A Functional Genomics Approach to Understand Variation in Cytokine Production in Humans.

As part of the Human Functional Genomics Project, which aims to understand the factors that determine the variability of immune responses, we investigated genetic variants affecting cytokine production in response to ex vivo stimulation in two independent cohorts of 500 and 200 healthy individuals. We demonstrate a strong impact of genetic heritability on cytokine production capacity after challenge with bacterial, fungal, viral, and non-microbial stimuli. In addition to 17 novel genome-wide significant cytokine QTLs (cQTLs), our study provides a comprehensive picture of the genetic variants that influence six different cytokines in whole blood, blood mononuclear cells, and macrophages. Important biological pathways that contain cytokine QTLs map to pattern recognition receptors (TLR1-6-10 cluster), cytokine and complement inhibitors, and the kallikrein system. The cytokine QTLs show enrichment for monocyte-specific enhancers, are more often located in regions under positive selection, and are significantly enriched among SNPs associated with infections and immune-mediated diseases. PAPERCLIP.

Viable Coxiella burnetii Induces Differential Cytokine Responses in Chronic Q Fever Patients Compared to Heat-Killed Coxiella burnetii.

Cytokine responses of chronic Q fever patients to the intracellular bacterium Coxiella burnetii have mostly been studied using ex vivo stimulation of immune cells with heat-killed C. burnetii due to the extensive measures needed to work with viable biosafety level 3 agents. Whether research with heat-killed C. burnetii can be translated to immune responses to viable C. burnetii is imperative for the interpretation of previous and future studies with heat-killed C. burnetii Peripheral blood mononuclear cells (PBMCs) of chronic Q fever patients (n = 10) and healthy controls (n = 10) were stimulated with heat-killed or viable C. burnetii of two strains, Nine Mile and the Dutch outbreak strain 3262, for 24 h, 48 h, and 7 days in the absence or presence of serum containing anti-C. burnetii antibodies. When stimulated with viable C. burnetii, PBMCs of chronic Q fever patients and controls produced fewer proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha, and IL-1ß) after 24 h than after stimulation with heat-killed C. burnetii In the presence of Q fever seronegative serum, IL-10 production was higher after stimulation with viable rather than heat-killed C. burnetii; however, when incubating with anti-C. burnetii antibody serum, the effect on IL-10 production was reduced. Levels of adaptive, merely T-cell-derived cytokine (gamma interferon, IL-17, and IL-22) and CXCL9 production were not different between heat-killed and viable C. burnetii stimulatory conditions. Results from previous and future research with heat-killed C. burnetii should be interpreted with caution for innate cytokines, but heat-killed C. burnetii-induced adaptive cytokine production is representative of stimulation with viable bacteria.

Interferon-γ and CXCL10 responses related to complaints in patients with Q fever fatigue syndrome.

Approximately 20% of patients with acute Q fever develop Q fever fatigue syndrome (QFS), a debilitating fatigue syndrome. This study further investigates the role of C. burnetii-specific IFNγ, but also IL-2, CXCL9, CXCL10, and CXLC11 production in QFS patients. C. burnetii-specific IFNy, IL-2, CXCL9, CXCL10, and CXCL11 production were tested in ex vivo stimulated whole blood of QFS patients who recovered from their complaints (n = 8), QFS patients with persisting complaints (n = 27), and asymptomatic Q fever seropositive controls (n = 10). With the exclusion of one outlier, stimulation with C. burnetii revealed significantly higher IFNy and CXCL10 production in QFS patients with persisting complaints (medians 288.0 and 176.0 pg/mL, respectively) than in QFS patients who recovered from their complaints (medians 93.0 and 85.5 pg/mL, respectively) (p = 0.041 and 0.045, respectively). No significant differences between groups were found for C. burnetii-specific IL-2, CXCL9, and CXCL11 production. These findings point towards a difference in cell-mediated immunity in QFS patients with persisting complaints compared to those who recovered from their complaints. Such a difference may aid to eventually diagnose QFS more objectively and might serve as an indicator of its underlying etiology.

CXCL9, a promising biomarker in the diagnosis of chronic Q fever.

BACKGROUND: In the aftermath of the largest Q fever outbreak in the world, diagnosing the potentially lethal complication chronic Q fever remains challenging. PCR, Coxiella burnetii IgG phase I antibodies, CRP and 18F-FDG-PET/CT scan are used for diagnosis and monitoring in clinical practice. We aimed to identify and test biomarkers in order to improve discriminative power of the diagnostic tests and monitoring of chronic Q fever. METHODS: We performed a transcriptome analysis on C. burnetii stimulated PBMCs of 4 healthy controls and 6 chronic Q fever patients and identified genes that were most differentially expressed. The gene products were determined using Luminex technology in whole blood samples stimulated with heat-killed C. burnetii and serum samples from chronic Q fever patients and control subjects. RESULTS: Gene expression of the chemokines CXCL9, CXCL10, CXCL11 and CCL8 was strongly up-regulated in C. burnetii stimulated PBMCs of chronic Q fever patients, in contrast to healthy controls. In whole blood cultures of chronic Q fever patients, production of all four chemokines was increased upon C. burnetii stimulation, but also healthy controls and past Q fever individuals showed increased production of CXCL9, CXCL10 and CCL8. However, CXCL9 and CXCL11 production was significantly higher for chronic Q fever patients compared to past Q fever individuals. In addition, CXCL9 serum concentrations in chronic Q fever patients were higher than in past Q fever individuals. CONCLUSION: CXCL9 protein, measured in serum or as C. burnetii stimulated production, is a promising biomarker for the diagnosis of chronic Q fever.

Genetic variation in TLR10 is not associated with chronic Q fever, despite the inhibitory effect of TLR10 on Coxiella burnetii-induced cytokines in vitro.

Coxiella burnetii, the causative agent of Q fever, is recognized by TLR2. TLR10 can act as an inhibitory receptor on TLR2-derived immune responses. Therefore, we investigated the role of TLR10 on C. burnetii-induced cytokine production and assessed whether genetic polymorphisms in TLR10 influences the development of chronic Q fever. HEK293 cells, transfected with TLR2, TLR10 or TLR2/TLR10, and human peripheral blood mononuclear cells (PBMCs) in the presence of anti-TLR10, were stimulated with C. burnetii. In both assays, the absence of TLR10 resulted in increased cytokine responses after C. burnetii stimulation. In addition, the effect of single nucleotide polymorphisms (SNPs) in TLR10 was examined in healthy volunteers whose PBMCs were stimulated with C. burnetii Nine Mile or the Dutch outbreak isolate C. burnetii 3262. Individuals bearing SNPs in TLR10 displayed increased cytokine production upon C. burnetii 3262 stimulation. Furthermore, 139 chronic Q fever patients and 220 controls were genotyped for TLR10 N241H, I775V and I369L. None of these polymorphisms were associated with increased susceptibility to chronic Q fever. In conclusion, TLR10 has an inhibitory effect on in vitro cytokine production by C. burnetii, but the presence of TLR10 polymorphisms does not lead to an increased risk of developing chronic Q fever.

A fatal case of disseminated chronic Q fever: a case report and brief review of the literature.

BACKGROUND: Chronic Q fever is a rare infection, which mainly manifests as endocarditis, infection of vascular prostheses or aortic aneurysms. We present the case of a 74-year-old immunocompromised man with a haematologically disseminated Coxiella burnetii infection, which has never been reported before. CASE REPORT: He was diagnosed with a chronic Q fever infection of an aneurysm with an endovascular prosthesis in 2015, but he died despite optimal treatment. Autopsy revealed a disseminated C. burnetii infection, confirmed by a positive PCR on samples from several organs. Retrospectively, he already had complaints and signs of inflammation since 2012, for which he had already been admitted in February 2014. At that time, Q fever diagnostics using PCR, complement fixation assay, and enzyme-linked immunosorbent assay on serum were all negative. In retrospect however, retesting available samples from February 2014 using immunofluorescence assay (IFA) already revealed serology compatible with chronic Q fever. CONCLUSION: Clinicians should be aware of this silent killer, especially in case of risk factors, and perform an appropriate diagnostic work-up for Q fever including IFA serology and PCR.

Recognition of Coxiella burnetii by toll-like receptors and nucleotide-binding oligomerization domain-like receptors.

BACKGROUND: Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against microorganisms, but little is known about their role in the recognition of C. burnetii in humans. METHODS: Human peripheral blood mononuclear cells (PBMCs) were stimulated with C. burnetii Nine Mile and the Dutch outbreak isolate C. burnetii 3262. TLRs were inhibited using specific antibodies or antagonists. Additionally, the influence of human polymorphisms in TLRs and Nod-like receptors (NLRs) on C. burnetii-induced cytokine production was assessed. RESULTS: Inhibition of TLR2, p38, JNK, and ERK led to decreased cytokine responses in C. burnetii-stimulated human PBMCs. Humans with polymorphisms in TLR1 and NOD2 had reduced cytokine production, compared with humans with wild-type genotypes, after stimulation. Interestingly, polymorphisms in TLR6 led to decreased cytokine production after C. burnetii 3262 stimulation but not after C. burnetii Nine Mile stimulation. CONCLUSIONS: The TLR1/TLR2 heterodimer and NOD2 are important recognition receptors for the induction of cytokine responses against C. burnetii in humans. Furthermore, an interesting finding was the divergent recognition of C. burnetii Nine Mile and C. burnetii 3262.

Genetic Variation in Pattern Recognition Receptors and Adaptor Proteins Associated With Development of Chronic Q Fever.

BACKGROUND: Q fever is an infection caused by Coxiella burnetii. Persistent infection (chronic Q fever) develops in 1%-5% of patients. We hypothesize that inefficient recognition of C. burnetii and/or activation of host-defense in individuals carrying genetic variants in pattern recognition receptors or adaptors would result in an increased likelihood to develop chronic Q fever. METHODS: Twenty-four single-nucleotide polymorphisms in genes encoding Toll-like receptors, nucleotide-binding oligomerization domain-like receptor-2, αvß3 integrin, CR3, and adaptors myeloid differentiation primary response protein 88 (MyD88), and Toll interleukin 1 receptor domain-containing adaptor protein (TIRAP) were genotyped in 139 patients with chronic Q fever and in 220 controls with cardiovascular risk-factors and previous exposure to C. burnetii. Associations between these single-nucleotide polymorphisms and chronic Q fever were assessed by means of univariate logistic regression models. Cytokine production in whole-blood stimulation assays was correlated with relevant genotypes. RESULTS: Polymorphisms in TLR1 (R80T), NOD2 (1007fsX1), and MYD88 (-938C>A) were associated with chronic Q fever. No association was observed for polymorphisms in TLR2, TLR4, TLR6, TLR8, ITGAV, ITGB3, ITGAM, and TIRAP. No correction for multiple testing was performed because only genes with a known role in initial recognition of C. burnetii were included. In the whole-blood assays, individuals carrying the TLR1 80R-allele showed increased interleukin 10 production with C. burnetii exposure. CONCLUSIONS: Polymorphisms in TLR1 (R80T), NOD2 (L1007fsX1), and MYD88 (-938C>A) are associated with predisposition to development of chronic Q fever. For TLR1, increased interleukin 10 responses to C. burnetii in individuals carrying the risk allele may contribute to the increased risk of chronic Q fever.

Acute and probable chronic Q fever during anti-TNFα and anti B-cell immunotherapy: a case report.

BACKGROUND: Q fever is caused by the intracellular bacterium Coxiella burnetii. Initial infection can present as acute Q fever, while a minority of infected individuals develops chronic Q fever endocarditis or vascular infection months to years after initial infection. Serology is an important diagnostic tool for both acute and chronic Q fever. However, since immunosuppressive drugs may hamper the humoral immune response, diagnosis of Q fever might be blurred when these drugs are used. CASE PRESENTATION: A 71-year-old Caucasian male was diagnosed with symptomatic acute Q fever (based on positive C. burnetii PCR followed by seroconversion) while using anti-tumor necrosis factor-α (anti-TNFα) drugs for rheumatoid arthritis (RA). He was treated for two weeks with moxifloxacin. After 24 months of follow-up, the diagnosis of probable chronic Q fever was established based on increasing anti-C. burnetii phase I IgG antibody titres in a immunocompromised patient combined with clinical suspicion of endocarditis. At the time of chronic Q fever diagnosis, he had been treated with anti B-cell therapy for 16 months. Antibiotic therapy consisting of 1.5 years doxycycline and hydroxychloroquine was started and successfully completed and no signs of relapse were seen after more than one year of follow-up. CONCLUSION: The use of anti-TNFα agents for RA in the acute phase of Q fever did not hamper the C. burnetii-specific serological response as measured by immunofluorescence assay. However, in the presented case, an intact humoral response did not prevent progression to probable chronic C. burnetii infection, most likely because essential cellular immune responses were suppressed during the acute phase of the infection. Despite the start of anti-B-cell therapy with rituximab after the acute Q fever episode, an increase in anti-C. burnetii phase I IgG antibodies was observed, supporting the notion that C. burnetii specific CD20-negative memory B-cells are responsible for this rise in antibody titres.

European society of clinical microbiology and infectious diseases guidelines for antimicrobial stewardship in emergency departments (endorsed by European association of hospital pharmacists).

SCOPE: This European Society of Clinical Microbiology and Infectious Diseases guideline provides evidence-based recommendations to support a selection of appropriate antibiotic use practices for patients seen in the emergency department (ED) and guidance for their implementation. The topics addressed in this guideline are (a) Do biomarkers or rapid pathogen tests improve antibiotic prescribing and/or clinical outcomes? (b) Does taking blood cultures in common infectious syndromes improve antibiotic prescribing and/or clinical outcomes? (c) Does watchful waiting without antibacterial therapy or with delayed antibiotic prescribing reduce antibiotic prescribing without worsening clinical outcomes in patients with specific infectious syndromes? (d) Do structured culture follow-up programs in patients discharged from the ED with cultures pending improve antibiotic prescribing? METHODS: An expert panel was convened by European Society of Clinical Microbiology and Infectious Diseases and the guideline chair. The panel selected in consensus the four most relevant antimicrobial stewardship topics according to pre-defined relevance criteria. For each main question for the four topics, a systematic review was performed, including randomized controlled trials and observational studies. Both clinical outcomes and stewardship process outcomes related to antibiotic use were deemed relevant. The literature searches were conducted between May 2021 and March 2022. In April 2022, the panel members were formally asked to suggest additional studies that were not identified in the initial searches. Data were summarized in a meta-analysis if possible or otherwise summarized narratively. The certainty of the evidence was classified according to the Grading of Recommendations Assessment, Development and Evaluation criteria. The guideline panel reviewed the evidence per topic critically appraising the evidence and formulated recommendations through a consensus-based process. The strength of the recommendations was classified as strong or weak. To substantiate the implementation process, implementation trials or observational studies describing facilitators/barriers for implementation were identified from the same searches and were summarized narratively. RECOMMENDATIONS: The recommendations on the use of biomarkers and rapid pathogen diagnostic tests focus on the initiation of antibiotics in patients admitted through the ED. Their effect on the discontinuation or de-escalation of antibiotics during hospital stay was not reported, neither was their effect on hospital infection prevention and control practices. The recommendations on watchful waiting (i.e. withholding antibiotics with some form of follow-up) focus on specific infectious syndromes for which the primary care literature was also included. The recommendations on blood cultures focus on the indication in three common infectious syndromes in the ED explicitly excluding patients with sepsis or septic shock. Most recommendations are based on very low and low certainty of evidence, leading to weak recommendations or, when no evidence was available, to best practice statements. Implementation of these recommendations needs to be adapted to the specific settings and circumstances of the ED. The scarcity of high-quality studies in the area of antimicrobial stewardship in the ED highlights the need for future research in this field.

Specific interferon γ detection for the diagnosis of previous Q fever.

BACKGROUND: Current practice for diagnosis of Q fever, caused by the intracellular pathogen Coxiella burnetii, relies mainly on serology and, in prevaccination assessment, on skin tests (STs), which both have drawbacks. In this study, C. burnetii-specific interferon γ (IFN-γ) production was used as a new diagnostic tool for previous Q fever, circumventing most of these drawbacks. Our aim was to compare this test to serology and ST. METHODS: One thousand five hundred twenty-five individuals from an endemic area with a risk for chronic Q fever were enrolled. IFN-γ production was measured after in vitro stimulation of whole blood with C. burnetii antigens. Various formats using different C. burnetii antigens were tested. Serology and ST were performed in all individuals. RESULTS: In all assay formats, C. burnetii-specific IFN-γ production was higher (P < .0001) in seropositive or ST-positive subjects than in seronegative and ST-negative subjects. Whole blood incubated for 24 hours with C. burnetii Nine Mile showed optimal performance. After excluding subjects with equivocal serology and/or borderline ST results, IFN-γ production was 449 ± 82 pg/mL in the positive individuals (n = 219) but only 21 ± 3 pg/mL in negative subjects (n = 908). Using Bayesian analysis, sensitivity and specificity (87.0% and 90.2%, respectively) were similar to the combination of serology and ST (83.0% and 95.6%, respectively). Agreement with the combination of serology and ST was moderate (84% concordance; κ = 0.542). CONCLUSIONS: Specific IFN-γ detection is a novel diagnostic assay for previous C. burnetii infection and shows similar performance and practical advantages over serology and ST. Future studies to investigate the clinical value in practice are warranted.

Differences in local immune cell landscape between Q fever and atherosclerotic abdominal aortic aneurysms identified by multiplex immunohistochemistry.

Background: Chronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue. Methods: Multiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune systems. Results: Q fever AAAs and atherosclerotic AAAs contained similar numbers of CD68+ macrophages and CD3+ T cells. However, in Q fever AAAs, the number of CD68+CD206+ M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8+ cytotoxic T cells and CD3+CD8-FoxP3+ regulatory T cells. Finally, Q fever AAAs did not contain any well-defined granulomas. Conclusions: These findings demonstrate that despite the presence of pro-inflammatory effector cells, persistent local infection with C. burnetii is associated with an immune-suppressed microenvironment. Funding: This work was supported by SCAN consortium: European Research Area - CardioVascualar Diseases (ERA-CVD) grant [JTC2017-044] and TTW-NWO open technology grant [STW-14716].

Antimicrobial stewardship in the emergency department: characteristics and evidence for effectiveness of interventions.

BACKGROUND: Emergency departments (EDs) are the entrance gates for patients presenting with infectious diseases into the hospital, yet most antimicrobial stewardship programmes are primarily focused on inpatient management. With equally high rates of inappropriate antibiotic use, the ED is a frequently overlooked yet important unit for targeted antimicrobial stewardship (AMS) interventions. OBJECTIVES: We aimed to (a) describe the specific aspects of antimicrobial stewardship in the ED and (b) summarize the findings from improvement studies that have investigated the effectiveness of antimicrobial stewardship interventions in the ED setting. SOURCES: (a) a PubMed search for 'antimicrobial stewardship' and 'emergency department', and (b) published reviews on effectiveness combined with publications from the first source. CONTENT: (a) An in depth analysis of selected publications provided four key antimicrobial use processes typically performed by front-line healthcare professionals in the ED: making a (tentative) clinical diagnosis, starting empirical therapy based on that diagnosis, performing microbiological tests before starting that therapy and following up patients who are discharged from the ED. (b) Further, we discuss the literature on improvement strategies in the ED focusing on guidelines and clinical pathways and multifaceted improvement strategies. We also summarize the evidence of microbiologic culture review. IMPLICATIONS: Based on our review of the literature, we describe four essential elements of antimicrobial use in the ED. Studying the various interventions targeting these care processes, we have found them to be of a variable degree of success. Nonetheless, while there is a paucity of AS studies specifically targeting the ED, there is a growing body of evidence that AS programmes in the ED are effective with modifications to the ED setting. We present key questions for future research.

Quality indicators for appropriate antimicrobial therapy in the emergency department: a pragmatic Delphi procedure.

OBJECTIVES: Antimicrobial stewardship (AMS) has established its importance for inpatient care. AMS is, however, also urgently needed in emergency departments (ED), where many antimicrobial prescriptions are initiated. It is currently unclear what metrics stewardship teams can use to measure and improve the appropriateness of antimicrobial prescription in the ED. In this study we develop quality indicators (QIs) for antimicrobial use in the ED. METHODS: A RAND-modified Delphi procedure was used to develop a set of QIs applicable to adult patients who present at the ED with a potential infection. First, pragmatically using two recent papers of the international expert-group DRIVE-AB, potential ED-specific QIs for appropriate antimicrobial use were retrieved. Thereafter, an international multidisciplinary expert panel appraised these QIs during two questionnaire rounds with a meeting in between. RESULTS: Thirty-three potential QIs were extracted from the DRIVE-AB papers. After appraisal by 13 experts, 22 QIs describing appropriate antimicrobial use in the ED were selected. These indicators provide recommendations within five domains: stewardship prerequisites (six QIs); diagnostics (one QI); empirical treatment (ten QIs); documentation of information (four QIs); and patient discharge (one QI). CONCLUSIONS: We pragmatically developed a set of 22 QIs that can be used by stewardship teams to measure the appropriateness of antimicrobial prescription in the ED. There is probably room for additional QI development to cover all key aspects of AMS in the ED. Measuring QIs can be a first step for stewardship teams to, in collaboration with ED professionals, choose targets for improvement and optimize antimicrobial use.

Altered interferon-γ response in patients with Q-fever fatigue syndrome.

OBJECTIVES: Whether immunological mechanisms underlie Q-fever fatigue syndrome (QFS) remains unclear. For acute Q-fever, the antigen-specific interferon-γ (IFNγ) response may be a useful tool for diagnosis, and the IFNγ/interleukin(IL)-2 production ratio may be a marker for chronic Q-fever and treatment monitoring. Here we explored the specific IFNγ production and IFNγ/IL-2 ratio in QFS patients. METHODS: IFNγ and IL-2 production were tested in ex-vivo stimulated whole blood of QFS patients (n = 20), and compared to those previously determined in seropositive controls (n = 135), and chronic Q-fever patients (n = 28). Also, the correlation between patient characteristics and IFNγ, IL-2, and IFNγ/IL-2 ratio was determined. RESULTS: QFS patients were younger (p < 0.001), but gender distribution was similar to seropositive controls and chronic Q-fever patients. Coxiella burnetii Nine Mile stimulation revealed a higher IFNγ production in QFS (median 319.5 pg/ml) than in seropositive controls (120 pg/ml, p < 0.01), but comparable to chronic Q-fever (2846 pg/ml). The IFNγ/IL-2 ratio was similar to that in seropositive controls, but lower than in chronic Q-fever patients (p < 0.01). Symptom duration was positively correlated with IL-2 production, and negatively correlated with the IFNγ/IL-2 ratio. CONCLUSIONS: These results point to an altered cell-mediated immunity in QFS, and suggest a different immune response than in chronic Q-fever.

Early cytokine and antibody responses against Coxiella burnetii in aerosol infection of BALB/c mice.

Coxiella burnetii, a Gram-negative intracellular bacterium, can give rise to Q fever in humans and is transmitted mainly by inhalation of infected aerosols from animal reservoirs. Serology is commonly used to diagnose Q fever, but the early cellular immune response-i.e., C. burnetii-specific interferon γ (IFN-γ) production in response to antigen challenge-might be an additional diagnostic. Detection of IFN-γ responses has been used to identify past and chronic Q fever infections, but the IFN-γ response in acute Q fever has not been described. By challenging immunocompetent BALB/c mice with aerosols containing phase I C. burnetii, the timing and extent of IFN-γ recall responses were evaluated in an acute C. burnetii infection. Other cytokines were also measured in an effort to identify other potential diagnostic markers. The data show that after initial expansion of bacteria first in lungs and then in other tissues, the infection was cleared from day 10 onwards as reflected by the decreasing number of bacteria. The antigen-induced IFN-γ production by splenocytes coincided with emergence of IgM phase II antibodies at day 10 postinfection and preceded appearance of IgG antibodies. This was accompanied by the production of proinflammatory cytokines including interleukin (IL) 6, keratinocyte-derived cytokine, and IFN-γ-induced protein 10, followed by monocyte chemotactic protein 1, but not by IL-1ß and tumor necrosis factor α, and only very low production of the anti-inflammatory cytokine IL-10. These data suggest that analysis of antigen-specific IFN-γ responses could be a useful tool for diagnosis of acute Q fever. Moreover, the current model of C. burnetii infection could be used to give new insights into immunological factors that predispose to development of persistent infection.

Coverage of the 2011 Q fever vaccination campaign in the Netherlands, using retrospective population-based prevalence estimation of cardiovascular risk-conditions for chronic Q fever.

BACKGROUND: In 2011, a unique Q fever vaccination campaign targeted people at risk for chronic Q fever in the southeast of the Netherlands. General practitioners referred patients with defined cardiovascular risk-conditions (age >15 years). Prevalence rates of those risk-conditions were lacking, standing in the way of adequate planning and coverage estimation. We aimed to obtain prevalence rates retrospectively in order to estimate coverage of the Q fever vaccination campaign. METHODS: With broad search terms for these predefined risk-conditions, we extracted patient-records from a large longitudinal general-practice research-database in the Netherlands (IPCI-database). After validation of these records, obtained prevalence rates (stratified for age and sex) extrapolated to the Q fever high-incidence area population, gave an approximation of the size of the targeted patient-group. Coverage calculation addressed people actually screened by a pre-vaccination Q fever skin test and serology (coverage) and patients referred by their general practitioners (adjusted-coverage) in the 2011 campaign. RESULTS: Our prevalence estimate of any risk-condition was 3.1% (lower-upper limits 2.9-3.3%). For heart valve defects, aorta aneurysm/prosthesis, congenital anomalies and endocarditis, prevalence was 2.4%, 0.6%, 0.4% and 0.1%, respectively. Estimated number of eligible people in the Q fever high-incidence area was 11,724 (10,965-12,532). With 1330 people screened for vaccination, coverage of the vaccination campaign was 11%. For referred people, the adjusted coverage was 18%. Coverage was lowest among the very-old and highest for people aged 50-70 years. CONCLUSION: The estimated coverage of the vaccination campaign was limited. This should be interpreted in the light of the complexity of this target-group with much co-morbidity, and of the vaccine that required invasive pre-vaccination screening. Calculation of prevalence rates of risk-conditions based on the IPCI-database was feasible. This procedure proved an efficient tool for future use, when prevalence estimates for policy, implementation or surveillance of subgroup-vaccination or other health-care interventions are needed.

Specific in vitro interferon-gamma and IL-2 production as biomarkers during treatment of chronic Q fever.

BACKGROUND: Antibiotic treatment of chronic Q fever is cumbersome and of long duration. To monitor treatment, there is a need for alternative biomarkers. Coxiella burnetii-specific interferon (IFN)-γ and interleukin (IL)-2 production reflect the type of effector and memory T-cell response. In chronic Q fever, C. burnetii-specific IFN-γ production is higher and IL-2 production is lower than in individuals with past Q fever. Here we explore whether C. burnetii-specific IFN-γ and IL-2 production correlate to treatment response. METHODS: We studied the longitudinal C. burnetii-specific IFN-γ/IL-2 ratio in fifteen proven chronic Q fever patients. All patients were followed for at least 18 months during antibiotic treatment. Treatment was considered successful when clinical recovery was observed, a positive PCR for C. burnetii DNA in blood became persistently negative, anti-phase I IgG showed a fourfold decrease or more, and imaging techniques showed disappearance of infectious foci. RESULTS: Overall, the IFN-γ/IL-2 ratio declined when patients experienced a successful treatment outcome. When treatment failed, IFN-γ/IL-2 ratios did not significantly decrease. The median (±IQR) slope of the longitudinal IFN-γ/IL-2 ratio with successful treatment was -2.10 (-7.02 to -0.06), and -0.15 (-1.13 to 0.25) with unsuccessful treatment (P = 0.19). Q fever endocarditis patients had higher IFN-γ/IL-2 ratios than patients with endovascular infections. CONCLUSION: We propose that the IFN-γ/IL-2 ratio can be used as an additional biomarker for monitoring chronic Q fever treatment, with declining ratios being indicative of successful treatment.

The effect of C. burnetii infection on the cytokine response of PBMCs from pregnant goats.

In humans, infection with Coxiella burnetii, the causative agent of Q fever, leads to acute or chronic infection, both associated with specific clinical symptoms. In contrast, no symptoms are observed in goats during C. burnetii infection, although infection of the placenta eventually leads to premature delivery, stillbirth and abortion. It is unknown whether these differences in clinical outcome are due to the early immune responses of the goats. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from pregnant goats. In total, 17 goats were included in the study. Six goats remained naive, while eleven goats were infected with C. burnetii. Toll-like receptor (TLR) and cytokine mRNA expression were measured after in vitro stimulation with heat-killed C. burnetii at different time points (prior infection, day 7, 35 and 56 after infection). In naive goats an increased expression of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-10 and interferon (IFN)-γ mRNA upon C. burnetii stimulation was detected. In addition, TLR2 expression was strongly up-regulated. In goats infected with C. burnetii, PBMCs re-stimulated in vitro with C. burnetii, expressed significantly more TNF-α mRNA and IFN-γ mRNA compared to naive goats. In contrast, IL-10 mRNA production capacity was down-regulated during C. burnetii infection. Interestingly, at day 7 after inoculation a decreased IFN-γ protein level was observed in stimulated leukocytes in whole blood from infected goats, whereas at other time-points increased production of IFN-γ protein was seen. Our study shows that goats initiate a robust pro-inflammatory immune response against C. burnetii in vitro. Furthermore, PBMCs from C. burnetii infected goats show augmented pro-inflammatory cytokine responses compared to PBMCs from non-infected goats. However, despite this pro-inflammatory response, goats are not capable of clearing the C. burnetii infection.

Adverse events and association with age, sex and immunological parameters of Q fever vaccination in patients at risk for chronic Q fever in the Netherlands 2011.

BACKGROUND: Following a large Q fever outbreak in the Netherlands, patients at risk for chronic Q fever received a whole-cell Q fever vaccine. Sensitized people were excluded based on pre-vaccination screening with skin test (ST) and serology. An investigational IFN-γ-production assay was added. No previous experience existed for Q fever vaccination in this patient risk-group with predefined cardiac valvular anomalies or aortic aneurysm/prosthesis and many co-morbidities. We studied the adverse events (AE) and their association with patient characteristics and immunological parameters. METHODS: AE registration covered the week after skin test and 90 days following vaccination, with the use of diaries, interviews and spontaneous reports. Serious (S)AE were assessed immediately to ensure safety. We coded AE according to reported severity. Univariate and multivariate analysis addressed associations. RESULTS: Pre-vaccination screening led to exclusion of 182 patients with positive serology and 207 patients with positive skin test-reading. The skin test did not lead to any causally related SAE. Subsequent vaccination of 1370 patients did not reveal unexpected AE; however, 80% of vaccinees reported local AE (in 26% of these pronounced or extensive). The two causally related SAE (0.1%) both concerned a persistent subcutaneous injection site mass. AE were more frequent in women, younger patients, and those without immunosuppressive co-morbidity/medication. The occurrence of local AE after skin test was associated with pre-vaccination positive serology and high IFN-γ production. This was also true for local AE following vaccination, with a strong association with local AE after skin test as well. The proportion of vaccinees with positive serology and positive IFN-γ values 6 months after vaccination was higher in those with local AE after skin test or after vaccination (non-significant, probably due to small numbers). CONCLUSION: Q fever vaccination was safe but reactogenic in this high-risk patient-group. Rates of local AE were higher in women, younger age groups and in those with positive immunological parameters. Vaccinees with local AE after skin test or after vaccination appear to have more pronounced post-vaccination immune responses.