Bartonella quintana Endocarditis of the Aortic Valve: First case report in Turkey

Endocarditis caused by Bartonella strains has been increasingly reported. While a few sporadic case reports associated with B. henselae were published in Turkey, no endocarditis cases caused by Bartonella spp. have been reported yet. Herein, a case of first B. quintana endocarditis in Turkey, diagnosed using molecular methods, was presented.


INTRODUCTION
Bartonella species are gram-negative, facultative and intracellular bacteria. They can cause bacteremia, endocarditis, trench fever, and bacillary angiomatosis (1). Reported Bartonella related infections in humans are associated with B.hanseleae in Turkey (2)(3)(4). B. quintana positivity has not been shown in animals and humans in Turkey.
B. quintana is responsible for approximately 75% of endocarditis with Bartonella species in humans (5). Direct examination, culture, serological and molecular methods (PCR assays) are used in the diagnosis of diseases caused by Bartonella species (1). Due to the slow growth of Bartonella species, cultures are not routinely recommended for the diagnosis of Bartonella infections (1). For the diagnosis of Bartonella infections, the gold standard serological test is an immunofluorescence assay (IFA) with a sensitivity of 82% and 93% in people with acute and chronic infection, respectively (1). In recent years, molecular techniques have been used effectively for the identification of Bartonella species, and DNA sequence analysis of polymerase chain reaction (PCR) and PCR amplification products of genus-specific gene regions such as gltA, ssrA, rib C, groEL have been used for molecular identification of Bartonella species (6).
Herein, a case of first B. quintana endocarditis in Turkey, diagnosed using serological and molecular methods, was presented.

CASE REPORT
A 64-year-old Turkish man, from the southeast of Anatolia, was hospitalized with a history of weakness, fatigue and weight loss for seven months. He had a history of diabetes mellitus and hypertension and a moderate aortic regurgitation. In the physical examination of the patient, body temperature, blood pressure, heart rate, and respiratory rate were 36.3 o C, 130/63 mmHg, 86/min, 16/min, respectively. A grade 3 diastolic heart murmur was detected at the left upper sternal border. Initial laboratory investigations demonstrated white blood cell count 10 700 cells/micro L (66 % neutrophils ), hemoglobin 9.0 g/ dL aspartate aminotransferase 25 U/L, alanine transaminase 27 U/L, blood urea nitrogen 22 mg/dl, creatinine 0.94 mg/dl, C-reactive protein 85 mg/dl and erythrocyte sedimentation rate: 95 mm/h. Microscopic hematuria was detected in urinalysis. Transthoracic echocardiography was performed, followed by transesophageal echocardiography. It revealed a severe aortic regurgitation (grade 3) and mobile aortic vegetation measuring 15 x 7 mm on the right coronary leaflet. Roth spots were detected in the ophthalmologic examination. Empirical ampicillinsulbactam treatment was started after two sets of aerobic and anaerobic blood cultures were obtained. Blood cultures yielded no bacteria. Serological tests for Coxiella burnetii, Brucella and Bartonella spp. were performed to evaluate the etiology of culture-negative endocarditis. An indirect fluorescent antibody test (IFA) was performed in Bartonella serology (6). B. henselae and B. quintana binary antigen closed commercial kit (Focus Diagnostic, California USA) was used for IFA test. IFA test result was positive for B. henselae IgG (1/1024 titer) and B. quintana IgG (1/1024 titer). Following this result, oral doxycycline and intravenous gentamicin treatment were started. Aortic valve replacement was performed after four weeks of treatment. The tissue was obtained from the aortic valve during the surgery. Bartonella was found to be positive in polymerase chain reaction (PCR) testing.
For DNA extraction, the aortic valve was homogenized in a Magnalyser homogenization device (Roche, Rotkreuz, Switzerland) by adding 500µl of phosphate-buffered saline to the tissue sample. 100 µl of the homogenized tissue sample was taken and DNA extraction was performed using the tissue extraction kit (Qiagen, Hilden, Germany). Real-time PCR was performed using ssrA gene-specific primer and probe to determine Bartonella species (7). The tissue sample was found to be Bartonella spp. positive in real-time PCR. Conventional PCR of the ssrA gene region and the PCR amplification product were sequenced to determine the species. DNA sequence analysis data were compared with GenBank data using the basic Local Alignment Search Tool (Blast version 2.0). The ssrA sequence was 100% compatible with the B. quintana strain Toulouse Accession number HG518998.1 and the B. quintana strain NCTC12899 Accession number LS483373.1. Using the DNA sequence analysis data and data from other Bartonella species at Genbank, the phylogenetic tree was created using the Maximum likelihood statistical method in MAGA 5.1 program and the B. quintana Bootstrap phylogeny value was determined to be highly reliable with 96% (Figure 1.)

DISCUSSION
This is the first case of aortic valve endocarditis (BCNE) caused by B. quintana in Turkey. Bartonella spp. are fastidious bacteria that cause blood culturenegative endocarditis (BCNE) and have been increasingly reported (8). The estimated incidence of Bartonella spp. endocarditis is ranging from 1.0 to 15.6%, depending on the series (8). Bartonella endocarditis has shown worldwide distribution, and a lot of case series of Bartonella endocarditis is reported from Europe (8,9). While a few seroprevalence studies and sporadic case reports associated with B. henselae were published in Turkey, no endocarditis cases caused by Bartonella spp. have been reported yet (3,4). This might be because of the specific laboratory assessment (serological testing or molecular assays) for Bartonella spp. are rarely performed in only a few reference laboratories in our country. B.quintana is responsible for three-fourths of Bartonella endocarditis cases (5). Male gender, immunosuppression including HIV infection, alcoholism, previous valvulopathy, and some epidemiologic aspects such as low socioeconomic status or homelessness are defined as risk factors for B. quintana endocarditis (5,8,9). Our patient had a higher socioeconomic condition. Diabetes mellitus and aortic regurgitation can be considered as risk factors for Bartonella endocarditis in our patient. Bartonella endocarditis usually presents with prolonged non-specific symptoms such as fever, fatigue, weight loss, or signs of heart failure, such as exertional dyspnea or hypoxia. This may lead to a delay in diagnosis and treatment, as in our patient (9).
Currently, there is a lack of criteria for the diagnosis of Bartonella endocarditis (8,9). Detection of IgG antibodies using the micro immunofluorescence technique has been used for the diagnosis of Bartonella endocarditis in many studies (8,9). Bartonella IgG titer of ≥1:800 is recommended as the cut off for a positive test result (8,9). However, serological test results, except for C. burnetii, are not incorporated into the modified Duke criteria (8,10). This might be because of the cross-reactivity between various antibodies in serological tests. Bartonella serological assays may demonstrate cross-reactivity with Epstein-Barr virus, cytomegalovirus, Toxoplasma gondii, Streptococcus pyogenes, Chlamydia and Coxiella (11,12). Also, cross-reactions can be seen among the Bartonella species, as in our case (11,12). For these reasons, molecular methods, are increasingly utilized to aid in the diagnosis of culture-negative endocarditis. Testing of cardiac valve tissue with Bartonella -specific PCR assays is more sensitive than testing blood or serum (8). Amplification of Bartonella DNA from the valve tissue has been shown to have high sensitivity and specificity ranging from 72 -98% (9). Edouard et al. (8) suggested that a positive PCR result from a valvular biopsy specimen can be considered as a definitive criterion for Bartonella endocarditis.
Histopathology can confirm the diagnosis by showing valvular inflammation and remains the gold standard for the diagnosis of endocarditis (8,9). Histopathological examination is generally non-specific in Bartonella endocarditis and primarily shows chronic inflammation with macrophage and lymphocytic infiltration (8)(9)(10). In our patient, in addition to serological test results, chronic inflammation with marked fibrosis and PCR positivity in valvular valve tissue were detected. The diagnosis was confirmed with all diagnostic steps suggested above.

CONCLUSION
Bartonella spp is fastidious bacteria that cause BCNE and have been increasingly reported. Due to the lack of diagnostic criteria, different methods should be used for diagnosis. The combination of serological testing and DNA sequence analysis of PCR increases the chance of diagnosing bartonella-related endocarditis.