, respectivamente, para INH. El análisis estadístico de los resultados permitió concluir que el M-MTT es un método seguro para la rápida detección de MDR. Por su sencillez y bajo costo, podría ser aplicado en los países en vías de desarrollo.

Multidrug-resistant tuberculosis (MDR) caused by strains resistant to both isoniazid and rifampin is now considered a serious sanitary problem worldwide. New technical tools for the early detection of these strains are urgently needed to avoid their spread within the community. We have evaluated a microplate colorimetric-based method to determine the minimal inhibitory concentration (MIC) of first-line antituberculosis drugs by using 3-(4, 5 dimethylthiazolyl 1-2 yl)-2,5 diphenyl tetrazolium bromide as a bacterial growth indicator (MTT) (M-MTT). A total of 603 clinical isolates, 507 from respiratory cases (84.1%) and 96 from non-respiratory cases (15.9%) were processed. The proportion method on a Löwenstein-Jensen medium (PM) with isoniazid (INH), 0.20 µg/ml; streptomycin (SM), 4.00 µg/ml; ethambutol (EMB), 2.00 µg/ml and rifampin (RMP), 40.00 µg/ml, was used as the gold standard. The drugs and the concentration range tested were: INH, 1.00-0.03 µg/ml; SM, 8.00-0.25 µg/ml; EMB, 32.00-1.00 µg/ml and RMP, 2.00-0.06 µg/ml. MIC results were obtained on an average of 8 days (range: 7-12). The cut-off values for each drug, calculated by the ROC curve method, were: INH, 0.25 µg/ml, RMP, 0.50µg/ml, SM, 4.00 µg/ml and EMB, 4.00 µg/ml. Sensitivity and specificity for RMP were 100 %, while for INH, they were 97.8% and 99.5% respectively. The results obtained suggested that M-MTT is a low cost and easy to set up method that could be applied to MDR clinical diagnosis in developing countries.

First description of Mycobacterium tuberculosis Beijing genotype in Argentina.

During a population-based study to genotype isolates of Mycobacterium tuberculosis from Buenos Aires Northern suburbs, we found isolates with molecular patterns related to those of the Beijing genotype. Five out of 590 (0.85%) patients had isolates with spoligopattern identical to that of the Beijing family. Since two of these isolates showed identical IS6110RFLP pattern, we found only four different patterns containing 11 to 19 bands. The isolates were obtained from young people (including a 7 years-old child) who were born in Argentina, and were living in a small area of our region. However, conventional contact tracing did not prove epidemiological linkage among them. These isolates were fully drug-susceptible to the first-line drugs. The comparison of the IS6110RFLP patterns from our isolates against a set of 19 reference Beijing patterns from the RIVM (The Netherlands) confirmed that the strains belonged to the Beijing lineage. These findings might be partially explained by the important migration phenomena occurred during the last decade. Further surveillance studies would help in the following of Beijing family strain dissemination in our community.

First description of Mycobacterium tuberculosis Beijing genotype in Argentina

During a population-based study to genotype isolates of Mycobacterium tuberculosis from Buenos Aires Northern suburbs, we found isolates with molecular patterns related to those of the Beijing genotype. Five out of 590 (0.85

) patients had isolates with spoligopattern identical to that of the Beijing family. Since two of these isolates showed identical IS6110RFLP pattern, we found only four different patterns containing 11 to 19 bands. The isolates were obtained from young people (including a 7 years-old child) who were born in Argentina, and were living in a small area of our region. However, conventional contact tracing did not prove epidemiological linkage among them. These isolates were fully drug-susceptible to the first-line drugs. The comparison of the IS6110RFLP patterns from our isolates against a set of 19 reference Beijing patterns from the RIVM (The Netherlands) confirmed that the strains belonged to the Beijing lineage. These findings might be partially explained by the important migration phenomena occurred during the last decade. Further surveillance studies would help in the following of Beijing family strain dissemination in our community.

First description of Mycobacterium tuberculosis Beijing genotype in Argentina.

During a population-based study to genotype isolates of Mycobacterium tuberculosis from Buenos Aires Northern suburbs, we found isolates with molecular patterns related to those of the Beijing genotype. Five out of 590 (0.85

) patients had isolates with spoligopattern identical to that of the Beijing family. Since two of these isolates showed identical IS6110RFLP pattern, we found only four different patterns containing 11 to 19 bands. The isolates were obtained from young people (including a 7 years-old child) who were born in Argentina, and were living in a small area of our region. However, conventional contact tracing did not prove epidemiological linkage among them. These isolates were fully drug-susceptible to the first-line drugs. The comparison of the IS6110RFLP patterns from our isolates against a set of 19 reference Beijing patterns from the RIVM (The Netherlands) confirmed that the strains belonged to the Beijing lineage. These findings might be partially explained by the important migration phenomena occurred during the last decade. Further surveillance studies would help in the following of Beijing family strain dissemination in our community.

A microplate indicator-based method for determining the susceptibility of multidrug-resistant Mycobacterium tuberculosis to antimicrobial agents.

SETTING: Reference Laboratory, Buenos Aires Province Tuberculosis Control Program, Dr Cetrangolo Hospital, Argentina. OBJECTIVE: To obtain a rapid, inexpensive method of determining minimal inhibitory concentrations (MIC) of several drugs acting on multidrug-resistant Mycobacterium tuberculosis (MDR-TB), a colorimetric, microplate-based assay (M-MTT) was developed. DESIGN: One hundred and one clinical isolates were studied. Drugs were placed in a microtiter plate, and several two fold dilutions were made in situ. Kanamycin, capreomycin, ethionamide, para-aminosalicylic acid and clarithromycin were tested in a range concentration of 8.0-0.25 microg/ml, cycloserine 60.0-1.9 microg/ml, clofazimine 3.0-0.10 microg/ml, levofloxacin 4.0-0.13 microg/ml and rifabutin 1.0-0.13 microg/ml. General indicator 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) at 5.0 mg/ml was used for visualising cellular growth and viability. The proportion method with Middlebrook 7H11 was used as the gold standard. RESULTS: MICs by M-MTT were obtained at on average 8 days and correlated with those obtained using the proportion method. In our conditions, the total cost of MIC determination for nine drugs was 5.00 US dollars. CONCLUSION: M-MTT could be used as a simple, rapid, low-cost technology to test the susceptibility of MDR-TB strains to several second-line and alternative drugs, with the objective of orienting future treatment regimens.