Serotyping Coxiella burnetii isolates from acute and chronic Q fever patients by using monoclonal antibodies

Abstract Four mouse monoclonal antibodies reacting with Coxiella burnetii lipopolysaccharide antigens were produced and used in serotyping 17 C. burnetii isolates from acute Q fever and Q fever endocarditis patients in France. Two monoclonal antibodies (1B2 and 3B6) were considered specific for the Priscilla strain, a representative of Q fever endocarditis isolates, and did not react with the Nine Mile strain, which is representative of acute Q fever isolates. Monoclonal antibodies Nos. 1B2 and 3B6 reacted with 75% (3/4) acute Q fever isolates and 85% (11/13) of endocarditis isolates from France. It is reasonable to conclude that Priscilla-like strains cause both acute Q fever and Q fever endocarditis. The hypothesis that Priscilla-like strains only are associated with Q fever endocarditis should be reconsidered.     

The ligands of Numb proteins X1 and X2 are specific markers for chronic Q fever

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is spontaneously resolutive and is characterized by an efficient immune response. In contrast, chronic Q fever is characterized by dysregulated immune response, as demonstrated by the failure of C. burnetii to induce lymphoproliferation and the lack of granulomas. Recently, it has been demonstrated that when co-expressed in heterologous mammalian cell lines, the ligands of Numb proteins X1 and X2 (LNX1 and LNX2) regulate the level of the T-cell co-receptor CD8, which plays an essential role in T-cell-mediated immune response. We decided to investigate the expression of LNX1 and LNX2 genes in patients with acute or chronic Q fever. Interestingly, we found a high level of LNX1 and LNX2 mRNAs in endocarditis, the principal manifestation of chronic Q fever, but not in acute Q fever. Our data suggest that LNXs may be used as complementary biomarkers to follow the prognosis of chronic Q fever.     

Protein array of Coxiella burnetii probed with Q fever sera

Coxiella burnetii is the etiological agent of Q fever.To identify its major seroreactive proteins,a subgenomic protein array was developed.A total of 101 assumed virulence-associated recombinant proteins of C.burnetii were probed with sera from mice experimentally infected with C.burnetii and sera from Q fever patients.Sixteen proteins were recognized as major seroreactive antigens by the mouse sera.Seven of these 16 proteins reacted positively with at least 45% of Q fever patient sera.Notably,HspB had the highest fluorescence intensity value and positive frequency of all the proteins on the array when probed with both Q fever patient sera and mouse sera.These results suggest that these seven major seroreactive proteins,particularly HspB,are potential serodiagnostic and subunit vaccine antigens of Q fever.     

Diverse and atypical manifestations of Q fever in a metropolitan city hospital: Emerging role of next-generation sequencing for laboratory diagnosis of Coxiella burnetii

Although Q fever has been widely reported in the rural areas of China, there is a paucity of data on the epidemiology and clinical characteristics of this disease in large metropolitan cities. In this study, we profile the epidemiology and clinical manifestations of Q fever from a tertiary hospital in Shenzhen, a Southern Chinese metropolitan city with a large immigrant population from other parts of China. A total of 14 patients were confirmed to have Q fever during a nine-year-and-six-month period, five of whom were retrospectively diagnosed during case review or incidentally picked up because of another research project on unexplained fever without localizing features. Some patients had the typical exposure histories and clinical features, while a few other patients had rare manifestations of Q fever, including one with heart failure and diffuse intracapillary proliferative glomerulonephritis, a patient presenting with a spontaneous bacterial peritonitis-like syndrome, and another one with concomitant Q fever and brucellosis. Using a combination of clinical manifestation, inflammatory marker levels, echocardiographic findings and serological or molecular test results, nine, three and two patients were diagnosed to have acute, chronic and convalescent Q fever, respectively. Seven, five and two patients were diagnosed to have Q fever by serological test, nested real-time PCR and next-generation sequencing respectively. Diverse and atypical manifestations are associated with Q fever. The incidence of Q fever is likely to be underestimated. Next-generation sequencing is becoming an important diagnostic modality for culture-negative infections, particularly those that the physicians fail to recognize clinically, such as Q fever.     

Q fever in maritime Canada.

Only nine cases of Q fever were recorded in Canada in the 20 years prior to 1978. In the 18 months from August 1979 to January 1981 the disease was diagnosed serologically in six patients from the Maritime provinces. All were epidemiologically unrelated and none had been exposed to animals. Five had pneumonia and one had chronic Q fever with probable prosthetic valve endocarditis. Three of the five pneumonia patients presented with signs and symptoms of an acute lower respiratory tract infection and were indistinguishable clinically from other patients with atypical pneumonias. The other two with pneumonia presented with nonresolving pulmonary infiltrates and complained of decreased energy. Four of the five pneumonia patients responded well to treatment with erythromycin; the fifth required two courses of tetracycline. The patient with chronic Q fever had a large amount of cryoglobulins in his serum and evidence of immune complex disease. These cases indicate that Q fever should be considered as a possible cause of atypical pneumonia in Canada.     

Profiling the humoral immune response of acute and chronic Q fever by protein microarray

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.     

Cellular immunity in Q fever: modulation of responsiveness by a suppressor T cell-monocyte circuit

Human infection with the rickettsia Coxiella burnetii presents as an acute flulike primary Q fever, as a subacute granulomatous hepatitis, or, rarely, as chronic endocarditis. We have previously described lymphocyte unresponsiveness to Coxiella antigen in patients with Q fever endocarditis. This unresponsiveness was antigen specific and was mediated in part by adherent suppressor cells. In this report we show that the adherent suppressor cells work via prostaglandin E2 (PGE2)4 production. Addition of the cyclooxygenase inhibitor indomethacin to cultures of PBMC from patients with endocarditis or chronic laboratory exposure resulted in consistent increases in Coxiella-specific lymphocyte proliferation. The degree of increase in proliferation induced by indomethacin correlated strongly with the amount of PGE2 produced in a 4-hr culture stimulated by Coxiella antigen, but it also correlated with the sensitivity to inhibition of mitogenesis by PGE2. The suppressor mechanism was antigen nonspecific, because induction of suppression in vitro by Coxiella antigen also suppressed Candida-induced proliferation when both antigens were present in the same culture. Addition of indomethacin to these antigen cocultures totally reversed the Coxiella-induced suppression, confirming the evidence above that the nonspecific effector mechanism of suppression was prostaglandin (PG)-mediated. Elicitation of suppression, however, was antigen specific and involved a T cell-monocyte suppressor circuit. Supernatants from Coxiella-stimulated immune T cells and from the suppressor subset (OKT8+-enriched) of those T cells, but not unstimulated immune cells, induced augmented PGE2 production by unrelated nonimmune PBMC. We conclude that the lymphocyte unresponsiveness characterizing patients with Q fever endocarditis is modulated in part by an antigen-specific T suppressor cell which secretes a lymphokine to stimulate PGE2 production by adherent cells.     

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

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

Foxp3(+)CD4(+)CD25(+) regulatory T cells are increased in patients with Coxiella burnetii endocarditis

Chronic Q fever, which principally manifests as endocarditis, is characterized by Coxiella burnetii persistence and an impaired cell-mediated immune response. The long-term persistence of pathogens has been associated with the expansion of regulatory T cells (Tregs), the CD4(+) T-cell subset that is characterized by the expression of CD25 and Foxp3. We investigated the presence of Tregs in patients with acute Q fever (n?=?17), known to exhibit an efficient immune response, patients with Q fever endocarditis (n?=?54) and controls (n?=?27) by flow cytometry. The proportion of CD3(+) , CD4(+) and CD8(+) T cells was similar in controls and patients with Q fever. The percentage of CD4(+) T cells that expressed CD25 was similar in controls and patients with Q fever. The population of CD4(+) T cells that expressed both CD25 and Foxp3 was significantly (P?相似文献   

Imbalance of Circulating Monocyte Subsets and PD-1 Dysregulation in Q Fever Endocarditis: The Role of IL-10 in PD-1 Modulation

Q fever endocarditis, a severe complication of Q fever, is associated with a defective immune response, the mechanisms of which are poorly understood. We hypothesized that Q fever immune deficiency is related to altered distribution and activation of circulating monocyte subsets. Monocyte subsets were analyzed by flow cytometry in peripheral blood mononuclear cells from patients with Q fever endocarditis and controls. The proportion of classical monocytes (CD14⁺CD16⁻ monocytes) was similar in patients and controls. In contrast, the patients with Q fever endocarditis exhibited a decrease in the non-classical and intermediate subsets of monocytes (CD16⁺ monocytes). The altered distribution of monocyte subsets in Q fever endocarditis was associated with changes in their activation profile. Indeed, the expression of HLA-DR, a canonical activation molecule, and PD-1, a co-inhibitory molecule, was increased in intermediate monocytes. This profile was not restricted to CD16⁺ monocytes because CD4⁺ T cells also overexpressed PD-1. The mechanism leading to the overexpression of PD-1 did not require the LPS from C. burnetii but involved interleukin-10, an immunosuppressive cytokine. Indeed, the incubation of control monocytes with interleukin-10 led to a higher expression of PD-1 and neutralizing interleukin-10 prevented C. burnetii-stimulated PD-1 expression. Taken together, these results show that the immune suppression of Q fever endocarditis involves a cross-talk between monocytes and CD4⁺ T cells expressing PD-1. The expression of PD-1 may be useful to assess chronic immune alterations in Q fever endocarditis.     

A proteomic approach to investigate the differential antigenic profile of two Coxiella burnetii strains

Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Current diagnostics of Q fever is based on serological testing of patient serum. Biological distinction among C. burnetii strains has been referred at the genetic level as well as in virulence in animal models of Q fever. Disclosure of strain specific antigens might show insight into the biology and pathogenesis of this query pathogen, as well as it can provide the literature with potential serodiagnostic markers. In the present study, we sought to obtain an outer membrane enriched fraction of two C. burnetii reference strains, which originate from different sources, in order to investigate the way in which their antigenic profile is differentiated against a patient serum. We systematically analyzed the sarcosyl-insoluble fraction, enriched in outer membrane proteins, of the two C. burnetii strains using doubled SDS-PAGE combined with MS/MS analysis. In total, twenty-two outer membrane proteins were identified, representing 26% of the overall 86 identified proteins. The sarcosyl-insoluble fraction was then separated on 2DE IEF/SDS-PAGE and probed with serum from an infected patient. Different immuno-reactive proteins between the two C. burnetii strains were identified and included 2 outer membrane proteins, a hypothetical protein (CBU_0937) with unknown function and OmpH (CBU_0612), a previously identified marker for Q fever endocarditis. This approach can be used to reveal strain-specific proteins involved in pathogenesis and new serodiagnostic markers.     

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.     

First case of Q fever endocarditis in Croatia and a short review

We present a 70-year-old man from Dalmatia, Croatia, with a history of prolonged high fever diagnosed as Q fever endocarditis. As far as we know, this is the first case of chronic Q fever in Croatia. The treatment was started as for culture-negative endocarditis, but was without clinical response. After significantly high anti-phase I IgG plus IgA antibodies titers to Coxiella burnetii were shown, the initial treatment with doxycycline was changed and ciprofloxacin was started with good clinical response.     

Detection of IgG binding to Schistosoma mansoni recombinant protein RP26 is a sensitive and specific method for acute schistosomiasis diagnosis

We recently described the first recombinant Schistosoma mansoni protein RP26, which was capable of acute infection diagnosis. The aim of the present work was to further characterize the RP26 diagnostic properties in immunoblot and enzyme-linked immunosorbent (ELISA) assays. Testing sera from uninfected donors and sera from patients with acute or chronic Schistosoma infection by Western blot immunoassay revealed 100% specificity and 100% sensitivity for acute infection identification. Sera from uninfected, acute, and chronic schistosomiasis were also probed for IgG, IgG4, IgA, and IgM reactivity to RP26 plus soluble egg antigens (SEA) in ELISA. The mean IgG reactivity to RP26 by sera from acute schistosomiasis patients was significantly higher than the chronic ones. The IgG4, IgA, and IgM reactivities to RP26 were low and similar in both infected groups. The mean IgA and IgM reactivities to SEA were significantly higher in the group of acute compared to chronic group, whereas mean IgG4 reactivity was higher in chronic group. To estimate the specificity of Schistosoma infection diagnosis sera from patients infected with other different parasites were tested to detect IgG reactivity to RP26 and IgA and IgM reactivity to SEA. For IgA against SEA detection, 72% of sera were positive and 48% of sera were positive for IgM detection. Based on these results we can suggest that detection of sera IgG binding to RP26 is a sensitive and specific method for acute schistosomiasis diagnosis. Therefore, RP26 is a candidate for immunodiagnostic kit development.     

Euthermic Endocarditis

Background

Most patients with infective endocarditis (IE) manifest fever. Comparison of endocarditis patients with and without fever, and whether the lack of fever in IE is a marker for poorer outcomes, such as demonstrated in other severe infectious diseases, have not been defined.

Methods and Results

Cases from the Mayo Clinic, Rochester, Minnesota, Division of Infectious Diseases IE registry, a single-center database that contains all cases of IE treated at our center. Diagnosis date between 1970 and 2006, which met the modified Duke criteria for definite endocarditis, without fever was included. There were 240 euthermic endocarditis cases included in this analysis, with 282 febrile controls selected by frequency matching on gender and decade of diagnosis. Euthermic patients had a median age of 63.6 years (±16.1) as compared to 59.0 years (±16.4) in the febrile control group (p=0.001). Median (IQR) symptom duration prior to diagnosis was 4.0 (1.0, 12.0) weeks in the euthermic group compared to 3.0 (1.0, 8.0) weeks in the febrile controls (p= 0.006). From unadjusted analyses, survival rates were 87% in euthermic cases versus 83% in febrile controls across 28-day follow-up (p=0.164), and 72% in euthermic group cases versus 69% in febrile controls across 1-year follow-up (p=0.345). Also unadjusted, the 1-year cumulative incidence rate of valve surgery was higher in euthermic cases versus febrile controls (50% vs. 39%, p= 0.004).

Conclusions

Patients with euthermic endocarditis are older, and lack of fever was associated with longer symptom duration and delayed diagnosis prior to IE diagnosis. Despite a higher unadjusted rate of valve surgery in euthermic patients, the result was not significant when adjusting for baseline confounders. Differences in survival rates at both 28-days and 365-days were not statistically significant between the two groups.     

Antigenic epitope mapping of the M24 protein of Streptococcus pyogenes: implications for serodiagnosis of rheumatic fever

Abstract Rheumatic fever continues to be a significant problem in Australian Aboriginal communities and developing countries worldwide. Early diagnosis could facilitate the institution of penicillin prophylaxis resulting in the prevention of recurrences of rheumatic fever. An overlapping biotinylated peptide bank of 82 peptides, based on the known sequence of Streptococcus pyogenes M24 protein, was used in a standard enzyme immunoassay. A total of 82 sera were tested from both aboriginal and non-aboriginal subjects with clinically proven rheumatic fever, rheumatic heart disease and matched controls. Two peptides with significant sequence homology at the C-terminal end were found to be discriminatory between aboriginal cases and controls. It is proposed that these peptides could be the basis of a serological test for rheumatic fever.     

Epidemiological investigation and physician awareness regarding the diagnosis and management of Q fever in South Korea, 2011 to 2017

BackgroundIn South Korea, the number of Q fever cases has rapidly increased since 2015. Therefore, this study aimed to characterize the epidemiological and clinical features of Q fever in South Korea between 2011 and 2017.Methods/Principal findingsWe analyzed the epidemiological investigations and reviewed the medical records from all hospitals that had reported at least one case of Q fever from 2011 to 2017. We also conducted an online survey to investigate physicians’ awareness regarding how to appropriately diagnose and manage Q fever. The nationwide incidence rate of Q fever was annually 0.07 cases per 100,000 persons. However, there has been a sharp increase in its incidence, reaching up to 0.19 cases per 100,000 persons in 2017. Q fever sporadically occurred across the country, with the highest incidences in Chungbuk (0.53 cases per 100,000 persons per year) and Chungnam (0.27 cases per 100,000 persons per year) areas. Patients with acute Q fever primarily presented with mild illnesses such as hepatitis (64.5%) and isolated febrile illness (24.0%), whereas those with chronic Q fever were likely to undergo surgery (41.2%) and had a high mortality rate (23.5%). Follow-up for 6 months after acute Q fever was performed by 24.0% of the physician respondents, and only 22.3% of them reported that clinical and serological evaluations were required after acute Q fever diagnosis.ConclusionsQ fever is becoming an endemic disease in the midwestern area of South Korea. Given the clinical severity and mortality of chronic Q fever, physicians should be made aware of appropriate diagnosis and management strategies for Q fever.     

Identification of a streptococcal octapeptide motif involved in acute rheumatic fever

Acute rheumatic fever is a serious autoimmune sequela of pharyngitis caused by certain group A streptococci. One mechanism applied by streptococcal strains capable of causing acute rheumatic fever is formation of an autoantigenic complex with human collagen IV. In some geographic regions with a high incidence of acute rheumatic fever pharyngeal carriage of group C and group G streptococci prevails. Examination of such strains revealed the presence of M-like surface proteins that bind human collagen. Using a peptide array and recombinant proteins with targeted amino acid substitutions, we could demonstrate that formation of collagen complexes during streptococcal infections depends on an octapeptide motif, which is present in collagen binding M and M-like proteins of different beta-hemolytic streptococcal species. Mice immunized with streptococcal proteins that contain the collagen binding octapeptide motif developed high serum titers of anti-collagen antibodies. In sera of rheumatic fever patients such a collagen autoimmune response was accompanied by specific reactivity against the collagen-binding proteins, linking the observed effect to clinical cases. Taken together, the data demonstrate that the identified octapeptide motif through its action on collagen plays a crucial role in the pathogenesis of rheumatic fever. Eradication of streptococci that express proteins with the collagen binding motif appears advisable for controlling rheumatic fever.     

Persistent High IgG Phase I Antibody Levels against Coxiella burnetii among Veterinarians Compared to Patients Previously Diagnosed with Acute Q Fever after Three Years of Follow-Up

Background

Little is known about the development of chronic Q fever in occupational risk groups. The aim of this study was to perform long-term follow-up of Coxiella burnetii seropositive veterinarians and investigate the course of IgG phase I and phase II antibodies against C. burnetii antigens and to compare this course with that in patients previously diagnosed with acute Q fever.

Methods

Veterinarians with IgG phase I ≥1:256 (immunofluorescence assay) that participated in a previous seroprevalence study were asked to provide a second blood sample three years later. IgG antibody profiles were compared to a group of acute Q fever patients who had IgG phase I ≥1:256 twelve months after diagnosis.

Results

IgG phase I was detected in all veterinarians (n = 76) and in 85% of Q fever patients (n = 98) after three years (p<0.001). IgG phase I ≥1:1,024, indicating possible chronic Q fever, was found in 36% of veterinarians and 12% of patients (OR 3.95, 95% CI: 1.84–8.49).

Conclusions

IgG phase I persists among veterinarians presumably because of continuous exposure to C. burnetii during their work. Serological and clinical follow-up of occupationally exposed risk groups should be considered.     

Anti-human cardiac myosin autoantibodies in Kawasaki syndrome.

Kawasaki syndrome (KS) is the major cause of acquired heart disease in children. Although acute myocarditis is observed in most patients with KS, its pathogenesis is unknown. Because antimyosin autoantibodies are present in autoimmune myocarditis and rheumatic carditis, the purpose of the current study was to determine whether anticardiac myosin Abs might be present during the acute stage of KS. Sera from KS patients as well as age-matched febrile controls and normal adults were compared for reactivity with human cardiac myosin in ELISAs and Western blot assays. A total of 5 of 13 KS sera, as compared with 5 of 8 acute rheumatic fever sera, contained Ab titers to human cardiac myosin that were significantly higher than those found in control sera. Both cardiac and skeletal myosins were recognized in the ELISA by KS sera, although stronger reactivity was observed to human cardiac myosin. Only IgM antimyosin Abs from KS sera were significantly elevated relative to control sera. KS sera containing antimyosin Abs recognized synthetic peptides from the light meromyosin region of the human cardiac myosin molecule and had a different pattern of reactivity than acute rheumatic fever sera, further supporting the association of antimyosin Ab with KS. These Abs may contribute to the pathogenesis of acute myocarditis found in patients with KS.