Praveen Kumar Balne

Persistence of Zika virus in conjunctival fluid of convalescence patients

A widespread epidemic of Zika fever, caused by Zika virus (ZIKAV) has spread throughout the Pacific islands, the Americas and Southeast Asia. The increased incidences of ocular anomalies observed in ZIKAV-infected infants and adults may be associated with the rapid spread of ZIKAV. The objective of this study was to check if ZIKAV could be detected in human tears after the first week of infection. Twenty-nine patients with PCR confirmed ZIKAV infection during the Singapore August 2016 ZIKAV outbreak were enrolled for the study. Detection and quantification of ZIKAV RNA was performed on conjunctival swabs collected from both eyes of these patients at the late convalescent phase (30 days post-illness). Efficiency of viral isolation from swab samples was confirmed by the limit of detection (as low as 0.1 PFU/µL, equivalent to copy number of 4.9) in spiked swabs with different concentrations of ZIKAV (PFU/µL). Samples from three patients were found positive by qRT-PCR for ZIKAV and the viral RNA copy numbers detected in conjunctival swabs ranged from 5.2 to 9.3 copies respectively. ZIKAV could persist in the tears of infected patients for up to 30 days post-illness, and may therefore possess a potential public health risk of transmission.

Zika Virus and Eye

ABSTRACT Zika virus (ZIKV), a mosquito-borne flavivirus, is the latest global health concern. Transmission is mainly via Aedes mosquitoes and the infection can be diagnosed on molecular or serologic testings. It typically causes a mild self-remitting illness of low-grade fever, maculopapular rash, and myalgia, but when severe, it is associated with neurological deficits and congenital structural defects. Ocular manifestations are usually mild like nonpurulent conjunctivitis in adults, though it may be linked to uveitis, maculopathy, and hypertensive iridocyclitis. Ocular signs seem to be more significant in congenital ZIKV—macular pigment mottling, neuroretinal atrophy with macular involvement, iris coloboma, and changes in retinal vasculature are noted in infants with infected mothers. Risk factors include ZIKV infection in first trimester and smaller cephalic diameter at birth. Hence, ophthalmic examination in newborns is now recommended. Currently, prevention and active surveillance are integral as there is no known vaccine, and treatment is only symptomatic.

A distinct cytokines profile in tear film of dry eye disease (DED) patients with HIV infection.

PURPOSE To investigate the tear cytokine profile in HIV patients with dry eye disease (DED) and study the association between the severity of ocular inflammatory complications and tear cytokines levels. We postulate that HIV-mediated inflammation may be the underlying pathogenic mechanism for HIV-associated DED. METHODS The current prospective case-control study compared tear film cytokine profiles in DED patients with HIV infection (n=34) and age/gender-matched DED patients without HIV infection [controls (n=32)]. Participants were recruited from tertiary referral eye care centre and communicable disease clinics, Singapore. Ocular surface health was documented using tear film, Schirmers test, corneal staining, and conjunctival injection measurements. Tear samples were collected using Schirmers strips and analysed for the levels of 41 cytokines using Luminex bead assay. Logistic regression models were performed to determine correlation and significance. RESULTS Among the 41 cytokines analysed, statistically significant differences were observed in the mean values of epithelial growth factor (EGF), growth related oncogene (GRO) and interferon gamma-induced protein 10 (IP-10). EGF and IP-10 levels were higher and GRO levels were lower in the tears of DED patients with HIV infection compared to DED patients without HIV infection. No significant association was found between varying levels of ocular surface parameters and cytokine concentrations in HIV patients with DED (p>0.05). CONCLUSIONS EGF and IP-10 were significantly elevated and GRO levels were lower in the tear profile of HIV patients with DED compared to immunocompetent patients with DED. This study suggests a novel cytokine driven paradigm for ocular inflammatory complications of HIV infection. Additional studies in large organised cohorts can validate the results.

Aqueous humor immune factors and cytomegalovirus (CMV) levels in CMV retinitis through treatment - The CRIGSS study.

PURPOSE This study aims to perform comprehensive longitudinal immune factor analysis of aqueous humor in relation to the aqueous CMV viral load and systemic CD4 counts during treatment of patients with co-infection of HIV and CMVR. METHODS Aqueous humor samples were collected from 17 HIV-positive patients with CMVR scheduled to undergo weekly intravitreal ganciclovir therapy as part of the prospective CMV Retinitis Intravitreal Ganciclovir Singapore Study (CRIGSS) over the course of 1year. Full data across all the 4 time points was obtained and analyzed for CMV DNA viral load, 41 cytokine and chemokine factors using real-time PCR with the FlexMAP 3D (Luminex®) platform and assessed using the Milliplex Human Cytokine® kit. RESULTS The following immune factors (Spearman correlation coefficient r value in parenthesis, p<0.05) showed strong correlation with CMV DNA load in the aqueous - MCP-1 (0.80, IFN-g (0.83), IP-10 (0.82), IL-8 (0.81), fractalkine (0.73), RANTES (0.68) - while the following showed moderate correlation - PDGF-AA (0.58), Flt-3L (0.59) and G-CSF (0.53). Only PDGF-AA revealed a statistically significant negative correlation with serum CD4 levels (r=-0.74). CONCLUSION Immune factors that correlate with intraocular CMV DNA load are identified. They are indicative of a Th1 and monocyte-macrophage mediated response, and exhibit a decreasing trend longitudinally through the course of treatment. These factors may be an important new consideration in individualizing the treatment of patients with CMVR.

Dataset of tear film cytokine levels in dry eye disease (DED) patients with and without HIV infection

The tear film cytokine profiling data in this article was obtained from a prospective case-control study with a sample size of 34 dry eye disease (DED) patients with HIV infection and 32 DED patients without HIV infection, see “A distinct cytokines profile in tear film of dry eye disease (DED) patients with HIV infection” (R. Agrawal, P.K. Balne, A. Veerappan, V.B. Au, B. Lee, E. Loo, A. Ghosh, L. Tong, S.C. Teoh, J. Connolly, P. Tan, 2016) [1]. Tear samples were collected from all the subjects using Schirmer׳s strips and cytokine profiling was done using the Luminex bead based multiplex assay with a panel of 41 analytes. The cytokine level differences in each group of subjects were analyzed using logistic regression models.

Author’s Reply: Zika Virus Infection and Ophthalmic Examination in Newborn

Dear Editor, We would like to thank Beuy Joob and Viroj Wiwanitkit for their interest in our work. They have raised pertinent questions in their article which we would like to address. We concur with the authors’ point that a full routine ophthalmic screening for all newborns is not costeffective. Hence, in our article, what we recommended was an ophthalmic evaluation for infants with suspected congenital Zika Virus (ZIKV) infection within the first month of life. If normal, a follow-up examination (with retinal assessment) should be done at 3 months of age, after which any abnormalities picked up should be referred to an ophthalmologist. This is based on the latest interim guidance for evaluation and management of congenital ZIKV by the United States Centers for Disease Control and Prevention (CDC). While it is challenging to diagnose ZIKV infection due to its mild and self-limiting nature, clinicians should have heightened suspicion to include ZIKV screening in pregnant mothers presenting with nonspecific viral illness and possible exposure, especially if they are in the first trimester of pregnancy. In fact, it is recommended to conduct ZIKV screening in all pregnant patients even if they are asymptomatic, as more than half of infected mothers are asymptomatic. We derived our recommendation after reviewing several reports of congenital ZIKV causing severe damage to the retina, choroid, and macula. Most of these cases have isolated ocular manifestations in infants of mothers with ZIKV infection. Recent articles suggest that ocular anomalies are more likely in the presence of other anomalies constituting congenital Zika syndrome (CZS) and this could highlight the value of ophthalmic screening in such newborns. Moore et al. defined five unique features of CZS which include (1) severe microcephaly with partially collapsed skull, (2) cerebral cortical thinning with subcortical calcifications, (3) macular scarring and focal retinal pigment mottling, (4) congenital contractures, and (5) marked early hypertonia with symptoms of extrapyramidal involvement. Ventura et al. evaluated the retina of 8 infants with CZS (7 infants had positive ZIKV serology on cerebrospinal fluid) and showed that 11 out of 16 eyes (69%) had retinal alterations, and main optical coherence tomography findings in 9 eyes were discontinuation and hyperreflectivity of retinal pigment epithelium in all 9 eyes (100%), retinal thinning in 8 eyes (89%), and choroidal thinning in 7 eyes (78%). Hence, regarding the full ophthalmic screening in newborns, we recommend for infants with suspected congenital ZIKV infection and newborns from mothers with positive ZIKV infection and CZS.

Incidence of Endophthalmitis after Intravitreal Injections: Risk Factors, Microbiology Profile, and Clinical Outcomes

ABSTRACT Purpose: To report the incidence and characteristics of endophthalmitis after intravitreal injections (IVI) of antivascular endothelial growth factor agents and triamcinolone acetonide. Methods: Patients’ medical records were retrospectively reviewed from January 2009 to June 2016, and the incidence, risk factors, clinical and microbiological characteristics of post-IVI endophthalmitis were evaluated. Results: The total number of intravitreal injections given, which included ranibizumab, bevacizumab, and triamcinolone acetonide, was 20,566, of which 27 cases developed endophthalmitis, giving an overall incidence of 0.131%. Significant reduction (p < 0.003) in incidence of endophthalmitis was observed in patients who received prefilled compounded bevacizumab injections (0.050%) compared to multiple bevacizumab injections from a single vial (0.235%). In the triamcinolone acetonide group, the incidence was 0.26%. Staphylococcus species were isolated from 18 cases (67%), and all strains were sensitive to vancomycin. Conclusions: Adherence to strict aseptic protocols and use of prefilled compounded bevacizumab injections reduces the rate of post-IVI endophthalmitis.

Non-Occlusive Retinal Vascular Inflammation and Role of Red Blood Cell Deformability in Birdshot Chorioretinopathy

ABSTRACT Purpose: To investigate differences in red blood cell (RBC) deformability between birdshot chorioretinopathy (BCR) subjects and matched controls, and to postulate its relationship with lack of vascular occlusion in BCR. Methods: In a single center, prospective, non-randomized mechanistic study, blood samples were collected from eight healthy controls and nine BCR patients, and subjected to biochemical and hematological tests, as well as RBC indices assessment using dual-beam optical tweezers. Results: The mean age of the controls was 52.37 ± 10.70 years and BCR patients was 53.44 ± 12.39 years. Initial cell size (Io) for the controls was 8.48 ± 0.25 μm and 8.87 ± 0.31 μm for BCR RBCs (p = 0.014). The deformability index (DI) for the controls was 0.066 ± 0.02 and that for BCR RBCs was 0.063 ± 0.03 (p = 0.441). Conclusion: There was no statistically significant difference in DI between RBCs from BCR and healthy controls. This may explain the rare occurrence of retinal vascular occlusion despite the underlying vasculitic pathophysiology of BCR.

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Fluorescein Labeled Leukocytes for in vivo Imaging of Retinal Vascular Inflammation and Infiltrating Leukocytes in Laser-Induced Choroidal Neovascularization Model

ABSTRACT Purpose: To study the effect of anti-VEGF treatment on retinal inflammation in a laser-induced CNV rodent model. Methods: Leukocytes labeled with 1% sodium fluorescein were injected into the laser-induced CNV (wild type C57BL/6) mice at days 4 (baseline), 7, 14, and 19. At baseline intravitreally 3 mice received 1× PBS, and 3 mice received anti-VEGF. FFA, OCT, and SLO were performed at each time point to assess the CNV pathophysiology and inflammatory response. Results: Fluorescein leakage, SRF, and leukocyte infiltration were observed at baseline in both the groups before injection. From days 7 to 19, leukocyte infiltration and SRF were noted in the 1× PBS group, but limited or no SRF and leukocyte infiltration was observed in the anti-VEGF group. Conclusions: Leukocyte infiltration was established as an in vivo imaging inflammatory marker and along with FFA and OCT showed response to anti-VEGF therapy in laser-induced CNV model.