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Applications of the polymerase chain reaction in clinical ophthalmology
Applications of the polymerase chain reaction in clinical ophthalmology Sonia N. Yeung, MD, PhD; Andrea Butler, BSc; Paul J. Mackenzie, MD, PhD !"342!#4 s 2»35-» Molecular biology has become a valuable component in many areas of medicine, including ophthalmology. Polymerase chain reaction (PCR) is the most widely used tool. It has proven to be a powerful technique in diagnosis and quantification of microorganisms and antibiotic resistance screening. For a growing number of ophthalmic conditions PCR testing can be conducted. It is therefore important that clinicians be knowledgeable about the indications, strengths, and limitations of the technique. The purpose of this review is to explore the current role of PCR in the diagnosis and management of eye disease. La biologie moléculaire est devenue un élément utile pour plusieurs secteurs de la médecine, y compris l’ophtalmologie. La polymérisation en chaîne (PCR) est l’outil médical le plus utilisé. C’est vraiment une puissante technique de diagnostic et de quantification des micro-organismes et de dépistage de la résistance aux antibiotiques. Le test PCR peut servir pour un nombre de plus en plus grand de maladies ophtalmiques. Il est donc important que les cliniciens en connaissent les indications, la puissance et les limites techniques. La présente revue a pour objet d’examiner le rôle actuel de la PCR pour le diagnostic et le traitement de la maladie oculaire.
P
olymerase chain reaction (PCR) is a powerful tool that involves in vitro amplification of specific nucleic acid sequences of DNA or RNA. It produces a large quantity of genetic material from a minute sample, which can then be examined by various means in order to detect, quantify, and analyze specific products. First described by Saiki et al. in 1985,1 the technique was perfected by Kary Mullis,2 who was awarded the Nobel Prize in 1993 in recognition of the impact of this technology on the scientific community. Initially developed as a research tool, PCR is now an integral part of diagnostics in many areas of medicine. It is therefore important that practicing ophthalmologists be familiar with the indications, strengths, and limitations of this technique in order to secure the full benefits and avoid the pitfalls of the tool. This review serves as an update to previous articles on the topic3–8 and discusses the basic principles of PCR, the different types and their indications, the strengths and limitations, and current applications of PCR in ophthalmic practice. METHOD OF LITERATURE SEARCH
The OVID Medline database was searched from 1966 to 2008 for the following terms: polymerase chain reaction or PCR, and uveitis, retinitis, endophthalmitis, lymphoma, keratitis, ocular inflammation, and ocular infection. The articles retrieved were limited to those published in English From the Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, B.C. Originally received June 1, 2008. Revised Sep. 1, 2008 Accepted for publication Sep. 15, 2008 Published online Jan. 23, 2009
or with English abstracts. Relevant cited references of these papers were also reviewed. BASIC PRINCIPLES OF PCR
Application of this technique depends on the availability of complementary specific DNA oligonucleotide sequences (known as primers) that flank areas of interest. The interactions between the primer sequence and the DNA to be studied are highly specific. Amplification of DNA sequences will only occur at the site defined by the primers. To perform PCR, several elements are required: a DNA template (aqueous or vitreous sample), a pair of short, synthetic oligonucleotide DNA primers, a thermostable DNA polymerase enzyme, a buffer system, nucleotide triphosphates, and a thermal cycling machine. There are 3 basic steps in PCR: denaturation, annealing, and extension (Fig. 1). Nucleic acid (e.g., DNA) is extracted from the clinical specimen of interest and used as the starting material for the process. In the first step, denaturation, heat (90–95 °C) is used to separate the extracted doublestranded DNA into single strands. During annealing the thermal cycler is cooled to a temperature that allows each oligonucleotide primer to bind to its associated sequence within the target strand of DNA (between 35 and 60 °C). The process of annealing is highly specific at optimal temperature, which is a value that is determined for each target Correspondence to Sonia N. Yeung, MD, Department of Ophthalmology and Visual Sciences, University of British Columbia, 2550 Willow St., Vancouver BC V5Z 3N9; [email protected] This article has been peer-reviewed. Cet article a été évalué par les pairs. Can J Ophthalmol 2009;44:23–30 doi:10.3129/i08-161 CAN J OPHTHALMOL—VOL. 44, NO. 1, 2009
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PCR in ophthalmology—Yeung et al. sequence empirically. In the last step, the temperature is elevated to the optimal level (typically 72–74 °C) for the thermally stable DNA polymerase to synthesize the target sequence starting from the annealed primers. A new strand of DNA that is complementary to the original target sequence is synthesized. The sequence of denaturation, annealing, and extension is termed a cycle. The newly synthesized DNA resulting from this cycle acts as target sequences for the next cycle. As this process cycles, the target sequence is amplified in an exponential fashion. Theoretically, amplification yields of 2N will occur after N thermal cycles (usually 30–40 cycles). Once all the primers and nucleotide triphosphates have been exhausted, production reaches a plateau. After the PCR reaction, gene products are visualized by separating DNA molecules by size, using electrophoresis (agarose or acrylamide gel) to determine whether the fragment of expected size has been synthesized. Higher sensitivity for detection can be obtained using more sensitive dyes or autoradiography. PCR products are also compared with a negative control, which includes all reagents but substitutes pure distilled water for the patient’s DNA sample.
Confirmation of the identity of the synthesized product can be achieved by fingerprinting. One method of fingerprinting involves evaluating the pattern of products following enzymatic cleavage at a specific sequence of DNA. Hybridization with a labeled DNA that anneals only to a specific product can also be used to identify product sequences. Finally, the products can be directly sequenced. TYPES OF PCR Universal primer
The genes that encode ribosomal RNA (rRNA) are highly repeated in the genome and have not significantly changed across much of evolution. Primers that detect these conserved regions take advantage of the universality of the genes. The 16S rRNA genes are conserved among bacteria; therefore, a primer for this sequence can be used to screen for any bacterium.9–12 Fungi, on the other hand, have highly conserved 18S and 28S rRNA genes, which can be used for the diagnosis of fungal disease.13 Multiplex
In multiplex PCR, several primer pairs are used that are specific to different DNA targets. This allows for the amplification and detection of a number of different sequences at the same time (e.g., 2 infectious agents from a single sample). However, for this procedure to work, the PCR conditions must be ideal for both reactions. Also, the primers must result in amplified products of different sizes for each target sequence, so that they appear distinct on the visualization method of choice.14,15 Reverse transcriptase
Reverse transcriptase PCR (RT-PCR) can be used to differentiate between active and inactive infection. Since RNA is produced during replication, active gene expression in tissues and cells can be identified by amplification. To detect RNA, it must first be converted to DNA by the enzyme reverse transcriptase. The resulting DNA is called complementary DNA (cDNA). PCR is then performed on the cDNA to amplify the target sequence. To ensure that the procedure is not amplifying contaminated genomic DNA, it is particularly important to include a control in which the reverse transcriptase enzyme is omitted. Fig. 1—One cycle of PCR. Denaturation: nucleic acid from a patient sample is heated to form single strands. Annealing: temperature is cooled to allow for oligonucleotide primers to bind. Extension: temperature is elevated to the optimal temperature for the thermostable DNA polymerase, which uses the annealed oligonucleotides as primers and the nucleotide triphosphate raw materials for DNA synthesis. At the end of 1 cycle, 2 target sequences of DNA are created from the original template. These can serve as templates for subsequent cycles.
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Real-time
Another means of characterizing active infection versus low-grade pathogenicity is to quantify the number of pathogen genomes in a sample. Low levels of genomes may reflect the low-level presence of that pathogen. For this technique, PCR is performed in a special thermocycler that has a real-time fluorescence detection unit in each well.16–18 The degree of fluorescence in the sample is compared with standard curves of known pathogens, allowing for the number of pathogen genomes to be determined.
PCR in ophthalmology—Yeung et al. Nested
Nested PCR (nPCR) increases both the sensitivity and specificity of the general PCR technique, as 2 different amplification processes are performed sequentially. The first amplification uses a set of primers that produces a larger segment, which then becomes the template for the next amplification. The second amplification uses a different set of primers that bind within the large segment. The sensitivity of nPCR is increased because of the 2 cycles of amplification, and the specificity is increased because of the binding of 2 separate pairs of primers for amplification.19–21 Inverse
Whereas standard PCR amplifies a known sequence of nucleic acids situated between 2 primer annealing sites, inverse PCR makes it possible to amplify an unknown sequence situated beside a known sequence (Fig. 2).22,23 A restriction enzyme (which cuts DNA at locations with a specific sequence of base pairs) cuts the DNA strand at points flanking the known and unknown sequences. Both sequences do not
Fig. 2—Inverse PCR: amplification of an unknown sequence situated beside a known sequence. A restriction enzyme cuts the DNA strand on either side of the known and unknown sequence. A different enzyme ligates the ends of this fragment, circularizing it. Finally, another restriction enzyme cuts the ring at a point within the known sequence to reform a linear fragment. The resulting DNA fragment includes the unknown sequence sandwiched between terminal known sequences, which can undergo amplification using the standard PCR approach.
contain any sites for this enzyme; therefore, the fragment remains intact. It then circularizes with the help of an enzyme that ligates the ends. A second enzyme cuts this ring at a point within the known sequence to reform a linear fragment that includes the unknown sequence sandwiched between terminal known sequences. The unknown sequence is amplified using the standard PCR approach. STRENGTHS AND LIMITATIONS OF PCR
A major strength of PCR is that the DNA segment of interest does not need to be first purified from the background DNA. Almost any tissue or body fluid can be used, including aqueous (anterior chamber paracentesis of ≥50 μL) and vitreous samples (preinfusion aspirate of ≥100 μL), which is placed in a sterile microfuge tube and snap-frozen in liquid nitrogen or dry ice.3,4,8 The exponential amplification of the segment of interest provides high sensitivity, and the use of unique primers provides high specificity. Using the Goldmann-Witmer coefficient in combination with PCR can further increase the sensitivity.24 PCR can provide results within a few hours. The high sensitivity of PCR makes it prone to falsepositive results. Specimen contamination during laboratory processing can arise from previous amplification procedures or cross-contamination from other samples.25 Careful specimen handling and processing must be conducted in conjunction with the use of rigorous negative controls. Since PCR can detect DNA in dead and latent samples (i.e., herpesviruses), it can be difficult to differentiate between expected organisms (i.e., normal flora) and those responsible for infection. RT-PCR can help differentiate between residual DNA of a pathogenic organism after successful treatment and active organisms. Finally, the specificity of this technology can be compromised by the selection of nonspecific primers, as well as suboptimal PCR conditions that allow nonspecific products to amplify. False-negative results may also occur in PCR. The assays may lack sensitivity if there is a low inoculum in the clinical specimen or an inadequate sample. This can be addressed by increasing the number of cycles performed. The specimen may contain substances that inhibit nucleic acid extraction or amplification. The high specificity can also lead to falsenegative results when primers are chosen for a polymorphic region of the pathogen’s DNA where sequence variations exist between strains. The use of several primer sets for each organism can help in this regard. For PCR to be an effective tool, it requires a well-defined differential diagnosis. Culture or staining methods can detect a wide range of organisms with a single test. PCR can only detect an organism for which a primer set has been selected. In 2006, Acharya et al.26 compared PCR results with presumptive clinical diagnosis in patients with vitritis. They determined that PCR for any infectious etiology was unlikely to be positive if the clinical suspicion of a specific diagnosis is low. PCR must be considered in clinical context. CAN J OPHTHALMOL—VOL. 44, NO. 1, 2009
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PCR in ophthalmology—Yeung et al. CURRENT CLINICAL APPLICATIONS OF PCR IN OPHTHALMOLOGY
The general applications of PCR are extensive.3,4,6 It can be used in the diagnosis of infection caused by either slow-growing or fastidious microorganisms, detection of infectious agents that cannot be cultured, recognition of newly emerging infectious diseases, and discovery of novel microorganisms. Additionally, the procedure improves the accuracy of subtyping pathogens in epidemiological studies, quantifies viral load (real-time PCR), and allows rapid identification of antimicrobial resistance. The next sections will briefly summarize the current applications of PCR in clinical ophthalmology with a focus on new advances in this area. Anterior uveitis
The diagnosis of anterior uveitis associated with herpes simplex virus and varicella zoster virus is commonly based on clinical presentation. In atypical cases, several groups have used PCR to confirm the presence of these pathogens.27–30 In several cases in which treatment of presumed herpetic infection failed, cytomegalovirus (CMV) infection was established by PCR.31–33 Fuchs’ heterochromic iridocyclitis (FHI) and PosnerSchlossman syndrome (PSS) bear many similarities to the clinical hallmarks of herpetic disease. In 2004, Quentin and Reiber34 determined that all patients with FHI in a European population had elevated intraocular antibody titers (modified Goldmann-Witmer coefficient), and the majority had positive RT-PCR for rubella virus.34 Interestingly, Chee et al.35 demonstrated positive CMV PCR for 36% of the patients in their study with either clinical PSS or FHI. Genetic variations in cytokines have been associated with disease susceptibility and severity in autoimmune disease. In 2006, El-Shabrawi et al.36 reported that individuals positive for HLA-B27 show a higher susceptibility to the development of anterior uveitis in the presence of singlenucleotide polymorphisms of the tumor necrosis factor alpha promoter at specified positions. In the same year, Yeo et al.37 suggested that a single nucleotide polymorphism of the chemokine gene MCP-1 may be a protective marker for idiopathic acute recurrent anterior uveitis. Posterior uveitis
The first application of PCR in eye disease was associated with the diagnosis of viral uveitis.20,38–42 In cases in which media opacity (cataract or dense vitritis) or atypical presentations precluded clinical diagnosis in posterior uveitis, several groups found that the clinical course mirrored the PCR results.20,40 The sensitivity of PCR for varicella zoster virus (VZV), herpes simplex virus (HSV), and CMV in vitreous fluid is greater than 90%, and its specificity greater than 95%.20,39,43–45 PCR has been used to subtype viral infections,43 to screen a substantial differential diagnosis of posterior uveitis in
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a single reaction (multiplex PCR),43,46 and to quantitate infectious pathogens (real-time PCR).7 Siqueira et al.47 demonstrated that PCR could detect CMV in aqueous humor with high specificity in patients infected with human immunodeficiency virus (HIV) who had active lesions in the retina.47 Often, both aqueous and vitreous fluid analysis is helpful in posterior uveitis. Improved primer designs allowed the detection of Toxoplasma DNA in 83% of patients with suspected ocular toxoplasmosis and positive serologic test results.48,49 Intraocular antibody production combined with PCR has further contributed significantly to the diagnosis of toxoplasmosis.50 Interestingly, Toxoplasma gondii DNA has been detected in the peripheral blood of patients with active disease51 and in retinochoroidal toxoplasmic scar specimens from eye bank and enucleated eyes.52 The identification of several unique strains of T. gondii were shown to have different antibiotic sensitivities to sulfa medications, which may explain the heterogeneity in response to treatment.53 Some bacterial causes of posterior segment disease cannot readily be cultured. The ocular manifestations of tuberculosis are commonly associated with difficulties in diagnosis and therapy, complicated by the slow rate of growth in culture. An nPCR assay was recently found to be useful in the confirmation of Mycobacterium tuberculosis in aqueous and vitreous samples.54 Whipple’s disease (Tropheryma whippelii) and Bartonella can now also be amplified from vitreous fluid55,56 and orbital granulomas.57 In addition to these diagnostic applications, PCR has been used to measure viral load in order to monitor response to therapy. This has been applied in the care of patients with HIV, CMV, and hepatitis C virus infection.58–61 PCR may also aid in the management of viral retinitis by the detection of antiviral-resistant strains, as many CMV patients acquire resistance to ganciclovir.62–65 The use of PCR may have a role in deciding whether to start, continue, or stop certain therapies. Recently, PCR was used to identify polymorphisms in chemokines and chemokine receptors that were shown to influence the clinical outcome in patients with idiopathic immune-mediated posterior segment uveitis.66 B-cell lymphoma
B-cell lymphoma often mimics a posterior uveitis, presenting as an ocular inflammation in older patients.67 PCR has been used to determine clonality68 and to detect IgH gene rearrangements in intraocular lymphoma.69 Coupeland et al.70 further illustrated the practicality of PCR for the detection of IgH gene rearrangements in chorioretinal biopsies. External eye disease
Although many bacterial causes of conjunctivitis and keratitis are readily detected by Gram-staining or culture, there are several organisms that still present a challenge.71,72 Adenovirus conjunctivitis outbreaks continue to be a
PCR in ophthalmology—Yeung et al. public health challenge. The organism is not readily cultured, and serotyping is generally not possible. PCR has been shown to increase the speed and accuracy of diagnosis73 and offers the advantage of potential serotype specificity,74 which has significant prognostic value. Herpetic infections often present diagnostic dilemmas. In 2004, 2 groups compared Giemsa staining, immunofluorescence assay, and PCR in the detection of HSV-1 and found that combined PCR and immunofluorescence provides the highest sensitivity and specificity for the diagnosis of HSV-1.75,76 PCR has also been explored as an adjunct to intraocular anti-HSV antibody in the detection of HSV in recipient corneal buttons with the hope of starting treatment early to prevent graft failure.77,78 A herpetic etiology for Thygeson’s superficial punctate keratitis has been investigated with PCR.79,80 Connell et al.79 reported the results from 8 patients with this condition to be negative for HSV-1, HSV-2, VZV, and adenovirus. PCR has also been helpful in identifying infections caused by Chlamydia with 88% sensitivity and 100% specificity,81 and Acanthamoeba with 84% sensitivity (53% for culture) and 95% specificity.82 Kumar et al.83 reported that early detection of mycotic keratitis by PCR resulted in successful treatment of 4 patients. Recently, Ghosh et al.84 used PCR and sequencing of rDNA to detect pathogenic strains in fungal keratitis. They found that PCR rDNA sequencing results correlated with culture-based results, and they further identified fungal strains that could not be cultured in routine medium. Endophthalmitis
The Endophthalmitis Vitrectomy Study reported that endophthalmitis following cataract surgery was often culture negative.85 With the use of PCR, bacterial DNA has been detected in the majority of cases of postoperative acute endophthalmitis.86–88 In 2007, Chiquet et al.87 used eubacterial PCR amplification with direct sequencing to identify the causative agents in aqueous humor samples from 30 patients with postcataract endophthalmitis. Cultures were positive in 32% of cases, and PCR was positive in 61% of cases with aqueous humor samples; when combined, a diagnosis was made in 71% of cases. Chiquet et al.89 later confirmed these findings in a prospective multicenter study. They concluded that culture and eubacterial PCR are complementary techniques, and PCR was particularly useful in the setting of previous administration of intravitreal antibiotics. The causative organisms responsible for delayed-onset endophthalmitis are often present in low numbers and difficult to culture. PCR has demonstrated that these infections were mainly caused by Propionibacterium acnes, Staphylococcus epidermidis, or Actinomyces israelii.90 Recently, Palani et al.91 applied PCR-based restriction fragment length polymorphism analysis to identify nontuberculous mycobacteria in 3 cases of delayed-onset endophthalmitis.91 PCR has also been used to identify Fusarium, Candida,
and Aspergillus,13 and has proven to be a more sensitive and rapid diagnostic tool than conventional methods for fungal endophthalmitis.92 FUTURE DIRECTIONS
Molecular technology is a powerful tool for the rapid and accurate diagnosis of a variety of infectious ocular diseases. Novel organisms have also been discovered since the advent of PCR, particularly those that have been historically difficult to culture and identify by traditional means. PCR has been used to measure viral load and to monitor response to therapy, and has shown itself to be a valuable instrument for the detection and confirmation of antimicrobial resistance. Etiologies for ocular disease once thought to be idiopathic may eventually be elucidated with this tool. Although PCR will not replace conventional methods, it has certainly proved to be a useful adjunct to existing techniques. As we continue to learn more about the genetic basis of disease, there will undoubtedly be an additional role for PCR in designing patient-specific treatment regimens for many diseases. The authors have no proprietary or commericial interest in any materials discussed in this article.
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50. De Groot-Mijnes JD, Rothova A, Van Loon AM, et al. Polymerase chain reaction and Goldmann-Witmer coefficient analysis are complimentary for the diagnosis of infectious uveitis. Am J Ophthalmol 2006;141:313–8.
64. Jabs DA, Enger C, Dunn JP, Forman M. Cytomegalovirus retinitis and viral resistance: ganciclovir resistance. CMV Retinitis and Viral Resistance Study Group. J Infect Dis 1998;177:770–3.
51. Bou G, Figueroa MS, Marti-Belda P, Navas E, Guerrero A. Value of PCR for detection of Toxoplasma gondii in aqueous humor and blood samples from immunocompetent patients with ocular toxoplasmosis. J Clin Microbiol 1999;37:3465–8. 52. Vallochi AL, Muccioli C, Martins MC, Silveira C, Belfort R Jr, Rizzo LV. The genotype of Toxoplasma gondii strains causing ocular toxoplasmosis in humans in Brazil. Am J Ophthalmol 2005;139:350–1. 53. Sibley LD, Boothroyd JC. Construction of a molecular karyotype for Toxoplasma gondii. Mol Biochem Parasitol 1992;51:291–300. 54. Ortega-Larrocea G, Bobadilla-del-Valle M, Ponce-de-Leon A, Sifuentes-Osornio J. Nested polymerase chain reaction for Mycobacterium tuberculosis DNA detection in aqueous and vitreous of patients with uveitis. Arch Med Res 2003;34:116–9. 55. Relman DA, Schmidt TM, MacDermott RP, Falkow S. Identification of the uncultured bacillus of Whipple’s disease. N Engl J Med 1992;327:293–301. 56. Kerkhoff FT, Bergmans AM, van Der Zee A, Rothova A. Demonstration of Bartonella grahamii DNA in ocular fluids of a patient with neuroretinitis. J Clin Microbiol 1999;37:4034–8.
65. Martin BK, Ricks MO, Forman MS, Jabs DA. Change over time in incidence of ganciclovir resistance in patients with cytomegalovirus retinitis. Clin Infect Dis 2007;44:1001–8. 66. Ahad MA, Missotten T, Abdallah A, Lympany PA, Lightman S. Polymorphisms of chemokine and chemokine receptor genes in idiopathic immune-mediated posterior segment uveitis. Mol Vis 2007;13:388–96. 67. Cuchacovich R. Clinical applications of the polymerase chain reaction: an update. Infect Dis Clin North Am 2006;20:735–58. 68. Gorochov G, Parizot C, Bodaghi B, et al. Characterization of vitreous B-cell infiltrates in patients with primary ocular lymphoma, using CDR3 size polymorphism analysis of antibody transcripts. Invest Ophthalmol Vis Sci 2003;44:5235–41. 69. Shen DF, Zhuang Z, LeHoang P, et al. Utility of microdissection and polymerase chain reaction for the detection of immunoglobulin gene rearrangement and translocation in primary intraocular lymphoma. Ophthalmology 1998;105:1664–9. 70. Coupland SE, Bechrakis NE, Anastassiou G, et al. Evaluation of vitrectomy specimens and chorioretinal biopsies in the diagnosis of primary intraocular lymphoma in patients with Masquerade syndrome. Graefes Arch Clin Exp Ophthalmol 2003; 241:860–70. CAN J OPHTHALMOL—VOL. 44, NO. 1, 2009
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PCR in ophthalmology—Yeung et al. 71. Cooper RJ, Yeo AC, Bailey AS, Tullo AB. Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis. Invest Ophthalmol Vis Sci 1999;40:90–5. 72. O’Duffy JD, Griffing WL, Li CY, Abdelmalek MF, Persing DH. Whipple’s arthritis: direct detection of Tropheryma whippelii in synovial fluid and tissue. Arthritis Rheum 1999;42:812–7. 73. Hidalgo F, Melon S, de Ona M, et al. Diagnosis of herpetic keratoconjunctivitis by nested polymerase chain reaction in human tear film. Eur J Clin Microbiol Infect Dis 1998;17:120–3. 74. Takeuchi S, Itoh N, Uchio E, Aoki K, Ohno S. Serotyping of adenoviruses on conjunctival scrapings by PCR and sequence analysis. J Clin Microbiol 1999;37:1839–45. 75. Subhan S, Jose RJ, Duggirala A, et al. Diagnosis of herpes simplex virus-1 keratitis: comparison of Giemsa stain, immunofluorescence assay and polymerase chain reaction. Curr Eye Res 2004;29:209–13. 76. Farhatullah S, Kaza S, Athmanathan S, Garg P, Reddy SB, Sharma S. Diagnosis of herpes simplex virus-1 keratitis using Giemsa stain, immunofluorescence assay, and polymerase chain reaction assay on corneal scrapings. Br J Ophthalmol 2004;88:142–4. 77. van Gelderen BE, Van der Lelij A, Treffers WF, van der Gaag R. Detection of herpes simplex virus type 1, 2 and varicella zoster virus DNA in recipient corneal buttons. Br J Ophthalmol 2000;84:1238–43. 78. Shimomura Y, Deai T, Fukuda M, Higaki S, Hooper LC, Hayashi K. Corneal buttons obtained from patients with HSK harbor high copy numbers of the HSV genome. Cornea 2007;26:190–3. 79. Connell PP, O’Reilly J, Coughlan S, Collum LM, Power WJ. The role of common viral ocular pathogens in Thygeson’s superficial punctate keratitis. Br J Ophthalmol 2007;91:1038–41. 80. Reinhard T, Roggendorf M, Fengler I, Sundmacher R. PCR for varicella zoster virus genome negative in corneal epithelial cells of patients with Thygeson’s superficial punctate keratitis. Eye 2004;18:304–5. 81. Kowalski RP, Uhrin M, Karenchak LM, Sweet RL, Gordon YJ. Evaluation of the polymerase chain reaction test for detecting chlamydial DNA in adult chlamydial conjunctivitis. Ophthalmology 1995;102:1016–9. 82. Lehmann OJ, Green SM, Morlet N, et al. Polymerase chain reaction analysis of corneal epithelial and tear samples in the
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diagnosis of Acanthamoeba keratitis. Invest Ophthalmol Vis Sci 1998;39:1261–5. 83. Kumar M, Mishra NK, Shukla PK. Sensitive and rapid polymerase chain reaction based diagnosis of mycotic keratitis through single stranded conformation polymorphism. Am J Ophthalmol 2005;140:851–7. 84. Ghosh A, Basu S, Datta H, Chattopadhyay D. Evaluation of polymerase chain reaction-based ribosomal DNA sequencing technique for the diagnosis of mycotic keratitis. Am J Ophthalmol 2007;144:396–403. 85. Johnson MW, Doft BH, Kelsey SF, et al. The Endophthalmitis Vitrectomy Study. Relationship between clinical presentation and microbiologic spectrum. Ophthalmology 1997;104:261–72. 86. Therese KL, Anand AR, Madhavan HN. Polymerase chain reaction in the diagnosis of bacterial endophthalmitis. Br J Ophthalmol 1998;82:1078–82. 87. Chiquet C, Lina G, Benito Y, et al. Polymerase chain reaction identification in aqueous humor of patients with postoperative endophthalmitis. J Cataract Refract Surg 2007;33:635–41. 88. Okhravi N, Adamson P, Carroll N, et al. PCR-based evidence of bacterial involvement in eyes with suspected intraocular infection. Invest Ophthalmol Vis Sci 2000;41:3474–9. 89. Chiquet C, Cornut PL, Benito Y, et al. Eubacterial PCR for bacterial detection and identification in 100 acute postcataract surgery endophthalmitis. Invest Ophthalmol Vis Sci 2008;49:1971–8. 90. Lohmann CP, Linde HJ, Reischl U. Improved detection of microorganisms by polymerase chain reaction in delayed endophthalmitis after cataract surgery. Ophthalmology 2000;107:1047–51. 91. Palani D, Kulandai LT, Naraharirao MH, Guruswami S, Ramendra B. Application of polymerase chain reaction-based restriction fragment length polymorphism in typing ocular rapid-growing nontuberculous mycobacterial isolates from three patients with postoperative endophthalmitis. Cornea 2007;26:729–35. 92. Anand A, Madhavan H, Neelam V, Lily T. Use of polymerase chain reaction in the diagnosis of fungal endophthalmitis. Ophthalmology 2001;108:326–30.
Keywords: polymerase chain reaction, ocular infection, ocular disease, diagnosis
P
olymerase chain reaction (PCR) is a powerful tool that involves in vitro amplification of specific nucleic acid sequences of DNA or RNA. It produces a large quantity of genetic material from a minute sample, which can then be examined by various means in order to detect, quantify, and analyze specific products. First described by Saiki et al. in 1985,1 the technique was perfected by Kary Mullis,2 who was awarded the Nobel Prize in 1993 in recognition of the impact of this technology on the scientific community. Initially developed as a research tool, PCR is now an integral part of diagnostics in many areas of medicine. It is therefore important that practicing ophthalmologists be familiar with the indications, strengths, and limitations of this technique in order to secure the full benefits and avoid the pitfalls of the tool. This review serves as an update to previous articles on the topic3–8 and discusses the basic principles of PCR, the different types and their indications, the strengths and limitations, and current applications of PCR in ophthalmic practice. METHOD OF LITERATURE SEARCH
The OVID Medline database was searched from 1966 to 2008 for the following terms: polymerase chain reaction or PCR, and uveitis, retinitis, endophthalmitis, lymphoma, keratitis, ocular inflammation, and ocular infection. The articles retrieved were limited to those published in English From the Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, B.C. Originally received June 1, 2008. Revised Sep. 1, 2008 Accepted for publication Sep. 15, 2008 Published online Jan. 23, 2009
or with English abstracts. Relevant cited references of these papers were also reviewed. BASIC PRINCIPLES OF PCR
Application of this technique depends on the availability of complementary specific DNA oligonucleotide sequences (known as primers) that flank areas of interest. The interactions between the primer sequence and the DNA to be studied are highly specific. Amplification of DNA sequences will only occur at the site defined by the primers. To perform PCR, several elements are required: a DNA template (aqueous or vitreous sample), a pair of short, synthetic oligonucleotide DNA primers, a thermostable DNA polymerase enzyme, a buffer system, nucleotide triphosphates, and a thermal cycling machine. There are 3 basic steps in PCR: denaturation, annealing, and extension (Fig. 1). Nucleic acid (e.g., DNA) is extracted from the clinical specimen of interest and used as the starting material for the process. In the first step, denaturation, heat (90–95 °C) is used to separate the extracted doublestranded DNA into single strands. During annealing the thermal cycler is cooled to a temperature that allows each oligonucleotide primer to bind to its associated sequence within the target strand of DNA (between 35 and 60 °C). The process of annealing is highly specific at optimal temperature, which is a value that is determined for each target Correspondence to Sonia N. Yeung, MD, Department of Ophthalmology and Visual Sciences, University of British Columbia, 2550 Willow St., Vancouver BC V5Z 3N9; [email protected] This article has been peer-reviewed. Cet article a été évalué par les pairs. Can J Ophthalmol 2009;44:23–30 doi:10.3129/i08-161 CAN J OPHTHALMOL—VOL. 44, NO. 1, 2009
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PCR in ophthalmology—Yeung et al. sequence empirically. In the last step, the temperature is elevated to the optimal level (typically 72–74 °C) for the thermally stable DNA polymerase to synthesize the target sequence starting from the annealed primers. A new strand of DNA that is complementary to the original target sequence is synthesized. The sequence of denaturation, annealing, and extension is termed a cycle. The newly synthesized DNA resulting from this cycle acts as target sequences for the next cycle. As this process cycles, the target sequence is amplified in an exponential fashion. Theoretically, amplification yields of 2N will occur after N thermal cycles (usually 30–40 cycles). Once all the primers and nucleotide triphosphates have been exhausted, production reaches a plateau. After the PCR reaction, gene products are visualized by separating DNA molecules by size, using electrophoresis (agarose or acrylamide gel) to determine whether the fragment of expected size has been synthesized. Higher sensitivity for detection can be obtained using more sensitive dyes or autoradiography. PCR products are also compared with a negative control, which includes all reagents but substitutes pure distilled water for the patient’s DNA sample.
Confirmation of the identity of the synthesized product can be achieved by fingerprinting. One method of fingerprinting involves evaluating the pattern of products following enzymatic cleavage at a specific sequence of DNA. Hybridization with a labeled DNA that anneals only to a specific product can also be used to identify product sequences. Finally, the products can be directly sequenced. TYPES OF PCR Universal primer
The genes that encode ribosomal RNA (rRNA) are highly repeated in the genome and have not significantly changed across much of evolution. Primers that detect these conserved regions take advantage of the universality of the genes. The 16S rRNA genes are conserved among bacteria; therefore, a primer for this sequence can be used to screen for any bacterium.9–12 Fungi, on the other hand, have highly conserved 18S and 28S rRNA genes, which can be used for the diagnosis of fungal disease.13 Multiplex
In multiplex PCR, several primer pairs are used that are specific to different DNA targets. This allows for the amplification and detection of a number of different sequences at the same time (e.g., 2 infectious agents from a single sample). However, for this procedure to work, the PCR conditions must be ideal for both reactions. Also, the primers must result in amplified products of different sizes for each target sequence, so that they appear distinct on the visualization method of choice.14,15 Reverse transcriptase
Reverse transcriptase PCR (RT-PCR) can be used to differentiate between active and inactive infection. Since RNA is produced during replication, active gene expression in tissues and cells can be identified by amplification. To detect RNA, it must first be converted to DNA by the enzyme reverse transcriptase. The resulting DNA is called complementary DNA (cDNA). PCR is then performed on the cDNA to amplify the target sequence. To ensure that the procedure is not amplifying contaminated genomic DNA, it is particularly important to include a control in which the reverse transcriptase enzyme is omitted. Fig. 1—One cycle of PCR. Denaturation: nucleic acid from a patient sample is heated to form single strands. Annealing: temperature is cooled to allow for oligonucleotide primers to bind. Extension: temperature is elevated to the optimal temperature for the thermostable DNA polymerase, which uses the annealed oligonucleotides as primers and the nucleotide triphosphate raw materials for DNA synthesis. At the end of 1 cycle, 2 target sequences of DNA are created from the original template. These can serve as templates for subsequent cycles.
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Real-time
Another means of characterizing active infection versus low-grade pathogenicity is to quantify the number of pathogen genomes in a sample. Low levels of genomes may reflect the low-level presence of that pathogen. For this technique, PCR is performed in a special thermocycler that has a real-time fluorescence detection unit in each well.16–18 The degree of fluorescence in the sample is compared with standard curves of known pathogens, allowing for the number of pathogen genomes to be determined.
PCR in ophthalmology—Yeung et al. Nested
Nested PCR (nPCR) increases both the sensitivity and specificity of the general PCR technique, as 2 different amplification processes are performed sequentially. The first amplification uses a set of primers that produces a larger segment, which then becomes the template for the next amplification. The second amplification uses a different set of primers that bind within the large segment. The sensitivity of nPCR is increased because of the 2 cycles of amplification, and the specificity is increased because of the binding of 2 separate pairs of primers for amplification.19–21 Inverse
Whereas standard PCR amplifies a known sequence of nucleic acids situated between 2 primer annealing sites, inverse PCR makes it possible to amplify an unknown sequence situated beside a known sequence (Fig. 2).22,23 A restriction enzyme (which cuts DNA at locations with a specific sequence of base pairs) cuts the DNA strand at points flanking the known and unknown sequences. Both sequences do not
Fig. 2—Inverse PCR: amplification of an unknown sequence situated beside a known sequence. A restriction enzyme cuts the DNA strand on either side of the known and unknown sequence. A different enzyme ligates the ends of this fragment, circularizing it. Finally, another restriction enzyme cuts the ring at a point within the known sequence to reform a linear fragment. The resulting DNA fragment includes the unknown sequence sandwiched between terminal known sequences, which can undergo amplification using the standard PCR approach.
contain any sites for this enzyme; therefore, the fragment remains intact. It then circularizes with the help of an enzyme that ligates the ends. A second enzyme cuts this ring at a point within the known sequence to reform a linear fragment that includes the unknown sequence sandwiched between terminal known sequences. The unknown sequence is amplified using the standard PCR approach. STRENGTHS AND LIMITATIONS OF PCR
A major strength of PCR is that the DNA segment of interest does not need to be first purified from the background DNA. Almost any tissue or body fluid can be used, including aqueous (anterior chamber paracentesis of ≥50 μL) and vitreous samples (preinfusion aspirate of ≥100 μL), which is placed in a sterile microfuge tube and snap-frozen in liquid nitrogen or dry ice.3,4,8 The exponential amplification of the segment of interest provides high sensitivity, and the use of unique primers provides high specificity. Using the Goldmann-Witmer coefficient in combination with PCR can further increase the sensitivity.24 PCR can provide results within a few hours. The high sensitivity of PCR makes it prone to falsepositive results. Specimen contamination during laboratory processing can arise from previous amplification procedures or cross-contamination from other samples.25 Careful specimen handling and processing must be conducted in conjunction with the use of rigorous negative controls. Since PCR can detect DNA in dead and latent samples (i.e., herpesviruses), it can be difficult to differentiate between expected organisms (i.e., normal flora) and those responsible for infection. RT-PCR can help differentiate between residual DNA of a pathogenic organism after successful treatment and active organisms. Finally, the specificity of this technology can be compromised by the selection of nonspecific primers, as well as suboptimal PCR conditions that allow nonspecific products to amplify. False-negative results may also occur in PCR. The assays may lack sensitivity if there is a low inoculum in the clinical specimen or an inadequate sample. This can be addressed by increasing the number of cycles performed. The specimen may contain substances that inhibit nucleic acid extraction or amplification. The high specificity can also lead to falsenegative results when primers are chosen for a polymorphic region of the pathogen’s DNA where sequence variations exist between strains. The use of several primer sets for each organism can help in this regard. For PCR to be an effective tool, it requires a well-defined differential diagnosis. Culture or staining methods can detect a wide range of organisms with a single test. PCR can only detect an organism for which a primer set has been selected. In 2006, Acharya et al.26 compared PCR results with presumptive clinical diagnosis in patients with vitritis. They determined that PCR for any infectious etiology was unlikely to be positive if the clinical suspicion of a specific diagnosis is low. PCR must be considered in clinical context. CAN J OPHTHALMOL—VOL. 44, NO. 1, 2009
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PCR in ophthalmology—Yeung et al. CURRENT CLINICAL APPLICATIONS OF PCR IN OPHTHALMOLOGY
The general applications of PCR are extensive.3,4,6 It can be used in the diagnosis of infection caused by either slow-growing or fastidious microorganisms, detection of infectious agents that cannot be cultured, recognition of newly emerging infectious diseases, and discovery of novel microorganisms. Additionally, the procedure improves the accuracy of subtyping pathogens in epidemiological studies, quantifies viral load (real-time PCR), and allows rapid identification of antimicrobial resistance. The next sections will briefly summarize the current applications of PCR in clinical ophthalmology with a focus on new advances in this area. Anterior uveitis
The diagnosis of anterior uveitis associated with herpes simplex virus and varicella zoster virus is commonly based on clinical presentation. In atypical cases, several groups have used PCR to confirm the presence of these pathogens.27–30 In several cases in which treatment of presumed herpetic infection failed, cytomegalovirus (CMV) infection was established by PCR.31–33 Fuchs’ heterochromic iridocyclitis (FHI) and PosnerSchlossman syndrome (PSS) bear many similarities to the clinical hallmarks of herpetic disease. In 2004, Quentin and Reiber34 determined that all patients with FHI in a European population had elevated intraocular antibody titers (modified Goldmann-Witmer coefficient), and the majority had positive RT-PCR for rubella virus.34 Interestingly, Chee et al.35 demonstrated positive CMV PCR for 36% of the patients in their study with either clinical PSS or FHI. Genetic variations in cytokines have been associated with disease susceptibility and severity in autoimmune disease. In 2006, El-Shabrawi et al.36 reported that individuals positive for HLA-B27 show a higher susceptibility to the development of anterior uveitis in the presence of singlenucleotide polymorphisms of the tumor necrosis factor alpha promoter at specified positions. In the same year, Yeo et al.37 suggested that a single nucleotide polymorphism of the chemokine gene MCP-1 may be a protective marker for idiopathic acute recurrent anterior uveitis. Posterior uveitis
The first application of PCR in eye disease was associated with the diagnosis of viral uveitis.20,38–42 In cases in which media opacity (cataract or dense vitritis) or atypical presentations precluded clinical diagnosis in posterior uveitis, several groups found that the clinical course mirrored the PCR results.20,40 The sensitivity of PCR for varicella zoster virus (VZV), herpes simplex virus (HSV), and CMV in vitreous fluid is greater than 90%, and its specificity greater than 95%.20,39,43–45 PCR has been used to subtype viral infections,43 to screen a substantial differential diagnosis of posterior uveitis in
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a single reaction (multiplex PCR),43,46 and to quantitate infectious pathogens (real-time PCR).7 Siqueira et al.47 demonstrated that PCR could detect CMV in aqueous humor with high specificity in patients infected with human immunodeficiency virus (HIV) who had active lesions in the retina.47 Often, both aqueous and vitreous fluid analysis is helpful in posterior uveitis. Improved primer designs allowed the detection of Toxoplasma DNA in 83% of patients with suspected ocular toxoplasmosis and positive serologic test results.48,49 Intraocular antibody production combined with PCR has further contributed significantly to the diagnosis of toxoplasmosis.50 Interestingly, Toxoplasma gondii DNA has been detected in the peripheral blood of patients with active disease51 and in retinochoroidal toxoplasmic scar specimens from eye bank and enucleated eyes.52 The identification of several unique strains of T. gondii were shown to have different antibiotic sensitivities to sulfa medications, which may explain the heterogeneity in response to treatment.53 Some bacterial causes of posterior segment disease cannot readily be cultured. The ocular manifestations of tuberculosis are commonly associated with difficulties in diagnosis and therapy, complicated by the slow rate of growth in culture. An nPCR assay was recently found to be useful in the confirmation of Mycobacterium tuberculosis in aqueous and vitreous samples.54 Whipple’s disease (Tropheryma whippelii) and Bartonella can now also be amplified from vitreous fluid55,56 and orbital granulomas.57 In addition to these diagnostic applications, PCR has been used to measure viral load in order to monitor response to therapy. This has been applied in the care of patients with HIV, CMV, and hepatitis C virus infection.58–61 PCR may also aid in the management of viral retinitis by the detection of antiviral-resistant strains, as many CMV patients acquire resistance to ganciclovir.62–65 The use of PCR may have a role in deciding whether to start, continue, or stop certain therapies. Recently, PCR was used to identify polymorphisms in chemokines and chemokine receptors that were shown to influence the clinical outcome in patients with idiopathic immune-mediated posterior segment uveitis.66 B-cell lymphoma
B-cell lymphoma often mimics a posterior uveitis, presenting as an ocular inflammation in older patients.67 PCR has been used to determine clonality68 and to detect IgH gene rearrangements in intraocular lymphoma.69 Coupeland et al.70 further illustrated the practicality of PCR for the detection of IgH gene rearrangements in chorioretinal biopsies. External eye disease
Although many bacterial causes of conjunctivitis and keratitis are readily detected by Gram-staining or culture, there are several organisms that still present a challenge.71,72 Adenovirus conjunctivitis outbreaks continue to be a
PCR in ophthalmology—Yeung et al. public health challenge. The organism is not readily cultured, and serotyping is generally not possible. PCR has been shown to increase the speed and accuracy of diagnosis73 and offers the advantage of potential serotype specificity,74 which has significant prognostic value. Herpetic infections often present diagnostic dilemmas. In 2004, 2 groups compared Giemsa staining, immunofluorescence assay, and PCR in the detection of HSV-1 and found that combined PCR and immunofluorescence provides the highest sensitivity and specificity for the diagnosis of HSV-1.75,76 PCR has also been explored as an adjunct to intraocular anti-HSV antibody in the detection of HSV in recipient corneal buttons with the hope of starting treatment early to prevent graft failure.77,78 A herpetic etiology for Thygeson’s superficial punctate keratitis has been investigated with PCR.79,80 Connell et al.79 reported the results from 8 patients with this condition to be negative for HSV-1, HSV-2, VZV, and adenovirus. PCR has also been helpful in identifying infections caused by Chlamydia with 88% sensitivity and 100% specificity,81 and Acanthamoeba with 84% sensitivity (53% for culture) and 95% specificity.82 Kumar et al.83 reported that early detection of mycotic keratitis by PCR resulted in successful treatment of 4 patients. Recently, Ghosh et al.84 used PCR and sequencing of rDNA to detect pathogenic strains in fungal keratitis. They found that PCR rDNA sequencing results correlated with culture-based results, and they further identified fungal strains that could not be cultured in routine medium. Endophthalmitis
The Endophthalmitis Vitrectomy Study reported that endophthalmitis following cataract surgery was often culture negative.85 With the use of PCR, bacterial DNA has been detected in the majority of cases of postoperative acute endophthalmitis.86–88 In 2007, Chiquet et al.87 used eubacterial PCR amplification with direct sequencing to identify the causative agents in aqueous humor samples from 30 patients with postcataract endophthalmitis. Cultures were positive in 32% of cases, and PCR was positive in 61% of cases with aqueous humor samples; when combined, a diagnosis was made in 71% of cases. Chiquet et al.89 later confirmed these findings in a prospective multicenter study. They concluded that culture and eubacterial PCR are complementary techniques, and PCR was particularly useful in the setting of previous administration of intravitreal antibiotics. The causative organisms responsible for delayed-onset endophthalmitis are often present in low numbers and difficult to culture. PCR has demonstrated that these infections were mainly caused by Propionibacterium acnes, Staphylococcus epidermidis, or Actinomyces israelii.90 Recently, Palani et al.91 applied PCR-based restriction fragment length polymorphism analysis to identify nontuberculous mycobacteria in 3 cases of delayed-onset endophthalmitis.91 PCR has also been used to identify Fusarium, Candida,
and Aspergillus,13 and has proven to be a more sensitive and rapid diagnostic tool than conventional methods for fungal endophthalmitis.92 FUTURE DIRECTIONS
Molecular technology is a powerful tool for the rapid and accurate diagnosis of a variety of infectious ocular diseases. Novel organisms have also been discovered since the advent of PCR, particularly those that have been historically difficult to culture and identify by traditional means. PCR has been used to measure viral load and to monitor response to therapy, and has shown itself to be a valuable instrument for the detection and confirmation of antimicrobial resistance. Etiologies for ocular disease once thought to be idiopathic may eventually be elucidated with this tool. Although PCR will not replace conventional methods, it has certainly proved to be a useful adjunct to existing techniques. As we continue to learn more about the genetic basis of disease, there will undoubtedly be an additional role for PCR in designing patient-specific treatment regimens for many diseases. The authors have no proprietary or commericial interest in any materials discussed in this article.
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14. Baumforth KR, Nelson PN, Digby JE, O’Neil JD, Murray PG. Demystified ... the polymerase chain reaction. Mol Pathol 1999; 52:1–10. 15. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 2000;13:559–70. 16. Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HR. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 2005;5:209–19. 17. Bell AS, Ranford-Cartwright LC. Real-time quantitative PCR in parasitology. Trends Parasitol 2002;18:337–42. 18. Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med 1998;36:255–69. 19. Miller RF, Howard MR, Frith P, Perrons CJ, Pecorella I, Lucas SB. Herpesvirus infection of eye and brain in HIV infected patients. Sex Transm Infect 2000;76:282–6. 20. Mitchell SM, Fox JD, Tedder RS, Gazzard BG, Lightman S. Vitreous fluid sampling and viral genome detection for the diagnosis of viral retinitis in patients with AIDS. J Med Virol 1994;43:336–40. 21. Severini GM, Mestroni L, Falaschi A, Camerini F, Giacca M. Nested polymerase chain reaction for high-sensitivity detection of enteroviral RNA in biological samples. J Clin Microbiol 1993;31:1345–9. 22. Ochman H, Gerber AS, Hartl DL. Genetic applications of an inverse polymerase chain reaction. Genetics 1988;120:621–3. 23. Triglia T, Peterson MG, Kemp DJ. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res 1988;16:8186. 24. Fekkar A, Bodaghi B, Touafek F, Le Hoang P, Mazier D, Paris L. Comparison of immunoblotting, calculation of the GoldmannWitmer coefficient, and real-time PCR using aqueous humor samples for diagnosis of ocular toxoplasmosis. J Clin Microbiol 2008;46:1965–7. 25. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 1990;93:125–8. 26. Acharya N, Lietman T, Cevallos V, et al. Correlation between clinical suspicion and polymerase chain reaction verification of infectious vitritis. Am J Ophthalmol 2006;141:584–5.
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Keywords: polymerase chain reaction, ocular infection, ocular disease, diagnosis