Detection of two simultaneous outbreaks of Klebsiella pneumoniae coproducing OXA-48 and NDM-1 carbapenemases in a tertiary-care hospital in Valencia, Spain.

Klebsiella pneumoniae is one of the most common hospital-acquired Gram-negative pathogens. During the last decade, the emergence of strains with reduced susceptibility or resistance to carbapenems is becoming a therapeutic challenge. This study takes place after the isolation of 14 strains of carbapenem-resistant K. pneumoniae with similar susceptibility patterns and carriage of OXA-48 and NDM-1 carbapenemases genes. Fourteen patients were found to be colonized (faecal carriage) and/or infected by two different clones of carbapenemase-coproducing K. pneumoniae during a 1-year period of time. Some of the patients had shared a hospital ward and continued to be colonized several months after the outbreak.

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.

Separation of two forms of neuropathy target esterase in the soluble fraction of the hen sciatic nerve.

Neuropathy target esterase (NTE) activity is operatively defined in this paper as the phenyl valerate esterase activity resistant to 40 microM paraoxon but sensitive to 250 microM mipafox. Molecular exclusion column chromatography with Sephacryl S-300 of the soluble (S) fraction from chick sciatic nerve demonstrated two NTE activity peaks. The first eluted with the front, thus indicating a mol. wt. of over 700 kDa (peak Vo), while the second peak eluted with kd = 0.36, suggesting a mol. wt. of about 100 kDa. The curve of total phenyl valerate (PVase) activity inhibition with paraoxon (0.19-200 microM) shows that at a concentration of 40 microM the esterases highly sensitive to paraoxon are inhibited in the Vo and 100-kDa peaks. The NTE activity in these two peaks in turn represented 31% and 44% of the 40 microM paraoxon resistant activity, respectively. The mipafox inhibition curves (1.0-250 microM) revealed different sensitivities to mipafox, with I50 values (t = 30 min) of approximately 1.47 and 63 microM, for Vo and 100-kDa peaks respectively. Mipafox sensitivity of the Vo and 100-kDa peaks correlates with the two components, that had been deduced from the kinetic properties of the S-fraction.

Organophosphorus inhibition and heat inactivation kinetics of particulate and soluble forms of peripheral nerve neuropathy target esterase.

Neuropathy target esterase (NTE) is the proposed target site for the mechanism of initiation of the so-called organophosphorus-induced delayed polyneuropathy (OPIDP). NTE is operationally defined in this article as the phenylvalerate esterase activity which is resistant to inhibition by 40 microM paraoxon and sensitive to 250 microM mipafox. Soluble (S-NTE) and particulate (P-NTE) forms of NTE had first been identified in hen sciatic nerve [E. Vilanova, J. Barril, V. Carrera, and M. C. Pellín (1990). J. Neurochem., 55, 1258-1265]. P-NTE and S-NTE showed different sensitivities to the inhibition by several organophosphorus compounds over a range of inhibitor concentrations for a 30 or 120 minute fixed inhibition time at 37 degrees C. S-NTE was less sensitive to the inhibition by O,O'-diisopropyl phosphorofluoridate (DFP), hexyl 2,5-dichlorophenyl phosphoramidate (H-DCP), and mipafox than P-NTE and brain NTE, while the opposite was true for O,S-dimethyl phosphoroamidothioate (methamidophos). For each of the four inhibitors assayed, S-NTE showed two components of different sensitivity according to the inhibition curves fitted with exponential models. However, the inhibition of P-NTE by mipafox, DFP, and HDCP did not show the presence of a considerable proportion of a second component. The kinetics of heat inactivation showed that P-NTE inactivated faster and to a greater extent than S-NTE. It is concluded that (1) sciatic nerve S-NTE is more different from brain NTE than P-NTE; (2) P-NTE and S-NTE have different sensitivities to the inhibition by the studied organophosphorous compounds; (3) the inhibition curves suggest that S-NTE has two different enzymatic components while these are not so evident for P-NTE.

Soluble and particulate organophosphorus neuropathy target esterase in brain and sciatic nerve of the hen, cat, rat, and chick.

Considerable evidence exists suggesting that the so-called neuropathy target esterase (NTE) is involved in the mechanisms responsible for organophosphorus-induced delayed polyneuropathy (OPIDP). Earlier studies in the adult hen, the habitually employed experimental model in OPIDP, have shown that most NTE activity in the brain is centered in particulate fractions, whereas approximately 50% of this activity in the sciatic nerve is encountered in soluble form, with the rest being particulate NTE. In the present work, we have studied the particulate and soluble fractional distribution of paraoxon-resistant phenylvalerate esterase activity (B activity), paraoxon- and mipafox-resistant phenylvalerate esterase activity (C activity), and NTE activity (B-C) according to ultracentrifugation criteria (100,000 g for 1 h). To this effect, two sensitive (adult hen and cat) and two scarcely sensitive (rat and chick) models were used. In all four experimental models, the distribution pattern was qualitatively similar: B activity and total NTE were much greater in brain (900-2,300 nmol/min/g of tissue) than in sciatic nerve (50-100 nmol/min/g of tissue). The proportion of soluble NTE in brain was very low (< 2%), whereas its presence in sciatic nerve was substantial (30-50%). The NTE/B ratio in brain was high for the particulate fraction (> 60%) and low in the soluble fraction (7-30%); in sciatic nerve the ratio was about 50% in both fractions.(ABSTRACT TRUNCATED AT 250 WORDS)