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1.
J Clin Microbiol ; 50(7): 2194-202, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22535987

RESUMEN

Rifampin resistance in Mycobacterium tuberculosis is largely determined by mutations in an 80-bp rifampin resistance determining region (RRDR) of the rpoB gene. We developed a rapid single-well PCR assay to identify RRDR mutations. The assay uses sloppy molecular beacons to probe an asymmetric PCR of the M. tuberculosis RRDR by melting temperature (T(m)) analysis. A three-point T(m) code is generated which distinguishes wild-type from mutant RRDR DNA sequences in approximately 2 h. The assay was validated on synthetic oligonucleotide targets containing the 44 most common RRDR mutations. It was then tested on a panel of DNA extracted from 589 geographically diverse clinical M. tuberculosis cultures, including isolates with wild-type RRDR sequences and 25 different RRDR mutations. The assay detected 236/236 RRDR mutant sequences as mutant (sensitivity, 100%; 95% confidence interval [CI], 98 to 100%) and 353/353 RRDR wild-type sequences as wild type (specificity, 100%; 95% CI, 98.7 to 100%). The assay identified 222/225 rifampin-resistant isolates as rifampin resistant (sensitivity, 98.7%; 95% CI, 95.8 to 99.6%) and 335/336 rifampin-susceptible isolates as rifampin susceptible (specificity, 99.7%; 95% CI, 95.8 to 99.6%). All mutations were either individually identified or clustered into small mutation groups using the triple T(m) code. The assay accurately identified mixed (heteroresistant) samples and was shown analytically to detect RRDR mutations when present in at least 40% of the total M. tuberculosis DNA. This was at least as accurate as Sanger DNA sequencing. The assay was easy to use and well suited for high-throughput applications. This new sloppy molecular beacon assay should greatly simplify rifampin resistance testing in clinical laboratories.


Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Factores de Tiempo , Temperatura de Transición , Tuberculosis/microbiología
2.
J Clin Microbiol ; 49(3): 932-40, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21191047

RESUMEN

Fluoroquinolones (FQ) are important second-line drugs to treat tuberculosis; however, FQ resistance is an emerging problem. Resistance has been mainly attributed to mutations in a 21-bp region of the Mycobacterium tuberculosis gyrA gene, often called the quinolone resistance-determining region (QRDR). We have developed a simple, rapid, and specific assay to detect FQ resistance-determining QRDR mutations. The assay amplifies the M. tuberculosis gyrA QRDR in an asymmetrical PCR followed by probing with two sloppy molecular beacons (SMBs) spanning the entire QRDR. Mutations are detected by melting temperature (T(m)) shifts that occur when the SMBs bind to mismatched sequences. By testing DNA targets corresponding to all known QRDR mutations, we found that one or both of the SMBs produced a T(m) shift of at least 3.6°C for each mutation, making mutation detection very robust. The assay was also able to identify mixtures of wild-type and mutant DNA, with QRDR mutants identified in samples containing as little as 5 to 10% mutant DNA. The assay was blindly validated for its ability to identify the QRDR mutations on DNA extracted from clinical M. tuberculosis strains. Fifty QRDR wild-type samples, 34 QRDR mutant samples, and 8 heteroresistant samples containing mixtures of wild-type and mutant DNA were analyzed. The results showed 100% concordance to conventional DNA sequencing, including a complete identification of all of the mixtures. This SMB T(m) shift assay will be a valuable molecular tool to rapidly detect FQ resistance and to detect the emergence of FQ heteroresistance in clinical samples from tuberculosis patients.


Asunto(s)
Antibacterianos/farmacología , Técnicas Bacteriológicas/métodos , Fluoroquinolonas/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Reacción en Cadena de la Polimerasa/métodos , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Girasa de ADN/genética , Cartilla de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Humanos , Mycobacterium tuberculosis/aislamiento & purificación , Sondas de Oligonucleótidos/genética , Temperatura de Transición
3.
J Clin Microbiol ; 48(2): 387-94, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20018816

RESUMEN

Tuberculosis (TB) remains an immense public health problem in the Republic of Korea despite a more than fivefold decrease in the prevalence of the disease over the last 3 decades. The rise in drug-resistant TB has compounded the situation. We analyzed 208 clinical isolates of M. tuberculosis from the National Masan Tuberculosis Hospital by spoligotyping, IS6110 restriction fragment length polymorphism (RFLP), and 24-locus-based mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing to assess the diversity and transmission dynamics of the tubercle bacilli in the Republic of Korea. The majority of the isolates (97.1%) belonged to the Beijing genotype. Cluster analysis by MIRU-VNTR yielded a low clustering rate of 22.3%, with most of the clusters comprising isolates with diverse drug resistance patterns. The discriminatory capacity of the typing methods was high for RFLP and MIRU-VNTR (allelic diversity [h] = 0.99) but low for spoligotyping (h = 0.31). Although analysis of 19 MIRU-VNTR loci was needed to achieve maximum discrimination, an informative set of 8 loci (960, 1955, 2163b, 2165, 2996, 3192, 4052, and 4348) (h = 0.98) that was able to differentiate most of the closely related strains was identified. These findings suggest that 24-locus-based MIRU-VNTR typing is a likely suitable alternative to RFLP to differentiate clinical isolates in this setting, which is dominated by M. tuberculosis Beijing strains. Within the study limits, our results also suggest that the problem of drug-resistant TB in the Republic of Korea may be largely due to acquired resistance as opposed to transmission.


Asunto(s)
Variación Genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Adulto , Anciano , Anciano de 80 o más Años , Antituberculosos/farmacología , Técnicas de Tipificación Bacteriana/métodos , Análisis por Conglomerados , Dermatoglifia del ADN/métodos , ADN Bacteriano/genética , Femenino , Genotipo , Hospitales de Enfermedades Crónicas , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Repeticiones de Minisatélite , Epidemiología Molecular , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo de Longitud del Fragmento de Restricción , República de Corea , Adulto Joven
4.
Infect Immun ; 76(6): 2333-40, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18347040

RESUMEN

Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow the targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in four animal models of disease: mouse, guinea pig, rabbit, and nonhuman primate. Following infusion of pimonidazole into animals with established infections, lung tissues from the guinea pig, rabbit, and nonhuman primate showed discrete areas of pimonidazole adduct formation surrounding necrotic and caseous regions of pulmonary granulomas by immunohistochemical staining. This labeling could be substantially reduced by housing the animal under an atmosphere of 95% O(2). Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3-mm) pulmonary lesions were severely hypoxic (1.6 +/- 0.7 mm Hg). Finally, metronidazole, which has potent bactericidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total-lung bacterial burdens in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.


Asunto(s)
Granuloma/metabolismo , Hipoxia/diagnóstico , Tuberculosis Pulmonar/metabolismo , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Modelos Animales de Enfermedad , Granuloma/tratamiento farmacológico , Granuloma/patología , Cobayas , Humanos , Hipoxia/metabolismo , Hipoxia/patología , Inmunohistoquímica , Pulmón/patología , Macaca fascicularis , Metronidazol/farmacología , Metronidazol/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/efectos de los fármacos , Nitroimidazoles , Oxígeno/análisis , Oxígeno/metabolismo , Conejos , Fármacos Sensibilizantes a Radiaciones , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/patología
5.
J Vet Sci ; 9(2): 145-53, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18487935

RESUMEN

Bovine tuberculosis (TB) is a major zoonosis that's caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis- like lesions. Twenty-four published MIRUVNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.


Asunto(s)
Variación Genética , Repeticiones de Minisatélite/genética , Mycobacterium bovis/genética , Tuberculosis Bovina/epidemiología , Tuberculosis Bovina/microbiología , Animales , Bovinos , Cartilla de ADN/genética , Genotipo , Corea (Geográfico)/epidemiología , Prevalencia
6.
Vet Microbiol ; 114(3-4): 292-7, 2006 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-16414212

RESUMEN

Skin ulcers, scoliosis, and dropsy-like scale edema were observed in laboratory-maintained zebrafish. Affected fish had multifocal granulomas not only in internal organs such as the liver, intestine, genital organs, kidney, muscle, and spleen but also in the fin, epithelium, gills, and sclera of the eyes. Large numbers of acid-fast-rod-shaped bacteria were observed within the necrotic centers of well-demarcated, multifocal granulomas with Gram's stain and Ziehl-Neelson's stain. The size of the Mycobacterium spp. was 1-2 microm x 2-3 microm with a double-layered cell wall, based upon electron-microscopical features. Definitive diagnosis of these outbreaks was obtained by culture on selective media followed by PCR-restriction fragment length polymorphism analysis (PRA) of the rpoB gene for species identification. The amplified 360-bp products of the rpoB gene of mycobacteria isolated from zebrafish were digested with MspI restriction enzyme, which revealed unique band patterns matching those of Mycobacterium abscessus and Mycobacterium chelonae which are responsible for skin and soft tissue infection caused by rapidly growing mycobacteria in humans. This is the first documentation of the precise identification of zoonotic non-tuberculous mycobacteria isolated from laboratory-maintained zebrafish by the PRA of the rpoB gene; this study thus provides a great deal of useful epidemiological information and reduces the likelihood that epizootics will occur.


Asunto(s)
Enfermedades de los Peces/microbiología , Infecciones por Mycobacterium/veterinaria , Mycobacterium chelonae/aislamiento & purificación , Mycobacterium/aislamiento & purificación , Polimorfismo de Longitud del Fragmento de Restricción , Pez Cebra , Animales , ARN Polimerasas Dirigidas por ADN/genética , Brotes de Enfermedades/veterinaria , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/patología , Amplificación de Genes , Mycobacterium/genética , Infecciones por Mycobacterium/diagnóstico , Infecciones por Mycobacterium/microbiología , Infecciones por Mycobacterium/patología , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium chelonae/genética , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Zoonosis/microbiología
7.
Autoimmunity ; 49(2): 90-101, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26699759

RESUMEN

Tolerogenic dendritic cells (tDCs) play an important role in inducing peripheral tolerance; however, few tDC-specific markers have been identified. The aims of this study were to examine whether tDCs show a different gene expression profile from that of immunogenic DCs and identify specific gene markers of each cell type, in DBA/1 mice. tDCs were generated by treating immature DCs (imDCs) with TNF-α and type II collagen. The gene expression profiles of mature (m)DCs and tDCs were then investigated by microarray analysis and candidate markers were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Supervised selection identified 75 gene signatures, 63 of which were consistently upregulated in mDCs and 12 of which were upregulated only in tDCs. Additionally, 10 genes were overexpressed or equally expressed in both tDCs and mDCs. Scin (tDC-specific genes) and Orm1, Pdlim4 and Enpp2 (mDC-specific genes) were validated by real-time qRT-PCR. Taken together, these results clearly show that tDCs and mDCs can be identified according to their expression of specific gene markers.


Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Tolerancia Inmunológica/genética , Transcriptoma , Animales , Biomarcadores , Células Cultivadas , Análisis por Conglomerados , Técnicas de Cocultivo , Biología Computacional/métodos , Perfilación de la Expresión Génica , Ontología de Genes , Inmunofenotipificación , Ratones , Ratones Endogámicos DBA , Reproducibilidad de los Resultados , Linfocitos T/inmunología , Linfocitos T/metabolismo
8.
Cardiovasc Res ; 101(2): 203-10, 2014 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-24189626

RESUMEN

AIMS: Autoimmunity plays an important role in the pathogenesis of viral myocarditis and giant cell myocarditis. Experimental autoimmune myocarditis (EAM) is a mouse model of myocarditis that is induced by cardiac myosin. Tolerogenic dendritic cells (tDCs) are used as anti-inflammatory and immunosuppressive targets in a number of autoimmune disease models, but their effect on EAM has not been addressed. The aim of this study was to investigate whether tDC therapy in an EAM mouse model can suppress inflammatory myocarditis, a potential precursor of dilated cardiomyopathy. METHODS AND RESULTS: tDCs were generated by treating immature DCs (imDCs) with TNF-α and cardiac myosin. Mice with EAM were injected twice with tDCs (with a 1-week interval) at three doses (2 × 10(5), 1 × 10(6), or 2 × 10(6)). The severity of myocarditis was histopathologically assessed. The phenotypes of the DC and regulatory T (Treg) cell populations were determined by flow cytometry and the effect of tDCs on autoimmunity-inducing cytokines was examined by ELISA. Myosin-pulsed tDCs displayed lower levels of DC-related surface markers and expressed higher levels of indoleamine 2, 3-dioxygenase (IDO) than mature DCs (mDCs). Histopathological examination revealed that hearts from tDC-treated mice showed markedly reduced myocardial inflammation compared with those of untreated EAM mice. These therapeutic effects by tDCs were mediated at least by enhanced myosin-specific Treg cell induction and anti-inflammatory cytokine secretion. CONCLUSION: Taken together, these results show for the first time that myosin-pulsed tDCs ameliorate EAM, and that this occurs most likely via the induction of antigen-specific Treg cells.


Asunto(s)
Traslado Adoptivo , Enfermedades Autoinmunes/prevención & control , Autoinmunidad , Miosinas Cardíacas/inmunología , Células Dendríticas/trasplante , Tolerancia Inmunológica , Miocarditis/prevención & control , Miocardio/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Miocarditis/inmunología , Miocarditis/metabolismo , Miocarditis/patología , Miocardio/metabolismo , Miocardio/patología , Fenotipo , Linfocitos T Reguladores/inmunología , Factores de Tiempo , Vacunación
10.
Infect Immun ; 73(9): 5666-74, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16113284

RESUMEN

In spite of its many other benefits, DNA vaccine is limited in its application by its insufficient immunogenicity. One promising approach for enhancing its immunogenicity is to maximize its expression in the immunized host. In the current study, we investigated whether codon optimization of the mycobacterial antigen Ag85B gene could enhance the expression and immunogenicity of the Ag85B DNA vaccine. We generated a synthetic humanized Ag85B (hAg85B) gene in which codon usage was optimized for expression in human cells. DNA plasmids with codon-optimized hAg85B increased the level of protein expression in vitro and in vivo. DNA vaccine with hAg85B induced stronger Th1-like and cytotoxic T-cell immune responses in BALB/c mice and generated higher protective immunity in a BALB/c mouse model of Mycobacterium tuberculosis aerosol infection than did the DNA vaccine with wild-type Ag85B. Therefore, our results suggest that codon optimization of mycobacterial antigens (e.g., Ag85B) could improve protein expression and thereby enhance the immunogenicity of DNA vaccines against M. tuberculosis.


Asunto(s)
Aciltransferasas/genética , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Codón/biosíntesis , Mycobacterium tuberculosis/inmunología , Tuberculosis/prevención & control , Vacunas de ADN/inmunología , Aciltransferasas/administración & dosificación , Aciltransferasas/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos/efectos de los fármacos , Formación de Anticuerpos/inmunología , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/inmunología , Secuencia de Bases , Femenino , Interferón gamma/biosíntesis , Interferón gamma/genética , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Tuberculosis/inmunología , Vacunas de ADN/administración & dosificación
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