Identification of Streptococcus bovis biotype I strains among S. bovis clinical isolates by PCR

Streptococcus bovis causes 24% of all streptococcal infective endocarditis cases. There are many reports linking both S. bovis bacteremia and endocarditis with various forms of gastrointestinal disease (primarily colonic cancers). S. bovis is divided into two biotypes: I and II. The biotype I strain is much more frequently isolated from patients with endocarditis, gastrointestinal disease, or both. We describe here the isolation of biotype I-specific DNA sequences and the development of a PCR test which can identify S. bovis biotype I strains among S. bovis clinical isolates.     

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.     

High frequency of Tropheryma whipplei in culture-negative endocarditis

"Classical" Whipple's disease (cWD) is caused by Tropheryma whipplei and is characterized by arthropathy, weight loss, and diarrhea. T. whipplei infectious endocarditis (TWIE) is rarely reported, either in the context of cWD or as isolated TWIE without signs of systemic infection. The frequency of TWIE is unknown, and systematic studies are lacking. Here, we performed an observational cohort study on the incidence of T. whipplei infection in explanted heart valves in two German university centers. Cardiac valves from 1,135 patients were analyzed for bacterial infection using conventional culture techniques, PCR amplification of the bacterial 16S rRNA gene, and subsequent sequencing. T. whipplei-positive heart valves were confirmed by specific PCR, fluorescence in situ hybridization, immunohistochemistry, histological examination, and culture for T. whipplei. Bacterial endocarditis was diagnosed in 255 patients, with streptococci, staphylococci, and enterococci being the main pathogens. T. whipplei was the fourth most frequent pathogen, found in 16 (6.3%) cases, and clearly outnumbered Bartonella quintana, Coxiella burnetii, and members of the HACEK group (Haemophilus species, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae). In this cohort, T. whipplei was the most commonly found pathogen associated with culture-negative infective endocarditis.     

Chronic Q fever in the United States

Infections due to Coxiella burnetii, the causative agent of Q fever, are uncommon in the United States. Cases of chronic Q fever are extremely rare and most often manifest as culture-negative endocarditis in patients with underlying valvular heart disease. We describe a 31-year-old farmer from West Virginia with a history of congenital heart disease and recurrent fevers for 14 months who was diagnosed with Q fever endocarditis based on an extremely high antibody titer against Coxiella burnetii phase I antigen. Despite treatment with doxycycline, he continued to have markedly elevated Coxiella burnetii phase I antibody titers for 10 years after the initial diagnosis. To our knowledge, this case represents the longest follow-up period for a patient with chronic Q fever in the United States. We review all cases of chronic Q fever reported in the United States and discuss important issues pertaining to epidemiology, diagnosis, and management of this disease.     

Contribution of systematic serological testing in diagnosis of infective endocarditis

Despite progress with diagnostic criteria, the type and timing of laboratory tests used to diagnose infective endocarditis (IE) have not been standardized. This is especially true with serological testing. Patients with suspected IE were evaluated by a standard diagnostic protocol. This protocol mandated an evaluation of the patients according to the modified Duke criteria and used a battery of laboratory investigations, including three sets of blood cultures and systematic serological testing for Coxiella burnetii, Bartonella spp., Aspergillus spp., Legionella pneumophila, and rheumatoid factor. In addition, cardiac valvular materials obtained at surgery were subjected to a comprehensive diagnostic evaluation, including PCR aimed at documenting the presence of fastidious organisms. The study included 1,998 suspected cases of IE seen over a 9-year period from April 1994 to December 2004 in Marseilles, France. They were evaluated prospectively. A total of 427 (21.4%) patients were diagnosed as having definite endocarditis. Possible endocarditis was diagnosed in 261 (13%) cases. The etiologic diagnosis was established in 397 (93%) cases by blood cultures, serological tests, and examination of the materials obtained from cardiac valves, respectively, in 348 (81.5%), 34 (8%), and 15 (3.5%) definite cases of IE. Concomitant infection with streptococci and C. burnetii was seen in two cases. The results of serological and rheumatoid factor evaluation reclassified 38 (8.9%) possible cases of IE as definite cases. Systematic serological testing improved the performance of the modified Duke criteria and was instrumental in establishing the etiologic diagnosis in 8% (34/427) cases of IE.     

Serological cross-reactions between Bartonella quintana, Bartonella henselae, and Coxiella burnetii.

The clinical manifestations of Q fever and bartonelloses can be confused, especially in cases of infectious endocarditis. Differential diagnosis of the diseases is important because the treatments required for Q fever and bartonelloses are different. Laboratory confirmation of a suspected case of either Q fever or bartonelloses is most commonly made by antibody estimation with an indirect immunofluorescence assay. With an indirect immunofluorescence assay, 258 serum samples from patients with Q fever were tested against Bartonella henselae and Bartonella quintana antigens, and 77 serum samples from patients with infection by Bartonella sp. were tested against Coxiella burnetii antigen. Cross-reactivity was observed: more than 50% of the chronic Q fever patients tested had antibodies which reacted against B. henselae antigen to a significant level. This cross-reaction was confirmed by a cross-adsorption study and protein immunoblotting. However, because the levels of specific antibody titers in cases of Bartonella endocarditis are typically extremely high, low-level cross-reaction between C. burnetii antibodies and B. henselae antigen in cases of Q fever endocarditis should not lead to misdiagnosis, provided serology testing for both agents is performed.     

Clinical evaluation of a fluorescent antibody test for the serological diagnosis of streptococcal endocarditis.

Serum fluorescent streptococcal antibody tests were carried out on 71 patients with clinically suspected infective endocarditis, and a final diagnosis of endocarditis was obtained in 46 patients. A serological diagnosis of streptococcal endocarditis was obtained in 10 patients who had persistently negative blood cultures, as fluorescent streptococcal antibody titres equal to or greater than 400 were detected against at least one of four strains of streptococci used as heterologous antigens. There were no false positive fluorescent antibody results with heterologous antigens during tests on 29 patients who had either non-streptococcal endocarditis, a final diagnosis other than endocarditis, or streptococcal sepsis not associated with endocarditis. A negative result with the heterologous antibody test could not, however, exclude a diagnosis of streptococcal endocarditis as six of 11 patients with endocarditis due to Streptococcus viridans or Str bovis confirmed on blood culture had serum fluorescent antibody titres less than 400 against all the heterologous streptococcal antigens tested. Homologous fluorescent streptococcal antibody titres equal to or greater than 400, using the patient's own blood culture isolate as the antigen, were found in the serum samples of 14 of 15 patients with endocarditis caused by viridans streptococci, three patients with enterococcal endocarditis, two patients with endocarditis caused by Str pneumoniae, and one patient with Str bovis endocarditis. In contrast, all five patients who had clinically insignificant streptococcal bacteraemias had serum fluorescent homologous antibody titres of only 100 or less. These results showed that the homologous serum fluorescent streptococcal antibody test could help to decide the clinical importance of a streptococcus which is initially isolated from only one or two of a number of inoculated blood culture bottles.     

Seroprevalence of Coxiella burnetii and Brucella abortus among pregnant women

Coxiella burnetii and Brucella abortus are two intracellular bacteria implicated in zoonotic miscarriage. In the present study, C. burnetii and B. abortus seroprevalence was compared among women from London with and without miscarriage. Coxiella burnetii seroprevalence was high (4.6%, 95% CI 2.8–7.1) despite the rare apparent exposure of this urban population. Only two patients exhibited anti- B. abortus antibodies. As a result of the risk of chronic Q fever with endocarditis and/or hepatitis, the mode of Coxiella burnetii infection in this population merits further investigation.     

Chronic Q fever-related dual-pathogen endocarditis: case series of three patients

Following Coxiella burnetii infection, there is a 1 to 5% risk of chronic Q fever. Endocarditis, mycotic aneurysm, and vascular prosthesis infection are common manifestations. We present three patients with endocarditis by C. burnetii concomitant with another bacterial pathogen. Chronic Q fever should therefore be considered in all endocarditis patients in regions where Q fever is endemic.     

Tropheryma whipplei infective endocarditis as the only manifestation of Whipple's disease

Here we describe a case of infective endocarditis caused by Tropheryma whipplei in a patient with no other symptoms of Whipple's disease. The case was diagnosed using broad-range PCR and confirmed by specific PCRs. We review the cases of infective endocarditis presenting as the only manifestation of Whipple's disease reported in the literature.     

Retrospective survey of chronic Q fever in Japan by using PCR to detect Coxiella burnetii DNA in paraffin-embedded clinical samples.

We used PCR to detect Coxiella burnetii DNA in paraffin-embedded tissues obtained from patients with chronic endocarditis in which the etiological agent had been unknown. On the basis of the published nucleotide sequence of the C. burnetii htpB gene, primers were chosen to produce an amplified fragment of 285 bp. A total of 60 samples from 56 patients were tested for the presence of C. burnetii DNA. Five samples from four patients were found to be positive. All of the amplified DNA fragments possessed a TthHB8I restriction site, as predicted from the published sequence of C. burnetii. In one of the four positive patients, rickettsia-like particles were found in sections of tissue stained by Gimenez's method. This is the first report of chronic Q fever in Japan.     

Prevalence of chronic Q fever in patients with a history of cardiac valve surgery in an area where Coxiella burnetii is epidemic

Chronic Q fever develops in 1 to 5% of patients infected with Coxiella burnetii. The risk for chronic Q fever endocarditis has been estimated to be ≈ 39% in case of preexisting valvulopathy and is potentially even higher for valvular prostheses. Since 2007, The Netherlands has faced the largest Q fever outbreak ever reported, allowing a more precise risk estimate of chronic Q fever in high-risk groups. Patients with a history of cardiac valve surgery were selected for microbiological screening through a cardiology outpatient clinic in the area where Q fever is epidemic. Blood samples were analyzed for phase I and II IgG against C. burnetii, and if titers were above a defined cutoff level, C. burnetii PCR was performed. Chronic Q fever was considered proven if C. burnetii PCR was positive and probable if the phase I IgG titer was ≥ 1:1,024. Among 568 patients, the seroprevalence of C. burnetii antibodies (IgG titer greater than or equal to 1:32) was 20.4% (n = 116). Proven or probable chronic Q fever was identified among 7.8% of seropositive patients (n = 9). Valve characteristics did not influence the risk for chronic Q fever. Patients with chronic Q fever were significantly older than patients with past Q fever. In conclusion, screening of high-risk groups is a proper instrument for early detection of chronic Q fever cases. The estimated prevalence of chronic Q fever is 7.8% among seropositive patients with a history of cardiac valve surgery, which is substantially higher than that in nonselected populations but lower than that previously reported. Older age seems to increase vulnerability to chronic Q fever in this population.     

Development and evaluation of a multiplex test for the detection of atypical bacterial DNA in community-acquired pneumonia during childhood

An incorrect or late diagnosis can lead to an increase in the morbidity and mortality caused by pneumonia, and the availability of a rapid and accurate microbiological test to verify the aetiology is imperative. This study evaluated a molecular test for the identification of the bacterial cause of atypical community-acquired pneumonia (ACAP). Fifty-four children with pneumonia were studied using bacteriological cultures, Mycoplasma pneumoniae , Coxiella burnetii , Chlamydophila pneumoniae and Legionella spp. serology, and Streptococcus pneumoniae and Legionella antigens. Simultaneously, the presence of bacterial and fungal DNA was tested for in respiratory secretion samples using the Vircell SL kit, including multiplex PCR and amplicon detection by means of line blots. There were 14 cases of ACAP caused by M. pneumoniae , with positive kit results for 13 of them, and two cases of Q-fever, with negative kit results for Coxiella burnetii . The test was negative in the remaining 38 cases (one staphylococcal pneumonia, 20 Streptococcus pneumoniae pneumonias, and 17 probable viral pneumonias). The sensitivity of the test for the detection of M. pneumoniae was 92.8% and the specificity was 100%. The Vircell SL kit allows detection of M. pneumoniae DNA in respiratory secretion samples from children with ACAP.     

In vivo endogenous spore formation by Coxiella burnetii in Q fever endocarditis.

AIMS--To determine whether Coxiella burnetii, the aetiological agent of Q fever, undergoes endogenous spore-like formation, the crucial stage of the developmental cycle, in the infected cardiac valves of patients with chronic Q fever endocarditis. METHODS--Surgically removed valves from three cases of Q fever endocarditis were processed for electron microscopy. Sections were stained with potassium permanganate and uranyl acetate before being extensively examined by transmission electron microscopy. RESULTS--In all three cases endogenous spore-like formation was seen in the infected cardiac valves. CONCLUSIONS--As the factors that govern sporogenesis in C burnetii are still largely unknown, it is uncertain how important are the implications of the discovery of endogenous spore-like formation in Q fever endocarditis. However, this finding may add new dimensions to current thinking about the treatment of chronic Q fever.     

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.     

Detection of Coxiella burnetti by DNA amplification using polymerase chain reaction.

The polymerase chain reaction (PCR) was used for the detection of Coxiella burnetti, an obligate intracellular bacterium and the etiologic agent of Q fever. A pair of primers derived from the C. burnetii superoxide dismutase gene served to amplify a targeted 257-bp fragment of genomic DNA. These primers were chosen on the basis of GenBank analysis, G + C ratio, and absence of secondary structure. This technique allowed the detection of as few as 10 C. burnetii organisms. C. burnetti was detected in tissue culture and in specimens from patients (heart valves). In all, 8 reference isolates and 22 new isolates of C. burnetii from France were successfully amplified. No amplification products were found when PCR was performed with 25 bacterial species that had been isolated in a clinical laboratory from patients with clinically similar infections. Amplification products of C. burnetii were confirmed by restriction enzyme digestion and dot blot hybridization. The method used here, a combination of PCR and restriction analysis, is a faster and more sensitive assay for C. burnetii than standard culture techniques.     

Endocarditis due to rare and fastidious bacteria

The etiologic diagnosis of infective endocarditis is easily made in the presence of continuous bacteremia with gram-positive cocci. However, the blood culture may contain a bacterium rarely associated with endocarditis, such as Lactobacillus spp., Klebsiella spp., or nontoxigenic Corynebacterium, Salmonella, Gemella, Campylobacter, Aeromonas, Yersinia, Nocardia, Pasteurella, Listeria, or Erysipelothrix spp., that requires further investigation to establish the relationship with endocarditis, or the blood culture may be uninformative despite a supportive clinical evaluation. In the latter case, the etiologic agents are either fastidious extracellular or intracellular bacteria. Fastidious extracellular bacteria such as Abiotrophia, HACEK group bacteria, Clostridium, Brucella, Legionella, Mycobacterium, and Bartonella spp. need supplemented media, prolonged incubation time, and special culture conditions. Intracellular bacteria such as Coxiella burnetii cannot be isolated routinely. The two most prevalent etiologic agents of culture-negative endocarditis are C. burnetti and Bartonella spp. Their diagnosis is usually carried out serologically. A systemic pathologic examination of excised heart valves including periodic acid-Schiff (PAS) staining and molecular methods has allowed the identification of Whipple's bacillus endocarditis. Pathologic examination of the valve using special staining, such as Warthin-Starry, Gimenez, and PAS, and broad-spectrum PCR should be performed systematically when no etiologic diagnosis is evident through routine laboratory evaluation.     

Streptococcus bovis infectious endocarditis: clinical and epidemiological characteristics

This paper shows our experience concerning the study of Streptococcus bovis infectious endocarditis (EI): 47 patients, with no prior history of IV drug abuse (NTD), who suffered EI caused by Streptococcus bovis were excerpted from a case record of 1053 cases with diagnosis of EI defined in accordance with Duke's Hospital criteria. For each patient we considered age, sex, complications, echocardiographic findings, antibiotic therapy, eventual heart-surgery and final outcome of the disease. We then compared the parameters of our patients, with the ones of 216 NTD patients suffering non Streptococcus bovis EI, selected according to age correspondence and concomitant onset of the disease. The characteristics of Streptococcus bovis EI are analogous to the ones of EIs caused by other micro-organisms in NTD patients, except for a non statistically significant trend of higher frequency in old age and in males. Concerning possible predisposing conditions, we considered the association extensively described in the literature between Streptococcus bovis EI and gastroenteric pathology (above all colon neoplasms): this association was not frequently observed in our study because appropriate instrumental investigations of the digestive tract were carried out only in a minority of patients.     

Molecular epidemiology of Streptococcus bovis causing endocarditis and bacteraemia in Italian patients

Streptococcus bovis is being recognised increasingly as a cause of infective endocarditis, and has also been associated with underlying gastrointestinal malignancy. This study evaluated the molecular epidemiology of S. bovis isolates responsible for endocarditis or bacteraemia in Italian patients between January 1990 and August 2003. S. bovis isolates were classified on the basis of their biochemical profiles, antimicrobial susceptibilities and genotypes. Of 25 isolates studied, 20 were S. bovis I and five were S. bovis II. Seven biochemical profiles were identified. Pulsed-field gel electrophoresis (PFGE) analysis identified 22 profiles that differed by at least two DNA fragments and showed a similarity of < 87%. Most PFGE patterns represented single isolates that differed in antimicrobial susceptibility, but three PFGE types were observed, with identical profiles and antibiotypes, in isolates from two different patients. S. bovis I and II isolates grouped into two distinct genetic clusters (I and II) with a similarity coefficient of 38%. Two sub-clusters (Ia and Ib), with a similarity coefficient of 47%, included 17 S. bovis I isolates with similar biochemical profiles (15 with biotype A, and two with biotype B), but different resistance phenotypes. Based on the phenotypic and genotypic heterogeneity of the isolates, it is postulated that the increase in S. bovis endocarditis in this geographical area might have been caused by the selection of sporadic endemic clones from the endogenous intestinal flora.     

Aortic valve endocarditis in a dog due to Bartonella clarridgeiae

We report the first documented case of endocarditis associated with Bartonella clarridgeiae in any species. B. clarridgeiae was identified as a possible etiological agent of human cat scratch disease. Infective vegetative valvular aortic endocarditis was diagnosed in a 2.5-year-old male neutered boxer. Historically, the dog had been diagnosed with a systolic murmur at 16 months of age and underwent balloon valvuloplasty for severe valvular aortic stenosis. Six months later, the dog was brought to a veterinary hospital with an acute third-degree atrioventricular block and was diagnosed with infective endocarditis. The dog died of cardiopulmonary arrest prior to pacemaker implantation. Necropsy confirmed severe aortic vegetative endocarditis. Blood culture grew a fastidious, gram-negative organism 8 days after being plated. Phenotypic and genotypic characterization of the isolate, including partial sequencing of the citrate synthase (gltA) and 16S rRNA genes indicated that this organism was B. clarridgeiae. DNA extraction from the deformed aortic valve and the healthy pulmonic valve revealed the presence of B. clarridgeiae DNA only from the diseased valve. No Borrelia burgdorferi or Ehrlichia sp. DNA could be identified. Using indirect immunofluorescence tests, the dog was seropositive for B. clarridgeiae and had antibodies against Ehrlichia phagocytophila but not against Ehrlichia canis, Ehrlichia ewingii, B. burgdorferi, or Coxiella burnetii.