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. 2023 Apr 25;23(1):177.
doi: 10.1186/s12886-023-02938-w.

Rapid detection and diagnosis of herpetic keratitis using quantitative microfluidic polymerase chain reaction system for herpes simplex and varicella-zoster virus DNA: a case series

Akira Hirota  1 , Jun Shoji  1 , Noriko Inada  1 , Rumi Adachi  1 , Yukiko Tonozuka  1 , Satoru Yamagami  2
Affiliations

Rapid detection and diagnosis of herpetic keratitis using quantitative microfluidic polymerase chain reaction system for herpes simplex and varicella-zoster virus DNA: a case series

Akira Hirota et al. BMC Ophthalmol. .

Abstract

Background: A microfluidic real-time polymerase chain reaction (PCR) system can rapidly detect the viral DNA in specimens. Detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA in tears is a useful diagnostic tool for herpes simplex virus keratitis (HSK) and herpes zoster ophthalmicus (HZO).

Methods: In total, 20 patients were included in this cross-sectional study. Among them, 8 patients with infectious epithelial HSK and 12 patients with HZO were included in HSK and HZO groups, respectively. In addition, 8 patients with non-herpetic keratitis and 4 healthy individuals without keratitis were included in the control group. Numbers of HSV and VZV DNA copies in tears of all patients and individuals were evaluated using a microfluidic real-time PCR system. Regarding HSV/VZV DNA test, tear specimens were collected by filter paper method using Schirmer's test paper, and subsequently, DNA was extracted from the filter paper using an automated nucleic acid extractor. Afterward, quantitative PCR was performed using a microfluidic real-time PCR system.

Results: From tear collection to real-time PCR result determination, the HSV/VZV DNA test took approximately 40 min. In the HSK group, the sensitivity and specificity of the HSV DNA tests were 100% each. The median value (range) of number of HSV DNA copies for affected eyes was 3.4 × 105 copies/μL (under a lower detection limit of 7.6). In the HZO group, the sensitivity and specificity of the VZV DNA tests were 100% each. The median value (range) of number of VZV DNA copies for affected eyes was 5.3 × 105 copies/μL (under a lower detection limit of 5.6 × 10-2).

Conclusion: In conclusion, quantitative PCR for HSV and VZV DNA in tears using a microfluidic real-time PCR system is useful for diagnosing and monitoring HSK and HZO.

Keywords: Conjunctivitis; Herpes simplex virus; Keratitis; Quantitative polymerase chain reaction; Varicella-zoster virus.

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Conflict of interest statement

J.S. has previously received honoraria from Santen Pharmaceutical Co., Ltd., Senju Pharmaceutical Co., Ltd., AbbVie GK, and Rohto Nitten Co., Ltd., outside the submitted work. H.A., Y.S., N.I., Y.T., and R.A. declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Calibration curve in quantitative PCR for HSV and VZV DNA in tears. We developed a calibration curve to determine the amount of HSV and VZV DNA in tears. a. The measurement ranges for HSV DNA tests in tears were 7.6 × 101–7.6 × 105 copies/μL. b. The measurement ranges for VZV DNA tests in tears were 5.6 × 10–3–5.6 × 105 copies/μL. PCR, polymerase chain reaction; HSV, herpes simplex virus; VZV, varicella-zoster virus
Fig. 2
Fig. 2
Comparison of HSV and VZV DNA levels in tears between affected and unaffected eyes. a. HSV DNA levels in tears of affected eyes are significantly higher than those of unaffected eyes. HSV DNA levels in all tear samples in unaffected eyes are under the detection limit. b. VZV DNA levels in tears of affected eyes are significantly higher than those of unaffected eyes. **, p < 0.01, Mann–Whitney U-test; HSV, herpes simplex virus; VZV, varicella-zoster virus; ND, not detected
Fig. 3
Fig. 3
Clinical course and features of the patient with HSV keratitis. Slit-lamp examination photographs of a patient with HSV keratitis at Days 0, 42, and 49. a, and b: Photographs at Day 0 show corneal leucoma with fluorescein staining positive superficial punctate keratopathy. c, and d: Photographs at Day 42 show geographic keratitis with a dendritic tail. e, and f: Photographs at Day 49 show improvement in geographic keratitis. The area of epithelial defect with fluorescein staining gradually decreased. HSV, herpes simplex virus
Fig. 4
Fig. 4
Clinical course and features of the patient with VZV keratitis. Slit-lamp examination photographs of the patients with varicella-zoster ophthalmicus. a, and b: Clinical findings of case 1 on Day 1 show multiple pseudodendritic keratitis with positive fluorescein staining and scleritis. c, and d: Clinical findings of case 1 on Day 22 show that varicella-zoster ophthalmicus improved. e, and f: Clinical findings of case 2 on Day 35 show improved keratitis and continued scleritis. VZV, varicella-zoster virus
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