Polymerase Chain Reaction in Intraocular Inflammation

Krishnendu Nandi, Prabhat Ranjan, Lily Therese , Jyotirmay Biswas*
Vision Research Foundation, Sankara Nethralaya, 18 College Road, Chennai 600 006, India

Article Metrics

CrossRef Citations:
Total Statistics:

Full-Text HTML Views: 3178
Abstract HTML Views: 2019
PDF Downloads: 688
Total Views/Downloads: 5885
Unique Statistics:

Full-Text HTML Views: 1356
Abstract HTML Views: 1222
PDF Downloads: 492
Total Views/Downloads: 3070

Creative Commons License
© Nandi et al..; Licensee Bentham Open.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.

* Address correspondence to this author at the Vision Research Foundation, Sankara Nethralaya, 18 College Road, Chennai 600 006, India; Tel: 044-28271616; Fax: 044-28254180; E-mail:


Polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences in repeated cycles of denaturation, oligonucleotide annealing and DNA polymerase extension. It is a powerful molecular biologic tool that allows the rapid production of analytic quantities of DNA from small amounts of starting material. PCR can be performed on nearly any ocular specimen or biopsy. For diagnosis of uveitis, the obtained sample is usually an anterior chamber paracentesis or vitreous tap. PCR potentially is more sensitive than culture for detection of many organisms. By utilizing a secondary detection system in concert with the initial PCR reaction, perfect specificity can be assured. The initial application of PCR diagnostics to ophthalmic disease was in the detection of viral uveitis. PCR has also been implicated in studies of noninfectious uveitis. The most common application is HLA typing. A universal bacterial PCR can be very helpful for the diagnosis of bacterial endophthalmitis at an early stage of the disease.


The polymerase chain reaction (PCR) is a powerful molecular biologic tool that allows the rapid production of analytic quantities of DNA from small amounts of starting material. Since the introduction of its modern form in 1988 [1], PCR has revolutionized much of molecular biology and has greatly accelerated the development of molecular diagnostics. Kary B. Mullis from USA received a Nobel Prize in 1993 for inventing this technique. This powerful technique has numerous applications in diagnostic pathology, especially in the fields of microbiology and genetics. All practicing ophthalmologists should have a working knowledge of the uses of PCR. PCR has been used to diagnose uveitis, infectious endophthalmitis and protozoal eye diseases [2]. This review discusses the use of PCR in the analysis of uveitis, and ways in which PCR is improving our knowledge of understanding of the mechanisms of uveitis.


Polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences in repeated cycles of denaturation, oligonucleotide annealing and DNA polymerase extension [3].The PCR uses in vitro enzymatic synthesis to amplify specific DNA sequence within few hours. The PCR consists of repetitive cycles of specific DNA synthesis, defined by short stretches of preselected DNA. With each cycle there is a doubling of the final, desired DNA product such that million-fold amplification is possible [4].

PCR is performed using two specific primers that flank the DNA region of interest. After enzymatic synthesis of the replicated strand is complete, the DNA is denatured into single strands. This allows the newly synthesized strand to serve as template for subsequent synthesis of new strands. Using an automated thermal heat block, 30 to 40 rounds of replication can be performed in just a few hours. Theoretically, the molar amount of PCR product doubles with each round of replication. Thirty-five cycles are typically used for diagnostic PCR. In order to perform PCR, we must have a source of DNA (DNA extracted either from an aqueous or vitreous specimen). It begins with the initial sample containing the target DNA and mixes in the appropriate primers, DNA polymerase, nucleotide triphosphates, and buffered salts. Following performance of PCR in the thermal cycler, the products may be detected in one of several ways. Generally, gel electrophoresis, with use of acrylamide or agarose, is employed to determine if a DNA fragment of expected size has been produced. Confirmation of the identity of the PCR product can be achieved by digesting the product with restriction endonuclease and observing the restriction digest pattern, a technique called fingerprinting. Ultimate identification of a DNA fragment can be achieved by sequencing the PCR product DNA.

Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction is used to determine whether or not a specific sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR), itself a modification of polymerase chain reaction.

The polymerase chain reaction is an effective tool for amplifying DNA, however for this to be adapted to measure RNA, the RNA sample first needs to be reverse transcribed to DNA via an enzyme known as a reverse transcriptase. This transcribed DNA is known as cDNA or complementary DNA. This method, known as RT-PCR, required extensive optimisation of the number of PCR cycles, so as to obtain results during logarithmic DNA amplification. Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites. Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product.

PCR can be performed on nearly any ocular specimen or biopsy. For diagnosis of uveitis, the obtained sample is usually an anterior chamber paracentesis or vitreous tap. Anterior chamber paracentesis of 50 microlitres is usually sufficient for diagnostic purposes. For vitrectomy specimens, the initial preinfusion aspirate (100-500 microlitre) is preferred. Specimens should be aseptically transferred to a sterile, capped tube (i.e., a 1.5-ml microfuge tube) and quick-frozen on dry ice or in liquid nitrogen. The sample should remain frozen until processed by the accepting laboratory; freeze thaw cycles will release nucleases that will degrade all RNA and some DNA [5].

The sensitivity for detection of foreign DNA is very high. Thus PCR potentially is more sensitive than culture for detection of many organisms. By utilizing a secondary detection system in concert with the initial PCR reaction, perfect specificity can be assured. Although PCR would seem to have nearly ideal characteristics for a diagnostic test, the high sensitivity and specificity can cause significant pitfalls.


PCR has had a major impact on our ability to detect infectious agents. Since the first detection of Toxoplasma gondii DNA in ocular tissue with the use of PCR in 1990 [6]. PCR has been applied to the detection and diagnosis of various infective uveitis [7].

The initial application of PCR diagnostics to ophthalmic disease was in the detection of viral uveitis [5, 8-10]. Knox et al. [10] performed PCR on aqueous or vitreous samples of 37 eyes of 38 patients, with “diagnostic dilemmas” in posterior uveitis. Of these cases, a definitive diagnosis of a viral infection could be made by PCR in 25 eyes. Of the PCR-negative cases, a number were ultimately diagnosed to be toxoplasmosis, and the remainder had natural histories inconsistent with viral retinitis. Thus, both positive and negative PCR results likely had diagnostic significance in this study. Probably the most common indication for performing diagnostic PCR for posterior uveitis is the presence of media opacity. Significant media opacity from cataract or dense vitritis can make otherwise straightforward diagnoses difficult. Mitchell et al. developed PCR primers with a sensitivity of 93% and specificity of 98% for the detection of Cytomegalovirus (CMV) [11]. Of the nine patients tested, four tested positive for CMV, and three for Varicella Zoster virus (VZV). The remaining two were subsequently judged to have toxoplasmosis. In all cases, the clinical course was consistent with the PCR-based diagnosis. The clinical diagnosis of atypical toxoplasmosis can also be problematic. Classical reactivation toxoplasmosis can be diagnosed by clinical examination, but primary toxoplasmosis can resemble a number of other infectious acute retinitis [12, 13]. Initial studies of PCR diagnosis of Toxoplasma gondii were disappointing, showing sensitivities less than 50% [14, 15]. In 1993, Aouizerate et al. performed PCR on the aqueous of 59 eyes with suspected or confirmed infection with Toxoplasma gondii; the parasite was demonstrated in 20 cases (33.8%) [14]. However, recent advances in primer design, utilizing highly repetitive pathogen DNA sequences, have greatly improved yields for PCR of T. gondii. Montoya et al. [15] were able to detect Toxoplasma DNA in nearly 80% of patients with suspected ocular toxoplasmosis and positive serum IgG titers. Using a similar PCR assay, Bou et al. [16] were able to detect Toxoplasma gondii DNA in the peripheral blood of most patients with active ocular toxoplasmosis, raising the possibility that in the future, reactivation disease could be diagnosed via a blood test. Biswas et al. performed PCR on the aqueous in a case of suspected miliary tuberculosis of choroids (Fig. 1) and Mycobacterium tuberculosis genome (Fig. 2) was found in PCR analysis [17].PCR is also helpful in detecting Leptospira related uveitis [18].

Fig. (1).

A case of Subretinal abscess.

Fig. (2).

Ethidium bromide stained 2% agarose gel with amplification products from a case of subretinal abscess suspected to be tuberculous. Lane 1: Reagent control of the first round. Lane 2: Reagent control of the second round. Lane 3: Aqueous humor - negative, Lane 4: FNAB specimen - positive, Lane 5: Blood - positive. Lane 6: Positive control M. tuberculosis (H37Rv). Lane 7: Phi x 174 DNA/Hinf l digest.


Madhavan et al. reviewed their experience using PCR to tissue sections obtained from formalin-fixed and paraffin embedded tissues of epiretinal membrane (ERM) from 23 patients of Eales’ disease [19]. 11 out of 23 (47.8%) were positive for Mycobacterium tuberculosis genome, indicating association of this bacterium with Eales’ disease. Gupta A et al. reported tubercular retinal vasculitis with varied fundus findings, and diagnosis was confirmed by doing PCR from the aqueous or vitreous humor [20].


PCR has also been implicated in studies of noninfectious uveitis. The most common application is HLA typing. Saiki et al. used PCR to enzymatically amplify a specific segment of beta-globin or HLA-DQ alpha gene in human genomic DNA [21].

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology is applied to HLA-DR, -DQ and -DW typing at the nucleotide level, eliminating the need for radioisotopes as well as allele specific oligonucleotide probes [22].

Using this technique, Shino et al. reported complete association of the HLA-DRB104 and -DQB104 alleles with Vogt-Koyanagi-Harada (VKH) disease [23].and it is more common for Asians. Polymerase chain reaction-sequencing-based typing (PCR-SBT) is used for HLA-B51 alleles. Evaluation of intraocular cytokines and other inflammatory mediators and makers provides important information, particularly in noninfectious uveitis [24]. Cytokines and inflammatory related transcripts are usually detected via reverse transcription PCR (RT-PCR) [24-26].The results from RT-PCR are complementary to data from Western blotting and/or immunohistochemistry.


The Masquerade syndrome is consisting of group of disorders that occurs with intra-ocular inflammation (most commonly malignancy) and is often misdiagnosed as uveitis. PCR can be useful for diagnosing masquerade syndrome. Primary intraocular lymphoma is a subtype of central nervous system lymphoma involving the eye. It can often mimic chronic uveitis. Utilization of PCR has become a practical tool for the detection of IgH gene rearrangements and provides a helpful adjunct for the diagnosis of B-cell lymphoma in the eye [27].


Although direct microscopy is the easiest and most rapid method to detect bacterial etiologies of endophthalmitis, its sensitivity is very low, with positive result varying from 4.2% to 46.5% for vitreous samples, which decreases further in aqueous fluid [28, 29]. More sensitive than microscopy, culture is considered “ the gold standard”. However, there have been no significant improvements in the yield of culture methods [30]. Postoperative endophthalmitis is a vision-threatening complication of cataract surgery and presents even further diagnostic challenges. The organisms are frequently present in low numbers, and they can be difficult to culture. Yields from diagnostic vitreous biopsies in this condition are less than 50%. The Endophthalmitis Vitrectomy Study reported culture yields of only 70% [31]. Culture results are also slow to return, thus requiring patients be treated with broad-spectrum antibiotics for several days, even for relatively indolent bacteria. In cases where conventional techniques have low sensitivity, PCR, characterized by its high sensitivity and specificity, would be an ideal technique to detect bacterial pathogens in the eye. All bacteria share common, highly repetitive DNA sequences for their 16S ribosomal RNA. By designing primers to these conserved 16S sequences, PCR can be performed on biopsy material from eyes with suspected endophthalmitis, with the results available within 6-8 hours. Therese et al. demonstrated the utility of this approach for culture-negative endophthalmitis [29]. They were able to determine a bacterial cause for endophthalmitis in 100% of culture-positive and 44% of culture-negative cases. Of the remaining culture-negative cases, one-third was found to be fungal. Lohmann et al. [32]. used 16S ribosomal primers as well as fungal PCR primers, along with culture and stain for 25 eyes with delayed-onset endophthalmitis. Aqueous culture and microscopy each had a 0% yield, but vitreous culture had a 24% yield in these patients. PCR of the aqueous yielded a diagnosis in 84% of the cases and PCR of a vitreous biopsy yielded a diagnosis in 92%. PCR thus has clear superiority to any other available diagnostic technique for diagnosis of endophthalmitis.

Biswas et al. demonstrated Aspergillus fumigatus fungus by PCR-based RFLP (Restriction Fragment Length Polymorphism) technique from paraffin section of an eyeball of an eight-month-old child removed for endogenous endophthalmitis [33].

Compared to the conventional technique, PCR for detection of fungal DNA was found to be a rapid and more sensitive method in the early diagnosis of postoperative fungal Endophthalmitis [34, 35]. Semi-nested polymerase chain reaction is also helpful for rapid detection of panfungal genome directly from ocular specimens [36].

Polymerase chain reaction-based technology is a useful adjunct to conventional culture because when used with aqueous humor samples only, the association of both techniques allowed for a microbiological diagnosis in 71% of cases of postoperative acute and delayed-onset endophthalmitis [37]. A universal bacterial PCR can be very helpful for the diagnosis of endogenous bacterial endophthalmitis at an early stage of the disease [38].


PCR is a powerful molecular technique for evaluation of very small amounts of DNA and RNA. PCR can be a simple, rapid, sensitive and specific tool for the diagnosis of infection, autoimmunity and masquerade syndromes of the eye.


[1] Saiki RK, Gelfand DH, Stoffel S. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 1988; 239: 487-91.
[2] Van Gelder RN. Application of polymerase chain reaction to diagnosis of ophthalmic disease Surv Ophthalmol 2001; 46: 248-58.
[3] Erlich HA, Gelfand D, Sninsky JJ. Recent advances in the polymerase chain reaction Science 1991; 252: 1643-51.
[4] Remick DG, Kunkel SL, Holbrook EA, et al. Theory and application of polymerase chain reaction Am J Clin Pathol 1990; 93: S49-54.
[5] Cunningham ET Jr, Short GA, Irvine AR. Acquired immunodeficiency syndrome-associated herpes simplex virus retinitis Clinical description and use of a polymerase chain reaction -based assay as a diagnostic tool Arch Ophthalmol 1996; 114: 834-40.
[6] Brezin AP, Egwuagu CE, Burnier M Jr, et al. Identification of toxoplasma gondii in paraffin-embedded sections by the polymerase chain reaction Am J Ophthalmol 1990; 110: 599-604.
[7] Gerling J, Neumann-Haefelin D, Seuffert HM, et al. Diagnosis and management of the acute retinal necrosis syndrome Ger J Opthalmol 1992; 1: 388-93.
[8] Abe T, Sato M, Tamai M. Correlation of varicella-zoster virus copies and final visual acuities of acute retinal necrosis syndrome Graefes Arch Clin Exp Ophthalmol 1998; 236: 747-52.
[9] Abe T, Tsuchida K, Tamai M. A comparative study of the polymerase chain reaction and local antibody production in acute retinal necrosis syndrome and cytomegalovirus retinitis Graefes Arch Clin Exp Ophthalmol 1996; 234: 419-24.
[10] Knox CM, Chandler D, Short GA, Margolis TP. Polymerase chain reaction-based assays of vitreous samples for the diagnosis of viral retinitis Use in diagnostic dilemmas Ophthalmology 1998; 105: 37-44.
[11] Mitchell SM, Fox JD, Tedder RS. Vitreous fluid sampling and viral genome detection for the diagnosis of viral retinitis in patients with AIDS J Med Virol 1994; 43: 336-40.
[12] Holland GN, Muccioli C, Silveira C. Intraocular inflammatory reactions without focal necrotizing retinochoroiditis in patients with acquired systemic toxoplasmosis Am J Ophthalmol 1999; 128: 413-20.
[13] Ronday MJ, Ongkosuwito JV, Rothova A, Kijlstra A. Intraocular anti-Toxoplasma gondii IgA antibody production in patients with ocular toxoplasmosis Am J Ophthalmol 1999; 127: 294-300.
[14] Aouizerate F, Cazenave J, Poirier L. Detection of Toxoplasma gondii in aqueous humour by the polymerase chain reaction Br J Ophthalmol 1993; 77: 107-9.
[15] Garweg J, Boehnke M, Koerner F. Restricted applicability of the polymerase chain reaction for the diagnosis of ocular toxoplasmosis Ger J Ophthalmol 1996; 5: 104-8.
[16] Bou G, Figueroa MS, Marti-Belda P. 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.
[17] Biswas J, Shome D. Choroidal tubercles in disseminated tuberculosis diagnosed by the polymerase chain reaction of aqueous humor A case report and review of the literature Ocul Immunol Inflamm 2002; 10: 293-8.
[18] Rathinam SR. Leptospirosis Curr Opin Ophthalmol 2002; 13: 381-6.
[19] Madhavan HN, Therese KL, Gunisha P, Jayanthi U, Biswas J. Polymerase chain reaction for detection of Mycobacterium tuberculosis in epiretinal membrane in Eales' disease Invest Ophthalmol Vis Sci 2000; 41: 822-5.
[20] Gupta A, Gupta V, Arora S, Dogra MR, Bambery P. PCR-positive tubercular retinal vasculitis: clinical characteristics and management Retina 2001; 21: 435-4.
[21] Saiki RK, Bugawan TL, Horn GT, et al. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele specific oligonucleotide probes Nature 1986; 324: 163-6.
[22] Uryu N, Maeda M, Ota M, et al. A simple and rapid method for HLA-DRB and -DQB typing by digestion of PCR amplified DNA with allele specific restriction endonuclease Tissue Antigens 1990; 35: 20-31.
[23] Shindo Y, Ohno S, Yamamoto T, et al. Complete association of the HLA-DRB104 and -DQB104 allele with Vogt-Koyanagi-Harada's disease Hum Immunol 1994; 39: 169-76.
[24] Murray PI, Clay CD, Mappin C, et al. Molecular analysis of resolving immune responses in uveitis Clin Exp Immunol 1999; 117: 455-61.
[25] Li B, Yang P, Zhou H, et al. T-bet expression is upregulated in Behcet's disease Br J Ophthalmol 2003; 87: 1264-7.
[26] Siolverman MD, Zamora DO, Pan Y, et al. Constitutive and inflammatory mediator-regulated fractaline expression in human ocular tissue and cultured cells Invest Ophthalmol Vis Sci 2003; 44: 1608-5.
[27] Coupland Se, Bechrakis NE, Anatassiou G, et al. Evaluation of vitrectomy specimens and choriorenital biopsies in the diagnosis of primary intraocular lymphoma in patients with masquerade syndrome Graefes Arch Clin Exp Ophthalmol 2003; 241: 860-7.
[28] Barza M, Pavan PR, Doft BH, et al. Evaluation of microbiological diagnostic techniques in postoperative Endophthalmitis in the Endophthalmitis Vitrectomy Study Arch Ophthalmol 1997; 115: 1142-50.
[29] Therese KL, Anand AR, Madhavan HN. Polymerase chain reaction in the diagnosis of bacterial Endophthalmitis Br J Ophthalmol 1998; 82: 1078-82.
[30] Anand AR, Madhavan HN, Therese KL. Use of Polymerase chain reaction and DNA probe hybridization to determine the Gram reaction of the infecting bacterium in the intraocular fluids of the patients with Endophthalmitis J Infect 2000; 41: 221-6.
[31] Durand M, et al. Microbiologic factors and visual outcome in the Endophthalmitis vitrectomy study Am J Ophthalmol 1996; 122: 830-46.
[32] 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.
[33] Biwas J, Bagyalakshmi R, Therese LK. Diagnosis of Aspergillus fumigatus endophthalmitis from formalin fixed paraffin-embedded tissue by polymerase chain reaction-based restriction fragment length polymorphism Indian J Ophthalmol 2008; 56: 65-.
[34] Tarai B, Gupta A, Ray P, Shivaprakash MR, Chakrabarti A. Polymerase chain reaction for early diagnosis of post-operative fungal endophthalmitis Indian J Med Res 2006; 123: 671-8.
[35] Anand A, Madhavan H, Neelam V, Lily T. Use of polymerase chain reaction in the diagnosis of fungal endophthalmitis Ophthalmology 2001; 108: 326-0.
[36] Bagyalakshmi R, Therese KL, Madhavan HN. Application of semi-nested polymerase chain reaction targeting internal transcribed spacer region for rapid detection of panfungal genome directly from ocular specimens Indian J Ophthalmol 2007; 55: 261-5.
[37] 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.
[38] Kerkhoff FT, van der Zee A, Bergmans AM, Rothova A. Polymerase chain reaction detection of Neisseria meningitidis in the intraocular fluid of a patient with endogenous endophthalmitis but without associated meningitis Ophthalmology 2003; 110: 2134-6.