Newer technological progressions in molecular science have paved the way for a better understanding of infectious diseases. DNA-based molecular diagnostic techniques which are specific, sensitive, and rapid in the identification of the pathogen in the clinical specimen has been developed extensively over the past decades The rapid identiﬁcation of speciﬁc infectious agents is very important for the diagnosis of an ocular infection because the course of treatment and disease prognosis could be different from non-infectious diseases. Despite the availability of a wide variety of antimicrobial agents to treat these infections, specific treatment is delayed due to the inability to identify ocular pathogens quickly and accurately. PCR has emerged as an essential powerful rapid laboratory diagnostic technique complement the conventional gold standard “culture “, with special reference to difficult to grow or slow-growing microbial agents like viruses, Mycobacterium tuberculosis, anaerobic bacteria, or non-cultivable microbial agents. Rapid and accurate diagnosis of infectious agents of the eye is particularly difficult and problematic. The reasons are:
1. Because of intrinsic biological properties of the organisms themselves (e.g., latency)
2. Ocular infectious agents are frequently present in only small numbers making identification on smears, touch preparations, or tissue biopsies unreliable
3. Often the clinical sample size is inherently small (e.g., anterior chamber paracentesis) reducing identification potential
4. Many ocular agents are extremely fastidious or slow-growing in culture further delaying diagnosis
5. In many infections, the generation of an immune response lags behind the infectious process or is localized, making standard serologic surveillance (biopsy, aspiration, etc.) complicates the diagnostic procedures.
The ‘molecular footprints’ of many infectious agents can now be identified, thus obviating the need for rigorous and time-consuming serological microbiological techniques.
Dos and Don’ts in PCR every ophthalmologist should know
- Any ocular specimen or biopsy usually conjunctival swab, anterior chamber aspiration, or vitreous tap can be subjected to PCR testing. Specimens should be aseptically transferred to a sterile, capped tube and placed on ice or quick-frozen on dry ice if the sample is must transported but it is very important that the samples must not be subjected to freeze-thaw cycles because that will release nucleases that will degrade all RNA and some DNA. Considering the high sensitivity of the PCR, minimum of 10 microlitres of nucleic acid extracted from the sample will be the required volume for running a PCR reaction. Therefore a minimal volume of sample will lead to less concentration of the genomic material available for detection of pathogen
- The method of sample collection should be guided by disease suspicion. A part of the aqueous aspirates should be obtained in cases of bacterial uveitis and endophthalmitis. A vitreous biopsy is taken in cases of suspected intraocular infection, intraocular lymphoma, atypical intraocular inflammation that is not responding to conventional therapy, and other conditions that necessitate a larger sample. A retinal biopsy is used to determine the cause of atypical or unexplained retinitis (1).
- Specimens must be collected with care to ensure that they are free of contamination. For example, when diagnosing viral conjunctivitis, the conjunctival swab should not be contaminated with tears because patients may shed herpes simplex virus in tears (2), resulting in a false positive result.
- The high sensitivity PCR can increase the risk of false positive results. Single genome of pathogens from laboratory contamination or commensal DNA amplification in the sample may potentially be amplified, resulting in false positives. To detect PCR contamination, controls should be used in PCR runs. Using nested PCR procedures for routine detection of viruses is more susceptible to amplicon contamination and should be avoided if possible.
- Since PCR can detect the DNA of previous infections or dead microbes, the results must be interpreted in the context of the clinical situation. Most Americans, for example, have been exposed to EBV, and the virus remains dormant in the host (3). PCR reaction to detect EBV manifested with ocular samples containing white blood cells of the patient will definitely yield a positive result.
- It is best to perform PCR prior to antibiotic or antiprotozoal drug administration or at some time point following treatment to confirm active infection. In certain cases, taking glucocorticoids increases the number of infectious species in the blood. As a result, corticosteroid administration, particularly at immunosuppressive doses, can improve PCR detection of a pathogen.
- Point mutations lead to sequence polymorphism between the strains which enables poor priming and result in false negative results. Clinicians should be aware that prolonged antiviral therapy for example in case of CMV in severely immunocompromised patients can result in drug-resistant strains (4). Novel in-house PCR assays must be developed for the detection of pathogens example like MTB which is prone to frequent genetic mutation rather than using ready-made available PCR diagnostic kits that are validated on different geographical populations.
- Differential diagnosis method of analysis is important after identifying true clinical conditions that might produce a patient's symptoms and signs in order to confirm the final diagnosis.
- It's worth noting that reactivation disease can be diagnosed using a blood sample. In the case of ocular toxoplasmosis, for example, PCR testing of blood samples from patients with ocular toxoplasmosis produced the same results as PCR testing of aqueous humor samples (5)
- Majumder, P. D., Sudharshan, S., & Biswas, J. (2013). Laboratory support in the diagnosis of uveitis. Indian journal of ophthalmology, 61(6), 269–276. https://doi.org/10.4103/0301-4738.114095
- Kaufman, H. E., Azcuy, A. M., Varnell, E. D., Sloop, G. D., Thompson, H. W., & Hill, J. M. (2005). HSV-1 DNA in tears and saliva of normal adults. Investigative ophthalmology & visual science, 46(1), 241–247.
- Dowd, J. B., Palermo, T., Brite, J., McDade, T. W., & Aiello, A. (2013). Seroprevalence of Epstein-Barr virus infection in U.S. children ages 6-19, 2003-2010. PloS one, 8(5), e64921
- Carmichael A. (2012). Cytomegalovirus and the eye. Eye (London, England), 26(2), 237–240.
- Bou, G., Figueroa, M. S., Martí-Belda, P., Navas, E., & Guerrero, A. (1999). Value of PCR for detection of Toxoplasma gondii in aqueous humor and blood samples from immunocompetent patients with ocular toxoplasmosis. Journal of clinical microbiology, 37(11), 3465–3468.