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, CA2 Ophthalmology and Visual Sciences, Federal University of Sao Paulo, Sao Paulo, Brazil3 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

Week 1 Week 2-4

Species Identification (Day 3-4)

Sample collection

Gram Stain + Culture (Day 1-3)

Antibiotic resistance (4-5 days)

Pulsed-Field GelElectrophoresis

PCR + DNA Sequencing

Current Approach to Bacterial Keratitis

Start antibiotics Change to correct antibiotics

Identification, virulence, antibiotic resistance

Genotyping, strain identification

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

• 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 l DMEM.

– Sample immediately stored at -80 C and thawed at the time of analysis.

IRB approved prospective evaluation of patients with suspected bacterial keratitis presenting to the Cornea Service at

- UC Davis- Federal University Sao Paulo

How the Biosensor System works?

1. PCR Amplification of bacterial DNAPrimers recognize conserved regionsand amplify variable regions

2. Electrospray ionization mass spectrometryTo determine precise mass of amplified DNA

3. Bioinformatics database analysisTo “triangulate” the identification of pathogens

S. Pyogenes [A38 G31 C29 T23]

PCR Calibrant [A34 G29 C27 T26]

Primer 356: rplB

S. Pyogenes [A24 G37 C30 T25]

H influenzae[A23 G37 C26 T27]

Neisseria meningitidis[A27 G34 C27 T27]

Primer 347: 16S rDNA

Multi-primer Triangulation: allows identification and quantification

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 Molecular Diagnostics MismatchBoth PosBoth Neg

Moraxella spp. Moraxella spp. XHaemophilus influenzae Haemophilus influenzae X

CNS Negative XFungus-Mold Negative X

CNS/Corynebacterium Negative XCNS Negative X

Corynebacterium Negative XCNS Moraxella spp X

Serratia Serratia marcescens XSerratia Serratia marcescens X

Fusarium dimerum Negative XFusarium solani Negative XSteptococcus Streptococcus oralis X

CNS Negative XCNS Negative X

Negative Negative XNegative Negative X

CNS Negative XCNS Negative X

Steptococcus pneumoniae Steptococcus pneumoniae XCNS/Serratia Negative X

Serratia Serratia marcescens XE.Coli E.Coli XCNS Negative XCNS Negative X

Negative Negative XCNS Negative XCNS Negative XCNS Negative XCNS Negative X

Negative Negative XSerratia Negative X

CNS Negative XCNS Negative X

Steptococcus pneumoniae Steptococcus pneumoniae X

Match

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 detectCoagulase 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