RESUMEN
Group A streptococcus (GAS) species cause bacterial pharyngitis in both adults and children. Early and accurate diagnosis of GAS is important for appropriate antibiotic therapy to prevent GAS sequalae. The Revogene Strep A molecular assay (Meridian Bioscience Canada Inc, Quebec City, QC, Canada) is an automated real-time PCR assay for GAS detection from throat swab specimens within approximately 70 min. This multicenter prospective study evaluated the performance of the Revogene Strep A molecular assay compared to that of bacterial culture. Dual throat swab specimens in either liquid Amies or Stuart medium were collected from eligible subjects (pediatric population and adults) enrolled across 7 sites (USA and Canada). Revogene Strep A and reference testing was performed within 7 days and 48 h of sample collection, respectively. Of the 604 evaluable specimens, GAS was detected in 154 (25.5%) samples by the reference method and in 175 (29%) samples by the Revogene Strep A assay. Revogene Strep A assay sensitivity and specificity were reported to be 98.1% (95% confidence interval [CI], 94.4 to 99.3) and 94.7% (95% CI, 92.2 to 96.4), respectively. The positive predictive value was 86.3% (95% CI, 80.4 to 90.6), negative predictive value was 99.3% (95% CI, 98.0 to 99.8) with a 1.0% invalid rate. Discrepant analysis with alternative PCR/bidirectional sequencing was performed for 24 false-positive (FP) and 3 false-negative (FN) specimens. Concordant results were reported for 17 (FP only) of 27 discordant specimens. The Revogene Strep A assay had high sensitivity and specificity for GAS detection and provides a faster alternative for GAS diagnosis.
Asunto(s)
Faringitis , Infecciones Estreptocócicas , Adulto , Canadá , Niño , Humanos , Faringitis/diagnóstico , Faringe , Estudios Prospectivos , Quebec , Sensibilidad y Especificidad , Infecciones Estreptocócicas/diagnóstico , Streptococcus pyogenes/genéticaRESUMEN
BACKGROUND: Cytomegalovirus (CMV) causes significant morbidity and mortality in solid organ and bone marrow transplant recipients. DNA vaccines can provide both humoral and cellular immunity without exposing immune-compromised persons to replication-competent CMV. METHODS: We studied the kinetics of CMV vaccine DNA in plasma. The samples were obtained from vaccine recipients who were enrolled in a double-blinded, placebo-controlled clinical trial of an intramuscular, plasmid-based, bivalent DNA vaccine for CMV in stem cell transplant recipients. Residual specimens on patients enrolled in the vaccine trial were saved until the trial was unblinded and published. Quantitative real-time polymerase chain reaction (PCR) was used to detect and quantify CMV glycoprotein B (gB) DNA in plasma from 4 recipients of the vaccine. The melting temperature of the vaccine gB amplicon was 62.4°C, compared to 68.8°C, which is seen with the wild-type virus. RESULTS: Sequence analysis revealed that there were 3 mismatches between the fluorescent resonance energy transfer probe and the vaccine DNA sequence. CONCLUSION: Because preemptive treatment of CMV disease in stem cell transplant patients is based on quantitative PCR analysis of viral sequences in plasma, it is important that vaccine sequences not be confused with those in wild-type virus. Confusion could lead to treatment with toxic medications, potentially compromising the transplant. Effects of PCR target choice and amplicon detection techniques on patient management and vaccine trials are discussed.
Asunto(s)
Vacunas contra Citomegalovirus/inmunología , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , ADN Viral/aislamiento & purificación , Trasplante de Células Madre Hematopoyéticas , Secuencia de Bases , Citomegalovirus/metabolismo , Vacunas contra Citomegalovirus/administración & dosificación , ADN Viral/genética , ADN Viral/metabolismo , Femenino , Humanos , Inyecciones Intramusculares , Masculino , Persona de Mediana Edad , Carga ViralRESUMEN
Sepsis caused by Staphylococcus aureus is a major health problem worldwide. Better outcomes are achieved when rapid diagnosis and determination of methicillin susceptibility enable early optimization of antimicrobial therapy. Eight large clinical laboratories, seven from the United States and one from Scotland, evaluated the combination of the Staphylococcus QuickFISH BC and the new mecA XpressFISH assay (both AdvanDx, Woburn, MA, USA) for the detection of methicillin-resistant S. aureus in positive blood cultures. Blood cultures flagged as positive by automated blood culture instruments and demonstrating only Gram-positive cocci in clusters on Gram stain were tested by QuickFISH, a 20-min assay. If only S. aureus was detected, mecA XpressFISH testing followed. The recovered S. aureus isolates were tested by cefoxitin disk diffusion as the reference method. The QuickFISH assay results were concordant with the routine phenotypic testing methods of the testing laboratories in 1,211/1,221 (99.1%) samples and detected 488/491 S. aureus organisms (sensitivity, 99.4%; specificity, 99.6%). Approximately 60% of the samples (730) contained coagulase-negative staphylococci or nonstaphylococci as assessed by the QuickFISH assay and were not tested further. The 458 compliant samples positive exclusively for S. aureus by the QuickFISH assay were tested by the mecA XpressFISH assay, which detected 209 of 211 methicillin-resistant S. aureus organisms (sensitivity, 99.1%; specificity, 99.6%). The mecA XpressFISH assay also showed high reproducibility, with 534/540 tests performed by 6 operators over 5 days achieving reproducible results (98.9% agreement). The combination of the Staphylococcus QuickFISH BC and mecA XpressFISH assays is sensitive, specific, and reproducible for the detection of methicillin-resistant S. aureus and yields complete results in 2 h after the blood culture turns positive.
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Sangre/microbiología , Hibridación Fluorescente in Situ/métodos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Sepsis/diagnóstico , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Técnicas Bacteriológicas/métodos , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Reproducibilidad de los Resultados , Escocia , Sensibilidad y Especificidad , Sepsis/microbiología , Estados UnidosRESUMEN
The recent progress made in developing laser-induced breakdown spectroscopy (LIBS) has transformed LIBS from an elemental analysis technique to one that can be applied for the reagentless analysis of molecularly complex biological materials or clinical specimens. Rapid advances in the LIBS technology have spawned a growing number of recently published articles in peer-reviewed journals which have consistently demonstrated the capability of LIBS to rapidly detect, biochemically characterize and analyse, and/or accurately identify various biological, biomedical or clinical samples. These analyses are inherently real-time, require no sample preparation, and offer high sensitivity and specificity. This overview of the biomedical applications of LIBS is meant to summarize the research that has been performed to date, as well as to suggest to health care providers several possible specific future applications which, if successfully implemented, would be significantly beneficial to humankind.
Asunto(s)
Rayos Láser , Análisis Espectral/instrumentación , Análisis Espectral/métodos , Bacterias/química , Tecnología Biomédica , Humanos , Técnicas Microbiológicas , Diente/químicaRESUMEN
Molecular method of 16S rRNA sequencing is reported to be helpful in the accurate identification of organisms with ambiguous phenotypic profiles. We analyzed the use of 16S rRNA sequencing method to identify clinically significant, "difficult-to-identify" bacteria recovered from clinical specimens, and evaluated its role in patient management and consequent clinical outcome. Among the 172 "difficult-to-identify" bacteria recovered over a 4-year period, 140 were gram-positive cocci or gram-negative bacilli; identification by 16S rRNA did not play a role in the management of patients infected with these bacteria. From 32 patients, 33 "difficult-to-identify" gram-positive bacilli were identified; the organisms were mycobacteria, Nocardia, Tsukamurella, Rhodococcus, and Gordonia. In 24 patients for whom clinical data were available, results from the 16S rRNA sequencing method led to treatment change in 14 immunocompromised patients (including 7 hematopoietic stem cell recipients and 1 liver transplant recipient). Therapy was modified in 9 patients, initiated in 3 patients, and discontinued in 2 patients. Most patients' therapy was switched to oral antibiotics with discontinuation of intravascular catheters, facilitating early hospital discharge. All 14 patients were alive 30 days after infection onset. The present study demonstrates the clinical application of 16S rRNA sequencing method to identify "difficult-to-identify" mycobacteria and other gram-positive bacilli in clinical specimens, particularly in immunocompromised hosts.
Asunto(s)
Bacterias/clasificación , Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , ARN Ribosómico 16S/genética , Adolescente , Adulto , Anciano , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/inmunología , Femenino , Humanos , Huésped Inmunocomprometido , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
We report the first case of Weissella confusa bacteremia in an allogeneic hematopoietic stem cell transplant patient. After engraftment and discharge, the patient returned with fever and graft failure and was started on an empiric regimen of aztreonam and vancomycin. A blood culture grew an alpha-hemolytic, gram-positive coccus forming pairs and chains, originally thought to be a viridans Streptococcus and a skin contaminant. The isolation of the organism from multiple blood cultures, and the presence of vancomycin resistance prompted identification and additional susceptibility testing. The RapID(™) Str panel, which has W. confusa in its database, provided multiple incorrect identifications. The MicroScan WalkAway 96 SI, using PC-20 or -29 panels, also did not identify this bacterium, because it is not in their database. The organism was identified as W. confusa by 16S rDNA sequencing. Antibiotic susceptibility determination by Etest revealed vancomycin resistance and daptomycin susceptibility. Therapy was changed to daptomycin, and the infection resolved. Additionally, W. confusa sepsis, with multiple positive blood cultures, developed in a patient in the burn unit at our medical center. The patient's blood cultures remained positive until vancomycin was discontinued and daptomycin therapy initiated. Infections with vancomycin-resistant, gram-positive cocci are emerging among immuno compromised hosts. Under appropriate circumstances, clinicians need to request that the laboratory perform susceptibility testing and accurate identification, by nucleic acid sequencing if necessary. Sequencing of 16S rDNA is an important tool in the accurate identification of unusual pathogens.
Asunto(s)
Bacteriemia/microbiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Resistencia a la Vancomicina , Weissella/genética , Weissella/aislamiento & purificación , Adulto , Antibacterianos/farmacología , ADN Bacteriano/análisis , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Huésped Inmunocomprometido , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/métodos , Trasplante Homólogo/efectos adversos , Vancomicina/farmacología , Weissella/clasificación , Weissella/efectos de los fármacosRESUMEN
We report the first case of Listeria grayi bacteremia in a stem cell transplant recipient. The patient developed bacteremia with a gram-positive rod that was initially thought to be Corynebacterium species and a skin contaminant. The organism grew in multiple blood cultures and therapy with vancomycin was initiated. The API Coryne (version 3.0) identified the organism as L. grayi. Susceptibility testing by Etest suggested that the organism was resistant to vancomycin, but susceptible to ampicillin. After therapeutic change from vancomycin to ampicillin, the bacteremia cleared. Empiric therapy with vancomycin for all gram-positive bacterial infections is not appropriate. Accurate identification and antibiotic susceptibility is important, particularly in those with persistent bacteremia.
Asunto(s)
Antibacterianos/farmacología , Bacteriemia/microbiología , Farmacorresistencia Bacteriana , Listeria/efectos de los fármacos , Trasplante de Células Madre/efectos adversos , Vancomicina/farmacología , Adulto , Ampicilina/uso terapéutico , Antibacterianos/uso terapéutico , Femenino , Humanos , Listeria/clasificación , Listeria/aislamiento & purificación , Listeriosis/microbiología , Pruebas de Sensibilidad Microbiana , Resultado del Tratamiento , Vancomicina/uso terapéutico , Adulto JovenRESUMEN
The molecular analysis of methicillin-resistant Staphylococcus aureus (MRSA) from 98 children admitted to the Children's Hospital of Michigan, Detroit, MI, with serious MRSA infections during 2006-2007 was correlated with risk factors, clinical features, and antibiotic susceptibility testing (ABST) results. Isolates were characterized by staphylococcal cassette chromosome (SCC) mec type, the presence of Panton-Valentine leukocidin (PVL) genes, repetitive sequence (rep) polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE), requirement for surgical intervention, antibiograms, and response to therapy. rep-PCR was more rapid than PFGE typing and correlated well. SCCmec type IV-containing isolates caused 92.8% of all infections, but the demographics and diseases associated with subtypes IVa and IVd differed. Subtype IVa (all PFGE type USA300 and PVL-positive) was identified in 81/93 (87.1%) of patients with community-onset (CO) MRSA, including 21/35 of those with risk factors for health care-associated (HA) infection. All other clones were PVL-negative. Subtype IVd (10 isolates; 9 USA800 and 1 eMRSA15) caused mainly HA-MRSA and no skin and soft tissue infections (SSTI). Seven classic HA-MRSA strains (SCCmec types II [6; 3 USA100 and 3 USA600] and III [1; USA200]) caused HA and hospital-onset (HO) infections. Surgical intervention was required in 68/81 patients infected with USA300 and 8/17 of the others. Most USA300 were susceptible (S) to clindamycin (CD) and patients were treated with CD alone or in combination. The other isolates were generally treated with vancomycin (VA) alone or in combination.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Infecciones Estafilocócicas/microbiología , Adolescente , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Niño , Niño Hospitalizado , Preescolar , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/genética , Exotoxinas/genética , Femenino , Humanos , Lactante , Recién Nacido , Leucocidinas/genética , Masculino , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/genética , Michigan , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Factores de Riesgo , Infecciones Estafilocócicas/patología , Infecciones Estafilocócicas/fisiopatologíaRESUMEN
The activity of the promoter regions of the cell division genes ftsZ, ftsE, minC, minD and minE from Neisseria gonorrhoeae (Ng) was studied under different environmental conditions using lacZ translational fusions. The promoters of the minNg genes have not been previously determined and we identified promoter regions upstream of each gene (minCp, minDp and minEp). We determined that minDp had the strongest activity. Expression of the promoter regions of ftSZ(Ng) and ftsE(Ng), which we had previously identified, as well as minD(Ng), were then studied under conditions reflecting the environment of the genitourinary tract. These conditions included anaerobiosis, presence of isoleucine or urea (3 mM and 400 mM, respectively) and acidity of pH 6. Both beta-galactosidase expression and northern blot analysis indicated that all three genes were upregulated under anaerobiosis. The addition of isoleucine as well as media at pH 6 did not have any significant effects on the promoter activity of these genes while the presence of urea significantly decreased ftsZ(Ng) promoter activity. The expression of the minD(Ng) promoter region was analyzed during different growth phases and shown to follow the growth behavior of the culture. By contrast, the ftSZ(Ng) promoter activity continued to rise after the onset of the stationary phase. When gonococcal ftsZ promoter 1, (Pz1) was altered by site-directed mutagenesis, a significant decrease in the expression of ftsZ(Ng) was observed under both aerobic and anaerobic conditions. These data infer that gonococci regulate their cell division in response to different environments.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas del Citoesqueleto , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Neisseria gonorrhoeae/genética , Adenosina Trifosfatasas/metabolismo , Anaerobiosis , Proteínas Bacterianas/metabolismo , División Celular/genética , Medios de Cultivo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Concentración de Iones de Hidrógeno , Isoleucina/metabolismo , Neisseria gonorrhoeae/citología , Neisseria gonorrhoeae/crecimiento & desarrollo , Neisseria gonorrhoeae/metabolismo , Regiones Promotoras Genéticas , Urea/metabolismoRESUMEN
A cluster of genes involved in cell division and cell wall (dcw) biosynthesis was identified in Neisseria gonorrhoeae using genomic analysis and through verification of gene order by polymerase chain reaction (PCR) analysis. The gonococcal dcw cluster consists of 17 genes, in the order 5'-mraZ-mraW-ftsI-murE-hyp1-murF- mraY-hyp2-murD-ftsW-murG-murC-ddl -ft sQ-ftsA-ftsZ-hyp3-3'. The gene organization of the dcw cluster of N. gonorrhoeae is more similar to that observed in Gram-negative rods such as Escherichia coli and Haemophilus influenzae than in Gram-positive bacteria. The cluster is characterized by several intergenic spaces. Compared with E. coli, two genes, ftsL and envA, are absent in the gonococcal dcw cluster and three hypothetical genes are novel to the cluster. The cluster is flanked by two transcriptional terminators consisting of paired neisserial uptake sequences and also includes four internal terminators, three of which are paired neisserial uptake sequences. We also found that a repeated sequence on the gonococcal genome, commonly called a Correia element, acts as the fourth transcriptional terminator. All termination sequences were shown to be fully functional by using reverse transcription PCR experiments. Transcriptional start sites upstream of ftsQ, ftsA and ftsZ were determined by primer extension and six promoters were identified; three promoters were located upstream of ftsZ in the intergenic space, two were upstream of ftsA within ftsQ and one was upstream of ftsQ within ddl. Some of these promoters were preferentially used under anaerobic conditions. The location of these promoters differed from those described in E. coli indicating dissimilar transcriptional regulation.
Asunto(s)
Genes Bacterianos/genética , Familia de Multigenes , Neisseria gonorrhoeae/genética , Secuencia de Bases , División Celular/genética , Pared Celular/metabolismo , ADN Bacteriano/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Regiones Terminadoras Genéticas , Transcripción GenéticaRESUMEN
Although ftsE and ftsX are not universally present in bacteria, they are present in various Neisseria species as determined by Southern hybridization. The ftsE and ftsX genes of Neisseria gonorrhoeae (Ng) CH811 were cloned, sequenced and were shown to be co-transcribed from two promoters (P(E)1 and P(E)2) which were identified upstream of ftsE(Ng) by primer extension. Sequence analysis of FtsE(Ng) and alignment with other FtsE indicated that it contained the conserved motifs of ABC domains while sequence alignment of FtsX(Ng) with other published FtsX sequences predicted that they all contain four transmembrane segments and a conserved motif (Leu-hydrophobic aa-Gly-Ala/Gly) which may prove to be important for FtsX function. The viability of ftsE(Ng) and ftsX(Ng) mutants that were constructed by insertional inactivation indicated that these genes are not essential. The role of FtsE and FtsX is controversial. Analysis of ftsE(Ng) and ftsX(Ng) mutants by transmission electron microscopy showed that both exhibited morphological abnormalities indicative of defective division sites and in some cases aberrant condensation of DNA.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas de Escherichia coli , Neisseria gonorrhoeae/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Southern Blotting , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Datos de Secuencia Molecular , Mutagénesis Insercional , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/ultraestructura , Fenotipo , Regiones Promotoras Genéticas , ARN Bacteriano/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
An origin of replication (ori) was obtained from a naturally occurring beta-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.
Asunto(s)
Vectores Genéticos/genética , Haemophilus/genética , Neisseria gonorrhoeae/genética , Origen de Réplica , Bacterias/genética , ADN Recombinante , Escherichia coli/genética , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Recombinantes de Fusión/genética , Transformación GenéticaRESUMEN
We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ(Ng) encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZ(Ng) is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ(Ng) on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZ(Ng) was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ(Ng) gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZ(Ng)-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZ(Ng)-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize at the middle of the cell. Expression of the FtsZ(Ng)-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZ(Ng)-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector.