Fluorescence in situ hybridization for detecting Coxiella burnetii in tissue samples from chronic Q fever patients

ObjectiveDetection of the intracellular bacterium Coxiella burnetii, causative agent of chronic Q fever, is notoriously difficult. Diagnosis of and duration of antibiotic treatment for chronic Q fever is partly determined by detection of the bacterium with polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) might be a promising technique for detecting C. burnetii in tissue samples from chronic Q fever patients, but its value in comparison with PCR is uncertain. We aim to assess the value of FISH for detecting C. burnetii in tissue of chronic Q fever patients.MethodsFISH and PCR were performed on tissue samples from Dutch chronic Q fever patients collected during surgery or autopsy. Sensitivity, specificity, and overall diagnostic accuracy were calculated. Additionally, data on patient and disease characteristics were collected from electronic medical records.ResultsIn total, 49 tissue samples from mainly vascular walls, heart valves, or placentas, obtained from 39 chronic Q fever patients, were examined by FISH and PCR. The sensitivity and specificity of FISH compared to PCR for detecting C. burnetii in tissue samples from chronic Q fever patients was 45.2% (95% confidence interval (CI), 27.3% – 64.0%) and 84.6% (95% CI, 54.6% – 98.1%), respectively. The overall diagnostic accuracy was 56.8% (95% CI, 42.2% - 72.3%). Two C. burnetii PCR negative placentas were FISH positive. Four FISH results (8.2%) were deemed inconclusive because of autofluorescence.ConclusionWith an overall diagnostic accuracy of 57.8%, we conclude that FISH has limited value in the routine diagnostics of chronic Q fever.     

Human abdominal aortic aneurysms. Immunophenotypic analysis suggesting an immune-mediated response.

Cellular immunity may play a role in the pathogenesis of atherosclerosis. In this report the potential role of these cells in the formation of abdominal aortic aneurysms by immunohistochemistry was investigated. Aortic tissues from 32 patients were examined: 4 normal aortas, 6 aortas with occlusive atherosclerotic disease, 17 abdominal aortic aneurysms, and 5 inflammatory abdominal aneurysms. Using monoclonal anti-CD3 (T cells), anti-CD19 (B cells), anti-CD11c (macrophages), anti-CD4 (T helper cells), and anti-CD8 (T suppressor cells), several distinctions among these groups were found. The amount of inflammatory cell infiltrate was as follows: inflammatory aneurysms more than abdominal aortic aneurysms more than occlusive aortas more than normal aortas. CD3-positive T lymphocytes rarely were found in the adventitia of normal or occlusive aortas. In contrast, abdominal aortic aneurysms and inflammatory aneurysms exhibited most of the CD3-positive infiltrates in the adventitia. CD19-positive B lymphocytes were present mainly in the adventitia of all pathologic tissues. The CD4-positive:CD8-positive ratio was greater in abdominal aortic aneurysms and inflammatory aneurysms than in the other groups, both in the adventitia and in the media of the aortas. CD11c-positive macrophages were present throughout the diseased tissues, often surrounded by lymphoid aggregates; the greatest numbers of macrophages were found in the inflammatory aneurysm group. Our data suggests that the aneurysmal disease may progress from occlusive disease and is accompanied by an increase in chronic inflammatory cells as well as a redistribution of these cell types. Therefore it is suggested that aneurysmal disease may represent an immune-mediated event.     

Q fever osteoarticular infection: four new cases and a review of the literature

Q fever is a worldwide-occurring zoonosis caused by Coxiella burnetii. Better knowledge of the disease and of evolving diagnostics can enable recognition of unusual manifestations. Reported here are four cases of Q fever osteoarticular infections in adults: two cases of Q fever tenosynovitis, which represent the first two reports of this infection, and two cases of Q fever spondylodiscitis complicated by paravertebral abscess. In addition, the literature is reviewed on the 15 previously reported cases of Q fever osteoarticular infection, six of which were vertebral infections. Osteomyelitis is the usual manifestation Q fever osteoarticular infection. Because its onset is frequently insidious, diagnosis is usually delayed. The main differential diagnosis is mycobacterial infection, based on the histological granulomatous presentation of lesions. Whereas serology is the reference diagnostic method for Q fever, detection of C. burnetii in tissue specimens by PCR and cell culture provides useful additional evidence of infection. Culture-negative osteoarticular samples with granulomatous presentation upon histological examination should raise suspicion of Q fever.     

Tortoise tick Hyalomma aegyptium as long term carrier of Q fever agent Coxiella burnetii—evidence from experimental infection

The experimental study investigated the ability of tortoise tick Hyalomma aegyptium to play a role in forming and maintaining natural foci of Q fever. We tested the competence of H. aegyptium larvae to acquire Coxiella burnetii infection from mammals, serve as a C. burnetii vector between mammalian hosts, and be a long-term carrier of C. burnetii, including interstadial transmission. H. aegyptium larvae were allowed to feed on guinea pigs experimentally infected with C. burnetii. Engorged larvae molted to nymphs, some of which were preserved in 96% ethanol and later examined by polymerase chain reaction (PCR) using C. burnetii-specific primers (CBCOS, CBCOE). Prevalence of C. burnetii among these nymphs was 5.6% (n = 235). Remaining nymphs then fed on other, C. burnetii-negative guinea pigs; and according to results of both, micro-agglutination reaction, and ELISA, they successfully transmitted C. burnetii to those new hosts. Detached engorged nymphs molted to adults, which were kept alive long term and then placed in 96% ethanol 383 days post-infection. Thereafter, they were examined by PCR in the same manner as were the nymphs. Prevalence of C. burnetii among adult H. aegyptium was 28.9% (n = 90). According to our results, tortoise-specific ticks have indisputable potential in the epidemiology of Q fever natural foci.     

Immunohistochemical characterization of inflammatory cells associated with advanced atherosclerosis

During repair of 12 atherosclerotic abdominal aortic aneurysms, fresh samples of aneurysm wall were obtained. Histology confirmed the presence of advanced atherosclerosis associated with medial thinning and a variable aortic adventitial chronic inflammatory cell infiltrate. Monoclonal antibodies were used to identify the inflammatory cells throughout the aortic wall. The majority of lymphocytes in the aortic adventitia were B-cells. B-cells were not present in atheromatous plaques. T-cells, predominantly T-helper cells, were found in atheromatous plaques and in aortic adventitia. The majority of lymphocytes and macrophages in aortic adventitia and most vascular endothelial cells were HLA-DR positive. Ki-67 staining was found in B-cells and T-helper cells, indicating that these cells were proliferating. Occasional lymphocytes were BerH2 positive, indicating that some lymphocytes were activated. These findings suggest that chronic periaortitis is an active, immunologically mediated, local complication of advanced human atherosclerosis.     

Acute Acalculous Cholecystitis Associated with Q Fever: Report of Seven Cases and Review of the Literature

Q fever is a worldwide-occurring zoonosis caused by Coxiella burnetii. There are various clinical manifestations of acute Q fever, of which acute cholecystitis is a very rare clinical presentation. This study reports seven cases of acute cholecystitis associated with Coxiella burnetii and reviews two other cases from the literature. All patients were admitted to hospital for fever and abdominal pain in the right upper quadrant. Abdominal echography showed a distended gallbladder with biliary sludge without concrements in eight cases and with a single stone in one case. Diagnosis was made by specific serological investigation (microimmunofluorescence assay) for Coxiella burnetii. All nine patients were cured, six after laparoscopic cholecystectomy and three with antibiotics only. Histological examination of the gallbladders showed inflammation in five cases, although Coxiella burnetii was not detected by immunohistochemistry. The results show that laboratory investigations in patients admitted to hospital for symptoms consistent with acute acalculous cholecystitis should include a systematic search for Coxiella burnetii. Electronic Publication     

Identification of protein candidates for the serodiagnosis of Q fever endocarditis by an immunoproteomic approach

Q fever is a worldwide zoonosis caused by Coxiella burnetii bacterium. Two clinical forms are present: acute Q fever and chronic disease, including endocarditis. Currently, the diagnosis of Q fever endocarditis is based on the detection of anti-phase I antibodies. The objective of the study was to identify candidate proteins for the serological diagnosis of endocarditis due to C. burnetii. The immunoreactivities of sera from 12 patients with C. burnetii infections, including the sera from patients with endocarditis and with the acute clinical form of Q fever, were compared with those of three control subjects who did not have Q fever. We identified 29 candidate antigenic proteins by mass spectrometry. Two proteins, arginine repressor and OmpH, were recognised exclusively by the sera of patients with Q fever endocarditis. These proteins are promising candidates for the development of serodiagnostic assays for Q fever endocarditis.     

Reactivation of Q fever following cardiac surgery

To determine whether reactivation of Q fever occurs following cardiac surgery 43 patients who had antibodies toCoxiella burnetii were identified pre-operatively. Serum samples were collected 1, 4 to 6, and 24 weeks post-operatively. One patient had active Q fever endocarditis. Seven of the remaining 42 (17 %) had a fourfold rise in titres for antibodies toCoxiella burnetii post-operatively. Only two of these seven had an increase in antibody titres for other agents suggesting that the fourfold titre rise forCoxiella burnetii was not part of a polyclonal response. It is concluded that self-limited reactivation of infection may occur amongCoxiella burnetii seropositive patients undergoing open heart surgery.     

Epstein-Barr virus-containing T-cell lymphoma and atherosclerotic abdominal aortic aneurysm in a young adult.

Malignant lymphoma infiltrating the abdominal aorta and resulting in an aortic aneurysm has never been documented. We report here a case of angiocentric T-cell lymphoma in a 33-year-old man who for months presented intermittent fever, splenomegaly, and an abdominal pulsatile mass. Angiography revealed extensive aneurysmal dilatation of the infrarenal abdominal aorta, bilateral iliac artery, and right common femoral artery. Splenic abscess and infected abdominal aortic aneurysm were initially suspected. An urgent splenectomy and aneurysmectomy with an aortic bifemoral bypass were performed. Pathological examination of the aortic aneurysm showed extensive necrosis, severe atherosclerosis, and lymphoma cell infiltration of the aortic wall. The lymphoid cells in the aorta and spleen were stained positive for CD45RO, CD56, and CD8, but negative for CD4 and CD19. Double-labeling immunohistochemistry and in situ hybridization using EBER1 for Epstein-Barr virus (EBV) revealed positive nuclear staining in the atypical T-lymphoid cells. This is the first definitive proof of peripheral T-cell lymphoma involving the abdominal aorta. Our evidence also supports that the EBV infection of T cells could be responsible for the atherosclerosis and hypertriglyceridemia, and the angiocentricity of the tumor cells apparently results in the presenting atherosclerotic aortic wall destruction, providing an additional causative concept for abdominal aortic aneurysm.     

Q fever seroprevalence in metropolitan samples is similar to rural/remote samples in Queensland,Australia

Q fever is a vaccine preventable disease; however, despite this, high notification numbers are still recorded annually in Australia. We investigated the seroprevalence of Coxiella burnetii, the Q fever agent, in a Queensland sample population. Notification data (N = 6425) from 1984–2008 were collated, identifying high risk areas of Q fever exposure. Of these 177 were recorded in children. Serum samples were collected from Queensland and screened using both an immunoflourescence assay at 1:10 dilution and a commercially available ELISA kit. Results were collated based on age, geographical location and sex. From 1988 Queensland samples screened, 103 were identified as Q fever IgG-positive, giving a seroprevalence of 5.2% (95% CI 4.3–6.2%). Seroprevalence in the rural/remote population was 5.3% (95% CI 4.6–6.6%), and the metropolitan Brisbane population, which is considered not at risk, was 5.0% (95% CI 3.7–6.7%). Sixty-three seropositive males and 40 females were identified, along with an increase in seropositivity with increasing age. The seropositivity of children was 1.3% (95% CI 0.7–2.3%) from 844 samples. We have shown that both metropolitan and paediatric populations which are considered low risk of Coxiella exposure have surprisingly high seropositivity. These emerging groups require further investigation and consideration for the introduction of preventive measures.     

Long-term serological follow-up after primary Coxiella burnetii infection in patients with vascular risk factors for chronic Q fever

We evaluated the long-term serological follow-up of patients with vascular risk factors for chronic Q fever that were previously Coxiella burnetii seropositive. C. burnetii phase I IgG titers were reevaluated in patients that gave informed consent or retrospectively collected in patients already deceased or lost to follow-up. Of 107 patients, 25 (23.4%) became seronegative, 77 (72.0%) retained a profile of past resolved Q fever infection, and five (4.7%) developed chronic Q fever. We urge clinicians to stay vigilant for chronic Q fever beyond two years after primary infection and perform serological testing based on clinical presentation.

     

Q fever and lymphadenopathy: report of four new cases and review

Coxiella burnetii, the causative agent of Q fever, is responsible for various clinical syndromes, but lymphadenitis has been described during Q fever in only three recent case reports. Four new cases of acute Q fever associated with lymphadenopathy are reported here, and these cases are discussed along with the three previously reported cases. Coxiella burnetii was isolated for the first time from a lymph node. Q fever should be considered an etiologic agent of lymphadenitis.     

Epidemiological study of Q fever in humans, ruminant animals, and ticks in Cyprus using a geographical information system

A cross-sectional study of Q fever was conducted in a representative sample of the human and animal population in Cyprus in order to assess the seroprevalence of Q fever and the prevalence of related risk factors. A total of 583 human and 974 ruminant animal serum samples were collected and tested for the detection of antibodies against Coxiella burnetii phase II antigen using an indirect immunofluorescent assay. One hundred forty-one ticks were collected from the infested animals examined; the polymerase chain reaction and the shell-vial technique were used to detect and isolate C. burnetii. Standardized questionnaires were used to obtain information concerning inhabitants and their animals. A geographical information system was used to identify high-risk regions. The prevalence of IgG antibodies against C. burnetii phase II antigen was estimated at 52.7% for humans, 48.2% for goats, 18.9% for sheep, and 24% for bovines. C. burnetii was detected in 11 (7.8%) ticks. Using the geographical information system, two villages were identified as high-risk regions on the basis of high seroprevalence rates of IgG antibodies in humans and animals. Risk factors related to Q fever seropositivity were identified by logistic regression analysis and included age, residence, occupation, use of manure in the garden, ownership of animals (especially goats), and the presence of tick-infested or aborting animals. Q fever poses an occupational hazard to humans living in close contact with sheep and/or goats. In parallel, ticks should be considered an important aspect in the epidemiology of Q fever and should be further studied to better elucidate their role.     

Increased expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and MMP10, MMP23 in inflammatory bowel disease: Cross‐sectional study

It has been reported that EMMPRIN is involved in the regulation of immune response and the induction of MMPs production by fibroblasts. The aim of this study was to describe the intestinal gene expression and protein production of EMMPRIN, MMP23 and MMP10 in patients with ulcerative colitis (UC) and Crohn’s disease (CD) and compared them with a control group. Gene expression of EMMPRIN, MMP10 and MMP23B was measured by RT‐PCR. In order to determine EMMPRIN and MMP protein expression, colonic tissues were immunostained. The results of the study showed EMMPRIN gene expression was upregulated in rectal mucosa from active (a)UC versus aCD patients (P = .045), remission (r)CD group (P = .0009) and controls (P < .0001). We detected differences between rUC and aCD (P = .004), rCD (P < .0001) or control group (P < .0001). EMMPRIN showed a higher expression in mucosa (intraepithelial lymphocytes), submucosa and adventitia (endothelial cells) from aCD patients. MMP23 levels were increased in aUC and aCD compared to rUC and rCD and the control group (P = .0001). EMMPRIN+/MMP23+─expressing cells were localized mainly in mucosa, muscular and adventitia from active UC patients. MMP10 gene expression was increased in aUC versus CD patients and the control group (P = .0001). MMP10 gene expression is associated with inflammation in UC patients (P = .0001, r² = .585). EMMPRIN+/MMP10+─producing cells were found mainly in all intestinal layers and perivascular inflammatory infiltrates from aUC patients. In conclusion, EMMPRIN, MMP23 and MMP10 were upregulated in patients with active UC versus remission UC , CD and control groups suggesting that, they are involved in the inflammatory process.     

Comparison of ELISA and indirect immunofluorescent antibody assay detecting <Emphasis Type="Italic">Coxiella burnetii</Emphasis> IgM phase II for the diagnosis of acute Q fever

A commercially available enzyme-linked immunosorbent assay (ELISA) detecting Coxiella burnetii phase II-specific IgM for the diagnosis of acute Q fever was compared with indirect immunofluorescent antibody assay (IFA). IFA is the current reference method for the detection of antibodies against C. burnetii, but has disadvantages because the judgment of fluorescence is subjective and tiring, and the test is expensive and automation is not possible. To examine whether phase II IgM ELISA could be used as a screening assay for acute Q fever, we compared the sensitivity and specificity of IFA and ELISA. The sensitivity of the IFA and ELISA tests were 100 and 85.7%, respectively, with a specificity of 95.3 and 97.6%, respectively. Because of the high sensitivity and specificity of the ELISA in combination with the practical disadvantages of the IFA, we introduced a new algorithm to screen samples of patients with symptoms of acute Q fever infection.     

Immunological arousal during acute Q fever infection

Physicians often encounter patients who present with a vague clinical syndrome. A wide serological workup is often ordered, which may include tests for Coxiella burnetii in endemic areas. Often, the results of these tests pose new dilemma, with overlapping positive laboratory assays. The objective of this investigation was to characterise the serological overlap between acute Q fever and other infectious and immunological diseases. We retrospectively scanned the files of patients with a positive or equivocal immunoglobulin (Ig) M for C. burnetii phase II over a period of 8 years in a general hospital. Clinical and laboratory data, including antibodies to infectious agents and antibodies related to immunological states, were recorded. Anti-nuclear antibody (ANA), smooth muscle antibody (SMA) and rheumatoid factor were positive in 38%, 33.3% and 22.2% of the cases, respectively. In patients with acute Q fever, elevated IgM levels for Epstein–Barr Virus (EBV), cytomegalovirus (CMV), Mycoplasma pneumoniae, parvovirus, Bordetella pertussis, Rickettsia conorii and R. typhi were noted in 13.8%, 8.3%, 12.12%, 22.2%, 25%, 13% and 21.7% of cases, respectively. Acute Q fever induces a non-specific immunological arousal in a significant number of patients. This may interfere with diagnosis and delay treatment. Caution, clinical judgment and serological follow-up is warranted in such conditions.     

Association between serological evidence of past Coxiella burnetii infection and atherosclerotic cardiovascular disease in elderly patients

Q fever, caused by Coxiella burnetii, may cause vascular complications, but the role that this infection may play in the development of atherosclerotic cardiovascular disease remains unknown. This study examined the association between Q fever serology and cardiovascular disease in a region where Q fever is endemic. A case-control study was conducted in the Hospital Universitario de Burgos (Spain) between February 2011 and June 2012. A total of 513 samples were tested, from 454 hospitalized patients ≥65 years old, of whom 164 were cases (patients with prevalent or incident coronary heart, cerebrovascular or peripheral artery, disease) and 290 controls (patients without cardiovascular disease). Serum IgG antibody phase II titres against Q fever were determined by immunofluorescence assay. Seropositivity (titres ≥1:256) was detected in 84/164 (51.2%) cases and in 109/290 (37.6%) controls (p = 0.005; OR, 1.7; 95% CI, 1.1–2.5). This ratio increases when adjusted for sex, hypertension, dyslipidaemia, smoking, diabetes and atrial fibrillation (OR, 2.6; 95% CI, 1.5–4.7). The geometric mean titre (GMT) for C. burnetii phase II assay was higher in cases than in controls (p = 0.004). We found no significant relationship between cardiovascular disease and C. pneumoniae, and Cytomegalovirus seropositivity (both determined by the IgG ELISA method). In conclusion, serological evidence of past Q fever is associated with atherosclerotic cardiovascular disease in elderly patients in an endemic region.     

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

ObjectivesChronic 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.MethodsThe 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.ResultsRAB7A (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.ConclusionsRAB7A (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.     

A Comparison of Diameter,Wall Stress,and Rupture Potential Index for Abdominal Aortic Aneurysm Rupture Risk Prediction

An abdominal aortic aneurysm (AAA) is a balloon-like dilation of the aorta, which is potentially fatal in case of rupture. Computational finite element (FE) analysis is a promising approach to a more accurate and patient-specific rupture risk prediction. AAA wall strength and rupture potential index (RPI) calculation are implemented in our FE software. Static structural FE simulations are performed on n = 30 non-ruptured asymptomatic, n = 9 non-ruptured symptomatic, and n = 14 ruptured AAAs. We calculate maximum values for diameter, wall displacement, strain, stress, and RPI as well as minimum wall strength for every AAA. All investigated quantities, except minimum strength, show statistically significant differences between non-ruptured asymptomatic and symptomatic/ruptured AAAs. Maximum wall stress and especially the RPI are notably increased for symptomatic and ruptured AAAs. The biggest difference is found to be the RPI (Δ = 44.9%, p = 8.0e−5). Lowest RPI obtained for symptomatic or ruptured AAAs is 0.3. The RPI of more than 55% of the investigated asymptomatic AAAs falls below this value. Maximum wall stress and maximum RPI criteria enable a reliable rupture risk evaluation for AAAs. Especially in the diameter range where surgical indication is not obvious, the RPI holds great potential for improvement of clinical decisions.