A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa.

CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RNA binding by CsrA/RsmA has been studied in detail for some genes, it is anticipated that there are numerous additional, as yet undiscovered, direct targets that mediate its global regulation. To assist in the discovery of these targets, we propose a sequence-based approach to predict genes directly regulated by these regulators. In this work, we develop a computer code (CSRA_TARGET) implementing this approach, which leads to predictions for several novel targets in Escherichia coli and Pseudomonas aeruginosa. The predicted targets in other bacteria, specifically Salmonella enterica serovar Typhimurium, Pectobacterium carotovorum and Legionella pneumophila, also include global regulators that control virulence in these pathogens, unraveling intricate indirect regulatory roles for CsrA/RsmA. We have experimentally validated four predicted RsmA targets in P. aeruginosa. The sequence-based approach developed in this work can thus lead to several testable predictions for direct targets of CsrA homologs, thereby complementing and accelerating efforts to unravel global regulation by this important family of proteins.

Utility of real-time PCR for detection of Exserohilum rostratum in body and tissue fluids during the multistate outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range and E. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection of E. rostratum in body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive for E. rostratum to 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-d-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data.

Update on Multistate Outbreak of Fungal Infections Associated with Contaminated Methylprednisolone Injections, 2012-2014.

During September 2012, CDC, in collaboration with state and local health departments and the Food and Drug Administration (FDA), investigated a multistate outbreak of fungal meningitis and other infections caused by injections of contaminated methylprednisolone acetate solution (MPA). After this unprecedented outbreak, scientists in the CDC Mycotic Diseases Branch, along with infectious diseases specialists who cared for patients from the outbreak, clinical experts, and public health officials from affected states, have continued to monitor the recovery of affected patients. A long-term follow-up study involving these patients was initiated and is being conducted by the Mycoses Study Group Education and Research Consortium (MSGERC). This update summarizes subsequent information about the current state of the outbreak.

Utility of (1-3)-ß-D-glucan testing for diagnostics and monitoring response to treatment during the multistate outbreak of fungal meningitis and other infections.

BACKGROUND: The 2012 outbreak of fungal meningitis associated with contaminated methylprednisolone produced by a compounding pharmacy has resulted in >750 infections. An important question facing patients and clinicians is the duration of antifungal therapy. We evaluated (1-3)-ß-d-glucan (BDG) as a marker for monitoring response to treatment. METHODS: We determined sensitivity and specificity of BDG testing using the Fungitell assay, by testing 41 cerebrospinal fluid (CSF) specimens from confirmed cases of fungal meningitis and 66 negative control CSF specimens. We also assessed whether BDG levels correlate with clinical status by using incident samples from 108 case patients with meningitis and 20 patients with serially collected CSF. RESULTS: A cutoff value of 138 pg/mL provided 100% sensitivity and 98% specificity for diagnosis of fungal meningitis in this outbreak. Patients with serially collected CSF were divided into 2 groups: those in whom BDG levels declined with treatment and those in whom BDG remained elevated. Whereas most patients with a decline in CSF BDG had clinical improvement, all 3 patients with continually elevated BDG had poor clinical outcomes (stroke, meningitis relapse, or development of new disease). CONCLUSIONS: Our data suggest that measuring BDG in CSF is a highly sensitive test for diagnosis of fungal meningitis in this outbreak. Analysis of BDG levels in serially collected CSF demonstrated that BDG may correlate with clinical response. Routine measurement of BDG in CSF may provide useful adjunctive data for the clinical management of patients with outbreak-associated meningitis.

Early clinical observations in prospectively followed patients with fungal meningitis related to contaminated epidural steroid injections.

UNLABELLED: Chinese translation BACKGROUND: Administration of epidural steroid injections (ESIs) with contaminated methylprednisolone resulted in an outbreak of fungal meningitis in many locations in the United States. OBJECTIVE: To characterize early clinical findings and initial response to treatment. DESIGN: Case series with standardized observation studied from 4 October to 31 October 2012. SETTING: An 800-bed hospital in Virginia. PATIENTS: 172 patients who presented to the hospital with exposure to contaminated ESI. INTERVENTION: Standardized approach to screening, case definition, treatment, and data collection. MEASUREMENTS: Clinical findings, cerebrospinal fluid (CSF) values, magnetic resonance imaging (MRI), serum and CSF voriconazole concentrations, and clinician assessment of response to therapy. RESULTS: Of 172 patients presenting to the hospital who had had ESI, 131 had lumbar puncture because of symptoms or signs consistent with central nervous system disease. Twenty-five (19%) had neutrophilic meningitis. All were started on voriconazole therapy alone. Three patients developed stroke during treatment. Ten patients had arachnoiditis, another had an epidural abscess, and 9 had urine retention. Fifteen continued to receive voriconazole, and 10 were switched to amphotericin B. Cerebrospinal fluid leukocyte counts began to decrease by day 13 of treatment. Findings on MRI included ventriculitis, leptomeningeal enhancement, infarction, hemorrhage, and arachnoiditis. Serum voriconazole levels varied, and CSF concentrations of voriconazole were approximately 50% those of serum. Exserohilum rostratum and Cladosporium species have been cultured. LIMITATIONS: This is an observational study of an evolving outbreak. Not all exposed patients presented for evaluation. Follow-up is too short to determine final outcomes. CONCLUSION: Meningitis after receipt of contaminated ESI has been diagnosed in many exposed patients presenting to 1 hospital. Most patients have improved on receipt of empirical voriconazole therapy. The full natural history and long-term sequelae of this infection are currently unknown. PRIMARY FUNDING SOURCE: None.

Complete Genome Sequence of Pseudomonas aeruginosa CMC-097, Isolated from a Ventilator-Associated Pneumonia Patient, Containing a Novel Carbapenem Resistance Class 1 Integron.

We report the complete genome of a clinical strain of Pseudomonas aeruginosa CMC-097, which was isolated from a ventilator-associated pneumonia patient with a chronic infection. Illumina sequence reads were assembled using Geneious to yield a 7,044,064-bp circular chromosome containing a carbapenem resistance integron, In2020.

Complete Genome Sequence of Pseudomonas aeruginosa CMC-115, a Clinical Strain from an Acute Ventilator-Associated Pneumonia Patient.

We report the complete genome of clinical strain Pseudomonas aeruginosa CMC-115, which was isolated from an acute ventilator-associated pneumonia patient. Illumina sequencing reads were assembled using Geneious to yield a 6,375,262-bp circular chromosome that exhibited an unusual ferrichrome receptor in the pyoverdine synthesis locus and the absence of type 3 secretion system genes.

Long-term Outcomes of Patients With Fungal Infections Associated With Contaminated Methylprednisolone Injections.

BACKGROUND: The largest health care-associated infection outbreak in the United States occurred during 2012-2013. Following injection of contaminated methylprednisolone, 753 patients developed infection with a dematiaceous mold, Exserohilum rostratum. The long-term outcomes of these infections have not been described. METHODS: This retrospective cohort study of 440 of a total of 753 patients with proven or probable Exserohilum infection evaluated clinical and radiographic findings, antifungal therapy and associated adverse effects, and outcomes at 6 weeks, 3, 6, 9, and 12 months after diagnosis. Patients were grouped into 4 disease categories: meningitis with/without stroke, spinal or paraspinal infections, meningitis/stroke plus spinal/paraspinal infections, and osteoarticular infections. RESULTS: Among the 440 patients, 223 (51%) had spinal/paraspinal infection, 82 (19%) meningitis/stroke, 123 (28%) both, and 12 (3%) osteoarticular infection. Of 82 patients with meningitis/stroke, 18 (22%) died; among those surviving, 87% were cured at 12 months. Only 7 (3%) of 223 patients with spinal/paraspinal infection died, but at 12 months, 68% had persistent or worsening pain and only 47% were cured. For the 123 patients with both meningitis/stroke and spinal/paraspinal infection, 10 (8%) died, pain persisted in 72%, and 52% were cured at 12 months. Only 37% of those with osteoarticular infection were cured at 12 months. Adverse events from antifungal therapy were noted at 6 weeks in 71% of patients on voriconazole and 81% on amphotericin B. CONCLUSIONS: Fungal infections related to contaminated methylprednisolone injections culminated in death in 8% of patients. Persistent pain and disability were seen at 12 months in most patients with spinal/paraspinal infections.

Epidemiological Effectiveness and Cost of a Fungal Meningitis Outbreak Response in New River Valley, Virginia: Local Health Department and Clinical Perspectives.

OBJECTIVE: We evaluated the effectiveness and cost of a fungal meningitis outbreak response in the New River Valley of Virginia during 2012-2013 from the perspective of the local public health department and clinical facilities. The fungal meningitis outbreak affected 23 states in the United States with 751 cases and 64 deaths in 20 states; there were 56 cases and 5 deaths in Virginia. METHODS: We conducted a partial economic evaluation of the fungal meningitis outbreak response in New River Valley. We collected costs associated with the local health department and clinical facilities in the outbreak response and estimated the epidemiological effectiveness by using disability-adjusted life years (DALYs) averted. RESULTS: We estimated the epidemiological effectiveness of this outbreak response to be 153 DALYs averted among the patients, and the costs incurred by the local health department and clinical facilities to be $30,413 and $39,580, respectively. CONCLUSIONS: We estimated the incremental cost-effectiveness ratio of $198 per DALY averted and $258 per DALY averted from the local health department and clinical perspectives, respectively, thereby assisting in impact evaluation of the outbreak response by the local health department and clinical facilities. (Disaster Med Public Health Preparedness. 2018;12:38-46).

Tn2008-driven carbapenem resistance in Acinetobacter baumannii isolates from a period of increased incidence of infections in a Southwest Virginia hospital (USA).

OBJECTIVES: The objectives of this study were (i) to determine the genetic basis for carbapenem resistance in multidrug-resistant (MDR) Acinetobacter baumannii strains isolated from patients affected by a sudden increase in the incidence of infections by such organisms in a tertiary care hospital in Virginia, USA, in 2009-2010 and (ii) to examine whether such strains are commonly encountered in the hospital setting. METHODS: The whole genomes of one outbreak strain as well as one carbapenem-resistant and one carbapenem-sensitive strain from sporadic infections in 2010-2012 were sequenced and analysed. Then, 5 outbreak isolates and 57 sporadic isolates (of which 39 were carbapenem-resistant) were screened by PCR for relevant DNA elements identified in the genomics investigation. RESULTS: All three strains for which whole-genome sequences were obtained carried resistance genes linked to MDR phenotypes and a ca. 111-kbp plasmid (pCMCVTAb1) without drug resistance genes. Of these, the two carbapenem-resistant strains possessed a ca. 74-kbp plasmid (pCMCVTAb2) carrying a Tn2008 transposon that provides high-level carbapenem resistance. PCR analysis showed that all of the outbreak isolates carried both plasmids and Tn2008, and of the sporadic isolates 88% carried pCMCVTAb1, 25% contained pCMCVTAb2 and 50% of the latter group carried Tn2008. CONCLUSIONS: Carbapenem resistance in outbreak strains and 12% of sporadic isolates was due to the pCMCVTAb2-borne Tn2008. This is the first report of a Tn2008-driven outbreak of carbapenem-resistant A. baumannii infections in the Commonwealth of Virginia, which followed similar cases in Pennsylvania and Ohio.

Clinical Response, Outbreak Investigation, and Epidemiology of the Fungal Meningitis Epidemic in the United States: Systematic Review.

We conducted a systematic review of the 2012-2013 multistate fungal meningitis epidemic in the United States from the perspectives of clinical response, outbreak investigation, and epidemiology. Articles focused on clinical response, outbreak investigation, and epidemiology were included, whereas articles focused on compounding pharmacies, legislation and litigation, diagnostics, microbiology, and pathogenesis were excluded. We reviewed 19 articles by use of the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) framework. The source of the fungal meningitis outbreak was traced to the New England Compounding Center in Massachusetts, where injectable methylprednisolone acetate products were contaminated with the predominant pathogen, Exserohilum rostratum. As of October 23, 2013, the final case count stood at 751 patients and 64 deaths, and no additional cases are anticipated. The multisectoral public health response to the fungal meningitis epidemic from the hospitals, clinics, pharmacies, and the public health system at the local, state, and federal levels led to an efficient epidemiological investigation to trace the outbreak source and rapid implementation of multiple response plans. This systematic review reaffirms the effective execution of a multisectoral public health response and efficient delivery of the core functions of public health assessment, policy development, and service assurances to improve population health.

Economic Evaluation of Fungal Meningitis Outbreak Response in New River Valley: Local Health Department Perspective.

BACKGROUND: The multi-state fungal meningitis outbreak started in September 2012 in Tennessee. The cause of the outbreak was injection of contaminated lots of methylprednisolone acetate used in epidural spinal injections. Roanoke and New River Valley were the epicenter of this outbreak in Virginia, with two clinical centers having administered the contaminated injections to their patients. New River Health District, in coordination with hospitals, and state and federal agencies, deployed its resources to control the local impact of the outbreak. PURPOSE: The objective of this study was to conduct an economic evaluation of the fungal meningitis outbreak response in New River Valley of Virginia, from the local public health department perspective. METHODS: The health department conducted the outbreak investigation from October 2012 until March 2013 to ascertain that all possible cases were identified and treated. Data were collected on the costs associated with the local health department in the outbreak response, and the epidemiologic effectiveness estimated, using the metric of disability adjusted life years (DALYs). RESULTS: The cost incurred by the local health department was estimated to be $30,493; the epidemiologic effectiveness was estimated to be 138 DALYs averted among the patients, for an incremental cost-effectiveness ratio of $221 per DALY averted. IMPLICATIONS: The incremental cost effectiveness ratio of the fungal meningitis outbreak response in New River Valley assists the local health department to analyze the costs and epidemiologic effectiveness of the outbreak response.