Transcript Bartonella Case

Getting to the HEART of the matter
Mr Khoi Nguyen
Dr Indy Sandaradura
Monsieur ET
 49y male
 CAFAT patient from
Gaston Bourret Hospital,
Noumea
 Born in Tahiti, but lived in
Noumea for >40y
 Works as a gardener
 Direct admission under
Cardiothoracics
Background
 Valvular Heart disease
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?Rheumatic
Dx Oct 2010 on TTE
Mod-Sev. AI, Mod. MR
 Chronic schizophrenia
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On depot haloperidol monthly
 L) elbow abscess 2009
 Heavy smoker, Heavy EtOH
Current presentation
 Admitted to Gaston Bourret Hospital Nov
2010
 ‘Unwell’, ?no fevers
 Advanced chronic renal failure [Cr 350]
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Anaemia, hypocalcemia
Normal kidneys on US
Renal bx; ‘extra-capillary GN’ [crescentic GN]
Cardiac investigations
 TOE: severe AI, Aortic valve vegetation
 BC taken, and started on amoxicillin/gent.
 ‘Bilateral otitis’ treated topically, no
sinus/dental problems
Cardiac investigations
 TOE: severe AI, Aortic valve vegetation
 BC taken, and started on amoxicillin/gent.
 ‘Bilateral otitis’ treated topically, no
sinus/dental problems
 Changed to ceftriaxone/doxy./gent. to cover
‘HACEK’ endocarditis
Cardiac investigations
 TOE: severe AI, Aortic valve vegetation
 BC taken, and started on amoxicillin/gent.
 ‘Bilateral otitis’ treated topically, no
sinus/dental problems
 Changed to ceftriaxone/doxy./gent. to cover
‘HACEK’ endocarditis
 BC negative after 3 weeks
Transferred to RPAH for valve replacement
Progress
 Bioprosthetic AVR + MVR
18/1/11
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Aortic valve abN, thickened
Explanted valve
 Only part of Aortic valve sent
 Micro
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Gram stain; no organisms
No growth after 1 week
Histopathology
MICROSCOPIC REPORT
1. "AORTIC VALVE".
The sections show markedly thickened and fibrotic valve cusps with heavy calcium deposition. There is focal
neovascularisation and a moderate inflammatory infiltrate present, consisting mainly of lymphocytes, histiocytes and
plasma cells. A small vegetation is present with fibrin deposition and a heavy mixed underlying inflammatory infiltrate,
including neutrophils. No Aschoff bodies are seen.
2. "MITRAL VALVE".
The sections show thickened valve leaflets in which there are areas of prominent myxoid change.
No significant inflammatory infiltrate is present. No neovascularisation or calcium deposition is seen. No vegetations are
present.
SUMMARY
1. AORTIC VALVE:
- Active chronic valvulitis, small vegetation.
2. MITRAL VALVE:
- Prominent myxoid changes.
SUPPLEMENTARY REPORT
1. No organisms are seen in the dPAS or Gram stained sections.
ET 16s on valve
Aortic valve Warthin-Starry stain
SUPPLEMENTARY REPORT:
•The Warthin-Starry stained sections show numerous bacilli
within the vegetation.
Some additional tests:
 B.henselae serology (IFA @ ICPMR): pos
(1:512)
 B.henselae PCR on blood: neg
The 16S Test
(Or how to pull the rabbit out of the hat)
Khoi Nguyen
Bartonella facts
 Short, facultative intracellular pleomorphic (weakly) gram-negative
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coccobacillary or bacilliary 0.2-0.6 by 0.5-1.0 µm.
Can induce acute and persistent intravascular infections in healthy people and
animals.
Slow growth ~24hrs/generation.
Oxidase & catalase: neg, no acid from sugars, 15-45 days visible growth on
blood agar at 5% CO2, 35-370C for B. henselae as they are highly dependent
on hemin.
Colonially white, rough, dry, raised appearance that pit the medium to smooth
(loss of trimeric autotransporter adhesin, TAA).
Twitching motility on wet mount, presence of TAA.
Cat scratch disease frequent self-limiting infection in healthy host.
 Worldwide distribution, cat reservoir, vector flea (Ctenocephalides felis).
Which one is better?
No, too many choices!?!
16S test
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16S rRNA gene (rDNA) sequence analysis is a proven useful supplement test
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Detect and identify all bacteria including fastidious, damaged, slowinggrowing and atypical phenotype bacteria.
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16S rRNA is part of the small subunit of ribosome with a sedimentation coefficient of 16.
Highly conserved among all bacteria.
High Variable Regions (HVRs).
Sequence analysis to identify .
First 500 base pair often used.
Acceptable ID (2 HDRs).
Full 1500 bp better + extra cost.
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3 sets of blood culture negative including the aortic valve tissue.
From Histo results -> Brain Heart Infusion broth was sent to Molecular for 16S testing with no
visible growth (after two weeks incubation).
A portion was separated and extracted using the QIAamp DNA Mini Kit (QIAGEN).
16S amplification is using real-time with SYBR green (Bioline Inc.).
Products cleaned using Exosapit (Millennium Science).
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Sequencing
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RPAH Molecular Genetics department
performs the sequencing step and send
back the raw data.
Applied Biosystems 3730xl DNA
launched in 2002 using Sanger method,
96-capillary, high-throughput sequencing.
Dideoxy bases PCR ->
different lengths products.
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Electrophoresis for size separation,
detection and recording of dye fluorescence, and data output as
fluorescent peak trace chromatogram.
Using Sequence Scanner v1.0, Applied Science 2005, software to trim
low-quality DNA sequence. This program scores the quality of each
peak.
Integrity is colour coded: blue excellent, yellow good to red poor quality.
Sequencing continued
 Common challenges of DNA sequencing
 poor quality in the first 15–40 bases of the sequence.
 deteriorating quality of sequencing traces after 700–900 bases.
 Mixed products.
 Often high concentration of clean-up products.
 Blast search: http://wwww.ncbi.nlm.gov/blast.
 Nucleotide-nucleotide BLAST, Other, blastn.
 Databases for comparison both commercial and non-
commercial
 GenBank is the one RPA Micro using.
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Non-commercial.
Good numbers of organisms coverage.
Sequence ‘fidelity’.
 Check with Clinical & Bacto details => issue report.
Acknowledgement
 Thank you to
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Dehua Chen (Liverpool Microbiology,
Molecular section)
Asc. Prof. Bing Yu (RPA Molecular Genetics)
Sally Dubedat (RPA Microbiology)
Belinda Mercorella (RPA Molecular Genetics)
Bobby Dimitrijovski (RPA Microbiology)
Australian Society for Microbiology
NSW-ACT Branch
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