**3.4 Optical coherence tomography**

Anterior eye segment imaging with 830 nm optical coherence tomography (AS OCT) was first demonstrated and published in 1994. Changing the light wavelength from 830 nm to 1310 nm allowed the direct transcleral anterior eye segment structures including trabecular-iris angle visualization in 2000. OCT provides in vivo anterior eye segment imaging with the axial resolution from 18 μm with time domain OCT (TD OCT) to 5 μm with spectral domain OCT (SD OCT) and to 5 μm with ultra high resolution spectral domain OCT. OCT is proven to provide reliable anterior eye segment morphology and morphometry results with high reproducibility and repeatability. Application of OCT in herpetic keratitis patients include: assisting in diagnosis of patients at active stage and assessing the scars in patients qualified for laser or surgical interventions. Active keratitis could be characterized

*Recent Advances in the Diagnosis and Management of Herpetic Keratitis DOI: http://dx.doi.org/10.5772/intechopen.96898*

#### **Figure 6.**

*Representative images of the confocal microscopy scans revealing significant features characteristic for HSV keratitis. (A) Epithelial, healed dendritic ulcer with noticeable fibrotic borders (arrows). (B) Multiple infiltration of small dendritic structures as the level of the epithelium. Clusters of inflammation cells (arrows). (C) Multiple infiltration of pronounced dendritic cells forming a lattice pattern (arrows) at the level of the basal epithelial cells. (D) Marked fibrosis at the level of the Bowman layer (arrow) with inflammation cells infiltration (stars). (E) Excessive fibrosis and inflammation cells infiltration forming clusters at the level of the Bowman layer (arrows). Dendritic structures visible (star). (F) Anterior stromal keratocytes activation with accompanying haze (arrow). (G) Stromal infiltration and haze accompanied by multiple crystalline structures due to the lipid degeneration (stars). (H) Multiple endothelial opacities. Examples marked with stars.*

by the presence of the ulceration, stromal edema and inflammatory hyperreflective infiltrates. Corneas with inactive keratitis are characterized by stromal scarring and thinning, and epithelial remodeling [17–22]. Characteristic OCT features are presented in the **Figure 7**.

#### **3.5 Laboratory testing**

There are several laboratory techniques, which may help in the diagnostic process. Clinical samples for the analysis may be obtained through collection of tears, corneal epithelial cells, and conjunctival cells. Tear samples are usually obtained using Schirmer test. Epithelial or conjunctival cells may be collected through corneal scrapings, corneal impression membranes (CIM) or using conjunctival or corneal swab. The less invasive the technique the lesser probability of obtaining a clinically detectable material.

The isolation of the HSV from the cornea and performing a viral culture remains a conventional, gold standard technique, however the main disadvantages of this methods are low sensitivity and a time consuming process. Giemsa staining of the epithelial corneal cells may visualize multinucleated giant cells, resulting from coalescence of HSV infected epithelial cells and intranuclear HSV inclusions. Immunofluorescence assay (IFA) is one of the modern techniques developed to diagnose HSV keratitis. The principle of the method is to introduce antibodies, that bind to HSV antigens specifically to gain fluorescence based immunological detection of HSV-1 antigen through color visualization under microscopy. Disadvantages of the method include: required subjective interpretation by an experienced technician and the risk of obtaining false positives results due to cross-reactivity between other microorganisms.

#### **Figure 7.**

*Representative images of the anterior segment swept source optical coherence scans revealing significant features characteristic for HSV corneal scars. (A) Slit lamp photograph of the central post herpetic keratitis scar. (B) High resolution scan. Hyperreflective tissue within corneal stroma with irregular borders (arrows). (C) Pachymetry map. Marked paracentral corneal thinning to 398* μ*m. (D) Slit lamp photograph of the central post herpetic keratitis scar. (E) High resolution scan. Hyperreflective tissue within corneal stroma with irregular borders. Note the relatively smooth corneal surface and epithelial compensation over the irregular corneal stroma (arrows). (F) Pachymetry map. Marked irregular, paracentral corneal thinning to 382* μ*m.*

Advanced diagnostic techniques include: Polymerase Chain Reaction (PCR) conventional PCR, reverse transcriptase PCR (RT-PCR), real-time PCR (qPCR) and multiplex PCR. qPCR overcomes the disadvantages of conventional PCR by acquiring more rapid and sensitive results. Guda SJM. et al. assessed sensitivity and specificity of the conventional and real-time PCR compared to IFA performed on corneal scrapings. The sensitivity and specificity of conventional PCR was 100% and 76.9% and 100% and 28.2% of qPCR respectively. Satpathy et al. assessed and concluded, that specificity and positive predictive value (PPV) of PCR was higher in tear (90.6% and 37.5%). compared to cornea scrapings (71.3% and 30.3%). Moreover, Akbarian A. et al. reported, that conventional PCR with added internal amplification control (IAC) had higher sensitivity (100%) vs. culture method (66.66%), while the specificity was 100% for both diagnostic methods.

Also novel methods, such as multiplex dot hybridization (MDH) assay, immunochromatographic assay (ICGA, AmpliVue) or Infected cell protein 0 (ICP0) detection in tears are either tested or incorporated into a clinical practice. AmpliVue is a commercially available immunochromatographic assay, office-based diagnostic test characterized by a 64.7% positive detection rate. Sensitivity and specificity of AmpliVue was assessed as 84% and 100% respectively, based on true positives from culture and PCR combined. The MDH assay is a rapid technique, that involves a series of oligonucleotide probes specific for HSV genes. Compared to the real-time PCR, the MDH assay is characterized by very high values of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 93.3%, 100%, 100% and 98.4%, respectively. The infected cell protein 0 (ICP0) is an acute phase protein during HSV infection and plays a significant role in the virus gene expression activation. ICP0 could be potentially defected in tears of affected subjects [23–28].
