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Abstract Background Mechanisms involved in the biological control of plant diseases are varied and complex. Hormones, including the auxin indole acetic acid (IAA) and abscisic acid (ABA), are essential regulators of a multitude of biological functions, including plant responses to biotic and abiotic stressors. This study set out to determine what hormones might play a role in Pseudomonas fluorescens –mediated control of Fusarium head blight (FHB) disease of barley and to determine if biocontrol-associated hormones directly affect disease development. Results A previous study distinguished bacterium-responsive genes from bacterium-primed genes, distinguished by the fact that the latter are only up-regulated when both P. fluorescens and the pathogen Fusarium culmorum are present. In silico analysis of the promoter sequences available for a subset of the bacterium-primed genes identified several hormones, including IAA and ABA as potential regulators of transcription. Treatment with the bacterium or pathogen resulted in increased IAA and ABA levels in head tissue; both microbes had additive effects on the accumulation of IAA but not of ABA. The microbe-induced accumulation of ABA preceded that of IAA. Gene expression analysis showed that both hormones up-regulated the accumulation of bacterium-primed genes. But IAA, more than ABA up-regulated the transcription of the ABA biosynthesis gene NCED or the signalling gene Pi2, both of which were previously shown to be bacterium-responsive rather than primed. Application of IAA, but not of ABA reduced both disease severity and yield loss caused by F. culmorum, but neither hormone affect in vitro fungal growth. Conclusions Both IAA and ABA are involved in the P. fluorescens-mediated control of FHB disease of barley. Gene expression studies also support the hypothesis that IAA plays a role in the primed response to F. culmorum. This hypothesis was validated by the fact that pre-application of IAA reduced both symptoms and yield loss asssociated with the disease. This is the first evidence that IAA plays a role in the control of FHB disease and in the bacterial priming of host defences.

Introduction: The COVID-19 pandemic focused attention on healthcare disparities and inequities faced by individuals within marginalized and structurally disadvantaged groups in the United States. These individuals bore the heaviest burden across this pandemic as they faced increased risk of infection and difficulty in accessing testing and medical care. Individuals experiencing housing insecurity are a particularly vulnerable population given the additional barriers they face. In this scoping review, we identify some of the barriers this high-risk group experienced during the early days of the pandemic and assess novel solutions to overcome these barriers., Methods: A scoping review was performed following PRISMA-Sc guidelines looking for studies focusing on COVID-19 testing among individuals experiencing housing insecurity. Barriers as well as solutions to barriers were identified as applicable and summarized using qualitative methods, highlighting particular ways that proved effective in facilitating access to testing access and delivery., Results: Ultimately, 42 studies were included in the scoping review, with 143 barriers grouped into four categories: lack of cultural understanding, systemic racism, and stigma; medical care cost, insurance, and logistics; immigration policies, language, and fear of deportation; and other. Out of these 42 studies, 30 of these studies also suggested solutions to address them., Conclusion: A paucity of studies have analyzed COVID-19 testing barriers among those experiencing housing insecurity, and this is even more pronounced in terms of solutions to address those barriers. Expanding resources and supporting investigators within this space is necessary to ensure equitable healthcare delivery., Competing Interests: CP was employed by the Healthspring Global Inc. CP reports receiving consulting fees from Abbott Molecular and Rapid Diagnostics. ET has been a consultant for Biomeme, Inc., has a patent pending for Methods to Diagnose and Treat Acute Respiratory Infections (US20180245154A1), and is currently employed by Danaher Corp. MC-W reports receiving support for research from the NIH (1U24-MD016258), National Institute of Allergy and Infectious Diseases (HHSN272201500006I, 1K24-AI143971), U.S. Food and Drug Administration (5U18-FD006298), and industry for drug development in adults and children. CW reports grants or contracts from DARPA, NIH-ARLG/NIAID/VTEU/NIMHO/NIGMS, Sanofi, Najit, CDC, Patient-Centered Outcomes Research Institute, USAMRAA, DOD, Abbott, and Pfizer; consulting fees from Arena Pharmaceuticals, BioFire, FHI Clinical, Giner, Karius, and SeLux Diagnostics; support for attending meetings and/or travel from American Society for Microbiology; patents planned, issued or pending for: biomarkers for the molecular classification of bacterial infection, methods to diagnose and treat acute respiratory infections, gene expression signatures useful to predict or diagnose sepsis and methods of using the same, host based molecular signatures of human infection with SARS-CoV-2 (COVID19), methods of identifying infectious disease and assays for identifying infectious disease, and nasopharyngeal protein biomarkers of acute respiratory virus infection and methods of using same; participation on a Data Safety Monitoring Board or Advisory Board for IDbyDNA, Janssen, Regeneron, Roche Molecular Sciences; leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid for American Society for Microbiology and American Society of Tropical Medicine and Hygiene; and other financial or non-financial interests with Biomeme and Predigen, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Johannesson, Glover, Petti, Veldman, Tsalik, Taylor, Hendren, Neighbors, Tillekeratne, Kennedy, Harper, Kibbe, Corbie, Cohen-Wolkowiez, Woods and Lee.)

Research on the COVID-19 pandemic revealed a disproportionate burden of COVID-19 infection and death among underserved populations and exposed low rates of SARS-CoV-2 testing in these communities. A landmark National Institutes of Health (NIH) funding initiative, the Rapid Acceleration of Diagnostics-Underserved Populations (RADx-UP) program, was developed to address the research gap in understanding the adoption of COVID-19 testing in underserved populations. This program is the single largest investment in health disparities and community-engaged research in the history of the NIH. The RADx-UP Testing Core (TC) provides community-based investigators with essential scientific expertise and guidance on COVID-19 diagnostics. This commentary describes the first 2 years of the TC's experience, highlighting the challenges faced and insights gained to safely and effectively deploy large-scale diagnostics for community-initiated research in underserved populations during a pandemic. The success of RADx-UP shows that community-based research to increase access and uptake of testing among underserved populations can be accomplished during a pandemic with tools, resources, and multidisciplinary expertise provided by a centralized testing-specific coordinating center. We developed adaptive tools to support individual testing strategies and frameworks for these diverse studies and ensured continuous monitoring of testing strategies and use of study data. In a rapidly evolving setting of tremendous uncertainty, the TC provided essential and real-time technical expertise to support safe, effective, and adaptive testing. The lessons learned go beyond this pandemic and can serve as a framework for rapid deployment of testing in response to future crises, especially when populations are affected inequitably., Competing Interests: The authors declare a conflict of interest. Ephraim Tsalik has been a consultant for Biomeme, Inc.; has a patent pending for Methods to Diagnose and Treat Acute Respiratory Infections (US20180245154A1); and is currently employed by Danaher Corp. Michael Cohen-Wolkowiez receives support for research from the NIH [1U24-MD016258]; National Institute of Allergy and Infectious Diseases [HHSN272201500006I, 1K24-AI143971]; U.S. Food and Drug Administration [5U18-FD006298]; and industry for drug development in adults and children. Cathy A Petti receives consulting fees from Abbott Molecular and Rapid Diagnostics. Christopher Woods reports consulting fees from Arena Pharmaceuticals, BioFire, FHI Clinical, Giner, Karius, and SeLux Diagnostics. All other authors report no conflicts of interests.

The microbiology, epidemiology, diagnostics, and treatment of infective endocarditis (IE) have changed significantly since the Duke Criteria were published in 1994 and modified in 2000. The International Society for Cardiovascular Infectious Diseases (ISCVID) convened a multidisciplinary Working Group to update the diagnostic criteria for IE. The resulting 2023 Duke-ISCVID IE Criteria propose significant changes, including new microbiology diagnostics (enzyme immunoassay for Bartonella species, polymerase chain reaction, amplicon/metagenomic sequencing, in situ hybridization), imaging (positron emission computed tomography with 18F-fluorodeoxyglucose, cardiac computed tomography), and inclusion of intraoperative inspection as a new Major Clinical Criterion. The list of "typical" microorganisms causing IE was expanded and includes pathogens to be considered as typical only in the presence of intracardiac prostheses. The requirements for timing and separate venipunctures for blood cultures were removed. Last, additional predisposing conditions (transcatheter valve implants, endovascular cardiac implantable electronic devices, prior IE) were clarified. These diagnostic criteria should be updated periodically by making the Duke-ISCVID Criteria available online as a "Living Document.", Competing Interests: Potential conflicts of interest. V. G. F. reports personal consulting fees from Novartis, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., MedImmune, Bayer, Basilea, Affinergy, Janssen, Contrafect, Regeneron, Destiny, Amphliphi Biosciences, Integrated Biotherapeutics; C3J, Armata, and Valanbio; Akagera, Aridis, Roche, and Pfizer (paid to author); grants from the National Institutes of Health, MedImmune, Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, and Merck, Medical Biosurfaces, Locus, Affinergy, Contrafect, Karius, Genentech, Regeneron, Deep Blue, Basilea, and Janssen; royalties from UpToDate; stock options from Valanbio and ArcBio; honoraria from the Infectious Diseases Society of America for his service as Associate Editor of Clinical Infectious Diseases; travel support from Contrafect to 2019 ECCMID; and a sepsis diagnostics patent pending. D. T. D. reports payment for expert testimony for 2 worker's compensation claims and 1 medico-legal claim (paid to author); membership on the ISCVID Council; stock in Becton Dickinson Company, Moderna, Merck, and Illumina; and stock options in Magnolia Medical, Inc. A. S. B. reports research grants from the National Institute of Allergy and Infectious Diseases, Akagera Medicines, ContraFect Corporation, and Riovant Pharmaceuticals; and payment for expert testimony from Hanson, Skemp & Sleik, La Crosse, Wisconsin. A. D. reports research grant support from the Lundbeck Foundation (unrelated to this work). L. D. reports payment for expert testimony in inferior vena cava filter litigation for Cordis, Braun, and Bard; and stock or stock options from Asensus Surgical (Transenterix). E. D. M. reports research funding for his institution from MSD, Pfizer, Angelini, Infectopharm, Advanz pharma, Menarini, and Shionogi; and personal consulting fees from Genentech, Roche, Angelini, Trx, Medtronic, and Abbvie; fees to participate in advisory boards for Pfizer; role as Secretary of the International Society for Cardiovascular Infectious Diseases; speaker's honoraria from Pfizer, Shionogi, and Advanz pharma; as well as personal fees from Bio-Merieux, Sanofi-Aventis, and DiaSorin. X. D. reports grants or contracts with Pfizer and Sanofi Pasteur (paid to institution). All other authors report no potential conflicts. E. F. reports independent research grants for valvular heart diseases from the Novo Nordisk Foundation. M. M. H. reports payment from Up To Date (paid to author) and participation on an Advisory Board for Takeda Island. A. W. K. reports a research grant from Karius; honoraria from Pfizer, Data Safety Monitoring Board; personal fees consulting from Debio Pharma; royalties from Up To Date; common stock in Pfizer, Abbvie, and Johnson and Johnson; and participation in Winter Course in Infectious Diseases. C. A. M. reports personal fees from Edwards Lifesciences (Clinical Events Committee) and Cytosorbents Corp. S. D. P. reports event payment from Agilent and Cariomyopathy UK (paid to institution); role as councilor for Association for European Cardiovascular Pathology (unpaid). F. V. reports research funding outside the scope of the present study by bioMérieux; 2 patents pending in antimicrobial resistance detection (PCT/EP2022/069857 July 2022; FR 2200268 Jan 2022); and shares in Weezion. J. M. M. reports consulting honoraria and/or research grants from Angelini, Contrafect, Cubist, Genentech, Gilead Sciences, Jansen, Lysovant, Medtronic, MSD, Novartis, Pfizer, and ViiV Healthcare, outside the submitted work. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We aimed to describe utilization and indication(s) for long-term central venous catheters (CVCs) in a pediatric intensive care unit (PICU) and identify potential strategies to decrease CVC utilization., Methods: We conducted a single-center prospective quality improvement initiative at a 30-bed PICU in a large, freestanding, academic children's hospital. We created an electronic report to identify patients with an indwelling CVC for 7 days and older (defined as long term). We discussed the ongoing need for each long-term CVC with PICU clinicians at weekly interdisciplinary structured "CVC stewardship rounds." We then made recommendations around expedited removal of CVCs. We conducted multiple Plan-Do-Study-Act cycles to categorize CVC indications, identify modifiable factors, and educate PICU clinicians. We hypothesized that CVC stewardship rounds would decrease long-term CVC utilization in our PICU., Results: From October 2016 to September 2017, 607 long-term CVCs were eligible for the stewardship intervention. Compared to the preintervention period, we recorded a significant increase in peripherally inserted central catheters and a decrease in nontunneled CVCs ( P < 0.001). Most patients had single- or double-lumen CVCs in both the preintervention and intervention periods (86% and 91%, respectively). The utilization of overall long-term CVC devices, and those with modifiable indications, decreased during the intervention period., Conclusions: A single-center QI intervention focused on PICU CVC stewardship was associated with a decrease in CVC utilization., Competing Interests: The authors have no financial interest to declare in relation to the content of this article., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

We evaluated the performance of an early prototype core molecular mirroring nuclear magnetic resonance detection platform (Mentor-100) to detect toxigenic Clostridium difficile from stool. This technology uses customized nanoparticles bound to target specific oligonucleotide probes that form binaries in the presence of nucleic acid from the target microorganism. Liquid patient stool specimens were seeded with C. difficile or other Clostridium species to determine the analytical sensitivity and specificity. Samples underwent nucleic acid extraction and target amplification with probes conjugated with iron nanoparticles. Signal from nuclear magnetic resonance spin-spin relaxation time was measured to detect the presence or absence of toxigenic C. difficile. The limit of detection was <180 colony forming units per reaction of toxigenic C. difficile. No cross-reactivity was observed with nontoxigenic C. difficile, Clostridium sordellii, Clostridium perfringens, Bacillus subtilis, or Paenibacillus polymyxa at 10 8 colony forming units/mL. Correlation studies using frozen stool samples yielded a sensitivity of 88.4% (61 of 69) and a specificity of 87.0% (40 of 46) as compared with a commercial PCR assay for C. difficile. The area under the curve in the receiver operating characteristic curve analysis was 0.922. The prototype molecular mirroring platform showed promising performance for pathogen detection from clinical specimens. The platform design has the potential to offer a novel, low-cost alternative to currently available nucleic acid-based tests., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The phenotypic expression of methicillin resistance among coagulase-negative staphylococci (CoNS) is heterogeneous regardless of the presence of the mecA gene. The potential discordance between phenotypic and genotypic results has led to the use of vancomycin for the treatment of CoNS infective endocarditis (IE) regardless of methicillin MIC values. In this study, we assessed the outcome of methicillin-susceptible CoNS IE among patients treated with antistaphylococcal β-lactams (ASB) versus vancomycin (VAN) in a multicenter cohort study based on data from the International Collaboration on Endocarditis (ICE) Prospective Cohort Study (PCS) and the ICE-Plus databases. The ICE-PCS database contains prospective data on 5,568 patients with IE collected between 2000 and 2006, while the ICE-Plus database contains prospective data on 2,019 patients with IE collected between 2008 and 2012. The primary endpoint was in-hospital mortality. Secondary endpoints were 6-month mortality and survival time. Of the 7,587 patients in the two databases, there were 280 patients with methicillin-susceptible CoNS IE. Detailed treatment and outcome data were available for 180 patients. Eighty-eight patients received ASB, while 36 were treated with VAN. In-hospital mortality (19.3% versus 11.1%; P = 0.27), 6-month mortality (31.6% versus 25.9%; P = 0.58), and survival time after discharge (P = 0.26) did not significantly differ between the two cohorts. Cox regression analysis did not show any significant association between ASB use and the survival time (hazard ratio, 1.7; P = 0.22); this result was not affected by adjustment for confounders. This study provides no evidence for a difference in outcome with the use of VAN versus ASB for methicillin-susceptible CoNS IE., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Consensus definitions have emerged for the discrimination between infected and uninfected prosthetic joints but diagnostic uncertainty often occurs. We examined the accuracy of orthopaedic surgeons' assessments to diagnose the infected prosthetic hip or knee and elucidated the added value of laboratory parameters. A prospective cohort study of patients undergoing revision arthroplasty of hip or knee was conducted over a one-year period. Orthopaedic surgeons' determinations prior to arthroplasty were recorded. A reference diagnostic standard was determined retrospectively by independent review from 3 infectious diseases physicians. Patients were followed up to 12 months. For 198 patients enrolled, 228 surgical encounters (110 knee, 118 hip) were classified by independent reviewers as 176 uninfected and 52 infected. Orthopaedic surgeons' preoperative diagnoses of infection had high diagnostic accuracy (sensitivity 89%, specificity 99%, PPV 98%, NPV 97%). Addition of intraoperative findings and histopathology improved their diagnostic accuracy. Addition of culture and PCR results improved sensitivity of diagnostic determinations but not specificity. We provide evidence that clinical acumen has high diagnostic accuracy using routine preoperative parameters. Histopathology from intraoperative specimens would improve surgeons' diagnostic accuracy but culture and PCR from intraoperative specimens could create greater diagnostic uncertainty. This study is critical to further our understanding of the added value, if any, of laboratory testing to support clinical decision making for the suspected infected joint and allow us to identify diagnostic gaps for emerging technologies to fill that will improve our ability to diagnose the infected prosthetic joint.

A number of exciting new technologies have emerged to detect infectious diseases with greater accuracy and provide faster times to result in hopes of improving the provision of care and patient outcomes. However, the challenge in evaluating new methods lies not in the technical performance of tests but in (1) defining the specific advantages of new methods over the present gold standards in a practicable way and (2) understanding how advanced technologies will prompt changes in medical and public health decisions. With rising costs to deliver care, enthusiasm for innovative technologies should be balanced with a comprehensive understanding of clinical and laboratory ecosystems and how such factors influence the success or failure of test implementation. Selecting bloodstream infections as an exemplar, we provide a 6-step model for test adoption that will help clinicians and laboratorians better define the value of a new technology specific to their clinical practices.

Background: Physicians rely on blood culture to diagnose bloodstream infections (BSI) despite its limitations. As new technologies emerge for rapid BSI diagnosis, optimization of their application to patient care requires an understanding of clinicians' perspectives on BSI diagnosis and how a rapid test would influence medical decisions., Methods: We administered a 26-question survey to practitioners in infectious diseases/microbiology, critical care, internal medicine, and hematology/oncology services in USA and Germany about current standards in diagnosing and treating BSI and a hypothetical rapid BSI test., Results: Responses from 242 providers had roughly equal representation across specialties. For suspected BSI patients, 78% of practitioners would administer empiric broad spectrum antibiotics although they estimated, on average, that 31% of patients received incorrect antibiotics while awaiting blood culture results. The ability of blood culture to rule in or rule out infection was very/extremely acceptable in 67% and 36%, respectively. Given rapid test results, 60-87% of practitioners would narrow the spectrum of antimicrobial therapy depending on the microorganism detected, with significantly higher percentages when resistance determinants were also tested. Over half of respondents felt a rapid test would be very/extremely influential on clinical practice., Conclusions: Limitations of blood culture were perceived as a barrier to patient care. A rapid test to diagnose BSI would impact clinical practice, but the extent of impact may be limited by prevailing attitudes and practices. Opportunities exist for interventions to influence practitioners' behaviors in BSI management particularly with emergence of newer diagnostic tests.

Members of the Mycobacterium chelonae-abscessus complex represent Mycobacterium species that cause invasive infections in immunocompetent and immunocompromised hosts. We report the detection of a new pathogen that had been misidentified as M. chelonae with an atypical antimicrobial drug susceptibility profile. The discovery prompted a multicenter investigation of 26 patients. Almost all patients were from the northeastern United States, and most had underlying sinus or pulmonary disease. Infected patients had clinical features similar to those with M. abscessus infections. Taxonomically, the new pathogen shared molecular identity with members of the M. chelonae-abscessus complex. Multilocus DNA target sequencing, DNA-DNA hybridization, and deep multilocus sequencing (43 full-length genes) support a new taxon for these microorganisms. Because most isolates originated in Pennsylvania, we propose the name M. franklinii sp. nov. This investigation underscores the need for accurate identification of Mycobacterium spp. to detect new pathogens implicated in human disease.

"Pseudomonas andersonii" is a Gram-negative bacillus initially isolated from a granulomatous lung lesion. Novel species status has not been validated for this single strain. We report four additional cases of pulmonary granuloma involving P. andersonii and further characterize the organism. These patients had pulmonary nodules that were surgically resected and which grew P. andersonii on routine culture. Mycobacterium avium complex was concomitantly isolated in two cases, and fungal structures were identified histopathologically in two other cases. The five P. andersonii strains described to date were similar in growth characteristics, biochemical reactions, matrix-assisted laser desorption ionization-time of flight mass spectrometry protein profiles, and susceptibility to antimicrobial agents. Their 16S rRNA genes were 99.9 to 100% identical but less than 95.0% similar to those of all other known bacteria. The gyrA genes of these strains were 99.5 to 100% identical. These shared features illustrate P. andersonii as a unique and distinct bacterium and support the novel species status of the organism.

Parasitic infection is uncommon in the United States, but surveys suggest that physicians test when the presence of parasites is unlikely and fail to order appropriate testing when suspicion is high. Numerous studies confirm that immunoassays are more sensitive for Giardia and Cryptosporidium detection, but our experience was that physicians preferentially used ovum and parasite examination (O&P). We conducted a retrospective study of fecal parasite testing at a referral laboratory nationally (1997 to 2006) and during a Cryptosporidium outbreak (Utah, 2007) to correlate physician use of O&P and enzyme immunoassays (EIAs) with the yield of parasites detected. Nationally, of 170,671 episodes, 76.0% (n = 129,732) included O&P, 27.9% (n = 47,666) included Giardia EIA, and 5.7% (n = 9,754) included Cryptosporidium EIA. Most pathogens were Giardia or Cryptosporidium. More episodes were positive when EIA was performed (n = 1,860/54,483 [3.4%]) than when O&P only was performed (n = 1,667/116,188 [1.4%]; P < 0.001), and EIA was more sensitive than O&P. However, more O&P results were positive among patients with both O&P and EIA performed (2.5%) than among those with O&P only performed (1.4%; P < 0.001), suggesting that patients tested by O&P only may have been at lower risk. During the first 10 weeks of the outbreak, physicians also preferentially used O&P over EIA, but no Cryptosporidium cases were detected by O&P. We conclude that clinicians frequently use O&P testing when test performance and epidemiology support the use of immunoassays or no testing. We recommend that stool O&P be limited to patients with negative immunoassay results and persistent symptoms or individuals at increased risk for non-Giardia, non-Cryptosporidium infection. An evidence-based algorithm for the evaluation of patients with suspected intestinal parasitic infection is proposed.

Reference isolates of Mycobacterium neoaurum, Mycobacterium aurum, and the nonvalidated species "Mycobacterium lacticola" were the focus of two recent molecular taxonomic studies. On the basis of this grouping, we identified 46 clinical pigmented, rapidly growing mycobacterial isolates. By 16S rRNA gene sequencing, only two major taxa were identified: M. neoaurum and a previously uncharacterized "M. neoaurum-like" group. The M. neoaurum-like group exhibited only 99.7% identity to M. neoaurum by 16S rRNA gene sequencing and 96.5% identity to M. neoaurum by rpoB sequencing and was named M. bacteremicum. No clinical isolates of M. aurum or M. lacticola were identified. Of isolates with known sources, 4/8 (50%) of M. bacteremicum isolates and 22/34 (65%) of M. neoaurum isolates were recovered from blood, and 35% of these were known to be from patients with catheter-related sepsis. MIC and clinical data on these 46 isolates of M. neoaurum and M. bacteremicum along with a review of 16 previously reported cases of infection with the M. neoaurum-M. lacticola group demonstrated that the isolates were highly susceptible to all drugs tested except clarithromycin, and most clinical cases were successfully treated. The clarithromycin resistance suggested the presence of an inducible erm gene reported in other species of rapidly growing mycobacteria. Sequencing studies are currently required to identify these two species. Strain ATCC 25791 (originally submitted as an example of Mycobacterium aurum) is proposed to be the type strain of M. bacteremicum.

Percutaneous osseointegrated implant technology provides a potential alternative to current socket prosthetics for individuals with limb loss. However, similar to other percutaneous devices, there remain concerns of periprosthetic infection. To understand this process of infection, bacterial isolates were collected and characterized from a sheep model of osseointegration. CSA-13, a novel cationic steroid antimicrobial, was used at the skin/implant interface in an attempt to reduce the rate of infection. Results indicated that in this application, normal flora and environmental organisms continued to colonize the skin/implant interface as well as cause infection in the presence of CSA-13. Two factors are believed to have contributed to this outcome: the delivery of CSA-13 and the lack of a skin seal at the skin/implant interface, which would create a biological barrier to infection.

Context: The anthrax incident of 2001 in the United States prompted the College of American Pathologists (CAP), the Association of Public Health Laboratories, and the Centers for Disease Control and Prevention to develop exercises for Laboratory Response Network (LRN) sentinel laboratories., Objective: To provide an overview of the results of the CAP bioterrorism Laboratory Preparedness Survey (LPS, 2007) and Laboratory Preparedness Exercise (LPX, 2008) and assist LRN sentinel laboratories and public health agencies in planning for bioterrorism events., Design: Bioterrorism agents and nonbiothreat mimic organisms were provided in 2 mailings per year (2007 and 2008, 20 total challenges). Within each mailing, 2 to 3 agents were category A or category B bioterrorism agents (total of 10 categoric challenges). Some category A/B isolates were modified/vaccine strains. The total number of laboratories participating in these exercises ranged from 1316 to 1381. Isolate characteristics used to identify the organisms were compiled along with the participants' reporting actions. Educational commentary was provided with each exercise., Results: Acceptable identification responses were as follows: Bacillus anthracis, 90% (2007) and 99.9% (2008); Yersinia pestis, 83.8% (2007) and 87.6% (2008); and Francisella tularensis subsp Holarctica, 86.6% (2007) and 91.6% (2008). The time interval between specimen receipt and notification of results to an LRN reference laboratory decreased from more than 10 days in 2007 to 3 or 4 days in 2008 for some challenges., Conclusions: The bioterrorism challenge program (LPS, LPX) provides important comparative data from more than 1300 sentinel laboratories that can be used by individual laboratories to evaluate their identification and LRN reporting performance.

Large amounts of respiratory viruses are shed in nasal secretions by children. Nasal mucus was compared with nasopharyngeal swabs as a source for respiratory virus testing. Multiplex reverse transcription-polymerase chain reaction detected virus in nasal mucus specimens in 73% (11/15) of positive cases, demonstrating the potential utility of less invasive specimens when a highly sensitive method is used for respiratory virus detection.

We assessed the utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae to diagnose respiratory tract infections. Compared to PCR and IgM serology, culture was less sensitive and had extremely low yield. Culture is not recommended for these pathogens, and this method should be eliminated from routine practice.

The 16S rRNA gene is commonly used to identify Mycobacterium spp., but alternative DNA targets can provide better resolution to the species level. We evaluated a novel system that enables simultaneous amplification, sequencing, and analysis of two different DNA targets in a single tube to identify clinical isolates of Mycobacterium spp. For 139 clinical isolates, we found that the 16S rRNA gene alone identified 67 (48%) isolates as single species, 68 (49%) isolates to the complex or group level, and 4 (3%) isolates to the genus level only. The rpoB gene alone identified 117 (84%) isolates as single species, 10 (7%) isolates to the complex or group level, and 12 (8%) isolates to the genus level only. Combining the separate analyses for sequencing of two gene targets, 119 (86%) isolates were identified as single species and 10 (7%) isolates were identified to the complex or group level. Seven (5%) isolates were identified as novel species within established groups, and 3 (2%) were identified to the genus level only. Dual-locus identification identified 110 (79%) isolates as single species and 22 (16%) isolates to the complex or group level. Six (4%) were identified as novel species within established groups, and 1 (1%) was identified to the genus level only. Identifications were more accurate when both the 16S rRNA and rpoB genes were screened, and reliance on a single gene target was suboptimal. RipSeq dual-locus software provides an accurate alternative method for laboratories using two different gene targets for microorganism identification.

The performance of 4 laboratory methods for diagnosis of viral respiratory tract infections (RTI) in older adults was evaluated. Seventy-four nasopharyngeal (NP) swab specimens were obtained from 60 patients with RTI at a long-term care facility over 2 respiratory seasons. Sixteen specimens were positive for a respiratory virus by at least 1 method. Multiplex reverse transcriptase polymerase chain reaction (RT-PCR) by the Luminex xTAG Respiratory Viral Panel (RVP) detected 16 (100%) of the positive specimens, RVP of 24-h culture supernatant detected 8 (50%), direct fluorescent antibody testing detected 4 (25%), rapid culture detected 2 (12.5%), and rapid antigen testing detected none. For a comparison group, RVP was performed on NP swabs from 20 outpatient children with RTI. The mean fluorescence intensity by RVP was significantly lower for positive adult patients than pediatric patients (P = 0.0373). Our data suggest that older adult patients shed lower titers of viruses, necessitating a highly sensitive assay such as RT-PCR to reliably detect respiratory viral pathogens.

Differentiation of Mycobacterium tuberculosis complex (MTC) species is important for patient management. We developed a genomic deletion assay based on multiplex polymerase chain reaction with melting temperature analysis that correctly identified 124 (96%) of 129 MTC isolates. This assay is a fast single-tube method to differentiate members of MTC., ((c) 2010 Elsevier Inc. All rights reserved.)

Culture and serotyping of human enteroviruses are time-consuming and labor-intensive. Targeted nucleic acid sequencing has emerged as a powerful alternative to conventional methods. Many published genotyping assays use two-step reverse transcription and polymerase chain reaction (RT-PCR), nested PCR protocols, and/or reflexive testing algorithms. The performance of a one-step RT-PCR protocol, a more clinically practical approach, was evaluated. The VP1 and/or VP2 region of archived enterovirus isolates (n=36, representing 32 serotypes), patient enterovirus isolates not typeable by immunofluorescence antibodies (n=50), and enterovirus from direct patient specimens (48 cerebrospinal fluid, 2 plasma/serum, 1 blood) were amplified and sequenced for genotype identification. The analytical sensitivity of the genotyping assays was 100-fold less than detection by RT-PCR of the 5'-untranslated region. Thirty-four of 36 archived isolates could be genotyped by combining results of VP1 and VP2 target sequencing. Non-typeable isolates included 17 echovirus 18, 6 enterovirus 68, 6 rhinovirus, and 7 which could not be classified further. From clinical specimens, 23 of 51 (45%) could be identified using VP2 typing and the most common types were coxsackievirus B1, echovirus 30, and echovirus 6. Using a one-step RT-PCR without nesting, most enterovirus isolates and a subset of clinical samples with high viral titer could be genotyped., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Transcutaneous osseointegrated implants can improve function for select amputee patients, but infection serves as a significant limitation of implantable transcutaneous devices. This study examined the efficacy of an antimicrobial, pexiganan acetate (SUPONEX), and a porous tantalum implant material (Trabecular Metal) in preventing pin tract infection of osseointegrated implants in a rabbit model. Thirty-seven rabbits were randomized to three groups: Ti-control group (n = 11) with titanium alloy implant and no antimicrobial, Ti-Pexiganan group (n = 8) with titanium alloy implant and topical pexiganan acetate 1% applied daily at the skin/implant interface, and Ta-control group (n = 18) with porous tantalum implant and no antimicrobial. All implants were placed transcutaneously through skin, muscle, and bone. Rabbits were monitored for infection for 24 weeks. We observed a 75% reduction in rates of pin tract infection in the Ti-Pexiganan group compared to that observed in the Ti-control group (p = 0.019). No difference in rates of infection was observed between the Ta-control group and the Ti-control group (p = 0.230). In conclusion, pexiganan acetate may be an important antimicrobial for transcutaneous osseointegrated implants. Porous tantalum will not likely prevent pin tract infection without additional methods of soft tissue immobilization around the implant site., ((c) 2009 Wiley Periodicals, Inc.)

Mycobacterium abscessus is resistant to multiple antibiotics, creating treatment challenges. Carbapenems are tested to increase therapeutic alternatives. We performed in vitro susceptibility testing by Etest of four carbapenems for M. abscessus isolates. Imipenem demonstrated the most in vitro activity, and testing of other carbapenems provided no additional value.

Culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. Flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Kinetic studies of coxsackievirus B1 and echovirus 30 infection of PMK cells were performed on days 1-4 after inoculation. Flow cytometry results for echovirus 6, 9, 11, and 30 and coxsackievirus B1 correlated with IFA in all cases. Coxsackievirus B1 and echovirus 30 infections were detected 1 day earlier by flow cytometry than IFA. Flow cytometry can be effectively used for detecting enterovirus-infected cells in a clinical laboratory with the advantages of better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems.

The INFINITI HPV-QUAD assay is a commercially available genotyping platform for human papillomavirus (HPV) that uses multiplex PCR, followed by automated processing for primer extension, hybridization, and detection. The analytical performance of the HPV-QUAD assay was evaluated using liquid cervical cytology specimens, and the results were compared with those results obtained using the digene High-Risk HPV hc2 Test (HC2). The specimen types included Surepath and PreservCyt transport media, as well as residual SurePath and HC2 transport media from the HC2 assay. The overall concordance of positive and negative results following the resolution of indeterminate and intermediate results was 83% among the 197 specimens tested. HC2 positive (+) and HPV-QUAD negative (-) results were noted in 24 specimens that were shown by real-time PCR and sequence analysis to contain no HPV, HPV types that were cross-reactive in the HC2 assay, or low virus levels. Conversely, HC2 (-) and HPV-QUAD (+) results were noted in four specimens and were subsequently attributed to cross-contamination. The most common HPV types to be identified in this study were HPV16, HPV18, HPV52/58, and HPV39/56. We show that the HPV-QUAD assay is a user friendly, automated system for the identification of distinct HPV genotypes. Based on its analytical performance, future studies with this platform are warranted to assess its clinical utility for HPV detection and genotyping.

Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs). NETs are lattices of extracellular DNA, chromatin, and antibacterial proteins that mediate extracellular killing of microorganisms and are thought to form via a unique death pathway signaled by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated reactive oxygen species (ROS). We found that neutrophils from term and preterm infants fail to form NETs when activated by inflammatory agonists-in contrast to leukocytes from healthy adults. The deficiency in NET formation is paralleled by a previously unrecognized deficit in extracellular bacterial killing. Generation of ROSs did not complement the defect in NET formation by neonatal neutrophils, as it did in adult cells with inactivated NADPH oxidase, demonstrating that ROSs are necessary but not sufficient signaling intermediaries and identifying a deficiency in linked or downstream pathways in neonatal leukocytes. Impaired NET formation may be a critical facet of a common developmental immunodeficiency that predisposes newborn infants to infection.

Phenotypic identification of AmpC, KPC and extended-spectrum beta-lactamases (ESBLs) among members of the Enterobacteriaceae remains challenging. This study compared the Phoenix Automated Microbiology System (BD Diagnostics) with the Clinical and Laboratory Standards Institute confirmatory method to identify ESBL production among 200 Escherichia coli and Klebsiella pneumoniae clinical isolates. The Phoenix system misclassified nearly half of the isolates as ESBL-positive, requiring manual testing for confirmation. Inclusion of aztreonam +/- clavulanic acid (CA) and cefpodoxime +/- CA in the testing algorithm increased the ESBL detection rate by 6 %. Boronic acid-based screening identified 24 isolates as AmpC(+), but in a subset of genotypically characterized isolates, appeared to have a high false-positivity rate. PCR screening revealed eight KPC(+) isolates, all of which tested as ESBL(+) or ESBL(+) AmpC(+) by phenotypic methods, but half were reported as carbapenem-susceptible by the Phoenix system. Overall, these results indicate that laboratories should use the Phoenix ESBL results only as an initial screen followed by confirmation with an alternative method.

We examined American Type Culture Collection (ATCC) strains of Mycobacterium aurum and Mycobacterium neoaurum by using multilocus DNA target sequencing. Apart from the type strain, all 10 ATCC M. aurum strains examined were classified incorrectly, with most being reclassified as belonging to the M. neoaurum-'Mycobacterium lacticola' relatedness group. All four M. neoaurum strains were tightly clustered, but heterogeneity was observed within the cluster. As a result of the incorrect annotation of the M. aurum strains, two commonly used methods of identification are compromised and two case reports implicating M. aurum as a human pathogen are probably incorrect, with the isolates probably belonging to the M. neoaurum-'M. lacticola' relatedness group. These findings together with a review of isolates identified at two large reference laboratories suggest that M. aurum is not a clinically significant isolate.

Seven international laboratories tested the recently proposed single-locus typing strategy for Aspergillus fumigatus subtyping for interlaboratory reproducibility. Comparative sequence analyses of portions of the locus AFUA&#95;3G08990, encoding a putative cell surface protein (denoted CSP), was performed with a panel of Aspergillus isolates. Each laboratory followed very different protocols for extraction of DNA, PCR, and sequencing. Results revealed that the CSP typing method was a reproducible and portable strain typing method.

Mollicutes can cause a wide spectrum of disease, especially in neonates. To better define their disease spectrum in the United States, we reviewed the results of >14,000 mollicute isolates, including 1346 from neonates. When mollicute infection is suspected, clinicians should alert laboratories, which will optimize methods of detection.

Objectives: To report an outbreak of respiratory synctyial virus (RSV) in a long-term care facility (LTCF) during ongoing routine respiratory illness surveillance., Design: Rapid antigen testing, viral culture, direct fluorescent antibody (DFA) testing, and reverse transcriptase polymerase chain reaction (RT-PCR) testing for up to 15 viruses in symptomatic residents and chart review., Setting: A 120-bed LTCF., Measurements: Comparison of rapid antigen testing, respiratory viral cultures, and DFA testing and RT-PCR in residents with symptoms of a respiratory tract infection., Results: Twenty-two of 52 residents developed symptoms of a respiratory tract infection between January 29, 2008, and February 26, 2008. RSV was detected using RT-PCR in seven (32%) of the 22 cases. None of the seven cases had positive RSV rapid antigen testing, and only two had positive culture or DFA results. This outbreak occurred during a time when state wide RSV rates were rapidly declining. One patient was admitted to the hospital during the infection and subsequently died., Conclusion: RSV may cause outbreaks in LTCFs that traditional diagnostic methods do not detect. RT-PCR can provide a more timely and accurate diagnosis of outbreaks, which allows for early symptomatic treatment, rational use of antibiotics, and improved infection control.

Stool culture for Clostridium difficile, while necessary for strain typing and antimicrobial surveillance, cannot determine toxin production. We prospectively tested in triplicate 91 C difficile cultured isolates for toxin production by 2 enzyme immunoassays (EIAs) (Meridian Premier Toxins A&B, Meridian Bioscience, Cincinnati, OH; and TechLab Tox A/B II, TechLab, Blacksburg, VA) and cytotoxin neutralization bioassay (CTN). By CTN, 88% (80/91) were toxigenic. Reproducibility was 93% (85/91) for CTN, 80% (73/91) for Meridian EIA, and 79% (72/91) for TechLab EIA. Compared with CTN, sensitivities were 87.1% and 89.2% for the Meridian and TechLab EIAs, respectively. In an additional 115 stool specimens, CTN detected toxin more frequently from cultured isolates (96/115) than stool (84/115). For C difficile toxin detection from isolates, EIA was less reproducible than CTN. EIA methods can be falsely negative in 10% to 12% of isolates, and these should be tested by CTN or polymerase chain reaction. When positive, EIA is fast and reliable for detecting C difficile toxin from culture.

Background: Many laboratories using R-Mix cell lines inoculate other shell vial cultures to improve the recovery of viruses, and in particular, perform terminal hemadsorption (THad) following 10-14 days of incubation to improve detection of respiratory viruses. We explored the cost-effectiveness and added benefits of THad on conventional shell vial cultures from respiratory samples for laboratories using R-Mix cell lines., Objectives: To determine if eliminating the practice of THad from conventional shell vial culture when R-Mix cultures are negative, would result in a significant reduction in the number of hemadsorbing respiratory viruses detected., Study Design: THad results were retrospectively reviewed for 41,129 respiratory shell vial cultures that were set up concurrently with R-Mix cultures., Results: Greater than 95% of hemadsorbing respiratory viruses were recovered by R-Mix standard protocol within 24h of inoculation, and only 5% were detected by THad at 10-14 days., Conclusion: The practice of hemadsorption at days 10-14 for conventional shell vial cultures from respiratory specimens should be discontinued for laboratories using R-Mix due to its low yield, questionable clinical impact of delayed results and additional costs.

Identification of viridans group streptococci (VGS) to the species level is difficult because VGS exchange genetic material. We performed multilocus DNA target sequencing to assess phylogenetic concordance of VGS for a well-defined clinical syndrome. The hierarchy of sequence data was often discordant, underscoring the importance of establishing biological relevance for finer phylogenetic distinctions.

We report the use of 16S ribosomal RNA gene amplification and sequencing to diagnose culture-negative intracerebral abscesses in younger patients. These 3 cases demonstrate the optimal application of gene sequencing from direct specimens for patients with negative culture results compromised by antibacterial therapy but histories highly suggestive of acute bacterial infection.

Accurate species determination for anaerobes from blood culture bottles has become increasingly important with the reemergence of anaerobic bacteremia and prevalence of multiple-drug-resistant microorganisms. Our knowledge of the taxonomical diversity of anaerobes that cause bloodstream infections is extremely limited, because identification historically has relied on conventional methods. Over a 5-year period, we profiled anaerobic bacteremia at a large tertiary care hospital with 16S rRNA gene sequencing to gain a better understanding of the taxonomical diversity of the bacteria. Of 316 isolates, 16S rRNA gene sequencing and phylogenetic analysis identified 316 (100%) to the genus or taxonomical group level and 289 (91%) to the species level. Conventional methods identified 279 (88%) to the genus level and 208 (66%) to the species level; 75 (24%) were misidentified at the species level, and 33 (10%) results were inconclusive. High intragenus variability was observed for Bacteroides and Clostridium species, and high intraspecies variability was observed for Bacteroides thetaiotaomicron and Fusobacterium nucleatum. Sequence-based identification has potential benefits in comparison to conventional methods, because it more accurately characterizes anaerobes within taxonomically related clusters and thereby may enable better correlation with specific clinical syndromes and antibiotic resistance patterns.

Coagulase-negative staphylococci (CNS) are important causes of infective endocarditis (IE), but their microbiological profiles are poorly described. We performed DNA target sequencing and susceptibility testing for 91 patients with definite CNS IE who were identified from the International Collaboration on Endocarditis-Microbiology, a large, multicenter, multinational consortium. A hierarchy of gene sequences demonstrated great genetic diversity within CNS from patients with definite endocarditis that represented diverse geographic regions. In particular, rpoB sequence data demonstrated unique genetic signatures with the potential to serve as an important tool for global surveillance.

Staphylococcus aureus is a common infecting agent of many surgical sites. As a commensal organism to humans and rabbits, the infection process may occur due to native or exogenous S. aureus. We applied exogenous S. aureus ATCC 49230 once weekly to the surgical site of an osseointegrated pin in 20 New Zealand white rabbits. Clinical signs of infection resulted in euthanasia and at necropsy samples were collected from putatively infected sites. The predominant organism cultured was S. aureus. We observed various beta-hemolysis patterns of S. aureus on culture media and used pulsed field gel electrophoresis (PFGE) to determine whether there were distinct strains of S. aureus collected from various sites of the rabbits. On the basis of PFGE results, we found that the exogenous S. aureus ATCC 49230 was not the S. aureus cultured during necropsy, but that S. aureus native to the rabbits was in fact the infecting agent. We conclude that this rabbit model for S. aureus infection, which has not been described previously, may contribute to understanding the pathogenesis of S. aureus infections in future studies with simulated osseointegrated pin infections secondary to S. aureus., (Copyright 2007 Wiley Periodicals, Inc.)

Leptotrichia species typically colonize the oral cavity and genitourinary tract. We report the first two cases of endocarditis secondary to L. goodfellowii sp. nov. Both cases were identified using 16S rRNA gene sequencing. Review of the English literature revealed only two other cases of Leptotrichia sp. endocarditis.

Gordonia species are emerging pathogens that are often misidentified as Rhodococcus or Nocardia species but are reliably distinguished by 16S ribosomal RNA gene sequencing. We present a case series of 6 episodes of catheter-associated infection caused by Gordonia species in 5 patients seen at a tertiary care pediatric hospital and describe the management and outcomes of this infection in adults and children.