Reproducible measurement of vancomycin MICs within the susceptible range in Staphylococcus aureus by a broth microdilution method with a "quasi-continuum" gradient of antibiotic concentrations.

The availability of reproducible broth microdilution (BMD) methods including inter log2 antibiotic dilutions for measuring Staphylococcus aureus (SA) vancomycin minimum inhibitory concentrations (MICs) within the susceptible range is needed to elucidate the impact of vancomycin MICs on clinical outcomes of invasive SA infections. Here, we report on the development of a very precise BMD method that incorporates the following incremental antibiotic concentrations: 0.50, 0.62, 0.75, 0.87, 1.0, 1.25, 1.40, 1.50, 1.60, 1.75, and 2.0 µg/mL. The intra- and inter-assay coefficients of variation of this method were around 20%. The mean of the differences in MIC values for all isolates obtained across two independent runs performed at one center was 0.04 µg/mL [95% confidence interval (CI), 0.011-0.07 µg/mL] and that for ten isolates measured at two different centers was 0.04 µg/mL (95% CI, 0-13 µg/mL). Vancomycin MIC values differed by less than 0.1 µg/mL between runs for most isolates. Storage of isolates at -20 °C for up to 3 months had no impact on the vancomycin MIC values. The mean vancomycin MIC values obtained by the Etest using a standard inoculum (0.5 McFarland) were significantly higher (p ≤ 0.001) than those measured by BMD and the MIC values measured by the two methods correlated poorly (Rho, 0.319; p = 0.148). Nevertheless, the mean MIC values measured by the Etest using lower inocula (107 or 106 CFU/mL) and those measured by BMD were comparable and correlated significantly (p = 0.004 for 107 CFU/mL and p = 0.029 for 106 CFU/mL).

Reconstitution of CMV pp65 and IE-1-specific IFN-γ CD8(+) and CD4(+) T-cell responses affording protection from CMV DNAemia following allogeneic hematopoietic SCT.

Threshold levels of CMV-specific T-cell populations presumably affording protection from active CMV infection in allo-SCT recipients have been proposed, but lack extensive validation. We quantified CMV pp65 and immediate-early 1-specific IFN-γ CD8(+) and CD4(+) T cell responses at days +30, +60 and +90 after transplantation in 133 patients, and established cutoff cell levels protecting from CMV DNAemia within the first 120 days after transplantation. No patients showing IFN-γ CD8(+) or IFN-γ CD4(+) T-cell counts >1.0 and >1.2 cells/µL, respectively, developed a subsequent episode of CMV DNAemia. Initial or recurrent episodes of CMV DNAemia occurred in the face of IFN-γ T-cell levels below defined thresholds. Negative predictive values at day +30 for the IFN-γ CD8(+) and CD4(+) T-cell markers were 68.1 and 61.8%, respectively. Recipients of grafts from CMV seropositive, related or HLA-matched donors, or receiving non-myeloablative conditioning had nonsignificant tendencies to reach more frequently protective levels of both T-cell subsets at early and late (day +365) times after transplantation. The use of anti-thymocyte globulin and umbilical cord blood transplantation were associated with impaired CMV-specific T-cell reconstitution. CMV-specific IFN-γ CD8(+) and CD4(+) T-cell recovery occurred irrespective of detectable CMV DNAemia.

Lack of prompt expansion of cytomegalovirus pp65 and IE-1-specific IFNgamma CD8+ and CD4+ T cells is associated with rising levels of pp65 antigenemia and DNAemia during pre-emptive therapy in allogeneic hematopoietic stem cell transplant recipients.

Rising levels of cytomegalovirus (CMV) DNAemia and/or pp65 antigenemia have been observed during pre-emptive ganciclovir therapy in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT). We assessed the incidence of this event in our series, and investigated whether its occurrence was associated with an impairment in the CMV-specific T-cell response. A total of 36 allo-SCT recipients experienced one or more episodes of active CMV infection (n=68) that were pre-emptively treated with val(ganciclovir). Rising levels of antigenemia and DNAemia, and an isolated increase in antigenemia, were observed in 39.7 and 2.9% of all episodes, respectively. Receipt of corticosteroids was associated with rising levels of antigenemia and DNAemia. Median increases of 12- and 6.8-fold of IFNgamma CD8(+) T and IFNgamma CD4(+) T cells, respectively, were observed at a median of 16.5 days after initiation of therapy in episodes with decreasing levels in antigenemia and DNAemia. In contrast, the numbers of both T-cell subsets at a median of 13.5 days after initiation of therapy did not differ significantly from those of pre-treatment samples in episodes with rising levels of antigenemia and DNAemia. Lack of prompt expansion of CMV pp65 and IE-1-specific IFNgamma CD8(+) and CD4(+) T cells is associated with rising levels in antigenemia and DNAemia during pre-emptive therapy.