Yoshiharu Sugamoto

Use of multiplex PCR and real-time PCR to detect human herpes virus genome in ocular fluids of patients with uveitis

Aim: To measure the genomic DNA of human herpes viruses (HHV) in the ocular fluids and to analyse the clinical relevance of HHV in uveitis. Methods: After informed consent was obtained, a total of 111 ocular fluid samples (68 aqueous humour and 43 vitreous fluid samples) were collected from 100 patients with uveitis. The samples were assayed for HHV-DNA (HHV1–8) by using two different polymerase chain reaction (PCR) assays, qualitative PCR (multiplex PCR) and quantitative PCR (real-time PCR). Results: In all of the patients with acute retinal necrosis (n = 16) that were tested, either the HSV1 (n = 2), HSV2 (n = 3), or VZV (n = 11) genome was detected. In all patients, high copy numbers of the viral DNA were also noted, indicating the presence of viral replication. In another 10 patients with anterior uveitis with iris atrophy, the VZV genome was detected. When using multiplex PCR, EBV-DNA was detected in 19 of 111 samples (17%). However, real-time PCR analysis of EBV-DNA indicated that there were only six of the 19 samples that had significantly high copy numbers. The cytomegalovirus (CMV) genome was detected in three patients with anterior uveitis of immunocompetent patients and in one immunocompromised CMV retinitis patient. In addition, one patient with severe unilateral panuveitis had a high copy number of HHV6-DNA. There was no HHV7- or HHV8-DNA detected in any of the samples. Conclusions: A qualitative multiplex PCR is useful in the screening of viral infections. However, the clinical relevance of the virus infection needs to be evaluated by quantitative real-time PCR.

Fovea-Sparing Internal Limiting Membrane Peeling for Myopic Traction Maculopathy

PURPOSE To investigate the effectiveness and safety of a new surgical technique of fovea-sparing internal limiting membrane (ILM) peeling for the treatment of foveal retinal detachments (RDs) in eyes with myopic traction maculopathy. DESIGN Retrospective, consecutive, interventional case series. METHODS Forty-five eyes of 45 consecutive patients who underwent vitrectomy and ILM peeling for the treatment of a foveal RD attributable to myopic traction maculopathy were studied. The patients were divided into 2 groups by the area of ILM peeled: complete macular ILM peeled group (30 eyes) and fovea-sparing ILM peeled group (15 eyes). A gas tamponade was used in all of the eyes. The main outcome measures were the rate of development of a full-thickness macular hole (MH) and the best-corrected visual acuity (BCVA). All of the patients were followed for more than 6 months. RESULTS A full-thickness MH developed in 5 of 30 eyes (16.7%) in the complete ILM peeled group and in none of the 15 eyes in the fovea-sparing ILM peeled group. Postoperative OCT examination showed a contraction of the residual ILM on the fovea and reduction of the outer lamellar holes in the fovea-sparing ILM peeled group. The postoperative BCVA was significantly better than the preoperative BCVA in the fovea-sparing ILM peeled group (P = .04), but not in the complete ILM peeled group. CONCLUSIONS Fovea-sparing ILM peeling results in better visual and anatomic outcomes for the treatment of foveal RD attributable to myopic traction maculopathy. These were accomplished by reducing the development of a full-thickness MH.

Progression from macular retinoschisis to retinal detachment in highly myopic eyes is associated with outer lamellar hole formation

Aims: To investigate the morphological changes that occur during the development of an early retinal detachment (RD) from a myopic macular retinoschisis (MRS) by optical coherence tomography (OCT). Methods: The OCT images of five eyes of five consecutive patients with myopic MRS who developed an RD during the follow-up period were studied. Results: The progression from MRS to early RDs went through four stages. In stage 1, OCT images appeared to show a focal irregularity of the thickness of external retina. In stage 2, an outer lamellar hole developed within the thickened area and a small RD developed. In stage 3, the column-like structures overlying the hole seemed to be separated horizontally, and the outer lamellar hole appeared to be larger vertically. In stage 4, the upper edge of the external retina was further elevated and attached to the upper part of the retinoschisis layer accompanied by further enlargement of the RD. Conclusions: This longitudinal study showed that the progression from myopic MRS to RD passes through four stages, and the formation of an outer lamellar hole predisposes the retina to a RD. These OCT findings might be useful for considering the surgical indication for eyes with a myopic MRS.

Evaluation of Characteristic Ocular Signs and Systemic Investigations in Ocular Sarcoidosis Patients

PurposeTo evaluate the diagnostic values of ocular signs and systemic investigations in ocular sarcoidosis, in a retrospective case–control study.MethodsSubjects were 67 consecutive uveitis patients with biopsy-proven sarcoidosis and 111 control patients with other clinical uveitis entities. The predictive values analyzed were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The five ocular signs for ocular sarcoidosis are (1) mutton fat keratic precipitates and iris nodules; (2) nodules at the trabecular meshwork and tent-shaped peripheral anterior synechia; (3) snowball vitreous opacities; (4) nodular periphlebitis, and (5) multiple chorioretinal lesions (active or atrophic) in the peripheral fundus. In addition, the results of the following five systemic investigations were considered: (1) negative tuberculin skin test; (2) elevated serum angiotensin-converting enzyme; (3) elevated serum lysozyme; (4) elevated serum γ-globulin; and (5) bilateral hilar lymphadenopathy on chest X-ray.ResultsThe incidence of all ocular signs and positive results for the systemic investigations were significantly higher in sarcoidosis patients than in controls (P < 0.001). The presence of two or three of the five ocular signs were indicative of a positive finding in the diagnostic parameters. The presence of two positive results among the five systemic investigations showed values higher than 0.800 for all diagnostic parameters.ConclusionsCombinations of the specified ocular signs and the results of systemic investigations can be used for the diagnosis of ocular sarcoidosis. Jpn J Ophthalmol 2007;51:121–126

Diagnosis of intraocular lymphoma by polymerase chain reaction analysis and cytokine profiling of the vitreous fluid

PurposeTo determine whether a diagnosis of intraocular lymphoma (IOL) can be made using a combination of polymerase chain reaction (PCR) analysis to detect gene rearrangement of immunoglobulin and cytokine concentrations in the vitreous fluid.MethodsVitreous samples from 22 patients with clinically suspected IOL and ten control patients with acute retinal necrosis or cytomegalovirus retinitis were examined by PCR analysis and cytokine measurements. Genomic DNA was extracted from the cells in the vitreous, and the immunoglobulin heavy chain (IgH) gene was amplified by two PCR procedures: (1) microdissection and PCR to detect IgH gene rearrangement and (2) qualitative PCR to detect IgH VDJ gene rearrangement. The supernatants of the vitreous samples were used for enzyme-linked immunosorbent assay to determine interleukin (IL)-10 and IL-6 levels.ResultsPCR examinations detected IgH rearrangement in the vitreous in 21 of the 22 IOL patients (95.5%) and in none of the ten control patients. Elevated IL-10 concentrations (>100 pg/ml) and the IL-10/IL-6 ratio (>1.0) were positive in 18 of the 22 IOL patients (81.8%), but negative in all of the control patients. Sensitivity, specificity, positive predictive value, and negative predictive value of PCR for the diagnosis of IOL were calculated to be 0.955, 1.000, 1.000, and 0.909, respectively, and those of the cytokine concentration assay to be 0.818, 1.000, 1.000, and 0.714, respectively. When both the intravitreal cytokine assay and PCR analysis of the vitreous samples are used, as well as diagnostic criteria of IOL defined as a positive outcome from one of the two assays together with clinical signs, the sensitivity and specificity of the criteria were 1.000.ConclusionsA combination of PCR assay to detect gene rearrangement of IgH and cytokine profiling (IL-10 and IL-6) is extremely useful for the diagnosis of intraocular lymphoma.

Diagnosis of bacterial endophthalmitis by broad-range quantitative PCR

Aim To measure the bacterial genome in ocular fluids and to analyse the clinical relevance of infectious endophthalmitis. Methods Nineteen ocular fluid samples (eight aqueous humour and 11 vitreous fluid samples) were collected from 19 patients with suspected bacterial endophthalmitis. Fifty ocular samples from uveitis patients were also collected along with 40 samples from patients without ocular inflammation and used as controls. Bacterial ribosomal DNA (16S rDNA) was measured by a quantitative PCR assay. Results Bacterial 16S rDNA was detected in patients with clinically suspected bacterial endophthalmitis (18/19, 95%). With the exception of one case, high copy numbers of bacterial DNA were detected (1.7×103–1.7×109 copies/ml) in these patients. There were 10 samples (53%) with positive bacterial cultures while there were nine samples (47%) with positive Gram-staining. Real-time PCR detected bacterial 16S rDNA in three (6%) of the 50 samples from the control uveitis patients. In addition, none of the samples from the control patients without intraocular inflammation were positive. Conclusions Quantitative broad-range PCR of bacterial 16S rDNA is a useful tool for diagnosing bacterial endophthalmitis.

Prophylactic vitrectomy for acute retinal necrosis

PurposeTo evaluate the efficacy of prophylactic vitrectomy for acute retinal necrosis.MethodsThe clinical charts of 17 patients (18 eyes) with acute retinal necrosis and no retinal break or rhegmatogenous retinal detachment (RRD) were retrospectively analyzed for the efficacy of prophylactic vitrectomy. The retinal necrotic lesions at the initial presentation were classified into three groups according to the lesion site as described by Holland: zone 1 (posterior pole; n = 3), zone 2 (midperiphery; n = 12), and zone 3 (periphery; n = 3). All patients were treated with intravenous antiviral therapy. Three zone 1 eyes and eight zone 2 eyes underwent prophylactic vitrectomy. Four zone 2 eyes and three zone 3 eyes did not receive prophylactic vitrectomy.ResultsAll zone 1 eyes developed RRD despite prophylactic vitrectomy. Among the 12 zone 2 eyes, eight of the eyes that underwent prophylactic vitrectomy did not develop RRD, whereas three of the four zone 2 eyes without prophylactic vitrectomy developed RRD. All zone 3 eyes were cured with only antiviral medication.ConclusionsProphylactic vitrectomy is effective in preventing the development of RRD in eyes where necrotic lesions do not extend beyond the midperiphery (zone 2).

Quantitative PCR for the detection of genomic DNA of Epstein-Barr virus in ocular fluids of patients with uveitis

PurposeTo measure the genomic DNA from Epstein-Barr virus (EBV) in ocular fluids and to analyze the clinical relevance of EBV in uveitis.MethodsIntraocular fluids (30 aqueous humor and 30 vitreous fluid samples) were taken from 55 patients with uveitis after informed consent was obtained. Samples were assayed for EBV DNA using qualitative multiplex polymerase chain reaction (PCR) and quantitative real-time PCR. Antibodies to EBV were examined using a complement fixation test.ResultsEBV DNA was detected in 17 of 60 samples (28%) and 16 of 55 patients (29%) using multiplex PCR. However, only three of the 17 samples showed significantly high copy numbers of EBV DNA with real-time PCR. EBV DNA was not detected in the serum of all patients. EBV-specific antibodies were positive in the serum of all patients, but not in the vitreous fluid. Vitreous anti-EBV antibodies were positive only in patients displaying genomic DNA of EBV in the vitreous samples.ConclusionsEBV DNA was detected by qualitative PCR in ocular fluids of many uveitis patients, but only a small proportion of patients showed high viral loads on quantitative real-time PCR, indicating that replication of the virus takes place only in a few patients.

Intraocular soluble IL-2 receptor alpha in a patient with adult T cell leukaemia with intraocular invasion

It has been reported that human T cell lymphotropic virus type I (HTLV-I) infection is related to a wide range of ocular disorders, such as intraocular lymphoma,1,2 uveitis,3 and cytomegalovirus (CNV) retinitis.4 The diagnosis of adult T cell leukaemia (ATL) cell infiltration in the eye is often difficult, even when characteristic ocular findings are present and cytological examinations of intraocular fluids are performed. It is well known that determination of patient serum interleukin 2 receptor alpha (sIL-2Rα) levels is critical in the evaluation of the clinical status of the disease.5 We report here a patient with systemic ATL who developed vitreous opacities and subretinal lesions and in whom vitreous measurement of the soluble form of sIL-2Rα provided information that could be used in making a diagnosis and in treating associated ocular disorders. A 69 year old man with a 2 year history of systemic ATL developed a sudden onset of decreased vision and vitreous floaters in the left eye. Funduscopic examination of the left eye revealed dense vitreous opacities and whitish retinal exudates along with superior vascular arcade (fig 1A). Based on the ocular manifestations and …