HIGH-THROUGHPUT MOLECULAR DIAGNOSTICS FOR RAPID DETECTION OF PATHOGENS IN CORNEAL ULCERS

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HIGH-THROUGHPUT MOLECULAR DIAGNOSTICS FOR RAPID
DETECTION OF PATHOGENS IN CORNEAL ULCERS
LA Oliveira1,2, MI Rosenblatt1, IR Schwab1, RT
Kashwiabushi2, MCZ Yu3, R Sampath3, LB Blyn3, MJ
Mannis1, LB de Sousa2, DJ Ecker3 1 Department of
Ophthalmology and Vision Science, University of
California-Davis, Davis, CA 2 Ophthalmology and
Visual Sciences, Federal University of Sao Paulo,
Sao Paulo, Brazil 3 Ibis Division of Isis
Pharmaceutical, Carlsbad, CA
Authors have no financial interest. R Sampath, LB
Blyn, and DJ Ecker work for Ibis Division of Isis
Pharmaceutical.
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Current Diagnostic Techniques

• Culture
• Identifies some pathogens
• Slow, labor intensive
• PCR analysis
• Identifies some pathogens
• One test for each agent

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Current Approach to Bacterial Keratitis
Identification, virulence, antibiotic resistance
Genotyping, strain identification
Antibiotic resistance (4-5 days)
Pulsed-Field Gel Electrophoresis
PCR DNA Sequencing
Species Identification (Day 3-4)
Sample collection
Start antibiotics
Change to correct antibiotics
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Purpose of Study

• To evaluate a novel molecular diagnostic
  technique for the RAPID identification of
  pathogens in patients with bacterial keratitis
• The goal is to allow earlier intervention with
  TAILORED antibiotic therapy, and thereby reduce
  sight-threatening complications

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Methods
IRB approved prospective evaluation of patients
with suspected bacterial keratitis presenting to
the Cornea Service at - UC Davis - Federal
University Sao Paulo

• Additional collection for molecular diagnosis
• Single calcium alginate swab immersed for 10
  seconds in sterile vial containing 200 ml DMEM.
• Sample immediately stored at -80 C and thawed at
  the time of analysis.

• Collection of microbiological samples
• Gram stain
• Giemsa stain
• Blood agar
• Chocolate agar
• Thioglycollate broth
• Sabourauds agar
• Acanthamoeba identification

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How the Biosensor System works?

• PCR Amplification of bacterial DNA
• Primers recognize conserved regions
• and amplify variable regions
• Electrospray ionization mass spectrometry
• To determine precise mass of amplified DNA
• Bioinformatics database analysis
• To triangulate the identification of pathogens

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Multi-primer Triangulation allows identification
and quantification
Primer 356 rplB
Primer 347 16S rDNA
H influenzae A23 G37 C26 T27
S. Pyogenes A38 G31 C29 T23
S. Pyogenes A24 G37 C30 T25
Neisseria meningitidis A27 G34 C27 T27
PCR Calibrant A34 G29 C27 T26
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Results

• 35 Samples (03 fungi excluded)
• 32 cultures
• 28 positive cultures for bacterial pathogens
  (80)
• Molecular Diagnostics
• Sensitivity 33
• Specificity 100
• Positive predictive value 100
• Negative predictive value 18.2
• Concordance Kappa0.600, p0.009

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Coagulase Negative Staphylococcus (CNS)

• Pathogen vs. Contaminant?
• Sampling Bias?
• Poor Detection by Ibis T5000 Biosensor?
• What is the Gold Standard?
• Molecular Diagnostics Excluding CNS
• Sensitivity 75
• Specificity 100
• Positive predictive value 100
• Negative predictive value 57.1

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Conclusions

• A molecular diagnostic approach which combines
  PCR, mass spectrometry, and bioinformatics can
  detect pathogens in bacterial keratitis
• This can be performed on a small sample and
  provide a rapid diagnosis
• This test has high specificity
• The sensitivity was limited by failure to detect
• Coagulase Negative Staphylococcus

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Future Directions

• Improved sampling to increase sensitivity
• Creation of a corneal pathogen panel
• (including bacteria, fungi, viruses, protozoa)
• Detection of antibiotic resistance genes
• Improved instrument design