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Identification of Cardiobacterium hominisby Broad-Range Bacterial Polymerase Chain Reaction Analysis in a Case of Culture-Negative Endocarditis

Identification of Cardiobacterium hominisby Broad-Range Bacterial Polymerase Chain Reaction... Culture-negative bacterial endocarditis may be attributed to fastidious microorganisms, prior institution of antibiotic treatment, or both. We describe a case of culture-negative endocarditis in which a modified Steiner stain revealed bacterial structures in the resected heart valve material. Prompted by this finding, broad-range polymerase chain reaction (PCR) amplification of small-subunit ribosomal DNA (16S rDNA) was performed, and Cardiobacterium hominissequences were detected. This case demonstrates the usefulness of both the Steiner stain and broad-range direct molecular amplification as supplemental diagnostic tools in identification of otherwise unexplained infections.REPORT OF A CASEA previously healthy 48-year-old man initially sought medical advice because of leg pain. The symptoms and signs progressed slowly over 5 months to include chest discomfort and, ultimately, clinical evidence of congestive heart failure, and he was admitted to a local hospital in March 2000. He worked as a repairman of office equipment and had no pets or farm exposure. His dental history was unremarkable. He had received several courses of oral antibiotics but could not provide specific details. Six days after admission, he went into septic shock, and therapy with imipenem, ciprofloxacin, and vancomycin was initiated. No prior antimicrobial treatment had been provided during his hospitalization. He was transferred on the eighth day of hospitalization to Geisinger Medical Center, Danville, Pa, where a transesophageal echocardiogram revealed a large aortic vegetation and a possible annular abscess. He had high levels of anticardiolipin and antiphospholipid antibodies. Two days after admission to Geisinger Medical Center, an aortic valve homograft was placed, as were Carpentier valves in the mitral and tricuspid positions. The patient was treated with ceftriaxone, gentamicin, and ampicillin for 4 weeks after surgery. At the time of his last clinic visit, which was 4 weeks after surgery, he appeared to be recovering well.At the beginning of the patient's stay at Geisinger Medical Center, 13 sets of blood cultures had been collected within a 10-day period using commercially available aerobic (BacT/Alert FAN; Organon Teknika, Durham, NC) and anaerobic (BacT/Alert FAN; Organon Teknika) blood culture bottles. Four of these blood culture sets were collected before the initiation of any hospital-administered antibiotic therapy. All blood cultures were negative for organisms after 5 days of routine incubation. The results of culture of surgically resected aortic tissue samples remained negative. Macroscopic vegetations measuring up to 6 mm in diameter were present on the resected valvular tissue. Brightfield microscopy of hematoxylin-eosin–stained sections revealed active chronic valvulitis. The results of routine histologic staining of the valvular tissue samples, including tissue gram, Grocott methenamine-silver for fungi, auramine-rhodamine for mycobacteria, and a modified acid-fast method for Nocardiaspecies, were negative. However, when a modified Steiner stain was used, bacterial rodlike structures were seen.Some of the stained organisms were filamentous; a few were branched; and rare forms were curved with loops (Figure 1).Figure 1.Aortic valve tissue specimen from a 48-year-old man with culture-negative endocarditis. Pleomorphic argyrophilic rods and filaments are seen in panel A and clusters of bacilli in panel B (modified Steiner stain, original magnification ×1000).Formalin-fixed, paraffin-embedded aortic valve tissue samples were subjected to bacterial broad-range 16S rDNA PCR amplification. DNA purification was performed essentially as described earlier.The presence of DNA suitable for PCR analysis was confirmed by amplification of human β-globin sequences from the tissue digests. After the bacterial 16S rDNA primers 516F and 806R were used, PCR products of the expected length (approximately 300 base pairs [bp]) were visualized with agarose gel electrophoresis (Figure 2).Direct sequencing of the PCR product, as well as 3 recombinant clones generated from amplification products from 2 separate tissue digests, provided identical sequences. All were 100% similar (253/253 bp), with the C hominis(ATCC strain 16826) 16S rRNA gene sequence (GenBank accession No. M35014). In 2 additional clones, a single nucleotide mismatch was observed. This mismatch may be explained by PCR misincorporation error or by possible intragenomic or intergenomic differences in C hominisrDNA sequences. The consensus sequence from the directly sequenced PCR product and the 5 recombinant clones, created by using a commercially available software system (AutoAssembler; Applied Biosystems, Foster City, Calif), was only 92.9% (235/253 bp) similar to the 16S rDNA sequence of Suttonella(formerly Kingella) indologenes(ATCC strain 25869, GenBank accession No. M35015), which is the next most closely related species, among those with entries submitted to GenBank. Negative control specimens failed to provide bacterial rDNA sequences.Figure 2.Agarose gel electrophoresis of polymerase chain reaction (PCR) products. Twenty-six thermal cycles with primers 516F and 806R were performed. The M lanes indicate molecular-weight markers; lane 1, 5-µL heart valve digestion product used as PCR template; lane 2, 0.5-µL heart valve digestion product; lane 3, sterile water processed in a manner similar to the heart valve specimen (digestion control); lane 4, sterile water as template (negative PCR control); lane 5, Escherichia coliDNA (equivalent to 500 recombinant ribsomal RNA gene copies, positive PCR control); and bp, base pairs.COMMENTCardiobacterium hominisis a known, but rare cause of endocarditis.It is a notorious cause of culture-negative endocarditis, as are the other members of the HACEKgroup (ie, Haemophilusspecies, Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Kingella kingae). The HACEKgroup is responsible for approximately 3% of cases of infective endocarditis.Although these organisms have historically been considered to be fastidious, it is our impression that modern continuous-monitoring blood culture systems recover these organisms more easily than similar systems that were used in the past. Indeed, using the FAN aerobic bottle, C hominiswas detected in a blood sample from another endocarditis patient seen at Geisinger Medical Center in 1998 within 2 days of incubation. In the case reported herein, it is likely that the patient's prior antibiotic treatment contributed to the negative blood culture results. Based on light microscopy of cells cultivated in vitro, C hominisis a pleomorphic and filamentous rod; sometimes a tearlike appearance and rosettes can be observed.It is considered to be gram stain negative. Molecular phylogenetic analysis places this organism within the gamma subdivision of Proteobacteria.The Steiner stain was initially developed for detection of spirochetes, but it can be used for the detection of other microorganisms, including those found in formalin-fixed, paraffin-embedded tissues.Although nonspecific, a positive Steiner stain result, coupled with a negative culture result, should prompt additional studies, as in the present case. The bacterial broad-range 16S rDNA PCR approach has been previously used successfully to identify putative bacterial pathogens, including Bartonella quintana, Streptococcusspecies, and Tropheryma whippelii, directly from heart valve tissue samples from patients with culture-negative endocarditis.Recently, C hominissequences were detected using this approach on an arterioembolic tissue sample from a patient with endocarditis.Resected valve material can be used for molecular diagnostics even after antibiotic treatment.The latter is the most common reason for negative culture results in cases of infective endocarditis and may have contributed to those in the present case as well.In this case, the microbiological diagnosis was available 3 months after surgical resection of the aortic valve. However, if the broad-range PCR method were performed on a routine basis, specific results could be available within a day or two after a fresh tissue specimen is obtained.Fresh-frozen tissue is preferable as template material for molecular amplification, as formalin fixation can prevent amplification of target DNA sequences longer than 300 to 400 bp.This case demonstrates the benefit of using the modified Steiner stain and broad-range 16S rDNA PCR on formalin-fixed paraffin-embedded tissue samples as supplemental tools for microbial detection and identification. If these tools were applied more frequently in clinical practice, the benefits of more rapid, sensitive, and specific microbiological diagnoses could be further assessed and balanced against the technical difficulties, yield, and costs of this approach.ESaizJLubinMJRobinsonThe modified Steiner stain: a new use for an old stain? staining cytomegalovirus-infected cells in gastrointestinal biopsies.Histochem J.1998;30:549-552.DARelmanUniversal bacterial 16S rDNA amplification and sequencing.In: Persing DH, Smith TF, Tenover FC, White TT, eds. Diagnostic Molecular Microbiology: Principles and Applications.Washington, DC: American Society of Microbiology; 1993:489-495.DARelmanTMSchmidtRPMacDermottSFalkowIdentification of the uncultured bacillus of Whipple's disease.N Engl J Med.1992;327:293-301.GPWormserEJBottoneCardiobacterium hominis: review of microbiologic and clinical features.Rev Infect Dis.1983;5:680-691.MDasADBadleyFRCockerillJMSteckelbergWRWilsonInfective endocarditis caused by HACEKmicroorganisms.Annu Rev Med.1997;48:25-33.JJalavaPKotilainenSNikkariUse of the polymerase chain reaction and DNA sequencing for detection of Bartonella quintanain the aortic valve of a patient with culture-negative infective endocarditis.Clin Infect Dis.1995;21:891-896.DGoldenbergerAKunzliPVogtRZbindenMAltweggMolecular diagnosis of bacterial endocarditis by broad-range PCR amplification and direct sequencing.J Clin Microbiol.1997;35: 2733-2739.NJMuellerVKaplanRZbindenMAltweggDiagnosis of Cardiobacterium hominisendocarditis by broad-range PCR from arterio-embolic tissue.Infection.1999;27:278-279.KRantakokko-JalavaSNikkariJJalavaDirect amplification of rRNA genes in diagnosis of bacterial infections.J Clin Microbiol.2000;38:32-39.PDBarnesDWCrookCulture negative endocarditis.J Infect.1997;35:209-213.Accepted for publication June 6, 2001.Corresponding author: Simo Nikkari, MD, PhD, MoBiDiag Oy, Biomedicum, Haartmaninkatu 8, PO Box 63, FIN-00290 Helsinki, Finland (e-mail: simo.nikkari@mobidiag.com). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JAMA Internal Medicine American Medical Association

Identification of Cardiobacterium hominisby Broad-Range Bacterial Polymerase Chain Reaction Analysis in a Case of Culture-Negative Endocarditis

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Publisher
American Medical Association
Copyright
Copyright 2002 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.
ISSN
2168-6106
eISSN
2168-6114
DOI
10.1001/archinte.162.4.477
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Abstract

Culture-negative bacterial endocarditis may be attributed to fastidious microorganisms, prior institution of antibiotic treatment, or both. We describe a case of culture-negative endocarditis in which a modified Steiner stain revealed bacterial structures in the resected heart valve material. Prompted by this finding, broad-range polymerase chain reaction (PCR) amplification of small-subunit ribosomal DNA (16S rDNA) was performed, and Cardiobacterium hominissequences were detected. This case demonstrates the usefulness of both the Steiner stain and broad-range direct molecular amplification as supplemental diagnostic tools in identification of otherwise unexplained infections.REPORT OF A CASEA previously healthy 48-year-old man initially sought medical advice because of leg pain. The symptoms and signs progressed slowly over 5 months to include chest discomfort and, ultimately, clinical evidence of congestive heart failure, and he was admitted to a local hospital in March 2000. He worked as a repairman of office equipment and had no pets or farm exposure. His dental history was unremarkable. He had received several courses of oral antibiotics but could not provide specific details. Six days after admission, he went into septic shock, and therapy with imipenem, ciprofloxacin, and vancomycin was initiated. No prior antimicrobial treatment had been provided during his hospitalization. He was transferred on the eighth day of hospitalization to Geisinger Medical Center, Danville, Pa, where a transesophageal echocardiogram revealed a large aortic vegetation and a possible annular abscess. He had high levels of anticardiolipin and antiphospholipid antibodies. Two days after admission to Geisinger Medical Center, an aortic valve homograft was placed, as were Carpentier valves in the mitral and tricuspid positions. The patient was treated with ceftriaxone, gentamicin, and ampicillin for 4 weeks after surgery. At the time of his last clinic visit, which was 4 weeks after surgery, he appeared to be recovering well.At the beginning of the patient's stay at Geisinger Medical Center, 13 sets of blood cultures had been collected within a 10-day period using commercially available aerobic (BacT/Alert FAN; Organon Teknika, Durham, NC) and anaerobic (BacT/Alert FAN; Organon Teknika) blood culture bottles. Four of these blood culture sets were collected before the initiation of any hospital-administered antibiotic therapy. All blood cultures were negative for organisms after 5 days of routine incubation. The results of culture of surgically resected aortic tissue samples remained negative. Macroscopic vegetations measuring up to 6 mm in diameter were present on the resected valvular tissue. Brightfield microscopy of hematoxylin-eosin–stained sections revealed active chronic valvulitis. The results of routine histologic staining of the valvular tissue samples, including tissue gram, Grocott methenamine-silver for fungi, auramine-rhodamine for mycobacteria, and a modified acid-fast method for Nocardiaspecies, were negative. However, when a modified Steiner stain was used, bacterial rodlike structures were seen.Some of the stained organisms were filamentous; a few were branched; and rare forms were curved with loops (Figure 1).Figure 1.Aortic valve tissue specimen from a 48-year-old man with culture-negative endocarditis. Pleomorphic argyrophilic rods and filaments are seen in panel A and clusters of bacilli in panel B (modified Steiner stain, original magnification ×1000).Formalin-fixed, paraffin-embedded aortic valve tissue samples were subjected to bacterial broad-range 16S rDNA PCR amplification. DNA purification was performed essentially as described earlier.The presence of DNA suitable for PCR analysis was confirmed by amplification of human β-globin sequences from the tissue digests. After the bacterial 16S rDNA primers 516F and 806R were used, PCR products of the expected length (approximately 300 base pairs [bp]) were visualized with agarose gel electrophoresis (Figure 2).Direct sequencing of the PCR product, as well as 3 recombinant clones generated from amplification products from 2 separate tissue digests, provided identical sequences. All were 100% similar (253/253 bp), with the C hominis(ATCC strain 16826) 16S rRNA gene sequence (GenBank accession No. M35014). In 2 additional clones, a single nucleotide mismatch was observed. This mismatch may be explained by PCR misincorporation error or by possible intragenomic or intergenomic differences in C hominisrDNA sequences. The consensus sequence from the directly sequenced PCR product and the 5 recombinant clones, created by using a commercially available software system (AutoAssembler; Applied Biosystems, Foster City, Calif), was only 92.9% (235/253 bp) similar to the 16S rDNA sequence of Suttonella(formerly Kingella) indologenes(ATCC strain 25869, GenBank accession No. M35015), which is the next most closely related species, among those with entries submitted to GenBank. Negative control specimens failed to provide bacterial rDNA sequences.Figure 2.Agarose gel electrophoresis of polymerase chain reaction (PCR) products. Twenty-six thermal cycles with primers 516F and 806R were performed. The M lanes indicate molecular-weight markers; lane 1, 5-µL heart valve digestion product used as PCR template; lane 2, 0.5-µL heart valve digestion product; lane 3, sterile water processed in a manner similar to the heart valve specimen (digestion control); lane 4, sterile water as template (negative PCR control); lane 5, Escherichia coliDNA (equivalent to 500 recombinant ribsomal RNA gene copies, positive PCR control); and bp, base pairs.COMMENTCardiobacterium hominisis a known, but rare cause of endocarditis.It is a notorious cause of culture-negative endocarditis, as are the other members of the HACEKgroup (ie, Haemophilusspecies, Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Kingella kingae). The HACEKgroup is responsible for approximately 3% of cases of infective endocarditis.Although these organisms have historically been considered to be fastidious, it is our impression that modern continuous-monitoring blood culture systems recover these organisms more easily than similar systems that were used in the past. Indeed, using the FAN aerobic bottle, C hominiswas detected in a blood sample from another endocarditis patient seen at Geisinger Medical Center in 1998 within 2 days of incubation. In the case reported herein, it is likely that the patient's prior antibiotic treatment contributed to the negative blood culture results. Based on light microscopy of cells cultivated in vitro, C hominisis a pleomorphic and filamentous rod; sometimes a tearlike appearance and rosettes can be observed.It is considered to be gram stain negative. Molecular phylogenetic analysis places this organism within the gamma subdivision of Proteobacteria.The Steiner stain was initially developed for detection of spirochetes, but it can be used for the detection of other microorganisms, including those found in formalin-fixed, paraffin-embedded tissues.Although nonspecific, a positive Steiner stain result, coupled with a negative culture result, should prompt additional studies, as in the present case. The bacterial broad-range 16S rDNA PCR approach has been previously used successfully to identify putative bacterial pathogens, including Bartonella quintana, Streptococcusspecies, and Tropheryma whippelii, directly from heart valve tissue samples from patients with culture-negative endocarditis.Recently, C hominissequences were detected using this approach on an arterioembolic tissue sample from a patient with endocarditis.Resected valve material can be used for molecular diagnostics even after antibiotic treatment.The latter is the most common reason for negative culture results in cases of infective endocarditis and may have contributed to those in the present case as well.In this case, the microbiological diagnosis was available 3 months after surgical resection of the aortic valve. However, if the broad-range PCR method were performed on a routine basis, specific results could be available within a day or two after a fresh tissue specimen is obtained.Fresh-frozen tissue is preferable as template material for molecular amplification, as formalin fixation can prevent amplification of target DNA sequences longer than 300 to 400 bp.This case demonstrates the benefit of using the modified Steiner stain and broad-range 16S rDNA PCR on formalin-fixed paraffin-embedded tissue samples as supplemental tools for microbial detection and identification. If these tools were applied more frequently in clinical practice, the benefits of more rapid, sensitive, and specific microbiological diagnoses could be further assessed and balanced against the technical difficulties, yield, and costs of this approach.ESaizJLubinMJRobinsonThe modified Steiner stain: a new use for an old stain? staining cytomegalovirus-infected cells in gastrointestinal biopsies.Histochem J.1998;30:549-552.DARelmanUniversal bacterial 16S rDNA amplification and sequencing.In: Persing DH, Smith TF, Tenover FC, White TT, eds. Diagnostic Molecular Microbiology: Principles and Applications.Washington, DC: American Society of Microbiology; 1993:489-495.DARelmanTMSchmidtRPMacDermottSFalkowIdentification of the uncultured bacillus of Whipple's disease.N Engl J Med.1992;327:293-301.GPWormserEJBottoneCardiobacterium hominis: review of microbiologic and clinical features.Rev Infect Dis.1983;5:680-691.MDasADBadleyFRCockerillJMSteckelbergWRWilsonInfective endocarditis caused by HACEKmicroorganisms.Annu Rev Med.1997;48:25-33.JJalavaPKotilainenSNikkariUse of the polymerase chain reaction and DNA sequencing for detection of Bartonella quintanain the aortic valve of a patient with culture-negative infective endocarditis.Clin Infect Dis.1995;21:891-896.DGoldenbergerAKunzliPVogtRZbindenMAltweggMolecular diagnosis of bacterial endocarditis by broad-range PCR amplification and direct sequencing.J Clin Microbiol.1997;35: 2733-2739.NJMuellerVKaplanRZbindenMAltweggDiagnosis of Cardiobacterium hominisendocarditis by broad-range PCR from arterio-embolic tissue.Infection.1999;27:278-279.KRantakokko-JalavaSNikkariJJalavaDirect amplification of rRNA genes in diagnosis of bacterial infections.J Clin Microbiol.2000;38:32-39.PDBarnesDWCrookCulture negative endocarditis.J Infect.1997;35:209-213.Accepted for publication June 6, 2001.Corresponding author: Simo Nikkari, MD, PhD, MoBiDiag Oy, Biomedicum, Haartmaninkatu 8, PO Box 63, FIN-00290 Helsinki, Finland (e-mail: simo.nikkari@mobidiag.com).

Journal

JAMA Internal MedicineAmerican Medical Association

Published: Feb 25, 2002

There are no references for this article.