A Case of Q Fever Prosthetic Joint Infection and Description of an Assay for Detection of Coxiella burnetii

We present the first published case of Coxiella burnetii prosthetic joint infection. Diagnosis was established with PCR and culture of periprosthetic tissue and synovial fluid (and serology). A novel PCR assay is described herein. Q fever should be considered in patients with prosthetic joint infection without an identified pathogen.     

Clinical Evaluation of a New PCR Assay for Detection of Coxiella burnetii in Human Serum Samples

A nested PCR method was developed for the detection of Coxiella burnetii in human serum samples. Two pairs of oligonucleotide primers were designed to amplify a 438-bp fragment of the com1 gene encoding a 27-kDa outer membrane protein of C. burnetii. The primers amplified the predicted fragments of 21 various strains of C. burnetii but did not react with DNA samples from other microorganisms. The 438-bp amplification products could be digested with restriction enzymes SspI and SalI. The utility of the nested PCR was evaluated by testing human serum samples. The com1 gene fragment was amplified from 135 (87%) of 155 indirect immunofluorescence test (IF)-positive serum samples and from 11 (11%) of 100 IF-negative serum samples. The nested PCR with primers targeted to the com1 gene appeared to be a sensitive, specific, and useful method for the detection of C. burnetii in serum samples.     

Isolation of Coxiella burnetii from heart valves of patients treated for Q fever endocarditis.

Coxiella burnetii was isolated from the valve material of two patients who underwent valvectomy because of progressive congestive heart failure due to endocarditis. In each case antibiotic therapy was administered for several months prior to valvectomy. Classical histopathological examination of the valves did not reveal an etiology. However, coxiella-like organisms were demonstrated in valvular material with Köster, Stamp, and Giemsa stains, and the organisms were grown in cell culture. Antibody titers were consistent with the diagnosis of chronic C. burnetii infection. This report illustrates the advantage of simple and fast staining techniques and cell culture for the demonstration and isolation of C. burnetii in the heart valve tissue of patients with Q fever endocarditis.     

Predominant Immunoglobulin A Response to Phase II Antigen of Coxiella burnetii in Acute Q Fever

Diagnosis of acute Q fever is usually confirmed by serology, on the basis of anti-phase II antigen immunoglobulin M (IgM) titers of ≥1:50 and IgG titers of ≥1:200. Phase I antibodies, especially IgG and IgA, are predominant in chronic forms of the disease. However, between January 1982 and June 1998, we observed anti-phase II antigen IgA titers of ≥1:200 as the sole or main antibody response in 10 of 1,034 (0.96%) patients with acute Q fever for whom information was available. In order to determine whether specific epidemiological or clinical factors were associated with these serological profiles, we conducted a retrospective case-control study that included completion of a standardized questionnaire, which was given to 40 matched controls who also suffered from acute Q fever. The mean age of patients with elevated phase II IgA titers was significantly higher than that usually observed for patients with acute Q fever (P = 0.026); the patients were also more likely than controls to live in rural areas (P = 0.026) and to have increased levels of transaminase in blood (P = 0.03). Elevated IgA titers are usually associated with chronic Q fever and are directed mainly at phase I antigens. Although the significance of our findings is unexplained, we herein emphasize the fact that IgA antibodies are not specific for chronic forms of Q fever and that they may occasionally be observed in patients with acute disease. Moreover, as such antibody profiles may not be determined by most laboratories, which test only for total antibody titers to phase I and II antigens, the three isotype-specific Ig titers should be determined as the first step in diagnosing Q fever.     

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.     

Molecular detection of Coxiella burnetii in the sera of patients with Q fever endocarditis or vascular infection

In the absence of a specific diagnosis based on serology, chronic Q fever is inevitably fatal. However, diagnosis is often delayed because the test is not widely available. To shorten the diagnostic delay, we adapted a nested-PCR assay with serum as a template and the LightCycler as a thermal cycler, termed LCN-PCR. We retrospectively and prospectively applied this method to samples from 48 patients diagnosed with Q fever endocarditis or vascular infection and to samples from 100 controls with endocarditis caused by other microorganisms. We also prospectively applied this technique to samples from 30 patients treated for a Q fever endocarditis and to samples from 13 patients with a convalescent acute Q fever with ambiguous immunoglobulin G (IgG) phase I titer. LCN-PCR had a specificity of 100%. It was positive only in samples from patients with evolutive Q fever, as none of the samples from patients with a treated chronic Q fever or with a convalescent acute Q fever presented positive results. When performed prospectively on recently stored sera, the sensitivity of LCN-PCR is 64% (7 of 11 samples; P = 0.004), but the efficiency of LCN-PCR was dramatically altered by the storage of specimens at -20 degrees C. High IgG phase I titers decreased the sensitivity of LCN-PCR. A significant difference was observed among LCN-PCR results for sera with IgG phase I titers of > or =1:25,600 compared to sera with IgG phase I titers of <1:25,600 (0 of 15 samples versus 13 of 33 samples; P = 0.004). In patient samples with titers below 1:25,600 tested prospectively, sensitivity was 100% (7 of 7). The LCN-PCR assay may be helpful in establishing an early diagnosis of chronic Q fever.     

Isolation and characterization of a plasmid from phase I Coxiella burnetii.

The DNA from the Nine Mile phase I strain of Coxiella burnetti, the etiological agent of Q fever, has been isolated and purified by cesium chloride-ethidium bromide density gradient centrifugation. A fraction of this DNA has a density characteristic of plasmid DNA. The plasmid DNA was cut with 20 different restriction endonucleases and shown to be a discrete entity. The plasmid, designated QpH1, is approximately 36 kilobases in size and has a molecular mass of 2.4 x 10(7) daltons. A partial restriction map of QpH1 has been constructed by using the restriction endonucleases SalI, KpnI, PstI, and XbaI. QpH1 DNA radioactively labeled by nick translation was used to show that sequences similar to the plasmid are also present in the phase II antigenic variant of C. burnetii.     

Correlations between Peripheral Blood Coxiella burnetii DNA Load,Interleukin-6 Levels,and C-Reactive Protein Levels in Patients with Acute Q Fever

From 2007 to 2010, the Netherlands experienced the largest reported Q fever outbreak, with >4,000 notified cases. We showed previously that C-reactive protein is the only traditional infection marker reflecting disease activity in acute Q fever. Interleukin-6 is the principal inducer of C-reactive protein. We questioned whether increased C-reactive protein levels in acute Q fever patients coincide with increased interleukin-6 levels and if these levels correlate with the Coxiella burnetii DNA load in serum. In addition, we studied their correlation with disease severity, expressed by hospital admission and the development of fatigue. Interleukin-6 and C-reactive protein levels were analyzed in sera from 102 patients diagnosed with seronegative PCR-positive acute Q fever. Significant but weak negative correlations were observed between bacterial DNA loads expressed as cycle threshold values and interleukin-6 and C-reactive protein levels, while a significant moderate-strong positive correlation was present between interleukin-6 and C-reactive protein levels. Furthermore, significantly higher interleukin-6 and C-reactive protein levels were observed in hospitalized acute Q fever patients in comparison to those in nonhospitalized patients, while bacterial DNA loads were the same in the two groups. No marker was prognostic for the development of fatigue. In conclusion, the correlation between interleukin-6 and C-reactive protein levels in acute Q fever patients points to an immune activation pathway in which interleukin-6 induces the production of C-reactive protein. Significant differences in interleukin-6 and C-reactive protein levels between hospitalized and nonhospitalized patients despite identical bacterial DNA loads suggest an important role for host factors in disease presentation. Higher interleukin-6 and C-reactive protein levels seem predictive of more severe disease.     

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.     

Coxiella burnetii survives in monocytes from patients with Q fever endocarditis: involvement of tumor necrosis factor

Endocarditis is the most frequent form of chronic Q fever, an infectious disease caused by Coxiella burnetii. As this obligate intracellular bacterium inhabits monocytes and macrophages, we wondered if pathogenesis of Q fever endocarditis is related to defective intracellular killing of C. burnetii by monocytes. Monocytes from healthy controls eliminated virulent C. burnetii within 3 days. In contrast, monocytes from patients with ongoing Q fever endocarditis were unable to eliminate bacteria even after 6 days. In patients who were cured of endocarditis, the monocyte infection was close to that of control monocytes. This killing deficiency was not the consequence of generalized functional impairment, since patient monocytes eliminated avirulent C. burnetii as did control cells. The addition of supernatants of C. burnetii-stimulated monocytes from patients with ongoing endocarditis to control monocytes enabled them to support C. burnetii survival, suggesting that some soluble factor is responsible for bacterial survival. This factor was related to tumor necrosis factor (TNF): expression of TNF mRNA and TNF release were increased in response to C. burnetii in patients with ongoing endocarditis compared to cured patients and healthy controls. In addition, neutralizing anti-TNF antibodies decreased C. burnetii internalization, an early step of bacterial killing, in monocytes from patients with ongoing endocarditis but did not affect delayed steps of intracellular killing. We suggest that Q fever-associated activation of monocytes allows the survival of C. burnetii by modulating early phases of microbial killing.     

Primary humoral antibody response to Coxiella burnetii, the causative agent of Q fever.

Of 147 patients with acute Q fever diagnosed during a major outbreak in Birmingham, England, in early summer 1989, 41 provided sets of sera which allowed us to make a detailed analysis of the primary humoral immune response. Antibody titers specific for Coxiella burnetii were measured by the complement fixation test and by an immunoglobulin M (IgM)- and IgG-specific indirect immunofluorescence test. The relative avidity of specific IgGs was determined by the indirect immunofluorescence test with and without treatment of antigen-antibody complexes with 8 M urea. The IgG subclass responses after primary infection and their avidities were also determined for a limited number of paired serum specimens. Specific IgM titers persisted for more than 6 months in the majority of cases and were therefore not a sufficient criterion for the diagnosis of recent infection. However, for serial samples the antibody titer ratios (IgG/IgM) and the ratios (IgG titer with treatment/IgG titer without treatment) that indicated relative avidity changed significantly, depending on the time postinfection. Within the IgG class, the C. burnetii-specific antibody response over time was almost exclusively represented by subclass 1 molecules, which thus showed affinity maturation.     

Isolation, Identification, and Molecular Characterization of Strains of Photorhabdus luminescens from Infected Humans in Australia

We describe the isolation of Photorhabdus (Xenorhabdus) luminescens from four Australian patients: two with multiple skin lesions, one with bacteremia only, and one with disseminated infection. One of the patients had multiple skin lesions following the bite of a spider, while the lesions in the other patient were possibly associated with a spider bite. The source of infection for the remaining two patients is unknown. As a member of the family Enterobacteriaceae, P. luminescens is unusual in that it fails to reduce nitrate and ferments only glucose and mannose. It gives negative reactions for lysine decarboxylase, arginine dihydrolase, and ornithine decarboxylase (Moeller). The species is motile, utilizes citrate, hydrolyzes urea, and usually produces a unique type of annular hemolysis on sheep blood agar plates incubated at 25 degrees C. A weak bioluminescence is the defining characteristic. P. luminescens is an insect pathogen and is symbiotically associated with entomopathogenic nematodes. Its isolation from human clinical specimens has been reported previously from the United States. Restriction fragment length polymorphism-PCR analysis of the 16S rRNA gene demonstrated a high level of similarity among the Australian clinical strains and significant differences between the Australian clinical strains and the U.S. clinical strains. However, numerical analyses of the data suggest that the two groups of clinical strains are more similar to each other than they are to the symbiotic strains found in nematodes. This is the first report of the isolation of P. luminescens from infected humans in Australia and the second report of the isolation of this species from infected humans worldwide.     

Q fever pneumonia: virulence of Coxiella burnetii pathovars in a murine model of aerosol infection

Q fever is a worldwide zoonosis caused by Coxiella burnetii, a strictly intracellular bacterium that is a potential bioweapon. Humans usually acquires Q fever after inhalation of dust infected by subclinical animals. We used an aerosol exposure apparatus to challenge immunocompetent (BALB/c) and severe combined immunodeficient (SCID) mice with two different strains (strain Nine Mile and strain Q 212) of C. burnetii at two different inocula. Pathological lesions and dissemination of the bacteria were related to the size of the inoculum. SCID mice showed major pulmonary lesions, whereas similarly infected BALB/c mice were more able to eliminate the bacteria. Pathological differences were found between the strains, with Nine Mile showing more severe histological lesions and quantified spread of bacteria. Our animal model could provide a new tool for the study of acute Q fever pneumonia, the development of Q fever in immunodeficient hosts, and the differentiation of pathogenicity among C. burnetii isolates.     

Coxiella burnetii, the agent of Q fever, stimulates an atypical M2 activation program in human macrophages

Coxiella burnetii is an obligate intracellular bacterium, responsible for Q fever, which survives in macrophages by interfering with their microbicidal competence. As functional polarization of macrophages is critical for their microbicidal activity, we studied the activation program of monocyte-derived macrophages (MDM) stimulated with C. burnetii. This program was markedly distinct from that induced by lipopolysaccharides (LPS), a canonical inducer of M1 polarization. Indeed, C. burnetii up-regulated the expression of genes associated with M2 polarization, including TGF-beta1, IL-1 receptor antagonist (IL-1ra), CCL18, the mannose receptor and arginase-1, and only up-regulated the expression of two genes associated with M1 polarization, namely IL-6 and CXCL8. In contrast, C. burnetii down-regulated the expression of genes associated with M1 polarization such as TNF, CD80, CCR7 and TLR-2. Functional analyses showed that C. burnetii-stimulated MDM produced high levels of TGF-beta1 and CCL18, and expressed the mannose receptor and arginase-1, the latter being associated with the prevention of nitric oxide production by MDM. Finally, C. burnetii induced the release of IL-6 and CXCL8 at a lower level than LPS-stimulated MDM. Our results suggest that C. burnetii stimulated an atypical M2 activation program that may account for the persistence of C. burnetii in macrophages.     

Automated Broad-Range Molecular Detection of Bacteria in Clinical Samples

Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice.     

Development of Molecular Methods for Identification of Schizophyllum commune from Clinical Samples

In the last 50 years, to our knowledge, only 16 cases of diseases caused by Schizophyllum commune in humans have been reported. Within only 6 months, we found four isolates of this basidiomycetous fungus, obtained from patients suffering from chronic sinusitis. The cultures of the isolated fungi showed neither clamp connections nor fruiting bodies (basidiocarps), which are distinctive features for S. commune, but fast-growing cottony white mycelium only. This was harvested, and DNA was extracted. The internal transcribed spacer region of the ribosomal DNA (rDNA) was amplified with fungus-specific primers, and the PCR products were sequenced. Two strains of S. commune, collected from branches of a European hornbeam (Carpinus betulus) and a tree of heaven (Ailanthus altissima), respectively; four specimens from the herbarium of the Institute of Botany, Karl-Franzens-University Graz; and two strains from internationally known culture collections (CBS 340.81 [ATCC 44201] and CBS 405.96) were investigated in the same way. The sequence data of all strains were compared and showed homology of over 99% in this 660-bp-long fragment of rDNA. With these results, a map of restriction enzyme cutting sites and a primer set specific for S. commune were created for reliable identification of this human pathogenic fungus.     

Genotypic diversity of Coxiella burnetii in the 2007-2010 Q fever outbreak episodes in The Netherlands

The genotypic diversity of Coxiella burnetii in clinical samples obtained from the Dutch Q fever outbreak episodes of 2007-2010 was determined by using a 6-locus variable-number tandem repeat analysis panel. The results are consistent with the introduction of one founder genotype that is gradually diversifying over time while spreading throughout The Netherlands.