Detection of Ehrlichia spp. and Anaplasma phagocytophilum in whole blood specimens using a duplex real-time PCR assay on the ARIES instrument.

Cases of tick-borne diseases are increasing in the United States, and new tick-borne pathogen species causing human illness are being discovered. The specific etiology is generally difficult to diagnose based on clinical signs and symptoms alone, because of their generalized nature and often lack of a known tick bite. For some infections, such as Lyme disease and spotted fever group rickettsioses, serology remains the most appropriate laboratory diagnostic tool, but for others such as anaplasmosis, ehrlichiosis, and babesiosis, direct detection in the blood is preferred for rapid diagnosis.

Role of Atypical Pathogens in the Etiology of Community-Acquired Pneumonia.

Atypical pneumonia has been described for over 100 years, but some of the pathogens attributed to it have been identified only in the past decades. The most common pathogens are , , and . The epidemiology and pathophysiology of these three pathogens have been studied since their discovery, and are reviewed herein to provide better insight when evaluating these patients, which hopefully translates into improved care.

Mixed cryoglobulinemia and secondary membranoproliferative glomerulonephritis associated with ehrlichiosis.

Ehrlichiosis is a tick-borne disease with diverse clinical presentations, ranging in severity from a flu-like illness with fever and myalgias to a serious systemic disease with multisystem organ failure. Nephrotic syndrome has been reported previously in two cases of human ehrlichiosis. A kidney biopsy revealed minimal change disease in one of those patients.

Phenotypic differences in virulence and immune response in closely related clinical isolates of influenza A 2009 H1N1 pandemic viruses in mice.

To capture the possible genotypic and phenotypic differences of the 2009 influenza A virus H1N1 pandemic (H1N1pdm) strains circulating in adult hospitalized patients, we isolated and sequenced nine H1N1pdm viruses from patients hospitalized during 2009-2010 with severe influenza pneumonia in Kentucky. Each viral isolate was characterized in mice along with two additional H1N1 pandemic strains and one seasonal strain to assess replication and virulence. All isolates showed similar levels of replication in nasal turbinates and lung, but varied in their ability to cause morbidity.

Antigens of persistent Chlamydia pneumoniae within coronary atheroma from patients undergoing heart transplantation.

Aims: In order for Chlamydia pneumoniae to play a causative role in chronic human disease, it would need to persist within infected tissue for extended periods of time. Current theory suggests that C pneumoniae may persist at the site of infection via an alternative replicative form, known as an aberrant body.

Methods: A panel of C pneumoniae-specific antibodies upregulated by the aberrant body was used to probe tissue specimens from the coronary atheroma from 13 explanted hearts to identify patterns of reactivity in these tissues, as well as to determine the presence and prevalence of C pneumoniae aberrant bodies.

Immunoproteomic identification and serological responses to novel Chlamydia pneumoniae antigens that are associated with persistent C. pneumoniae infections.

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C.

Evidence for persistent Chlamydia pneumoniae infection of human coronary atheromas.

To date, structures representing developmental stages of Chlamydia pneumoniae, especially persistent forms of this intracellular bacteria, have not been described in human atherosclerotic tissues using specific antibody labeling and transmission electron microscopy. Staining of atherosclerotic tissue from five patients seeking heart transplantation with gold-labeled antibodies specific for up-regulated chlamydial heat shock proteins, GroEL and GroES, and visualisation via transmission electron microscopy revealed intracellular, atypical, round to oval structures of variable diameter. These structures resembled reticulate bodies of Chlamydia, were surrounded by membranes and were located within smooth muscle cells, macrophages or fibroblasts.

A worldwide perspective of atypical pathogens in community-acquired pneumonia.

Rationale: Controversy still exists in the international literature regarding the need to use antimicrobials covering atypical pathogens when initially treating hospitalized patients with community-acquired pneumonia (CAP). In different regions of the world, monotherapy with a beta-lactam antimicrobial is common.

Objectives: We sought to correlate the incidence of CAP due to atypical pathogens in different regions of the world with the proportion of patients treated with an atypical regimen in those same regions.

Tissue MicroArray (TMA) analysis of normal and persistent Chlamydophila pneumoniae infection.

Background: Chlamydophila pneumoniae infection has been implicated as a potential risk factor for atherosclerosis, however the mechanism leading to persistent infection and its role in the disease process remains to be elucidated.

Methods: We validated the use of tissue microarray (TMA) technology, in combination with immunohistochemistry (IHC), to test antibodies (GroEL, GroES, GspD, Ndk and Pyk) raised against differentially expressed proteins under an interferon-gamma (IFN-gamma) induced model of chlamydial persistence.

Results: In the cell pellet array, we were able to identify differences in protein expression patterns between untreated and IFN-gamma treated samples.

Identification of Chlamydia pneumoniae proteins in the transition from reticulate to elementary body formation.

Chlamydia pneumoniae is an important human respiratory pathogen that is responsible for an estimated 10% of community-acquired pneumonia and 5% of bronchitis and sinusitis cases. We examined changes in global protein expression profiles associated with the redifferentiation of reticulate body (RB) to elementary body (EB) as C. pneumoniae cells progressed from 24 to 48 h postinfection in HEp2 cells.

Protein expression profiles of Chlamydia pneumoniae in models of persistence versus those of heat shock stress response.

Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C.

Differential expression of chlamydial signal transduction genes in normal and interferon gamma-induced persistent Chlamydophila pneumoniae infections.

Characteristic features of the persistent chlamydial developmental cycle, associated with chronic infections in both humans and animals, include the generation of non-replicative, morphologically aberrant bodies which are distinct from normal propagating reticulate bodies. Previous studies have correlated these morphological and metabolic changes with differential expression of diverse functional subsets of chlamydial genes. To further investigate these correlations, we compared mRNA expression of predicted chlamydial signal transduction genes between normal Chlamydophila pneumoniae A-03 infections in HEp-2 cells and those treated with gamma interferon (IFN-gamma) by using real-time RT-PCR.

Analysis of altered protein expression patterns of Chlamydia pneumoniae by an integrated proteome-works system.

We have identified, analyzed, and quantified differential protein expression profile of five C. pneumoniae proteins, Adk (adenylate kinase), AhpC (thiol-specific antioxidant), CrpA (15 KD cysteine rich protein), Map (methionine aminopeptidae), and Cpn0710 (hypothetical protein) under normal versus persistent growth conditions induced by interferon-gamma, at different time intervals of their replicative cycle by successfully employing the latest proteomic analysis tool, PDQuest 2-D analysis software. We have also determined that this software represents a reliable analytical tool for mapping protein expression patterns in C.

Detailed protocol for purification of Chlamydia pneumoniae elementary bodies.

A detailed protocol for the growth and harvest of purified elementary bodies of Chlamydia pneumoniae is presented. This procedure utilizes a flask-to-flask passage scheme designed to achieve high bacterial titers in a short period of time.

Effects of fluoroquinolones on the migration of human phagocytes through Chlamydia pneumoniae-infected and tumor necrosis factor alpha-stimulated endothelial cells.

The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF-alpha). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic.

Increases in c-Jun N-terminal kinase/stress-activated protein kinase and p38 activity in monocyte-derived macrophages following the uptake of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, infects and replicates within a variety of eukaryotic cells. The purpose of the current study was to examine host cell signaling events immediately following uptake and early in the endocytic process (less than 1 h) following the phagocytosis of L. pneumophila.

Effect of macrolide antibiotics on human endothelial cells activated by Chlamydia pneumoniae infection and tumor necrosis factor-alpha.

This study investigated the potential anti-inflammatory activity of 3 macrolide antibiotics, clarithromycin, roxithromycin, and azithromycin, in an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVECs) were seeded in Transwell inserts, treated with serial dilutions of the antibiotics, and infected with Chlamydia pneumoniae or stimulated with tumor necrosis factor (TNF)-alpha. In HUVECs infected with C.

Proteomic analysis of differentially expressed Chlamydia pneumoniae genes during persistent infection of HEp-2 cells.

Recent data have shown that the respiratory pathogen Chlamydia pneumoniae expresses an altered gene transcription profile during gamma interferon (IFN-gamma)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of C. pneumoniae proteins labeled intracellularly with [(35)S]methionine/cysteine under normal conditions or IFN-gamma-mediated persistence.

The use of heteroduplex analysis of polymerase chain reaction products to support the possible transmission of Legionella pneumophila from a malfunctioning automobile air conditioner.

Air conditioner condensates have not been previously associated with cases of Legionnaires' disease. We report the possible transmission of Legionella pneumophila serogroup 1 from a malfunctioning automobile air conditioning system's leaking water onto the floorboard of a car driven for a long distance by the patient. Heteroduplex analysis of polymerase chain reaction products was used to help establish an epidemiologic link between the water specimen and the patient.

Inhibition of oxidative burst and chemotaxis in human phagocytes by Legionella pneumophila zinc metalloprotease.

Legionella pneumophila produces several extracellular proteins, but their role in the pathogenesis of Legionnaires' disease is unclear. This study examined the effects of the L. pneumophila major secretory protein (Msp), a zinc metalloprotease, on the oxidative burst and chemotaxis of human phagocytes.