Quantitative Autofluorescence Intensities in Acute Zonal Occult Outer Retinopathy vs Healthy Eyes

Key Points

Question  Are fundus autofluorescence intensities elevated at the border of acute zonal occult outer retinopathy lesions compared with intensities at the same fundus positions in healthy eyes?

Findings  In a study of 6 patients diagnosed as having acute zonal occult outer retinopathy, measurement by quantitative fundus autofluorescence revealed that intensities were elevated compared with age and race/ethnicity–matched healthy eyes.

Meaning  These findings suggest that acute zonal occult outer retinopathy pathogenesis involves accelerated formation of bisretinoid fluorophores in impaired photoreceptor cells, leading to elevated fundus autofluorescence and potential for aggravated damage.

Importance  Acute zonal occult outer retinopathy (AZOOR) remains a challenging diagnosis. Early recognition of the disease depends on advances in imaging modalities that can improve phenotyping and contribute to the understanding of the underlying pathogenesis.

Objectives  To expand the range of approaches available to assist in the identification of AZOOR by multimodal imaging and to analyze the fundus lesions by quantifying short-wavelength fundus autofluorescence (quantitative fundus autofluorescence [qAF]) and spectral-domain optical coherence tomography.

Design, Setting, and Participants  In this observational study, patients underwent imaging at Columbia University Medical Center between November 2010 and March 2016 and were analyzed between September 2015 and August 2016. Six patients diagnosed as having AZOOR were studied by qAF and spectral-domain optical coherence tomography and were compared with 30 age and race/ethnicity–matched controls from a database of 277 healthy control eyes.

Main Outcomes and Measures  In unaffected regions of the macula, qAF was calculated within predetermined circularly arranged segments (qAF₈). In addition, qAF was measured within specified regions of interest positioned at the autofluorescent lesion border (AZOOR line). Electroretinograms and electro-oculograms were recorded in 5 of 6 patients.

Results  Among 6 patients (age range, 26-61 years; 4 female; 4 of white race/ethnicity, 1 Asian, and 1 Hispanic), 5 exhibited an autofluorescent AZOOR line in short-wavelength fundus autofluorescence images, delineating the peripapillary lesion. The mean (SD) region-of-interest qAF measured on the AZOOR line was 60 (26) times higher than in healthy control eyes (P = .03) at equivalent fundus locations. The qAF₈ within nondiseased macular regions were within the normal range. At the lesion border, spectral-domain optical coherence tomography revealed a loss of outer retinal integrity in all patients. Single-flash cone b-wave latency and 30-Hz flicker latency responses were significantly delayed bilaterally. Lesions with smooth, homogeneous borders exhibited only minimal expansion in size over time, while the lesion in a patient with a heterogeneous border progressed more rapidly.

Conclusions and Relevance  The finding that qAF is elevated at the border between diseased and nondiseased retina in patients with AZOOR contributes to the understanding of the natural history of the disease.

While the etiology of acute zonal occult outer retinopathy (AZOOR) is not known, efforts have been made to achieve consensus on diagnostic criteria.^1-4 Patients with AZOOR are typically young and female and have a history of photopsia and a blind spot in the temporal field. Lesions can be unilateral or bilateral. Central visual acuity is typically good in patients with AZOOR owing to foveal sparing. Recent studies have assigned a diagnosis of AZOOR based on the presence of distinctive fundus features.⁴ In short-wavelength fundus autofluorescence (SW-AF) images, these patients manifest diffuse patches of hyperautofluorescence outside of the central macula and a peripapillary hypoautofluorescence indicative of RPE atrophy. The border between lesion and nonlesion retina can be demarcated by a distinctly hyperautofluorescent line (AZOOR line).^1,4 The lesions in SW-AF images are associated with altered photoreceptor cell–attributable layers in optical coherence tomography scans that can include disruptions of the ellipsoid zone and interdigitation zone.⁴ While in most cases AZOOR lesions exhibit peripapillary involvement with centrifugal progression, patients infrequently are seen with peripheral concentric zonal atrophy that progresses centripetally.⁵ Investigators have endeavored to differentiate AZOOR from other similar disorders (white spot syndromes, multifocal choroiditis, and acute macular neuroretinopathy) that are not a single entity and can be defined as the AZOOR complex.^6-11

The present observational study focused on patients diagnosed as having AZOOR primarily on the basis of peripapillary fundus lesions conferring abnormal SW-AF patterns and anomalous findings in outer retinal layers of spectral-domain optical coherence tomography (SD-OCT) scans. Other conditions, such as infections or inherited retinal degeneration, that could also cause outer retinopathies were excluded on the basis of the history and presentation.

The aim of the study was to use quantitative fundus autofluorescence (qAF) to objectively measure SW-AF intensities and compare these intensities with healthy eyes over time.^12,13 The SW-AF signal has been shown to originate from bisretinoid lipofuscin that forms in photoreceptor cells as a product of inadvertent reactivity of retinaldehyde of the visual cycle.^14,15 Bisretinoids accumulate throughout life and undergo greater synthesis in some retinal disorders. While hyperautofluorescence is observed at the junction of diseased and nondiseased areas in the AZOOR fundus, whether the fluorescence is actually increased relative to healthy eyes is not known to date. Because elevated levels of bisretinoid lipofuscin are toxic,¹⁶ this question is relevant to the disease process.

Patients underwent imaging at Columbia University Medical Center between November 2010 and March 2016 and were analyzed between September 2015 and August 2016. Inclusion criteria were patients seen with the following: (1) unilateral or bilateral visual field loss associated with (2) a peripapillary hypoautofluorescent lesion on fundus autofluorescence imaging, (3) a loss of outer retinal layers on SD-OCT and (4) findings not suggestive of an inherited retinal disease, and (5) negative infectious workup (including Treponema pallidum, Mycobacterium tuberculosis, and Borrelia burgdorferi infections). Exclusion criteria were the presence of media opacities that would lower qAF measurements (ie, cataracts, corneal scars, or severe vitreous opacities).

The study was approved by the institutional review boards at Columbia University Medical Center and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants before acquisition of images.

The SD-OCT and SW-AF images were acquired by confocal scanning laser ophthalmoscopy. For qAF, the confocal scanning laser ophthalmoscope was equipped with an internal fluorescent reference. The qAF was calculated in nondiseased fundus and in nonaffected eyes by measuring the mean gray levels within 8 concentric segments (eccentricity, 7° to 9°).¹⁷

Region-of-interest (ROI) (rectangular areas) analysis was used to measure gray levels at 3 positions within the AZOOR line and nasal and temporal to these positions (9 ROI-qAF values, including 3 within the AZOOR line, 3 nasal, and 3 temporal, in patients 2 through 5 [P2-P5)]. The AZOOR line was identified as brightness at the lesion border. The 3 ROI locations within the AZOOR line were traversed by SD-OCT line scans to correlate qAF with retinal structure. Follow-up was available for P5 during 6 years; therefore, ROI-qAF measurements were repeated at the same locus. For comparison with each patient, ROI-qAF unit intensities were measured in 5 age and race/ethnicity–matched healthy control eyes (±5 years).¹⁸ Details are given in the eAppendix in the Supplement).

For patients having serial visits (P2, P3, P5, and P6), we examined for changes in the lesion over time. The SW-AF images from the first and last visits were aligned and superimposed. Detailed information about image acquisition and analysis is provided in the eAppendix in the Supplement.

Full-field scotopic and photopic electroretinograms and electro-oculograms were recorded in 5 of 6 patients following International Society for Clinical Electrophysiology of Vision standards.^19,20 Details of the testing are available in the eAppendix in the Supplement.

Linear mixed models were fit with a software package (Stata Data Analysis and Statistical Software; StataCorp LLC). The model provided the mean qAF units for each eye calculated at 3 ROIs positioned on the AZOOR line. Two-sided P < .05 was considered statistically significant. At each ROI-qAF site (within the AZOOR line, nasal, and temporal), the mean ROI-qAF was calculated and compared with the mean qAF (95% CI) of the ROI-qAF determined in 5 age and race/ethnicity–matched healthy control eyes at the same location. The mean qAF₈ (qAF calculated within predetermined circularly arranged segments) value of each eye was also calculated and compared with a previously published normative database from the group.¹⁸

Paired t test was used to compare the mean difference in electroretinogram results between the affected eye and the healthy eye of the same patient. Affected eyes (mean [SD]) of the patients with AZOOR were also compared with an age-matched control from a normative database of 82 healthy control eye patients for cone responses and 86 healthy control eye patients for rod responses (Diagnosys LLC normative database) (eAppendix in the Supplement).

Six patients (age range, 26-61 years; 4 female; 4 of white race/ethnicity, 1 Asian, and 1 Hispanic) diagnosed as having unilateral (P1, P2, and P4-P6 [5 eyes]) or bilateral (P3 [2 eyes]) AZOOR were studied. Clinical and demographic characteristics are summarized in eTable 1 in the Supplement. The interval between disease onset (acute visual field loss with photopsia) and the first clinical visit ranged from 4 weeks to 8 years (median, 48 months) with the exception of P1 and P5, who were asymptomatic. Four patients (P2, P3, P5, and P6) were followed up for 3 to 5.25 years; of those who were initially seen with unilateral disease, none developed lesions in the contralateral eye. Two patients (P1 and P2) reported a history of systemic autoimmune disorder. In P5, a history of unspecified white dot syndrome had manifested as visible residual yellowish punctate macular lesions bilaterally 10 years before the diagnosis of AZOOR.

Affected eyes of all patients exhibited peripapillary lesions of varying severity extending temporally but often sparing the macula. Although a single continuous lesion was observed in 5 patients, P1 exhibited multiple disconnected patches, with the largest situated along the inferior arcades (Figure 1A). The appearance of affected regions within the lesion also varied, appearing transparent with visibility of the underlying choroidal vessels (P2) (Figure 1B) or pale and opaque on ophthalmoscopy (P5 and P6) (Figure 1C and D). The SW-AF imaging revealed the presence of a readily discernible hyperautofluorescent AZOOR line that demarcated affected and nonaffected retina in a trizonal presentation that has been previously described.⁴ Two other distinct patterns were noted. In the first pattern, the AZOOR line was continuous and uniform in 4 patients (P3-P6) (Figure 1C and D, Figure 2A and B, and Figure 3C and F). In the second pattern, the AZOOR line was scalloped and speckled (P1 and P2) (Figure 1B, Figure 2A and B, and Figure 3A and B). The AZOOR line was also less pronounced in P1 (Figure 1A). The appearance of the affected region in SW-AF images ranged from dark to moderately hypoautofluorescent and granular (Figure 1).

The qAF₈ values were generated as the mean of 8 scaled segments positioned over nondiseased macular regions of each affected eye (Figure 2A), and color-coded maps were generated (Figure 2B) according to the qAF unit scale. Compared with color maps acquired from healthy control eyes (Figure 2C), the lesions were distinct in affected eyes. In nonlesion fundus of the patients, qAF units plotted as a function of age were within the normal range as indicated by the mean (95% CI) of 277 age and race/ethnicity–matched healthy control eyes (Figure 2D-F). The mean qAF₈ values acquired from the fellow eyes of the patients with AZOOR are also shown (Figure 2D-F).

The linear regression model for ROI-qAF intensities measured directly on the AZOOR line revealed that in patients with AZOOR the mean (SD) ROI-qAF at this position was 60 (26) times higher than in healthy control eyes (P = .03). Because the lesion border was not distinct, P1 was not included in this ROI-qAF analysis. The linear regression model for ROI-qAF nasal and temporal to the border showed that the mean (SD) qAF units for the patients with AZOOR were 33 (27) (P = .23) and 13 (23) (P = .59) lower, respectively, than in healthy control eyes (eTable 2 in the Supplement).

The ROI locations were positioned within the AZOOR line (Figure 3A-F), and the mean qAF values were plotted as a function of the mean (95% CI) of corresponding areas in 5 healthy control eyes. Six of 21 measurements exhibited ROI-qAF levels above the normal range (P3, P4, and P5) (Figure 4G). No distinct AZOOR line was observed in P1, and P2 had normal ROI-qAF units at the border of the lesion compared with healthy control eyes. Although P2 exhibited scattered autofluorescent dots at the lesion border (Figure 1), averaging of high and low gray levels within the ROI probably contributed to normal ROI-qAF measurements. In P5, a follow-up visit allowed measurements to be performed 6 years after the first measurements. Measurement at the same positions indicated a decrease in ROI-qAF to within the normal range. The SD-OCT imaging revealed that enlargement of the lesion had not occurred. An AZOOR line in SW-AF images was observed in P6, but qAF unit levels were within the 95% CIs of age and race/ethnicity–matched healthy control eyes. The absence of increased qAF in P6 could be attributable to nonvisible floaters that scattered the signal.

Analysis of SD-OCT scans through the ROI positions on the AZOOR line revealed abrupt disruptions of outer retinal reflectivity layers at these locations. The outer nuclear layer was typically thinned, and retinal pigment epithelium (RPE) atrophy was indicated by thinning of the Bruch membrane or RPE reflectivity layer or by transmission of signal into the choroid (Figure 4C, D, and E). In both eyes of P2, the edge of the less distinct AZOOR line corresponded to the positions of abrupt ellipsoid zone loss (Figure 4A and B). In almost all scans, the external limiting membrane appeared to persist after ellipsoid zone disruption.

Progression of relative lesion size was followed up in 4 patients within various intervals. In P2, lesion growth was noted within the inner hypoautofluorescent region (Figure 5A, red arrowheads) and at the external speckled (apparently hyperautofluorescent) border (Figure 5A, blue arrowheads), which extended into the central macula after 2.5 years (Figure 5B and C, with the dotted trace denoting the initial AZOOR line and the solid trace denoting subsequent growth). Because atrophy was present in more than 50% of the central 30° in the SW-AF image, further qAF imaging was not performed. In comparison, expansion of the external hyperautofluorescent border of the lesions in P6 (3.6-year interval), P2 (4-year interval), and P5 (5.25-year interval) was less pronounced and encompassed a small area (Figure 5D-F).

Full-field electroretinograms and electro-oculograms were analyzed in 5 patients (eTable 3, eTable 4, and eFigure in the Supplement). Generalized rod and cone amplitudes were unaffected in all patients compared with age and race/ethnicity–matched healthy control eyes, even on repeated electroretinogram testing at 3-year follow-up in P2. Although there was a tendency for amplitude asymmetry between the healthy eye and the affected eye, with the affected eye showing lower responses, the difference was not significant, even for maximum a-wave amplitudes (eTable 4 in the Supplement). The 30-Hz flicker and single-flash photopic b-wave latencies were delayed for more than 2 milliseconds bilaterally in all patients compared with age and race/ethnicity–matched healthy controls (Diagnosys LLC normative database). Lesions with smooth, homogeneous borders exhibited only minimal expansion in size over time, while the lesion in a patient with a heterogeneous border (P3) progressed more rapidly. Only one patient (P6) showed a decreased Arden ratio in electro-oculograms that was out of proportion to the electroretinogram asymmetry. In P5, asymmetry was demonstrated in Arden ratios but was in proportion to electroretinogram abnormalities.

The patients we studied were initially seen with the trizonal pattern of AZOOR that involved photoreceptor cell degeneration and RPE and choroidal atrophy.^4,21 Our results demonstrated that P3, P4, and P5 exhibited high ROI-qAF levels within the border of AZOOR lesions. The qAF levels in P5 returned to normal when remeasured after 6 years. This observation indicates that elevated SW-AF intensities at the AZOOR line can be impermanent. While P6 had an SW-AF pattern similar to P3, P4, and P5, ROI-qAF intensities in P6 were within the range of healthy control eyes. Autofluorescence intensities may have been higher at an earlier stage of the disease. Nevertheless, these findings indicate that qualitatively visible hyperautofluorescence in SW-AF images is not always indicative of an actual elevation in qAF relative to qAF at similar positions in healthy eyes. Herein lies the advantage of measuring by qAF rather than relying on contrast within individual images to evaluate the disease process. At the positions of elevated qAF within the AZOOR line, photoreceptor cell degeneration was evidenced by ellipsoid zone loss and outer nuclear layer thinning. This finding indicates that the process of photoreceptor cell degeneration may involve increased bisretinoid formation. This result complements our group’s recent report indicating that qAF within the autofluorescent rings typical of retinitis pigmentosa can be increased compared with an equivalent position in the healthy fundus.²²

All of our patients showed consistent delays in the 30-Hz flicker electroretinogram response and single-flash photopic b-wave response compared with healthy control eyes. Delayed 30-Hz flicker has been associated with generalized retinal dysfunction, particularly in patients with white dot syndrome, and has been shown to be a useful parameter to detect progression.²³ None of our patients with unilateral involvement reported seeing new phosphene, exhibited visual field defects or abnormalities in SW-AF images, or showed progressive electroretinogram dysfunction in the contralateral healthy normal eye when follow-up was performed. Given that other electroretinogram parameters, including a-wave and b-wave maximum amplitudes, were not different than healthy control eyes and that the delay was mild (2 milliseconds) and present bilaterally, we supposed that the delayed 30-Hz flicker was unlikely to be clinically significant. A study with more patients would be needed to verify whether bilateral increased latency is a consistent finding in patients with AZOOR lesions, as shown in this study. At the longest follow-up (5.25 years), bilateral delayed cone responses were not associated with increased risk of contralateral involvement or worsening visual function in the affected eye in P5.

The underlying pathologic process (autoimmune, infectious, or inflammatory) triggering the development of AZOOR is still under debate.²⁴ Whether the disease affects photoreceptor cells or RPE first is not known to date. Photopsia is common early on in AZOOR, and a loss of outer retinal layers occurs.⁴ It has been proposed that photoreceptor cell impairment can lead to ineffective handling of vitamin A aldehyde, resulting in excessive production of bisretinoid fluorophores in photoreceptor cells.¹⁶ The high qAF levels measured at the border of expanding lesions in our patients is consistent with this postulate and with the possibility that photoreceptor cell dysfunctioning is an early event in the disease process. In addition, given the phototoxicity associated with bisretinoids, excessive bisretinoid production could contribute to photoreceptor cell damage.²⁵ Few studies have addressed the light-rise of the electro-oculograms in patients seen with AZOOR or the AZOOR complex.⁶

A precise rate of recurrence or progression in patients with typical fundus lesions is not well documented in the literature, but many patients develop relapses or a chronic progressive disease.^4,26 So far, there have been no proven definitive criteria to predict whether AZOOR will progress or recur. However, it has been documented that patients with borders delineated by an AZOOR line at presentation showed more frequent progression over time.⁴ This finding could also indicate that enhanced formation of lipofuscin at the transition zone is critical in the disease process. Calculation of the ROI-qAF levels in a longitudinal study using predetermined intervals and locations and with a larger cohort would have been required for us to provide more data on these trends.

While the study involved a small number of patients, AZOOR is a rare condition, and the sample size was consistent with that in other reports of AZOOR.^8,26-30 The patients were not all tested at the same interval after disease onset, and none of the patients were in the acute stage of the disease at first presentation. In addition, the use of the ROI-qAF approach necessitated an analysis of discrete areas of the hyperautofluorescent border and not the border as a whole. Nevertheless, despite these limitations, statistically significant differences in ROI-qAF between patients and controls were observed, comparisons with the unaffected eyes were obtained, and qAF measurements were acquired from nonlesion retina.

We have considered whether there are other explanations for the visibility of the autofluorescent AZOOR line at the lesion border. Because our imaging protocol includes photopigment bleaching before image acquisition, the unmasking of RPE SW-AF due to photoreceptor cell degeneration cannot account for the increased SW-AF within the AZOOR line. The abnormal SW-AF within the AZOOR line cannot be attributed to accelerated phagocytosis of photoreceptor outer segments, as has been suggested,^31-33 because bisretinoid fluorophore formation occurs in photoreceptor cells before outer segment shedding and subsequent phagocytosis.

The diagnosis of AZOOR should be considered in patients with a history of photopsia, abnormal visual fields with corresponding peripapillary lesions visible by fundus autofluorescence, and a transition zone of optical coherence tomography–detectable retinal degeneration. Our results confirmed that most patients with AZOOR are initially seen with abnormally high fundus autofluorescence (qAF) at the border of the lesion. Given the origin of SW-AF from bisretinoid lipofuscin, the findings of this study suggest an association between bisretinoid lipofuscin and the pathogenesis of AZOOR. Further studies are necessary to determine whether the lipofuscin aggravates the disease.

Accepted for Publication: September 11, 2017.

Corresponding Author: Janet R. Sparrow, PhD, Department of Ophthalmology, Columbia University, 635 W 165th St, New York, NY 10032 (jrs88@cumc.columbia.edu).

Published Online: October 26, 2017. doi:10.1001/jamaophthalmol.2017.4499

Author Contributions: Drs Boudreault and Sparrow had full access to all of the data in the study and take responsibility for the integrity of the data and accuracy of the data analysis.

Study concept and design: Boudreault, Cabral, Yannuzzi, Tsang, Sparrow.

Acquisition, analysis, or interpretation of data: Boudreault, Schuerch, Zhao, Lee, Cabral, Tsang, Sparrow.

Drafting of the manuscript: Boudreault, Cabral, Sparrow.

Critical revision of the manuscript for important intellectual content: Boudreault, Schuerch, Lee, Cabral, Yannuzzi, Tsang, Sparrow.

Obtained funding: Tsang, Sparrow.

Administrative, technical, or material support: Schuerch, Lee, Cabral, Yannuzzi, Tsang, Sparrow.

Study supervision: Boudreault, Schuerch, Tsang, Sparrow.

Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported.

Funding/Support: This study was supported by award EY024091 from the National Eye Institute and by a grant from Research to Prevent Blindness to the Department of Ophthalmology at Columbia University (both to Dr Sparrow).

Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.