Infectious endocarditis by Bartonella species. Report of two cases

Infectious endocarditis (IE) by Bartonella species is an emerging problem worldwide. We report two cases of native valve Bartonella-associated IE events, both affecting adult male patients with a history of alcohol abuse and a low socioeconomic status. Admissions were due to pancytopenia and bleeding in one case and embolic stroke in the other. Blood cultures were negative and IgG indirect immunofluorescence assays (IFA) were positive for B. henselae/B. quintana in high titers (1/16,384-1/16,384, and 1/32,768 -1/16,384, respectively). Cases were classified as definitive IE events according to modified Duke criteria due to the presence of valve vegetations with at least three minor criteria. One patient required aortic mechanical valve replacement and survived, and the other died after a massive hemorrhagic transformation of his stroke. PCR amplification and sequencing of the 16S ribosomal bacterial DNA from a valve tissue sample obtained at surgery in the patient who survived, confirmed B. quintana as the etiological agent. Bartonella-associated IE is an emerging problem in Chile, present in disadvantaged populations. It should be suspected in patients with culture-negative IE. IFA does not discriminate between B. henselae and B. quintana infection, but high titers suggest IE. Complementary PCR techniques may help to elucidate the final causative agent. (Rev Med Chile 2019; 147: 1340-1345)

Bacteria of the genus Bartonella includes facultative Gram-negative intracellular pathogens, able to form biofilms, and its diagnosis has been made possible by serology or molecular methods such as detection of Bartonella 16S rRNA gene sequences or other PCR gene targets in tissue or blood samples 3,8 .
Serology methods involve indirect fluorescent antibody assay (IFA) or ELISA tests. Both have problems of cross-reactivity between B. quintana and B. henselae 3 . Of note, IFA titers ≥ 1/3,200 have a high positive predictive value for IE by these agents (100%) 4 . Western blotting with cross-adsorption circumvents problems of cross-reactivity and increases both sensitivity and specificity 4 . In large clinical series dedicated to IE secondary to Bartonella sp, B. quintana lightly surpasses B. henselae in frequency 4 .
B. quintana, the agent of trench fever, has a human reservoir with worldwide distribution and nowadays is mostly associated to alcoholic and homeless populations in urban settings 3 . Infection between humans is transmitted by body lice and possible by fleas 11,12 . B. henselae is endemic in Chile, and transmission to humans has been linked to cats 3,13 . Infections have mainly involved children with cat scratch disease and other Barto-nella infections have been rarely described in our country [14][15][16] .
In recent years, we have observed two patients with IE by Bartonella species, a condition not reported in Chile before. Patients gave their informed consent for this report.

Case 1
A 57-year-old male patient, with a past medical history of chronic renal failure (CRF) stage 3, tobacco smoking, and alcohol and drug abuse (cannabis and cocaine pasta base),was initially hospitalized during April 2017 by weight loss, gastrointestinal bleeding, epistaxis, gross hematuria, pancytopenia (hemoglobin 7.8 g/dL, WBC count 3,980/µL; platelets count 14,000 µL), and hypoalbuminemia. A mild midsystolic murmur was registered. He received a short course of ceftriaxone and clindamycin, did not require transfusions and was discharged after being stabilized. No blood cultures were taken, and murmur was attributed to anemia. He was readmitted one month later with effort dyspnea, cough with hemoptoic sputum but without fever.
Exams revealed the persistence of macrocytic anemia, thrombocytopenia, and appearance of microscopic hematuria with dysmorphic red blood cells (100%). CT imaging detected bilateral pleural effusion and fluid analysis showed a transudate. Erythrocyte sedimentation rate (ESR) was 66 mm/h. Microbiological analysis from a bronchoalveolar lavage discarded tuberculosis and fungal infections. A bone marrow examination discarded a lymphoproliferative disorder. Not a definitive diagnosis was reached and was discharged with vitamin B12-folic acid therapy and follow-up controls. After three weeks, he was readmitted, with constipation, lower limb edema, hemoptysis, abdominal pain due to hepatomegaly and Janeway lesions. Pancytopenia persisted, and a direct Coombs test was positive. Bilateral pleural effusion and cardiomegaly was detected by chest-x-ray. A transthoracic echocardiogram (TTE) indicated global contractile dysfunction (ejection fraction 35%) and a degenerative aortic valve disease with two vegetations.
After blood cultures, empirical treatment with ceftriaxone and ampicillin was initiated as part of the global management of his heart failure. He evolved with hypotension, fever, tachypnea, and persistent heart failure being transferred to the ICU where he received vasoactive drugs and non-invasive mechanical ventilation. As blood cultures were negative, serological studies for Bartonella sp were requested and resulted positive at

Case 2
A 67-year-old male patient with heavy alcohol consumption was admitted on June 2019 by a cerebrovascular accident with left hemiparesis and effort dyspnea in the preceding three months. He was conscious, had fever (37.5ºC) and no arrhythmia. There was not past history of smoking or obesity. Laboratory evaluation showed mild anemia (hemoglobin 13.1 g/dL) without leukocytosis and an elevated ESR (61 mm/h). Total cholesterol and triglycerides were normal. Brain tomography indicated a right frontoparietal hypodensity suggesting a right posterior frontal infarction. (Figure 1A). Due to a mild increase in hemiparesis, a new brain CT obtained at 72 h showed a hemorrhagic zone in the infarcted area ( Figure 1B). A new holosystolic mitral murmur was detected, and IE was suspected.
No dental procedures, urinary, intestinal or skin disorders were detected. Blood cultures remained negative, and empirical treatment with cloxacillin, ampicillin and gentamicin was started. Additional exams showed microhematuria (5-10 per high power field) of dysmorphic predominance (90%), hypocomplementemia (C3 81. at one lower dilution (1/16,384). Treatment was changed to doxycycline 100 mg BID, rifampin 300 mg BID both by the oral route, maintaining iv gentamicin. An attempt to amplify gene targets of B. henselae from a blood sample a week after gentamicin started was unsuccessful. The patient admitted contact with cats from his neighbor that he fed every night but without touching them. He remained in stable conditions, but unfortunately, he suddenly presented a massive intracerebral hemorrhage on the 29th day of admission, out of surgical scope ( Figure 1C), and died. He was not receiving anticoagulants nor antiaggregant drugs. The event was classified as a definitive IE case (Table 1). HIV test was not studied, and serological tests for C. burnetii serology were negative. His monthly income was low (Table 1).

Discussion
Cases presented here illustrate the emergence of IE by Bartonella species in Chile, enlarging the list of possible etiological agents involved in this condition in our country [17][18][19] . To the best of our knowledge, Bartonella-associated IE has not been described in Chile before. Patients shared similar features: both were males, had a history of alcohol consumption, and a low socioeconomic status, a profile that has been described in large series 3,4 . For B. quintana, a pathogen of human reservoir, these conditions may be a proxy of debilitated patients living in urban trench conditions 3 . IFA does not discriminate between B. henselae and B. quintana infection, but high titers, as in our patients, are only associated with IE, and rarely to disseminated disease 4,20 .
Our small series also illustrates the relevance of including studies beyond classical blood cultures in pursuing the etiological agent involved in IE cases, especially when they are negative. Serological studies, together with PCR amplification and sequencing of different gene targets of blood or valve samples, may enhance the diagnostic yield 4,8,9 . This strategy may recognize not only Bartonella infections but also those provoked by Brucella and Coxiella burnetii 2 . All of these techniques are currently available in Chile. Specific living conditions, habits, and zoonotic exposures may improve the clinical suspicion.
Clinical manifestations and prognosis of IE by Bartonella species are somewhat similar to other causes. Diagnostic limitations already commented, together with special therapeutic requirement, are the most relevant differences. Only observational studies are available that have compared different compounds and outcomes for Bartonella IE, so a definitive therapeutic regimen is not yet defined 21 .
Gentamycin has been associated with higher recovery rates when compared to other antibiotics and less mortality if at least 14 days are completed 21 . Doxycycline has a lower performance if not accompanied by aminoglycosides. One of our patients died despite receiving this compound but after neurological complications.
In conclusion, Bartonella species provoke IE in Chile, and serological and PCR techniques are available for diagnosis. These agents must be searched when blood cultures remain negative or when specific behaviors or zoonotic exposures are detected. Prognosis is similar to other causes of IE, but therapy appears to rely on aminoglycosides compounds.

Aknowledgments:
To Eugenio Montaner, MD, Servicio de Cardiología, Hospital Base de Valdivia, for his kindly technical helps with echocardiograms.