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Redondoviruses are circular Rep-encoding single-stranded DNA (CRESS) viruses of high prevalence in healthy humans. Redondovirus abundance is increased in oro-respiratory samples from individuals with periodontitis, acute illness, and severe COVID-19. We investigated potential host cells supporting redondovirus replication in oro-respiratory samples and uncovered the oral amoeba Entamoeba gingivalis as a likely host. Redondoviruses are closely related to viruses of Entamoeba and contain reduced GC nucleotide content, consistent with Entamoeba hosts. Redondovirus and E. gingivalis co-occur in metagenomic data from oral disease and healthy human cohorts. When grown in xenic cultures with feeder bacteria, E. gingivalis was robustly positive for redondovirus RNA and DNA. A DNA proximity-ligation assay (Hi-C) on xenic culture cells showed enriched cross-linking of redondovirus and Entamoeba DNA, supporting E. gingivalis as the redondovirus host. While bacteria are established hosts for bacteriophages within the human virome, this work shows that eukaryotic commensals also contribute an abundant human-associated virus.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.ImportanceSARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in South Eastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a three-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. IMPORTANCE COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.

Chimeric antigen receptor–engineered T cells targeting CD19 (CART19) provide an effective treatment for pediatric acute lymphoblastic leukemia but are less effective for chronic lymphocytic leukemia (CLL), focusing attention on improving efficacy. CART19 harbor an engineered receptor, which is delivered through lentiviral vector integration, thereby marking cell lineages and modifying the cellular genome by insertional mutagenesis. We recently reported that vector integration within the host TET2 gene was associated with CLL remission. Here, we investigated clonal population structure and therapeutic outcomes in another 39 patients by high-throughput sequencing of vector-integration sites. Genes at integration sites enriched in responders were commonly found in cell-signaling and chromatin modification pathways, suggesting that insertional mutagenesis in these genes promoted therapeutic T cell proliferation. We also developed a multivariate model based on integration-site distributions and found that data from preinfusion products forecasted response in CLL successfully in discovery and validation cohorts and, in day 28 samples, reported responders to CLL therapy with high accuracy. These data clarify how insertional mutagenesis can modulate cell proliferation in CART19 therapy and how data on integration-site distributions can be linked to treatment outcomes.

Genome engineering methods have advanced greatly with the development of programmable nucleases, but methods for quantifying on- and off-target cleavage sites and associated deletions remain nascent. Here, we report an improvement of the GUIDE-seq method, iGUIDE, which allows filtering of mispriming events to clarify the true cleavage signal. Using iGUIDE, we specify the locations of Cas9-guided cleavage for four guide RNAs, characterize associated deletions, and show that naturally occurring background DNA double-strand breaks are associated with open chromatin, gene dense regions, and chromosomal fragile sites. iGUIDE is available from https://github.com/cnobles/iGUIDE. Electronic supplementary material The online version of this article (10.1186/s13059-019-1625-3) contains supplementary material, which is available to authorized users.

Rapid whole genome sequencing of SARS-CoV-2 has presented the ability to detect new emerging variants of concern in near real time. Here we report the genome of a virus isolated in Pennsylvania in March 2021 that was identified as lineage B.1.1.7 (VOC-202012/01) that also harbors the E484K spike mutation, which has been shown to promote “escape” from neutralizing antibodies in vitro. We compare this sequence to the only 5 other B.1.1.7+E484K genomes from Pennsylvania, all of which were isolated in mid March. Beginning in February 2021, only a small number (n=60) of isolates with this profile have been detected in the US, and only a total of 253 have been reported globally (first in the UK in December 2020). Comparative genomics of all currently available high coverage B.1.1.7+E484K genomes (n=235) available on GISAID suggested the existence of 7 distinct groups or clonal complexes (CC; as defined by GNUVID) bearing the E484K mutation raising the possibility of 7 independent acquisitions of the E484K spike mutation in each background. Phylogenetic analysis suggested the presence of at least 3 distinct clades of B.1.1.7+E484K circulating in the US, with the Pennsylvanian isolates belonging to two distinct clades. Increased genomic surveillance will be crucial for detection of emerging variants of concern that can escape natural and vaccine induced immunity.

Rapid whole genome sequencing of SARS-CoV-2 has presented the ability to detect new emerging variants of concern in near real time. Here we report the genome of a virus isolated in Pennsylvania in March 2021 that was identified as lineage B.1.1.7 (VOC-202012/01) that also harbors the E484K spike mutation, which has been shown to promote “escape” from neutralizing antibodies in vitro. We compare this sequence to the only 5 other B.1.1.7+E484K genomes from Pennsylvania, all of which were isolated in mid March. Beginning in February 2021, only a small number (n=60) of isolates with this profile have been detected in the US, and only a total of 253 have been reported globally (first in the UK in December 2020). Comparative genomics of all currently available high coverage B.1.1.7+E484K genomes (n=235) available on GISAID suggested the existence of 7 distinct groups or clonal complexes (CC; as defined by GNUVID) bearing the E484K mutation raising the possibility of 7 independent acquisitions of the E484K spike mutation in each background. Phylogenetic analysis suggested the presence of at least 3 distinct clades of B.1.1.7+E484K circulating in the US, with the Pennsylvanian isolates belonging to two distinct clades. Increased genomic surveillance will be crucial for detection of emerging variants of concern that can escape natural and vaccine induced immunity.

Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability.

Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location., The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)–ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions insilico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

Automated iGUIDE Summary Report. This reproducible report includes specifics on the samples sequenced and annotated data for each, generated by a standardized software pipeline. (PDF 1035 kb)

Figure S1. Frequency of dsODN incorporation surrounding the expected Cas9 cleavage sites for B2M, TRAC5, and VEGFA sgRNAs. â Cells Observedâ were quantified using lengths of flanking DNA fragments after sonication as a measure of independent isolation events. (PDF 169 kb)

A Standard Operating Procedure for carrying out iGUIDE analysis. This report provides detailed protocols for carrying out the iGUIDE procedure. (PDF 107 kb)

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

Lineage analysis of autoreactive B cells can reveal the origins of autoimmunity. In the autoimmune disease pemphigus vulgaris (PV), desmoglein 3 (DSG3) and DSG1 autoantibodies are predominantly of the IgG4 subclass and less frequently of IgG1 and IgA subclasses, prompting us to investigate whether anti-DSG IgG4 B cells share lineages with IgG1, IgA1, and IgA2. Combining subclass-specific B cell deep sequencing with high-throughput antibody screening, we identified 80 DSG-reactive lineages from 4 PV patients. Most anti-DSG IgG4 B cells lacked clonal relationships to other subclasses and preferentially targeted DSG adhesion domains, whereas anti-DSG IgA frequently evolved from or to other subclasses and recognized a broader range of epitopes. Our findings suggest that anti-DSG IgG4 B cells predominantly evolve independently or diverge early from other subclasses and that IgA is most often not the origin of IgG autoreactivity in PV. These data provide insight into how autoreactivity diversifies across B cell subclasses.

Leuconostoc spp. (LS1and LI1) isolated from sauerkraut and idli batter was selected for dextran production. To enhance the yield of dextran, effects of various parameters such as sucrose concentration, pH, temperature, incubation and inoculum percentage were analyzed. The optimum sucrose concentration for the Leuconostoc spp. (LS1 and LI1) was found to be 15% and 25% respectively. Isolates produced maximum dextran after 20 h of incubation at 29° C and the optimum pH was found between 8 and 8.5. The inoculum concentration of 7.5% was more favorable for the production of dextran by Leuconostoc spp. (LS1 and LI1). The growth kinetic parameters were studied and compared for the strains LS1 and LI1. Mass production of dextran was carried out using a stirred tank batch reactor. FTIR analysis was done to determine the functional groups of dextran. sephadex is prepared by cross linking dextran using epichlorohydrin and the functional groups are determined by FTIR analysis.

BACKGROUND AND OBJECTIVE: To determine the effectiveness of spectral-domain optical coherence tomography (SD-OCT) as a screening tool for the evaluation of chloroquine or hydroxychloroquine retinal toxicity. PATIENTS AND METHODS: This is a prospective, case–control study. Subject eyes were divided into four groups (group I = eyes with bull’s eye maculopathy, group II = eyes with early changes of toxicity, group III = eyes with exposure but no signs of toxicity, and group IV = eyes of age-matched controls). Retinal thickness was measured via SD-OCT 0.5 and 1.0 mm from the foveal center. RESULTS: Mean retinal thickness 1.0 mm from the fovea in group I eyes was significantly thinner when compared to group IV. Eyes in group II also showed retinal thinning 1.0 mm from the foveal center when compared to both groups III and IV. Mean retinal thickness 0.5 mm from the foveal center did not differ significantly between any groups. CONCLUSION: Significant retinal thinning occurred 1.0 mm, but not 0.5 mm, from the foveal center in patients with early and late chloroquine or hydroxychloroquine toxicity. Measuring retinal thickness 1.0 mm from the foveal center in patients receiving these medications may help screen for early toxicity.

CD19-specific Chimeric Antigen Receptor (CTL019)-engineered T-cells provide a breakthrough for personalized cancer therapy. An anti-CD19 CAR gene with 41BB costimulatory domain is delivered into patient T-cells ex vivo using a lentiviral vector, expanded in culture and then reinfused into patients. While dramatically successful for some treatment-refractory cancers, a significant proportion of patients do not experience therapeutic levels of CAR T cell expansion - thus it is important to investigate factors driving successful expansion in responders in more detail. Here we have analyzed sites of lentiviral vector integration in CAR T cells from trials to ALL and CLL, comparing successful and unsuccessful therapy in longitudinal data sets for 40 subjects. The location of each integrated vector marks a cell lineage uniquely allowing the fate mapping of individual CAR-engineered T cells in the infusion product and after adoptive transfer. We found that 81.4% of integrations had occurred in annotated transcription units which is consistent with previous reports for lentiviral vector integration sites. Relatively larger and more diverse populations of CAR-modified T-cells were associated with improved outcome (Chao1 index, p=0.043). Population sizes were also significantly more diverse in the infusion product compared with day 28 post-infusion, and more diverse at this time point when comparing responders with non-responders, or even partial responders with non-responders (p Disclosures Fraietta: Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Porter:Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board; Genentech: Other: Spouse employment. Frey:Servier Consultancy: Consultancy; Novartis: Consultancy. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Siegel:Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Tmunity: Research Funding; Parker Foundation: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees. Melenhorst:Shanghai UNICAR Therapy, Inc: Consultancy; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding.

The authors describe a case of nasal and macular retinoschisis in a patient with open angle glaucoma. A 75 year-old female with optic nerve head damage secondary to chronic open angle glaucoma developed macular schisis and a separate area of retinoschisis nasal to her optic disk. There were no other identifiable causes for her retinoschisis. Glaucoma related structural defects offer a plausible explanation for multiple cavities of retinoschisis in favor of multiple occult congenital pits of the optic nerve head.

Background Vitrectomy systems are now available with 20-, 23-, and 25-gauge vitreous cutters and are designed to be used with conventional or new generation pneumatic actuation technology. Methods Five vitreous cutters using pneumatic actuation technology were studied. Flow rates were evaluated using porcine vitreous. A high-speed camera timed the cutting phases (open, closing, closed, and opening), and duty cycle was calculated. The cutting port surface area and internal shaft surface areas were calculated. Results Increasing cut rate decreased the average open phase duration without affecting other cutting phases. The opening and closing phases of vitreous cutters using new generation pneumatic actuation technology were longer than those for other vitreous cutters. Flow rate was correlated to internal shaft surface area. Conclusion In addition to confirming the importance of duty cycle at high cut rates, this study demonstrated that the transition phases should also be considered when one is evaluating the efficiency of a vitreous cutter.

Aim: To review the outcomes of eyes with neovascular age-related macular degeneration (AMD) treated with photodynamic therapy (PDT) with verteporfin and intravitreal triamcinolone acetonide injection. Materials and Methods: We retrospectively reviewed the outcomes of consecutive eyes with neovascular AMD that received an intravitreal triamcinolone injection within 1 week of their first PDT and had at least 6 months of follow-up. Eyes were retreated with PDT at 3-month intervals if angiographic leakage was present. Results: Twenty-six eyes from 24 patients were identified. The mean visual acuity at baseline was 20/118 (median 20/112). The mean visual acuity decreased to 20/138 at 9 months (P = 0.24, n = 15) and to 20/174 at 12 months (P = 0.23, n = 8). The change in visual acuity from baseline was not statistically significant at any time point. The mean central foveal thickness by OCT measured 342 µm at baseline and decreased to 296 µm at 12 months (P = 0.31). Sixty-two per cent of eyes required no additional PDT at 12 months. Nineteen per cent of 26 eyes had a rise in intraocular pressure that was controlled with topical medication alone. Conclusion: Photodynamic therapy with verteporfin combined with intravitreal triamcinolone injection in the treatment of neovascular AMD may be superior to PDT alone by decreasing visual loss and reducing the number or retreatments. Key words: Choroidal neovascularisation, Macular degeneration, Photodynamic therapy, Triamcinolone, Verteporfin

Purpose To analyze the in vivo morphology of the sea fan in proliferative sickle cell retinopathy and compare it with the neovascularization in proliferative diabetic retinopathy. Methods Brief case report of spectral-domain optical coherence tomography imaging of a sea fan in a patient with sickle cell anemia and newly diagnosed proliferative sickle cell retinopathy, and morphologic comparison with spectral-domain optical coherence tomography imaging of a neovascular membrane in a diabetic patient with proliferative diabetic retinopathy. Results Spectral-domain optical coherence tomography imaging revealed that the sea fan is a thicker caliber preretinal fibrovascular membrane involving primarily the retinal nerve fiber and ganglion cell layers. The diabetic membrane has more vitreous adhesions, and it is more closely intertwined with the retina, involving all the retinal layers down to the outer plexiform layer. Conclusion Spectral-domain optical coherence tomography imaging identified important in vivo morphologic differences between neovascularization in proliferative sickle cell retinopathy and proliferative diabetic retinopathy. These differences were consistent with previous histological studies and may explain the increased risk of tractional retinal detachment in patients with diabetes.

Optic disk pits are a congenital optic disk anomaly frequently associated with serous macular detachment.A case of optic disk pits associated with serous maculopathy in a patient with incontinentia pigmenti is presented. The patient was treated with oral acetazolamide.Over the course of 1 year, treatment with oral acetazolamide resulted in the complete resolution of subretinal fluid and improvement in vision.An association between optic disk pits and incontinentia pigmenti is presented. The case demonstrates the utility of oral acetazolamide in the treatment of serous maculopathy associated with optic disk pits.

Purpose To describe a case of choroidal infarction as illustrated by the "triangular sign of Amalric" after cocaine inhalation. Methods Observational case report. Results A 52-year-old male presented with an acute loss of vision in his left eye immediately after cocaine inhalation. Color photography and fluorescein angiography revealed triangular areas of choroidal ischemia following the distribution of the posterior ciliary arteries. Conclusion Cocaine-induced vasospasm, specifically of the distal short posterior ciliary arteries, may explain the triangular wedge-shaped choroidal infarcts in our patient. A thorough drug history may circumvent unnecessary and costly investigations in such cases.

Purpose: To describe a fundus autofluorescent photographic pattern in a case of bilateral diffuse uveal melanocytic proliferation (BDUMP) with occult esophageal carcinoma. Methods: Observational case report. Results: Color photography captured multiple round areas of discoloration throughout the fundus in a background of orange pigmentation that may have represented a lipofuscin-laden retinal pigment epithelium (RPE). Autofluorescence photography showed extensive nummular areas of hypoautofluorescence interspersed between areas of hyperautofluorescence as a result of extensive deposition of lipofuscin within the RPE. Conclusion: Our case attests to the utility of autofluorescence photography as a sensitive, noninvasive imaging modality for the early detection of BDUMP.

PURPOSE We report two cases of macular infarction as a presenting sign of systemic lupus erythematosus (SLE). METHODS Ophthalmic examination and intravenous fluorescein angiography were supplemented by rheumatology consultations and imaging. RESULTS Two patients presented with complaints of decreased vision in one or both eyes. Systemic manifestations included fever, rash, and arthralgias, while serologic tests revealed an elevated erythrocyte sedimentation rate and positive antinuclear antibody titers in both cases, confirming the diagnosis of SLE in each case. Ophthalmoscopic changes included cotton-wool spots, intraretinal hemorrhages, and retinal edema. Fluorescein angiography revealed macular infarction with extensive retinal capillary nonperfusion in both patients. CONCLUSION Macular infarction is an uncommon but recognized complication of vasculitis associated with SLE. We report two additional cases of newly diagnosed SLE where vision loss secondary to macular infarction was the presenting sign of the disease. SLE should be considered in all patients who present with macular infarction. Visual prognosis is usually poor.

Purpose To describe a case of a pigment epithelial detachment (PED) that likely led to a branch retinal vein occlusion (BRVO). Methods Color photography, fluorescein angiography, and three-dimensional spectral optical coherence tomography (3D-spectral OCT) were performed. Results Fluorescein angiography identified a PED at the site of a BRVO, and 3D-spectral OCT illustrated compression and distortion of the overlying retina at the location of the venous obstruction. Conclusion It is well known that retinal arterial disease can compress an underlying vein and lead to a BRVO. A theoretically possible, but previously unrecognized cause of a BRVO is compression of an overlying vein by an underlying PED. Our case reminds us to continue to explore local anatomical factors in addition to systemic factors in patients with venous occlusive disease.

Backround Retinal cavernous hemangiomas are rare vascular anomalies, and can be associated with cerebral cavernous malformations (CCM). Distinct mutations have been reported in patients who have both CCMs and retinal cavernous hemangiomas. Methods Fluorescein angiography, spectral domain optical coherence tomography, and genetic testing were performed on a patient with a retinal cavernous hemangioma and a CCM. Results Our patient was heterozygous in the KRIT1/CCM1 gene for a frameshift mutation, c.1088delC. This would be predicted to result in premature protein termination. Discussion We have identified a novel mutation in the KRIT1/CCM1 gene in a patient with both CCM and retinal cavernous hemangioma. We hypothesize that the occurrence of retinal cavernous hemangiomas and CCMs is underlaid by a common mechanism present in the KRIT1/CCM1 gene.

Aim: To determine the prevalence and clinical characteristics of the Inflammatory ocular hypertension syndrome (IOHS) in patients with uveitis and serologic evidence of syphilis. Methods: We performed a retrospective, observational case review of 39 consecutive patients with uveitis and serologic evidence of syphilis between January, 1977, and December, 2001, at The Francis I. Proctor Foundation of the University of California, San Francisco. History, examination, and directed laboratory testing were used to exclude other causes of uveitis. The prevalence and clinical characteristics of IOHS among patients with uveitis and serologic evidence of syphilis were documented. Inflammatory ocular hypertension syndrome was defined as intraocular pressure (IOP) elevation greater than 21mmHg that began at the onset of acute, recurrent, or chronic anterior chamber inflammation, and which reversed promptly with appropriate anti-inflammatory and/or antimicrobial therapy. Results: Of the 39 patients with uveitis and serologic evidence of syphilis, 8 eyes of 7 patients (18%) presented with IOHS. This was significantly higher than the prevalence of IOHS in the uveitis population at large (2.3%; P

PURPOSE To describe the fundus autofluorescence photographic pattern in a case of deferoxamine retinopathy. METHODS Observational case report. RESULTS A 91-year-old woman receiving intravenous deferoxamine treatment presented with a history of deteriorating vision in both eyes. Autofluorescence photography revealed well circumscribed areas of hyperautofluorescence that correlated with areas of pigmentation shown by color photography. Deferoxamine retinopathy was diagnosed. CONCLUSION Fundus autofluorescence photography is a noninvasive imaging method for early detection of deferoxamine retinopathy and can be used to monitor the status of the macula in patients at risk for this toxicity.

A 42-year-old man with a history of human immunodeficiency virus and previous immune reconstitution after initiating a highly active antiretroviral therapy regimen was referred to rule out cytomegalovirus retinitis. He had an unusual juxtapapillary collection of white superficial material and multiple pre-retinal collections resting on a peripheral ground glass opacification of the retina. These lesions quickly resolved after initiating treatment for syphilitic retinitis. The patient appeared to have developed an exaggerated response to the ocular syphilitic infection that may be related to the augmentation of his impaired immune system by highly active antiretroviral therapy. [Ophthalmic Surg Lasers Imaging 2006;37:429-431.] AUTHORS From the Department of Ophthalmology (SR, ETC, RFS), New York University School of Medicine; Vitreous-Retina-Macula Consultants of New York (ETC, RFS); and the LuEsther T. Mertz Retina Research Center (ETC, RFS), Manhattan Eye, Ear and Throat Hospital, New York, New York. Accepted for publication April 20, 2006. Address correspondence to Richard F. Spaide, MD, Vitreous-Retina-Macula Consultants of New York, 460 Park Avenue, 5th Floor, New York, NY 10022.

This study was conducted to investigate a case of reactive lymphoid hyperplasia following laser assisted in situ keratomileusis (LASIK).A 31-year-old man who underwent LASIK presented 1 month later with a fleshy conjunctival (plical) tumor in the left eye. An excision biopsy of the tumor was performed.Histopathology of the excised tumor revealed reactive lymphoid hyperplasia.Conjunctival lymphomas can masquerade as chronic conjunctivitis and can be preceded by reactive lymphoid hyperplasia. It is important to identify and differentiate these tumors. This report describes the unusual occurrence of a lymphoid conjunctival tumor after LASIK eye surgery.

Plant phospholipids and glycolipids can be analyzed by direct infusion electrospray ionization triple-quadrupole mass spectrometry. A biological extract is introduced in solvent by continuous infusion into the mass spectrometer's electrospray ionization source, where ions are produced from the lipids. For analysis of membrane lipids, a series of precursor and neutral loss scans, each specific for lipids containing a common head group, are obtained sequentially. The mass spectral data are processed and combined, using the Web application LipidomeDB Data Calculation Environment, to create a lipid profile.

The association between retinal hemorrhages and meningitis has been previously documented; however, all of these cases were among critically ill patients with clinically progressive bacterial meningitis. This case report describes a patient with aseptic meningitis who presented with retinal hemorrhage. Physicians should consider meningitis in the work-up of patients presenting with retinal hemorrhage.