LABORATORY DIAGNOSIS OF ACANTHAMOEBA KERATITIS USING THE CEPHEID SMARTCYCLER® II AND THE EFFECTS OF TOPICAL OPHTHALMIC DRUGS ON REAL-TIME PCR by Paul Thompson BMedSc, Charles Sturt University, Australia, 2000 Submitted to the Graduate Faculty of Graduate School of Public Health in partial fulfillment of the requirements for the degree of Master of Science University of Pittsburgh 2007 UNIVERSITY OF PITTSBURGH GRADUATE SCHOOL OF PUBLIC HEALTH This thesis was presented by Paul Thompson It was defended on August 23rd, 2007 and approved by Thesis Advisor: Regis P. Kowalski, MS, [M] ASCP Associate Professor Department Ophthalmology School of Medicine University of Pittsburgh Thesis Co-advisor: Velpandi Ayyavoo, Ph. D Assistant Professor Department of Infectious Diseases and Microbiology Graduate School of Public Health University of Pittsburgh ii UNIVERSITY OF PITTSBURGH GRADUATE SCHOOL OF PUBLIC HEALTH Committee Member: Paul R. Kinchington, Ph. D Associate Professor Departments of Ophthalmology and Molecular Genetic and Biochemistry School of Medicine University of Pittsburgh Committee Member: Jeremy J. Martinson, Ph. D CopyrighAssistantt © by Paul Professor Thomps on Departments of Infectious Disease and Microbiology and Human Genetics Graduate School2007 of Public Health University of Pittsburgh Committee Member: Robert M. Wadowsky, Sc. D, Professor Departments of Pathology and Infectious Disease and Microbiology School of Medicine and Graduate School of Public Health University of Pittsburgh iii Regis P Kowalski, MS, [M] ASCP LABORATORY DIAGNOSIS OF ACANTHAMOEBA KERATITIS USING THE CEPHEID SMARTCYCLER® II AND THE EFFECTS OF TOPICAL OPHTHALMIC DRUGS ON REAL-TIME PCR Paul Thompson, MS University of Pittsburgh, 2007 Introduction: Acanthamoeba keratitis (AK) infection needs to be diagnosed definitively to optimize therapy in order to avoid possible visual impairment. Aims: 1) To optimize two noted Real-time PCR (RT-PCR) TaqMan methods (Rivière and Qvarnstrom) using the Cepheid SmartCycler® II system. 2) To identify potential inhibitory effects from topical drugs on RT-PCR. 3) To validate and compare the two assays using ocular clinical samples. Methods: 1) Primers and probes were optimized for both assays to detect genus-specific Acanthamoeba 18S rDNA. 2) Thirteen topical ophthalmic drugs were diluted to determine the level of inhibitory effect present. The lowest non-inhibitory concentrations were then used to determine RT-PCR amplification efficiency. 3) Excess clinical samples (139) were processed for culture and assayed by both assays on the SmartCycler® II and the results were compared. Results: 1) The Rivière RT-PCR plasmid DNA, cyst and trophozoite limits of detection and amplification efficiency were 10.13 copies/10μl, 0.7/300µl, 2.3/300µl, 94% respectively. The Qvarnstrom RT-PCR plasmid DNA, cyst and trophozoite limits of detection and amplification efficiency were 43.8 copies/10μl, 0.7/300µl, 2.3/300µl, 92% respectively. 2) Out of the thirteen topical drugs, the most noteworthy result was that of Polyhexamethylene biguanide (PHMB). iv The non-inhibitory dilution and RT-PCR efficiency were 1/2560 and 72.7%. 3) The results of the clinical validation indicated that 134/139 (96.4%) results correlated between the two assays of which 4/134 samples were culture negative but RT-PCR positive. Conclusions: The two RT-PCR assays were optimized successfully on the SmartCycler® II system with comparable results in detecting genus - specific Acanthamoeba DNA. In examining the effects of thirteen topical drugs on RT-PCR, PHMB was demonstrated to both inhibit the reaction at a high dilution and reduce amplification efficiency substantially. Ocular samples (139) were tested using both assays and results thus far indicate that both could be used to diagnose AK in the laboratory. Public health relevance: RT-PCR can be used to rapidly diagnose AK. Commencement of AK specific therapy earlier will substantially reduce the patients the pain and suffering. Also by examining the effects of topical ophthalmic drugs on RT-PCR, the potential for false negative results and result delays could be minimized. v TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................................xii 1.0 INTRODUCTION........................................................................................................ 1 1.1 ACANTHAMOEBA BIOLOGY.......................................................................... 1 1.2 ACANTHAMOEBA KERATITIS....................................................................... 5 1.3 ACANTHAMEOBA MECHANISMS OF PATHOPHYSIOLOGY................ 7 1.4 ACANTHAMOEBA KERATITIS INCIDENCE............................................... 8 1.5 ACANTHAMOEBA TREATMENT................................................................... 9 1.6 ACANTHAMOEBA DIAGNOSIS .................................................................... 10 1.7 CEPHEID SMARTCYCLER® II SYSTEM ................................................... 14 1.8 LOCKED NUCLEIC ACID PROBE............................................................... 14 1.9 PCR INHIBITION............................................................................................. 15 2.0 GOALS AND SPECIFIC AIMS............................................................................... 17 2.1 SPECIFIC AIM 1: TO OPTIMIZE TWO REAL-TIME PCR ASSAYS TO DETECT ACANTHAMOEBA DNA USING THE CEPHEID SMARTCYCLER® II SYSTEM ...................................................................... 17 2.2 SPECIFIC AIM 2: TO EVALUATE THE EFFECTS OF COMMONLY USED TOPICAL OPHTHALMIC DRUGS ON REAL-TIME PCR PERFORMANCE.............................................................................................. 18 vi 2.3 SPECIFIC AIM 3: TO VALIDATE AND COMPARE THE TWO REAL- TIME PCR ASSAYS BY TESTING A NUMBER OF OCULAR CLINICAL SAMPLES .......................................................................................................... 18 3.0 MATERIALS AND METHODS .............................................................................. 20 3.1 SAMPLE COLLECTION................................................................................. 20 3.2 DNA EXTRACTION......................................................................................... 21 3.3 PREPARATION OF CONTROLS.................................................................. 21 3.3.1 Negative Controls........................................................................................ 21 3.3.2 Positive controls .......................................................................................... 22 3.4 PRIMERS AND PROBES ................................................................................ 23 3.5 SMARTCYCLER® II REACTION MIX ........................................................ 24 3.6 SMARTCYCLER® II PCR THERMAL CYCLING ..................................... 24 3.7 ACANTHAMOEBA SEQUENCING................................................................ 25 3.8 SMARTCYCLER® II RESULT INTERPRETATION ................................. 26 3.9 PLASMID DNA PREPARATION................................................................... 26 3.10 TROPHOZOITE DILUTION .......................................................................... 28 3.11 CYST DILUTION ............................................................................................. 28 3.12 EFFECTS ON RT-PCR EFFICIENCY BY NON-INHIBITORY DRUG TITER................................................................................................................. 29 3.12.1 Determination of Non-Inhibitory Drug Concentration using RT-PCR.29 3.12.2 Effect of Non-Inhibitory Drug Concentration on RT-PCR Amplification Efficiency ..................................................................................................... 30 4.0 RESULTS ................................................................................................................... 32 vii 4.1 THE OPTIMIZATION OF TWO REAL-TIME PCR ASSAYS TO DETECT ACANTHAMOEBA DNA USING THE CEPHEID SMARTCYCLER® II SYSTEM ...................................................................... 32 4.1.1 Rivière RT-PCR Assay: Optimization, Limit of Detection and Amplification Efficiency............................................................................. 32 4.1.2 Qvarnstrom RT-PCR Assay: Optimization, Limit of Detection and Amplification Efficiency............................................................................. 34 4.1.3 Rivière and Qvarnstrom RT-PCR: Trophozoite Limit of Detection ..... 36 4.1.4 Rivière and Qvarnstrom RT-PCR: Cyst Limit of Detection .................. 37 4.1.5 Negative Controls........................................................................................ 38 4.1.6 Positive Controls ......................................................................................... 39 4.2 THE EVALUATATION OF COMMONLY USED TOPICAL OPHTHALMIC DRUGS ON REAL-TIME PCR PERFORMANCE ......... 39 4.2.1 Determination of Non-Inhibitory Concentration using RT-PCR .......... 39 4.2.2 Effect of Non-Inhibitory Drug Concentration on RT-PCR Amplification Efficiency ..................................................................................................... 40 4.3 THE VALIDATION AND COMPARISON OF REAL-TIME PCR ASSAYS USING OCULAR CLINICAL SAMPLES ..................................................... 41 5.0 DISCUSSION ............................................................................................................