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Culture-negative endocarditis caused by Bartonella henselae
C
Culture-negative endocarditis caused by Bartonella henselae
Elizabeth Baorto, MD, R. Mark Payne, MD, Leonard N. Slater, MD, Fred Lopez, MD, David A. Relman, MD, Kyung-Whan Min, MD, and Joseph W. St. Geme III, MD
A 4-year-old girl presented with clinical evidence of infective endocarditis involving her aortic valve, but blood cultures were sterile. Serologic studies and analysis of resected valve by immunohistochemistry and polymerase chain reaction established the diagnosis of Bartonella henselae endocarditis. Clinicians should be aware that B. henselae can cause apparent culture-negative endocarditis in children. (J Pediatr 1998;132:1051-4.)
Bartonella henselae is a pleomorphic gram-negative bacillus that was first reported in 1990 in association with bacillary angiomatosis, relapsing bacteremia with fever, and peliosis hepatis.1-3 All three of these clinical entities occur predominantly in patients who are immunocompromised, usually from infection with human immunodeficiency virus. Recent evidence indicates that B. henselae produces disease in immunocompetent hosts as well.4 The most common manifestation in healthy children is catscratch disease, which usually is characterized by a self-limited regional lymphadenitis. On occasion, cat-scratch disease is complicated by ocular involvement (Parinaud’s oculoglandular syndrome, retinitis, or optic neuritis), meningoencephalitis, or disseminated infection with persistent fever and visceral involvement.5
In this report, we describe B. henselae endocarditis in a child; the diagnosis was suggested by serologic studies. It was confirmed by serial serologic measurements, by polymerase chain reaction, and by a novel application of immunohistochemistry performed on the diseased valve.
METHODS Enzyme Immunosorbent Assay for Anti-Bartonella Antibodies Enzyme immunosorbent assays were performed by using outer membrane protein preparations from B. henselae strain 87-66 (ATCC 49793) and B. quintana (“Oklahoma”) strain 90-268 (ATCC 51694) as the antigens. IgG and IgM were detected by means of a 1:80,000 dilution of goat antihuman IgG or goat antihuman IgM antibody conjugated to horseradish peroxidase (Jackson Im-
From the Divisions of Infectious Diseases and Cardiology, Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri; the Department of Medicine and the Veterans Affairs Medical Center, University of Oklahoma Health Sciences Center, Oklahoma City; the Departments of Medicine and Microbiology & Immunology and the Veterans Affairs Palo Alto Health Care System, Stanford University School of Medicine, Stanford, California; and Deaconess Medical Center in Oklahoma City. Supported in part by the Lucille P. Markey Charitable Trust (Dr. Relman) and the Department of Veterans Affairs (Dr. Slater). Dr. Relman is a Lucille P. Markey Biomedical Scholar. Submitted for publication May 22, 1997; revision received Aug. 29, 1997; accepted Oct. 15, 1997. Reprint requests: Joseph W. St. Geme, III, Division of Infectious Diseases, Department of Pediatrics, Washington University School of Medicine, One Children’s Place, St. Louis, MO 63110. Copyright © 1998 by Mosby, Inc. 0022-3476/98/$5.00 + 0 9/22/87264
munoResearch Laboratories, West Grove, PA). K-Blue TMB (Neogen Corp., Lexington, KY) was used as the colorimetric substrate, and optical densities were read at 450 nm. All measurements were performed in triplicate, allowing calculation of mean ODs. Cut-off values for determining elevated antibody levels were established from 100-fold dilutions of serum from 203 healthy adult blood donors from central Oklahoma, whose mean OD + 2 standard deviations were as follows: anti-B. henselae IgM, 0.108; anti-B. henselae IgG, 0.102; anti-B. quintana IgM, 0.105; anti-B. quintana IgG, 0.181.
OD PCR
Optical density Polymerase chain reaction
Immunohistochemistry for B. henselae To demonstrate B. henselae in valve tissue by immunohistochemistry, tissue sections initially were treated with citrate buffer in a microwave oven to facilitate “antigen retrieval.”6 Subsequently, they were incubated with a 1:4000 dilution of a polyclonal goat antiserum specific for either B. henselae or B. quintana, as described previously with rabbit antisera.7 Antibody labeling was detected by means of the avidin-biotin-peroxidase complex technique.8 Sections known to contain B. henselae were used as positive controls, and sections known to lack B. henselae were used as negative controls.
PCR and Amplicon Sequence Analysis Sections of paraffin-embedded, formalin-fixed aortic valve from the case patient were digested as previously de1051
BAORTO ET AL.
THE JOURNAL OF PEDIATRICS JUNE 1998 um bromide. PCR products were purified with Centricon-100 concentrators (Amicon, Beverly, MA) and sequenced directly with a Dye Terminator Cycle Sequencing kit by means of primers p24E and p12B and an automated DNA sequencer (373A; Applied Biosystems, Foster City, CA). The amplicon sequence was compared with other 16S rDNA sequences in the GenBank/ EMBL database.
CASE REPORT
Fig. 1. Histologic examination of resected aortic valve after A, staining with Warthin-Starry silver or B, treatment with antibody specific for B. henselae and then a secondary antibody conjugated to horseradish peroxidase.Warthin-Starry staining reveals small and often slightly curved bacilli predominantly at edge of tissue (white arrow). Immunohistochemical analysis reveals strong labeling at same location (note punctate black staining); although morphology is not as clear, labeling is consistent with that seen in cases of catscratch disease lymphadenitis and other forms of proven B. henselae disease (×1000).
scribed.9 Subsequently, 1 to 10 µl aliquots (approximately 1% to 5%) of the tissue digestion supernatants were added to each PCR. Alpha-Proteobacteria range-restricted primers, p24E and p12B, were used to amplify Bartonella 16S ribosomal DNA.1 Primers PCO4 and GH20 (Perkin-Elmer, Norwalk, CT) were used to amplify human beta1052
globin gene DNA. Negative controls included reaction mixtures with PCR reagents alone and reaction mixtures with digestions of paraffin-embedded liver tissue from a patient with a pulmonary embolus and no evidence of infection. PCR products were detected by electrophoresis in 1.5% to 3% agarose gels and subsequent staining with ethidi-
A previously healthy 4-year-old white female with a ventricular septal defect, a bicuspid aortic valve, and repaired coarctation of the aorta (performed at age 14 days) had 3 weeks of fever, fatigue, bilateral foot and lower extremity swelling, and epistaxis. She and her family lived in rural southern Missouri, having recently moved from Florida. The family had two cats and six kittens, and the patient played with the animals often. On physical examination, the patient was chronically ill-appearing, pale, and febrile with a temperature of 38.5°C. She had a harsh grade 3/6 systolic murmur heard best along the left sternal border. Liver was palpable 3 cm below the right costal margin with a span of 10 cm, and spleen was palpable 5 cm below the left costal margin. Feet and ankles were swollen and tender, with multiple petechiae, some palpable, extending from her feet to her buttocks. Laboratory values included a white blood cell count of 6,500/mm3 (31% neutrophils, 64% lymphocytes, 3% monocytes, 2% eosinophils), hemoglobin of 9.3 gm/dl (MCV 72), and platelet count of 87,000/mm3. Urinalysis was notable for 40 to 50 red blood cells per high power field. The erythrocyte sedimentation rate was 33 mm/hr; rheumatoid factor was positive at a dilution of 1:1280. A chest roentgenogram revealed cardiomegaly with normal pulmonary vasculature. Echocardiography demonstrated aortic insufficiency, aortic stenosis, and a bicuspid aortic valve with outer leaflet motion restricted by a large vegetation measuring 4 by 9 mm. The leaflet prolapsed through a large perimembranous ventricular septal defect.
THE JOURNAL OF PEDIATRICS VOLUME 132, NUMBER 6 At the time of admission, three blood cultures were obtained, and therapy was initiated with intravenous oxacillin and gentamicin. The patient defervesced within 24 hours, but blood cultures remained negative, and new palpable purpura continued to appear. On hospital day four, she experienced sudden swelling and discoloration of her left hand. Three days later, she underwent a Ross procedure (aortic valve resection and replacement with a pulmonary homograft) and repair of her ventricular septal defect. The resected valve and vegetation were cultured routinely, and for Legionella, Mycobacteria, Chlamydia, and Bartonella; all cultures were sterile. Pathologic examination revealed acute and chronic inflammation with calcifications. Gram, Gomori methenamine-silver, and Warthin-Starry stains were considered negative. Serology for Coxiella burnetti was negative, whereas serum antibody titers against C. pneumoniae, C. trachomatis, and C. psittaci were 1:128, 1:64, and 1:128, respectively. Postoperatively, the patient’s antibiotic regimen was changed to ceftriaxone and gentamicin. On hospital day 14, gentamicin was discontinued, and she was discharged on treatment with ceftriaxone for culture-negative endocarditis. One week later, it was learned that a sample of serum from day three of hospitalization contained markedly elevated levels of antibody reactive with Bartonella henselae (IgG OD450 1.698, IgM OD450 0.225), moderately elevated levels of IgG antibody reactive with B. quintana (OD450 0.551), and no detectable IgM antibody reactive with B. quintana (OD450 0.079). As a result, intravenous erythromycin and oral rifampin were added to the antibiotic regimen. She continued on treatment with ceftriaxone, erythromycin, and rifampin for the next 4 weeks, and then rifampin plus clarithromycin for another 3 months. She remained afebrile throughout her course of outpatient therapy. The erythrocyte sedimentation rate normalized, and rheumatoid factor decreased to undetectable levels. IgM reactive with B. henselae declined to background within 1 month, whereas IgG reactive with B. henselae persisted at very high levels for at least 5 months
BAORTO ET AL.
Fig. 2. Polymerase chain reaction products generated with alpha-Proteobacteria range-restricted primers, p24E and p12B.1 A 296-base pair product is predicted by 16S rDNA sequences of Bartonella spp. Lane 1, PCR reagents alone; lane 2, negative control liver tissue; lanes 3 and 4, different reactions from aortic valve of case patient; lane 5, B. henselae chromosomal DNA. 1-kb DNA ladder (Life Technologies, Gaithersburg, MD) was used for standards.
after initiation of treatment. IgG reactive with B. quintana declined significantly by 2 months and to background by 3 months. Eighteen months after diagnosis, the patient remains clinically stable.
RESULTS Given the elevated antibody levels against Bartonella, fresh sections of valve and vegetation were prepared and stained again with Warthin-Starry stain. As shown in Fig. 1, A, examination revealed multiple bacilli. To confirm the diagnosis of B. henselae endocarditis, immunohistochemical studies were performed. Goat antiserum raised against B. henselae and adsorbed with B. quintana resulted in strong labeling of the bacillary forms (Fig. 1, B), whereas antiserum raised against B. quintana and adsorbed with B. henselae was associated with minimal labeling (not shown). To obtain further evidence for B. henselae endocarditis, valve tissue was examined by PCR by means of alpha-Proteobacteria range-restricted primers. As depicted in Fig. 2, a PCR product of approximately 300 base pairs was consistently detected in digests from the heart valve from the patient (lanes 3 and 4) and from the positive control, B. henselae chromosomal DNA (lane 5). No other PCR products were detected in these samples or in negative control tissue (Fig. 2, lane 2). The amplicon from the patient’s heart valve contained 241 base pairs of sequence internal to the two primers, and the sequence was identical
to the corresponding region of 16S rDNA of Bartonella (formerly Rochalimaea) henselae strain BA-TF (GenBank accession number M59459). This sequence differed by four base pairs from the corresponding sequences available for three different strains of B. quintana. In control reactions all tissue digests yielded an amplicon of the expected size with beta-globin gene primers.
DISCUSSION B. henselae is a recently described fastidious bacterium.1-3 With improvements in diagnostic techniques, this organism is being associated with an expanding spectrum of clinical manifestations, which tend to vary according to the immune status of the host.5 Endocarditis caused by B. henselae has now been reported in at least seven adults, all apparently immunocompetent.10-12 To our knowledge, endocarditis caused by any of the Bartonella species has not been previously reported in children. In our patient, the diagnosis of Bartonella endocarditis was initially suggested by elevated levels of IgG and IgM reactive with B. henselae and of IgG reactive with B. quintana. Upon serial measurement, IgG reactive with B. henselae persisted without significant change, whereas IgM against B. henselae and IgG reactive with B. quintana declined rapidly to background levels. Given that adults with Bartonella endocarditis have serum antibodies that are cross-reactive with other Bartonella species, we suspected in1053
BAORTO ET AL.
fection with B. henselae and cross-reactive antibodies against B. quintana.12,13 Immunohistochemistry performed on the resected valve confirmed Bartonella as the source of endocarditis, and PCR analysis with subsequent nucleotide sequencing implicated B. henselae. In parts of Canada and France, B. henselae and B. quintana are estimated to account for approximately 3% of all cases of infective endocarditis in adults.12 Most patients with disease caused by B. henselae, including ours, have underlying valvular disease and a history of contact with cats or kittens, whereas individuals with B. quintana endocarditis typically lack valvulopathy or cat exposure and are often homeless and alcoholic.12 In adults, the diagnosis of Bartonella endocarditis has been established by serologic studies or by combining Warthin-Starry silver staining and PCR analysis of resected valve.10-12 In our patient, initial histologic examination of the valve after staining with Warthin-Starry silver revealed no organisms. As a result, when repeat staining demonstrated bacillary forms, we felt compelled to confirm the result by immunohistochemistry. Using an antiserum raised against B. henselae, we detected strong specific labeling that corresponded in location to silver-stained organisms. In future cases, immunohistochemistry with anti-Bartonella antibody might be especially useful when staining with Warthin-Starry silver, which can be difficult technically, is indeterminate. The very high levels of anti-B. henselae IgG and IgM in our patient are noteworthy. Raoult et al.12 found that their patients with Bartonella endocarditis also had very high concentrations of antibody against Bartonella. Indeed, by means of a microimmunofluorescence assay, a titer of 1:1600 or more had a positive predictive value of 0.884 for Bartonella endocarditis. Our patient had antibody titers of 1:64 to 1:128 against C. pneumoniae, C.
1054
THE JOURNAL OF PEDIATRICS JUNE 1998 trachomatis, and C. psittaci as well, likely reflecting cross-reactivity between B. henselae and Chlamydia antigens, a phenomenon that has been described for B. quintana and B. bacilliformis.13,14 Definitive recommendations for therapy and its duration cannot be made. All adults reported with this infection have required valve replacement. The combination of gentamicin, ceftriaxone, rifampin, and macrolides was chosen for our patient on the basis of the published in vitro susceptibility data. Given the uncertainty regarding in vivo antibacterial activity, therapy was administered for a total of 5 months. In addition, because of possible recurrent embolic events, the aortic valve was replaced. Among children with clinical evidence of infective endocarditis, blood cultures are negative in approximately 13% of cases.15 It is interesting to speculate that B. henselae may be responsible for a number of these cases. Careful attention to epidemiologic factors associated with B. henselae and expanded use of improved diagnostic techniques is certain to uncover additional cases of Bartonella endocarditis in the pediatric population. We wish to thank A. Robinson for the performance of the serologic assays.
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REFERENCES 1. Relman DA, Loutit JS, Schmidt TM, Falkow S, Tompkins LS. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med 1990;323: 1573-80. 2. Slater LN, Welch DF, Hensel D, Coody DW. A newly recognized fastidious gram-negative pathogen as a cause of fever and bacteremia. N Engl J Med 1990;323:1587-93. 3. Perkocha LA, Geaghan SM, Benedict Yen TS, Nishimura SL, Chan SP, Garcia-Kennedy R, et al. Clinical and pathological features of bacillary peliosis hepatis in association with human immunodeficiency virus infection. N Engl J Med 1990;323:1581-6. 4. Dolan MJ, Wong MT, Regnery RL,
12.
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15.
Jorgensen JH, Garcia M, Peters J, et al. Syndrome of Rochalimaea henselae adenitis suggesting cat scratch disease. Ann Intern Med 1993;118:331-6. Bass JW, Vincent JM, Person DA. The expanding spectrum of Bartonella infections: II. Cat-scratch disease. Pediatr Infect Dis J 1997;16:163-79. Beckstead JH. Improved antigen retrieval in formalin-fixed, paraffin-embedded tissues. Appl Immunohistochem 1994;2:274-81. Reed JA, Brigati DJ, Flynn SD, McNutt NS, Min K-W, Welch DF, Slater LN. Immunocytochemical identification of Rochalimaea henselae in bacillary (epithelioid) angiomatosis, parenchymal bacillary peliosis, and fever with bacteremia. Am J Surg Pathol 1992;16:650-7. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1982;29:577-80. Relman DA. Universal bacterial 16S rDNA amplification and sequencing. In: Persing DH, Smith TF, Tenover FC, White TT, editors. Diagnostic molecular microbiology: principles andapplications. Washington, DC: American Society of Microbiology; 1993. p. 489-95. Hadfield TL, Warren R, Kass M, Brun E, Levy C. Endocarditis caused by Rochalimaea henselae. Hum Pathol 1993; 24:1140-1. Holmes AH, Greenough TC, Balady GJ, Regnery RL, Anderson BE, O’Keane JC, et al. Bartonella henselae endocarditis in an immunocompetent adult. Clin Infect Dis 1995;21:1004-7. Raoult D, Fournier PE, Drancourt M, Marrie TJ, Etienne J, Cosserat J, et al. Diagnosis of 22 new cases of Bartonella endocarditis. Ann Intern Med 1996;125: 646-52. Drancourt M, Mainardi J, Brouqui P, Vandenesch F, Carta A, Lehnert F, et al. Bartonella (Rochalimaea) quintana endocarditis in three homeless men. N Engl J Med 1995;332:419-23. Knobloch J, Bialek R, Muller G, Asmus P. Common surface epitope of Bartonella bacilliformis and Chlamydia psittaci. Am J Trop Med Hyg 1988;39:427-33. Starke JR. Infective endocarditis. In: Feigin RD, Cherry JD, editors. Textbook of pediatric infectious diseases. 4th ed. Philadelphia: Saunders; 1998. p. 31538.
Culture-negative endocarditis caused by Bartonella henselae
Elizabeth Baorto, MD, R. Mark Payne, MD, Leonard N. Slater, MD, Fred Lopez, MD, David A. Relman, MD, Kyung-Whan Min, MD, and Joseph W. St. Geme III, MD
A 4-year-old girl presented with clinical evidence of infective endocarditis involving her aortic valve, but blood cultures were sterile. Serologic studies and analysis of resected valve by immunohistochemistry and polymerase chain reaction established the diagnosis of Bartonella henselae endocarditis. Clinicians should be aware that B. henselae can cause apparent culture-negative endocarditis in children. (J Pediatr 1998;132:1051-4.)
Bartonella henselae is a pleomorphic gram-negative bacillus that was first reported in 1990 in association with bacillary angiomatosis, relapsing bacteremia with fever, and peliosis hepatis.1-3 All three of these clinical entities occur predominantly in patients who are immunocompromised, usually from infection with human immunodeficiency virus. Recent evidence indicates that B. henselae produces disease in immunocompetent hosts as well.4 The most common manifestation in healthy children is catscratch disease, which usually is characterized by a self-limited regional lymphadenitis. On occasion, cat-scratch disease is complicated by ocular involvement (Parinaud’s oculoglandular syndrome, retinitis, or optic neuritis), meningoencephalitis, or disseminated infection with persistent fever and visceral involvement.5
In this report, we describe B. henselae endocarditis in a child; the diagnosis was suggested by serologic studies. It was confirmed by serial serologic measurements, by polymerase chain reaction, and by a novel application of immunohistochemistry performed on the diseased valve.
METHODS Enzyme Immunosorbent Assay for Anti-Bartonella Antibodies Enzyme immunosorbent assays were performed by using outer membrane protein preparations from B. henselae strain 87-66 (ATCC 49793) and B. quintana (“Oklahoma”) strain 90-268 (ATCC 51694) as the antigens. IgG and IgM were detected by means of a 1:80,000 dilution of goat antihuman IgG or goat antihuman IgM antibody conjugated to horseradish peroxidase (Jackson Im-
From the Divisions of Infectious Diseases and Cardiology, Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri; the Department of Medicine and the Veterans Affairs Medical Center, University of Oklahoma Health Sciences Center, Oklahoma City; the Departments of Medicine and Microbiology & Immunology and the Veterans Affairs Palo Alto Health Care System, Stanford University School of Medicine, Stanford, California; and Deaconess Medical Center in Oklahoma City. Supported in part by the Lucille P. Markey Charitable Trust (Dr. Relman) and the Department of Veterans Affairs (Dr. Slater). Dr. Relman is a Lucille P. Markey Biomedical Scholar. Submitted for publication May 22, 1997; revision received Aug. 29, 1997; accepted Oct. 15, 1997. Reprint requests: Joseph W. St. Geme, III, Division of Infectious Diseases, Department of Pediatrics, Washington University School of Medicine, One Children’s Place, St. Louis, MO 63110. Copyright © 1998 by Mosby, Inc. 0022-3476/98/$5.00 + 0 9/22/87264
munoResearch Laboratories, West Grove, PA). K-Blue TMB (Neogen Corp., Lexington, KY) was used as the colorimetric substrate, and optical densities were read at 450 nm. All measurements were performed in triplicate, allowing calculation of mean ODs. Cut-off values for determining elevated antibody levels were established from 100-fold dilutions of serum from 203 healthy adult blood donors from central Oklahoma, whose mean OD + 2 standard deviations were as follows: anti-B. henselae IgM, 0.108; anti-B. henselae IgG, 0.102; anti-B. quintana IgM, 0.105; anti-B. quintana IgG, 0.181.
OD PCR
Optical density Polymerase chain reaction
Immunohistochemistry for B. henselae To demonstrate B. henselae in valve tissue by immunohistochemistry, tissue sections initially were treated with citrate buffer in a microwave oven to facilitate “antigen retrieval.”6 Subsequently, they were incubated with a 1:4000 dilution of a polyclonal goat antiserum specific for either B. henselae or B. quintana, as described previously with rabbit antisera.7 Antibody labeling was detected by means of the avidin-biotin-peroxidase complex technique.8 Sections known to contain B. henselae were used as positive controls, and sections known to lack B. henselae were used as negative controls.
PCR and Amplicon Sequence Analysis Sections of paraffin-embedded, formalin-fixed aortic valve from the case patient were digested as previously de1051
BAORTO ET AL.
THE JOURNAL OF PEDIATRICS JUNE 1998 um bromide. PCR products were purified with Centricon-100 concentrators (Amicon, Beverly, MA) and sequenced directly with a Dye Terminator Cycle Sequencing kit by means of primers p24E and p12B and an automated DNA sequencer (373A; Applied Biosystems, Foster City, CA). The amplicon sequence was compared with other 16S rDNA sequences in the GenBank/ EMBL database.
CASE REPORT
Fig. 1. Histologic examination of resected aortic valve after A, staining with Warthin-Starry silver or B, treatment with antibody specific for B. henselae and then a secondary antibody conjugated to horseradish peroxidase.Warthin-Starry staining reveals small and often slightly curved bacilli predominantly at edge of tissue (white arrow). Immunohistochemical analysis reveals strong labeling at same location (note punctate black staining); although morphology is not as clear, labeling is consistent with that seen in cases of catscratch disease lymphadenitis and other forms of proven B. henselae disease (×1000).
scribed.9 Subsequently, 1 to 10 µl aliquots (approximately 1% to 5%) of the tissue digestion supernatants were added to each PCR. Alpha-Proteobacteria range-restricted primers, p24E and p12B, were used to amplify Bartonella 16S ribosomal DNA.1 Primers PCO4 and GH20 (Perkin-Elmer, Norwalk, CT) were used to amplify human beta1052
globin gene DNA. Negative controls included reaction mixtures with PCR reagents alone and reaction mixtures with digestions of paraffin-embedded liver tissue from a patient with a pulmonary embolus and no evidence of infection. PCR products were detected by electrophoresis in 1.5% to 3% agarose gels and subsequent staining with ethidi-
A previously healthy 4-year-old white female with a ventricular septal defect, a bicuspid aortic valve, and repaired coarctation of the aorta (performed at age 14 days) had 3 weeks of fever, fatigue, bilateral foot and lower extremity swelling, and epistaxis. She and her family lived in rural southern Missouri, having recently moved from Florida. The family had two cats and six kittens, and the patient played with the animals often. On physical examination, the patient was chronically ill-appearing, pale, and febrile with a temperature of 38.5°C. She had a harsh grade 3/6 systolic murmur heard best along the left sternal border. Liver was palpable 3 cm below the right costal margin with a span of 10 cm, and spleen was palpable 5 cm below the left costal margin. Feet and ankles were swollen and tender, with multiple petechiae, some palpable, extending from her feet to her buttocks. Laboratory values included a white blood cell count of 6,500/mm3 (31% neutrophils, 64% lymphocytes, 3% monocytes, 2% eosinophils), hemoglobin of 9.3 gm/dl (MCV 72), and platelet count of 87,000/mm3. Urinalysis was notable for 40 to 50 red blood cells per high power field. The erythrocyte sedimentation rate was 33 mm/hr; rheumatoid factor was positive at a dilution of 1:1280. A chest roentgenogram revealed cardiomegaly with normal pulmonary vasculature. Echocardiography demonstrated aortic insufficiency, aortic stenosis, and a bicuspid aortic valve with outer leaflet motion restricted by a large vegetation measuring 4 by 9 mm. The leaflet prolapsed through a large perimembranous ventricular septal defect.
THE JOURNAL OF PEDIATRICS VOLUME 132, NUMBER 6 At the time of admission, three blood cultures were obtained, and therapy was initiated with intravenous oxacillin and gentamicin. The patient defervesced within 24 hours, but blood cultures remained negative, and new palpable purpura continued to appear. On hospital day four, she experienced sudden swelling and discoloration of her left hand. Three days later, she underwent a Ross procedure (aortic valve resection and replacement with a pulmonary homograft) and repair of her ventricular septal defect. The resected valve and vegetation were cultured routinely, and for Legionella, Mycobacteria, Chlamydia, and Bartonella; all cultures were sterile. Pathologic examination revealed acute and chronic inflammation with calcifications. Gram, Gomori methenamine-silver, and Warthin-Starry stains were considered negative. Serology for Coxiella burnetti was negative, whereas serum antibody titers against C. pneumoniae, C. trachomatis, and C. psittaci were 1:128, 1:64, and 1:128, respectively. Postoperatively, the patient’s antibiotic regimen was changed to ceftriaxone and gentamicin. On hospital day 14, gentamicin was discontinued, and she was discharged on treatment with ceftriaxone for culture-negative endocarditis. One week later, it was learned that a sample of serum from day three of hospitalization contained markedly elevated levels of antibody reactive with Bartonella henselae (IgG OD450 1.698, IgM OD450 0.225), moderately elevated levels of IgG antibody reactive with B. quintana (OD450 0.551), and no detectable IgM antibody reactive with B. quintana (OD450 0.079). As a result, intravenous erythromycin and oral rifampin were added to the antibiotic regimen. She continued on treatment with ceftriaxone, erythromycin, and rifampin for the next 4 weeks, and then rifampin plus clarithromycin for another 3 months. She remained afebrile throughout her course of outpatient therapy. The erythrocyte sedimentation rate normalized, and rheumatoid factor decreased to undetectable levels. IgM reactive with B. henselae declined to background within 1 month, whereas IgG reactive with B. henselae persisted at very high levels for at least 5 months
BAORTO ET AL.
Fig. 2. Polymerase chain reaction products generated with alpha-Proteobacteria range-restricted primers, p24E and p12B.1 A 296-base pair product is predicted by 16S rDNA sequences of Bartonella spp. Lane 1, PCR reagents alone; lane 2, negative control liver tissue; lanes 3 and 4, different reactions from aortic valve of case patient; lane 5, B. henselae chromosomal DNA. 1-kb DNA ladder (Life Technologies, Gaithersburg, MD) was used for standards.
after initiation of treatment. IgG reactive with B. quintana declined significantly by 2 months and to background by 3 months. Eighteen months after diagnosis, the patient remains clinically stable.
RESULTS Given the elevated antibody levels against Bartonella, fresh sections of valve and vegetation were prepared and stained again with Warthin-Starry stain. As shown in Fig. 1, A, examination revealed multiple bacilli. To confirm the diagnosis of B. henselae endocarditis, immunohistochemical studies were performed. Goat antiserum raised against B. henselae and adsorbed with B. quintana resulted in strong labeling of the bacillary forms (Fig. 1, B), whereas antiserum raised against B. quintana and adsorbed with B. henselae was associated with minimal labeling (not shown). To obtain further evidence for B. henselae endocarditis, valve tissue was examined by PCR by means of alpha-Proteobacteria range-restricted primers. As depicted in Fig. 2, a PCR product of approximately 300 base pairs was consistently detected in digests from the heart valve from the patient (lanes 3 and 4) and from the positive control, B. henselae chromosomal DNA (lane 5). No other PCR products were detected in these samples or in negative control tissue (Fig. 2, lane 2). The amplicon from the patient’s heart valve contained 241 base pairs of sequence internal to the two primers, and the sequence was identical
to the corresponding region of 16S rDNA of Bartonella (formerly Rochalimaea) henselae strain BA-TF (GenBank accession number M59459). This sequence differed by four base pairs from the corresponding sequences available for three different strains of B. quintana. In control reactions all tissue digests yielded an amplicon of the expected size with beta-globin gene primers.
DISCUSSION B. henselae is a recently described fastidious bacterium.1-3 With improvements in diagnostic techniques, this organism is being associated with an expanding spectrum of clinical manifestations, which tend to vary according to the immune status of the host.5 Endocarditis caused by B. henselae has now been reported in at least seven adults, all apparently immunocompetent.10-12 To our knowledge, endocarditis caused by any of the Bartonella species has not been previously reported in children. In our patient, the diagnosis of Bartonella endocarditis was initially suggested by elevated levels of IgG and IgM reactive with B. henselae and of IgG reactive with B. quintana. Upon serial measurement, IgG reactive with B. henselae persisted without significant change, whereas IgM against B. henselae and IgG reactive with B. quintana declined rapidly to background levels. Given that adults with Bartonella endocarditis have serum antibodies that are cross-reactive with other Bartonella species, we suspected in1053
BAORTO ET AL.
fection with B. henselae and cross-reactive antibodies against B. quintana.12,13 Immunohistochemistry performed on the resected valve confirmed Bartonella as the source of endocarditis, and PCR analysis with subsequent nucleotide sequencing implicated B. henselae. In parts of Canada and France, B. henselae and B. quintana are estimated to account for approximately 3% of all cases of infective endocarditis in adults.12 Most patients with disease caused by B. henselae, including ours, have underlying valvular disease and a history of contact with cats or kittens, whereas individuals with B. quintana endocarditis typically lack valvulopathy or cat exposure and are often homeless and alcoholic.12 In adults, the diagnosis of Bartonella endocarditis has been established by serologic studies or by combining Warthin-Starry silver staining and PCR analysis of resected valve.10-12 In our patient, initial histologic examination of the valve after staining with Warthin-Starry silver revealed no organisms. As a result, when repeat staining demonstrated bacillary forms, we felt compelled to confirm the result by immunohistochemistry. Using an antiserum raised against B. henselae, we detected strong specific labeling that corresponded in location to silver-stained organisms. In future cases, immunohistochemistry with anti-Bartonella antibody might be especially useful when staining with Warthin-Starry silver, which can be difficult technically, is indeterminate. The very high levels of anti-B. henselae IgG and IgM in our patient are noteworthy. Raoult et al.12 found that their patients with Bartonella endocarditis also had very high concentrations of antibody against Bartonella. Indeed, by means of a microimmunofluorescence assay, a titer of 1:1600 or more had a positive predictive value of 0.884 for Bartonella endocarditis. Our patient had antibody titers of 1:64 to 1:128 against C. pneumoniae, C.
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THE JOURNAL OF PEDIATRICS JUNE 1998 trachomatis, and C. psittaci as well, likely reflecting cross-reactivity between B. henselae and Chlamydia antigens, a phenomenon that has been described for B. quintana and B. bacilliformis.13,14 Definitive recommendations for therapy and its duration cannot be made. All adults reported with this infection have required valve replacement. The combination of gentamicin, ceftriaxone, rifampin, and macrolides was chosen for our patient on the basis of the published in vitro susceptibility data. Given the uncertainty regarding in vivo antibacterial activity, therapy was administered for a total of 5 months. In addition, because of possible recurrent embolic events, the aortic valve was replaced. Among children with clinical evidence of infective endocarditis, blood cultures are negative in approximately 13% of cases.15 It is interesting to speculate that B. henselae may be responsible for a number of these cases. Careful attention to epidemiologic factors associated with B. henselae and expanded use of improved diagnostic techniques is certain to uncover additional cases of Bartonella endocarditis in the pediatric population. We wish to thank A. Robinson for the performance of the serologic assays.
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