High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.

Cardiobacterium valvarum, a fastidious Gram-negative bacterium, was detected in the aortic valve of a previously healthy 63-year-old man by broad-range PCR and 16S rRNA gene sequencing. In contrast to the patients in five previously published cases, our patient had neither a congenital bicuspid nor a prosthetic aortic valve. Here, we present a case of C. valvarum native tricuspid aortic valve infective endocarditis and a review of the literature. IntroductionMembers of the genus Cardiobacterium (Cardiobacterium hominis, Cardiobacterium valvarum) are a common part of the normal human oropharyngeal flora, but they can rarely act as pathogens if the mucosal integrity is disturbed (Gatselis et al., 2006). Since the first C. hominis isolation from a patient with infective endocarditis (IE) in 1962, only 87 cases have been reported in the literature (Brouqui & Raoult, 2001; Apisarnthanarak et al., 2002;Lesimple et al., 2002;Balcou-Leroy et al., 2003;Arnold et al., 2004;Walkty, 2005;Shivaprakasha et al., 2007;Jenssen et al., 2008). C. valvarum was first described in 2004, and five cases of IE caused by this species have been published so far (Han et al., 2004;Hoover et al., 2005; Bothelo et al., 2006; Geißdörfer et al., 2007;Gonzales et al., 2007). All of these C. valvarum IE patients had some form of pre-existing cardiac disease (congenital bicuspid aortic valve in four and prosthetic aortic valve in one), while 4/5 had histories of dental manipulation or poor teeth and 3/5 were afebrile. C. valvarum was not specifically identified in blood from any of the patients, but Cardiobacterium sp. was found in 2/5, HACEK in 1/5 and a Gram-negative bacterium in 1/5. In one case, C. valvarum was identified incorrectly as Eubacterium tenue. All cultures from resected valves were negative (3/3).Here, we report a case of C. valvarum IE affecting a normal (tricuspid) aortic valve in a patient with no history of recent dental procedure that was detected by broad-range PCR and 16S rRNA gene sequencing. Case reportA 63-year-old man was admitted to the University Hospital Brno with an approximate 1-month history of gradually developing resting breathlessness and repetitious episodes of cardiac decompensation. Chest pain, palpitation and weight loss were absent. The patient's personal history included type 2 diabetes, hypertension and cataract surgery, but no previously documented cardiac failure. He was not a drug addict and had not undergone any recent dental manipulations.On admission, his temperature was normal, his blood pressure was 96/30 mmHg and his heart rate was 90 beats min 21. An auscultation revealed both systolic and diastolic aortic murmurs and rales at the lung bases. A transoesophageal echocardiogram showed a mobile element of 2065 mm on the partially destroyed tricuspid aortic valve resulting in severe aortic valve regurgitation and mild functional mitral and tricuspid valve regurgitations. An angiogram showed no significant coronary stenosis, but right heart catheterization revealed severe lung hypertension. No oral ...

BackgroundThe presence of more than one bacterial agent is relatively rare in infective endocarditis, although more common in prosthetic cases. Molecular diagnosis from a removed heart tissue is considered a quick and effective way to diagnose fastidious or intracellular agents.Case presentationHere we describe the case of postpartum polymicrobial prosthetic valve endocarditis in a young woman. Sneathia sanguinegens and Mycoplasma hominis were simultaneously detected from the heart valve sample using broad range 16S rRNA polymerase chain reaction (PCR) followed by sequencing while culture remained negative. Results were confirmed by independent PCR combined with denaturing gradient gel electrophoresis. Before the final agent identification, the highly non-compliant patient left from the hospital against medical advice on empirical intravenous treatment with aminopenicillins, clavulanate and gentamicin switched to oral amoxycillin and clavulanate. Four months after surgery, no signs of inflammation were present despite new regurgitation and valve leaflet flail was detected. However, after another 5 months the patient died from sepsis and recurrent infective endocarditis of unclarified etiology.Conclusions Mycoplasma hominis is a rare causative agent of infective endocarditis. To the best of our knowledge, presented case is the first report of Sneathia sanguinegens detected in this condition. Molecular techniques were shown to be useful even in polymicrobial infective endocarditis samples.

The aim of the study was to establish a diagnostic value for broad-range polymerase chain reaction (br-PCR) and staphylococci-specific multiplex PCR (ssm-PCR) performed on surgical material from patients with staphylococcal infective endocarditis (IE). Data were analysed retrospectively from 60 patients with suspected staphylococcal IE and 59 controls who were surgically treated at three cardiosurgery centres over 4 years. Both PCR tests showed high agreement and could be aggregated. In patients with definite and rejected IE, the clinical sensitivity and specificity of PCR reached 89 and 95%, respectively. Tissue culture (TC) and PCR agreed with blood culture (BC) in 29% and 67% of IE cases. TC helped to determine aetiology in five BC negative cases while PCR aided in nine cases. Out of 52 patients with conclusive staphylococcal IE, 40 were diagnosed with S. aureus and 12 with coagulase-negative staphylococci. PCR was shown to be highly superior to TC in confirming preoperative diagnosis of IE. In addition to aid in culture negative patients, PCR helped to establish or refine aetiology in inconclusive cases. We suggest that simultaneous br-PCR and ssm-PCR performed on surgical material together with histopathology could significantly increase the performance of current Duke criteria.

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