Cell death induction facilitates egress of Coxiella burnetii from infected host cells at late stages of infection.

Intracellular bacteria have evolved mechanisms to invade host cells, establish an intracellular niche that allows survival and replication, produce progeny, and exit the host cell after completion of the replication cycle to infect new target cells. Bacteria exit their host cell by (i) initiation of apoptosis, (ii) lytic cell death, and (iii) exocytosis. While bacterial egress is essential for bacterial spreading and, thus, pathogenesis, we currently lack information about egress mechanisms for the obligate intracellular pathogen C. burnetii, the causative agent of the zoonosis Q fever. Here, we demonstrate that C. burnetii inhibits host cell apoptosis early during infection, but induces and/or increases apoptosis at later stages of infection. Only at later stages of infection did we observe C. burnetii egress, which depends on previously established large bacteria-filled vacuoles and a functional intrinsic apoptotic cascade. The released bacteria are not enclosed by a host cell membrane and can infect and replicate in new target cells. In summary, our data argue that C. burnetii egress in a non-synchronous way at late stages of infection. Apoptosis-induction is important for C. burnetii egress, but other pathways most likely contribute.

Impact of Q-fever on physical and psychosocial functioning until 8 years after Coxiella burnetii infection: An integrative data analysis.

BACKGROUND: This study aimed to determine short- and long-term physical and psychosocial impact of Coxiella burnetii infection in three distinct entities: Q-fever fatigue syndrome (QFS), chronic Q-fever, and patients with past acute Q-fever without QFS or chronic Q-fever. METHODS: Integrative data analysis was performed, combining original data from eight studies measuring quality of life (QoL), fatigue, physical and social functioning with identical validated questionnaires, from three months to eight years after onset infection. Linear trends in each outcome were compared between Q-fever groups using multilevel linear regression analyses to account for repeated measures within patients. RESULTS: Data included 3947 observations of 2313 individual patients (228 QFS, 135 chronic Q-fever and 1950 patients with past acute Q-fever). In the first years following infection, physical and psychosocial impact was highest among QFS patients, and remained high without significant improvements over time. In chronic Q-fever patients, QoL and physical functioning worsened significantly over time. Levels of fatigue and social participation in patients with past acute Q-fever improved significantly over time. CONCLUSION: The impact differs greatly between the three Q-fever groups. It is important that physicians are aware of these differences, in order to provide relevant care for each patient group.

Epidemiological, clinical and laboratory features of acute Q fever in a cohort of hospitalized patients in a regional hospital, Israel, 2012-2018.

INTRODUCTION: Acute Q fever is endemic in Israel, yet the clinical and laboratory picture is poorly defined. METHODS: A retrospective study reviewing the medical records of acute Q fever patients, conducted in a single hospital in the Sharon district, Israel. Serum samples from suspected cases were preliminary tested by a qualitative enzyme immunoassay (EIA). Confirmatory testing at the reference laboratory used an indirect immunofluorescence assay (IFA). Positive cases were defined as fever with at least one other symptom and accepted laboratory criteria such as a single-phase II immunoglobulin G (IgG) antibody titer ≥1:200. Cases not fulfilling these criteria and in which acute Q fever was excluded, served as a control group. RESULTS: Between January 2012 and May 2018, 484 patients tested positive. After confirmatory testing, 65 (13.4%) were positive for acute Q fever (with requisite clinical picture), 171 (35.3%) were definitely not infected, the remaining 248 were excluded because of past/chronic/undetermined infection. The average age was 58 years and 66% were males. Most resided in urban areas with rare animal exposure. Pneumonia was seen in 57% of cases and a combination with headache/hepatitis was highly suggestive of acute Q fever diagnosis. Syncope/presyncope, fall and arthritis were more common in acute Q fever cases. Laboratory indexes were similar to the control group, except for erythrocyte sedimentation rate (ESR) which was more common and higher in the study group. CONCLUSION: Acute Q fever in the Sharon district could be better diagnosed by using a syndromic approach in combination with improved rapid diagnostic testing.

CLINICAL FINDINGS, PATHOLOGY, BIOSECURITY, AND SEROSURVEILLANCE OF COXIELLOSIS IN WHITE RHINOCEROSES (CERATOTHERIUM SIMUM) AT A CONSERVATION CENTER: TWO CASES.

A primiparous white rhinoceros (Ceratotherium simum) gave birth to a calf overnight after approximately 16 mo of gestation. The calf was found dead in the morning. Necrosuppurative placentitis with bacterial inclusions suggestive of coxiellosis was diagnosed histologically, and Coxiella burnetii was identified in fetal tissues and placenta by polymerase chain reaction and immunohistochemistry. Another primiparous female from the same herd aborted later that year after approximately 15 mo of gestation, and coxiellosis was similarly diagnosed in fetal tissues and on vaginal shedding. Estimates of exposure time, duration of vaginal shedding, and phase I and phase II antibody dynamics were determined retrospectively and prospectively for the two confirmed cases. Biosecurity measures were put in place to prevent guests, staff, and conspecific exposure to the organism. No other confirmed cases have occurred in the collection 3 yr after the initial cases. Coxiellosis outbreaks could represent an emerging threat to conservation efforts and ex situ white rhinoceros breeding programs.

Case Report: Α Case of Endocarditis and Embolic Stroke in a Child, Suggestive of Acute Q Fever Infection.

Acute Q fever is usually asymptomatic or is associated with a mild self-limited course and a favorable outcome. The occurrence of endocarditis during acute infection by Coxiella burnetii is an emerging clinical entity observed in adults that has been attributed to an autoimmune complication of early infection. Herein, we report the first case of a previously healthy 2-year-old child with endocarditis complicated by septic embolic stroke, in which the identified microbiological evidence was suggestive of acute rather than chronic C. burnetii infection. The development of endocarditis in this case occurred in the absence of any autoimmune reaction, but in the context of a very mild form of congenital heart disease, a small ventricular septal defect, which might serve as a predisposing factor for endocarditis. This case suggests that acute Q fever endocarditis may affect children as well and can be attributed not only to autoimmune mechanisms but also to a potential effect of the infectious agent per se on the cardiac endothelium in patients with underlying heart defects, regardless of their severity.

Novel multiparameter correlates of Coxiella burnetii infection and vaccination identified by longitudinal deep immune profiling.

Q-fever is a flu-like illness caused by Coxiella burnetii (Cb), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb, consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb.

Pathologic changes and immune responses against Coxiella burnetii in mice following infection via non-invasive intratracheal inoculation.

Q fever is a worldwide zoonosis caused by Coxiella burnetii. Human Q fever is typically acquired through inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In this study, BALB/c mice were infected with C. burnetii via an intratracheal (IT) route using a non-invasive aerosol pulmonary delivery device to directly place the living C. burnetii organisms into the lungs of the mice. The bacterial loads, pathological lesions, and antibody and cellular responses were analyzed and compared with those of mice infected via an intraperitoneal (IP) route. Compared with mice infected via an IP route, mice infected via an IT route exhibited a higher bacterial load and more severe pathological lesions in the heart and lungs at days 3 and 7 post-infection (pi). The levels of interferon-γ and IL-12p70 in the serum of mice infected via the IT route were significantly higher than those of mice infected via the IP route at day 3 pi. In conclusion, this murine model of acute C. burnetii infection via IT inoculation closely resembles the natural route of C. burnetii infection than that of IP injection. Thus, this newly developed model will be useful for investigating the pathogenesis and immunity of C. burnetii aerosol infection, as well as for the evaluation of therapeutic drugs and preventive vaccines of Q fever.

Acute phase proteins, proinflammatory cytokines and oxidative stress biomarkers in sheep, goats and she-camels with Coxiella burnetii infection-induced abortion.

Acute phase proteins (APPs) and oxidative stress are helpful markers in diagnosis of several infectious diseases. APPs, proinflammatory cytokines and oxidative stress markers were evaluated for their role in the diagnosis of naturally acquired Coxiella burnetii (Q fever) associated with abortion in sheep, goats and she-camels. Blood, aborted materials and vaginal swabs were collected from mixed herds in the Eastern Province of Saudi Arabia. Antioxidant biomarkers showed significant decline in cases of abortion compared to control animals at delivery time. The correlation between disease status and all parameters ranged from moderate to high. The APPs, cytokines and the oxidative stress marker malondialdehyde (MDA) displayed a high degree of distinction between aborted sheep and goat and normal delivered animals (AUC > 0.90). However, only MDA showed a high degree of differentiation (AUC > 0.90) between aborted she-camels and normal delivered controls. In conclusion, results from our study allow us to recommend using APPs, cytokines and oxidative stress markers as an additional tool for diagnosis of naturally occurring C. burnetii infection in sheep, goats and she-camels. However, it does not replace standard procedures for detection of C. burnetii.

"Hairiness" is a Facsimile of Reorganized Cytoskeletons: A Cytopathic Effect of Coxiella burnetii.

In 1993, I reported that Coxiella burnetii transforms human B cells into hairy cells (cbHCs), the first hairy cell reported outside of hairy cell leukemia (HCL). Over last few decades, advances in molecular biology have provided evidence supporting that C. burnetii induces hairiness and inhibits the apoptosis of host cells. The present review summarizes new information in support of cbHC. C. burnetii was shown to induce reorganization of the cytoskeleton and to inhibit apoptosis in host cells. Peritoneal B1a cells were found to be permissive for virulent C. burnetii Nine Mile phase I (NMI) strains in mice. C. burnetii severely impaired E-cad expression in circulating cells of Q fever patients. B-cell non-Hodgkin lymphoma was linked to C. burnetii. Mutation of BRAF V600E was pronounced in HCL, but "hairiness" was not linked to the mutation. Risk factors shared among coxiellosis and HCL in humans and animals were reported in patients with Q-fever. Accordingly, I propose that C. burnetii induces reorganization of the cytoskeleton and inhibits apoptosis as cytopathic effects that are not target cell specific. The observed hairiness in cbHC appears to be a fixed image of dynamic nature, and hairy cells in HCL are distinct among lymphoid cells in circulation. As the cytoskeleton plays key roles in maintaining cell structural integrity in health and disease, the pathophysiology of similar cytopathic effects should be addressed in other diseases, such as myopathies, B-cell dyscrasias, and autoimmune syndromes.

Q Fever in Southern California: a Case Series of 20 Patients from a VA Medical Center.

Query fever (Q fever), caused by Coxiella burnetii, was first described in southern California in 1947. It was found to be endemic and enzoonotic to the region and associated with exposure to livestock. We describe a series of 20 patients diagnosed with Q fever at a Veterans Affairs hospital in southern California, with the aim of contributing toward the understanding of Q fever in this region. Demographics, laboratory data, diagnostic imaging, risk factors, and treatment regimens were collected via a retrospective chart review of patients diagnosed with Q fever at our institution between 2000 and 2016. Cases were categorized as acute or chronic and confirmed or probable. The majority presented with an acute febrile illness (90%). There was a delay in ordering diagnostic serology from the time of symptom onset (acute cases, average 31.9 days; chronic cases, average 63 days), and 15% progressed from acute to chronic infection. Of the chronic cases, 22.2% had endocarditis, 22.2% had endovascular infection, and 11.1% had both endocarditis and endovascular infection. The geographic distribution revealed that 20% resided in rural areas. Of the cases of Q fever that died, death attributed to Q fever was associated with an average diagnostic delay of 65.5 days. Acute Q fever is underreported in this region largely because of its often nonspecific clinical presentation.

Epidemiological, clinical and laboratory characteristics of acute Q fever in an endemic area in Israel, 2006-2016.

Our purpose was to describe the clinical, epidemiological and laboratory characteristics of patients hospitalised with acute Q fever in an endemic area of Israel. We conducted a historical cohort study of all patients hospitalised with a definite diagnosis of acute Q fever, and compared them to patients suspected to have acute Q fever, but diagnosis was ruled out. A total of 38 patients had a definitive diagnosis, 47% occurred during the autumn and winter seasons, only 18% lived in rural regions. Leucopaenia and thrombocytopaenia were uncommon (16% and 18%, respectively), but mild hepatitis was common (mean aspartate aminotransferase 76 U/l, mean alanine aminotransferase 81 U/l). We compared them with 74 patients in which acute Q fever was ruled out, and found that these parameters were not significantly different. Patients with acute Q fever had a shorter hospitalisation and they were treated more often with doxycycline than those without acute Q fever (6.4 vs. 14 days, P = 0.007, 71% vs. 38%, P = 0.001, respectively). In conclusion, acute Q fever can manifest as an unspecified febrile illness, with no seasonality. We suggest that in endemic areas, Q fever should be considered in the differential diagnosis in any febrile patient with risk factors for a persistent infection.

The sexual dimorphism of anticardiolipin autoantibodies in acute Q fever patients.

OBJECTIVES: Q fever is a zoonotic disease caused by Coxiella burnetii which affects men more than women (sex ratio men/women: 2.2). Acute Q fever complications are associated with elevation of anticardiolipin (aCL) antibodies. Here, we investigate the sexual dimorphism of aCL antibodies during acute C. burnetii infection. METHODS: IgG aCL antibodies were evaluated at the time of Q fever serological diagnosis with enzyme-linked immunosorbent assay. Results were analysed according to sex. RESULTS: Among the 1323 patients with Q fever tested for aCL, 1013 had acute Q fever (692 men/321 women) and 310 had persistent focalized infection (226 men/84 women). In cases of acute Q fever, men presented a significantly higher proportion of positive aCL antibodies (351/692, 50.7%) than women (113/321, 35.2%) (p <0.05). In addition, men had significantly higher aCL antibodies levels than women (p <0.001). CONCLUSIONS: We highlight a relationship between sex and markers of autoimmunity during Q fever. Further investigations are necessary to better understand the mechanisms of this sexual dimorphism.

Comparative virulence of diverse Coxiella burnetii strains.

Coxiella burnetii is an intracellular, gram-negative bacterium that causes the zoonosis Q fever. This disease typically presents as an acute flu-like illness with persistent, focalized infections occurring less frequently. Clinical outcomes of Q fever have been associated with distinct genomic groups of C. burnetii, suggesting that gene content is responsible for virulence potential. To investigate this hypothesis, the virulence of thirteen C. burnetii strains (representing genomic groups I-VI) was evaluated in a guinea pig infection model by intraperitoneal injection. Seven strains caused a sustained fever (at least two days ≥39.5°C) in at least half of the animals within each experimental group. At fourteen days post infection, animals were euthanized and additional endpoints were evaluated, including splenomegaly and serology. The magnitude of these endpoints roughly correlated with the onset, duration, and severity of fever. The most severe disease was caused by group I strains. Intermediate and no virulence were evidenced following infection with group II-V and group VI strains, respectively. Flow cytometric analysis of the mesenteric lymph nodes revealed decreased CD4+ T cell frequency following infection with highly virulent group I strains. These findings buttress the hypothesis that the pathogenic potential of C. burnetii strains correlates with genomic grouping. These data, combined with comparative genomics and genetic manipulation, will improve our understanding of C. burnetii virulence determinants.

Genetic variations in innate immunity genes affect response to Coxiella burnetii and are associated with susceptibility to chronic Q fever.

OBJECTIVES: Chronic Q fever is a persistent infection, mostly of aortic aneurysms, vascular prostheses or damaged heart valves, caused by the intracellular bacterium Coxiella burnetii. Only a fraction of C. burnetii-infected individuals at risk develop chronic Q fever. In these individuals, a defective innate immune response may contribute to the development of chronic Q fever. We assessed whether genetic variations in genes involved in the killing machinery for C. burnetii by macrophages, contribute to the progression to chronic Q fever. METHODS: The prevalence of 66 single nucleotide polymorphisms (SNPs) in 31 genes pivotal in phagolysosomal maturation, bacterial killing and autophagy, was determined in 173 chronic Q fever patients and 184 controls with risk factors for chronic Q fever and serological evidence of a C. burnetii infection. Associations were detected with univariate logistic regression models. To assess the effect of these SNPs on innate responses to C. burnetii, the C. burnetii-induced cytokine production and basal reactive oxygen species production of healthy volunteers was determined. RESULTS: RAB7A (rs13081864) and P2RX7 loss-of-function SNP (rs3751143) were more common in chronic Q fever patients than in controls. RAB5A (rs8682), P2RX7 gain-of-function SNP (rs1718119), MAP1LC3A (rs1040747) and ATG5 (rs2245214) were more common in controls. In healthy volunteers, RAB7A (rs13081864) and MAP1LC3A (rs1040747) influenced the C. burnetii-induced cytokine production. RAB7A (rs13081864) modulated basal reactive oxygen species production. CONCLUSIONS: RAB7A (rs13081864) and P2RX7 (rs3751143) are associated with the development of chronic Q fever, whereas RAB5A (rs8682), P2RX7 (rs1718119), MAP1LC3A (rs1040747) and ATG5 (rs2245214) may have protective effects.

Long-term effect of cognitive behavioural therapy and doxycycline treatment for patients with Q fever fatigue syndrome: One-year follow-up of the Qure study.

BACKGROUND: Previously, we reported a randomized placebo-controlled trial, the Qure study, showing that cognitive behavioural therapy (CBT), and not doxycycline, was significantly more effective than placebo in reducing fatigue severity in Q fever fatigue syndrome (QFS) patients. This follow-up study evaluates the long-term effect of these treatment regimens, 1 year after completion of the original trial. METHODS: All patients who completed the Qure study, CBT (n = 50), doxycycline (n = 52), and placebo (n = 52), were included in this follow-up study. Between twelve and fifteen months after end of treatment (EOT), patients filled out web-based questionnaires including the main outcome measure fatigue severity, assessed with the Checklist Individual Strength (CIS), subscale fatigue severity. RESULTS: Fatigue severity in the CBT, but not doxycycline or placebo, group was significantly increased at follow-up compared to EOT (respective means 39.5 [95% CI, 36.2-42.9] and 31.3 [95% CI, 27.5-35.1], mean difference 8.2 [95% CI, 4.9-11.6]; P < .001). Fatigue severity scores of CBT (adjusted mean 39.8 [95% CI, 36.1-43.4]) and doxycycline (adjusted mean 41.0 [95% CI, 37.5-44.6]) groups did not significantly differ from the placebo group (adjusted mean 37.1 [95% CI, 33.6-40.7]; P = .92 and P = .38, respectively). CONCLUSION: The beneficial effect of CBT on fatigue severity at EOT was not maintained 1 year thereafter. Due to its initial beneficial effect and side effects of long-term doxycycline use, we still recommend CBT as treatment for QFS. We suggest further investigation on tailoring CBT more to QFS, possibly followed by booster sessions.

Cytokine profiles in patients with Q fever fatigue syndrome.

BACKGROUND: Q fever fatigue syndrome (QFS) is a state of prolonged fatigue following around 20% of acute Q fever cases. It is thought that chronic inflammation plays a role in its etiology. To test this hypothesis we measured circulating cytokines and the ex-vivo cytokine production in patients with QFS and compared with various control groups. MATERIALS/METHODS: Peripheral blood mononuclear cells (PBMCs), whole blood, and serum were collected from 20 QFS patients, 19 chronic fatigue syndrome (CFS) patients, 19 Q fever seropositive controls, and 25 age- and sex-matched healthy controls. Coxiella-specific ex-vivo production of tumor necrosis factor (TNF)α, interleukin (IL)-1ß, IL-6, and interferon (IFN) was measured, together with a total of 92 circulating inflammatory proteins. RESULTS: PBMCs of QFS patients produced more IL-6 (P = 0.0001), TNFα (P = 0.0002), and IL-1ß (P = 0.0005) than the various control groups when stimulated with Coxiella antigen. QFS patients had distinct differences in circulating inflammatory markers compared to the other groups, including higher concentrations of circulating IL-6 and IFNγ. CONCLUSION: QFS patients showed signs of chronic inflammation compared to asymptomatic Q fever seropositive controls, CFS patients, and healthy controls, of which the monocyte-derived cytokines TNFα, IL-1ß, and especially IL-6, are likely crucial components.