Use of Polymerase Chain Reaction in Pediatric Bone and Joint Infections

1
Use of Polymerase Chain Reaction in Pediatric
Bone and Joint Infections

• Childrens Hospital and Regional Medical Center
  of Seattle
• Kit M. Song, MD Craig Boatright, MD William
  Nilsson, PhD Mark Strom PhD
• Funded by Grant from
• The Pediatric Society of North America

2
Microbiology

• The diagnosis and management of bacterial
  pediatric bone and joint infections is
  complicated by a high rate of negative culture
  results
• Gram Stain 50
• Synovial Fluid 40-50
• Bone Aspirates 30-50
• Blood 50-70
• Positive Cultures 2-3 Days

• Negative cultures may due to fastidious
  organisms, unusual organisms, revious antibiotic
  treatment, or growth inhibition by synovial sluid
• This may lead to empiric treatment and resistant
  organisms from overly broad antibiotic coverage
• Need for a sensitive and rapid means of bacterial
  identification
• Hypothsis Molecular methods are more sensitive
  and specific for detection and speciation of
  bacterial bone and joint infections in children.

3
Polymerase Chain Reaction based on DNA
replication and amplification of genomic material
5
3
Denature DNA 94? - 30s (Add dNTPs) Anneal
Primers 57? - 30s (Specific probe) Extend 72?
- 30s (Taq polymerase)
5
Primer R
3
Primer F
n cycles yields 2n copies of original DNA 30
cycles 1.1x109 copies
4
We aimed to use Universal Primers and restriction
endonucleases to aid in identification of
bacterial species in bone and joint infections in
children

• Procaryotic Specific
• Highly conserved regions
• Gene coding for 16s rRNA
• Detects pathogen presence
• 500 base pair amplicons produced
• Variable regions that vary depending on species

Restriction Endonucleases

• Cleave highly conserved genetic regions in
  predictable manner
• Fluorescence label primers
• Produces characteristic fragments

5

• Methods
• Create library of known organisms
• Determine sensitivity of technique using spiked
  specimens
• Subject unknowns to PCR and restriction
  endonuclease cleavage
• Identify unknown pathogens by characteristic
  electropherograms using gene analyzer.

• S. aureus,
• S. epidermidis
• S. pneumoniae
• S. pyogenes
• K. kingae
• H. influenza
• N. gonorrhoeae
• N. meningitidis
• E. coli
• P. aeurginosa

6

• Methods
• 36 Consecutive patients suspected of bone and
  joint infection underwent aspiration of bone or
  joint and blood cultures
• Clinical infection was defined as
• Positive culture
• Positive culture clinical/radiographic osteo
  response to antibiotics
• Five of six
• Temp gt 38.3 C
• Pain in joint worse with motion
• Swelling in joint
• Systemic symptoms
• Absence of other pathologic process
• Satisfactory response to antibiotics
• 5 control JRA patients synovial aspirate
• 4 control Arthroscopy patients
• 2 Osteotomy patients
• All samples subjected to standard cultures and
  molecular techniques
• Extraction of bacterial DNA from bone, blood,
  synovial fluid using commercially available kits
• PCR amplification with universal primers
• Restriction endonuclease digestion

7
Results

• 5 JRA patients
• All culture negative
• 4 PCR negative
• 1 PCR positive for Kingella sp.
• Arthroscopy/Osteotomy patients
• All culture negative
• All PCR negative
• 3 patients, inappropriate collection, 3 patients,
  reactive arthritis/transient synovitis
• 28 patients with infection
• Site culture positive 12/28
• 9/12 PCR positive for same organism
• 1 polymicrobial infection, culture negative
• 3/12 PCR negative
• S. Aureus few
• K. Kingae

8
Results

• 2/28 broth only culture PCR negative
• 16/28 culture negative
• 4 PCR positive
• 1 polymicrobial
• 3 organisms not in our library
• 27/28 blood cultures
• 7/27 positive
• 4/7 PCR positive

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Conclusions

• PCR techniques can detect and speciate pediatric
  bone and joint infections
• Polymicrobial infections
• Sensitivity
• Isolation of DNA Gm lt Gm -
• Polymerase inhibitors

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The Future?

• Replacement of microbiologic techniques
• Increased automation of molecular genetic
  techniques
• Direct Sequencing of amplicons