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

1. 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.

2. Single Gene PCR Amplification Current Diagnostic Techniques • Culture • Identifies some pathogens • Slow, labor intensive • PCR analysis • Identifies some pathogens • One test for each agent

3. Gram Stain + Culture (Day 1-3) Week 2-4 Week 1 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

4. 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

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

6. 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

7. 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] S. Pyogenes [A24 G37 C30 T25] Neisseria meningitidis [A27 G34 C27 T27] PCR Calibrant [A34 G29 C27 T26]

8. 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

10. 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%

11. 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

12. 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