The T2Bacteria Assay Is a Sensitive and Rapid Detector of Bacteremia That Can Be Initiated in the Emergency Department and Has Potential to Favorably Influence Subsequent Therapy.

BACKGROUND: Bacteremia causes a major worldwide burden, in terms of financial and productivity costs, as well the morbidity and mortality it can ultimately cause. Proper treatment of bacteremia is a challenge because of the species-dependent response to antibiotics. The T2Bacteria Panel is a U.S. Food and Drug Administration-cleared and culture-independent assay for detection of bacteremia, including common ESKAPE pathogens-Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa-and provides species identification in as little as 3.6 h directly from blood. OBJECTIVE: Our aim was to evaluate the T2Bacteria assay performance and potential to affect patient care in the emergency department (ED). METHODS: ED patients from a Louisiana and Florida center were enrolled as part of the T2Bacteria Panel clinical study, which was prospective and noninterventional. Blood samples for blood culture (BC) and T2Bacteria were matched in time and anatomic location. RESULTS: Data from 137 ED patients were evaluated. Relative to BC, T2Bacteria showed 100% positive percent agreement and 98.4% negative percent agreement. In addition, for species on the T2Bacteria Panel, the T2Bacteria assay detected 25% more positives associated with infection, and on average identified the infectious species 56.6 h faster. The T2Bacteria assay covered 70.5% of all species detected by BC. Finally, relative to actual care, the T2Bacteria assay could have potentially focused therapy in 8 patients, reduced time to a species-directed therapy in 4 patients, and reduced time to effective therapy in 4 patients. CONCLUSIONS: In this ED population, the T2Bacteria assay was a rapid and sensitive detector of bacteremia from common ESKAPE pathogens and showed the theoretical potential to influence subsequent patient therapy, ranging from antibiotic de-escalation to faster time to effective therapy.

Clinical Evaluation of the iCubate iC-GPC Assay for Detection of Gram-Positive Bacteria and Resistance Markers from Positive Blood Cultures.

The iC-GPC Assay (iCubate, Huntsville, AL) is a qualitative multiplex test for the detection of five of the most common Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) responsible for bacterial bloodstream infections, performed directly from positive blood cultures. The assay also detects the presence of the mecA, vanA, and vanB resistance determinants. This study comparatively evaluated the performance of the iC-GPC Assay against the Verigene Gram-positive blood culture (BC-GP) assay (Luminex Corp., Austin, TX) for 1,134 patient blood culture specimens positive for Gram-positive cocci. The iC-GPC Assay had an overall percent agreement with the BC-GP assay of 95.5%. Discordant specimens were further analyzed by PCR and a bidirectional sequencing method. The results indicate that the iC-GPC Assay together with the iCubate system is an accurate and reliable tool for the detection of the five most common Gram-positive bacteria and their resistance markers responsible for bloodstream infections.

Rapid detection of carbapenemase genes by multiplex real-time PCR.

OBJECTIVES: To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48). METHODS: A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA). RESULTS: Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. CONCLUSIONS: The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

Randomized controlled trial of mindfulness-based stress reduction (MBSR) for survivors of breast cancer.

OBJECTIVES: Considerable morbidity persists among survivors of breast cancer (BC) including high levels of psychological stress, anxiety, depression, fear of recurrence, and physical symptoms including pain, fatigue, and sleep disturbances, and impaired quality of life. Effective interventions are needed during this difficult transitional period. METHODS: We conducted a randomized controlled trial of 84 female BC survivors (Stages 0-III) recruited from the H. Lee Moffitt Cancer and Research Institute. All subjects were within 18 months of treatment completion with surgery and adjuvant radiation and/or chemotherapy. Subjects were randomly assigned to a 6-week Mindfulness-Based Stress Reduction (MBSR) program designed to self-regulate arousal to stressful circumstances or symptoms (n=41) or to usual care (n=43). Outcome measures compared at 6 weeks by random assignment included validated measures of psychological status (depression, anxiety, perceived stress, fear of recurrence, optimism, social support) and psychological and physical subscales of quality of life (SF-36). RESULTS: Compared with usual care, subjects assigned to MBSR(BC) had significantly lower (two-sided p<0.05) adjusted mean levels of depression (6.3 vs 9.6), anxiety (28.3 vs 33.0), and fear of recurrence (9.3 vs 11.6) at 6 weeks, along with higher energy (53.5 vs 49.2), physical functioning (50.1 vs 47.0), and physical role functioning (49.1 vs 42.8). In stratified analyses, subjects more compliant with MBSR tended to experience greater improvements in measures of energy and physical functioning. CONCLUSIONS: Among BC survivors within 18 months of treatment completion, a 6-week MBSR(BC) program resulted in significant improvements in psychological status and quality of life compared with usual care.

Epstein-Barr virus reactivation after superinfection of the BJAB-B1 and P3HR-1 cell lines with cytomegalovirus.

BACKGROUND: Studies examining herpesvirus-herpesvirus (cytomegalovirus (CMV)-Epstein-Barr virus (EBV)) interactions are limited, and many of the studies have been clinical observations suggesting such an interaction exists. This report aims to examine the in vitro susceptibilities of BJAB-B1 and P3HR-1 cells (EBV positive Burkitt's lymphoma B-cell lines) to a CMV superinfection; and show that EBV reactivation occurs after CMV superinfects these cell lines. RESULTS: The BJAB-B1 and P3HR-1 cells were observed to be susceptible to a CMV superinfection by detecting the major immediate early (MIE) viral transcript and protein (p52) expression. The BZLF1 transcript was observed in both cell lines superinfected with CMV, indicating EBV reactivation. BZLF1 protein was observed in the BJAB-B1 cells. Antigen detection was not performed in the P3HR-1 cells. CONCLUSION: The results from the in vitro superinfections support the in vivo studies suggesting a CMV infection is related to an EBV reactivation and suggests that CMV may be important as a co-factor in EBV pathogenesis in the immunocompromised patient.

Differentiation of Klebsiella pneumoniae carbapenemase (KPC) variants by pyrosequencing.

A fast and reliable protocol using the pyrosequencing technique was developed to identify 11 different types of the KPC enzyme. A total of 65 blaKPC positive bacterial isolates were tested and characterized. In the end, the pyrosequencing proved to be a powerful tool for epidemiological studies of KPC producer isolates.

Baseline immune biomarkers as predictors of MBSR(BC) treatment success in off-treatment breast cancer patients.

Researchers focused on patient-centered medicine are increasingly trying to identify baseline factors that predict treatment success. Because the quantity and function of lymphocyte subsets change during stress, we hypothesized that these subsets would serve as stress markers and therefore predict which breast cancer patients would benefit most from mindfulness-based stress reduction (MBSR)-facilitated stress relief. The purpose of this study was to assess whether baseline biomarker levels predicted symptom improvement following an MBSR intervention for breast cancer survivors (MBSR[BC]). This randomized controlled trial involved 41 patients assigned to either an MBSR(BC) intervention group or a no-treatment control group. Biomarkers were assessed at baseline, and symptom change was assessed 6 weeks later. Biomarkers included common lymphocyte subsets in the peripheral blood as well as the ability of T cells to become activated and secrete cytokines in response to stimulation with mitogens. Spearman correlations were used to identify univariate relationships between baseline biomarkers and 6-week improvement of symptoms. Next, backward elimination regression models were used to identify the strongest predictors from the univariate analyses. Multiple baseline biomarkers were significantly positively related to 6-week symptom improvement. The regression models identified B-lymphocytes and interferon-γ as the strongest predictors of gastrointestinal improvement (p < .01), +CD4+CD8 as the strongest predictor of cognitive/psychological (CP) improvement (p = .02), and lymphocytes and interleukin (IL)-4 as the strongest predictors of fatigue improvement (p < .01). These results provide preliminary evidence of the potential to use baseline biomarkers as predictors to identify the patients likely to benefit from this intervention.

Rapid diagnosis of Mycobacterium abscessus endophthalmitis.

Nontuberculous mycobacteria are widely distributed in the environment and have the potential to cause a wide spectrum of infections including pulmonary, bone, soft tissue or ocular infections. They are a rare cause of endophthalmitis, a potentially devastating condition, which may be acquired through contamination of water or antiseptic solutions. Diagnosis is often delayed due to low clinical suspicion, resulting in poor clinical outcomes. Newer laboratory techniques such as real-time PCR can be used for rapid detection, identification and speciation of mycobacteria and allow for initiation of focused antibiotic therapy. We describe a case of Mycobacterium abscessus endophthalmitis that developed 30 years after traumatic loss of cornea in a patient with diabetes mellitus.

Lymphocyte recovery after breast cancer treatment and mindfulness-based stress reduction (MBSR) therapy.

OBJECTIVES: This randomized controlled trial was conducted to examine immune recovery following breast cancer (BC) therapy and evaluate the effect of mindfulness-based stress reduction therapy (MBSR) on immune recovery with emphasis on lymphocyte subsets, T cell activation, and production of T-helper 1 (Th1; interferon [IFN]-γ) and T-helper 2 (Th2; interleukin-4 [IL-4]) cytokines. METHOD: Participants who completed the study consisted of 82 patients diagnosed with Stage 0-III BC, who received lumpectomy and adjuvant radiation ± chemotherapy. Patients were randomized into an MBSR(BC) intervention program or a control (usual care) group. Immune cell measures were assessed at baseline and within 2 weeks after the 6-week intervention. The numbers and percentages of lymphocyte subsets, activated T cells, and Th1 and Th2 cells in peripheral blood samples were determined by immunostaining and flow cytometry. RESULTS: Immune subset recovery after cancer treatment showed positive associations with time since treatment completion. The B and natural killer (NK) cells were more susceptible than T cells in being suppressed by cancer treatment. Women who received MBSR(BC) had T cells more readily activated by the mitogen phytohemagglutinin (PHA) and an increase in the Th1/Th2 ratio. Activation was also higher for the MBSR(BC) group if <12 weeks from the end of treatment and women in MBSR(BC) <12 weeks had higher T cell count for CD4(+). CONCLUSION: MBSR(BC) promotes a more rapid recovery of functional T cells capable of being activated by a mitogen with the Th1 phenotype, whereas substantial recovery of B and NK cells after completion of cancer treatment appears to occur independent of stress-reducing interventions.

Role of Toll-like receptor 9 in Legionella pneumophila-induced interleukin-12 p40 production in bone marrow-derived dendritic cells and macrophages from permissive and nonpermissive mice.

The progression of Legionella pneumophila infection in macrophages is controlled by the Lgn1 gene locus, which expresses the nonpermissive phenotype in cells from BALB/c mice but the permissive phenotype in cells from A/J mice. Activation of dendritic cells and macrophages by L. pneumophila is mediated by the pathogen recognition receptor Toll-like receptor 2 (TLR2); furthermore, Legionella induces innate and adaptive immune cytokines by the MyD88-dependent pathway. TLR9 is coupled to MyD88 and mediates the production of interleukin-12 (IL-12) in dendritic cells infected with other facultatively intracellular pathogens. In the current study, L. pneumophila growth in dendritic cells from BALB/c and A/J mice was examined along with the role of TLR9 in the induction of IL-12 in these cells. Dendritic cells from both strains were nonpermissive for L. pneumophila intracellular growth, suggesting that the products of the Lgn1 gene locus that control intracellular growth in macrophages do not control the growth of Legionella in dendritic cells. In addition, chloroquine treatment suppressed IL-12 p40 production in response to Legionella treatment in dendritic cells and macrophages from BALB/c and A/J mice. Furthermore, the TLR9 inhibitor ODN2088 suppressed the Legionella-induced IL-12 production in dendritic cells from both mouse strains. These results suggest that L. pneumophila is similar to other intracellular bacteria in that it stimulates the production of immune-transitioning cytokines, such as IL-12, through activation of TLR9 and that this receptor provides a common mechanism for sensing these types of microbes and inducing innate and adaptive immunity.