RESUMEN
The frequency of mutations causing drug resistance in MTB isolates were studied in the respiratory material obtained from TB-patients in the Moscow Region. In izoniazid-resistant isolates, the most prevalent mutation was found to be the Ser315Thr substitution in the katG gene (15.8%) whereas the most frequent mutations in multidrug-resistant isolates were Ser531Leu and Ser315Thr in the rpoB and katG genes (26.3%), or a combination of these two substitutions with a T15 mutation in the inhA gene (5.3%). We compared performance of three molecular assays--"TB-BIOCHIP" ("BIOCHIP-IMB", Ltd, Russia), Xpert MTB/RIF ("Cepheid", USA) and GenoType MTBDRplus ("Hain Life-science", Germany), with the efficiency of luminescent microscopy, and phenotypic drug-suscepibility testing in an automated system BACTEC MGIT 960 (Becton, Disckinson and Company, USA). Xpert MTB/RIF, TB-BIOCHIP and GenoType MTBDRplus detected MTB in sputum in 92, 78 and 49% of all culture-positive cases, respectively. The agreement between standard cultural data and molecular DST results for Xpert MTB/RIF (resistance towards rifampicin), for TB-BIOCHIP and GenoType MTBDRplus (resistance towards rifampicin and izoniazid) amounted to 100, 97 and 100% respectively. Summing up, Xpert MTB/RIF was concluded to be the most efficient assay for primary detection of MTB, whereas the TB-BIOCHIP was shown to be the only molecular assay sensitive enough for simultaneous detection of MTB DNA and for revealing multidrug resistance in sputum (i.e. resistance to both first-line anti-TB drugs, rifampicin and izoniazid).
Asunto(s)
Resistencia a Múltiples Medicamentos/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Esputo/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/genética , Sustitución de Aminoácidos/genética , Antituberculosos/uso terapéutico , Humanos , Isoniazida/uso terapéutico , Mutación , Mycobacterium tuberculosis/patogenicidad , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Two hundred and forty-two mycobacterial cultures isolated from clinical materials on the Lwenstein-Jensen solid medium and 208 cultures in the automatic Bactec MGIT 960 system (on the modified Middlebrook 7H9 liquid medium) were examined. Of them, there was M. tuberculosis (85 and 82, respectively), M. kansasii (26 and 24), MAC (46 and 38), M. xenopi (24 and 20), M. fortuitum (26 and 22), and M. chelonae/abscessus complex (20 and 18). Identification of mycobacterial cultures isolated on the solid medium by microbiological assay and high performance liquid chromatography (HPLC) showed the agreement of results of the cultures isolated on the liquid medium in 95.5 and 97.2%, respectively; that by these techniques revealed rapidly growing nontuberculosis mycobacteria in 95.8 and 95.2% of cases, respectively; slowing growing mycobacteria in 91.7 and 97.8%, and M. tuberculosis in 96.5 and 97.6%. Mycobacterial isolation on the Middlebrook 7H9 liquid medium in the automatic Bactec MGIT-960 system takes a shorter time than that on the solid (Lwenstein-Jensen) medium. The microbiological identification of mycobacteria lasts as long as 3 weeks while the use of HPLC reduces its time to 24 hours. The efficiency of HPCL does not depend on whether mycobacterial cultures are isolated on the solid or liquid media.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Mycobacterium/aislamiento & purificación , Tuberculosis/diagnóstico , Medios de Cultivo , Humanos , Mycobacterium/crecimiento & desarrollo , Sensibilidad y Especificidad , Tuberculosis/microbiologíaRESUMEN
AIM: To compare results of mycobacteria identification by bacteriologic methods as well as by high-performance liquid chromatography (HPLC). MATERIALS AND METHODS: Two hundred and eighty strains of mycobacteria isolated from respiratory specimens and identified by bacteriologic methods and HPLC were studied. RESULTS: It was established that results of HLPC use were highly correlated with results of microbiologic methods of mycobacteria identification: for identification of M. tuberculosis complex the correlation was 97.0%, for nontuberculous (NTM) slowly growing mycobacteria--95.3%, for quickly growing NTM--96.2% (overall--96.1%). Results of identification of mycobacteria by HPLC were ready in significantly shorter time-frame (during 24 hours). CONCLUSION: HLPC method could be recommended for identification of mycobacteria in bacteriologic reference laboratories.
Asunto(s)
Cromatografía Líquida de Alta Presión , Infecciones por Mycobacterium/diagnóstico , Mycobacterium/aislamiento & purificación , Diagnóstico Diferencial , Humanos , Mycobacterium/clasificación , Infecciones por Mycobacterium/microbiología , Sensibilidad y EspecificidadRESUMEN
We developed a method of identification of Mycobacterium tuberculosis with simultaneous evaluation of the sensitivity to fluoroquinolones on a biological microchip array. The method of multiplex two-staged PCR followed by hybridization of a biochip makes it possible to detect 8 mutant variants of gyrA gene occurring in fluoroquinolone-resistant strains (approximately 85% all resistant forms) within 1 day. Using this method we analyzed 107 cultures isolated from patients with tuberculosis and 78 sputum samples. Mutations in gyrA gene were detected in 48 (92%) resistant strains. Natural S95T polymorphism in gyrA gene was detected in all resistant and in 76% sensitive strains. The sensitivity and specificity of the proposed method calculated on the basis of the analysis of sputum samples (n=78) were 94 and 100%, respectively.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Fluoroquinolonas/farmacología , Análisis por Micromatrices/métodos , Mutación , Mycobacterium tuberculosis , Antituberculosos/uso terapéutico , Secuencia de Bases , Fluoroquinolonas/uso terapéutico , Humanos , Hibridación Genética , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
At present the left-handed "respiratory" quinolones such as moxifloxacin and levofloxacin are the most promising drugs for therapy of multidrug resistant tuberculosis (MDR). Fast and specific diagnostics of sensitivity of M. tuberculosis (MBT) with MDR to this group of drugs is required for timely prescription of adequate chemotherapy and its correction in case of MBT resistance to fluoroquinolones. A new generation of biological microchips - TB-BIOCHIP-2 makes possible to detect 9 mutation types in quinolones resistant determination region (QRDR) of gene. About 800 samples from 169 patients in Antituberculosis center were studied. In patients with new detected tuberculosis 23.5% MBT resistant to isoniazid and rifampicin (MDR) and sensitive to fluoroquinolones were revealed. In patients with chronic tuberculosis 65.5% MBT-MDR were revealed. Our results were confirmed with detecting ofloxacin resistance on Lowenstein - Jensen. In addition efficiency of TB-BIOCHIP-2 to control drug testing sensitivity of MBT-MDR on fluoroquinolones was confirmed.
Asunto(s)
Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple , Electroforesis por Microchip/métodos , Fluoroquinolonas/uso terapéutico , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiología , Enfermedad Crónica , Procesamiento Automatizado de Datos , Humanos , Procedimientos Analíticos en Microchip , Esputo/microbiologíaRESUMEN
The paper presents the main points of the authors' own concept of the centralization of mycobacteriological service in large towns of the Russian Federation. The main points of step-by-step organizational and methodological measures required to solve this problem are described in detail. Consecutive measures to realize the proposed mycobacteriological service centralization model originated in January 2004 on a model of the Moscow Eastern Administrative District with 1380 thousand inhabitants are described.
Asunto(s)
Bacteriología/organización & administración , Laboratorios/organización & administración , Infecciones por Mycobacterium/complicaciones , Infecciones por Mycobacterium/microbiología , Neumología/organización & administración , Tuberculosis/microbiología , Servicios Urbanos de Salud/organización & administración , Áreas de Influencia de Salud , Humanos , Infecciones por Mycobacterium/epidemiología , Neumología/normas , Federación de Rusia/epidemiología , Tuberculosis/epidemiología , Servicios Urbanos de Salud/estadística & datos numéricosRESUMEN
The genus Mycobacterium currently comprises more than 90 species of Mycobacterium, of which a third is able to induce human diseases. With a rise in the incidence of diseases induced by non-tuberculosis mycobacteria, tuberculosis caused by M. bovis that is characterized by a severe cause and a high frequency of poor outcomes cannot be remembered. The species of mycobacteria should be identified to establish a diagnosis and to prescribe adequate chemotherapy. For this purpose, cultural, biochemical, chromatographic, and molecular genetic studies are conducted. The present study using the hsp65 gene restriction fragment length polymorphism test on museum mycobacterial strains and strains isolated from the diagnostic material of patients with suspected tuberculosis by means of Hind61 restrictase has provided a clear differentiation of the restriction profiles of MAIS complex mycobacteria and some other species of non-tuberculosis mycobacteria. To determine the species of representatives of M. tuberculosis complex (M. tuberculosis, M. bovis, BCG M. bovis), the authors have successfully used the test system "TUB-dif" developed at the Institute of Molecular Genetics, Russian Academy of Sciences, by applying the chain polymerase reaction of the senX3-regX3 region.
Asunto(s)
Biología Molecular/métodos , Mycobacterium tuberculosis/genética , Mycobacterium/genética , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Restriction fragment length polymorphism analysis of hsp65 gene was performed on museum strains of mycobacteria using Hin6I restrictase. Study of restriction profiles allowed us to distinguish mycobacterial species of the MAIS complex and several strains of nontuberculous mycobacteria.
Asunto(s)
Proteínas Bacterianas/genética , Chaperoninas/genética , Mycobacterium/genética , Chaperonina 60 , Cartilla de ADN , Electroforesis en Gel de Agar , Polimorfismo de Longitud del Fragmento de Restricción , Especificidad de la EspecieRESUMEN
By using the diagnostic material (175 sputum samples and 103 bronchoalveolar lavage fluid samples) taken from 39 patients with suspected tuberculous infection during a 2.5-month follow-up, the authors traced the time course of changes in the composition and drug sensitivity of a mycobacterial population to rifampicin. Along with the traditional microbiological studies, the latest molecular biological studies, a TB-BIOCHIP test system (enzyme immunoassay) in particular, were employed to detect the bacterial and L-transformed forms of the causative agent. A molecular biological assay was first developed to detect the drug sensitivity of L-forms of Mycobacterium tuberculosis.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Formas L/aislamiento & purificación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Rifampin/farmacología , Líquido del Lavado Bronquioalveolar , Distribución de Chi-Cuadrado , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Formas L/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Esputo/microbiología , Factores de TiempoRESUMEN
Two hundred and two patients with different forms of pulmonary tuberculosis were examined to study the characteristics of sensitivity with the signs of multidrug resistance to rifampicin and isoniazid, by using a microbiological assay of the absolute concentrations and determining mutations in the genes rpoB, katG, inhA, oxyR, and kasA, by employing different molecular biological assays. Mycobacterium tuberculosis (MBT) DNA was isolated from both a diagnostic material (such as sputum, bronchial secretion), and clinical MBT isolates. By showing a higher sensitivity and a higher specificity, as cultural techniques, molecular biological assays of MBT drug sensitivity in patients with tuberculosis were ascertained to accelerate its diagnosis until the patient was admitted to a clinic.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Antituberculosos/farmacología , ADN Bacteriano/aislamiento & purificación , Genes Bacterianos/genética , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Tuberculosis Pulmonar/tratamiento farmacológico , Antibióticos Antituberculosos/uso terapéutico , Antituberculosos/uso terapéutico , Farmacorresistencia Bacteriana , Resistencia a Múltiples Medicamentos , Humanos , Isoniazida/uso terapéutico , Pruebas de Sensibilidad Microbiana , Mutación , Rifampin/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Pulmonar/microbiologíaRESUMEN
The results of the laboratory diagnosis of respiratory tract candidiasis in patients with tuberculosis of the respiratory organs within 2-year observation in a tuberculosis clinic were generalized. The species composition of 327 isolates of the pathogens of the respiratory tract mycotic infection and the frequency of their detection in the specimens of various clinical material from patients with pulmonary tuberculosis were determined. The data on the resistance of the Candida spp. isolates to 6 antifungal drugs are presented. The number of the strains resistant to azol drugs (intraconazol and fluconazol) amounted to 10% of the total number of the Candida spp. isolates.
Asunto(s)
Antifúngicos/farmacología , Candida/efectos de los fármacos , Candidiasis/microbiología , Farmacorresistencia Fúngica , Infecciones del Sistema Respiratorio/microbiología , Tuberculosis Pulmonar/complicaciones , Candida/aislamiento & purificación , Candidiasis/diagnóstico , Femenino , Fluconazol/farmacología , Humanos , Itraconazol/farmacología , Masculino , Pruebas de Sensibilidad Microbiana , Infecciones del Sistema Respiratorio/diagnósticoRESUMEN
A variety of mutations in the genes rpoB, katG, inhA, ahpC, kasA was studied by using different molecular biological methods (conformational polymorphism of single-chain fragments, heteroduplex analysis, biochips) in rifampicin- and isoniazid-resistant Mycobacterium tuberculosis (MBT) strains isolated from patients with pulmonary tuberculosis. Twenty-nine mutation combinations were identified in the MBT strains. The use of biochips is the most promising method for identifying the type of mutations responsible for the simultaneous resistance to rifampicin and isoniazid. Detection of several MBT strains in one patient requires the use a combination of molecular biological and microbiological studies.
Asunto(s)
Antituberculosos/farmacocinética , Antituberculosos/uso terapéutico , Farmacorresistencia Microbiana , Pruebas de Sensibilidad Microbiana/instrumentación , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/metabolismo , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/microbiología , Análisis Mutacional de ADN , Humanos , Mutación Puntual/genética , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Pulmonar/genéticaRESUMEN
The results of the laboratory diagnosis of secondary (mixed) infection of the respiratory tracts in patients with respiratory tract tuberculosis were summarized. The study was performed for 12 months in a Tuberculosis Clinic. The species of the pathogens and the frequency of their detection in various clinical specimens from pulmonary tuberculosis patients were determined. The data on resistance of the strains of Streptococcus viridans group isolated from the pulmonary tuberculosis patients to various antimicrobials including new fluoroquinolones are presented.
Asunto(s)
Infecciones Estreptocócicas/microbiología , Sobreinfección/microbiología , Tuberculosis Pulmonar/microbiología , Estreptococos Viridans , Farmacorresistencia Bacteriana , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Infecciones Estreptocócicas/etiología , Sobreinfección/etiología , Tuberculosis Pulmonar/complicacionesRESUMEN
Mutations in the rpoB, katG, inhA, oxyR/ahpC genes in rifampicin- and isoniazid-resistant M. tuberculosis strains isolated from residents of Moscow, Astrakhan', and Moldova Republic were studied by molecular biological methods (heteroduplex analysis, single strand conformational polymorphism, biochips). Twenty-five combinations of mutations were detected. Some differences in the type distribution of detected mutations were found. The use of biochips is the most perspective method for determining the type of mutation.
Asunto(s)
Técnicas de Tipificación Bacteriana , Isoniazida/farmacología , Mycobacterium tuberculosis/metabolismo , Rifampin/farmacología , Antibióticos Antituberculosos/farmacología , Antituberculosos/farmacología , Codón , ADN/metabolismo , Farmacorresistencia Microbiana , Humanos , Mutación , Ácidos Nucleicos Heterodúplex , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo Conformacional Retorcido-Simple , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
RCR-heteroduplex (GDA) and chip methods were used to detect rifampricin-resistant (RR) and rifampicin-sensitive (RS) Mycobacterium tuberculosis (MTB) in the samples from patients (sputum) and in the clinical isolates of MTB from these patients (MB/BacT liquid medium and Lowenstein Jensen's (LJ) solid medium. The efficiency of detecting RR and RS of MTB (from the sputum) is 100 and 92.3% in the chip and GDA tests, respectively. Correlations between GDA (sputum) and drug test (LJ) were 91.7%, that of chip (sputum) and drug test LJ, 88.5%, chip (sputum) and chip clinical isolates (LJ), 100%. The efficacy of GDA and chip in the detection of RR of MTB strains is under discussion.
Asunto(s)
Antituberculosos/uso terapéutico , Biología Molecular/métodos , Infecciones por Mycobacterium/genética , Infecciones por Mycobacterium/microbiología , Mycobacterium tuberculosis/genética , Rifampin/uso terapéutico , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Farmacorresistencia Microbiana , Humanos , Mutación Puntual/genética , Tuberculosis/diagnósticoRESUMEN
A hundred and seventy respiratory samples from patients with different forms of tuberculosis were used to test the efficiency of the automatic liquid culture systems BACTEC MGIT 960 and MB/BacT with inoculation into the standard dense media. All these media provided 47 M. tuberculous isolates, of them 41 (87.2%), 38 (80.9%), and 76.6% on the BACTER 960, MB/BacT, and dense media, respectively. The average time of detection of mycobacterial growth by means of automatic systems was much shorter and equal to 10.7 days on the BACTEC 960 and 18.7 days on the MB/BacT versus 33.2 days on the standard dense medium. In terms of their sensitivity and detection rate, the automatic systems were superior to the dense media widely used in laboratory practice.
Asunto(s)
Mycobacterium tuberculosis/aislamiento & purificación , Técnicas Bacteriológicas , Medios de Cultivo , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Two alternative methods for identification of rifampicin-resistant strains of Mycobacterium tuberculosis on biological microchips are developed. The methods are based on detection of point mutations and other rearrangements in the rpoB gene region determining rifampicin resistance. Hybridization on TB-microchip detects 30 mutant variants of DNA in rifampicin-resistant strains (about 95% of all resistant forms). Allele-specific microchip PCR shortens the duration of analysis to 1.5 h. These methods can be used in clinical diagnostic laboratories for evaluating drug resistance/sensitivity of tuberculosis agent and for monitoring of the efficiency of antibiotic therapy.
Asunto(s)
Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Rifampin/farmacología , Antibióticos Antituberculosos/farmacología , Farmacorresistencia Microbiana , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/genética , Oligonucleótidos/metabolismoAsunto(s)
Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Pulmonar/tratamiento farmacológico , Antituberculosos/administración & dosificación , Antituberculosos/uso terapéutico , Medios de Cultivo , Farmacorresistencia Microbiana , Humanos , Isoniazida/administración & dosificación , Isoniazida/uso terapéutico , Modelos Teóricos , Mycobacterium tuberculosis/crecimiento & desarrollo , Investigación , Factores de TiempoRESUMEN
A test-system based on amplification of IS 986 fragment (nested-PCR) was developed for the detection of Mycobacterium tuberculosis and M. bovis in different biological samples. We constructed external primers and selected appropriate amplifications parameters (annealing temperatures for states I and II, the number of cycles for each amplification stage, components of the amplification mixture, and pretreatment conditions for different biological samples). The developed parameters make the detection of mycobacteria more efficient and less expensive compared to commercial Cobas Amplicor system.