Download 297: High-Throughput Molecular Diagnostics for Rapid Detection of

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

History of molecular evolution wikipedia , lookup

Community fingerprinting wikipedia , lookup

Real-time polymerase chain reaction wikipedia , lookup

Transcript
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.
Current Diagnostic Techniques
• Culture
– Identifies some pathogens
– Slow, labor intensive
• PCR analysis
– Identifies some pathogens
– One test for each agent
Single Gene PCR Amplification
Current Approach to Bacterial Keratitis
Identification, virulence, antibiotic resistance
Antibiotic resistance (4-5 days)
Genotyping, strain identification
Pulsed-Field Gel
Electrophoresis
Species Identification (Day 3-4)
Gram Stain + Culture (Day 1-3)
Sample collection
Week 1
Start antibiotics
Week 2-4
Change to correct antibiotics
PCR + DNA
Sequencing
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
Methods
IRB approved prospective evaluation of patients with suspected
bacterial keratitis presenting to the Cornea Service at
- UC Davis
- Federal University Sao Paulo
• Collection of
microbiological samples
–
–
–
–
–
–
–
Gram stain
Giemsa stain
Blood agar
Chocolate agar
Thioglycollate broth
Sabouraud’s agar
Acanthamoeba identification
• 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.
How the Biosensor System works?
1. PCR Amplification of bacterial DNA
Primers recognize conserved regions
and amplify variable regions
2. Electrospray ionization mass spectrometry
To determine precise mass of amplified DNA
3. Bioinformatics database analysis
To “triangulate” the identification of pathogens
Multi-primer Triangulation: allows
identification and quantification
Primer 347: 16S rDNA
Primer 356: rplB
H influenzae
[A23 G37 C26 T27]
S. Pyogenes
[A38 G31 C29 T23]
Neisseria meningitidis
[A27 G34 C27 T27]
S. Pyogenes
[A24 G37 C30 T25]
PCR Calibrant
[A34 G29 C27 T26]
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: Kappa=0.600, p=0.009
Culture
Moraxella spp.
Haemophilus influenzae
CNS
Fungus-Mold
CNS/Corynebacterium
CNS
Corynebacterium
CNS
Serratia
Serratia
Fusarium dimerum
Fusarium solani
Steptococcus
CNS
CNS
Negative
Negative
CNS
CNS
Steptococcus pneumoniae
CNS/Serratia
Serratia
E.Coli
CNS
CNS
Negative
CNS
CNS
CNS
CNS
Negative
Serratia
CNS
CNS
Steptococcus pneumoniae
Molecular Diagnostics Match
Moraxella spp.
Haemophilus influenzae
Negative
Negative
Negative
Negative
Negative
Moraxella spp
Serratia marcescens
Serratia marcescens
Negative
Negative
Streptococcus oralis
Negative
Negative
Negative
Negative
Negative
Negative
Steptococcus pneumoniae
Negative
Serratia marcescens
E.Coli
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Steptococcus pneumoniae
Mismatch
Both Pos Both Neg
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
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%
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
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