Rapid Detection and Identification of Uveitis Pathogens by Qualitative Multiplex Real-Time PCR

Abstract

PurposeInfectious uveitis is a serious sight-threatening infection commonly caused by herpesviruses and Toxoplasma gondii. Etiologic diagnosis based on the clinical evaluation is often challenging. We developed and validated a multiplex real-time PCR assay coupled with high-resolution melting (HRM) for rapid detection and identification of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), and T. gondii.MethodsThe assay was designed to target pathogen genome regions that yield products with distinct melting temperatures. Analytical specificity, sensitivity, and precision of HRM identification were determined. Clinical validation was performed by testing 108 intraocular fluids collected from eyes suffering with infectious uveitis (n = 30) and controls (n = 78).ResultsA nonoverlapping high-precision profile for each pathogen was generated following HRM (coefficient of variation 0%). The assay was highly sensitive, with a limit of detection of 20 genome copies for herpesviruses and 200 genome copies for T. gondii. The intra- and interassay variability of cycle threshold (Ct) measurement was ≤4% and ≤6%, respectively. Thirteen intraocular specimens collected from suspected cases of infectious uveitis were positive (mean Ct values varied from 19.4 to 27.7). Melting profiles of positive cases were consistent with HSV-2 (n = 5), VZV (n = 5), CMV (n = 2), and T. gondii (n = 1). Amplicon identities were confirmed by sequencing. Control intraocular samples from patients without a clinical diagnosis of infectious uveitis were all negative.ConclusionsThis assay allows rapid, sensitive, and reliable detection and identification of the most common known causes of infectious uveitis, making early pathogen information-based intervention possible.

Evidence-based Management of Culture-negative Microbial Keratitis

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Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis

“…Culture-independent molecular techniques such as polymerase chain reaction (PCR) has been utilised in the diagnosis of ocular infections (Kim et al, 2008), however, this requires a priori knowledge of the likely pathogenic micro-organism to determine which specific primer sets to use and is limited by the number of species that can be detected simultaneously in a single PCR assay (Bispo et al, 2018;Ung et al, 2020). Newer, high-throughput sequencing approaches can be broadly classified into two major options: targeted amplicon sequencing (selective amplification of the specific genetic region of interest such as 16S rRNA in bacteria or 18S rRNA in fungi) and metagenomic sequencing (untargeted amplification of all genomic DNA) (Ung et al, 2020).…”

Multiplex PCR for Detection of Herpes Simplex Viruses Type-1 and Type-2, Cytomegalovirus, Varicella-zoster Virus, and Adenovirus in Ocular Viral Infections

“…They have targeted viruses HSV-1, HSV-2, VZV, CMV and parasite T. gondii and have found the multiplex real-time PCR to be highly specific with a limit of detection of 20 genome copies for viral pathogens and 200 genome copies for T. gondii. [16] There are a few limitations of the study. We have not found the detection limit in terms of genome copy number of uniplex and multiplex PCR for each virus, which would be a better marker of sensitivity of the test.…”