Anthony P. Cardile

Human plasma enhances the expression of Staphylococcal microbial surface components recognizing adhesive matrix molecules promoting biofilm formation and increases antimicrobial tolerance In Vitro.

BackgroundMicrobial biofilms have been associated with the development of chronic human infections and represent a clinical challenge given their increased antimicrobial tolerance. Staphylococcus aureus is a major human pathogen causing a diverse range of diseases, of which biofilms are often involved. Staphylococcal attachment and the formation of biofilms have been shown to be facilitated by host factors that accumulate on surfaces. To better understand how host factors enhance staphylococcal biofilm formation, we evaluated the effect of whole human plasma on biofilm formation in clinical isolates of S. aureus and the expression of seven microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) known to be involved in biofilm formation by quantitative real-time PCR. We also evaluated whether plasma augmented changes in S. aureus biofilm morphology and antimicrobial resistance.ResultsExposure of clinical isolates of S. aureus to human plasma (10%) within media, and to a lesser extent when coated onto plates, significantly enhanced biofilm formation in all of the clinical isolates tested. Compared to biofilms grown under non-supplemented conditions, plasma-augmented biofilms displayed significant changes in both the biofilm phenotype and cell morphology as determined by confocal scanning laser microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Exposure of bacteria to plasma resulted in a significant fold-increase in MSCRAMM expression in both a time and isolate-dependent manner. Additionally, plasma-augmented biofilms displayed an increased tolerance to vancomycin compared to biofilms grown in non-supplemented media.ConclusionsCollectively, these studies support previous findings demonstrating a role for host factors in biofilm formation and provide further insight into how plasma, a preferred growth medium for staphylococcal biofilm formation enhances as well as augments other intrinsic properties of S. aureus biofilms. Consequently, these findings indicate that incorporation of host factors may be necessary to better replicate in vivo conditions and for the best utility of a clinical biofilm assay to evaluate the process of biofilm formation and treatments.

Biofilm Formation by Clinical Isolates and Its Relevance to Clinical Infections

Reports of biofilms have increased exponentially in the scientific literature over the past two decades, yet the vast majority of these are basic science investigations with limited clinical relevance. Biofilm studies involving clinical isolates are most often surveys of isolate collections, but suffer from lack of standardization in methodologies for producing and assessing biofilms. In contrast, more informative clinical studies correlating biofilm formation to patient data have infrequently been reported. In this chapter, biofilm surveys of clinical isolates of aerobic and anaerobic bacteria, mycobacteria, and Candida are reviewed, as well as those pertaining to the unique situation of cystic fibrosis. In addition, the influence of host components on in vitro biofilm formation, as well as published studies documenting the clinical impact of biofilms in human infections, are presented.

Use of Peptide Thrombopoietin Receptor Agonist Romiplostim (Nplate) in a Case of Primary HIV-Associated Thrombocytopenia

Thrombocytopenia is frequently encountered in HIV-infected patients, and the predominant cause is primary HIV-associated thrombocytopenia (PHAT). Standard treatment regimens include optimization of antiretroviral therapy, intravenous immunoglobulin, anti-D, and corticosteroids. Retreatment due to the inability to sustain remission or inferior responses is common, and investigation into the safety and efficacy of alternative therapies is warranted. We describe novel and effective treatment of PHAT with the peptide thrombopoietin receptor agonist romiplostim in a patient with a minimal response to conventional therapy.

Operation United Assistance: Infectious Disease Threats to Deployed Military Personnel

As part of the international response to control the recent Ebola outbreak in West Africa, the Department of Defense has deployed military personnel to train Liberians to manage the disease and build treatment units and a hospital for health care volunteers. These steps have assisted in providing a robust medical system and augment Ebola diagnostic capability within the affected nations. In order to prepare for the deployment of U.S. military personnel, the infectious disease risks of the regions must be determined. This evaluation allows for the establishment of appropriate force health protection posture for personnel while deployed, as well as management plans for illnesses presenting after redeployment. Our objective was to detail the epidemiology and infectious disease risks for military personnel in West Africa, particularly for Liberia, along with lessons learned from prior deployments.

Activity of Norspermidine on Bacterial Biofilms of Multidrug-Resistant Clinical Isolates Associated with Persistent Extremity Wound Infections

Biofilm formation is a major virulence factor for numerous pathogenic bacteria and is cited as a central event in the pathogenesis of chronic human infections, which is in large part due to excessive extracellular matrix secretion and metabolic changes that occur within the biofilm rendering them highly tolerant to antimicrobial treatments. Polyamines, including norspermidine, play central roles in bacterial biofilm development, but have also recently been shown to inhibit biofilm formation in select strains of various pathogenic bacteria. The aim of this study was to evaluate in vitro the biofilm dispersive and inhibitory activities of norspermidine against multidrug-resistant clinical isolates of Acinetobacter baumannii(n = 4), Klebsiella pneumoniae (n = 3), Pseudomonas aeruginosa (n = 5) and Staphylococcus aureus (n = 4) associated with chronic extremity wound infections using the semi-quantitative 96-well plate method and confocal laser microscopy. In addition to the antibiofilm activity, biocompatibility of norspermidine was also evaluated by measuring toxicity in vitro to human cell lines and whole porcine tissue explants using MTT viability assay and histological analysis. Norspermidine (5-20 mM) had variable dispersive and inhibitory activity on biofilms which was dependent on both the strain and species. Of the clinical bacterial species evaluated herein, A. baumannii isolates were the most sensitive to the effect of norspermidine, which was in part due to the inhibitory effects of norspermidine on bacterial motility and expression of genes involved in the production of homoserine lactones and quorum sensing molecules both essential for biofilm formation. Importantly, exposure of cell lines and whole tissues to norspermidine for prolonged periods of time (≥24 h) was observed to reduce viability and alter tissue histology in a time and concentration dependent manner, with 20 mM exposure having the greatest negative effects on both tissues and individual cell lines. Collectively our findings demonstrate that, similar to other polyamines, norspermidine displays both inhibitory and dispersive activities on biofilms of clinical multidrug-resistant bacterial isolates, in particular for strains of A. baumannii. Additionally our findings suggest that direct application may be considered on tissues, albeit for limited exposure times.

Disease and Injury Epidemiology Among Military Personnel Deployed to West Africa During the 2014–2015 Ebola Outbreak

Abstract Background In November 2014, in response to the Ebola outbreak, the US government sent ~3000 military personnel to West Africa to construct Ebola treatment units, train health care workers and enhance laboratory testing capabilities. We describe the observed disease and injury epidemiology to identify trends, optimize preventive measures, and mitigate illness during future military operations. Methods Disease and injury visits were monitored daily from December 1, 2014 to February 25, 2015 and categorized by illness and injury type. The personnel were deployed to eight locations in Liberia and Senegal. Diagnostic testing was limited to routine laboratory testing, rapid malaria tests, Ebola PCR, but no rapid tests for other pathogens. Surveillance data were analyzed for disease trend associations (Mann–Whitney U-test, SPSS software). Results The number of people deployed ranged from 1057 to 2983. There were 2,493 visits (>50% in the first month), with a decline in number of patients, illnesses, injuries, and rates of disease and injury during the surveillance period (P < 0.001). Upper respiratory, gastrointestinal (GI) and dermatologic complaints accounted for the largest number of non-injury visits (373, 325, and 306, respectively) and declined over time (P < 0.001). Fifty-one percent and 73% of visits for injuries and GI complaints, respectively, were seen in the first month. Operational stress, musculoskeletal, and soft tissue injuries decreased from the first to third month (P < 0.001). Enterovirus meningitis, norovirus gastroenteritis, and Chikungunya were diagnosed in patients after medical evacuation or redeployment. No cases of malaria were identified in deployed personnel while in West Africa. Conclusion The disease and injury patterns seen in this humanitarian mission are consistent with surveillance data from combat operations in Iraq and Afghanistan, where GI, respiratory and dermatologic complaints accounted for most of the illnesses and rates declined over time. The high prevalence of upper respiratory and GI illness supports a need for rapid diagnostic platforms in the deployed setting to promptly identify threats to individuals and the mission, minimize communicable disease risks and avoid unnecessary medical evacuations. Disclosures All authors: No reported disclosures.

Deployment of the 1st Area Medical Laboratory in a Split-Based Configuration During the Largest Ebola Outbreak in History

BACKGROUND The U.S. Army 1st Area Medical Laboratory (1st AML) is currently the only deployable medical CBRNE (Chemical, Biological, Radiological, Nuclear, and Explosives) laboratory in the Armys Forces Command. In support of the United States Agency for International Development Ebola response, the U.S. military initiated Operation United Assistance (OUA), and deployed approximately 2,500 service members to support the Government of Liberias Ebola control efforts. Due to its unique molecular diagnostic and expeditionary capabilities, the 1st AML was ordered to deploy in October of 2014 in support of OUA via establishment of Ebola testing laboratories. To meet the unique mission requirements of OUA, the unit was re-organized to operate in a split-based configuration and sustain four separate Ebola testing laboratories. METHODS This article is a review of the 1st AMLs OUA participation in a split-based configuration. Topics highlighted include pre-deployment planning/training, operational/logistical considerations in fielding/withdrawing laboratories, laboratory testing results, disease and non-battle injuries, and lessons learned. FINDINGS Fielding the 1st AML in a split-based configuration required careful pre-deployment planning, additional training, optimal use of personnel, and the acquisition of additional laboratory equipment. Challenges in establishing and sustaining remote laboratories in Liberia included: difficulties in transportation of equipment due to poor road infrastructure, heavy equipment unloading, and equipment damage during transit. Between November 26, 2014 and February 18, 2015 the four 1st AML labs successfully tested blood samples from patients and oral swabs collected by burial teams in rural Liberia. The most significant equipment malfunction during laboratory operations was generators powering the labs, with the same problem impacting headquarters. Generator failures delayed laboratory operations/result reporting, and put temperature sensitive reagents at risk. None of the 22 1st AML soldiers (at remote labs or headquarters) had an Ebola exposure, none were infected with malaria or other tropical diseases, and none required evacuation from the time deployed to remote sites. The primary medical condition encountered was acute gastroenteritis, and within the first week of arrival to Liberia, 19 (86%) soldiers were affected. DISCUSSION/IMPACT/RECOMMENDATIONS With proper planning and training, the 1st AML can successfully conduct split-based operations in an outbreak setting, and this capability can be utilized in future operations. The performance of the 1st AML during the current Ebola outbreak highlights the value of this asset, and the need to continue its evolution to support U.S. military operations.