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.

A TaqMan Probe-Based Real-Time PCR Assay for the Rapid Identification of the Emerging Multidrug-Resistant Pathogen Candida auris on the BD Max System.

Candida auris is an emerging multidrug-resistant fungal pathogen that has been associated with nosocomial bloodstream and deep wound infections causing a high mortality rate mainly in intensive care unit (ICU) patients. Laboratories currently rely on phenotypic testing using commercial automated systems for identification of yeasts; however, this technique has often led to misidentification of C. auris to other closely related species. We developed and validated a TaqMan-based real-time PCR assay on the BD Max platform targeting ribosomal DNA (rDNA) region nucleotide sequences to quickly and accurately test for C. auris infection from culture and clinical specimens. The assay is highly specific, reproducible, and sensitive, allowing detection of as low as 1 C. auris CFU per reaction within 3 h.

Performance of the T2Bacteria Panel for Diagnosing Bloodstream Infections: A Diagnostic Accuracy Study.

Background: Blood cultures, the gold standard for diagnosing bloodstream infections (BSIs), are insensitive and limited by prolonged time to results. The T2Bacteria Panel (T2 Biosystems) is a direct-from-blood, nonculture test that identifies the most common ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli). Objective: To assess performance of the T2Bacteria Panel in diagnosing suspected BSIs in adults. Design: Prospective patient enrollment (8 December 2015 through 4 August 2017). Setting: Eleven U.S. hospitals. Patients: 1427 patients for whom blood cultures were ordered as standard of care. Intervention: Paired blood culture and T2Bacteria testing. Measurements: Performance of T2Bacteria compared with a single set of blood cultures in diagnosing proven, probable, and possible BSIs caused by T2Bacteria-targeted organisms. Results: Blood culture and T2Bacteria results were positive for targeted bacteria in 3% (39 of 1427) and 13% (181 of 1427) of patients, respectively. Mean times from start of blood culture incubation to positivity and species identification were 38.5 (SD, 32.8) and 71.7 (SD, 39.3) hours, respectively. Mean times to species identification with T2Bacteria were 3.61 (SD, 0.2) to 7.70 (SD, 1.38) hours, depending on the number of samples tested. Per-patient sensitivity and specificity of T2Bacteria for proven BSIs were 90% (95% CI, 76% to 96%) and 90% (CI, 88% to 91%), respectively; the negative predictive value was 99.7% (1242 of 1246). The rate of negative blood cultures with a positive T2Bacteria result was 10% (146 of 1427); 60% (88 of 146) of such results were associated with probable (n = 62) or possible (n = 26) BSIs. If probable BSIs and both probable and possible BSIs were assumed to be true positives missed by blood culture, per-patient specificity of T2Bacteria was 94% and 96%, respectively. Limitation: Low prevalence of positive blood cultures, collection of a single set of culture specimens, and inability of T2Bacteria to detect nontargeted pathogens. Conclusion: The T2Bacteria Panel rapidly and accurately diagnoses BSIs caused by 5 common bacteria. Primary Funding Source: T2 Biosystems.

Rapid detection of four non-fermenting Gram-negative bacteria directly from cystic fibrosis patient's respiratory samples on the BD MAX™ system.

The aim of this study was to develop a multiplex PCR test to detect Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) directly from CF patient's respiratory samples using the open mode of the BD MAX™ System. A total of 402 CF respiratory samples were evaluated by culture and PCR. Specific sets of primers and probes for each target were designed in-house. Out of 402 samples tested, 196 were identified as negative and 206 as positive by culture for AX, PSA, BC and SM. Among culture positive samples, PCR detected 21/27 AX, 4/5 BC, 138/140 PSA and 29/34 SM. In addition, PCR assay identified 35 samples as positive that were initially negative by culture for those 4 targets. The CF BDM test proved to be an excellent tool to detect AX, BC, PSA and SM by real-time PCR on an automated platform.

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.

Multicenter Evaluation of the Xpert MRSA NxG Assay for Detection of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs.

Health care-associated methicillin-resistant Staphylococcus aureus (MRSA) infections are a burden on the health care system. Clinical laboratories play a key role in reducing this burden, as the timely identification of MRSA colonization or infection facilitates infection control practices that are effective at limiting invasive MRSA infections. The Xpert MRSA NxG assay recently received FDA clearance for the direct detection of MRSA from nasal swabs. This multicenter study evaluated the clinical performance characteristics of the Xpert MRSA NxG assay with prospectively collected rayon nasal swabs (n = 1,103) and flocked swab (ESwab) nasal specimens (n = 846). Culture-based identification methods and antimicrobial susceptibility testing were used as the reference standards for comparison. According to the reference method, the positivity rates for MRSA in the population evaluated were 11.1% (122/1,103) for rayon swabs and 11.6% (98/846) for flocked swabs. The overall sensitivity and specificity of the rayon swabs were 91.0% (95% confidence interval [CI], 84.6 to 94.9%) and 96.9% (95% CI, 95.7 to 97.8%), respectively, across eight testing sites. The flocked swab specimens were 92.9% sensitive (95% CI, 86.0 to 96.5%) and 97.6% specific (95% CI, 96.2 to 98.5%) for MRSA detection across six testing sites. The sensitivity and specificity of the combined flocked and rayon swab data were 91.8% (95% CI, 87.4 to 94.8%) and 97.2% (95% CI, 96.3 to 97.9%), respectively. The positive predictive value (PPV) for rayon swabs was 78.7%, versus 83.5% for ESwabs. The negative predictive values (NPVs) for rayon swabs and ESwab specimens were 98.9% and 99.1%, respectively. In conclusion, the Xpert MRSA NxG assay is a sensitive and specific assay for the direct detection of MRSA from nasal swab specimens.

Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System.

A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform.

Detection of Group B Streptococcus Directly from Collected ESwab Samples by Use of the BD Max GBS Assay.

Group B Streptococcus detection directly from Copan ESwab collected samples, using the BD Max GBS assay, was evaluated on receipt in the laboratory and after 24 h at room temperature. Results were compared to those using Lim broth enrichment PCR and culture. No significant difference was observed between 24 h ESwab and Lim broth PCRs.

Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System.

A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

Evaluation of BD Max StaphSR and BD Max MRSAXT Assays Using ESwab-Collected Specimens.

The BD Max MRSAXT and the BD Max StaphSR assays were validated for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in ESwab samples. In addition, the BD Max StaphSR assay was evaluated for its ability to detect and differentiate S. aureus and MRSA in the same sample. A total of 255 ESwab samples collected from the anterior nares of patients were tested by each of three BD Max assays, including the BD Max MRSA first-generation assay. The results were compared to those of direct and enrichment culture. Additionally, a challenge panel comprising 14 control strains was evaluated to determine the ability of these assays to correctly identify MRSA and also appropriately differentiate S. aureus by the BD Max StaphSR assay. Out of 255 clinical samples tested, 161 were negative and 30 were positive for MRSA, and 45 were positive for S. aureus (by BD Max StaphSR) and negative for MRSA by all three PCR assays and culture. Nineteen samples had discrepant results; all of them were retested by additional laboratory testing. All strains from the challenge panel were correctly identified or excluded by the BD Max MRSAXT and BD Max StaphSR assays. The results showed that the BD Max StaphSR and the BD MRSAXT assays have excellent sensitivity (94.3%) and specificity (97.7%) for detecting MRSA. The BD Max StaphSR assay demonstrated excellent sensitivity (96.4%) and specificity (93.6%) for detecting S. aureus.

Laboratory evaluation of the BD MAX MRSA assay.

A comparison between the BD MAX MRSA and Xpert MRSA assays was performed using 239 nares samples. A 97.9% overall agreement between the two molecular assays was observed. The BD MAX MRSA assay proved to be a reliable alternative for a highly automated system to detect methicillin-resistant Staphylococcus aureus (MRSA) in patient nares samples.

Comparison of ESwab with traditional swabs for detection of methicillin-resistant Staphylococcus aureus using two different walk-away commercial real-time PCR methods.

The ESwab system (Copan Diagnostics) was evaluated as a nasopharyngeal specimen collection device to be used for methicillin-resistant Staphylococcus aureus (MRSA) detection by the GeneXpert and BD Max MRSA assays. Different MRSA strains and dilutions of each strain were tested in triplicate. ESwabs proved to be a suitable collection system for the two assays tested.

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.

Stemness of B-cell progenitors in multiple myeloma bone marrow.

PURPOSE: In myeloma, B cells and plasma cells show a clonal relationship. Clonotypic B cells may represent a tumor-initiating compartment or cancer stem cell responsible for minimal residual disease in myeloma. EXPERIMENTAL DESIGN: We report a study of 58 patients with myeloma at time of diagnosis or relapse. B cells in bone marrow were evaluated by multicolor flow cytometry and sorting. Clonality was determined by light chain and/or immunoglobulin chain gene rearrangement PCR. We also determined aldehyde dehydrogenase activity and colony formation growth. Drug sensitivity was tested with conventional and novel agents. RESULTS: Marrow CD19+ cells express a light chain identical to plasma cells and are therefore termed light chain restricted (LCR). The LCR B-cell mass is small in both newly diagnosed and relapsed patients (≤ 1%). Few marrow LCR B cells (~10%) are CD19+/CD34+, with the rest being more differentiated CD19+/CD34- B cells. Marrow LCR CD19+ B cells exhibit enhanced aldehyde dehydrogenase activity versus healthy controls. Both CD19+/CD34+ and CD19+/CD34- cells showed colony formation activity, with colony growth efficiency optimized when stroma-conditioned medium was used. B-cell progenitors showed resistance to melphalan, lenalidomide, and bortezomib. Panobinostat, a histone deacetylase inhibitor, induced apoptosis of LCR B cells and CD138+ cells. LCR B cells are CD117, survivin, and Notch positive. CONCLUSIONS: We propose that antigen-independent B-cell differentiation stages are involved in disease origination and progression in myeloma. Furthermore, investigations of myeloma putative stem cell progenitors may lead to novel treatments to eradicate the potential reservoir of minimal residual disease.

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.