Diagnostic efficacy of real-time PCR for ocular cytomegalovirus infections.

PURPOSE: The aim of this study is to determine the efficacy of quantitative real-time PCR (qPCR) and clinical characteristics to diagnose ocular cytomegalovirus (CMV) infections. METHODS: The technical factors were assessed by the outcomes of the qPCR assay at five institutions in Japan using the WHO International Standard of cytomegalovirus. The clinical factors were assessed by examining the aqueous humor samples of 197 eyes of 197 consecutive patients suspected of CMV using the receiver operating characteristics (ROCs). RESULTS: All of the institutions had excellent detection efficacy, although the copy number ranged from 0.82 to 4.66 copies/IU. In the clinical samples, CMV was detected in 51 eyes, and the amount of CMV DNA was significantly higher for CMV retinitis. In corneal diseases, the amount of CMV DNA was significantly associated with frequency of recurrences and IOP elevations. The sensitivity and specificity of qPCR for the diagnosis was 90.0 and 98.7%, respectively. For the corneal and anterior uveitis types of CMV diseases, the area under the curve (AUC) of qPCR was 0.95 and 0.96, followed by frequency of recurrences with AUC of 0.89 and 0.82, and IOP elevations with AUC of 0.78 and 0.76. Unclassified cytomegalovirus detection, which did not meet diagnostic criteria of CMV corneal endotheliitis, anterior uveitis, or retinitis, was 4.6%, and it was significantly associated with corneal diseases and history of corneal transplantation. CONCLUSIONS: qPCR with standardization is specific and accurate; however, the inclusion and knowledge of the clinical characteristics improve the diagnostic efficacy.

Effects of antiviral medications on herpetic epithelial keratitis in mice.

PURPOSE: Aciclovir (ACV), valaciclovir (VACV) and famciclovir (FCV) are used for systemic infections caused by herpes virus. In Japan, only topical ACV is permitted for use against herpetic keratitis. We investigated the effectiveness of topical ACV, oral VACV and oral FCV on mouse epithelial herpetic keratitis. METHODS: C57/BL76 mice were inoculated with HSV-1 McKrae strain in the cornea. Once infection was confirmed 4 days after inoculation, topical ACV, oral VACV and FCV were started and administered for 5 days. Control groups were given either topical or oral saline. On days 2, 4, 6 and 10 after medication started, tears, eyeballs, and trigeminal ganglia were examined using viral culture and real-time PCR. RESULTS: Viral culture of tears detected no HSV in the topical ACV group on day 4 after administration start; with similar results for the oral VACV group on day 4; and the oral FCV group on day 6. Real-time PCR of the eyeballs showed significant decrease of HSV DNA copy number in the topical ACV group on days 4 and 6 compared to the topical saline group. Real-time PCR of the trigeminal ganglia showed significant decrease of HSV DNA copy number in the oral VACV group on days 4 and 6, and in the oral FCV group on day 6 compared to the oral saline group. CONCLUSION: We suggest that 5-day administration of topical ACV, oral VACV and oral FCV are effective for mouse epithelial herpetic keratitis and sufficiently decrease HSV amounts in the ocular surface and eyeballs.

Virological and molecular biological evidence supporting herpes simplex virus type 1 corneal latency.

PURPOSE: Trigeminal and other ganglia are known as sites of latent infection by herpes simplex virus type 1 (HSV-1). In ophthalmology, HSV-1 remains latent in the trigeminal ganglia, and becomes reactivated by several factors, including stress, thermal stimulation, or immunosuppression, and may lead to herpetic keratitis. The purpose of this study was to demonstrate HSV corneal latent infection using molecular biology and virology techniques. METHODS: Six corneas obtained at penetrating keratoplasty were snap-frozen; three of them were with past history of herpetic keratitis. TaqMan Real-time PCR was used to show positive HSV DNA in the corneas. We proved negative homogenate and positive explant virologically. Using real-time RT-PCR, we showed that only latency-associated transcript (LAT) was detected and no transcriptional products of other virus genes (α, ß, γ) were detected. RESULTS: All three corneas with past history of herpetic keratitis had HSV DNA and showed negative homogenate and positive explant. LAT was detected in all three corneas. However, α, ß, or γ genes were not expressed. All the results of these corneas were consistent with the conditions of corneal latency. The other three corneas without history of herpetic keratitis showed negative homogenate and negative explant. None of them had LAT. CONCLUSION: We have shown a possibility that HSV can latently infect the cornea aside from the ganglion.

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.

Results of penetrating keratoplasty triple procedure with 25-gauge core vitrectomy.

PURPOSE: To report and compare the outcomes of penetrating keratoplasty (PKP) triple procedures [combined PKP, cataract extraction, and intraocular lens (IOL) implantation] with a 25-gauge (G) system, 20-G system, and without core vitrectomy. SUBJECTS AND METHODS: Subjects comprised the following 3 groups: the 25-G group including 12 eyes of 12 patients (4 men and 8 women) that underwent PKP with 25-G core vitrectomy, the 20-G group including 9 eyes of 9 patients (3 men and 6 women) that underwent PKP with 20-G core vitrectomy, and the non-core vitrectomy group including 9 eyes of 9 patients (1 man and 8 women) that underwent PKP without core vitrectomy. RESULTS: In the 25-G, 20-G, and non-core vitrectomy groups, the success rates of IOL implantation were 91.7% (11 of 12 eyes), 88.9% (8 of 9 eyes), and 66.7% (6 of 9 eyes), respectively; the average operation times were 69 minutes, 82 minutes, and 90 minutes, respectively. The 25-G group showed significantly shorter operation time than the other 2 groups. Although not statistically significant, a higher rate of capsular rupture was seen in the non-core vitrectomy group. CONCLUSIONS: A PKP triple procedure with the 25-G system can be a safe treatment that offers a better success rate of IOL implantation and a significantly shorter operation time.

Utility of real-time polymerase chain reaction in diagnosing and treating acanthamoeba keratitis.

PURPOSE: Using real-time polymerase chain reaction (PCR), we detected Acanthamoeba and monitored the changes in Acanthamoeba DNA copy number over the treatment course in patients suspected of Acanthamoeba keratitis (AK). METHODS: Subjects were 6 patients (average age, 26.2 years) suspected of AK at the Kinki University Outpatient Clinic. For detection of Acanthamoeba, patients' corneal scrapings were collected for smear analysis, culture, and real-time PCR. After the diagnosis of AK was confirmed, treatment was initiated based on the quantitative result of the real-time PCR. RESULTS: Both the smear and culture were positive for Acanthamoeba in 4 cases and negative in 2 cases (agreement in 3 cases and disagreement in 2 cases). By real-time PCR, all 6 cases were positive for Acanthamoeba with an average DNA copy number of 4.8 ± 9.1 × 10 copies per sample. We further monitored the variation in the Acanthamoeba DNA copy number over the treatment course and successfully treated all the patients. DNA copy number provided a parallel with other clinical features of AK. CONCLUSIONS: Real-time PCR can be a useful method for a rapid and precise diagnosis of AK. Moreover, utility of the Acanthamoeba DNA copy number obtained by real-time PCR can help ophthalmologists in making the best treatment decision.

The kinetics of herpes virus on the ocular surface and suppression of its reactivation.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in sensory neurons that can sometimes be reactivated. HSV-1 keratitis often recurs and can be vision threatening. Reactivation of the latent virus can be stimulated by stress, immunosuppression, trauma, adrenergic iontophoresis, and UV radiation. Healthy and asymptomatic individuals are known to shed HSV-1, and this is a major factor in the spread of the virus. We investigated the frequency of shedding of HSV-1 DNA in tears of dry eye patients and individuals with conjunctivitis. Subjects were divided into 3 groups: normal (12 eyes), dry eye (11 eyes), and conjunctivitis (15 eyes). Quantitative real-time polymerase chain reaction was used for HSV DNA detection. The incidences of HSV positivity in the normal, dry eye, and conjunctivitis groups were 1 of 12 (8.3%), 3 of 11 (27.3%), and 4 of 15 (26.7%), respectively. We have previously shown that bromfenac sodium eye drops, intramuscular adenosine monophosphate, and geldanamycin effectively lower HSV-1 recurrence rates in a mouse model. Recently, we also found that nuclear factor κ-B, an IκB kinase-ß inhibitor, could be a candidate for reducing HSV-1 reactivation. We sampled recipients' corneal buttons during keratoplasty and performed polymerase chain reaction. Cytomegalovirus (CMV) DNA was detected in corneas obtained from some patients, and the copy number of the detected CMV DNA was quantified. CMV DNA-positive samples were taken from 2 of the 3 patients with ocular pemphigoid; thus, in future work, the relationship between CMV in the cornea and the incidence/onset of ocular diseases of the anterior segment needs to be evaluated.

The quantitative detection of herpes simplex virus, varicella zoster virus, and cytomegalovirus DNAs in recipient corneal buttons.

PURPOSE: We detected herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV), and cytomegalovirus (CMV) DNAs in recipient corneal buttons taken at the time of penetrating keratoplasty. METHODS: Twenty-seven corneal buttons were obtained from 27 patients (10 men and 17 women), 7 of whom had a history of HSV keratitis. Excised corneal buttons were immediately frozen in liquid nitrogen in the operating theater and then stored at -80°C until DNA extraction. The detection of HSV-1, HSV-2, VZV, and CMV DNAs was carried out by a nested polymerase chain reaction (PCR) method. The genome copy numbers for the nested PCR-positive samples were subsequently quantified by real-time PCR. RESULTS: HSV-1, HSV-2, VZV, and CMV DNAs were detected in 10, 1, 9, and 2 of the 27 recipient corneal buttons, respectively. HSV-1 or HSV-2 DNAs were also detected in 5 of 7 patients with a history of HSV keratitis. Both CMV-positive patients (patients 2 and 3) had ocular pemphigoid. Among the nested PCR-positive samples, 2 HSV-1, 1 HSV-2, 1 VZV, and 1 CMV sample could be quantified by real-time PCR. Copy numbers ranged from 19 to 928 copies. CONCLUSIONS: All 4 herpesviruses, including CMV, were detected in the corneal buttons. The relationship between CMV in the cornea and ocular diseases of the anterior segment should be further evaluated.

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 patient's 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.

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.

Case of bilateral multiple herpetic epithelial keratitis manifested as dendriform epithelial edema during primary Kaposi's varicelliform eruption.

BACKGROUND: Bilateral herpetic keratitis has been reported in patients with atopy, measles, graft-versus-host disease, and altered immune status. We report a serologically verified case of primary, simultaneous onset, bilateral, atypical epithelial herpetic keratitis that manifested as dendriform epithelial edema during a generalized dermatitis incident. CASE: A 37-year-old man with chronic atopic dermatitis developed Kaposi's varicelliform eruption and bilateral dendritic epithelial keratitis with corneal epithelial edema. OBSERVATIONS: The pathogens isolated from both eyes were identified as herpes simplex virus type 1 (HSV-1) by a direct immunofluorescence method. Serological tests obtained on three different occasions over a 5-week period verified a primary HSV infection. CONCLUSIONS: With serological verification, we report a rare case of primary, simultaneous onset, bilateral, dendritic epithelial keratitis at a very early stage with complications of generalized herpetic disease in a patient with atopic dermatitis.

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 x 10(6) copies (detection rate, 81.1%), 8.9 x 10(5) copies (detection rate, 59.1%), and 9.2 x 10(4) 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 x 10(5) 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.

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.

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.

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 (

Longterm results of deep lamellar keratoplasty using grafts with endothelium.

PURPOSE: To report the longterm results of deep lamellar keratoplasty (DLK) using grafts with their own endothelia. METHODS: Fourteen eyes of 14 patients underwent DLK using grafts with endothelium. The average follow-up was approximately 80.0 months. Preoperative diagnoses included: corneal leukoma (five eyes); gelatinous drop-like corneal dystrophy (three eyes); Avellino corneal dystrophy (two eyes); corneal perforation (two eyes); corneal mucopolysaccharidosis (one eye), and keratoconus (one eye). RESULTS: Corrected visual acuity was improved in 13 eyes (93%), but ruptures of Descemet's membrane occurred in six eyes (43%) and a double anterior chamber was found in five eyes (36%) postoperatively. Despite this, all grafts remained clear as a result of their functioning endothelia. CONCLUSIONS: Deep lamellar keratoplasty using a graft with its own endothelium is a safe and valuable procedure with flexibility and feasibility that should suit corneal surgeons of all levels.

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 recipient's 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 x 10(4) 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 x 10(2) 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.