Shiro Higaki

Quantitative analysis of herpes simplex virus genome in tears from patients with herpetic keratitis.

Purpose Herpetic keratitis is manifested in various corneal disorders, for example, dendritic keratitis, persistent epithelial defect, disciform keratitis, and endotheliitis. In this paper, we report on the quantity of herpes simplex virus (HSV) genome in tears from patients with various types of herpetic keratitis in an attempt to understand the role of HSV in these conditions. Methods We collected tear samples from both eyes of 56 consecutive patients with herpetic keratitis who visited Kinki University Hospital between June 2000 and May 2002. All patients had unilateral herpetic keratitis: epithelial keratitis in 27 eyes; persistent epithelial defect in 6; active disciform stromal keratitis in 14; silent stromal keratitis in 6; and endotheliitis in 3. We measured levels of HSV genome in these tear samples using a real-time polymerase chain reaction (PCR) method. Results In epithelial keratitis, HSV DNA was detected in all 27 samples from affected eyes (6.4 ± 4.4 × 105copies/sample). In persistent epithelial defect, HSV DNA was detected in all 6 samples from affected eyes (8.5 ± 3.3 × 104copies/sample). In active disciform stromal keratitis, HSV DNA was detected in 8 of the 14 affected eyes (1.4 ± 1.1 × 105copies/sample including zero values in negative samples). HSV DNA was not detected in samples from unaffected eyes or eyes affected by silent stromal keratitis or endotheliitis. Conclusion Real-time PCR is a useful method for quantifying HSV DNA in tear samples from patients with herpetic keratitis. Using this method, we demonstrate that HSV reproduction occurs in persistent epithelial defect and disciform stromal keratitis.

Changes in the anterior and posterior radii of the corneal curvature and anterior chamber depth by orthokeratology.

Purpose. To investigate the mechanism of the refractive effect of orthokeratology by using measurements of the anterior and posterior radii of the corneal curvature and anterior chamber depth with a Pentacam analysis system. Methods. Nine women (18 eyes) with a mean age of 29.6 ± 3.8 years and low to moderate myopia (≤–4.00 diopters [D]) were recruited for a 53-week trial of overnight orthokeratology using RD171K lenses (hexafocon A). After wearing the orthokeratology lenses overnight, subjects were examined during the day. With a Pentacam analysis system, subjects were examined at 2, 4, 8, 12, 24, 36, and 53 weeks for the assessment of the anterior and posterior radii of the corneal curvature and anterior chamber depth. Results. Myopic refractive error significantly reduced during the trial (P<0.001, analysis of variance). The mean refractive error was –2.85 ± 0.46 D at baseline and significantly reduced to –0.28 ± 0.65 D at 2 weeks (P<0.01, Bonferroni–Dunn post hoc test). At any week, no significant differences were seen in the central posterior radius of the corneal curvature (P=0.55, analysis of variance) or the anterior chamber depth (P=0.69, analysis of variance). The amount of change in the central anterior radius of the corneal curvature significantly correlated with the change in refractive error at 24 weeks (r = 0.57, P<0.05, Pearson correlation coefficient). Conclusions. Overnight orthokeratology lens wear alters the anterior corneal shape rather than the posterior radius of the corneal curvature and the anterior chamber depth. This finding suggests that the primary factor in the refractive effect of orthokeratology is change in the anterior corneal shape rather than overall corneal bending.

Corneal buttons obtained from patients with HSK harbor high copy numbers of the HSV genome.

Purpose: To detect herpes simplex virus (HSV) genome in the cornea, we sampled the limbal corneas and scleras of the imported eye bank eyes and recipients corneal buttons and quantitated HSV genome in them by real-time polymerase chain reaction (PCR). Methods: Forty-four recipient corneas including 7 corneas with and 37 corneas without a history of herpetic keratitis, 70 eye bank donor limbal corneas, and 35 eye bank donor scleras were obtained. Primers for real-time PCR were synthesized using the HSV-1 and -2 common regions of the viral DNA polymerase. Primers for conventional PCR were designed to detect HSV-1 and -2 and varicella zoster virus (VZV). Results: Significantly higher copy number of HSV DNA was detected in corneas with a history of herpetic keratitis 85.7% (6/7), with an average of 1.6 × 104 copies/mg tissue weight than in corneas without a history of herpetic keratitis 10.8% (4/37), with an average of 8.7 copies/mg tissue weight (P < 0.05, Mann-Whitney U test). HSV DNA was detected in 5.7% (4/70) of the eye bank donor corneas, with an average of 4.9 × 102 copies/mg tissue weight, and in 8.6% (3/35) of the donor scleras, with an average of 10.6 copies/mg tissue weight. HSV-2 and VZV-DNA were not detected in these samples. Conclusions: Real-time PCR quantitated HSV genome in the cornea even at a quiescent phase of infection. HSV genome was detected in the corneas and scleras without a past history of herpetic keratitis by this method.

Detection and Quantification of Pathogenic Bacteria and Fungi Using Real-Time Polymerase Chain Reaction by Cycling Probe in Patients With Corneal Ulcer

OBJECTIVE To detect and quantitate the causative pathogens in patients with corneal ulcer using real-time polymerase chain reaction (PCR) by cycling probe. DESIGN Clinical and laboratory study of 40 eyes of 40 patients diagnosed with corneal ulcer. Two methods were used for pathogen detection: bacterial culture and real-time PCR with the patients corneal scrapings. Probes and primers of real-time PCR were designed to be pathogen specific for simultaneous detection of Staphylococcus aureus, Staphylococcus pneumoniae, Pseudomonas aeruginosa, methicillin-resistant S aureus, Candida species, and Fusarium species. Results by both methods were evaluated and compared. RESULTS Of 40 eyes, 20 eyes had the same pathogens detected by both methods and those were S aureus (3 eyes; mean [SE], 3.8 [1.3] x 10(1) copies/sample), S pneumoniae (5 eyes; mean [SE], 5.6 [5.1] x 10(3) copies/sample), P aeruginosa (8 eyes; 5.1 [4.0] x 10(3) copies/sample), methicillin-resistant S aureus (1 eye; 1.0 x 10(2) copies/sample), and Candida species (3 eyes; mean [SE], 8.8 [4.9] x 10(3) copies/sample). Six eyes showed negative results by both methods. Results of both methods disagreed in 14 eyes; specifically, 11 had positive PCR results only, 2 had positive culture results only, and 1 eye had positive results for different pathogens. CONCLUSIONS The real-time PCR assay can simultaneously detect and quantitate bacterial and fungal pathogens in patients with corneal ulcer. Real-time PCR can be a fast diagnostic tool and may be useful as an adjunct to identify potential pathogens.

Assessment of Real-Time Polymerase Chain Reaction Detection of Acanthamoeba and Prognosis Determinants of Acanthamoeba Keratitis

OBJECTIVE To evaluate the diagnostic value of real-time polymerase chain reaction (PCR) for detecting Acanthamoeba in eyes diagnosed with Acanthamoeba keratitis (AK) by conventional tests. In addition, to determine the preoperative prognosis-determining factors in eyes with AK. DESIGN Retrospective, cross-sectional study. PARTICIPANTS A total of 104 eyes of 103 patients who were diagnosed with AK or with bacterial or bacteria-associated keratitis (BK) by conventional tests. METHODS Twenty-nine eyes with AK and 75 eyes with BK were evaluated for Acanthamoeba and bacterial DNA by real-time PCR. The Acanthamoeba copy numbers, bacterial load, and clinical parameters in the patients with AK were assessed for those significantly associated with poor outcome, that is, final visual acuity of <20/50 or requiring keratoplasty, by logistic regression analysis. MAIN OUTCOME MEASURES Acanthamoeba DNA copy number, bacterial DNA copy number, and odds ratio (OR) for poor prognosis. RESULTS The detection of amoebic DNA was 50 times more sensitive by real-time PCR than by conventional cyst counting. The Acanthamoeba copy numbers at the first visit (mean: 4.7×10(5)±3.2×10(5) copies) were significantly correlated with the AK stage, and both were significant risk factors for a poor outcome. The Acanthamoeba DNA copy numbers at the first visit and AK stage had a significantly high risk for poor outcome (OR of Acanthamoeba DNA copy per logarithm of copy numbers: 3.48, 95% confidence interval [CI], 1.04-111.63, P<0.05; OR of AK stage: 2.8 per stage increase, 95% CI, 1.07-7.30, P<0.05, after adjustment of age). In the AK cases with poor outcome, the amoebic DNA was not reduced by more than 90% after 1 month of treatment. The weak amoebic reduction was significantly associated with advanced AK stages or previous use of steroids. Bacterial 16S rDNA was detected in 53.6% of the eyes with AK, but it was not associated with any risk for refractoriness. CONCLUSIONS Real-time PCR was effective in detecting and managing AK. The Acanthamoeba copy number and AK stage at the first visit were significantly associated with poor outcome. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Excimer Laser Photokeratectomy Reactivates Latent Herpes Simplex Virus

BackgroundIt has been reported that excimer laser irradiation might elicit herpes simplex virus (HSV) genome activation. We describe a clinical case in which HSV DNA sequences were detected quantitatively after phototherapeutic keratectomy (PTK).CaseA 90-year-old woman underwent excimer laser photokeratectomy for bilateral band-shaped keratopathy. Tear film was collected from both eyes using a Schirmer’s strip before and 3 and 7 days after phototherapeutic keratectomy.ObservationsHSV-DNA was quantified by a real-time polymerase chain reaction assay. HSV-DNA was detected only on the third day postoperatively in both eyes. The amount of viral DNA was 2.0 × 105 (OD) and 1.3 × 105 (OS) copies/sample, respectively.ConclusionsExcimer laser photokeratectomy stimulated viral shedding in the tear film. Ophthalmologists should be aware that laser irradiation can reactivate latent HSV. Jpn J Ophthalmol 2004;48:570–572

Herpes simplex virus genome quantification in two patients who developed herpetic epithelial keratitis during treatment with antiglaucoma medications

Purpose We quantitated herpes simplex virus (HSV) DNA in tear film obtained from 2 patients who developed herpetic epithelial keratitis (HEK) during treatment with latanoprost and a beta-blocker. Methods The patient in case 1 is a 77-year-old woman with bilateral open-angle glaucoma who had been treated with latanoprost and timolol for 11 months. She developed HEK in the right eye followed by HEK in the left eye 1 month later. Both eyes healed with administration of acyclovir. Ten months later, HEK recurred in the right eye. The patient in case 2 is a 45-year-old man with bilateral normal tension glaucoma who had been treated with latanoprost for 2 years. After concurrent treatment with nipradilol for 5 months, typical dendritic keratitis developed in the left eye. In both cases, a real-time PCR assay was used to quantify HSV-DNA in the tear film. Results In the patient in case 1, 71 copies of the HSV genome were detected in the tear film obtained from the right eye at the time of presentation with HEK. After 1 week of treatment with topical acyclovir ointment, the corneal epithelial defects healed and the number of HSV genome copies present in the tear film fell below the sensitivity limit of the assay. In the patient in case 2, 7.0 × 105 copies of the HSV genome were detected in the tear film from the left eye. After 3 days of topical acyclovir ointment, it healed and the HSV genome in the tear film became undetectable. Conclusions Herpetic keratitis may occur during treatment with latanoprost and beta-blockers. The amount of HSV DNA detected in the tear film paralleled with the activity of the corneal lesion.

Presence of a large amount of herpes simplex virus genome in tear fluid of herpetic stromal keratitis and persistent epithelial defect patients.

Herpetic eye diseases exhibit various clinical manifestations making a diagnosis difficult in some patients. We quantitated herpes simplex virus (HSV) genomes in the tear fluid and aqueous humor obtained from patients with various herpetic eye diseases by real time PCR. The resulting amounts of HSV-DNA in herpetic epithelial keratitis (HEK), herpetic stromal keratitis (HSK) in active phase, and persistent epithelial defects (PED) were 3.9 × 106 copies (detection rate, 81.1%), 8.9 × 105 copies (detection rate, 59.1%), and 9.2 × 104 copies (detection rate, 88.9%), respectively. In the tear samples obtained from quiescent phase of HSK and endotheliitis, no HSV-DNA was detected. In the aqueous humor of uveitis patients, HSV-DNA was found 3.8 × 105 copies/ml (detection rate, 16.7%). Previous studies have shown that active viral replication is not directly related to the persistent epithelial defects and progressive HSK. A relatively high level of HSV-DNA, however, was detected in the tear samples of these two disease forms, although the source of the viral replication was not identified. These findings might bring new ideas about the mechanisms of developments in HSK and PED.

CORNEAL INFILTRATION AND CMV RETINITIS IN A PATIENT WITH AIDS

PURPOSE To describe the clinical features of stromal keratitis in a patient with cytomegalovirus (CMV) retinitis and acquired immunodeficiency syndrome (AIDS). METHODS Case report. RESULTS Human CMV genome was detected in plasma, urine, and aqueous humor by polymerase chain reaction. CMV retinitis subsided and the corneal infiltrate was scarred within 4 weeks of systemic ganciclovir treatment. CONCLUSION This is the first report of the corneal infiltration seen in an AIDS patient with CMV retinitis. Etiological significance of the finding is discussed.

Cyclooxygenase (COX)-Inhibiting Drug Reduces HSV-1 Reactivation in the Mouse Eye Model

Purpose: To examine the effects of COX inhibitors on suppressing HSV-1 reactivation in a mouse model. Methods: BALB/c mice were latently infected with HSV-1 and treated by 0.1% bromfenac Na eye drops, 0.1% pranoprofen eye drops, 0.1 mg oral etodolac 4 times/day, and saline for 4 days. After reactivating the latent HSV-1, we swabbed the mouse ocular surface for the culture of the infectious virus and assessed the viral loads in the eyes and trigeminal ganglia (TGs) using real-time PCR to determine the treatment efficacies. Results: With stimulated reactivation, 10 of 24 (41.7%), 5 of 10 (50.0%), 17 of 25 (68%), and 16 of 22 eyes (72.7%) showed positive swab results in the bromfenac Na, etodolac, pranoprofen, and saline groups, respectively; and a significant difference was seen only between the bromfenac Na and saline groups (p = 0.033). None of the three drug-treated groups showed any significant difference from the saline group in the viral DNA in the eyes and TGs (p > 0.05). Conclusions: Bromfenac Na eye drops can suppress HSV-1 reactivation.