This study used a multistage stratified random sampling method. The sample size was calculated using the formula: n=Z² [p (1–p)]/B². At a 95% confidence level, Z=1.96, B=0.15p, and prevalence value (P)=18.2%. The initial sample size was adjusted by a sampling factor of 2.0, resulting in a minimum required survey population of at least 1534 individuals. Accounting for an estimated response rate of 90%, a minimum of 1705 individuals were deemed necessary.

Stratification of townships was performed based on the number of patients with diabetes registered with the Funing County CDC. Townships were stratified using a criterion of 1000 patients with diabetes per stratum. Townships with more than 2000 patients with diabetes were split into 2 or more strata, while those with fewer than 500 patients were combined into 1 stratum. Two townships were then selected using a random number table. Subsequently, villages within these 2 selected townships were further stratified using a criterion of 50 patients with diabetes per stratum. Villages with more than 100 patients with diabetes were counted as 2 or more strata, while those with fewer than 25 patients were combined into 1 stratum. Villages were similarly selected using a random number table.

It should be noted that, when a village was divided into multiple virtual strata based on geographical subunits (eg, streets or residential clusters), these strata were assigned to preserve local spatial information. In the actual sampling for this study, no two strata from the same original village were simultaneously selected, thus avoiding potential within-village clustering in the analysis.

Participants were notified by telephone to confirm the exact date, time, and screening location (township health centers). Upon arrival, staff at the health centers assisted participants in completing questionnaires covering basic and disease-related information. Nurses then collected blood samples and performed HbA_1c testing, with results provided to participants before the conclusion of the screening procedure. Following assessments of PVA and BCVA by optometrists, participants sequentially underwent axial length (AL) and intraocular pressure measurements performed by trained technicians. Subsequently, an ophthalmologist conducted slit-lamp examinations and, when conditions permitted, administered bilateral pupillary dilation. Vitreous degeneration was defined as the presence of vitreous liquefaction, posterior vitreous detachment, or visible vitreous opacities (eg, dust-like, spider web, or diffuse haze) on slit-lamp biomicroscopy. Participants then underwent 2-field digital color fundus photography (those receiving mydriasis waited approximately 20 minutes beforehand). Finally, participants submitted their completed questionnaires, thereby concluding the screening process.

Results for DR and VTDR were analyzed separately in each subgroup. Statistical analyses were performed using SPSS (version 27.0, IBM Corp) and R (version 4.4.2; R Foundation for Statistical Computing). Categorical data are presented as frequencies and percentages, with differences between the DR and non-DR groups assessed using chi-square tests, Mann-Whitney U tests, and Kruskal-Wallis tests.

Generalized estimating equations (GEEs) with a binomial distribution and logit link function were used to investigate the association between AL and the presence of tessellated fundus (TF). An exchangeable working correlation structure was specified to account for the correlation between two eyes of the same individual, and robust (sandwich) SEs were used. The analysis was performed separately for the DR group (556 eyes) and the non-DR group (2764 eyes).

For variables with 5% to 15% missing values (mainly AL), multiple imputation by chained equations (10 imputations and 10 iterations) was performed in R. A δ-adjustment sensitivity analysis (delta range –1 to 1; step 0.2) was conducted to assess the robustness of the results under different missing-not-at-random assumptions; the findings remained stable. The results of the GEEs, including the coefficient (B), SE, Wald chi-square, odds ratio (OR), 95% CI, and P value, are presented in Table S1 in Multimedia Appendix 1. Sensitivity analysis results are shown in Figure 1.

SPSS was used to calculate sensitivity, specificity, and their 95% CI for each subgroup.

This study adhered to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Affiliated Hospital of Nantong University (2024-K048-01). Data were collected anonymously to protect participants’ confidentiality, and only the research team had access to the dataset. All participants were fully informed before the investigation and signed written informed consent forms; no form of compensation was provided to the participants.

From October 21 to November 12, 2024, 1847 patients with diabetes (3694 eyes) met the inclusion criteria. Of these, 1748 (94.6%) patients (n=3496, 94.6% eyes) completed the ocular screening according to the protocol (Figure 2). A total of 99 (5.4%) patients were excluded due to various reasons for not completing the 2-field imaging required for analysis by the EVisionAI system. Among the eyes that completed screening with 2-field imaging, 104 (3.0%) eyes were excluded due to poor image quality (eg, resolution too low for discernment or significant eye position deviation affecting DR grading). Ultimately, 3392 (97.0%) eyes were eligible for DR analysis.

The mean age of participants was 67.17 (SD 8.52) years, with a mean diabetes duration of 9.13 (SD 6.97) years. Complete disease characteristics are presented in Table 1. Among all 1748 participants, DR was identified in 335 (19.2%) participants. Of these, 129 (38.5%) had VTDR (7.4% overall prevalence). The EVisionAI demonstrated an overall sensitivity of 90.61% (95% CI 87.89%‐92.79%) and specificity of 98.99% (95% CI 98.52%‐99.31%) for detecting any DR. While its sensitivity was substantially lower in early-stage DR, EVisionAI exhibited diagnostic performance nearly identical to that of human graders in referable cases (such as VTDR). Concurrently, pupillary dilation improved EVisionAI’s sensitivity for DR diagnosis (except in early-stage DR) but had minimal effect on specificity (Table 2).

To evaluate the impact of refractive media opacity in 2-field imaging on EVisionAI’s diagnostic performance, images were graded based on optic disc and macular clarity according to previous literature [16] and the average values were obtained (Table S2 in Multimedia Appendix 1).

The results showed that mild to moderate image clarity impairment, whether in the optic disc or macular region, did not affect EVisionAI’s diagnostic performance. However, severely compromised image clarity resulted in a modest reduction in EVisionAI’s diagnostic sensitivity, while specificity remained largely unaffected (Figure 3 and Table S3 in Multimedia Appendix 1). Notably, for overall DR, reduced clarity in the optic disc region appeared to cause more severe consequences (Figure 3A and Table S3 in Multimedia Appendix 1). In the least clear optic disc cohort (P3 group), sensitivity dropped to 80.95%, indicating potential missed diagnoses within this subset. Conversely, the impact of severely reduced macular clarity on sensitivity was relatively limited (sensitivity in the M3 group only decreased to 87.50%; Figure 3A and Table S3 in Multimedia Appendix 1). Further analysis revealed that the detection rate for early-stage DR was higher in the M3 group than in the P3 group (Table S3 and Table S4 in Multimedia Appendix 1).

In the VTDR subgroup, although severe image clarity impairment similarly affected the correct identification of DR, we observed that EVisionAI demonstrated diagnostic capabilities remarkably consistent with those of human graders (Figure 3D and Table S3 in Multimedia Appendix 1). Even within the subgroup with the poorest image clarity (whether in the optic disc or macular region), sensitivity remained above 90%. Further analysis of cases in this subgroup that were not correctly diagnosed (all subgroups where sensitivity failed to reach 100%) revealed that all these cases exhibited severe vitreous degeneration (Figure 4B). This degeneration obscured retinal hemorrhages, preventing their identification, or led to the misidentification of degenerated vitreous as retinal exudates (Figure 4D).

It is noteworthy that for early-stage DR, EVisionAI demonstrated very high specificity while sensitivity was maintained at only around 80% (Figure 3B). Severe defects in image clarity, particularly in the optic disc region, led to a further reduction in sensitivity (Figure 3B and Table S4 in Multimedia Appendix 1). We observed a higher proportion of TF in early-stage DR compared with the VTDR subgroup (VTDR: 52.17% and early DR: 62.24%). To clarify the impact of TF on DR diagnosis, particularly in early DR, we graded the severity of TF in participants according to the reported grading standards [24,25] (Table S2 in Multimedia Appendix 1).

Overall, similar to image clarity, severe TF caused a significant decrease in sensitivity, while specificity remained almost unaffected (Figure 5A). In the TF 3 group, sensitivity remained below 85% regardless of whether pupil dilation was performed, indicating a certain probability of missed diagnoses. Further analysis showed that in the TF 3 group, EVisionAI had a relatively low detection rate for early-stage DR (Table 3). Consistent with this, within the early-stage DR subgroup, severe TF led to a sharp decline in sensitivity (Figure 5B), which could not be compensated for even with pupil dilation.

For the VTDR subgroup, similar to the image clarity, severe TF changes did not affect EVisionAI’s diagnostic capability (Figure 5D). Further analysis of subgroups where sensitivity did not reach 100% revealed that all patients with misgraded images exhibited severe vitreous degeneration, with some overlapping with patients in the image clarity subgroup (Figure 4). Notably, the vast majority of patients with VTDR whose images were misgraded by EVisionAI simultaneously presented with severe TF changes, image clarity abnormalities, and severe vitreous degeneration. The combination of these factors will undoubtedly significantly compromise both image acquisition quality and EVisionAI’s ability to accurately identify retinal pathology.

In this cross-sectional study, EVisionAI was used for automated DR screening among people with diabetes in resource-limited regions in primary health care centers. Although EVisionAI achieved high sensitivity and specificity comparable to those of human graders and successfully identified nearly all VTDR cases, severe ocular factors could still diminish its diagnostic performance, particularly in the detection of early-stage DR. Given its advantages of low cost and high efficiency, as well as the potential optimization of the factors affecting its performance, EVisionAI still represents a cost-effective solution capable of replacing traditional manual grading–dependent DR screening models. Our study identified specific ocular factors that might influence EVisionAI’s diagnostic performance. Optimizing screening protocols for patients exhibiting these factors can significantly enhance recognition efficiency and contribute to further improving the accuracy and referral efficiency for DR in primary health care centers with limited specialists.

Our research indicated that the accurate identification of early-stage DR was significantly influenced by ocular factors, particularly the extent of TF changes. Subtle lesions, such as microaneurysms and dot hemorrhages, were highly susceptible to being obscured and confused by turbid refractive media and visible choroidal vessels. Consequently, patients at this stage were prone to being missed. From the perspective of diabetes management, screening for mild DR holds substantial clinical value. It enables more patients at this stage to receive earlier medical intervention and personalized diabetes management, thereby reducing or delaying the likelihood of permanent visual impairment [26,27]. Most existing deep learning systems for DR screening primarily focus on the accurate identification of referable DR. Accurately detecting very early-stage DR lesions, such as microaneurysms, remains challenging for most of these systems [11].

Severe TF is typically associated with longer AL and advanced age (Table S1 in Multimedia Appendix 1), with longer AL being a well-established protective factor against the development of DR [28]. Patients who nonetheless progress to VTDR under these conditions generally exhibit poorer glycemic control (Table S5 in Multimedia Appendix 1 and Figure 6), which results in more severe lens opacity and shallower ACD (precluding effective pupil dilation). A cloudy posterior lens capsule and constricted pupil size significantly reduce the clarity of fundus images (Figure 7). Therefore, for older patients with poor glycemic control who concurrently present with these compounding negative factors, meticulous manual re-examination of images can help identify obscured or missed lesions, serving as a valuable compensatory measure. This approach can mitigate the limitations of deep learning systems in these specific scenarios and effectively improve the detection of early-stage DR.

The decline in image clarity and the presence of TF changes, even when quite severe, almost never affected the correct identification and grading of patients with VTDR who required referral. It is worth noting that while pupil size was related to image quality, it did not significantly impact the correct identification of DR, especially VTDR. In large-scale DR screening conducted at primary health centers without specialized ophthalmologists, pupil dilation may carry significant risks, potentially causing transient increases in intraocular pressure or triggering acute glaucoma attacks. This is particularly critical for participants with severe diabetic optic neuropathy, as a sharp rise in intraocular pressure could cause irreversible vision impairment. In addition, the simplicity of the screening procedure is paramount in primary health care centers, where operators may include technicians and staff without specialized expertise. Our results demonstrated that screening under natural pupil size was already sufficient for the correct identification of DR by deep learning systems. We also acknowledge that our conservative mydriasis criterion (ACD <1/2 CT [corneal thickness]) was adopted to balance participant safety and screening efficiency in rural township settings. This threshold reduced the proportion of dilated participants, which may have confounded the observed effect of severe media opacity on AI performance. Nonetheless, the final image clarity input to the AI reflects real-world nonmydriatic conditions, and our main findings on ocular factors remain valid within this context.

Severe vitreous degeneration appeared to invariably hinder the accurate identification of retinopathy, even in cases where the retinopathy itself was relatively severe. The markedly turbid vitreous compromised correct image focusing, obscured hemorrhagic lesions, and was mistakenly identified as hard exudates. This interference was particularly pronounced when coexisting with other ocular confounding factors (such as severe image quality deficiency or significant TF). Severe vitreous opacity often indicates either previous vitreous hemorrhage or metabolic abnormalities resulting from prolonged poor glycemic control, both of which indicate a high likelihood of severe DR. Notably, even with excellent image clarity, misdiagnosis primarily caused by severe vitreous degeneration manifested as undergrading rather than overdiagnosis, which undoubtedly led to underestimation of patients with DR who require referral. While cost-effective, the primary goal of community health screening is to efficiently identify patients needing further referral. Therefore, for patients with diabetes presenting with multiple ocular confounding factors—especially severe vitreous degeneration and opacities—existing deep learning systems may not yet fully replace experienced professionals. For this group of patients, we recommend definitive diagnosis by a professional ophthalmologist.

Most previous studies on AI-assisted DR screening focus on diagnostic performance evaluation, with limited analysis of complex ocular confounding factors. Our study provides a detailed analysis of how retinal image clarity (simultaneously affected by refractive media opacity and pupil size) and the degree of TF changes impact AI diagnostic performance. Our findings offer optimization pathways to enhance AI systems’ recognition of DR—particularly early-stage DR with multiple ocular influencing factors—in large-scale screening.

We acknowledge that limitations due to confounding from other social, economic, and cultural factors cannot be entirely excluded. Our sample was recruited from patients with type 2 diabetes registered at the CDC in an economically resource-limited region. Participants were primarily middle-aged and older individuals with lower education levels from rural areas. Although we used stratified sampling as extensively as possible to cover nearly all townships in the local area, compared with the broader general population in China, our sample may exhibit certain differences from regions with lower rural residency and higher education levels. Additionally, our study used a single, albeit well-validated and widely used AI system for DR screening. The lack of cross-validation with other AI models (including commercially available or open-source systems) limits the direct generalizability of our findings to other AI algorithms. Future studies should deliberately include multiple AI systems in the same population to assess whether the identified ocular factors consistently influence diagnostic performance across different AI platforms.

In summary, our study identifies ocular factors affecting deep learning systems’ diagnostic performance in DR screening for patients with type 2 diabetes in resource-limited regions and proposes actionable optimizations. While generalizability to broader populations and other deep learning systems requires further validation, this work enhances DR diagnostic accuracy and referral efficiency by primary care providers in underserved areas, alleviating critical pressures on China’s DR screening programs. This approach expands essential primary health care access while reallocating resources to other medical domains. Future studies will validate findings across diverse populations, health care settings, and more universally applicable AI systems.