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Acute influenza virus (AIV) infection can manifest as a severe life-threating illness in patients who are not vaccinated, and furthermore, have comorbidities that place them at risk for rapid respiratory decompensation. Each year influenza causes death in individuals with high risk for contracting this infection, although the illness is preventable by vaccination. Complications of AIV infection, such as bacterial pneumonia are treatable, but other severe complications such as acute respiratory distress syndrome (ARDS) leading to diffuse alveolar damage (DAD) are limited to supportive therapy and self-resolution. In most cases, ARDS leading to DAD is fatal, due to the insidious severity of symptoms which lead to rapid oxygen desaturation without correction, and despite supportive therapy. Regardless of a poor prognosis, the clinical signs and symptoms are congruent with imaging and attest to the importance of vaccination, which protect against high mortality rates.

Context.—Previous studies suggest that training in pathology residency programs does not adequately prepare pathology residents to become competent in clinical chemistry.Objectives.—To define the beliefs of pathology residents in the United States regarding their preparation for practicing clinical chemistry in their career, their attitude toward the discipline, and the attractiveness of clinical chemistry as a career.Design.—The residents of all pathology residency programs in the United States were given the opportunity to participate in an online survey.Results.—Three hundred thirty-six pathology residents responded to the survey. Analysis of the survey results indicates that pathology residents are more likely to believe that their income may be lower if they select a career that has a clinical chemistry focus and that their faculty do not value clinical chemistry as much as the anatomic pathology part of the residency. Residents also report that clinical chemistry is not as enjoyable as anatomic pathology rotations during residency or preferable as a sole career path. A large proportion of residents also believe that they will be slightly prepared or not prepared to practice clinical chemistry by the end of their residency and that they do not have enough background and/or time to learn clinical chemistry during their residency programs to be able to practice this specialty effectively post graduation.Conclusions.—Our survey results suggest that many pathology residents do not have a positive attitude toward clinical chemistry and do not experience a supportive learning environment with an expectation that they will become competent in clinical chemistry with a residency alone.

To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log10 focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3&ndash, 4 days post-infection at a maximum value of 6.9 log10 genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed.

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light. Fluidic force discrimination is used to increase specificity of the assay, following which a difference imaging algorithm that can see single 3 μm magnetic particles without optical calibration is used to detect and quantify signal intensity from each sub-array of retroreflectors. We demonstrate the utility of embedded microretroreflectors as a new sensing modality through a proof-of-concept immunoassay for a small, obligate intracellular bacterial pathogen, Rickettsia conorii, the causative agent of Mediterranean Spotted Fever. The combination of large sensing area, optimized surface chemistry and microfluidic protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in a sensitive immunoassay with a limit of detection of roughly 4000 R. conorii per mL.

We present advancements in microfluidic technology for rapid detection of as few as ten rickettsial organisms in complex biological samples. An immuno-reactive filter, macroporous polyacrylamide monolith (PAM), fabricated within a microfluidic channel enhances solid-phase immuno-capture, staining and detection of targeted bacteria. Bacterial cells in samples flowing through the channel are forced to interact with the PAM filter surface due to size exclusion, overcoming common transport and kinetic limitations for rapid (minutes), high-efficiency (~100%) capture. In the process, targeted cells in sample volumes of 10 μl to >100 μl are concentrated within a sub-50 nl region at the PAM filter edge in the microchannel, thus concentrating them over 1,000-fold. This significantly increases sensitivity, as the hydrophilic PAM also yields low non-specific immuno-fluorescence backgrounds with samples including serum, blood and non-targeted bacteria. The concentrated target cells are detected using fluorescently-labeled antibodies. With a single 2.0 × 2.0 × 0.3 mm PAM filter, as few as 10 rickettsial organisms per 100 μl of lysed blood sample can be analyzed within 60 min, as compared to hours or even days needed for conventional detection methods. This method is highly relevant to rapid, multiplexed, low-cost point of care diagnostics at early stages of infection where diagnostics providing more immediate and actionable test results are needed to improve patient outcomes and mitigate potential natural and non-natural outbreaks or epidemics of rickettsial diseases.

International audience; Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p < 0.01 for all), and closely correlated (p < 0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p < 0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood. K. Otterdal, (C) 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11–12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14–19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/β, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions., Author Summary Scrub typhus is a life-threatening disease that presents as a severe acute febrile illness. It is caused by mite-transmitted Orientia tsutsugamushi, a Gram-negative, obligately intracellular bacterium. Every year, approximately one million people are infected globally; however, there is no vaccine for the control of this infection. Mechanistic studies of host immune responses have been few, partially due to the limited availability of suitable animal models and research facilities. Here, we report our development of an intradermal inoculation mouse model that mimics the natural infection. We also examined the kinetics of immune and inflammatory responses during acute and chronic stages of O. tsutsugamushi infection. As the first comprehensive report of an intradermal inoculation murine model of scrub typhus, this study improves our understanding of host immune responses following cutaneous exposure to the bacteria and opens new avenues for future vaccine-based investigations.

International audience; Background: The pathophysiological hallmark of Rickettsia conorii (R. ă conorii) infection comprises infection of endothelial cells with ă perivascular infiltration of T-cells and macrophages. Although ă interferon (IFN)-gamma-induced protein 10 (IP-10)/CXCL10 is induced ă during vascular inflammation, data on CXCL10 in R. conorii infection is ă scarce. ă Methods: Serum CXCL10 was analyzed in two cohorts of southern European ă patients with R. conorii infection using multiplex cytokine assays. The ă mechanism of R. conorii-induced CXCL10 release was examined ex vivo ă using human whole blood interacting with endothelial cells. ă Results: (i) At admission, R. conorii infected patients had excessively ă increased CXCL10 levels, similar in the Italian (n = 32, similar to ă 56-fold increase vs controls) and the Spanish cohort (n = 38, 68-fold ă increase vs controls), followed by a marked decrease after recovery. The ă massive CXCL10 increase was selective since it was not accompanied with ă similar changes in other cytokines. (ii) Heat-inactivated R. conorii ă induced a marked CXCL10 increase when whole blood and endothelial cells ă were co-cultured. Even plasma obtained from R. conorii-exposed whole ă blood induced a marked CXCL10 release from endothelial cells, comparable ă to the levels found in serum of R. conorii-infected patients. Bacteria ă alone did not induce CXCL10 production in endothelial cells, macrophages ă or smooth muscle cells. ă Conclusions: We show a massive and selective serum CXCL10 response in R. ă conorii-infected patients, likely reflecting release from infected ă endothelial cells characterized by infiltrating T cells and monocytes. ă The CXCL10 response could contribute to T-cell infiltration within the ă infected organ, but the pathologic consequences of CXCL10 in clinical R. ă conorii infection remain to be defined. (C) 2016 Elsevier Ltd. All ă rights reserved.

Ehrlichioses and anaplasmoses are caused by alpha-proteobacteria within the family of Anaplasmataceae. These diseases have been known for a long time in veterinary medicine and recently in human medicine. These tick-borne zoonoses are considered as emerging diseases. The first case of human monocytotropic ehrlichiosis occurred in 1986. Human granulocytic anaplasmosis was described as a separate entity in 1994 and ehrlichiosis caused by Ehrlichia ewingii was reported in humans in 1999. The number of cases has been rising steadily due to better diagnostic techniques and better surveillance worldwide. In this review, we will present human and animal ehrlichioses and anaplasmoses as emerging diseases and present candidate(s) for the future.

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU ofAeromonas hydrophilabound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease α2-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT)A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability ofA. hydrophilaSSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied intrans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase inA. hydrophilaSSU; the mutated forms of enolase were hyperexpressed inEscherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues (252FYDAEKKEY260) in theA. hydrophilaSSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis ofA. hydrophilaSSU infections and of any gram-negative bacteria in general.

More than 50 emerging and reemerging pathogens have been identified during the last 40 years. Until 1992 when the Institute of Medicine issued a report that defined emerging infectious diseases, medicine had been complacent about such infectious diseases despite the alarm bells of infections with human immunodeficiency virus. Molecular tools have proven useful in discovering and characterizing emerging viruses and bacteria such as Sin Nombre virus (hantaviral pulmonary syndrome), hepatitis C virus, Bartonella henselae (cat scratch disease, bacillary angiomatosis), and Anaplasma phagocytophilum (human granulocytotropic anaplasmosis). The feasibility of applying molecular diagnostics to dangerous, fastidious, and uncultivated agents for which conventional tests do not yield timely diagnoses has achieved proof of concept for many agents, but widespread use of cost-effective, validated commercial assays has yet to occur. This review presents representative emerging viral respiratory infections, hemorrhagic fevers, and hepatitides, as well as bacterial and parasitic zoonotic, gastrointestinal, and pulmonary infections. Agent characteristics, epidemiology, clinical manifestations, and diagnostic methods are tabulated for another 22 emerging viruses and five emerging bacteria. The ongoing challenge to the field of molecular diagnostics is to apply contemporary knowledge to facilitate agent diagnosis as well as to further discoveries of novel pathogens.

Yersiniae are gram-negative bacilli in the family Enterobacteriaceae, and three species in the genus Yersinia are human pathogens. Yersinia pseudotuberculosis and Y. enterocolitica cause enteropathogenic infections that are typically self-limiting and are characterized by diarrhea, fever, and abdominal pain (52). Y. pestis is transmitted to humans through the bite of an infected flea or inhalation of the organisms, resulting in bubonic, pneumonic, or septicemic forms of plague. Y. pestis is historically credited for 200 million deaths worldwide and is also a potential agent of biowarfare or bioterrorism (18, 31, 34). The pathogenesis of yersinia infections is complex and multifactorial. A number of virulence-associated systems (mechanisms) are common among the three human-pathogenic yersiniae. For example, the type three secretion system (T3SS), encoded on a 70-kb plasmid, exists in all three species, and this plasmid is designated pCD1 in Y. pestis, pYVe in Y. enterocolitica, and pYV in Y. pseudotuberculosis (30, 45). Through the T3SS, a variety of effector proteins called Yops (for Yersinia outer membrane proteins) are translocated into the host cytosol and are absolutely required for the virulence of the yersiniae (30). On the other hand, each Yersinia species possesses its own unique set of virulence factors (30, 45). In the two enteropathogenic species, the virulence determinants Yad (for Yersinia adherence protein, formerly designated YopA) and invasin protein (Inv) play crucial roles in bacterial colonization and invasion of the intestinal epithelium but are not functional in Y. pestis (45). In addition to plasmid pCD1, Y. pestis harbors another two plasmids that are absent in the enteropathogenic yersiniae. Plasmid pPCP1 encodes the virulence determinant plasminogen activator (Pla), while pMT1, or pFra, encodes capsular antigen F1 and the murine toxin (45). It has been shown that the deletion of the 102-kb pigmentation (pgm) locus from the chromosome of Y. pestis attenuates its virulence, while this pgm locus is usually silent in Y. pseudotuberculosis (30). Recently, a transcriptional regulator, RovA, was identified in all three Yersinia species. RovA regulates a variety of genes and is specifically required for the development of bubonic plague in mice (8). Interestingly, a new set of T3SS-dependent proteins has been identified in Y. pestis and includes insecticidal-like proteins that are thought to function as transmission factors, contributing to flea morbidity by promoting colonization of the midgut (26). Therefore, the continued investigation of new virulence factors and virulence-associated systems is warranted to fully elucidate the mechanisms of yersinia pathogenesis. The outer membrane of gram-negative bacteria is comprised of many different proteins that help maintain the structural integrity of the bacterial cell envelope. Some of these proteins are covalently modified by the addition of a lipid, N-acyl-S-diacylglyceryl cysteine, and such modified proteins are collectively known as lipoproteins (37). One particularly abundant lipoprotein, designated murein (or Braun) lipoprotein (Lpp), is associated with the outer membranes of bacteria within the family Enterobacteriaceae. Originally identified in Escherichia coli, Lpp links the murein (peptidoglycan) layer to the outer bacterial membrane (29). Lpp has previously been demonstrated to play a role in the host's immune response against infections with some gram-negative enteric pathogens, such as E. coli, Salmonella enterica serovar Typhimurium, and Y. enterocolitica (65). Our earlier studies indicated that Lpp (6.3 kDa) purified from E. coli and Y. enterocolitica not only synergized with lipopolysaccharide (LPS) to induce septic shock, but also evoked the production of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in both LPS-responsive and LPS-nonresponsive mice and in mouse peritoneal exudate macrophages, suggesting an alternative signaling mechanism for Lpp (66). In fact, a subsequent study showed that Lpp signals through Toll-like receptor 2 (TLR-2) and not TLR-4 (2), which LPS utilizes for cell signaling. In our most recent studies, we provided evidence that S. enterica serovar Typhimurium harbors two copies of the lipoprotein gene, lppA and lppB, separated by 82 bp on the chromosome (22). Both single and double lpp gene deletions resulted in attenuation of bacteria in terms of their motility, invasiveness, and cytokine/chemokine production, and such mutants were avirulent in mice when administered by systemic and oral routes compared to the wild-type (WT) bacterium (21, 22, 58). By searching the National Center for Biotechnology Information database, we identified a homolog of the lpp gene in the genomes of all three pathogenic yersiniae (10, 15, 44). However unlike in S. enterica serovar Typhimurium, only a single copy of the lpp gene is present on the chromosome in Yersinia species. Consequently, in this study, we generated lpp mutants of WT/parental yersiniae (Y. pseudotuberculosis YPIII and Y. pestis KIM/D27 [pgm locus minus] and Y. pestis CO92) and evaluated them in mouse models of infection. Blood chemistries and blood cell counts, as well as organ pathology, were examined in specimens from the pneumonic and bubonic plague mouse models after infection with WT and Δlpp mutant strains of Y. pestis. We also evaluated the abilities of WT and mutant yersiniae to disseminate to the distal organs after infection. Additionally, the intracellular survival of WT and mutant Y. pestis CO92 bacteria was assessed in murine macrophages. Our data provided evidence that lpp mutants of Y. pseudotuberculosis and Y. pestis were attenuated in mice, an effect that could be complemented. More importantly, deletion of the lpp gene from the Y. pestis KIM/D27 mutant further attenuated the strain, and immunization of mice with this mutant provided protection to animals against pneumonic plague invoked by intranasal (i.n.) inoculation of Y. pestis CO92. To our knowledge, this is the first systematic study illustrating the role of Lpp in the pathogenesis of yersinia infection.

In our previous study, we deleted the gene encoding Aeromonas outer membrane protein B (AopB), a structural component of the type III secretion system (T3SS) from a cytotoxic enterotoxin gene (act)-minus diarrheal isolate SSU of Aeromonas hydrophila. Our laboratory also molecularly characterized the cytotoxic enterotoxin (Act), which is secreted by the bacterium utilizing the type II secretion system (T2SS). The act/aopB mutant exhibited significantly reduced cytotoxicity to cultured cells (e.g. RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells) and was avirulent in mice. In this study, we developed additional A. hydrophila mutants in which T3SS-associated ascV and acrV genes were deleted, either individually or in combination with that of the act gene, to examine host-pathogen interactions. A significant reduction in the induction of inflammatory cytokines and chemokines was noted in the sera of mice infected with these mutants when compared to animals infected with wild-type (WT) A. hydrophila. After infection with the WT and act/aopB mutant, we performed microarray analyses on RNA from the above-mentioned murine macrophages and human colonic epithelial cells to examine global cellular transcriptional responses. Based on three independent experiments, WT A. hydrophila altered the expression of 434 genes in RAW 264.7 cells and 80 genes in HT-29 cells. Alteration in the expression of 209 macrophage and 32 epithelial cell genes was reduced when the act/aopB mutant was used, compared to when cells were infected with the WT bacterium, indicating the involvement of Act and/or AopB in transcriptional regulation of these genes. We verified up-regulation of 15 genes by real-time reverse transcriptase-polymerase chain reaction and confirmed A. hydrophila WT-versus mutant-induced production of cytokines/chemokines in supernatants from RAW 264.7 and HT-29 cells. This is the first description of host cell transcriptional responses to A. hydrophila infection.

We constructedSalmonella entericaserovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from anmsbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models ofSalmonellapathogenesis.

Human monocytotropic ehrlichiosis caused by Ehrlichia chaffeensis was reported in 1987. An animal model to study acute fatal ehrlichiosis in mice that has been developed closely resembles the fatal form of human monocytotropic ehrlichiosis. However, animal models for persistent infection in the genus Ehrlichia in immunocompetent mice have not been characterized. We report the histopathological progression of Ehrlichia muris infection in immunocompetent mice (AKR and C57BL/6 strains) correlated with their antibody response determined by indirect immunofluorescence and Western immunoblotting, and the distribution and quantity of the ehrlichial load by immunohistochemistry, polymerase chain reaction (PCR), and real-time PCR in lungs, liver, and spleen. Mild to moderate correlation was observed between histopathological grading in these organs and relative ehrlichial loads. The highest ehrlichial loads were present between days 4 and 14 after infection. E. muris was detected in tissues examined up to 150 days after infection by real-time PCR. Analysis of the serological response revealed several immunodominant antigens, including 200-, 180-, 100-, 73/75-, 45-, and 28-kd proteins. In conclusion, we have provided for the first time a complete histopathological, serological, immunohistochemical, and quantitative analysis of an animal model for the study of persistent ehrlichial infection.

An emphasis on cellular immunity againstRickettsiahas led to neglect of analysis of the role of antibody. The availability of an excellent mouse model of spotted fever rickettsiosis enabled investigation of a potential role of antibody in immunity toRickettsia conorii. C3H severe combined immunodeficiency (SCID) mice were passively transfused with monoclonal antibodies against rickettsial outer membrane protein A (OmpA), OmpB, or lipopolysaccharide (LPS), polyclonal anti-R. conoriiserum, Fab fragments of polyclonal antiserum, or no antibodies and then challenged 48 h later with 10 50% lethal doses (LD50) ofR. conorii. All mice that received monoclonal antibodies against OmpA and two of four mice that received monoclonal antibodies against OmpB or polyclonal antisera were completely protected, but the recipients of anti-LPS antibodies or the Fab fragments were not protected. Polyclonal antibody treatment of C3H SCID mice that had been infected with 10 LD50ofR. conorii4 or 5 days earlier prolonged the life of the infected mice from 10.4 to 22.5 days and resulted in decreased levels of infectious rickettsiae in the spleen and liver 24 and 48 h later. Treatment with protective antibodies resulted in the development of large aggregates ofR. conoriiantigens in splenic macrophages and intraphagolysosomal rickettsial death and digestion. The kinetics of development of antibodies toR. conoriidetermined by immunoblotting revealed antibodies to LPS on day 6 and antibodies to OmpA and OmpB on day 12, when recovery from the infection had already occurred. Antibodies to particular epitopes of OmpA and OmpB may protect against reinfection, but they may not play a key role in immunity against primary infection. Antibodies might be useful for treating infections with antibiotic-resistant organisms, and some B-cell epitopes should be included in a subunit vaccine.

The emergence and reemergence of a serious infectious disease are often associated with a high case-fatality rate because of misdiagnosis and inappropriate or delayed treatment. The current reemergence of spotted fever rickettsiosis caused by Rickettsia rickettsii in Brazil has resulted in a high proportion of fatal cases. We describe two familial clusters of Brazilian spotted fever in the state of Minas Gerais, involving six children 9 months to 15 years of age; five died. Immunohistochemical investigation of tissues obtained at necropsy of a child in each location, Novo Cruzeiro and Coronel Fabriciano municipalities, established the diagnosis by demonstration of disseminated endothelial infection with spotted fever group rickettsiae. The diagnosis in the two fatal cases from Coronel Fabriciano and the surviving patient from Novo Cruzeiro was further supported by immunofluorescence serologic tests.

Human ehrlichioses represent one of the best examples of newly emergent infectious diseases in which the classic triad of host, infectious agent, and environment are intertwined closely. These pathogens have existed for eons on the planet, and some were described as veterinary pathogens decades ago. Because of dramatic increases of deer and small mammal populations in certain areas and the subsequent increased populations of particular blood-feeding ticks, the risk of developing these diseases is higher than before. Increasing human populations in suburban areas and increased immunosuppressed populations (transplant patients, human immunodeficiency virus patients, and cancer survivors) also have increased risk of developing severe forms of these diseases.

Ehrlichiosis in lung transplant (LT) recipients is associated with severe outcomes. Ehrlichia ewingii is a less frequent cause of symptomatic ehrlichiosis, characterized by cytoplasmic inclusions (morulae) within circulating neutrophils. We report a case of E. ewingii infection in an LT recipient diagnosed promptly by blood smear exam and confirmed with molecular studies.

To the Editor: We report a case of Rickettsia sibirica mongolitimoniae strain infection associated with lymphangitis (1). A 44-year-old man was admitted to Sao Bernardo Hospital in Setubal, Portugal, on August 21, 2006. Twelve days previously while on vacation at Troia Peninsula, he noted malaise, insomnia, and dry buccal mucosa. The next day he observed a small erythematous pruritic lesion on the lower right forearm that 2 days later developed into an eschar. He also had fever and sought medical care. After treatment with topical bacitracin, floxacillin, and acetaminophen for 2 days, fever (38.7°C) continued with lymphangitis extending from the right wrist to the elbow. The medication was changed to nimesulide. Three days later a rash developed on the trunk and arms, and lymphangitis extended to the axilla. Fever and chills continued, leading to hospital admission. No history of tick exposure was reported. Physical examination showed blood pressure 128/73 mm Hg, pulse 96/min, and a rubbery, nontender right supraclavicular lymph node ≈1 cm in diameter. Several 5- to 10-mm maculopapular erythematous lesions were observed on the patient’s palms. He had inflammation on the right forearm suggestive of lymphangitis and an eschar with surrounding edema and erythema on the dorsal lower right forearm (Figure). Admission evaluation showed platelets 117,000/μL, total bilirubin 0.42 mg/dL, albumin 3.42 g/dL, creatinine 1.1 mg/dL, alanine aminotransferase 244 U/L, aspartate aminotransferase 54 U/L, alkaline phosphatase 1061 U/L, creatine phosphokinase 87 U/dL, lactate dehydrogenase 784 U/L, C-reactive protein 7.1 mg/dL, radiographic pulmonary diffuse reticular pattern, arterial pO2 68 mm Hg, O2 saturation 94%, pCO2 22 mm Hg, and arterial blood pH 7.35. The differential diagnoses included rickettsiosis, pneumonia, and cellulitis. Treatment with vancomycin, cefatriaxone, and 100 mg of doxycycline twice a day was begun. On the day after hospitalization, a heparinized blood sample and 2 skin biopsy samples were collected. Vancomycin and ceftriaxone were discontinued at 48 hours when rickettsial infection was confirmed by PCR on skin biopsy; 48 hours later, the patient was afebrile. Figure Lymphangitis extending from the right forearm to the axilla and (inset) eschar on right forearm, caused by Rickettsia sibirica mongolitimonae strain. Arrows indicate lymphangitis. Immunofluorescence assay for antibodies that used R. sibirica mongolitimonae strain as antigen demonstrated seroconversion with no antibodies in the serum sample collected on August 21 and immunoglobulin G (IgG) and IgM antibodies at a titer of 256 in serum collected on August 30. DNA was extracted from 1 skin biopsy sample by using a DNeasy Tissue Kit (QIAGEN, Hilden, Germany). The products of nested PCR showed 100% similarity with gltA (353/353) and ompA (350/350) nucleotide sequences of R. sibirica mongolitimonae strain (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"DQ423368.1","term_id":"90657239"}}DQ423368.1 and {"type":"entrez-nucleotide","attrs":{"text":"DQ423367.1","term_id":"90657237"}}DQ423367.1) (1). Cutaneous biopsy indicated epidermal and dermal necrosis with extensive lymphocyte- and macrophage-rich inflammatory infiltrates involving the papillary and reticular dermal blood vessels characteristic of rickettsial infection. Relatively scant intracellular organisms were observed in the reticular dermis by spotted fever group rickettsia–specific immunohistochemistry (2). Because of the presence of shared protein and lipopolysaccharide antigens in spotted fever group rickettsiae, distinguishing infections with closely related rickettsiae such as R. conorii, R. africae, and R. sibirica by serologic or immunohistochemical methods is very difficult. However, isolation and/or PCR detection followed by genetic characterization can determine the genotype of the organism to the level of genus, species, and strain. The incidence of R. sibirica mongolitimonae strain infection in Portugal is not known because the usual laboratory confirmation by serologic methods does not distinguish these cases from Mediterranean spotted fever. Both cases of R. sibirica infection that have been recognized in Portugal occurred in August during the seasonal peak of Mediterranean spotted fever (1). This epidemiology differs from that in other countries (3–6). Perhaps differences in seasonal activity, population dynamics, or species of the vectors are the basis for the varying epidemiology. In Portugal, R. sibirica has been detected in Rhipicephalus pusillus (1). Rickettsia sibirica mongolitimonae strain was first isolated from a Hyalomma asiaticum tick from Inner Mongolia in 1991 (7) and subsequently from H. truncatum in Niger and from H. excavatum removed from a Greek patient (4,8). Lymphangitis in some patients with R. sibirica mongolitimonae strain infection is a potentially useful diagnostic sign. Nevertheless, half of the patients with reported cases have not had lymphangitis, and infections caused by other Rickettsia spp. can also cause lymphangitis (e.g., patients with African tick bite fever and R. heilongjiangensis infections) (1,3–6,9,10). Thus, the diagnosis of any rickettsiosis should not be based solely on clinical manifestations. The pathogenic role of rickettsiae in lymphangitis remains to be determined. R. sibirica may possibly infect the endothelium of the lymphatic vessel along the pathway from the rickettsial portal of entry at the eschar inoculation site to regional lymph nodes. However, rickettsiae have not been observed in lymphatic vessels, and the lymphatic vessel lesion has not been characterized. The possibility of another agent or pathogenic effectors cannot be excluded. Currently, this clinical manifestation is the strongest evidence that rickettsiae may initially spread by a lymphogenous route before hematogenous dissemination.

We investigated 2 fatal cases of Rocky Mountain spotted fever that occurred in 2003 and 2004 near the same locality in Colombia where the disease was first reported in the 1930s. A retrospective serosurvey of febrile patients showed that >21% of the serum samples had antibodies against spotted fever group rickettsiae.

Well-documented cases of simultaneous human infection with more than one tick-borne pathogen are rare. To our knowledge only two dual infections have been reported: simultaneous human infection with the agent of human granulocytic ehrlichiosis and Borrelia burgdorferi and simultaneous human infection with B. burgdorferi and Babesia microti (1-2). Rocky Mountain spotted fever has long been known to be endemic in North Carolina; cases of human ehrlichial infection were recognized there soon after Ehrlichia chaffeensis was recognized as an important cause of tick-borne disease in the southeastern United States. Because both Rocky Mountain spotted fever and ehrlichiosis are prevalent in North Carolina, occasional cases of simultaneous human infection by rickettsial and ehrlichial agents would not be surprising; however, no such cases seem to have been reported.

Background: Based on their essential role in concerting immunological and inflammatory responses we hypothesized that the homeostatic chemokines CCL19 and CCL21 may play a pathogenic role in rickettsiae infection. Methods: Serum levels of CCL19 and CCL21 in patients with R. africae and R. conorii infection were analyzed by enzyme immunoassays. Lungs from R. conorii infected mice were examined for CCL19, CCL21 and CCR7 expression by immunohistochemistry. Results: We found that patients with R. africae infection (n = 15) and in particular those with R. conorii infection (n = 16) had elevated serum levels of CCL19 on admission, with a decline during follow-up. While a similar pattern was seen for CCL21 in R. africae infection, patients with R. conorii infection showed persistently increased CCL21 levels during follow-up. In experimental R. conorii infection, we found strong immunostaining of CCL19 and CCL21 in the lungs, particularly in individuals that had received lethal doses. Immunofluorescence showed co-localization of CCR7 to endothelial cells, macrophages and fibroblasts within the lung tissue of R. conorii infected mice. Conclusions: Our findings suggest that the CCL19/CCL21/CCR7 axis is up-regulated during R. africae and in particular during R. conorii infection, which may potentially contribute to the pathogenesis of these disorders. © 2014 Astrup et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Diseases caused by protozoans and helminthes are some of the most common afflicting humans around the world, and yet they are some of the most scientifically neglected regarding their pathogenesis, diagnosis, and treatment. In this chapter, principles of molecular pathogenesis of the most common protozoan and helminth infections are discussed. Emphasis is placed on immunopathology (innate and adaptive immune responses), parasitic proteases, and immunomodulation by parasites. Selected protozoan and helminth infections are discussed in detail.

Massive steatosis has recently been described among a few human immunodeficiency virus-seropositive patients who were receiving antiretroviral therapy. Although clinical and light-microscopic pathological findings were carefully described, no ultrastructural studies of the liver were performed in these cases. We report the light-microscopic and ultrastructural findings at autopsy of a 35-year-old woman with AIDS who developed severe lactic acidosis and hepatic failure. The patient had been receiving standard doses of zidovudine for 5 months when she was hospitalized because of the rapid onset of abdominal pain, nausea, and vomiting. The most significant findings at autopsy were massive hepatomegaly and steatosis. Ultrastructural examination of the liver and skeletal muscle showed slightly enlarged mitochondria in the liver but no mitochondrial changes in the skeletal muscle. The pathogenesis of mitochondrial toxicity associated with antiviral therapies is briefly discussed.

The pathophysiological hallmark of spotted fever group rickettsioses comprises vascular inflammation. Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized that Dickkopf-1 (DKK-1), as a major modulator of Wnt signaling, could be involved in the pathogenesis in rickettsial infections. Our major findings were: (i) While baseline concentration of DKK-1 in patients with R. conorii infection (n = 32) were not different from levels in controls (n = 24), DKK-1 rose significantly from presentation to first follow-up sample (median 7 days after baseline). (ii) In vitro experiments in human umbilical vein endothelial cells (HUVECs) showed that while heatinactivated R. conorii enhanced the release of interleukin-6 (IL-6) and IL-8, it down-regulated the release of endothelialderived DKK-1 in a time- and dose-dependent manner. (iii) Silencing of DKK-1 attenuated the release of IL-6, IL-8 and growth-related oncogene (GRO)a in R. conorii-exposed HUVECs, suggesting inflammatory effects of DKK-1. (iv) Silencing of DKK-1 attenuated the expression of tissue factor and enhanced the expression of thrombomodulin in R. conorii-exposed HUVECs suggesting pro-thrombotic effects of DKK-1. The capacity of R. conorii to down-regulate endothelial-derived DKK-1 and the ability of silencing DKK-1 to attenuate R. conorii-induced inflammation in endothelial cells could potentially reflect a novel mechanism by which R. conorii escapes the immune response at the site of infection. © 2012 Antonov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Context—Molecular diagnostics continues to evolve very rapidly, and its impact in the diagnosis of infectious diseases is undeniable. Molecular tools have played a pivotal role in discovering and characterizing several emerging infectious agents and have now become the gold standard for the diagnosis of infectious diseases caused by fastidious or uncultivable agents. Multiple challenges still remain for the widespread use of cost-effective, validated, and commercially available molecular tools. Automated instruments capable of sample processing and multiplex nucleic acid amplification and postamplification analysis have already been approved by the US Food and Drug Administration (FDA) for use in the clinical setting. Nanobiotechnology is beginning to impact laboratory diagnostics in the clinical setting. Objective—To address current nucleic acid techniques used in the clinical laboratory for diagnosis of infectious diseases. FDA-approved tests are listed, as well as molecular techniques (amplification and postamplification analysis). A comprehensive list of emerging pathogens during the last 4 decades is also presented. Biosurveillance systems are discussed in the context of molecular tools. The rapidly evolving field of nanobiotechnology is briefly addressed. Data Sources—Original publications, major reviews, and book chapters were used to present a comprehensive, yet short, review of molecular diagnostics in infectious diseases. Conclusions—We will continue to witness an exponential growth of molecular techniques used for the initial diagnosis of infectious diseases. Molecular tools will also continue to have an impact on disease prognosis and response to therapeutic interventions. Automation, multiplexing, and miniaturization will continue to be driving forces in the development of new instruments.

Background Until recently, Rickettsia rickettsii was the only substantiated cause of tick-borne spotted fever group (SFG) rickettsiosis in humans in the United States. Rickettsia parkeri , originally thought to be nonpathogenic in humans, was recently proved to be another cause of tick-borne SFG rickettsiosis. Observations We report 3 cases of SFG rickettsiosis and discuss the epidemiology, clinical presentation, histopathologic features, and laboratory findings that support confirmed or probable diagnoses of R parkeri infection and describe the expanding list of eschar-associated SFG rickettsioses recognized in US patients. Conclusions The SFG rickettsioses share many clinical manifestations and extensive antigenic cross-reactivity that may hamper specific confirmation of the causative agent. Published online April 19, 2010 (doi:10.1001/archdermatol.2010.48).

Rickettsiae are arthropod-borne intracellular bacterial pathogens that primarily infect the microvascular endothelium leading to systemic spread of the organisms and the major pathophysiological effect, increased microvascular permeability, and edema in vital organs such as the lung and brain. Much work has been done on mechanisms of immunity to rickettsiae, as well as the responses of endothelial cells to rickettsial invasion. However, to date, no one has described the mechanisms of increased microvascular permeability during acute rickettsiosis. We sought to establish an in vitro model of human endothelial-target rickettsial infection using the etiological agent of Rocky Mountain spotted fever, Rickettsia rickettsii, and human cerebral microvascular endothelial cells. Endothelial cells infected with R. rickettsii exhibited a dose-dependent decrease in trans-endothelial electrical resistance, which translates into increased monolayer permeability. Additionally, we showed that the addition of pro-inflammatory stimuli essential to rickettsial immunity dramatically enhanced this effect. This increase in permeability correlates with dissociation of adherens junctions between endothelial cells and is not dependent on the presence of nitric oxide. Taken together, these results demonstrate for the first time that increased microvascular permeability associated with rickettsial infection is partly attributable to intracellular rickettsiae and partly attributable to the immune defenses that have evolved to protect the host from rickettsial spread.

The diagnosis of acute rickettsioses during the acute phase of the disease is challenging. We present preliminary evidence that antigen-capture using streptavidin-coated magnetic beads with biotinylated anti-rickettsia typhi rabbit polyclonal antibodies followed by electrochemiluminescent detection with ruthenylated antibodies of the same specificity could be used for the diagnosis of rickettsial diseases in the acute phase.