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. In Kentucky, USA, the area served by our laboratory, the most commonly reported tick-borne illnesses include spotted fever group rickettsiosis, ehrlichiosis and Lyme disease, but of these three diseases, only ehrlichiosis is well-suited for direct detection using PCR methods during the acute stage of illness. We report here the validation of a duplex real-time PCR assay using whole blood specimens on the Luminex ARIES® instrument, combining DNA extraction, amplification and detection into a one-step process. This method allows for rapid and sensitive detection of acute infections with Ehrlichia spp. and Anaplasma phagocytophilum using whole blood specimens. We included A. phagocytophilum to monitor emergence of this pathogen in Kentucky, since surrounding states have reported many more cases than Kentucky.

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 Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila. 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. The incidence of atypical pathogens has been shown to be approximately 22% worldwide, but this probably varies with location. The history and physical exam of a patient with atypical pneumonia reveals how patients share many signs and symptoms with their counterpart patients who have typical pneumonias; therefore, the diagnosis primarily depends on laboratory identification, which is evolving and improving. What started out as simple, but difficult to yield cultures, has progressed to modern molecular-based testing assays. Treatment is missed if an empiric regimen includes only monotherapy with a ß-lactam antimicrobial; so, many country guidelines, including the Infectious Diseases Society of America/American Thoracic Society guidelines for community-acquired pneumonia, recommend using a regimen containing either a macrolide or a fluorinated quinolone. Once an atypical pathogen has been identified, evidence trends toward favoring a quinolone, but more data are needed to confirm. The concept of using combination therapy in severe patients is also explored.

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. Herein, we present the case of a 40-year-old man with ehrlichiosis who developed nephrotic syndrome, cryoglobulinemia, and secondary membranoproliferative glomerulonephritis (MPGN). The patient originally presented with shortness of breath, diffuse myalgias, headache, and lower extremity edema. He subsequently developed acute kidney injury and underwent kidney biopsy which showed MPGN and acute tubular injury. A tick-borne disease panel was positive for IgM and IgG to Ehrlichia chaffeensis. Serum testing revealed type 3 mixed cryoglobulinemia with no evidence of hepatitis C infection. The cryoprecipitate contained IgM and IgG antibodies to E. chaffeensis. Cryoglobulinemia is frequently associated with infections, particularly hepatitis C; however, our case is the first to describe ehrlichiosis associated with cryoglobulinemia and secondary MPGN.

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. Further differences were identified in cytokine and chemokine responses. IL-6 and KC were expressed early in mice infected with strains associated with higher virulence. Strains that showed lower pathogenicity in mice had greater IFNγ, MIG, and IL-10 responses. A principal component analysis (PCA) of the cytokine and chemokine profiles revealed 4 immune response phenotypes that correlated with the severity of disease. A/KY/180/10, which showed the greatest virulence with a rapid onset of disease progression, was compared in additional studies with A/KY/136/09, which showed low virulence in mice. Analyses comparing a low (KY/136) versus a high (KY/180) virulent isolate showed a significant difference in the kinetics of infection within the lower respiratory tract and immune responses. Notably by 4 DPI, virus titers within the lung, bronchoalveolar lavage fluid (BALf), and cells within the BAL (BALc) revealed that the KY/136 replicated in BALc, while KY/180 replication persisted in lungs and BALc. In summary, our studies suggest four phenotypic groups based on immune responses that result in different virulence outcomes in H1N1pdm isolates with a high degree of genetic similarity. In vitro studies with two of these isolates suggested that the more virulent isolate, KY/180, replicates productively in macrophages and this may be a key determinant in tipping the response toward a more severe disease progression.

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. RESULTS: Six of 13 patients had an ischaemic cardiomyopathy secondary to coronary atherosclerosis, while another six patients had an idiopathic, dilated cardiomyopathy. One additional patient, a young (24 years) woman with cardiomyopathy, had no history of atherosclerotic disease. Eleven patients were positive by immunohistochemistry with at least one antibody. Coronary arteries of the two other patients were negative by immunohistochemistry with all antibodies. One of these patients was the 24-year-old woman with grade I disease and no risk factors for coronary artery disease. CONCLUSIONS: The protein antigens of persistent C pneumoniae infection found in the atheromatous lesions from patients in this study could potentially be used as markers to detect such infections and some may be virulence factors or immunogens specific to C pneumoniae, thus serving as target molecules for diagnostic use or therapeutic intervention.

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. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

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. By using double immunogold electron microscopy technique (GroEL and GroES in combination with chlamydial LPS/MOMP antibodies), we demonstrated these structures were of chlamydial origin. In the current study, we demonstrated the presence of aberrant bodies of C. pneumoniae in vivo in archival coronary atheromatous heart tissues by the immunogold electron microscopy technique.

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. In addition, we sought to compare clinical outcomes of patients with CAP treated with and without atypical coverage. METHODS: A secondary analysis was performed using two comprehensive international databases. World regions were defined as North America (I), Europe (II), Latin America (III), and Asia and Africa (IV). Time to reach clinical stability, length of hospital stay, and mortality were compared between patients treated with and without atypical coverage. MEASUREMENTS AND MAIN RESULTS: The incidence of CAP due to atypical pathogens from 4,337 patients was 22, 28, 21, and 20% in regions I-IV, respectively. The proportion of patients treated with atypical coverage from 2,208 patients was 91, 74, 53, and 10% in regions I-IV, respectively. Patients treated with atypical coverage had decreased time to clinical stability (3.7 vs. 3.2 d, p < 0.001), decreased length of stay (7.1 vs. 6.1 d, p < 0.01), decreased total mortality (11.1 vs. 7%, p < 0.01), and decreased CAP-related mortality (6.4 vs. 3.8%, p = 0.05). CONCLUSIONS: The significant global presence of atypical pathogens and the better outcomes associated with antimicrobial regimens with atypical coverage support empiric therapy for all hospitalized patients with CAP with a regimen that covers atypical pathogens.

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. Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-gamma treated samples. The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-gamma persistence using proteomic analysis. Subsequently, it was shown in a second TMA including archival atheromatous heart tissues from 12 patients undergoing heart transplantation, that GroEL, GroES, GspD and Pyk were expressed in atheromatous heart tissue specimens as well, and were detectable morphologically within lesions by IHC. CONCLUSION: TMA technology proved useful in documenting functional proteomics data with the morphologic distribution of GroEL, GroES, GspD, Ndk and Pyk within formalin-fixed, paraffin-embedded cell pellets and tissues from patients with severe coronary atherosclerosis. The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients. These may be useful markers for the detection of persistent infection in vitro and in vivo.

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. Proteins corresponding to those showing the greatest changes in abundance in the beginning of the RB to EB transition were then identified from purified EBs. Among the 300 spots recognized, 35 proteins that were expressed at sufficiently high levels were identified by mass spectrometry. We identified C. pneumoniae proteins that showed more than 2-fold increases in abundance in the early stages of RB to EB transition, including several associated with amino acid and cofactor biosynthesis (Ndk, TrxA, Adk, PyrH, and BirA), maintenance of cytoplasmic protein function (GroEL/ES, DnaK, DksA, GrpE, HtrA, ClpP, ClpB, and Map), modification of the bacterial cell surface (CrpA, OmpA, and OmcB), energy metabolism (Tal and Pyk), and the putative transcriptional regulator TctD. This study identified C. pneumoniae proteins involved in the process of redifferentiation into mature, infective EBs and indicates bacterial metabolic pathways that may be involved in this transition. The proteins involved in RB to EB transition are key to C. pneumoniae infection and are perhaps suitable targets for therapeutic intervention.

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. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-gamma) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42 degrees C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.

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. Inspection of the Cp. pneumoniae genome revealed at least 39 candidate signal transduction genes, of which 30 were differentially expressed in Cp. pneumoniae mediated persistence. Functional sub-groups of differentially expressed signal transduction genes include chlamydial GTPases (hflX, ychF, yhbZ and yphC), linked to bacterial cellular processes such as cell cycle control and ribosome assembly and stability. Other up-regulated signal transduction genes sharing similarity to bacterial stress response genes (htrA, surE, lytB and hrcA) were also detected. The transcriptional changes observed for the majority of signal transduction genes appear to be unique for Cp. pneumoniae, as similar changes were not observed in recent whole genomic analysis of C. trachomatis IFN-gamma mediated persistence. These results suggest that chlamydial signal transduction genes play potentially important roles in the establishment and maintenance of Cp. pneumoniae persistence, likely as part of the IFN-gamma response stimulon as described for C. trachomatis, but with considerable differences in the transcriptional profile.

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. pneumoniae.

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. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-alpha, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF-alpha-stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively.

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. This examination focused on the protein kinase signal pathways to identify any aberrant signal(s) induced by L. pneumophila within its host, as a means to alter the normal endocytic pathway. The mitogen-activated protein kinase cascades are of interest due to their involvement in cellular regulation. The experiments were carried out with monocyte-derived macrophages (MDMs). All three mitogen-activated protein kinase cascades were activated when MDMs were inoculated with either Legionella strain (wild-type strain AA100 or dotA mutant GL10) or an Escherichia coli control. Whereas the avirulent treatments, GL10 and E. coli, exhibited a leveling off or a return to near basal levels of phosphorylation/activity of c-Jun N-terminal kinase by 60 min, the virulent strain AA100 exhibited a significantly increased level of activity through 60 min that was greater than that seen in GL10 (P = 0.025) and E. coli (P = 0.014). A similar trend was seen with p38 phosphorylation. Phosphorylation of mitogen-activated protein/ERK kinase (MEK) was decreased in strain AA100 compared to E. coli. Inhibition of the activity of either the stress-activated protein kinase/c-Jun N-terminal kinase or p38 pathway significantly decreased the ability of legionellae to replicate intracellularly, suggesting the necessity of these two pathways in its intracellular survival and replication.

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. pneumoniae or stimulated with TNF-alpha, both azithromycin and roxithromycin caused significant decreases in neutrophil and monocyte TEM, compared with antibiotic-free controls. Clarithromycin had no detectable effect in either group. Azithromycin caused significant decreases in interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1, whereas roxithromycin significantly decreased IL-8. This study indicates heterogeneity in the anti-inflammatory activity of these antibiotics. Mechanisms of monocyte and neutrophil TEM inhibition by azithromycin and roxithromycin are unclear but may be partially due to inhibition of IL-8 and MCP-1 production.

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 identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in C. pneumoniae elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-gamma per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in C. pneumoniae-infected cells due to IFN-gamma treatment. Upregulation of C. pneumoniae proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-gamma while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-gamma pressure to obtain a finer representation of the gene products involved in establishing persistence.