Early diagnosis of Ehrlichia ewingii infection in a lung transplant recipient by peripheral blood smear.

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.

High serum CXCL10 in Rickettsia conorii infection is endothelial cell mediated subsequent to whole blood activation.

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

Increased expression of the homeostatic chemokines CCL19 and CCL21 in clinical and experimental Rickettsia conorii infection.

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.

A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine.

The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing COVID-19, but they do not induce efficient mucosal immunity or prevent viral transmission. Here, we report the first noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free, mucosal vaccine harboring engineered Spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of two doses of this T4 SARS-CoV-2 vaccine 21 days apart induced robust mucosal immunity, in addition to strong systemic humoral and cellular immune responses. The intranasal vaccine induced broad virus neutralization antibody titers against multiple variants, Th1-biased cytokine responses, strong CD4+ and CD8+ T cell immunity, and high secretory IgA titers in sera and bronchoalveolar lavage specimens from vaccinated mice. All of these responses were much stronger in intranasally vaccinated mice than those induced by the injected vaccine. Furthermore, the nasal vaccine provided complete protection and sterilizing immunity against the mouse-adapted SARS-CoV-2 MA10 strain, the ancestral WA-1/2020 strain, and the most lethal Delta variant in both BALB/c and human angiotensin converting enzyme (hACE2) knock-in transgenic mouse models. In addition, the vaccine elicited virus-neutralizing antibodies against SARS-CoV-2 variants in bronchoalveolar lavage specimens, did not affect the gut microbiota, exhibited minimal lung lesions in vaccinated and challenged mice, and is completely stable at ambient temperature. This modular, needle-free, phage T4 mucosal vaccine delivery platform is therefore an excellent candidate for designing efficacious mucosal vaccines against other respiratory infections and for emergency preparedness against emerging epidemic and pandemic pathogens. IMPORTANCE According to the World Health Organization, COVID-19 may have caused ~15-million deaths across the globe and is still ravaging the world. Another wave of ~100 million infections is predicted in the United States due to the emergence of highly transmissible immune-escaped Omicron variants. The authorized vaccines would not prevent these transmissions since they do not trigger mucosal immunity. We circumvented this limitation by developing a needle-free, bacteriophage T4-based, mucosal vaccine. This intranasally administered vaccine generates superior mucosal immunity in mice, in addition to inducing robust humoral and cell-mediated immune responses, and provides complete protection and sterilizing immunity against SARS-CoV-2 variants. The vaccine is stable, adjuvant-free, and cost-effectively manufactured and distributed, making it a strategically important next-generation COVID vaccine for ending this pandemic.

Surface-expressed enolase contributes to the pathogenesis of clinical isolate SSU of Aeromonas hydrophila.

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound 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 alpha(2)-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 of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia 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 ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.

David Versus Goliath: The Clinical Consequences of an Influenza A Viral Infection in an Unvaccinated Patient.

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.

Braun lipoprotein (Lpp) contributes to virulence of yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague.

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 x 10(7) CFU of the Deltalpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Deltalpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD(50)). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Deltalpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Deltalpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Deltalpp mutant than in those infected with WT CO92. Additionally, the Deltalpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Deltalpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.

Emerging pathogens: challenges and successes of molecular diagnostics.

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.

ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaques.

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 3⁻4 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.

Attitudes and Beliefs of Pathology Residents Regarding the Subspecialty of Clinical Chemistry: Results of a Survey.

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.

Early diagnosis of rickettsioses by electrochemiluminscence.

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.

An Intradermal Inoculation Mouse Model for Immunological Investigations of Acute Scrub Typhus and Persistent Infection.

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/ß, 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.

Rickettsial infections.

Rickettsiae are obligate intracellular alpha-proteobacteria that primarily target the microvascular endothelium. In the last two decades, new rickettsial pathogens have been associated with human illness around the world. Clinically, the common denominator in all rickettsioses is the development of increased microvascular permeability, leading to cerebral and non-cardiogenic pulmonary edema. With the development of powerful research tools, advances in the understanding of rickettsial pathogenesis have been dramatic. Entry into the host cell is followed by rapid escape into the cytoplasm to avoid phagolysosomal fusion. Spotted fever group rickettsiae induce actin polymerization via a group of proteins called RickA, which promote nucleation of actin monomers via the Arp2/3 complex at one rickettsial pole, propelling the bacteria across the cytoplasm and into neighboring cells. Damage to the host cell is most likely multifactorial. The most extensively studied mechanism is the generation of reactive oxygen species (ROS) and downregulation of enzymes involved in protection against oxidative injury. The significance of ROS-mediated cellular damage in vivo is beginning to be elucidated. The main pathogenic mechanism is increased microvascular permeability leading to profound metabolic disturbances in the extravascular compartment. The underlying factors responsible for those changes are beginning to be elucidated in vitro and include direct effects of intracellular rickettsiae, cytokines, and possibly activated coagulation factors--all of which most likely modify interendothelial junctions. Our knowledge on rickettsial pathogenesis will continue to expand in the near future as new research tools become available.

Nitric oxide as a mediator of increased microvascular permeability during acute rickettsioses.

Rickettsiae primarily infect the microvascular endothelium, leading to changes in microvascular permeability that result in potentially severe pulmonary and cerebral edema. The mechanisms responsible for these changes are not well understood. One potential mechanism of increased vascular permeability is the anti-rickettsial nitric oxide response described by Walker and colleagues. We hypothesized that anti-rickettsial levels of nitric oxide adversely affects microvascular permeability in vitro. To this end we sought to describe the effects of exogenous nitric oxide on the proliferation of intracellular rickettsiae while monitoring the transendothelial electrical resistance as a measure of endothelial barrier integrity. It was determined that the addition of the NO-donor DETA NONOate at certain levels results in a dose-dependent change in electrical resistance across the monolayer while effectively limiting the number of intracellular rickettsiae in human microvascular endothelial cells. The data presented support the idea that nitric oxide produced by infected endothelial cells may be contributing to the changes in vascular permeability that occur during acute rickettsioses. Future experiments aim to elaborate on these results in a model that more clearly depicts the in vivo response as well as to describe the changes that occur with respect to interendothelial junctions.

Pathogenic mechanisms of diseases caused by Rickettsia.

The specter of bioterrorism employing genetically engineered Rickettsia resistant to all antibiotics should reawaken the world's desire to elucidate the pathogenesis of typhus and spotted fever rickettsioses in a search for mechanisms vulnerable to interdiction. The pathogenetic sequence includes rickettsial entry into the dermis, hematogenous dissemination to vascular endothelial cells (most critically in brain and lungs), increased vascular permeability, edema, and immunity mediated by NK cells, IFN-gamma, TNF-alpha, RANTES, antibodies, and cytotoxic T lymphocytes. Silverman has demonstrated the role of reactive oxygen species (ROS) produced by R. rickettsii-infected endothelial cells in peroxidative damage to cell membranes in vitro, and Heinzen has described actin-based rickettsial intracellular mobility and intercellular spread. At this point the availability of sequences of rickettsial genomes and excellent animal models of rickettsioses have yielded insufficient progress towards the identification of rickettsial virulence factors and knowledge of the importance of injury mediated by ROS, phospholipase A(2), protease(s) or other mechanisms in vivo. Attention to the rickettsiosis-associated procoagulant state led to determination that hemostatic mechanisms largely prevent major hemorrhage without disseminated intravascular coagulation or thrombosis-mediated ischemia. Particularly lacking is knowledge of early events in vivo at the portal of entry in skin (or lung), of the effects of the inoculum medium (arthropod saliva or feces), mediators produced by infected endothelium under conditions of flow and of the contributions in vivo of immune effectors to pathology, of the role of apoptosis in rickettsial infection, and of the endothelial cell alterations that account for increased vascular permeability. The host cell receptor for the Rickettsia ligand and the mechanism of rickettsial escape from the phagosome need to be elucidated.

Current status of immune mechanisms of killing of intracellular microorganisms.

The interaction between intracellular pathogens and the mammalian host follows different pathways that reflect evolved survival mechanisms of both the pathogen and the host to assure each one's own survival. From the host's perspective, different immune mechanisms predominate at different stages of infection. Both phagocytic and non-phagocytic target cells participate in microbial uptake and, in some cases, intracellular destruction. In addition, the development of specific immunity ensures sustained activation of intracellular microbicidal mechanisms in the target cells, and induction of apoptotic or lytic target cell death by cytotoxic T lymphocytes. From the pathogen's perspective, different evasion strategies are employed to counteract host defenses. Understanding microbial survival strategies and the immune mechanisms that result in killing of intracellular pathogens will deepen our insight into the pathogenesis of infection that could be applied towards the development of effective vaccination and immunotherapy.

Human ehrlichioses.

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.

Ehrlichia chaffeensis: a prevalent, life-threatening, emerging pathogen.

Ehrlichia chaffeensis are small, obligately intracellular, endosomal bacteria with tropism for macrophages. Persistent infection in reservoir white-tailed deer is transmitted by lone star ticks. Flu-like illness can progress to severe multisystem disease with toxic shock-like syndrome, meningitis, or ARDS. The case-fatality rate is 2.7%. Leukopenia and thrombocytopenia are diagnostically useful. Granulomas are associated with control of the infection. Ehrlichial proteins and glycoproteins have been sequenced and expressed for diagnostic serology and vaccine development. Mouse models (mild disease and persistent infection with E. muris and fatal monocytotropic ehrlichiosis with a Japanese tick isolate) revealed that CD4 and CD8 T type 1 lymphocyte responses, IFN-gamma, TNF-alpha, and antibodies play roles in protective immunity, while a weak CD4 T-helper response, overproduction of TNF-alpha, and very high IL-10 are associated with toxic shock-like mortality. Protection against fatal ehrlichiosis was achieved by prior infection with low virulence E. muris. Acute clinical diagnosis is difficult except by PCR. Response to doxycycline is dramatic.

Rapid detection of trace bacteria in biofluids using porous monoliths in microchannels.

We present advancements in microfluidic technology for rapid detection of as few as 10 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 (min), 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 1000-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.