Use of Postexposure Prophylaxis After Occupational Exposure to Zaire ebolavirus.

From September 2014 to April 2015, 6 persons who had occupational exposures to Zaire ebolavirus in West Africa received investigational agent rVSV-ZEBOV or TKM-100802 for postexposure prophylaxis and were monitored in the United States. All patients experienced self-limited symptoms after postexposure prophylaxis; none developed Ebola virus disease.

Characteristics and Clinical Management of a Cluster of 3 Patients With Ebola Virus Disease, Including the First Domestically Acquired Cases in the United States.

BACKGROUND: More than 26,000 cases of Ebola virus disease (EVD) have been reported in western Africa, with high mortality. Several patients have been medically evacuated to hospitals in the United States and Europe. Detailed clinical data are limited on the clinical course and management of patients with EVD outside western Africa. OBJECTIVE: To describe the clinical characteristics and management of a cluster of patients with EVD, including the first cases of Ebola virus (EBOV) infection acquired in the United States. DESIGN: Retrospective clinical case series. SETTING: Three U.S. hospitals in September and October 2014. PATIENTS: First imported EVD case identified in the United States and 2 secondary EVD cases acquired in the United States in critical care nurses who cared for the index case patient. MEASUREMENTS: Clinical recovery, EBOV RNA level, resolution of Ebola viremia, survival with discharge from hospital, or death. RESULTS: The index patient had high EBOV RNA levels, developed respiratory and renal failure requiring critical care support, and died. Both patients with secondary EBOV infection had nonspecific signs and symptoms and developed moderate illness; EBOV RNA levels were moderate, and both patients recovered. LIMITATION: Both surviving patients received uncontrolled treatment with multiple investigational agents, including convalescent plasma, which limits generalizability of the results. CONCLUSION: Early diagnosis, prompt initiation of supportive medical care, and moderate clinical illness likely contributed to successful outcomes in both survivors. The inability to determine the potential benefit of investigational therapies and the effect of patient-specific factors that may have contributed to less severe illness highlight the need for controlled clinical studies of these interventions, especially in the setting of a high level of supportive medical care. PRIMARY FUNDING SOURCE: None.

Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species.

BACKGROUND: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. RESULTS: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. CONCLUSIONS: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions.

Emergency postexposure vaccination with vesicular stomatitis virus-vectored Ebola vaccine after needlestick.

IMPORTANCE: Safe and effective vaccines and drugs are needed for the prevention and treatment of Ebola virus disease, including following a potentially high-risk exposure such as a needlestick. OBJECTIVE: To assess response to postexposure vaccination in a health care worker who was exposed to the Ebola virus. DESIGN AND SETTING: Case report of a physician who experienced a needlestick while working in an Ebola treatment unit in Sierra Leone on September 26, 2014. Medical evacuation to the United States was rapidly initiated. Given the concern about potentially lethal Ebola virus disease, the patient was offered, and provided his consent for, postexposure vaccination with an experimental vaccine available through an emergency Investigational New Drug application. He was vaccinated on September 28, 2014. INTERVENTIONS: The vaccine used was VSVΔG-ZEBOV, a replicating, attenuated, recombinant vesicular stomatitis virus (serotype Indiana) whose surface glycoprotein gene was replaced by the Zaire Ebola virus glycoprotein gene. This vaccine has entered a clinical trial for the prevention of Ebola in West Africa. RESULTS: The vaccine was administered 43 hours after the needlestick occurred. Fever and moderate to severe symptoms developed 12 hours after vaccination and diminished over 3 to 4 days. The real-time reverse transcription polymerase chain reaction results were transiently positive for vesicular stomatitis virus nucleoprotein gene and Ebola virus glycoprotein gene (both included in the vaccine) but consistently negative for Ebola virus nucleoprotein gene (not in the vaccine). Early postvaccination cytokine secretion and T lymphocyte and plasmablast activation were detected. Subsequently, Ebola virus glycoprotein-specific antibodies and T cells became detectable, but antibodies against Ebola viral matrix protein 40 (not in the vaccine) were not detected. CONCLUSIONS AND RELEVANCE: It is unknown if VSVΔG-ZEBOV is safe or effective for postexposure vaccination in humans who have experienced a high-risk occupational exposure to the Ebola virus, such as a needlestick. In this patient, postexposure vaccination with VSVΔG-ZEBOV induced a self-limited febrile syndrome that was associated with transient detection of the recombinant vesicular stomatitis vaccine virus in blood. Strong innate and Ebola-specific adaptive immune responses were detected after vaccination. The clinical syndrome and laboratory evidence were consistent with vaccination response, and no evidence of Ebola virus infection was detected.

Whole genome single nucleotide polymorphism based phylogeny of Francisella tularensis and its application to the development of a strain typing assay.

BACKGROUND: A low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates. RESULTS: We have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2). CONCLUSION: Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.

Pathological findings and diagnostic implications of a rhesus macaque (Macacca mulatta) model of aerosol exposure to Burkholderia mallei (glanders).

Burkholderia mallei is a Gram-negative bacillus that causes a pneumonic disease known as glanders in equids and humans, and a lymphatic infection known as farcy, primarily in equids. With the potential to infect humans by the respiratory route, aerosol exposure can result in severe, occasionally fatal, pneumonia. Today, glanders infections in humans are rare, likely due to less frequent contact with infected equids than in the past. Acutely ill humans often have non-specific clinical signs and in order to diagnose cases, especially in scenarios of multiple cases in an unexpected setting, rapid diagnostics for B. mallei may be critical. The pathogenesis of acute glanders in the rhesus macaque (Macaca mulatta) was studied as an initial effort to improve diagnostic methods. In the study described here, the diagnostic techniques of PCR, culture and histopathology were compared. The results indicated that PCR may provide rapid, non-invasive diagnosis of glanders in some cases. As expected, PCR results were positive in lung tissue in 11/12 acutely infected rhesus macaques, but more importantly in terms of diagnostic algorithm development, PCR results were frequently positive in non-invasive samples such as broncho-alveolar lavage or nasal swabs (7/12) and occasionally in blood (3/12). However, conventional bacterial culture failed to recover bacteria in many of these samples. The study showed that the clinical presentation of aerosol-exposed rhesus macaques is similar to descriptions of human glanders and that PCR has potential for rapid diagnosis of outbreaks, if not individual cases.

Finished Genome Assembly of Yersinia pestis EV76D and KIM 10v.

Here, we sequenced the completed genome of Yersinia pestis EV76D and KIM 10v, two genomes used as references in assay development, to improved high-quality draft status.

Finished Genome Assembly of Warm Spring Isolate Francisella novicida DPG 3A-IS.

We sequenced the complete genome of Francisella novicida DPG 3A-IS to closed and finished status. This is a warm spring isolate recovered from Hobo Warm Spring (Utah, USA). The final assembly is available in NCBI under accession number CP012037.

Finished Genome Sequence of Bacillus cereus Strain 03BB87, a Clinical Isolate with B. anthracis Virulence Genes.

Bacillus cereus strain 03BB87, a blood culture isolate, originated in a 56-year-old male muller operator with a fatal case of pneumonia in 2003. Here we present the finished genome sequence of that pathogen, including a 5.46-Mb chromosome and two plasmids (209 and 52 Kb, respectively).

Management of a pet dog after exposure to a human patient with Ebola virus disease.

In October 2014, a health-care worker who had been part of the treatment team for the first laboratory-confirmed case of Ebola virus disease imported to the United States developed symptoms of Ebola virus disease. A presumptive positive reverse transcription PCR assay result for Ebola virus RNA in a blood sample from the worker was confirmed by the CDC, making this the first documented occurrence of domestic transmission of Ebola virus in the United States. The Texas Department of State Health Services commissioner issued a control order requiring disinfection and decontamination of the health-care worker's residence. This process was delayed until the patient's pet dog (which, having been exposed to a human with Ebola virus disease, potentially posed a public health risk) was removed from the residence. This report describes the movement, quarantine, care, testing, and release of the pet dog, highlighting the interdisciplinary, one-health approach and extensive collaboration and communication across local, county, state, and federal agencies involved in the response.

Bacteraemia in free-ranging Hawaiian green turtles Chelonia mydas with fibropapillomatosis.

Past studies of free-ranging green turtles Chelonia mydas with fibropapillomatosis (FP) in Hawaii have shown that animals become immunosuppressed with increasing severity of this disease. Additionally, preliminary clinical examination of moribund turtles with FP revealed that some animals were also bacteraemic. We tested the hypothesis that bacteraemia in sea turtles is associated with the severity of FP. We captured free-ranging green turtles from areas in Hawaii where FP is absent, and areas where FP has been endemic since the late 1950s. Each turtle was given an FP severity score ranging from 0 (no tumours) to 3 (severely affected). A fifth category included turtles that were stranded ashore and moribund with FP. We found that the percentage of turtles with bacteraemia increased with the severity of FP, and that the majority of bacteria cultured were Vibrio spp. Turtles with severe FP were more susceptible to bactaeremia, probably in part due to immunosuppression. The pattern of bacteraemia in relation to severity of disease strengthens the hypothesis that immunosuppression is a sequel to FP.

Fatal spirochetosis due to a relapsing fever-like Borrelia sp. in a northern spotted owl.

Acute septicemic spirochetosis was diagnosed in an adult male northern spotted owl (Strix occidentalis caurina) found dead in Kittitas County, Washington, USA. Gross necropsy findings included marked enlargement of the liver and spleen and serofibrinous deposits on the serous membranes lining the body cavities and the pericardial and perihepatic sacs. Microscopic observations included macrophage infiltration in the liver and spleen with mild thrombosis and multifocal necrosis, as well as hemorrhage and acute inflammation in the choroid plexus of the brain. No viruses or pathogenic bacteria were isolated from brain, liver, or spleen, and no parasites were found in blood smears or impression smears of the liver. Chlamydial culture attempts were unsuccessful and no chlamydial antibodies were detected in serum. In silver-stained microscopic sections and by transmission electron microscopy of liver, numerous long, thin, spiral-shaped bacteria were seen in the liver, spleen, cerebral ventricles, and within blood vessels in many organs. The organism was identified as a member of the Borrelia genus by sequence analysis of the PCR-amplified 16S rRNA gene. The most closely related species is B. hermsii, an agent of relapsing fever in humans in the western United States. This is the first report of a relapsing fever-related Borrelia in a wild bird.

Pathological findings and diagnostic implications of a rhesus macaque (Macaca mulatta) model of aerosol-exposure melioidosis (Burkholderia pseudomallei).

Aerosolized Burkholderia pseudomallei, the causative agent of melioidosis, can infect many species of mammals (including humans), causing rapid, severe pneumonia with high mortality. Diagnosis in humans is challenging, as few organisms can be detected in blood or other non-invasive samples. Although it cannot be said that the model is established, studies to date indicate that rhesus macaques may represent a good model of human melioidosis. This is supported by the results of this study. The early progression of meliodosis in the rhesus macaque was studied in an effort to better understand the disease and the application of rapid diagnostic methods. Results indicate that a PCR analysis of key diagnostic samples such as nasal swabs, throat swabs, tracheo bronchial lymph node aspirates and broncho-alveolar lavage may be a useful component of a rapid diagnostic algorithm in case of aerosol exposure.

Biolog phenotype microarrays.

Phenotype microarrays nicely complement traditional genomic, transcriptomic, and proteomic analysis by offering opportunities for researchers to ground microbial systems analysis and modeling in a broad yet quantitative assessment of the organism's physiological response to different metabolites and environments. Biolog phenotype assays achieve this by coupling tetrazolium dyes with minimally defined nutrients to measure the impact of hundreds of carbon, nitrogen, phosphorous, and sulfur sources on redox reactions that result from compound-induced effects on the electron transport chain. Over the years, we have used Biolog's reproducible and highly sensitive assays to distinguish closely related bacterial isolates, to understand their metabolic differences, and to model their metabolic behavior using flux balance analysis. This chapter describes Biolog phenotype microarray system components, reagents, and methods, particularly as they apply to bacterial identification, characterization, and metabolic analysis.

Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness.

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA Master(PLUS) HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Real-time PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs.

Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry.

Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.

Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including shiga toxin encoding phage stx2.

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

A rhesus macaque (Macaca mulatta) model of aerosol-exposure brucellosis (Brucella suis): pathology and diagnostic implications.

The US Centers for Disease Control and Prevention lists Brucella as a potential bioterrorism threat requiring enhanced diagnostic capacity and surveillance (http://emergency.cdc.gov/bioterrorism/). Successful treatment and management of patients after exposure to biological threat agents depends on accurate and timely diagnosis, but many biothreat agents present with similar, vague clinical signs--commonly referred to as 'flu-like illness'. Diagnosis of brucellosis is notoriously challenging, especially early in infection, and definitive diagnosis may require invasive methods, e.g. bone marrow biopsy. We studied the pathogenesis of Brucella suis aerosol infection in rhesus macaques in an effort to guide the diagnostic algorithm in case of possible intentional exposure of humans. Rhesus proved to be an excellent model for human brucellosis; the data showed that PCR DNA amplification testing of non-invasive diagnostic samples has the potential to definitively detect a point-source outbreak immediately and for several days after exposure.

A novel brain heart infusion broth supports the study of common Francisella tularensis serotypes.

Francisella tularensis Schu S4, LVS and U112 have become model organisms for the study of Francisella pathogenesis, and represent a cross section of the different F. tularensis subspecies. Both Schu S4 and LVS are fastidious organisms, requiring medium fortified with supplements and nutrients for enhanced growth. Chamberlains defined medium, Tryptone Soy Broth supplemented with cysteine (TSBc), and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 2% IsoVitaleX are typically used in the cultivation of these bacteria. In this report, we describe a simple brain heart infusion broth formulation that can be used to obtain superior growth characteristics in all of these model organisms, and can support bacterial growth from low inoculum. Surprisingly, CAMHB, which is favored in the literature for culturing Schu S4 and LVS, induced the worst growth characteristics of the four formulations studied. To expand on these observations, an additional seven strains of F. tularensis, representing types A.I, A.II, and B were selected from the Department of Defense United Culture Collection (UCC) and a comparative analysis of their growth characteristics performed in the four broth formulations. Results demonstrate differences in the growth characteristics of Francisella species that are significantly influenced by both strain type and the choice of growth medium. Though four of the five additional Type A strains displayed superior growth characteristics in Chamberlain's defined medium, growth characteristics of all three model organisms, as well the Type B strains, were enhanced by the new BHI-based broth formulation. We conclude that this medium represents the optimal choice for cultivation of the three model organisms used for Francisella research.

DNA-Based Rapid Methods for the Detection of Foodborne Pathogens.

The reports of foodborne illnesses are increasing and rapid, sensitive and reliable methods to detect and identify foodborne microorganisms are needed. This review encompasses a description of recent technologies upon which DNA detection methods are based. This review also provides current information on hybridization assays developed or under study for application in food microbiology. Literature pertaining to some of the most common foodborne microorganisms, including Salmonella spp., Shigella spp., Listeria spp., Escherichia coli , and Yersinia enterocolitica is emphasized.