Motoki Itahashi

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.

Intraocular penetration of sequentially instilled topical moxifloxacin, gatifloxacin, and levofloxacin.

Purpose: The objective of the study was to compare the intraocular penetration levels of the newer fluoroquinolones, moxifloxacin, gatifloxacin, and levofloxacin in the rabbit’s cornea, aqueous humor, and conjunctiva after topical instillation. Methods: 0.5% moxifloxacin, 0.3% gatifloxacin, and 0.5% levofloxacin were instilled in random sequence in both eyes of nine New Zealand White rabbits at two-minute intervals. Instillation was repeated every 15 minutes for a total of three drops of each fluoroquinolone per eye. Three additional animals had only moxifloxacin instilled bilaterally using the same schedule. Sixty minutes after the final instillation, the rabbits were sacrificed for determination of corneal, aqueous humor, and conjunctival fluoroquinolone concentrations using high-performance liquid chromatography. Results: Moxifloxacin achieved significantly higher concentrations than levofloxacin and gatifloxacin in the cornea (P = 0.0102 and P = 0.0006, respectively), aqueous humor (P = 0.0015 and P < 0.0001, respectively), and conjunctiva (P = 0.0191 and P = 0.0236, respectively). Conclusions: 0.5% moxifloxacin eyedrops provided superior intraocular penetration in rabbits’ eyes compared with the two other fluoroquinolones, 0.5% levofloxacin and 0.3% gatifloxacin.

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.

Effects of Anti Herpetic Drugs on Mice with Herpetic Epithelial Keratitis After Reactivation of Herpes Simplex Virus Type 1

To compare the efficacies of valacyclovir (VCV) and acyclovir (ACV) on murine herpetic epithelial keratitis, mice inoculated with herpes simplex virus type 1(HSV-1) strain McKrae were divided into 6 treatment groups: oral VCV 50 mg/kg and 100 mg/kg, oral ACV 50 mg/kg, ACV eye ointment (EO), ACV eye drops (ED), and placebo. Keratitis scores showed that oral VCV 50 mg/kg, oral ACV, and ACV ED had equivalent efficacies, while oral VCV 100 mg/kg was as efficacious as ACV EO during acute infection. Each treatment group was further divided into the stimulated group with HSV-1 reactivation by immunosuppressant drugs and hyperthermia, and the non-stimulated group without reactivation. We assessed the virus titers in tissues by plaque assay and HSV DNA copy number in the trigeminal ganglia (TG) by real time polymerase chain reaction (PCR). Results showed that the virus titers in the tissues were lowered after reactivation, and the oral VCV group with reactivation had significantly reduced DNA copy number in the TG than the same treatment group without reactivation. In conclusion, oral VCV is as efficacious as ACV EO and significantly suppresses HSV-1 reactivation.

Effect of oral valaciclovir on herpetic keratitis

Purpose: To examine the efficacy of valaciclovir (VACV) oral formulation as an alternative to topical treatments in a case of herpetic keratitis. Methods: The patient was a 61-year-old man who presented with dendritic keratitis in his left eye. After recognizing his difficulty in using eye ointment, we prescribed oral VACV 500 mg tablets twice daily for 7 days. We also describe our experiments with orally administered VACV in a mouse model of this disease. In this study, 143 Balb/c mice were inoculated with herpes simplex virus type 1 (HSV)-1 in each eye and treated with oral VACV 50 or 100 mg/kg twice daily, oral acyclovir (ACV) 50 mg/kg 5 times/d, 3% ACV eye ointment (ACV-O) 5 times/d, 3% ACV eye drops 5 times/d, or control for 5 days. Results: After 7 days, the patients lesion was observed healed. Corrected left visual acuity was also improved, and HSV DNA was below detectable level. In the mouse study, slit-lamp examination on days 3, 4, 5, and 7 revealed that all 5 ACV and VACV treatment groups were significantly more effective in improving symptoms of herpetic epithelial keratitis versus control (P < 0.05). Moreover, VACV 100 mg/kg was superior to other treatments. Viral titers in mouse eyeball and trigeminal ganglia were lowest in the VACV 100 mg/kg group versus other treatment groups. Conclusion: Our case example suggests that when frequent application, blurred vision, and foreign body sensation after ACV-O application cause difficulty for patients to follow treatment, oral VACV might be an effective and safe option for patients with herpetic keratitis.

Regulatory Mechanism of Collagen Degradation by Keratocytes and Corneal Inflammation: The Role of Urokinase-Type Plasminogen Activator.

Abstract: Keratocytes, corneal resident cells in the corneal stroma, exist between collagen lamellae and maintain the corneal stromal structure. When the corneal stroma is damaged, keratocytes are transformed to myofibroblasts to aid corneal wound healing by phagocytizing debris. Keratocytes and extracellular collagen influence each other because keratocytes cultured in a 3D collagen gel undergo morphological changes and keratocytes produce metalloproteases that degrade extracellular collagen. IL-1 and plasminogen are critical mediators for collagen degradation. The plasminogen system contributes to tissue repair by activating matrix metalloproteinases (MMPs), releasing growth factors from the extracellular matrix and extracellular matrix degradation. Urokinase-type plasminogen activator (uPA) is thought to be involved in corneal disorders and regulates corneal wound healing. uPA is a serine protease synthesized by various cells such as corneal epithelial cells, corneal fibroblasts, vascular endothelial cells, smooth muscle cells, monocytes, macrophages, and malignant tumor cells of different origins. Here, we review the role of uPA in corneal stromal wound healing. uPA is expressed in leukocytes and corneal fibroblasts in the corneas of patients with corneal ulcerations suggesting it is a key regulator of corneal stromal wound healing. uPA is directly involved in plasmin-mediated collagen degradation induced by IL-1. Moreover, uPA is critically involved in promoting leukocyte infiltration in corneal inflammation by activating MMP-9. This activation is presumably directly and indirectly mediated by the plasminogen/plasmin cascade. Moreover, uPA mediates the release of inflammatory cytokines from corneal fibroblasts to promote leukocyte infiltration.

A-5021: a new acyclovir analogue inhibits murine herpetic keratitis.

Purpose: To determine the efficacy of A-5021, a new analogue of acyclovir, on murine herpetic keratitis. Methods: Herpes simplex virus type 1 (strain CHR3) was inoculated onto bilateral scarified BALB/c corneas. Clinical scores on the corneas treated with A-5021 eyedrops were compared with those obtained from the treatment with 3% acyclovir eye ointment by slit lamp microscopy. Virus titers of the trigeminal ganglia and eyeballs were quantitated on Vero cell monolayers. Mice treated with saline or a white petroleum jelly were used as controls. Results: A-5021 eyedrops significantly suppressed both corneal epithelial and stromal lesions at all concentrations used. Clinical scores on the epithelium and stroma treated with 0.1% A-5021 were equivalent to those with 3% acyclovir treatment. When compared with the non-drug-treated control mice, virus titers in the eyeballs and trigeminal ganglia in A-5021- and acyclovir-treated mice were significantly less than those in controls. Conclusions: A-5021 eyedrops, which are easily applied onto the affected cornea, ameliorated clinical scores and suppressed virus growth. It is a promising alternative treatment of herpetic keratitis.