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.

     

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.     

Serological follow-up in patients with aorto-iliac disease and evidence of Q fever infection

The aim of this study was to provide data on the risk of developing chronic Q fever in patients with aorto-iliac disease and evidence of previous Q fever infection. Patients with an aortic and/or iliac aneurysm or aorto-iliac reconstruction (aorto-iliac disease) and evidence of previous Q fever infection were included. The presence of phase I and II Coxiella burnetii IgG antibodies was assessed periodically using immunofluorescence assay. A total of 111 patients with aorto-iliac disease were divided into three groups, based upon the serological profile [mean follow-up: 16?±?9 months (mean?±?standard deviation)]. Group 1 consisted of 30 patients with a serological trace of C. burnetii infection (negative IgG phase I, IgG phase II titer of 1:32). Of these, 36.7 % converted to serological profile matching past resolved Q fever. Group 2 included 49 patients with negative IgG phase I titer and IgG phase II titer ≥1:64. No patients developed chronic Q fever, but 14.3 % converted to a positive IgG phase I titer. Group 3 consisted of 32 patients with positive IgG phase I and positive IgG phase II titers, of which 9.4 % developed chronic Q fever (significantly different from group 2, p?=?0.039). The IgG phase I titer increased in 28.1 % of patients (from 1:64 to 1:4,096). The risk of developing chronic Q fever in patients with aorto-iliac disease and previous Q fever infection with a positive IgG phase I titer was 9.4 %. The IgG phase I titer increases or becomes positive in a substantial number of patients. A standardized serological follow-up is proposed.     

Evaluation of Commonly Used Serological Tests for Detection of Coxiella burnetii Antibodies in Well-Defined Acute and Follow-Up Sera

In this study, we compared Coxiella burnetii IgG phase I, IgG phase II, and IgM phase II detection among a commercially available enzyme-linked immunosorbent assay (ELISA) (Virion/Serion), an indirect fluorescent antibody test (IFAT) (Focus Diagnostics), and a complement fixation test (CFT) (Virion/Serion). For this, we used a unique collection of acute- and convalescent-phase sera from 126 patients with acute Q fever diagnosed by positive Coxiella burnetii PCR of blood. We were able to establish a reliable date of onset of disease, since DNA is detectable within 2 weeks after the start of symptoms. In acute samples, at t = 0, IFAT demonstrated IgM phase II antibodies in significantly more sera than did ELISA (31.8% versus 19.7%), although the portion of solitary IgM phase II was equal for IFAT and for ELISA (18.2% and 16.7%, respectively). Twelve months after the diagnosis of acute Q fever, 83.5% and 62.2% of the sera were still positive for IgM phase II with IFAT and ELISA, respectively. At 12 months IFAT IgG phase II showed the slowest decline. Therefore, definitive serological evidence of acute Q fever cannot be based on a single serum sample in areas of epidemicity and should involve measurement of both IgM and IgG antibodies in paired serum. Based on IgG phase II antibody detection in paired samples (at 0 and 3 months) from 62 patients, IFAT confirmed more cases than ELISA and CFT, but the differences were not statically significant (100% for IFAT, 95.2% for ELISA, and 96.8% for CFT). This study demonstrated that the three serological tests are equally effective in diagnosing acute Q fever within 3 months of start of symptoms. In follow-up sera, more IgG antibodies were detected by IFAT than by ELISA or CFT, making IFAT more suitable for prevaccination screening programs.     

The sexual dimorphism of anticardiolipin autoantibodies in acute Q fever patients

ObjectivesQ 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.MethodsIgG aCL antibodies were evaluated at the time of Q fever serological diagnosis with enzyme-linked immunosorbent assay. Results were analysed according to sex.ResultsAmong 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).ConclusionsWe 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.     

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     

Real-Time PCR with Serum Samples Is Indispensable for Early Diagnosis of Acute Q Fever

The world''s largest Q fever outbreak is ongoing in The Netherlands with around 3,000 confirmed cases since the first half of 2007. Increased awareness has resulted in early referral of patients for diagnostics. An important drawback to serological diagnosis of acute Q fever is the lag phase in antibody response. Therefore, we evaluated the performance of a real-time PCR for detection of Coxiella burnetii DNA using serum samples from patients with acute Q fever. PCR, targeting IS1111, was retrospectively performed on acute-phase and follow-up convalescent-phase serum samples from 65 patients with acute Q fever as diagnosed by immunofluorescence assay. The results obtained by PCR were related to disease stage as defined by subsequent appearance of phase II IgM, phase II IgG, phase I IgM, and phase I IgG (IgM-II, IgG-II, IgM-I, and IgG-I, respectively) antibodies and time since onset of disease. In addition, we analyzed seronegative acute-phase serum samples from patients with inconclusive Q fever serology, because no convalescent-phase serum samples were available. PCR was scored positive in 49/50 (98%) seronegative sera, 9/10 (90%) sera with isolated IgM-II antibodies, 3/13 (23%) sera with IgM-II/IgG-II antibodies, 2/41 (5%) sera with IgM-II/IgG-II/IgM-I antibodies, 0/15 (0%) sera with IgM-II/IgG-II/IgM-I/IgG-I antibodies, and 0/1 (0%) serum sample with IgM-II/IgG-II/IgG-I antibodies. The latest time point after onset of disease in which C. burnetii DNA could be detected was at day 17. In patients with inconclusive Q fever serology, PCR was positive in 5/50 (10%) cases. We conclude that real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever. C. burnetii DNA becomes undetectable in serum as the serological response develops.Q fever, an infection caused by the bacterium Coxiella burnetii, results in a self-limiting disease in 40 to 50% of infected cases. Pneumonia is the predominant presenting symptom in acute Q fever, although fever and hepatitis are also frequently observed (9, 10). Failure to diagnose acute Q fever and delay in treatment may lead to prolonged morbidity and increased hospital admission rates (4, 7, 11, 14).During three consecutive years, large Q fever epidemics occurred in an area in the south of The Netherlands where the disease was formerly not prevalent (11). In 2007 there were a total of 191 confirmed cases reported, in 2008 a total of 998, and in 2009 more than 2,000 confirmed cases were reported, which ranks the outbreak as the largest Q fever epidemic recorded to date. The affected area has a large density of dairy goats, of which a number have tested positive for Q fever. Next to the differences in sizes of the epidemics, the interval between onset of disease and date of diagnosis decreased from a median of 77 days in 2007 to 29 days in 2008 and 17 days in 2009 (12). Moreover, the hospital admission rates were reduced from 40% in 2007 to 20% in 2008 (11). Both observations are most likely due to increased awareness among physicians in the affected area resulting in early submission of clinical samples to the laboratory, subsequent earlier diagnosis, and probably fewer undiagnosed cases. The majority of diagnostic samples from both epidemics were submitted to our laboratory, which lies in the center of the epidemic area and serves a catchment area of roughly 500,000 persons in a semirural district supporting two hospitals and surrounding general practitioners.The gold standard for serological diagnosis of an infectious disease is either a seroconversion or a 4-fold rise in antibody titer. The reference test for serological diagnosis of Q fever is the immunofluorescence assay (IFA) (8). Antibodies are expressed against phase II antigens during the acute infection and against phase I antigens in the established infection. For both antigens, IgM antibody production precedes IgG production, and thus three phases can be distinguished in acute Q fever: a seronegative phase followed by IgM/IgG phase II seroconversion during the acute infection and subsequent IgM/IgG phase I seroconversion in the established infection. However, an important drawback to serological diagnosis of acute Q fever is the lag phase in antibody response of 7 to 15 days after onset of clinical symptoms (8).Apart from serology, C. burnetii-specific PCR of serum samples can be an additional tool to diagnose Q fever in the early acute phase, but conflicting sensitivities have been reported (3, 13). Here, we evaluated the performance of an in-house-developed real-time PCR assay for detection of C. burnetii DNA in serum samples from patients with acute Q fever.     

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.     

Targeted screening as a tool for the early detection of chronic Q fever patients after a large outbreak

In the aftermath of the Dutch Q fever outbreak, an increasing number of patients are being diagnosed with chronic Q fever. Most of these patients are unaware of being infected with Coxiella burnetii, the causative agent of Q fever. To find patients in an earlier, asymptomatic stage, a targeted screening strategy (TSS) for patients with risk factors for chronic Q fever was started in the southeast region of Noord-Brabant. In total, 763 patients were tested using an IgG phase II indirect fluorescent antibody test (IFAT), of which 52 (7 %) patients tested positive. Ten of these 52 patients displayed a chronic Q fever serological profile. All of these 10 patients had a heart valve(s) or (endo-)vascular prosthesis. All except one were asymptomatic. Suggestive signs for chronic infections on positron emission tomography–computed tomography (PET-CT) were demonstrated in 5 (50 %) of these patients. Forty-two out of the 52 patients with a positive screening test showed a past Q fever serological profile. After a year of follow-up (every 3 months), none of these patients showed elevation of antibody titres and no new chronic Q fever patients were found in this group. A targeted screening programme is a useful instrument for detecting patients at risk of developing chronic Q fever.     

Conventional viral cultures and shell vial assay for diagnosis of apparently culture-negativeCoxiella burnetii endocarditis

A patient with culture-negative endocarditis was diagnosed with Q fever endocarditis based on the results of serological tests and positive leukocyte cultures obtained using conventional viral cultures and the shell vial technique. This case report suggests that isolation ofCoxiella burnetii from blood may allow better diagnostic and therapeutical evaluation of patients with Q fever endocarditis. The use of both conventional and shell vial viral cultures is recommended for the isolation ofCoxiella burnetii from the blood of patients with apparently culture-negative endocarditis.     

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.     

Coxiella burnetii infection among subjects infected with HIV type 1 in the Central African Republic

Sixty-six sera from HIV-1-seropositive adult African subjects and 49 sera from HIV-seronegative age and sex matched healthy African controls living in Bangui, Central African Republic, were screened forCoxiella burnetii antibody by an indirect immunofluorescent antibody test. 16.7 % of HIV-infected patients and 16.3 % of the HIV-negative controls had positive IgG titres, with no significant difference between the two groups. Two of the seven HIV-infected patients seropositive forCoxiella burnetii for whom clinical data was available had a medical history compatible with symptomatic Q fever. These findings indicate that there is a high degree of exposure toCoxiella burnetii infection in Bangui. In individuals co-infected with HIV andCoxiella burnetii, cellular immunosuppression could favour symptomatic Q fever. Physicians should be aware of the possibility of symptomaticCoxiella burnetii infection among HIV-infected people, particularly in endemic regions for both infections such as in sub-saharan Africa.     

Serology in chronic Q fever is still surrounded by question marks

Detection of antibodies using immunofluoresence tests (IFAT) is recommended for diagnosis of chronic Q fever, but other commercial antibody assays are also available. We compared an enzyme-linked immunosorbent assay (ELISA) (Virion/Serion) and a complement fixation test (CFT) (Virion/Serion) for the detection of Coxiella burnetii IgG phase I and IgA phase I in early- and follow-up serum samples from patients with chronic Q fever, diagnosed according to an algorithm that involves IFAT. For this, we tested sera of 49 patients, including 30 proven, 14 probable and five possible chronic Q fever cases. Sensitivity of CFT for diagnosis of chronic Q fever was suboptimal (85 %), as eight patients, including five with chronic Q fever, tested negative at time of diagnosis, whereas IgG phase I antibodies were detected in these five patients by ELISA. Sensitivity of ELISA was higher, although three probable patients were missed. No differences in ELISA IgA phase I detection between proven chronic Q fever and probable were observed; instead possible patients were in majority IgA negative (60 %). Serological examination using ELISA and CFT in follow-up sera from 26 patients on treatment was unsatisfactory. Like IFAT, all kinetic options were possible: decreasing, remaining stable or even increase during time. This study demonstrated that the sensitivity of CFT-based phase I antibody detection is low and therefore not recommended for diagnosis of chronic Q fever. Based on our results, serological follow-up to guide treatment decisions was of limited value.     

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.     

Frequency of anti-Coxiella burnetii antibodies in cattle with reproductive disorders

In this serological study, Q fever was detected in dairy cattle with reproductive problems. A total of 161 sera (74 samples from 9 dairy cattle with reproductive problems and 87 sera from 10 apparently healthy cattle herds) were collected randomly. The CHEKIT Q fever ELISA kit (IDEXX) was used to identify specific antibodies against Coxiella burnetii in dairy cattle. The results showed that 51.35% of dairy cattle with reproductive problems and 10.3% cattle without problems were positive (P?<?0.05). This first serosurvey suggests coxiellosis is an important cause in cattle with reproductive problems, and the disease may cause abortion and other reproductive problems in cattle.     

High Coxiella burnetii DNA Load in Serum during Acute Q Fever Is Associated with Progression to a Serologic Profile Indicative of Chronic Q Fever

PCR is very effective in diagnosing acute Q fever in the early stages of infection, when bacterial DNA is present in the bloodstream but antibodies have not yet developed. The objective of this study was to further analyze the diagnostic value of semiquantitative real-time PCR (qPCR) in diagnosing acute Q fever in an outbreak situation. At the Jeroen Bosch Hospital, in 2009, qPCR testing for Coxiella burnetii DNA was performed for 2,715 patients suspected of having acute Q fever (positive, n = 385; negative, n = 2,330). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the qPCR assay were calculated for patients with negative qPCR results with a follow-up sample obtained within 14 days (n = 305) and qPCR-positive patients with at least one follow-up sample (n = 369). The correctness of the qPCR result was based on immunofluorescence assay results for samples submitted for qPCR and follow-up testing. The sensitivity of the Q fever qPCR assay was 92.2%, specificity 98.9%, PPV 99.2%, and NPV 89.8%. Patients who later developed serologic profiles indicative of chronic Q fever infection had significantly higher C. burnetii DNA loads during the acute phase than did patients who did not (P < 0.001). qPCR testing is a valuable tool for the diagnosis of acute Q fever and should be used in outbreak situations when the onset of symptoms is <15 days earlier. Special attention is needed in the follow-up monitoring of patients with high C. burnetii DNA loads during the acute phase, as this might be an indicator for the development of a serologic profile indicative of chronic infection.     

Clinical and Serological Analysis of a Q Fever Outbreak in Western Slovakia with Four-Year Follow-Up

 In the spring of 1993, an outbreak of respiratory infection affected 113 persons (103 males) in Jedl'ové Kostol'any, a village located in a hilly area of western Slovakia. Q fever, manifested as a flu-like illness with atypical pneumonia and hepatic involvement, was diagnosed using four serological tests (microimmunofluorescence, microagglutination, complement fixation, and enzyme immunoassay). Aborting goats were proven as a source of infection. During a 4-year follow-up study, no chronic form of Q fever could be demonstrated, either clinically or by tests to detect phase I Coxiella burnetii antibodies.     

Comparison of immunofluorescence with enzyme immunoassay for detection of Q fever

To evaluate enzyme immunoassay (EIA) as an alternative to indirect immunofluorescence assay (IFA) to screen for Q fever in humans, 157 serum samples from patients suspected of having the disease were tested for immunoglobulin G antibodies toCoxiella burnetii. The agreement between the tests and the sensitivity of EIA were excellent (96.8% and 98.4%, respectively) when an IFAtiter of > 1/160 was considered positive. All serum samples with a titer of > 1/320 in the IFA were also positive by the EIA. The EIA seems to be an acceptable alternative to IFA for screening for Q fever.     

Early Diagnosis and Treatment of Patients with Symptomatic Acute Q Fever Do Not Prohibit IgG Antibody Responses to Coxiella burnetii

Little is known about the effect of timing of antibiotic treatment on development of IgG antibodies following acute Q fever. We studied IgG antibody responses in symptomatic patients diagnosed either before or during development of the serologic response to Coxiella burnetii. Between 15 and 31 May 2009, 186 patients presented with acute Q fever, of which 181 were included in this retrospective study: 91 early-diagnosed (ED) acute Q fever patients, defined as negative IgM phase II enzyme-linked immunosorbent assay (ELISA) and positive PCR, and 90 late-diagnosed (LD) acute Q fever patients, defined as positive/dubious IgM phase II ELISA and positive immunofluorescence assay (IFA). Follow-up serology at 3, 6, and 12 months was performed using IFA (IgG phase I and II). High IgG antibody titers were defined as IgG phase II titers of ≥1:1,024 together with IgG phase I titers of ≥1:256. At 12 months, 28.6% of ED patients and 19.5% of LD patients had high IgG antibody titers (P = 0.17). No statistically significant differences were found in frequencies of IgG phase I and IgG phase II antibody titers at all follow-up appointments for adequately and inadequately treated patients overall, as well as for ED and LD patients analyzed separately. Additionally, no significant difference was found in frequencies of high antibody titers and between early (treatment started within 7 days after seeking medical attention) and late timing of treatment. This study indicates that early diagnosis and antibiotic treatment of acute Q fever do not prohibit development of the IgG antibody response.     

Histological characteristics of the abdominal aortic wall in patients with vascular chronic Q fever

The aim of this study was to describe specific histological findings of the Coxiella burnetii‐infected aneurysmal abdominal aortic wall. Tissue samples of the aneurysmal abdominal aortic wall from seven patients with chronic Q fever and 15 patients without evidence of Q fever infection were analysed and compared. Chronic Q fever was diagnosed using serology and tissue PCR analysis. Histological sections were stained using haematoxylin and eosin staining, Elastica van Gieson staining and immunohistochemical staining for macrophages (CD68), T lymphocytes (CD3), T lymphocyte subsets (CD4 and CD8) and B lymphocytes (CD20). Samples were scored by one pathologist, blinded for Q fever status, using a standard score form. Seven tissue samples from patients with chronic Q fever and 15 tissue samples from patients without Q fever were collected. Four of seven chronic Q fever samples showed a necrotizing granulomatous response of the vascular wall, which was characterized by necrotic core of the arteriosclerotic plaque (P = 0.005) and a presence of high numbers of macrophages in the adventitia (P = 0.007) distributed in typical palisading formation (P = 0.005) and surrounded by the presence of high numbers of T lymphocytes located diffusely in media and adventitia. Necrotizing granulomas are a histological finding in the C. burnetii‐infected aneurysmal abdominal aortic wall. Chronic Q fever should be included in the list of infectious diseases with necrotizing granulomatous response, such as tuberculosis, cat scratch disease and syphilis.