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 Table of Contents  
REVIEW ARTICLE
Year : 2021  |  Volume : 59  |  Issue : 4  |  Page : 331-337

Polymerase chain reaction in ocular and adnexal inflammation: Our experience with review of literature


1 Medical Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India
2 Department of Uveitis and Ocular Pathology, Medical Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India

Date of Submission11-Sep-2021
Date of Decision16-Oct-2021
Date of Acceptance16-Oct-2021
Date of Web Publication21-Dec-2021

Correspondence Address:
Dr. Jyotirmay Biswas
Department of Uveitis and Ocular Pathology, Medical Research Foundation, Sankara Nethralaya, 18, College Road, Nungambakkam, Chennai - 600 006, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tjosr.tjosr_139_21

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  Abstract 


Polymerase chain reaction (PCR) is a revolutionary innovation involving enzymatic amplification of nucleic acid sequences in repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension. It is a unique molecular biologic tool that allows the rapid production of analytic quantities of DNA from small amounts of starting material. PCR can be performed on any ocular specimen or biopsy. The most common application of the PCR is human leukocyte antigen (HLA) typing. A universal bacterial PCR can be very helpful for the diagnosis of bacterial endophthalmitis at an early stage of the disease. There are also several advances in the application of PCR in the recent time, and this technology has opened a new era in the diagnosis and treatment of uveitis, enabling ophthalmologists to establish new clinical entities of uveitis caused by infectious microorganisms, identify pathogens in the eyes of many patients with uveitis, and determine prompt diagnosis and appropriate therapy.

Keywords: Cytomegalovirus retinitis, Eales' disease, endophthalmitis, intraocular inflammation, ocular tuberculosis, polymerase chain reaction, scleritis, toxoplasmosis


How to cite this article:
Pradhan A, Lakshmipathy DR, Biswas J. Polymerase chain reaction in ocular and adnexal inflammation: Our experience with review of literature. TNOA J Ophthalmic Sci Res 2021;59:331-7

How to cite this URL:
Pradhan A, Lakshmipathy DR, Biswas J. Polymerase chain reaction in ocular and adnexal inflammation: Our experience with review of literature. TNOA J Ophthalmic Sci Res [serial online] 2021 [cited 2022 Jan 24];59:331-7. Available from: https://www.tnoajosr.com/text.asp?2021/59/4/331/333158




  Introduction Top


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. In 1985, Kary B. Mullis from the USA invented the technique of PCR and received the Nobel Prize in 1993 for this invention. Since the introduction of its modern form in 1988, it has revolutionized much of molecular biology and has greatly accelerated the development of molecular diagnostics.[1] This is a powerful technique, which has important applications in diagnostic pathology, microbiology, and genetics. Remarkable advancements in molecular and immunological technologies have been made in the last decade. The diagnosis of uveitis in clinical practice has been greatly changed by the application of molecular and immunological investigations, particularly PCR. PCR can be performed on any ocular specimen or biopsy [Figure 1]. For diagnosis of uveitis, the obtained sample is usually an anterior chamber (AC) aspirate or vitreous aspirate. Infectious uveitis is particularly important because it causes tissue damage to the eye and may result in blindness unless treated. However, it can be treated if the pathogenic microorganisms are identified promptly and accurately. The initial application of PCR to the ophthalmic diseases was the detection of viral uveitis. PCR has also been implicated in studies of noninfectious uveitis.[2] The sensitivity for detection of foreign DNA is very high. 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 [Figure 2] and [Figure 3].
Figure 1: Sample can be taken from the various parts of the eyeball as shown in the above picture

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Figure 2: Schematic diagram of the steps of polymerase chain reaction

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Figure 3: Utility of polymerase chain reaction in uveitis

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  Methods Top


A search of MEDLINE database (2000–2020) was conducted. The following keywords were used: Polymerase chain reaction, Intra ocular inflammation, Ocular tuberculosis, Toxoplasmosis, Cytomegalo virus retinitis, Eales disease, Endophthalmitis, and Scleritis. Additional sources included publications which have been cited in other articles. Relevant articles were reviewed and included. One of the author's ophthalmic registries was used for photographs and images. Applications of PCR in various ocular inflammatory diseases are described below.


  Polymerase Chain Reaction in Infectious Uveitis Top


Polymerase chain reaction in tubercular uveitis

Infectious uveitis accounts for 50% of the causes of uveitis in developing countries like India. Among them, tuberculosis (TB) is the most common cause of uveitis. Disseminated TB occurs due to the hematogenous spread of pulmonary TB systemically. PCR of various body fluids has been found to be beneficial in the diagnosis of disseminated TB, including intraocular TB. Single-step PCR using primers coding for the IS6110 gene and nested PCR (nPCR) for the detection of the MPB64 gene were performed on the aqueous aspirate in a case of disseminated TB with miliary involvement of the choroid. On PCR, the aqueous aspirate showed the Mycobacterium tuberculosis (MTB) genome, thereby confirming the diagnosis of intraocular TB. The role of MTB in multifocal serpiginous choroiditis (MSC) has been studied by conventional PCR.[3]

Ampiginous choroiditis is a specific choroiditis with multiple small geographic lesions in the choroid of tubercular etiology. nPCR for MTB from the aqueous sample of one patient was found to be positive for MPB64 gene of MTB. Anti-TB treatment was continued, with which the patient responded and his choroiditis regressed.[4]

The role of MTB in MSC has been studied by conventional PCR before. Real time PCR AND Nested PCR can help to clinch the diagnosis of MSC. It can be used to quantify the bacillary load before starting therapy and to document the treatment response.[5]

A study was done with forty patients of choroiditis after dividing them into two groups – Group A (serpiginous-like choroiditis, ampiginous choroiditis, and multifocal choroiditis) and Group B (choroidal abscess, miliary TB, and choroidal tubercle), and they were analyzed retrospectively. In 27 controls (patients without uveitis undergoing phacoemulsification), AC aspirate was done and the sample was subjected to real-time PCR. Patients underwent nPCR for MTB using IS6110 and MPB64 primers from aqueous (n = 39) or vitreous (n = 1). PCR positivity rates were 41.3% (n = 12/29) and 81.82% (n = 9/11) in Groups A and B, respectively. No controls had PCR-positive result. Comparison of PCR positivity rates showed a statistically significant difference between Groups A and B (P = 0.028). This study showed that choroiditis of suspected/presumed tubercular origin can be subjected to PCR for diagnosis of TB and can be treated with ATT for prevention of recurrences.[6]

Shetty et al.[7] have reported a 42-year-old patient with diabetes with a known history of miliary TB on antitubercular therapy for 2 months who presented with complaints of pain and redness followed by diminution of vision in the right eye for 1 month. Visual acuity was counting fingers close to face in the right eye. AC showed Grade 4 cells and flared with a pigmented hypopyon measuring 2 mm. AC tap analysis was not helpful in diagnosis initially. AC wash was done, and the AC sample evaluation revealed acid-fast bacilli. PCR results confirmed it to be MTB. This case showed that pigmented hypopyon can be due to mycobacterial TB infection.

Choroidal granulomas in immunocompetent patients can pose difficulty in diagnosis, as in most cases, systemic examination may not reveal any evidence of TB. There was a case report of bilateral multiple choroidal granulomas with systemic vasculitis-like features in an immunocompetent patient without pulmonary involvement. AC tap was positive for mycobacterial TB by PCR. There was a good systemic and ocular response to antitubercular therapy with resolution of lesions.[8]

Subretinal abscess is a serious vision-threatening disorder which can cause panophthalmitis if not treated promptly and adequately. Clinical profile and PCR of 12 eyes of 12 patients with subretinal abscess were studied. A tuberculin skin test was positive in seven patients (58.3%). PCR for mycobacterial TB genome was positive from aqueous aspirants in 54% and from vitreous in 57% of cases. This study showed the usefulness of PCR in subretinal abscess.[9]

Rao et al.[10] have reported the use of real-time polymerase chain reaction in a case of herpes simplex keratouveitis in a 59-year-old Asian Indian male who presented with circumciliary congestion, corneal edema, mutton fat-pigmented keratic precipitates, and complicated cataract with raised intraocular pressure. Anterior chamber tap was done and real time polymerase chain reaction was positive for herpes simplex virus. He responded well to a course of valcyclovir and topical steroid.

Mahalakshmi et al.[11] have studied the role of real-time polymerase chain reaction (RT-PCR) for HIV RNA in confirming the diagnosis of HIV-induced uveitis and its usefulness in the management and follow-up of these patients. Three patients with an age range of 21–36 years, HIV disease duration of 1 day–10 years, and CD4 counts of 138–412 cells/μL and with intraocular inflammation were included. The CD4 count range was 138–412 cells/μL. In all patients, aqueous testing for RT-PCR for HSV, varicella-zoster virus (VZV), cytomegalovirus (CMV), MTB, and Toxoplasma was negative. None of the patients had any systemic opportunistic infection. Blood RPR/TPHA, ELISA for toxoplasmosis, and tests for cryptococci were negative. Real-time PCR for HIV RNA was positive in three patients with a range of 121–164,773 copies/ml. Chest X-ray was normal. Our study demonstrated that in real-time PCR, there is a corresponding decrease in HIV copies in aqueous and blood with resolution of inflammation, after initiation of Highly active antiretroviral therapy (HAART).

There was a case report of a 47-year-old male patient with diffuse choroiditis and testicular swelling. Aqueous tap for PCR analysis was positive for IS6110 gene of mycobacterial TB, and a biopsy of testicular sac was suggestive of tubercular epididymitis. Hence, identification of MTB in aqueous sample by PCR helped in clinching the diagnosis of tubercular epididymitis.[12] PCR of vitreous sample can also be used to confirm the clinical diagnosis of tubercular intermediate uveitis.[13]

Collaborative Ocular TB Study is a landmark study where they have evaluated the role of PCR in diagnosis and management of tubercular uveitis.[14]

Polymerase chain reaction in parasitic uveitis

Dirofilaria parasite can cause an extensive chorioretinal damage giving a diffuse unilateral subacute neuroretinitis-like picture wherein PCR was used in identification of filarial species.[15] Uveitis develops as a late complication of leptospirosis in 40% of patients and often occurs a year after acute illness. Nucleic acid-based molecular technique can detect the presence of genes that code that Lru A and Lru B proteins in aqueous humor to find out the associations of these genes with uveitis. Our study showed that PCR for Leptospira can be done for uveitis cases in Leptospira endemic areas.[16]

Garweg J et al.[13] 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.[17] 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.

Polymerase chain reaction in viral uveitis

It is found that the reliability of the PCR method is similar to in situ DNA hybridization for the detection of CMV, although morphologic correlation is provided only by in situ DNA hybirdization.[18] Mitchell et al. developed PCR primers with a sensitivity of 93% and specificity of 98% for the detection of Cytomegalovirus (CMV).[19]

Acute retinal necrosis following herpes simplex encephalitis in an immunocompetent patient is a rare condition. Quantitative real-time polymerase chain reaction has made it possible to identify and quantify viral genome.[20] Real-time PCR can confirm the clinical diagnosis of viral retinitis and progressive outer retinal necrosis [Figure 4] and [Figure 5].
Figure 4: Fundus photo of the left eye of a patient with progressive outer retinal necrosis

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Figure 5: Agarose gel photograph showing the amplified products of varicella-zoster virus of the above patient

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Chikungunya is a mosquito-borne virus that has been reported to cause retinitis. RT-PCR can be used for diagnosis for Chikungunya virus.[21]

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. RT-PCR has an important role in these conditions.

PCR can be performed for the detection of the genome of MTB, herpes virus family, Chikungunya virus, T. gondii, fungus, Eubacterium, and Propionibacterium acnes. The sensitivity, specificity, positive predictive value, and negative predictive value of PCR analysis were 90.2%, 93.9%, 93.9%, and 90.2%, respectively. The quantitative value of real-time MTB-positive PCR ranged from 32 c/ml to 2722 c/ml. PCR assay is an accurate technique with high sensitivity and specificity to diagnose the DNA genome in infectious uveitis.[22]

Polymerase chain reaction in retinal vasculitis

Madhavan et al.[16] studied the PCR to tissue sections obtained from formalin-fixed and paraffin-embedded tissues of epiretinal membrane from 23 patients of Eales' disease. Eleven out of 23 (47.8%) were positive for MTB genome, indicating association of this bacterium with Eales' disease. Gupta A et al.[21] reported tubercular retinal vasculitis with varied fundus findings, and diagnosis was confirmed by doing PCR from the aqueous or vitreous humor.

Polymerase chain reaction in noninfectious uveitis

PCR has also been implicated in studies of noninfectious uveitis. The most common application is HLA typing. Saiki et al.[22] used PCR to enzymatically amplify a specific segment of beta-globin or HLA-DQ alpha gene in human genomic DNA. PCR-restriction fragment length polymorphism (PCR-RFLP) methodology is applied to HLA-DR, HLA-DQ, and HLA-DW typing at the nucleotide level, eliminating the need for radioisotopes as well as allele-specific oligonucleotide probes.[23] Using this technique, Shindo et al.[20] reported complete association of the HLA-DRB104 and HLA-DQB104 alleles with Vogt–Koyanagi–Harada. It is more common for Asians. PCR-sequencing-based typing 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).[25],[26] The results from RT-PCR are complementary to data from Western blotting and/or immunohistochemistry.

Polymerase chain reaction for Masquerade syndrome

The Masquerade syndrome consists of a group of disorders that mimics intraocular 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]

Polymerase chain reaction for endophthalmitis

Although direct microscopy is the easiest and most rapid method to detect bacteria in 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] More sensitive than microscopy, culture is considered “the gold standard.” However, there have been no significant improvements in the yield of culture methods.[29] Postoperative endophthalmitis is a vision-threatening complication of intraocular 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%.[30] Culture results takes relatively longer time which makes the patients to be treated with broad-spectrum antibiotics for several days. PCR can solve the problem in the above situations and save the patient from taking long-term broad-spectrum antibiotics. In cases where conventional techniques have low sensitivity, PCR is a rapid technique which has high sensitivity and specificity and 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 h. Therese et al.[27] demonstrated the utility of this approach for culture-negative endophthalmitis. 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.[28] 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 other available diagnostic techniques for diagnosis of endophthalmitis. Aspergillus fumigatus was found by PCR-based RFLP technique from paraffin section of an eyeball of an 8-month-old child which was removed for endogenous endophthalmitis.[12] 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. Semi-nPCR is also helpful for rapid detection of panfungal genome directly from ocular specimens.[29]

PCR-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. A universal bacterial PCR can be very helpful for the diagnosis of endogenous bacterial endophthalmitis at an early stage of the disease.[30]

Polymerase chain reaction for scleritis

Scleritis is commonly noninfectious. Infectious scleritis can be a diagnostic challenge. Histopathological examination of an enucleated eye revealed tuberculous bacilli in retinal pigment epithelial cells in a scleritis patient. PCR showed that MTB DNA confirming nodular scleritis could be due to TB infection, which correlated with histopathology study infection.[24]

There is a case report showing the usefulness of PCR in the case of Nocardia scleritis. Culture report in a 32-year-old male following an accidental rice powder injury revealed the growth of Gram-positive branching filamentous bacilli suggestive of Nocardia sp. Scleral biopsy on histopathological examination showed granulomatous inflammation. PCR-based DNA sequencing identified the bacterium as Nocardia cyriacigeorgica. The patient responded to topical fortified amikacin (2.5%), fortified cefuroxime, oral sulfamethoxazole, and trimethoprim with complete healing of scleritis[12] [Figure 6], [Figure 7], [Figure 8].
Figure 6: Slit-lamp photo of tubercular nodular scleritis

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Figure 7: Agarose gel electrophoretogram showing II round amplified products of nested polymerase chain reaction targeting MPB64 and IS6110 gene of Mycobacterium tuberculosis complex genome

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Figure 8: Real-time quantitative polymerase chain reaction result for Mycobacterium tuberculosis complex genome showing 33 copies/ml of DNA

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Polymerase chain reaction for adnexal infection

Ocular adnexal TB is quite rare. Histopathology revealed a granulomatous inflammation with caseation necrosis in a patient of orbital TB. PCR showed amplification of the MTB genome. The patient responded to a course of antitubercular treatment.[21]


  Conclusion Top


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 various parts of the eye and adnexa.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Shetty SB, Biswas J,Murali S. Real-time and nested polymerase chain reaction inthe diagnosis of multifocal serpiginoid choroiditis caused by Mycobacterium tuberculosis - a case report. J Ophthalmic Inflamm Infect. 2014;4:29.  Back to cited text no. 5
    
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Rishi E, Rishi P, Therese KL, Ramasubban G, Biswas J, Sharma T, et al. Culture and reverse transcriptase polymerase chain reaction (RT-PCR) proven Mycobacterium tuberculosis endophthalmitis: A case series. Ocul Immunol Inflamm 2018;26:220 7.  Back to cited text no. 14
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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