Polymerase Chain Reaction and Goldmann-Witmer Coefficient to Examine the Role of Epstein-Barr Virus in Uveitis.

PURPOSE: To use polymerase chain reaction (PCR) and Goldmann-Witmer Coefficient (GWC) calculation to search for evidence that Epstein-Barr virus (EBV) causes uveitis. METHODS: A prospective cross-sectional study where participants with positive multiplex EBV PCR results were further investigated by: 1) real-time PCR for EBV viral loads (VL) and 2) EBV GWC. RESULTS: Eleven of 106 consecutive uveitis patients (10.4%) had positive multiplex PCR for EBV on aqueous humor sampling and 7/11 (63.6%) were HIV-positive. Only 4/10 (40%) cases had detectable intraocular EBV VLs which were always lower than the blood or plasma VL. EBV GWC was negative in all 10 cases tested. In 9/11 (81.8%) of these cases an alternative, more plausible cause of uveitis was identified. CONCLUSION: We found no evidence of active intraocular replication or antibody production to prove that EBV caused uveitis in these cases. In most cases an alternative treatable cause of uveitis was identified.

The Etiology of Intraocular Inflammation in HIV Positive and HIV Negative Adults at a Tertiary Hospital in Cape Town, South Africa.

PURPOSE: To describe the patterns of uveitis in South Africa. METHODS: Prospective cross-sectional study. RESULTS: One hundred and six patients were enrolled and 37.7% had human immune-deficiency virus (HIV) infection. Anterior and panuveitis occurred most frequently. Infectious, non-infectious and idiopathic uveitis were diagnosed in 66.0%, 17.0% and 17.0% of all cases, respectively. Eighty percent of HIV+ cases had infectious uveitis. Overall, intraocular tuberculosis (IOTB), herpetic and syphilitic uveitis were the commonest infectious causes. Sarcoidosis and HLA-B27-associated uveitis were the commonest non-infectious causes. In anterior uveitis, HIV+ cases most frequently had probable IOTB, syphilitic or idiopathic uveitis while HIV- cases had possible IOTB, idiopathic or HLA-B27-associated uveitis. In panuveitis, HIV+ cases mostly had syphilis, probable IOTB, toxoplasma and varicella-zoster virus whereas HIV- cases mostly had possible IOTB, sarcoidosis and idiopathic uveitis. CONCLUSION: Infectious uveitis is common in South Africa, especially amongst HIV+ patients. Causes of anterior and panuveitis differ between HIV+ and HIV- patients.

Polymerase Chain Reaction and Goldmann-Witmer Coefficient Testing in the Diagnosis of Infectious Uveitis in HIV-Positive and HIV-Negative Patients in South Africa.

PURPOSE: To use polymerase chain reaction (PCR) and Goldmann-Witmer coefficient (GWC) calculation to diagnose infectious uveitis. METHODS: Prospective cross-sectional study. RESULTS: Twenty-seven of 106 patients had positive PCR and/or GWC results on aqueous humor (AH) sampling and 15 of 27 (55.6%) were HIV-positive. Patients with non-anterior uveitis (NAU) were more likely to be HIV+ (p = 0.005). More than 1 possible pathogen was identified in 9 of 27 patients of whom 7 were HIV+. The final clinical diagnosis was discordant with AH findings in 9 of 27 cases. A positive EBV PCR result was associated with a discordant diagnosis (p = 0.001). All cases of herpetic anterior uveitis (42.9% HIV+) tested PCR-/GWC+ while all cases of herpetic NAU tested PCR+/GWC- (83.3% HIV+). All rubella virus cases were PCR+/GWC+. CONCLUSION: PCR is useful to diagnose herpetic NAU in HIV+ patients while GWC is useful to diagnose herpetic anterior uveitis.

Potential Diagnosis of Vitreoretinal Lymphoma by Detection of MYD88 Mutation in Aqueous Humor With Ultrasensitive Droplet Digital Polymerase Chain Reaction.

Importance: The diagnostic workup of patients suspected of having vitreoretinal lymphoma (VRL) is primarily based on vitreous fluid analysis, including the recently emerging myeloid differentiation primary response gene 88 (MYD88) mutation analysis. Aqueous humor paracentesis is a relatively less invasive and safer procedure than taking vitreous fluid specimens, and aqueous humor-based MYD88 mutation analysis would provide an additional liquid biopsy tool to diagnose and monitor patients with VRL. Objective: To investigate whether the detection of MYD88 L265P by highly sensitive droplet digital polymerase chain reaction (ddPCR) is feasible in the vitreous fluid and aqueous humor of patients with VRL. Design, Setting, and Participants: This cohort study includes aqueous humor and vitreous fluid samples from patients with VRL who were treated at the University Medical Center Utrecht, in Utrecht, the Netherlands, from August 2005 to August 2017. Ocular fluids were randomized and masked before MYD88 L265P analysis, which was performed using an in-house validated ddPCR platform. Patients with uveitis were included as a comparison group. Main Outcomes and Measures: The presence of MYD88 L265P mutation detected by ddPCR in AH and VF. Results: The study included 96 samples from 63 individuals, including 23 patients with VRL (of whom 10 were female and 13 male, with a mean [SD] age of 72 [7.3] years) and 40 individuals with uveitis (of whom 23 were female and 17 male, with a mean [SD] age of 58 [20.9] years). In 17 of 23 patients with VRL (74%), MYD88 L265P was detected; it was not detected in any of the patients with uveitis. It was detectable in both vitreous fluid and aqueous humor samples. In the paired samples, the mutation was detected in 8 of 9 aqueous humor samples (89%) of the MYD88 L265P-positive vitreous fluid samples. In vitreous fluid, the MYD88 ddPCR test showed a sensitivity of 75% (95% CI, 50%-92%) and a positive predictive value of 100%; in aqueous humor, sensitivity was 67% (95% CI, 42%-92%), and positive predictive value was 100%. Specificity was 100% in both fluids. After treatment, the mutation was no longer detectable in any ocular fluids. Conclusions and Relevance: The high concordance between aqueous humor and vitreous fluid samples suggests that use of the easily accessible aqueous humor is nearly as informative as vitreous fluid in the identification of key somatic mutations in patients with VRL. This approach may provide an additional minimally invasive tool for accurate diagnosis, detection of recurrence, and monitoring of treatment.

Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity.

Human beta1-coronavirus (ß1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal ß1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. ß1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus.

Intraocular and serum cytokine profiles in patients with intermediate uveitis.

PURPOSE: To study the intraocular and serum cytokine and chemokine profile in patients with intermediate uveitis (IU) at various stages of inflammatory activity. METHODS: Institutional, prospective association study. Paired aqueous humor (AqH) and serum samples were collected from 36 consecutive IU patients and 10 controls. The concentrations of interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor (TNF)-α, CC--chemokine ligand 5/regulated upon activation normal T-cell expressed, and secreted (CCL5/RANTES), CC--chemokine ligand 3/macrophage inflammatory protein 1alpha (CCL3/MIP-1α), CCL4/MIP-1ß, and CC--chemokine ligand 2/monocyte chemotactic protein--1 (CCL2/MCP-1) were measured in both AqH and serum by multiplex immunoassay. Main outcome measures were serum and intraocular levels of the analyzed cyto- and chemokines. RESULTS: Patients with IU had higher serum levels of TNF-α than non-uveitic controls (p<0.0001), whereas their AqH TNF-α levels did not show a difference (p=0.323). IU patients had higher intraocular levels of IL-1ß, IL-6, IL-8, IL-10, IL-12p70 and CCL2/MCP-1 than the controls (p=0.020, 0.001, <0.0001, 0.005, 0.003, and 0.003, respectively). Active stages of IU were characterized by higher levels of IL-6, IL-8, CCL5/RANTES and CCL2/MCP-1 (p=0.003, <0.0001, 0.033, and 0.033, respectively). Higher levels of IL-6 and IL-8 were found in IU patients with cystoid macular edema (CME) compared to non-CME IU patients (p=0.026 and 0.012, respectively). Significant positive correlations between various observed mediators were present in the AqH of IU patients only. CONCLUSIONS: Significantly elevated concentrations of multiple intraocular cytokines were found in IU patients, especially IL-6 and IL-8 in those with CME and active disease. In serum elevated TNF-α levels were observed in IU patients. Our findings improve the understanding of the pathogenesis of IU and contribute to the identification of factors which may contribute to the activity of IU.

Intraocular interleukin-17 and proinflammatory cytokines in HLA-A29-associated birdshot chorioretinopathy.

PURPOSE: To determine the levels of 23 immune mediators in paired aqueous humor (AqH) and serum samples from patients with birdshot chorioretinopathy (BSCR). DESIGN: Single-centre case-control study. METHODS: A multiplex immunoassay was used to determine the levels of 23 immune mediators (T-cell, proinflammatory, and vascular-active mediators) in paired AqH and serum of 16 BSCR patients. The AqH of 11 age-related cataract controls served as controls. RESULTS: AqH levels of the T-cell mediators interleukin (IL)-2 (P=.044) and IL-17 (P=.039) and proinflammatory mediators IL-1ß (P=.032), IL-6 (P=.034), and tumor necrosis factor α (P=.041) were elevated compared with that of age-related cataract controls. The elevated intraocular levels of IL-1ß, IL-17, and tumor necrosis factor α in BSCR samples were higher than their concurrent serum levels. A significant positive correlation of intraocular mediators was noted between IL-17 and both IL-2 (r=0.744; P<.0001) and IL-23 (r=0.921; P<.0001) and between IL-2 and IL-23 (r=0.776; P<.0001). AqH levels of vascular-active mediators were not distinct between the groups. CONCLUSIONS: BSCR patients have elevated intraocular levels of proinflammatory and T cell-associated cytokines. Our results suggest the novel pathogenic concept that BSCR is an autoimmune inflammatory disease restricted to the eye and associated with elevated IL-17.

Identification of new pathogens in the intraocular fluid of patients with uveitis.

PURPOSE: To determine infectious causes in patients with uveitis of unknown origin by intraocular fluids analysis. DESIGN: Case-control study. METHODS: Ocular fluids from 139 patients suspected of infectious uveitis, but negative for herpes simplex virus, varicella-zoster virus, cytomegalovirus, and Toxoplasma gondii by polymerase chain reaction and/or antibody analysis in intraocular fluids, were assessed for the presence of 18 viruses and 3 bacteria by real-time polymerase chain reaction (PCR). The ocular fluids from 48 patients with uveitis of known etiology or with cataract were included as controls. RESULTS: Positive PCR results were found for Epstein-Barr virus, for rubella virus, and for human herpesvirus 6 each in 1 patient and for human parechovirus in 4 patients. Of the human parechovirus-positive patients, 1 was immunocompromised and had panuveitis. The other 3 patients were immunocompetent and had anterior uveitis, all with corneal involvement. CONCLUSIONS: Human parechovirus might be associated with infectious (kerato)uveitis.

Distinct cytokine and chemokine profiles in the aqueous of patients with uveitis and cystoid macular edema.

PURPOSE: To investigate the global cytokine and chemokine expression pattern in the aqueous humor of uveitis patients and relate them to clinical features. DESIGN: Cross-sectional study. METHODS: In 31 aqueous humor samples from uveitis patients, the concentration of mediators was measured by a multiplex immunoassay. Eleven control samples were included. RESULTS: Uveitis samples had higher levels of interleukin-6, interleukin-8, soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule (sVCAM), and interferon-inducible protein-10 (IP-10) than nonuveitis controls. Active uveitis samples had higher levels of interleukin-6, interleukin-10, interferon-gamma, sVCAM, regulated on activation, normal T cell expressed and secreted (RANTES), and IP-10 than quiescent uveitis samples. Infectious uveitis was associated with higher levels of interleukin-10 than noninfectious uveitis (P < .03 for all subgroups). No significant differences were found between cystoid macular edema (CME) and non-CME samples. CONCLUSIONS: Elevated levels of specific mediators were found in active and in infectious uveitis, but not in CME. Mediator profiles might lead to a better understanding of the pathogenesis of uveitis.

Three-color flow cytometry detection of virus-specific CD4+ and CD8+ T cells in the cat.

We describe a three-color flow cytometry assay for the detection of virus-specific CD4+ and CD8+ T cells in the cat. The assay is based upon detection of intracellular TNFalpha using the cross-reactive mAb 6401.1111, raised against the human cytokine. Allophycocyanin-conjugated mAb 6401.1111 specifically stained feline TNFalpha-producing murine cells and also Staphylococcus aureus Enterotoxin B-stimulated feline T cells, thus providing formal evidence for cross-reactivity. By using the anti-TNFalpha mAb in combination with PE- and FITC-conjugated mAbs against feline CD4 and CD8, respectively, antiviral CD4+ and CD8+ T cells could be identified in the peripheral blood and in the spleens of feline infectious peritonitis virus-infected cats. Moreover, feline calicivirus (FCV)-specific CD4+ T cells were detected in the spleens of FCV-vaccinated cats. As antigen-presenting cells (APCs), we used immortalized autologous fibroblast cell lines, PBMC or splenocytes. A straightforward protocol, in which splenocyte preparations served both as APCs and effector cells, consistently yielded best results. The assay will permit further studies of the cellular immune responses in cats during natural and experimental viral infections. It will contribute to vaccine development against feline viruses by facilitating the identification of T cell antigens and epitopes, and by allowing the quantitative detection of virus-specific T cells after vaccination. Furthermore, the assay will add to the value of those systems in which viral infections of the cat serve as models for human disease.