Case 1. One month after a healthy 60-year-old male carpenter had an accident in which a foreign body entered his left eye while using a grinder, the patient noticed blurred vision of his left eye and visited a nearby hospital. The initial doctor who examined the patient suspected bacterial keratitis and prescribed levofloxacin and cefmenoxime eye drops. Due to the lack of improvement after 1 week in conjunction with negative bacterial culture results, he was referred to our department. Best corrected visual acuity (BCVA) at his first visit was 1.2 (right) and 0.04 (left). Intraocular pressures (IOPs) were within normal limits in both eyes. Slit-lamp examination of the left eye showed ciliary injection, cells in the anterior chamber and feathery infiltration in the upper nasal peripheral cornea with fibrin formation on the corneal endothelium (Fig. 1A). Small keratic precipitates (KPs) were seen in the lower cornea. The findings for the right eye and the fundus were normal. As we suspected fungal or bacterial keratitis, we performed corneal scraping. However, direct microscopy did not find any pathogens, including bacteria or fungi. Although we continued topical antibiotics, the feathery lesion expanded to the central cornea and exhibited a ring-shaped lesion with slight hypopyon after 2 weeks (Fig. 1B). Results for the Cochet-Bonnet esthesiometer were 60 and 50 mm in the right and left cornea, respectively. Even though no growth was observed in a bacterial and fungal culture, we clinically suspected a fungal infection and thus, we added hourly topical voriconazole 1% and miconazole 0.1%. Subsequently, we then performed corneal scraping again for multiplex real-time PCR5. At 3 weeks after his first visit, PCR detected 6.0 x 106 copies/μg glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of the herpes simplex virus type 1 (HSV-1) DNA per microgram of sample. As a result, we switched the patient from antifungals to topical corticosteroids and oral valacyclovir. Although there was gradual scarring due to the keratitis, the left BCVA improved to 1.2 at 2 months after his first visit (Fig. 1C).
Case 2. After a 52-year-old healthy man with herpes keratitis was treated with topical steroid at a nearby eye clinic, he was referred to our department due to the development of feathery infiltration in his left cornea. BCVA at his first visit was 1.2 (right) and hand motion (left), respectively. IOP of the right eye was 20 mmHg, while the left eye was elevated to 45 mmHg. Slit-lamp examination of the left eye showed ciliary injection, and full-thickness feathery corneal infiltration with round epithelial defect (Fig. 1D). In addition, anterior segment optical coherence tomography (ASOCT) demonstrated the presence of a retrocorneal plaque (Fig. 1E). After performing corneal scraping, direct microscopy did not find the presence of any pathogen. The residual sample was then evaluated by culture and multiplex real-time PCR5. Since we clinically suspected fungal keratitis, the patient was started on topical voriconazole 1% hourly, levofloxacin 1.5% 6 times per day, atropine 1% once per day and oral acetazolamide. After 1 week, there was no improvement in the corneal findings, and a hypopyon developed. As PCR detected 1.0 x 105 copies/μg GAPDH of HSV-1 DNA, we added topical acyclovir and oral valacyclovir. At 2 weeks after his first visit, there was no growth observed in his fungal culture. Based on these results, his antifungal was stopped, and topical betamethasone 0.1% twice a day was added. Small KPs were observed around the lesion during the resolution of the corneal edema. After 2 months, there was scarring of the lesion, with the BCVA improving to 0.2 (Fig. 1F). There has been no recurrence observed for 1 year.
Case 3. A 76-year-old healthy woman was being treated with eye drops that included topical fluorometholone 0.1% for dry eye syndrome. After developing pain in her right eye, she visited a hospital. The examining doctor treated her for 2 weeks with topical antibiotics and natamycin 1% ointment and oral itraconazole. However, as there was no improvement in her right eye, she was referred to our department. At her initial visit, BCVAs were hand motion (right) and 1.2 (left), respectively. IOPs were within normal range in both eyes. Slit-lamp examination showed deep stromal feathery infiltration in the right cornea with retrocorneal plaque, hypopyon and iris rubeosis (Fig. 1GH). Corneal scraping and a subsequent direct microscopy evaluation did not show either bacteria or fungi. The residual samples were then evaluated by culture and multiplex PCR5. Even though we did not detect fungi, we clinically suspected fungal keratitis and therefore started treatment with topical voriconazole 1% hourly, levofloxacin and cefmenoxime 6 times per day. However, while real-time PCR detected 5.0 x 105 copies/μg GAPDH of HSV-1 DNA on the fifth day after his first visit, there was no pathogen observed in the culture. As a result, we then tapered the antifungals and added topical acyclovir 3% ointment 5 times a day along with fluorometholone 0.1% twice a day and oral valacyclovir. After the treatment, KPs were seen around the lesion and there was corneal scarring observed. At 2 months after the treatment, the BCVA of the right eye had improved to 1.0 (Fig. 1I). There has been no recurrence observed for 6 months.
Real-time PCR of HSV-1. DNA of the corneal smear samples was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's protocol. The DNA was eluted with 40 to 100 μL elution buffer and quantified with NanoDrop 1000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
The genomic DNA of HSV-1 was measured using two independent PCR assays that included a combination of a qualitative multiplex PCR using the 24-pathogen strip PCR assay5, and a quantitative real-time PCR. The 24-pathogen strip PCR, which comprehensively detects 24 ocular infectious pathogens including HSV-1, HSV-2, varicella-zoster virus, adenovirus, Mycobacterium tuberculosis, bacterial 16S rRNA, Candida species, Aspergillus species, Fusarium species, fungal 18S rRNA, Acanthamoeba, and other pathogens was performed as previously described5. PCR reaction conditions were 95°C for 10 seconds, followed by 45 cycles at 95°C for 5 seconds, and at 60°C for 60 seconds. GAPDH was also used as a PCR monitoring control. Semiquantitative measurement with quantification cycle values after PCR provided an indication of the approximate amount of pathogen.
The real-time PCR assay was performed to quantitatively measure the genomic DNA of HSV-1. This experiment used GAPDH as an internal positive extraction and amplification control. Each PCR reaction (20 μL total volume) contained 10 μL buffer, 7.2 μL PCR-grade water, 2 μL DNA or DW (negative control), 0.6 μL primer-probe mix, and 0.2 μL enzyme. The calibration curves were generated using positive control DNA dilutions (106, 104, and 102 copies/μL). Primers and probes for HSV-1 have been previously described6. The products were subjected to 45 cycles of PCR amplification, with cycling conditions set at 95°C for 10 seconds, followed by 45 cycles at 95°C for 5 seconds, and at 60°C for 30 seconds. Mx3000P qPCR system (Agilent Technologies, Santa Clara, CA, USA) was used for both of the PCR assays. A copy number of greater than 50 copies/mL was used to define the clinical positivity as per a previous report7.