A 12-tube polymerase chain reaction strip can detect 24 ocular pathogens.
Prompt, accurate diagnosis of the pathogenic microorganisms that cause infectious uveitis is crucial: the infection may damage eye tissue and result in blindness unless clinicians begin appropriate treatment quickly. Diagnostic testing based on the polymerase chain reaction (PCR) technique has revolutionized the diagnosis and treatment of infectious uveitis because these tests are highly sensitive, specific, and quick. Moreover, because PCR-based tests amplify a specific region of DNA by many orders of magnitude, they require only a small sample of intraocular fluid.
These observations were made in a recent review by Manabu Mochizuki, MD, and a team at the Tokyo Medical and Dental University in Tokyo, Japan. The review also covered new developments in the application of PCR technology to uveitis.
In their review, the team noted that clinicians often treat infectious uveitis with antibiotics before collecting samples of ocular fluid for testing. Under these circumstances PCR testing, which may still be able to detect the DNA of dead microorganisms, is often more effective than cultures and smears in detecting the presence of bacteria in ocular fluid.
According to the review, multiplex PCR can measure many different target genomic DNAs simultaneously, so it enables the comprehensive measurement of the genomes of many different pathogens. However, positive results from qualitative techniques such as multiplex PCR can be falsely positive because of contamination. Therefore, quantifying the copy number of genomic DNA by real-time PCR is essential in determining whether the pathogens detected by multiplex PCR are causing uveitis.
The research team developed a comprehensive PCR system for detecting and measuring all eight human herpes viruses, parvovirus, the BK and JC viruses, and the parasite toxoplasma in intraocular fluid. This system is especially useful for evaluating uveitis in immunodeficient patients.
In this system, positive results generated by multiplex PCR are quantified with real-time PCR to measure the copy number of the genome in the sample and confirm the pathogenic role of the organism associated with this genome. A high copy number indicates active viral replication, and a negative result can exclude many common infectious agents and aid clinicians in deciding whether to use corticosteroids and immunosuppressants.
The Japanese team noted that broad-range PCR techniques can provide evidence of the presence of bacterial or fungal infection in the eye but cannot identify the species and strains of microorganisms causing the infection. However, they have developed real-time PCR primers and probes that can quantify copy numbers of bacteria or fungi by using the LightCycler 480 II System (Roche). With these primers and probes, this system can detect the genomic DNA of 16 strains of bacteria at levels as low as 1—10 colony-forming units in diluted vitreous samples.
According to the team, sampling vitreous fluid instead of aqueous humor is important for avoiding false-negative results in PCR testing. False-positive results can be avoided by setting an appropriate cut-off value of >100 copies/mL as a significant indicator of infection. When copy numbers are low, follow-up testing with cultures or smears may help to avoid misdiagnosis.
The researchers also developed a 12-tube multiplex PCR strip that can detect 24 common ocular infectious disease pathogens in one assay that takes just a few hours. The strip awaits a prospective, multicenter clinical trial to evaluate its usefulness.
The study also notes that PCR testing can detect rubella virus, which has been implicated in Fuchs heterochromic iridocyclitis, and can identify other uveitis-causing pathogens, including human T-cell leukemia virus type 1, dengue virus, West Nile virus, and Zika virus.
The review, “A new era of uveitis: impact of polymerase chain reaction in intraocular inflammatory diseases,” was published in late October of 2016 in the Japanese Journal of Ophthalmology.