Comparative evaluation of VITEK 2 for antimicrobial susceptibility testing of group B Streptococcus

OBJECTIVES:Intrapartum antibiotic prophylaxis is recommended to prevent neonatal group B streptococcal (GBS) disease in colonized women, and penicillin or aminopenicillin constitute the first-line antibiotics. Most isolates are resistant to tetracycline, and resistance to macrolide-lincosamide-streptogramin (MLS) antibiotics is increasing. Therefore, laboratory testing for MLS resistance in GBS is now recommended for penicillin-allergic patients. The aim of this study was to compare the antimicrobial susceptibility of GBS as determined by the VITEK 2 system (bioMérieux, Marcy l'Etoile, France), agar diffusion methods and PCR-genotypic detection of resistance genes.METHODS:One hundred and ten unrelated selected GBS clinical isolates were studied. The antibiotics tested (VITEK 2 and agar diffusion method) were benzylpenicillin, ampicillin, erythromycin, clindamycin, co-trimoxazole, tetracycline, kanamycin, streptomycin and vancomycin. A standardized double-disc (DD) diffusion test was performed for MLS antibiotics. Genotypic characterization of tetracycline, MLS and aminoglycoside resistance genes was performed by PCR.RESULTS:All strains were susceptible to benzylpenicillin, ampicillin and vancomycin [category agreement (CA) between VITEK 2 and the diffusion method was 100%]. Ninety-five (86%) strains were resistant to tetracycline (CA was 98.9%). Eighty-one strains (73.6%) harboured an MLS resistance phenotype; 50 (61.8%) an MLS(B)-constitutive phenotype, 25 (30.8%) an MLS(B)-inducible phenotype and 6 (7.4%) an M phenotype. The agreement between data of VITEK 2 and the DD diffusion test for the detection of MLS(B)-constitutive, MLS(B)-inducible and M phenotype isolates was 76%, 36% and 100%, respectively. Almost all discrepancies were due to failure to detect erythromycin resistance by VITEK 2.CONCLUSIONS:VITEK 2 allows accurate determination of GBS susceptibility for the majority of antibiotics, but has to be improved for erythromycin. Thus, the DD diffusion test remains the most simple and reliable method for macrolide resistance detection among this streptococcal species

Fast and Accurate Quantitative Analysis of Cytokine Gene Expression in Human Neutrophils by Reverse Transcription Real-Time PCR

Polymorphonuclear neutrophils, traditionally viewed as short-lived effector cells, are nowadays regarded as important components of effector and regulatory circuits in the innate and adaptive immune systems. Most of the physiological functions of neutrophils as crucial players in the host immune response, able not only to act in the early phases of acute inflammation but also to condition the progression of the inflammatory reaction and the subsequent initiation of the specific immune response, relies on their capacity to produce and release a number of proinflammatory and immunoregulatory cytokines. This fact has reevaluated the importance, the role, and the physiological and pathological significance of neutrophils in the pathogenesis of inflammatory, infectious, autoimmune, and neoplastic diseases and has identified neutrophils as an important potential target for selective pharmacological intervention to both promote and restrain inflammation. In this context, understanding the mechanisms of modulation of neutrophil-derived cytokines and chemokines represents a critical step toward a better understanding of how neutrophils may influence pathophysiological processes in vivo. Herein, we describe and discuss an updated version of the methods that we have developed to rapidly and precisely characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the reverse transcription quantitative real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool, in the neutrophil research area, to test many experimental conditions for gene expression analysis

Fast and Accurate Quantitative Analysis of Cytokine Gene Expression in Human NeutrophilsNeutrophil Methods and Protocols

Polymorphonuclear neutrophils, traditionally viewed as short-lived effector cells, are nowadays regarded as important components of effector and regulatory circuits in the innate and adaptive immune systems. Most of the physiological functions of neutrophils as crucial players in the host immune response, able not only to act in the early phases of acute inflammation but also to condition the progression of the inflammatory reaction and the subsequent initiation of the specific immune response, rely on their capacity to produce and release a number of proinflammatory and immunoregulatory cytokines. This fact has reevaluated the importance, role, and physiological and pathological significance of neutrophils in the pathogenesis of inflammatory, infectious, autoimmune, and neoplastic diseases and has identified neutrophils as an important potential target for selective pharmacological intervention to both promote and restrain inflammation. In this context, understanding the mechanisms of modulation of neutrophil-derived cytokines and chemokines represents a critical step toward a better understanding of how neutrophils may influence pathophysiological processes in vivo. Herein, we describe and discuss an updated version of the methods that we have developed to rapidly and precisely characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the reverse transcription quantitative real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool, in the neutrophil research area, to test many experimental conditions for gene expression analysis