RESUMEN
OBJECTIVES: We aimed to improve the microbial diagnosis of first episodes of febrile neutropenia (FEFNs) since <30% of episodes are microbiologically documented. Consequently patients are usually treated by empirical antibiotic therapy. METHODS: A prospective study evaluated a new workflow combining: (i) one 40 mL blood culture (BC) sampled from the central venous catheter; (ii) immediate incubation in an automated BC system on the ward; (iii) direct detection of microbial DNA in blood; and (iv) identification and susceptibility testing using rapid methods performed directly on positive BC bottles. Patients were also sampled for the standard workflow with two BC sets incubated in the central laboratory and assessed by classical procedures. RESULTS: One hundred and twenty consecutive FEFNs were included (February 2008-March 2009). The new workflow was as sensitive as the standard workflow, with BC positivity rates of 30% (36/120) and 28% (34/120), respectively (McNemar's χ(2)â=0.67, P=0.41). Direct DNA detection was positive in nine episodes (7.5%) that were also positive in BC. The new workflow provided microbiological results significantly earlier than the standard workflow, with a shorter time to BC positivity (median 12 h 31 min, range 7 h 55 min-25 h 37 min versus median 13 h 01 min, range 9 h 31 min-43 h 33 min, P=0.004) and shorter turnaround times for identification and susceptibility testing, with most of the results obtained <24 h after BC sampling. We retrospectively estimated that the new workflow would lead to earlier adequacy of antimicrobial therapy in 30% of documented cases. CONCLUSIONS: Our new process improved the microbiological diagnosis in FEFNs. Cost effectiveness needs to be tested.
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Infecciones Relacionadas con Catéteres/diagnóstico , Catéteres Venosos Centrales/efectos adversos , Fiebre de Origen Desconocido/diagnóstico , Técnicas Microbiológicas/métodos , Neutropenia/diagnóstico , Adulto , Anciano , Infecciones Relacionadas con Catéteres/microbiología , Femenino , Fiebre de Origen Desconocido/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Neutropenia/complicaciones , Estudios Prospectivos , Factores de Tiempo , Flujo de Trabajo , Adulto JovenRESUMEN
The eradication rate of Helicobacter pylori by standard therapy is decreasing due to antibiotic resistance, mainly to clarithromycin. Our aim was to provide a new molecular test to guide the treatment of new and relapsed cases. We first studied 126 H. pylori strains for phenotypic (MIC) and genotypic resistance to clarithromycin (rrl mutation) and levofloxacin (gyrA mutation) and then developed a DNA strip genotyping test on the basis of the correlation results and literature data. Clinical strains (n = 92) and gastric biopsy specimens containing H. pylori (n = 105) were tested blindly with the new molecular test GenoType HelicoDR. The presence of mutations or the absence of hybridization with wild-type sequences was predictive, in rrl for clarithromycin resistance in 91 cases (mostly the A2147G mutation) and in gyrA for levofloxacin resistance in 58 cases (mutations at codon 87 or 91). Genotyping revealed a mix of genotypes in 33% of the cases, reflecting a coinfection or selection for resistant mutants. The sensitivity and specificity of detecting resistance were 94% and 99% for clarithromycin and 87% and 98.5% for levofloxacin, respectively. The concordance scores were 0.96 for clarithromycin and 0.94 for levofloxacin. With global resistance rates of 46% for clarithromycin and 25% for levofloxacin, which were observed for consecutive positive biopsy specimens from 2007 and 2008, the positive and negative predictive values for detecting resistance were 99% and 94% for clarithromycin and 96% and 96% for fluoroquinolone. GenoType HelicoDR is efficient at detecting mutations predictive of antibiotic resistance in H. pylori when applied to strains or directly to gastric biopsy specimens.
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Antibacterianos/farmacología , Claritromicina/farmacología , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Levofloxacino , Pruebas de Sensibilidad Microbiana/métodos , Técnicas de Diagnóstico Molecular/métodos , Ofloxacino/farmacología , Proteínas Bacterianas/genética , Girasa de ADN/genética , Genotipo , Humanos , Hibridación de Ácido Nucleico/métodos , Mutación Puntual , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Eliminación de SecuenciaRESUMEN
OBJECTIVES: Besifloxacin is a new fluoroquinolone in development for ocular use. We investigated its mode of action and resistance in two major ocular pathogens, Streptococcus pneumoniae and Staphylococcus aureus, and in the reference species Escherichia coli. METHODS: Primary and secondary targets of besifloxacin were evaluated by: (i) mutant selection experiments; (ii) MIC testing of defined topoisomerase mutants; and (iii) inhibition and cleavable complex assays with purified S. pneumoniae and E. coli DNA gyrase and topoisomerase IV enzymes. RESULTS: Enzyme assays showed similar besifloxacin activity against S. pneumoniae gyrase and topoisomerase IV, with IC(50) and CC(25) of 2.5 and 1 microM, respectively. In contrast to ciprofloxacin and moxifloxacin, besifloxacin was equally potent against both S. pneumoniae and E. coli gyrases. DNA gyrase was the primary target in all three species, with substitutions observed at positions 81, 83 and 87 in GyrA and 426 and 466 in GyrB (E. coli numbering). Topoisomerase IV was the secondary target. Notably, resistant mutants were not recovered at 4-fold besifloxacin MICs for S. aureus and S. pneumoniae, and S. aureus topoisomerase mutants were only obtained after serial passage in liquid medium. Besifloxacin MICs were similarly affected by parC or gyrA mutations in S. aureus and S. pneumoniae and remained below 1 mg/L in gyrA-parC double mutants. CONCLUSIONS: Although mutant selection experiments indicated that gyrase is a primary target, further biochemical and genetic studies showed that besifloxacin has potent, relatively balanced activity against both essential DNA gyrase and topoisomerase IV targets in S. aureus and S. pneumoniae.