Matthew P. Muller

Complete Nucleotide Sequence of a 92-Kilobase Plasmid Harboring the CTX-M-15 Extended-Spectrum Beta-Lactamase Involved in an Outbreak in Long-Term-Care Facilities in Toronto, Canada

ABSTRACT A major outbreak involving an Escherichia coli strain that was resistant to expanded-spectrum cephalosporins occurred in Toronto and surrounding regions in 2000 to 2002. We report the complete sequence of a plasmid, pC15-1a, that was found associated with the outbreak strain. Plasmid pC15-1a is a circular molecule of 92,353 bp consisting of two distinct regions. The first is a 64-kb region that is essentially homologous to the non-R-determinant region of plasmid R100 except for several point mutations, a few small insertions and deletions, and the absence of Tn10. The second is a 28.4-kb multidrug resistance region (MDR) that has replaced the R-determinant region of the R100 progenitor and consists mostly of transposons or partial transposons and five copies of the insertion element IS26. All drug resistance genes found in pC15-1a, including the beta-lactamase genes blaCTX-M-15, blaOXA-1, and blaTEM-1, the tetracycline resistance gene tetA, and aminoglycoside resistance genes aac(6′)-Ib and aac(3)-II, are located in the MDR. The blaCTX-M-15 gene was found downstream of ISEcp1as part of a transposition unit, as determined from the surrounding sequence. Examination of the plasmids from CTX-M-15-harboring strains isolated from hospitals across Canada showed that pC15-1a was found in several strains isolated from a site in western Canada. Comparison of pC15-1a and pCTX15, found in an E. coli strain isolated in India in 1999, revealed that the plasmids had several features in common, including an R100 backbone and several of the resistance genes, including blaCTX-M-15, blaTEM-1, blaOXA-1, tetA, and aac(6′)-Ib.

Development of a triage protocol for critical care during an influenza pandemic

Background: The recent outbreaks of avian influenza (H5N1) have placed a renewed emphasis on preparing for an influenza pandemic in humans. Of particular concern in this planning is the allocation of resources, such as ventilators and antiviral medications, which will likely become scarce during a pandemic. Methods: We applied a collaborative process using best evidence, expert panels, stakeholder consultations and ethical principles to develop a triage protocol for prioritizing access to critical care resources, including mechanical ventilation, during a pandemic. Results: The triage protocol uses the Sequential Organ Failure Assessment score and has 4 main components: inclusion criteria, exclusion criteria, minimum qualifications for survival and a prioritization tool. Interpretation: This protocol is intended to provide guidance for making triage decisions during the initial days to weeks of an influenza pandemic if the critical care system becomes overwhelmed. Although we designed this protocol for use during an influenza pandemic, the triage protocol would apply to patients both with and without influenza, since all patients must share a single pool of critical care resources.

Interferon-Mediated Immunopathological Events Are Associated with Atypical Innate and Adaptive Immune Responses in Patients with Severe Acute Respiratory Syndrome

ABSTRACT It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-α, IFN-γ, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.

Severe Acute Respiratory Syndrome

Abstract The first cases of severe acute respiratory syndrome (SARS) occurred in China in November 2002. The agent causing this illness has been identified as a novel coronavirus, SARS-coronavirus. Since its introduction <1 year ago, this virus has infected 8098 people in 26 countries, killing 774 of them. We present an overview of the epidemiology, clinical presentation, diagnosis, and treatment of SARS based on the current state of knowledge derived from published studies and our own personal experience.

The Impact of Infection on Population Health: Results of the Ontario Burden of Infectious Diseases Study

Background Evidence-based priority setting is increasingly important for rationally distributing scarce health resources and for guiding future health research. We sought to quantify the contribution of a wide range of infectious diseases to the overall infectious disease burden in a high-income setting. Methodology/Principal Findings We used health-adjusted life years (HALYs), a composite measure comprising premature mortality and reduced functioning due to disease, to estimate the burden of 51 infectious diseases and associated syndromes in Ontario using 2005–2007 data. Deaths were estimated from vital statistics data and disease incidence was estimated from reportable disease, healthcare utilization, and cancer registry data, supplemented by local modeling studies and national and international epidemiologic studies. The 51 infectious agents and associated syndromes accounted for 729 lost HALYs, 44.2 deaths, and 58,987 incident cases per 100,000 population annually. The most burdensome infectious agents were: hepatitis C virus, Streptococcus pneumoniae, Escherichia coli, human papillomavirus, hepatitis B virus, human immunodeficiency virus, Staphylococcus aureus, influenza virus, Clostridium difficile, and rhinovirus. The top five, ten, and 20 pathogens accounted for 46%, 67%, and 75% of the total infectious disease burden, respectively. Marked sex-specific differences in disease burden were observed for some pathogens. The main limitations of this study were the exclusion of certain infectious diseases due to data availability issues, not considering the impact of co-infections and co-morbidity, and the inability to assess the burden of milder infections that do not result in healthcare utilization. Conclusions/Significance Infectious diseases continue to cause a substantial health burden in high-income settings such as Ontario. Most of this burden is attributable to a relatively small number of infectious agents, for which many effective interventions have been previously identified. Therefore, these findings should be used to guide public health policy, planning, and research.

Human immunopathogenesis of severe acute respiratory syndrome (SARS)

Abstract Progressive immune-associated injury is a hallmark of severe acute respiratory syndrome (SARS). Viral evasion of innate immunity, hypercytokinemia and systemic immunopathology in the SARS coronavirus (SARS CoV) infected host have been suggested as possible mechanisms for the cause of severe pathology and morbidity in SARS patients. The molecular and cellular basis for how SARS CoV impacts the host immune system resulting in severe SARS, however, has not been elucidated. The variable clinical course of SARS may be the result of complex programs of host responses against the infectious agent. Therefore, the systematic analysis of innate and adaptive immune responses to SARS CoV is imperative in building as complete an immunological model as possible of host immunity and inflammatory responses during illness. Here we review recent advances in SARS immunopathogenesis research and present a summary of our findings regarding host responses in SARS patients. We contend that dysregulated type I and II interferon (IFN) responses during SARS may culminate in a failure of the switch from hyper-innate immunity to protective adaptive immune responses in the human host.

Astrocytoma Adhesion to Extracellular Matrix: Functional Significance of Integrin and Focal Adhesion Kinase Expression

Evidence is accumulating implicating a role for integrins in the pathogenesis of cancer, a disease in which alterations in cellular growth, differentiation, and adhesive characteristics are defining features. In the present report we studied a panel of 8 human astrocytoma cell lines for their expression of integrin subunits by RT-PCR, and of integrin heterodimers by immunoprecipitation analyses. The functionality of integrin heterodimers was assessed using cell attachment assays to plastic or single matrix substrates. Downstream effects of integrin activation were studied by western blot analyses of FAK expression in human astrocytoma cell lines growing on plastic and on a fibronectin matrix, and in 13 primary human brain tumor specimens of varying histopathological grade. Furthermore, we studied tyrosine phosphorylation of FAK in astrocytoma cells growing on plastic versus fibronectin. Finally, we analyzed the effects of intermediate filament gene transfer on FAK phosphorylation in SF-126 astrocytoma cells. Our data show that astrocytoma cell lines express various integrin subunits by RT-PCR, and heterodimers by immunoprecipitation analyses. The beta1 and alphav integrin subunits were expressed by all astrocytoma cell lines. The alpha3 subunit was expressed by all cell lines except SF-188. By immunoprecipitation, the fibronectin receptor (alpha5beta1 integrin heterodimer) and the vitronectin receptor (alphavbeta3) were identified in several cell lines. Astrocytoma cell attachment studies to human matrix proteins suggested that these integrin heterodimers were functional. Using confocal laser microscopy, we showed that FAK was colocalized to actin stress fibers at sites of focal adhesion complexes. By western blot, FAK was variably but quite ubiquitously expressed in human astrocytoma cell lines, and in several primary human astrocytoma specimens. When U373 and U87 MG astrocytoma cells bind to a fibronectin matrix, FAK is phosphorylated. GFAP-transfected SF-126 human astrocytoma cells were shown to overexpress the phosphorylated form of FAK only when these cells were placed on a fibronectin matrix. This result is of interest because it suggests that manipulations of the astrocytoma cytoskeleton per se can bring about potential signaling changes that channel through integrins and focal adhesion sites leading to activation of key kinases such as FAK.

Invasive Pneumococcal Disease Among Immunocompromised Persons: Implications for Vaccination Programs

BACKGROUND In 2012/2013, a single dose of 13-valent pneumococcal conjugate vaccine (PCV13) was recommended for immunocompromised adults in the United States and Canada. To assess the potential benefits of this recommendation, we assessed the serotype-specific burden of invasive pneumococcal disease (IPD) among immunocompromised individuals. METHODS From 1995 to 2012, population-based surveillance for IPD was conducted in Metropolitan Toronto and Peel Region, Canada. Disease incidence and case fatality were measured in immunocompromised populations over time, and the contribution of different serotypes determined. RESULTS Overall, 2115/7604 (28%) episodes of IPD occurred in immunocompromised persons. IPD incidence was 12-fold higher (95% confidence interval [CI], 8.7-15) in immunocompromised compared to immunocompetent persons; the case fatality rate was elevated in both younger (odds ratio [OR] 1.8) and older (OR 1.3) adults. Use of immunosuppressive medications was associated with a 2.1-2.7 fold increase in the risk of IPD. Five years after PPV23 program implementation, IPD incidence had declined significantly in immunocompromised adults (IRR 0.57, 95% CI, .40-.82). Ten years after pediatric PCV7 authorization, IPD due to PCV7 serotypes had decreased by 90% (95% CI, 77%-96%) in immunocompromised persons of all ages. In 2011/2012, 37% of isolates causing IPD in immunocompromised persons were PCV13 serotypes and 27% were PPV23/not PCV13 serotypes. CONCLUSIONS Immunocompromised individuals comprised 28% of IPD. Both PPV23 and herd immunity from pediatric PCV7 were associated with reductions in IPD in immunocompromised populations. PCV13 vaccination of immunocompromised adults may substantially reduce the residual burden until herd immunity from pediatric PCV13 is fully established.

The efficacy of infection control interventions in reducing the incidence of extended-spectrum β-lactamase-producing Enterobacteriaceae in the nonoutbreak setting: A systematic review

We conducted a systematic review to examine the efficacy of infection control interventions for the control of ESBL-producing Enterobacteriaceae in hospitals in the non-outbreak setting. Although 4 uncontrolled, retrospective studies were included in the review, no well designed prospective studies capable of informing infection control practice were identified, underscoring the urgent need for research in this area.

Alternative methods of estimating an incubation distribution: examples from severe acute respiratory syndrome.

Background: Accurate and precise estimates of the incubation distribution of novel, emerging infectious diseases are vital to inform public health policy and to parameterize mathematical models. Methods: We discuss and compare different methods of estimating the incubation distribution allowing for interval censoring of exposures, using data from the severe acute respiratory syndrome (SARS) epidemic in 2003 as an example. Results: Combining data on unselected samples of 149 and 168 patients with defined exposure intervals from Toronto and Hong Kong, respectively, we estimated the mean and variance of the incubation period to be 5.1 day and 18.3 days and the 95th percentile to be 12.9 days. We conducted multiple linear regression on the log incubation times and found that incubation was significantly longer in Toronto than in Hong Kong and in older compared with younger patients, while it was significantly shorter in healthcare workers than in other patients. Conclusions: Our findings suggest subtle but important heterogeneities in the incubation period of SARS among different strata of patients. Robust estimation of the incubation period should be independently carried out in different settings and subgroups for novel human pathogens using valid statistical methods.