This is a retrospective single-center observational study. This study followed the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Karsa Husada General Hospital (Ref. No. 072/251/102.13/2022). Data were obtained from the ophthalmology clinic database in Karsa Husada General Hospital, Batu, Indonesia.

One hundred eight eyes belonging to 74 patients were treated with three doses of IVR injection, at one month intervals, for DME treatment at the Karsa Husada General Hospital ophthalmology clinic between January 2021 and December 2022. The inclusion criteria were the presence of DME before therapy, no history of ocular surgery/treatment within the previous 10 weeks, and no macular involvements (such as retinal vein occlusion and age-related macular degeneration).

Before injection and at monthly follow-ups, patients underwent comprehensive ophthalmologic examinations, including measurement of best-corrected visual acuity (BCVA), intraocular pressure (IOP) evaluation with Goldmann applanation tonometry, as well as biomicroscopic examination of the anterior segment and funduscopy. Intravitreal injections were conducted monthly with 0.5mg/0.05mL ranibizumab (Patizra, Novartis). A topical antibiotic was applied after the IVR injection for six days.

Both horizontal A and vertical B scan images of the macular region concentrated at the fovea were examined using spectral domain optical coherence tomography (SD-OCT) (Spectralis, Heidelberg Engineering, Heidelberg, Germany). Structural outcomes were examined by evaluating retinal layer thickness, choroidal layer thickness and subretinal fluid area. Individual retinal layer and choroidal layer thickness were measured manually according to the literature4,12. Briefly, the OCT image was loaded into ImageJ. The scale bar provided in the OCT image was used as a standard reference. The retinal layer thickness was then manually measured by outlining each retinal layer in the parafoveal ring (the average retinal thickness in the superior, inferior, nasal, and temporal regions was used), while choroidal layer thickness was quantified in the central fovea (Figure 1A). Basically, the scale bar was converted into the distance in pixels. Then unknown distance in pixels outlined in each layer will be automatically quantified to the corresponding scale by ImageJ.

Figure 1.

Retina and choroid segmentation

(A) Retina and choroid segmentation using SD-OCT; (B) Structural changes before and after treatment in patients who received 3 doses of IVR injections due to DME; (C) Receiver operating characteristics (ROC) curve analysis. ROC curve of INL, ONL, TRLT, ORLT, and FA for the assessment of ranibizumab effectiveness. FA, fluid area; IRLT, inner retinal layer thickness; ORLT, outer retinal layer thickness; TRLT, total retinal layer thickness; ICLT, inner choroidal layer thickness; OCLT, outer choroidal layer thickness; TCLT, total choroidal layer thickness; INL, inner retina layer; ONL, outer retina layer.

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The subretinal fluid area was evaluated by manually outlining the areas using ImageJ software (Figure 1A). The outer choroid layer thickness (OCLT, yellow line) was measured from the inner boundary of the choroid scleral junction to the innermost point of the choroidal large blood vessel observed closest to the fovea centralis. The inner choroidal layer thickness (ICLT, red line) was obtained by subtracting the OCLT from the total choroid layer thickness (TCLT, white line).

All continuous variables were expressed as the mean±standard deviation, while categorical variables were expressed as percentages. The variables between the two groups (baseline vs. final visit) were compared using either an independent or dependent t-test, depending on the nature of the data. Correlations between visual and structural outcomes were evaluated using Pearson correlation analysis. r>0.5 was considered positively correlated and r=0.3 to 0.5 as moderate positively correlated. The values of retinal changes were used to generate ROC (receiver operating characteristic) curves using the GraphPad Prism version 9 in order to determine the area under the curve (AUC). The optimal cut-off for each parameter was determined using Youden's index, and the corresponding sensitivity and specificity for the cut-off were also quantified. Individual clinical factors were subjected to univariate linear analysis and were subsequently entered into the multivariate analysis in a backward stepwise manner. The criterion for retention in the multivariate model was p<0.05. All statistical analyses were 2-tailed with a significance threshold of p<0.05 calculated with SPSS version 25.

Seventy-four patients with DME, including 32 males and 42 females, were examined and followed up for 3 months. The baseline characteristics are depicted in Table 1. The mean age of patients with DME was 53.17±8.42 years old, with a duration of diabetes of 9.95±7.21 years. All patients were treated with ranibizumab (Patizra, Novartis) monotherapy. The mean baseline visual acuity (LogMAR BCVA) of the treated eye was 0.93±0.61 and decreased by an average of 0.65±0.51 at month three (p=0.0007) (Table 2).

The total retinal layer thickness (TRLT) decreased from 410.40±115.00μm at baseline to 358.20±79.52μm after three months of ranibizumab treatment (p=0.010, Table 2, figure 1B). Further examination indicated that only the outer retinal layer thickness (ORLT) was significantly lower at the final visit (p=0.015, Table 2). Although each individual retinal layer thickness (RLT) tended to decrease after IVR injection within three months of treatment, the thickness changed more significantly within the inner nuclear layer (INL, p=0.007) and outer nuclear layer (ONL, p=0.007) (Table 2). In contrast to RLT, no changes were observed in the choroidal layer thickness (CLT) (Table 2). In addition, we observed that three months of ranibizumab treatment effectively decreased the fluid area in the subretinal region (p=0.0005, Table 2). ROC analysis was performed in parameters with significant changes after ranibizumab injection in order to identify a possible biomarker for treatment evaluation. The changes in the subretinal fluid area were relatively superior to the other parameters (AUC=0.76, cut-off=78,257 with sensitivity of 84% and specificity of 68%, p=0.001) (Figure 1C).

The correlations of structural changes with logMAR BCVA were explored (Table 3). We found a significant positive correlation between logMAR BCVA and retinal nerve fiber layer (RNFL), ONL, TRLT, ORLT, and subretinal fluid area (r=0.419, 0.477, 0.483, 0.540, and 0.683, respectively, Table 3) at baseline, while other layers did not show good correlation. At the final visit, no significant correlations were detected (Table 3). We then examined whether there was any correlation between changes in the logMAR BCVA (ΔVA) and structural changes (Δstructural changes) from the baseline to the final visit. Similarly, no correlations were observed (Table 3). A multivariate analysis was further conducted using data at the final visit. The decrease in the PRL was associated with better visual acuity in the univariate analysis. However, this result was not statistically significant in the multivariate analysis (Table 4).