We investigated the utility of the tear test for detecting HSV and VZV DNA in patients with herpetic ocular surface diseases as a clinical examination tool. In addition, in this study, herpetic keratitis including HSK and HZO was diagnosed using a microfluidic real-time PCR system. The current study demonstrated the following clinical advantages: 1) the time required for the detection of HSV and VZV DNA in tears with a microfluidic real-time PCR system was approximately 40 minutes; 2) the sensitivity and specificity of quantitative PCR test for HSV and VZV DNA in tears of the patients with infectious epithelial keratitis type of HSK and VZO were 100% each; 3) no cross-reaction between HSV and VZV DNA was observed.
Quantitative PCR for HSV DNA in tears is reportedly a useful diagnostic tool for HSK. Our previous multicenter study reported that the diagnosis of HSK using real-time PCR for HSV DNA had a sensitivity and specificity of 55.8% and 100%, respectively [5]. In particular, one of the factors that decreased the sensitivity of tear HSV DNA test in patients with HSK is the influence of the pre-test use of antiviral drugs such as acyclovir ophthalmic ointment, which reduces the number of HSV DNA copies. However, this study demonstrated no difference in sensitivity between the patients with acyclovir treatment and those without. Another factor that affects the sensitivity of the HSV DNA test is that the HSV DNA levels in tears vary depending on the clinical form and severity of HSK. The HSV DNA levels in tears are high in infectious epithelial keratitis including dendritic, geographic, and necrotizing keratitis, and low in disciform keratitis and endotheliitis [5, 10]. Therefore, it is important to set a suitable lower detection limit for the HSV DNA test to improve the sensitivity for the diagnosis of HSK. In the current study, the lower limit of detection of HSV DNA test was set at 7.6×101 copies/µL; furthermore, the sensitivity and specificity of HSV DNA test were 100% each in patients with and without acyclovir treatment. These results may indicate the usefulness of this HSV DNA test as a clinical diagnostic tool for infectious epithelial HSK. In addition, a high copy number of HSV DNA in tears was detected in an HSK patient with refractory keratitis that resisted topical acyclovir treatment. We considered this a severe case in which the viral load was not sufficiently reduced by topical acyclovir treatment. Therefore, systemic administration of antiviral drugs should be considered for such refractive cases. Accordingly, a clinical test that can rapidly measure the number of HSV DNA copies in the tears of the patients being treated with antiviral drugs is a useful tool for determining the clinical treatment strategy.
In the patients with HZO, corneal findings including pseudodendritic keratitis, corneal subepithelial opacity, disciform keratitis, and interstitial keratitis were observed. These corneal lesions sometimes recur, resist treatment, and become refractory. For these patients with VZV keratitis, the copy number of VZV DNA in tears has been reported to be useful not only in diagnosis but also in determining the severity of the keratitis [11]. In the current study, the copy number of VZV DNA in tears of patients with acute HZO as well as the diagnostic rate (sensitivity) was high. In addition, acute phase HZO was positive for VZV DNA in the tears of both eyes, although the VZV DNA levels in the affected eyes were significantly higher than the unaffected ones. These results suggest that in the treatment of HZO, the need for topical ocular administration of antiviral drugs, in addition to systemic administration of antiviral drugs, should be considered, taking into account the viral load in tears. On the other hand, in our reported patients who were followed up by monitoring copy number of VZV DNA in tears, re-elevation of copy number of VZV DNA in tears was observed. This re-elevation of VZV in tears of patients with HZO has not been studied previously. Therefore, in patients with refractory HZO, VZV DNA levels in tears should be monitored and the results of tear testing should be incorporated into therapeutic drug selection, especially in the indication of steroids.
This study has some limitations. First, the current study did not evaluate the usefulness of HSV DNA test in tears in patients with herpetic stromal keratitis. Herpetic stromal keratitis is divided into disciform and necrotizing keratitis. The copy number of HSV DNA is high in patients with necrotizing keratitis and almost under the detection limit in disciform keratitis in HSV DNA tests in tears [5, 10]. Therefore, the copy number of HSV DNA in the tears of patients with herpetic stromal keratitis may help select an appropriate medical treatment. In other words, steroid eye drops, which are the main drugs used in the treatment of herpetic stromal keratitis, should be administered with caution to patients with high copy numbers of HSV DNA in tears. If we examine a patient with herpetic stromal keratitis, a rapid detectable test for HSV DNA in tears may be a crucial clinical test tool in routine clinical practice. The usefulness of this HSV DNA test in patients with herpetic stromal keratitis should be evaluated in large-scale studies in the future. Second, the usefulness of the HSV and VZV DNA test for HSK is unclear because the present patient group did not include any cases of corneal endotheliitis. HSV and VZV DNA testing of the aqueous humor is useful in diagnosing herpetic corneal endotheliitis; however, the usefulness of the tear test should be accurately verified. In addition, examining the usefulness of this test system for detecting HSV DNA and VZV DNA in aqueous humor may provide more certainty in the differential diagnosis of corneal endotheliitis and infectious uveitis.