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1.
Int J Mycobacteriol ; 9(1): 24-28, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32474484

RESUMEN

Background: Drug-resistant tuberculosis (TB) is an ongoing health threat, and the greatest challenge to adequate control of TB in many countries lies in the lack of proper laboratory drug-susceptibility test. The aim of this study was to evaluate the activity-based costs (ABC) of Kit SIRE Nitratase® (Kit SIRE) and compare its values with the conventional drug-susceptibility test. Methods: The ABC was calculated for three different approaches: Kit SIRE (clinical samples and cultures), proportion methods in Lowenstein Jensen (PM-LJ), and the Bactec™ MGIT™ 960 system based on Mycobacterial Research Laboratory's routine. Results: The ABC of Kit SIRE from cultures was US$ 148.54, while from clinical samples was US$ 136.12. In the case of conventional tests, the ABC of Bactec™ MGIT™ 960 was US$ 227.63 and of the PM-LJ was US$ 132.64. The Kit SIRE has a lower ABC when clinical samples are used instead of cultures. Compared to conventional tests, the ABC is similar to the PM-LJ and lower the Bactec™ MGIT™ 960. Conclusion: The Kit SIRE should be used as a screening method in clinical specimens and in culture for laboratories that do not have Bactec™ MGIT™ 960. Therefore, it can be incorporated into the routine of laboratories in countries with low resources and a high burden of TB and drug-resistant TB.

2.
Mem Inst Oswaldo Cruz ; 115: e190407, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32321155

RESUMEN

BACKGROUND: Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES: To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS: Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS: The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS: qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.


Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Marcadores Genéticos/genética , Isoniazida/farmacología , Mutación/genética , Mycobacterium tuberculosis/genética , ADN Bacteriano/genética , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad
3.
Mem. Inst. Oswaldo Cruz ; 115: e190407, 2020. tab
Artículo en Inglés | LILACS-Express | LILACS | ID: biblio-1101275

RESUMEN

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

4.
BMC Infect Dis ; 19(1): 1047, 2019 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-31823734

RESUMEN

BACKGROUND: Molecular tests can allow the rapid detection of tuberculosis (TB) and multidrug-resistant TB (MDR-TB). TB-SPRINT 59-Plex Beamedex® is a microbead-based assay developed for the simultaneous spoligotyping and detection of MDR-TB. The accuracy and cost evaluation of new assays and technologies are of great importance for their routine use in clinics and in research laboratories. The aim of this study was to evaluate the performance of TB-SPRINT at three laboratory research centers in Brazil and calculate its mean cost (MC) and activity-based costing (ABC). METHODS: TB-SPRINT data were compared with the phenotypic and genotypic profiles obtained using Bactec™ MGIT™ 960 system and Genotype® MTBDRplus, respectively. RESULTS: Compared with MGIT, the accuracies of TB-SPRINT for the detection of rifampicin and isoniazid resistance ranged from 81 to 92% and 91.3 to 93.9%, respectively. Compared with MTBDRplus, the accuracies of TB-SPRINT for rifampicin and isoniazid were 99 and 94.2%, respectively. Moreover, the MC and ABC of TB-SPRINT were USD 127.78 and USD 109.94, respectively. CONCLUSION: TB-SPRINT showed good results for isoniazid and rifampicin resistance detection, but still needs improvement to achieve In Vitro Diagnostics standards.


Asunto(s)
Farmacorresistencia Bacteriana , Citometría de Flujo/métodos , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Catalasa/genética , Costos y Análisis de Costo , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Citometría de Flujo/economía , Genotipo , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Regiones Promotoras Genéticas , Juego de Reactivos para Diagnóstico , Rifampin , Sensibilidad y Especificidad , Tuberculosis/economía
5.
BMC Infect Dis ; 19(1): 556, 2019 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-31238885

RESUMEN

BACKGROUND: In recent decades, Mycobacterium tuberculosis with the RDRio genotype, frequently isolated from tuberculosis patients in Rio de Janeiro, has become part of the Latin American - Mediterranean (LAM) family and has been associated with multidrug-resistant tuberculosis (MDR-TB). The aim of this study was to investigate the frequency of M. tuberculosis RDRio in the state of Minas Gerais, Brazil, and its relationship with MDR-TB. METHODS: For convenience, 172 susceptible and 63 MDR M. tuberculosis isolates were taken from pulmonary samples from patients diagnosed between January 2007 and December 2011. The DNA extracted from these isolates was analyzed by spoligotyping, PCR-RFLP to characterize fbpC103/Ag85C103, multiplex PCR to detect RDRio and RD174, and MIRU-VNTR 24 loci. RESULTS: Among the 235 isolates, the RDRio pattern was identified in 122 (51.9%) isolates (IC 0.45-0.58), with 100 (42.5%) wild-type and 13 (5.5%) mixed pattern isolates, whereas RD174 was identified in 93 of the 122 RDRio positive samples (76.3%). The LAM family and the LAM9 lineage were the most frequently identified among the isolates in this study. Among the 63 MDR isolates, 41 (65.1%) were RDRio and 28 (44.4%) RD174. CONCLUSION: The association of both deletions with MDR proved to be statistically significant, corroborating the few reports that have associated RDRio with MDR.


Asunto(s)
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Técnicas de Tipificación Bacteriana , Brasil/epidemiología , Farmacorresistencia Bacteriana Múltiple/genética , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Repeticiones de Minisatélite , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Tuberculosis/epidemiología , Tuberculosis/microbiología
6.
BMC Infect Dis ; 15: 306, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-26231661

RESUMEN

BACKGROUND: We aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil. METHODS: One hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions). RESULTS: Fifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB. CONCLUSIONS: Although our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20-30 % of the isolates we analyzed might be MDR-TB transmission cases.


Asunto(s)
Variación Genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Alelos , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Brasil/epidemiología , Análisis por Conglomerados , ADN Bacteriano/análisis , ARN Polimerasas Dirigidas por ADN , Genotipo , Humanos , Isoniazida/uso terapéutico , Repeticiones de Minisatélite/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología
7.
Rev Argent Microbiol ; 47(1): 47-9, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25724341

RESUMEN

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3%, 100%, 100% and 89.7%, respectively whereas accuracy was 90.7%. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method.


Asunto(s)
Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , ADN Bacteriano/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa , Seguridad , Manejo de Especímenes
8.
Rev. argent. microbiol ; 47(1): 47-9, 2015 Jan-Mar.
Artículo en Español | BINACIS | ID: bin-133755

RESUMEN

The possibility to obtain DNA from smears is a valuable alternative to remedy the lack of samples when they are totally used for bacilloscopy; this technique solves the biosafety problem related to a possible accident with the transportation of flasks containing potentially transmissible clinical samples. Hence, the purpose of this study was to utilize the insertion sequence IS6110 for amplification of DNA from a smear-positive sample for tuberculosis (TB) diagnosis. Among the 52 positive bacilloscopies, sensitivity, specificity, positive predictive value and negative predictive value were 52.3


, 100


, 100


and 89.7


, respectively whereas accuracy was 90.7


. The IS6110-based PCR for TB diagnosis developed in DNA extracted from a positive smear is a fast, simple, specific, and safe method.

9.
BMC Res Notes ; 6: 561, 2013 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-24373461

RESUMEN

BACKGROUND: Developments in molecular detection and strain differentiation of members of Mycobacterium tuberculosis complex have proved to be useful. The DNA extraction method influences the amplification efficiency, causing interference on the sensitivity and respective inhibitors. The aim of this study was to standardize a simple and fast DNA extraction method, providing DNA amplification by IS6110-PCR effectively free from undue interferences. FINDINGS: The efficiency of the six different protocols tested in M. tuberculosis cultures has varied from 75% to 92.5%. This preliminary study evaluating the IS6110 PCR sensitivity and specificity was developed in DNA extracted from microscope slides, and achieved 100% of efficiency. CONCLUSIONS: DNA extraction by Chelex + NP-40 method from both, cultures of M. tuberculosis and smear slides, resulted in good quantity of interference free DNA, especially in samples with low concentrations of genetic material; therefore, such technique may be used for the molecular diagnosis of tuberculosis.


Asunto(s)
ADN Bacteriano/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/normas , Tuberculosis Pulmonar/diagnóstico , Técnicas de Tipificación Bacteriana , Femenino , Humanos , Masculino , Polietilenglicoles/química , Poliestirenos/química , Polivinilos/química , Sensibilidad y Especificidad , Tuberculosis Pulmonar/microbiología
10.
J Bras Pneumol ; 32(1): 43-7, 2006.
Artículo en Inglés, Portugués | MEDLINE | ID: mdl-17273568

RESUMEN

OBJECTIVE: To describe data related to the pulmonary function of patients with sequelae of pulmonary tuberculosis, pleural tuberculosis or both. METHODS: In the outpatient clinic of a university hospital, 218 patients were evaluated. Of those 218, 56 had sequelae of tuberculosis (pulmonary, pleural or both), and 162 had other types of tuberculosis. All patients were evaluated in the pulmonary function laboratory between February 2000 and July 2004, and 43 were found to be eligible for inclusion in the study. Patients with a history of asthma, chronic pulmonary obstructive disease, cardiac insufficiency, collagen diseases, silicosis or thoracic surgery, as well as those for whom spirometry yielded unacceptable results or was not performed, were excluded. The lung fields were divided into six zones, and radiographic results were classified by degree: I (involvement of only one zone with no cavitation); II (involvement of two or three zones or of one zone with cavitation); or III (extensive involvement of three or more zones with or without cavitation). RESULTS: The final study sample comprised 50 patients, 44 (88%) of whom had pulmonary tuberculosis. The most prevalent form (17/50; 34%) was mixed ventilatory disturbance. Severe disturbances were more significant in degree III radiographs (p = 0.0002) and normal pulmonary function was predominant among patients presenting degree I and II radiographs (p = 0.002). CONCLUSION: The early discovery and treatment of tuberculosis contribute to reduce the number of cases, as well as the incidence of tuberculosis sequelae, thereby improving the quality of life of tuberculosis patients. Further studies, involving longitudinal, sequential analysis and larger samples of patients with tuberculosis sequelae, should be conducted in referral centers in Brazil.


Asunto(s)
Insuficiencia Respiratoria/etiología , Tuberculosis Pleural/complicaciones , Tuberculosis Pulmonar/complicaciones , Adulto , Femenino , Hospitales Universitarios , Humanos , Masculino , Pruebas de Función Respiratoria , Insuficiencia Respiratoria/diagnóstico , Índice de Severidad de la Enfermedad
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