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

Abstract

Purpose: Infectious 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.

Methods: The 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).

Results: A 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.

Conclusions: This 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.

Figure 1

Melting curve profiles for each target included in the assay following HRM analysis using SYBR GreenER. T_m values corresponding to each peak are 79.1°C for T. gondii, 81.7°C for VZV, 85.7°C for CMV, 90.3°C for HSV-1, and 90.8°C for HSV-2.

Figure 2

Standard curves obtained by testing serial 10-log dilutions ranging from 2 × 10⁵ copies to 2 copies per reaction. Calculations of efficiency (E) for each reaction are shown. Efficiency was calculated from the slope given by regression analysis using the formula E = 10(−1/−slope) − 1. Coefficients of correlation (R²) are also shown.