Assessment of SARS-CoV-2 Immunity in Convalescent Children and Adolescents.

Persistence of protective immunity for SARS-CoV-2 is important against reinfection. Knowledge on SARS-CoV-2 immunity in pediatric patients is currently lacking. We opted to assess the SARS-CoV-2 adaptive immunity in recovered children and adolescents, addressing the pediatrics specific immunity towards COVID-19. Two independent assays were performed to investigate humoral and cellular immunological memory in pediatric convalescent COVID-19 patients. Specifically, RBD IgG, CD4+, and CD8+ T cell responses were identified and quantified in recovered children and adolescents. SARS-CoV-2-specific RBD IgG detected in recovered patients had a half-life of 121.6 days and estimated duration of 7.9 months compared with baseline levels in controls. The specific T cell response was shown to be independent of days after diagnosis. Both CD4+ and CD8+ T cells showed robust responses not only to spike (S) peptides (a main target of vaccine platforms) but were also similarly activated when stimulated by membrane (M) and nuclear (N) peptides. Importantly, we found the differences in the adaptive responses were correlated with the age of the recovered patients. The CD4+ T cell response to SARS-CoV-2 S peptide in children aged <12 years correlated with higher SARS-CoV-2 RBD IgG levels, suggesting the importance of a T cell-dependent humoral response in younger children under 12 years. Both cellular and humoral immunity against SARS-CoV-2 infections can be induced in pediatric patients. Our important findings provide fundamental knowledge on the immune memory responses to SARS-CoV-2 in recovered pediatric patients.

Association of Optical Coherence Tomography Angiography Metrics With Detection of Impaired Macular Microvasculature and Decreased Vision in Amblyopic Eyes: The Hong Kong Children Eye Study.

Importance: Microvascular abnormalities in amblyopia are becoming evident with high-resolution imaging, such as optical coherence tomography angiography (OCT-A); however, to our knowledge, the clinical significance and use of these findings are unknown. Objective: To assess changes in quantitative OCT-A metrics in amblyopic eyes and explore their association with visual acuity in children. Design, Setting, and Participants: This population-based nested case-control study included children aged 6 to 8 years who were consecutively recruited between January 2016 and July 2017 from the population-based Hong Kong Children Eye Study (HKCES) at the Chinese University of Hong Kong Eye Centre. All participants underwent OCT-A with a swept-source OCT and detailed ophthalmic investigations. Macular microvasculature of the superficial capillary plexus was quantified by a customized automated image analysis program. A multivariable linear regression was conducted to evaluate the differences in OCT-A metrics between amblyopic and nonamblyopic eyes after adjustment for all known confounders. Data analysis was conducted from September to November 2018. Main Outcomes and Measures: Differences in OCT-A metric (foveal avascular zone [FAZ]) area, FAZ circularity, vessel density, vessel diameter index, and fractal dimension between amblyopic and nonamblyopic eyes. Results: There were 30 participants with amblyopia (mean [SD] age, 7.57 [1.2] years; 16 girls [53.3%]) and 1045 controls (mean [SD] age, 7.65 [1.0] years; 580 girls [55.5%]) in this cohort. Compared with control eyes, amblyopic eyes had decreased FAZ circularity (-0.058; 95% CI, -0.096 to -0.021, P = .002), decreased fractal dimension (-0.014; 95% CI, -0.024 to -0.003; P = .01), and increased vessel diameter index (0.002; 95% CI, 0.002 to 0.003; P < .001). A difference was not identified between FAZ area and vessel density. LogMAR visual acuity was associated with FAZ circularity (sß, -0.133; P < .001) and vessel diameter index (sß, 0.097; P = .001) but not with vessel density nor FAZ area. Conclusions and Relevance: The results of this population-based study in children supports the presence of macular microvascular abnormalities in amblyopic eyes. Such changes as measured by OCT-A metrics are associated with visual acuity, inferring retinal involvement in the development of amblyopia and suggesting a potential role of quantitative OCT-A metrics in the diagnosis and recognition of amblyopia.

Isolation and identification of bioactive substance 1-hydroxyphenazine from Pseudomonas aeruginosa and its antimicrobial activity.

A strain named as Pseudomonas aeruginosa 2016NX1, which could produce phenazine and cereusitin, was isolated from the root of Millettia specisoa. Phenazines were extracted, isolated and purified by chloroform, thin-layer chromatography, column chromatography and high-performance liquid chromatography. Then the purified materials were identified by analysis of nuclear magnetic resonance. The major yellow component is 1-hydroxyphenazine and the minor blue component is cereusitin A. The tests of antimicrobial activity of yellow component showed that the growth of several common plant pathogenic fungi and bacteria (such as Cochliobolus miyabeanus, Diaporthe citri, Salmonella sp., Klebsiella oxytoca) could be strongly inhibited. This study suggested that Pseudomonas aeruginosa strain 2016NX1 had a significant potential for biological control of phytopathogenic fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, one bioactive substance from Pseudomonas aeruginosa 2016NX1 was identified and its antimicrobial activity was verified. This study demonstrated that one bioactive substance from P. aeruginosa can strongly inhibit the growth of plant pathogenic fungi and bacteria. This study suggested that P. aeruginosa strain 2016NX1 has a significant potential for biological control of phytopathogenic fungi.

Bilateral retinal microglial response to unilateral optic nerve transection in rats.

When retinal ganglion cells undergo apoptosis after optic nerve (ON) injury, microglial cells proliferate and promptly clear the degenerated debris in the ipsilateral retina. However, microglial changes in the contralateral retina have not been fully elucidated. This study characterized the long-term bilateral retinal microglial responses after unilateral ON transection. We analyzed the time course of proliferation and morphology changes of microglial cells, between 3 days and 12 weeks post ON transection, of undisturbed and reactive microglia in bilateral retinas of adult Fischer rats with unilateral ON transection. Microglia in retinas without ON transection were distributed homogeneously and possessed a highly ramified morphology, as judged by immunohistochemistry for ionized calcium-binding adapter molecule 1 (Iba1). After ON transection, microglia density in the ipsilateral retina increased gradually from 3 days to 2 weeks, and decreased from 3 weeks to 12 weeks, along with dramatic inverted alteration of process branch points of microglia in the ganglion cell layer (GCL). Transformation of ramified microglia into ameboid-like macrophages with few branching processes was observed in the ipsilateral retina from 1 week to 3 weeks. Though an increase in microglial density was weak in the contralateral retina and could only be statistically detected in the central retina, the morphological alteration over time was obvious and similar to that of the ipsilateral retina. In the inner plexiform layer (IPL), cell density and morphological changes of microglia in both the ipsilateral and contralateral retina were not prominent. These findings indicates that, though proliferation of microglial cells is weak in the contralateral retina after unilateral ON transection, conspicuous alterations in microglial morphology occur bilaterally. These suggest that using the contralateral retina as a control in studies of retinal degeneration should be considered with caution.

Rapid and cost-effective molecular diagnosis using exome sequencing of one proband with autosomal dominant congenital cataract.

PURPOSE: Due to high genetic heterogeneity, to exclude known mutations and map novel mutations in autosomal dominant congenital cataract (ADCC) using conventional candidate gene screening requires laborious laboratory work. We attempted to use a cost-effective exome sequencing strategy to identify disease-causing mutations in an ADCC pedigree. METHODS: An ADCC pedigree affected by nuclear cataract and 200 unrelated senile cataract controls were recruited and given comprehensive ophthalmic examination. Whole exome of the proband of the family was captured by the Illumina TruSeq Exome Enrichment Kit, followed by sequencing using Illumina HiSeq 2000 sequencer. Validation was performed by direct sequencing. RESULTS: The whole exome, including all exons of known ADCC disease-causing genes, was screened for possible disease-causing mutations. A recurrent missense mutation c.773C>T (p.S258F) in exon 2 of the gap junction protein alpha 8 gene (GJA8) was identified in the proband with nuclear cataract. The result was confirmed by direct sequencing. The mutation showed complete co-segregation with the disease phenotype in the family but was not observed in unrelated unaffected controls. CONCLUSION: By successfully sequencing whole exome of only one proband and identifying a GJA8 mutation in one ADCC pedigree, the current study demonstrated that exome sequencing could serve as a rapid, robust, and cost-effective approach in clinical diagnosis and disease-causing gene discovery for ADCC.

FPR1 interacts with CFH, HTRA1 and smoking in exudative age-related macular degeneration and polypoidal choroidal vasculopathy.

PURPOSE: To determine the genetic association of an inflammation-related gene, formyl peptide receptor 1 (FPR1), in exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). METHODS: The coding region of FPR1 gene was sequenced in 554 unrelated Chinese individuals: 155 exudative AMD patients, 179 PCV patients, and 220 controls. Interactions and combined effects of FPR1 with complement factor H (CFH), high temperature requirement factor A1 (HTRA1), and smoking were also investigated. RESULTS: A total of 28 polymorphisms in FPR1 were identified. Single nucleotide polymorphisms (SNP) rs78488639 increased the risk to exudative AMD (P=0.043) and PCV (P=0.016), whereas SNP rs867229 decreased the risk to exudative AMD (P=0.0026), but not PCV. Homozygous G allele of rs1042229 was associated with exudative AMD (P=0.0394, odds ratio (OR)=2.27, 95% confident interval: 1.08-4.74), but not with PCV. Exudative AMD, but not PCV, was associated with the heterozygous genotypes of rs2070746 (P=0.019, OR=0.57) and rs867229 (P=0.0082, OR=0.54). Significantly, interactions were identified among FPR1 rs78488639, CFH rs800292, and HTRA1 rs11200638 in both exudative AMD and PCV. Combined heterozygous risk alleles of CFH rs800292 GA and FPR1 rs78488639 CA were posed to PCV (P=2.22 × 10(-4), OR=10.47), but not exudative AMD. Furthermore, FPR1 rs78488639 CA combining with HTRA1 rs11200638 and smoking was also predisposed risks to exudative AMD and PCV. CONCLUSION: FPR1 is associated with exudative AMD and PCV in a Hong Kong Chinese cohort. FPR1 rs78488639 interacted with CFH rs800292, HTRA1 rs11200638, and smoking, enhancing risk to exudative AMD and PCV.

Multimodal retinal imaging in a Chinese kindred with familial amyloid polyneuropathy secondary to transthyretin Ile107Met mutation.

OBJECTIVE: To investigate the ocular phenotype and gene mutation of a Chinese pedigree with familial amyloid polyneuropathy (FAP) and vitreous amyloidosis. METHODS: A Chinese pedigree with familial amyloid polyneuropathy and vitreous amyloidosis was recruited. Combined phacoemulsification, vitrectomy and intraocular lens implantation were performed on the right eye of the index patient. Ophthalmic investigations were performed before and after surgery. The DNA from the pedigree was sequenced for the transthyretin (TTR) gene. RESULTS: After vitrectomy, the best-corrected visual acuity of the patient improved from counting finger to 20/20. Red-free confocal ophthalmoscopy demonstrated perifoveal ring and several perivessel white sheaths. Optical coherence tomography (OCT) revealed cotton wool like reflections on the vitreoretinal interface. Electroretinogram and autofluorescence was normal. Amyloid was present in the vitreous specimen. A substitution of T to G at nucleotide 381 in exon 4 of TTR DNA (Ile107Met) was found. This mutation co-segregated with phenotype in the pedigree and was not detected in 200 controls. CONCLUSIONS: TTR Ile107Met mutation is associated with vitreous amyloidosis and FAP. OCT and red-free imaging are helpful in identifying amyloid deposits in the retina.

Contribution of SNRNP200 sequence variations to retinitis pigmentosa.

PURPOSE: Mutations in the SNRNP200 gene have been reported to cause autosomal dominant retinitis pigmentosa (adRP). In this study, we evaluate the mutation profile of SNRNP200 in a cohort of southern Chinese RP patients. METHODS: Twenty adRP patients from 11 families and 165 index patients with non-syndromic RP with mixed inheritance patterns were screened for mutations in the mutation hotspots of SNRNP200. These included exons 12-16, 22-32, and 38-45, which covered the two helicase ATP-binding domains in DEAD-box and two sec-63 domains. The targeted regions were amplified by polymerase chain reaction and analyzed by direct DNA sequencing, followed by in silico analyses. RESULTS: Totally 26 variants were identified, 18 of which were novel. Three non-synonymous variants (p.C502R, p.R1779H and p.I698V) were found exclusively in patients. Two of them, p.C502R and p.R1779H, were each identified in one simplex RP patient, whereas p.I698V occurred in one patient with unknown inheritance pattern. All three residues are highly conserved in SNRNP200 orthologs. Nevertheless, only p.C502R and p.R1779H were predicted to affect protein function by in silico analyses, suggesting these two variants are likely to be disease-causing mutations. Notably, all mutations previously identified in other study populations were not detected in this study. CONCLUSIONS: Our results reveal a distinct mutation profile of the SNRNP200 gene in a southern Chinese cohort of RP patients. The identification of two novel candidate mutations in two respective patients affirmed that SNRNP200 contributes to a proportion of overall RP.

Skeletal muscle-specific overproduction of constitutively activated c-Jun N-terminal kinase (JNK) induces insulin resistance in mice.

AIMS/HYPOTHESIS: Although skeletal muscle insulin resistance has been associated with activation of c-Jun N-terminal kinase (JNK), whether increased JNK activity causes insulin resistance in this organ is not clear. In this study we examined the metabolic consequences of isolated JNK phosphorylation in muscle tissue. METHODS: Plasmids containing genes encoding a wild-type JNK1 (WT-JNK) or a JNK1/JNKK2 fusion protein (rendering JNK constitutively active; CA-Jnk) were electroporated into one tibialis anterior (TA) muscle of C57Bl/6 mice, with the contralateral TA injected with an empty vector (CON) to serve as a within-animal control. RESULTS: Overproduction of WT-JNK resulted in a modest (~25%) increase in phosphorylation (Thr(183)/Tyr(185)) of JNK, but no differences were observed in Ser(307) phosphorylation of insulin receptor substrate 1 (IRS-1) or total IRS-1 protein, nor in insulin-stimulated glucose clearance into the TA muscle when comparing WT-JNK with CON. By contrast, overexpression of CA-Jnk, which markedly increased the phosphorylation of CA-JNK, also increased serine phosphorylation of IRS-1, markedly decreased total IRS-1 protein, and decreased insulin-stimulated phosphorylation of the insulin receptor (Tyr(1361)) and phosphorylation of Akt at (Ser(473) and Thr(308)) compared with CON. Moreover, overexpression of CA-Jnk decreased insulin-stimulated glucose clearance into the TA muscle compared with CON and these effects were observed without changes in intramuscular lipid species. CONCLUSIONS/INTERPRETATION: Constitutive activation of JNK in skeletal muscle impairs insulin signalling at the level of IRS-1 and Akt, a process which results in the disruption of normal glucose clearance into the muscle.

Identification of hemopexin in tear film.

Human tear fluid is a complex mixture of aqueous lipids, proteins, enzymes, and other biochemical and cellular elements. By conventional comparative proteomic approaches, we investigated the proteome in human tear fluid and compared the tear protein profile of normal control subjects with that of patients suffering from the ocular inflammatory disease vernal keratoconjunctivitis (VKC). Collected tear samples were directed to two-dimensional polyacrylamide gel electrophoresis protein separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide identification. Six differentially expressed proteins-interleukin 4, phospholipase A2, albumin, lactoferrin, hemopexin, and lipocalin-were displayed. Hemopexin had not been reported previously in tear film. Enzyme-linked immunosorbent assay confirmed that hemopexin concentrations were significantly higher in VKC tear samples and increased with disease stages. The results implied clinical interest of hemopexin in the tear proteome and eye diseases.

Predictors of atypical birefringence pattern in scanning laser polarimetry.

AIM: To evaluate predictors of atypical birefringence patterns (ABP) observed in scanning laser polarimetry. METHODS: A total of 179 eyes from 82 normal subjects and 97 glaucoma patients were included. The retinal nerve fibre layer in each eye was imaged sequentially with GDx variable corneal compensation (VCC) and GDx enhanced corneal compensation (ECC) (Carl Zeiss Meditec, Dublin, California). The associations between the typical scan score (TSS) and age, axial length, spherical error, parapapillary atrophy (PPA) and visual-field mean deviation (MD) were evaluated with univariate and multivariate regression analyses. RESULTS: 23.5% (42/179) and 5.0% (9/179) of subjects had ABP (TSS<80) with GDx VCC and GDx ECC, respectively. For both GDx VCC and ECC, the TSS was significantly correlated with age, axial length, spherical error and PPA, but not with visual-field MD. After adjusting the effect of covariates, the axial length/spherical error and PPA were significantly associated with GDx VCC TSS, whereas the axial length/spherical error was the only predictor for GDx ECC TSS. Myopic eyes were more likely to develop ABP in both GDx VCC and ECC. CONCLUSIONS: Axial length or spherical error is a significant predictor for ABP with both GDx VCC and GDx ECC. Caution should be exercised in interpreting the results of scanning laser polarimetry in eyes with a long axial length or myopia.

Quantitative assessment of lens opacities with anterior segment optical coherence tomography.

AIM: To evaluate the reliability of lens density measurement with anterior segment optical coherence tomography (OCT) and its association with the Lens Opacity Classification System Version III (LOCS III) grading. METHODS: Fifty-five eyes from 55 age-related cataract patients were included. One eye from each subject was selected at random for lens evaluation. After dilation, lens photographs were taken with a slit lamp and graded against the LOCS III standardised condition. Anterior segment OCT imaging was performed on the same eyes with a high-resolution scan. The association between the anterior segment OCT nucleus density measurement and LOCS III nuclear opalescence (NO) and nuclear colour (NC) scores was evaluated with the Spearman correlation coefficient. Anterior segment OCT measurement precision, coefficient of variation (CVw), and intraclass correlation coefficient (ICC) were calculated. RESULTS: The mean NO and NC scores were 3.39 (SD 1.10) and 3.37 (SD 1.27), respectively. Significant correlations were found between anterior segment OCT nuclear density measurements and the LOCS III NO and NC scores (r = 0.77 and 0.60, respectively, both with p<0.001). The precision, CVw and ICC of anterior segment OCT measurement were 2.05 units, 4.55% and 0.98, respectively. CONCLUSION: Anterior segment OCT nucleus density measurement is reliable and correlates with the LOCS III NO and NC scores.

A novel mutation in CRYBB2 responsible for inherited coronary cataract.

PURPOSE: To study the molecular pathogenesis of a Chinese family with coronary form of cataract. METHODS: One Chinese three-generation family with inherited coronary cataract phenotype was recruited. Five affected and seven unaffected family members attended our study. Genome-wide linkage analysis was applied to map the disease loci, and two candidate genes from a locus on chromosome 1 and a locus on chromosome 22 were sequenced for mutation identification. Software at the Expasy proteomics server was utilized to predict the mutation effect on proteins. RESULTS: Whole genome linkage analysis indicated some regions on chromosome 1, 10, and 22, with LOD score values greater than 1. Within these loci, the GJA8 and CRYBB2 genes, located in the two loci with the highest LOD score of 1.51 on chromosomes 1 and 22, respectively, were sequenced. A novel mutation c.92C>G in exon 2 of CRYBB2 causing S31W was identified in all five patients. It was not found in 95 unrelated controls. This missense sequence alteration likely enhanced the local solubility. Around the mutation site, a lipocalin signature motif was predicted by ScanProsite. CONCLUSIONS: A novel disease-causing mutation S31W in CRYBB2 was identified in a Chinese cataract family. It is the first reported mutation for coronary cataract. Functional characterization should be carried out to evaluate the biological effects of this mutant.

Effects of scan circle displacement in optical coherence tomography retinal nerve fibre layer thickness measurement: a RNFL modelling study.

OBJECTIVE: To study the effect of optical coherence tomography (OCT) scan circle displacement on retinal nerve fibre layer (RNFL) measurement errors using cubic spline models. METHODS: Forty-nine normal subjects were included in the analysis. In one randomly selected eye in each subject, RNFL thickness around the optic disc was measured by taking 16 circular scans of different sizes (scan radius ranged from 1 to 2.5 mm). The RNFL profile in each eye was constructed with a mathematical model using a smoothing spline approximation. Scan circle (diameter 3.4 mm) RNFL measurements (total average, superior, nasal, inferior, and temporal RNFL thicknesses) obtained from eight directions (superior, superonasal, nasal, inferonasal, inferior, inferotemporal, temporal, and superotemporal) displaced at different distances (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mm) from the disc centre were then computed by a computer program and compared to the 'reference standard' where the scan circle is centred at the optic disc. RNFL measurement error was calculated as the absolute of (RNFL thickness(displaced) - RNFL thickness(reference standard)). RESULTS: The respective mean average, superior, nasal, inferior, and temporal RNFL measurement errors were 2.3+/-2.0, 4.9+/-4.5, 4.1+/-3.8, 6.2+/-7.6, and 3.8+/-3.5 microm upon 0.1 mm scan circle displacement, and 12.1+/-11.4, 27.8+/-18.4, 21.7+/-18.6, 34.8+/-22.9, and 15.2+/-10.7 microm upon 0.7 mm scan circle displacement. Significant differences of average and quadrant RNFL thicknesses were evident between centred and displaced scan circle measurements (all with P<0.001). RNFL measurement error increased in a monotonic fashion with increasing distance away from the disc and the change was direction-dependent. CONCLUSIONS: RNFL measurement error varies with the direction and distance of scan displacement. The superior and the inferior RNFL measurements are most vulnerable to scan displacement errors, whereas the average RNFL thickness is the least susceptible. Obtaining a well-centred scan is essential for reliable RNFL measurement in OCT.

Prenatal screening for retinoblastoma in Hong Kong.

We report the first use in Hong Kong of molecular techniques to screen prenatally for retinoblastoma and review 17 cases of retinoblastoma seen at the Hong Kong Eye Hospital from 2001 to 2006. A pregnant couple whose first child had retinoblastoma requested prenatal screening for retinoblastoma during their second pregnancy in 2000. Whole RB1 coding gene sequencing was performed on peripheral blood cells taken from family members and cultured amniocytes collected from the foetus during the 14th week of gestation. No RB1 gene mutations were found in the amniocyte samples and at birth the baby had no evidence of ocular tumours. During 5 years of follow-up the child remained healthy with intact visual function. Prenatal diagnosis of retinoblastoma alleviates parental stress and improves the perinatal care of affected family members.

Differential roles of phosphatidylinositol 3-kinase/akt pathway in retinal ganglion cell survival in rats with or without acute ocular hypertension.

Intraocular pressure (IOP) elevation has often been used as an experimental model to study mechanisms underlying retinal ganglion cell (RGC) death associated with ocular ischemic injury and glaucoma. The aim of the present study, using both in vitro and in vivo approaches, was to investigate the role of phosphatidylinositol 3-kinase (PI3K)/akt pathway in RGC viability in normal rats and rats following transient IOP elevation. For in vivo studies, pathway inhibitors were administered intravitreally on days 3, 9, and 15 post-2-h IOP elevation at 110 mm Hg. Toward the end of the 3-week examination period, the fluorescent dye Fluorogold was used to retrogradely label surviving RGCs. In order to examine the role of macrophages that were recruited into the eye following the pathway inhibition, clodronate liposomes were used to deplete phagocytic cells in the eye. PI3K/akt pathway activity and location in the retina were examined using Western blot and immunohistochemistry, respectively. Here we showed that PI3K/akt inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and KY12420 at low concentrations (2 microM or 20 microM) did not influence RGC survival but caused RGC loss at high concentration (200 muM) in retinal explants derived from intact rats. In contrast, both LY294002 and KY12420 at 20 microM led to RGC loss in retinal explants derived from IOP-elevated eyes. A detrimental action of phagocytic cells on RGC survival was also seen in these retinas. In vivo results confirmed the detrimental actions of PI3K/akt inhibition and macrophages on RGC survival in IOP-elevated, but not intact eyes even with high concentration of LY294002. Low level of PI3K/akt activity was detected in the ganglion cell layer (GCL) in intact retina. Acute IOP elevation activated PI3K/akt pathway in the inner nuclear layer and GCL including RGCs. This study thus demonstrates that PI3K/akt pathway mediates RGC survival after IOP elevation but not under normal condition.

An in vitro study on the compatibility and concentrations of combinations of vancomycin, amikacin, and dexamethasone in human vitreous.

PURPOSE: To investigate the precipitation process of a mixture of vancomycin, amikacin, and dexamethasone by equilibrium dialysis and its subsequent effect on the levels of available-free antibiotics and steroid. METHODS: Concentrations of amikacin, vancomycin, and dexamethasone in an equilibrium dialysis chamber were measured during the equilibrium process by high-performance liquid chromatography and fluorescence polarisation immunoassay. Vitreous were used as the medium of dialysis, with the three medications prepared in normal saline (NS) and balanced salt solution plus (BSS Plus) separately. RESULTS: Amikacin showed no measurable loss in NS or BSS Plus, either alone or when mixed with vancomycin or dexamethasone. Vancomycin showed minimal loss in BSS Plus, either alone or when mixed with amikacin or dexamethasone. Dexamethasone showed a median loss of 16 and 15% when incubated alone in NS and BSS Plus, respectively, at 48 h. When mixed with vancomycin or amikacin in BSS Plus, it showed a median loss of 13 and 12%, respectively, at 48 h. There was no statistically significant difference in the loss of dexamethasone under various conditions. In equilibrium dialysis in vitreous, amikacin, vancomycin, and dexamethasone reached equilibrium within 24 h and with no loss up to 192 h. There was no difference observed when the medications were prepared in NS or BSS Plus. CONCLUSIONS: Both amikacin and vancomycin did not show precipitation or decrease in concentration in NS or BSS Plus. Dexamethasone showed relatively small percentage loss. As a result, treatment of endophthalmitis with vancomycin and amikacin combination is preferred.