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LABORATORY DIAGNOSIS OF ACANTHAMOEBA KERATITIS USING THE CEPHEID SMARTCYCLER® II AND THE EFFECTS OF TOPICAL OPHTHALMIC DRUGS ON REAL-TIME PCR

Thompson, Paul P (2007) LABORATORY DIAGNOSIS OF ACANTHAMOEBA KERATITIS USING THE CEPHEID SMARTCYCLER® II AND THE EFFECTS OF TOPICAL OPHTHALMIC DRUGS ON REAL-TIME PCR. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

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). 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.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Thompson, Paul Ppaulpthompson@msn.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairKowalski, Regis Pkowalskirp@upmc.eduKOWALSK
Committee CoChairAyyavoo, Velpandivelpandi@pitt.eduVELPANDI
Committee MemberMartinson, Jeremyjmartins@pitt.eduJMARTINS
Committee MemberKinchington, Paulkinchingtonp@upmc.eduKINCH
Committee MemberWadowsky, Robertrobert.wadowsky@chp.edu
Date: 27 September 2007
Date Type: Completion
Defense Date: 23 August 2007
Approval Date: 27 September 2007
Submission Date: 1 September 2007
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Acanthamoeba; Keratitis; PCR inhibition; Real-time PCR
Other ID: http://etd.library.pitt.edu/ETD/available/etd-09012007-110049/, etd-09012007-110049
Date Deposited: 10 Nov 2011 20:01
Last Modified: 19 Dec 2016 14:37
URI: http://d-scholarship.pitt.edu/id/eprint/9312

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