Elisa Costa

Diagnostic accuracy and potential clinical value of the LightCycler SeptiFast assay in the management of bloodstream infections occurring in neutropenic and critically ill patients

OBJECTIVES The objectives of this study were to compare the performance of the LightCycler SeptiFast Test MGRADE and conventional blood culture in the etiological diagnosis of febrile episodes occurring in neutropenic and critically ill patients (in the intensive care unit; ICU), and to assess the potential clinical value of the SeptiFast test in patient management. METHODS A total of 86 febrile episodes occurring in 33 neutropenic patients and 53 ICU patients were analyzed. Blood samples for blood culture and SeptiFast testing were obtained at the onset of fever, before the implementation of empirical antimicrobial therapy. RESULTS The overall microorganism-to-isolate agreement between the SeptiFast test and blood culture was 69% (κ=0.37) in neutropenic patients and 75% (κ=0.56) in ICU patients. The sensitivity of the SeptiFast assay for clinically relevant episodes of bacteremia and fungemia was 62% in neutropenic patients and 70% in ICU patients. Based on SeptiFast results, empirical treatments were deemed adequate in all but one of the febrile episodes. Nevertheless, early antibiotic treatment readjustment was judged feasible in most of clinically significant episodes overall. CONCLUSIONS The SeptiFast assay is a valuable ancillary method for the diagnosis of bloodstream infections in neutropenic and ICU patients. In these clinical settings, results of the SeptiFast assay may lead to a more targeted antibiotic therapy early after the onset of fever.

Virological and immunological features of active cytomegalovirus infection in nonimmunosuppressed patients in a surgical and trauma intensive care unit.

Cytomegalovirus (CMV) reactivation occurs frequently in critically ill patients. The natural course of CMV infection and the interaction between CMV and the adaptive immune system in this setting remain poorly defined. Fifty‐three CMV‐seropositive patients in a surgical and trauma intensive care unit were included in this study. The CMV DNA load in tracheal aspirates (TA) and plasma (PL) was monitored by qPCR. CMV‐specific T‐cell immunity was assessed by intracellular cytokine staining. Plasma TNF‐α levels were determined by ELISA. CMV reactivation occurred in 39.7% of patients (23% had CMV DNA detected only in TA). The analysis of TA allowed an earlier diagnosis in 28% of patients. Clearance of CMV DNAemia preceded that of CMV DNA in TA in some episodes. Peak CMV DNA levels were significantly higher in TA than in PL (P = 0.02). CMV reactivation developed in the presence of CMV‐specific T cells. Termination of CMV reactivation was associated with an expansion of functional CMV‐specific T cells. Plasma levels of TNF‐α did not allow for the prediction of the occurrence of CMV reactivation. CMV‐specific T‐cell immunity is preserved in most critically ill patients experiencing CMV reactivation. Analysis of respiratory specimens is imperative for an optimal monitoring of CMV reactivation in this setting. J. Med. Virol. 82:1384–1391, 2010.

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.

Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quantitation of Plasma Cytomegalovirus DNAemia in Allogeneic Stem Cell Transplant Recipients

ABSTRACT Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.

Comparison of the new Abbott Real Time CMV assay and the Abbott CMV PCR Kit for the quantitation of plasma cytomegalovirus DNAemia

CMV DNA loads measured by the new Abbott RealTime CMV PCR were significantly higher than those quantitated by the Abbott CMV PCR kit (approximately 1 log(10)), and provided a better estimate of the actual CMV load present in plasma specimens as inferred by the use of the WHO standard.

Performance of the QuantiFERON-Cytomegalovirus (CMV) Assay for Detection and Estimation of the Magnitude and Functionality of the CMV-Specific Gamma Interferon-Producing CD8+ T-Cell Response in Allogeneic Stem Cell Transplant Recipients

ABSTRACT The performance of the QuantiFERON-cytomegalovirus (CMV) assay was compared to that of a flow cytometry intracellular cytokine staining (ICS) method for the detection of CMV-specific gamma interferon (IFN-γ)-producing CD8+ T-cell responses in allogeneic stem cell transplant (allo-SCT) recipients and for estimations of their magnitude and functionality. A total of 90 whole-blood specimens from 23 allo-SCT recipients was analyzed by both methods. Overall, the percentage of specimens that yielded concordant results by both methods was 68.8% (κ = 0.691; 95% confidence interval [CI], 0.548 to 0.835), and the sensitivity of the QuantiFERON-CMV assay for the detection of positive IFN-γ T-cell responses (>0.2 IU/ml), taking the ICS method as the reference, was 76.3%. The magnitude of IFN-γ-producing CD8+ T-cell responses to CMV-specific peptides measured with the QuantiFERON-CMV assay correlated significantly (σ = 0.695; P = <0.001) with that of the total IFN-γ-producing CD8+ T cells and dual-functional (IFN-γ/tumor necrosis factor alpha [TNF-α] [σ = 0.652; P = <0.001] and IFN-γ/CD107a [σ = 0.690; P = <0.001]) and trifunctional (IFN-γ/TNF-α/CD107a [σ = 0.679; P = >0.001]) CMV-specific CD8+ T-cell responses, as quantitated by ICS. In summary, the data indicated that the QuantiFERON-CMV assay is less sensitive than the ICS method for the detection of CMV-specific IFN-γ-producing CD8+ T-cell responses in the allo-SCT setting. Nevertheless, it allowed the estimation of the total and polyfunctional CMV-specific IFN-γ-producing CD8+ T-cell responses in specimens that tested positive by both methods.

Functional profile of cytomegalovirus (CMV)-specific CD8+ T cells and kinetics of NKG2C+ NK Cells associated with the resolution of CMV DNAemia in allogeneic stem cell transplant recipients

Immune mechanisms involved in control of cytomegalovirus (CMV) infection in the allogeneic stem cell transplantation setting have not been fully disclosed. CMV pp65 and IE‐1‐specific CD8+ T cells expressing IFN‐γ, TNF‐α, and CD107a, alone or in combination, and NKG2C+ NK cells were prospectively enumerated during 13 episodes of CMV DNAemia. The expansion of monofunctional and polyfunctional CD8+ T cells was associated with CMV DNAemia clearance. The size and functional diversity of the expanding CD8+ T‐cell population was greater in self‐resolved episodes than in episodes treated with antivirals. These differences were related to the magnitude of expansion of cognate antigen IFN‐γ CD4+ T cells. The resolution of CMV DNAemia was associated frequently with a marked expansion of both CD56dim/CD16+ NK cells and NKG2C+ CD56bright/CD16− NK cells. The data lend support to the role of polyfunctional CD8+ T cells in controlling CMV replication in the allogeneic stem cell transplantation setting, and suggest that NKG2C+ NK cells may be involved critically in the resolution of CMV DNAemia episodes. J. Med. Virol. 84:259–267, 2012.

Performance of the LightCycler SeptiFast Test Mgrade in Detecting Microbial Pathogens in Purulent Fluids

ABSTRACT The performance of the LightCycler SeptiFast (SF) assay was compared to that of culture methods in the detection of microorganisms in 43 purulent fluids from patients with pyogenic infections. The SF assay was more sensitive than the culture methods (86% versus 61%, respectively), irrespective of whether the infections were mono- or polymicrobial.

Immunological insights into the pathogenesis of active CMV infection in non-immunosuppressed critically ill patients.

Dissociation of cytomegalovirus (CMV) DNA loads between the lower respiratory tract and blood, with high levels in the former compartment and low or undetectable levels in the latter, commonly occurs during active CMV infection in critically ill patients despite the presence of high frequencies of CMV‐specific IFN‐γ‐producing CD8+ and CD4+ T cells in blood. Data presented in this case report suggest that inter‐compartmental differences in interleukin‐10 (IL‐10) levels may, in part, explain the pathobiology of this phenomenon. In the absence of ganciclovir treatment, a significant correlation was observed between IL‐10 levels and CMV DNA loads in lower respiratory tract specimens (P = 0.016), but not in plasma samples (P = 0.46). Comparable data were obtained during the course of active CMV infection episodes that developed in six CMV‐seropositive critically ill patients with no canonical immunosuppression. The presence of higher levels of IL‐10 in the lower respiratory tract than in plasma may result in increased impairment of CMV‐specific T‐cell effector responses in the lung compared to the systemic compartment, facilitating local CMV replication. J. Med. Virol. 83:1966–1971, 2011.

Comparison of the performance of 2 commercial multiplex PCR platforms for detection of respiratory viruses in upper and lower tract respiratory specimens

Abstract The performance of the CLART® PneumoVir system with that of the Luminex xTAG RVP Fast v1 assay for detection of most common respiratory viruses in upper and lower tract respiratory specimens (n=183) from unique patients with influenza-like syndrome or lower tract respiratory infection. Nested PCR coupled to automated sequencing was used for resolution of discrepancies. Fully concordant results were obtained for a total of 122 specimens, whereas 56 specimens gave partially (n=21) or fully discordant (n=35) results (Kappa coefficient, 0.62). The overall specificity of the Luminex xTAG RVP Fast v1 assay was slightly higher than that of the CLART® PneumoVir assay for human bocavirus, influenza A virus/H3N2, influenza B virus, human metapneumovirus, and parainfluenza virus, whereas the sensitivity of the latter was higher for most targeted viruses except, notably, for picornaviruses. This was irrespective of either the origin of the respiratory specimen or the age group to which the patients belonged.