Title Page:

Full Length Title: Fundus autofluorescence in rubella retinopathy: correlation with photoreceptor structure and function

Running Title: Autofluorescence in rubella retinopathy

Authors:*Danuta M Bukowska, PhD1

*Sue Ling Wan, MBBS (Hons), FRANZCO1,2

Avenell Chew, MBBS1

Enid Chelva, BSc (Hons)3

Ivy Tang, BSc (Hons) MOrth1

David A Mackey, MD, FRANZCO1

Fred K Chen, PhD, MBBS (Hons), FRANZCO1,2

* Equal first authors

Affiliations:1. Centre for Ophthalmology and Visual Science, Lions Eye Institute, The University of Western Australia,

Western Australia

2. Department of Ophthalmology, Royal Perth Hospital, Perth, Western Australia

3. Department of Medical Technology and Physics, Sir Charles Gairdner Hospital, Perth, Western Australia

Corresponding author: Fred K Chen, Centre for Ophthalmology and Visual Science

Lions Eye Institute, 2 Verdun Street, Nedlands, WA 6009

Email: fredchen@lei.org.au

Funding source: NH&MRC Early Career Fellowship (APP1054712 , FK Chen)

Ophthalmic Research Institute of Australia (DM Bukowska)

The authors report no conflicts of interest

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Keywords: adaptive optics, autofluorescence, congenital rubella syndrome, microperimetry, scanning laser

ophthalmoscopy, spectral domain optical coherence tomography

Summary Statement: We describe fundus autofluorescence features of 4 patients with rubella retinopathy. Hypoautofluorescent

lesions on blue and near-infrared excitation did not correlate with attenuation of ellipsoid or interdigitation

zone. There was no significant reduction in retinal sensitivity in regions with hypoautofluorescence. We

discuss the mechanisms for autofluorescence features in rubella retinopathy.

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ABSTRACT:

PurposeTo illustrate altered fundus autofluorescence in rubella retinopathy and to investigate their relationships with

photoreceptor structure and function using multimodal imaging.

MethodsWe report 4 cases of rubella retinopathy aged 8, 33, 42 and 50 years. All patients had dilated clinical fundus

examination; wide-field color photography; blue, green and near-infrared autofluorescence imaging and

spectral-domain optical coherence tomography (OCT). Two patients also underwent microperimetry and

adaptive optics imaging. En face OCT, cone mosaic and microperimetry were co-registered with

autofluorescence images. We explored the structure-function correlation.

ResultsAll 4 patients had a “salt-and-pepper” appearance on dilated fundus examination and wide-field color

photography. There were variable-sized patches of hypoautofluorescence on both blue and near-infrared

excitation in all 4 patients. Wave-guiding cones were visible and retinal sensitivity was intact over these

regions. There was no correlation between hypoautofluorescence and regions of attenuated ellipsoid and

interdigitation zones. Hyperautofluorescent lesions were also noted and some of these were pseudo-

vitelliform lesions.

Conclusions:Patchy hypoautofluorescence on near-infrared excitation can be a feature of rubella retinopathy. This may be

due to abnormal melanin production or loss of melanin within retinal pigment epithelium (RPE) cells

harboring persistent rubella virus infection. Preservation of the ellipsoid zone, wave-guiding cones and retinal

sensitivity within hypoautofluorescent lesions suggest that these RPE changes have only mild impact on

photoreceptor cell function.

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INTRODUCTION

Congenital rubella syndrome (CRS) imposes a heavy burden on both the affected individual and their

community. CRS affects almost 100,000 children every year worldwide despite successful vaccination

programs.1 Moreover, vaccination programs do not exist in all countries, leaving many fetuses exposed to

the risk of developing CRS.

A "salt-and-pepper" fundus appearance is commonly associated with CRS due to disruption of the

normal embryogenesis of the retinal pigment epithelium (RPE). Histologically, these lesions correspond to

RPE clumping and migration.2,3 These RPE abnormalities are particularly well-visualized on short wavelength

(blue light) fundus autofluorescence (AF) imaging as stippled signal within the macula.4 Loss of AF or

hypoautofluorescence (hypo-AF) is often associated with photoreceptor damage and retinal dysfunction.5

However, the "salt-and-pepper" retinopathy in CRS has been described as a non-progressive pigmentary

retinopathy that does not impair vision or retinal electrophysiology response.

the RPE and melanin metabolism.

METHODS

Four patients aged 8, 33, 42 and 50 years (Cases 1, 2, 3 and 4 respectively) with rubella retinopathy

underwent Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity testing, complete ophthalmic

examination by a retinal specialist (FKC) and multimodal imaging. The 50 year old patient was male and the

other 3 were female. The diagnosis of rubella retinopathy was based on positive clinical signs together with a

history of maternal infection during the first trimester. The 50yo male did not have this history available but

had congenital sensorineural deafness and clinical features consistent with rubella retinopathy. No patient in

this series had a history of using any medication known to cause retinal toxicity, including

hydroxychloroquine, deferoxamine and chlorpromazine.

Wide-field color and medium wavelength AF imaging (excitation: 532 nm; green light, detector: 570-

780 nm) were performed using a scanning laser ophthalmoscope (P200Tx, Optos plc, Dunfermline, UK).

Spectral domain optical coherence tomography (SD-OCT) and short wavelength (excitation: 486 nm; blue

light, detector: 500-750 nm) and long wavelength (excitation: 786 nm, near-infrared light, detector: 810-900

nm) AF imaging were performed using the Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg,

Germany). One patient also had retinal electrophysiology (incorporating the International Society for Clinical

Electrophysiology of Vision standards) and two patients had fundus-controlled microperimetry and adaptive

optics (AO) retinal imaging (see below).

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Spectralis images were captured in high-resolution mode (1,536 x 1,536 pixels). SD-OCT images

were acquired concurrently with near-infrared reflectance imaging by using the following protocols: (1) a

dense raster scan of the foveal region (20° x 10°) consisting of 97 horizontal lines (line separation of 30 µm)

and (2) macular volume scan of 30 x 25° consisting of 61 horizontal lines (lines separation of 61 µm). Each

B-scan line was obtained by averaging 9 single frames. The en-face minimum projection intensity reflectivity

maps of the ellipsoid and interdigitation zones within the foveal region were created using the HEYEX 3D

viewing module in Spectralis (version 6.0.9.0). We used the lexicon proposed by the International

Nomenclature for Optical Coherence Tomography Consensus to describe structural changes in the OCT

scan.6 AF images were acquired with a 30° x 30° field of view centered on the fovea. One hundred AF

frames were averaged in automated real time (ART) mode to acquire the final high definition AF images. If

near-infrared AF could not be acquired, combined angiography mode (the simultaneous fluorescein and

indocyanine green angiography mode) was used.7

Microperimetry was performed using the fundus-controlled microperimeter MAIA (Centervue,

Padova, Italy). Pupils were dilated with tropicamide 1% and phenylephrine 2.5%. The sessions were

conducted in a darkened room prior to retinal imaging. Each eye was tested with a 10-2 (68 loci) stimulus

grid using a 4-2 staircase strategy to determine retinal sensitivity thresholds to the nearest 1dB.

Adaptive optics (AO) retinal imaging was performed through dilated pupils using a commercially

available instrument, the rtx1 camera (Imagine Eyes, Orsay, France), based on a flood-illumination

ophthalmoscopy system. Each AO image frame is 4° x 4° and consecutive images were acquired with 50%

overlap in area of adjacent frames. MosaicJ plugin (ImageJ, Image Processing and Analysis in Java, NIH,

Bethesda, USA) was used to manually create a wide-field montage of the AO images. Cone density maps

were generated using AOdetect software, provided by Imagine Eyes. A wide-field cone density map was

created using coordinates describing the relative positions of AO image frames that were montaged using

the MosaicJ software.

RESULTS

Multimodal Imaging

Wide-field pseudo-color SLO imaging (Optos) demonstrated mottled pigmentary changes extending from the

macula to the temporal equator in all 4 patients. Green AF imaging (Optos) showed speckled hypo-AF in the

macular region extending to the temporal equator to variable degrees (Figure 1). Stippling of AF signal was

more prominent on near-infrared AF imaging (Spectralis) than on blue (Figure 2) or green AF imaging

(Figure 3). There were several hyperautofluorescent (hyper-AF) lesions scattered throughout the posterior

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pole on blue AF imaging (Spectralis) and some of these correlated with hyper-reflective sub-retinal pseudo-

vitelliform lesions or drusen-like lesions on SD-OCT (Figure 2). Case 4 also had hyper-reflective lesions

within the outer retina representing hard exudate from resolving diabetic macular edema. The hypo-AF

lesions seen with near-infrared excitation were hyper-reflective on near-infrared reflectance imaging (Figure

3). It was not possible to capture near-infrared AF images in one patient (case 3) despite using the combined

dual wavelength mode provided by the Spectralis device.

To investigate the relationship of the ellipsoid and interdigitation zone reflectivity profiles with hypo-

AF patches seen on near-infrared AF, en face intensity maps of the ellipsoid and interdigitation zones were

created (Figure 4). In general, the ellipsoid zone was intact except in the regions of pseudo-vitelliform

lesions, but there were separate patches of the retina affected by attenuation of the interdigitation zone. Co-

registration between these en face intensity maps and near-infrared AF images showed no obvious

correlation between regions of ellipsoid or interdigitation zone attenuation and loss of near-infrared AF signal.

Case 2 had microperimetry and AO imaging in the foveal region (Figure 5). Co-registration between

the cone mosaic montages with near-infrared AF images showed that the wave-guiding cone outer tips were

still visible in regions of hypo-AF. Retinal sensitivity was preserved over regions of hypo-AF (Figure 5). Case

2 also had normal light rise in electro-oculography and amplitudes and latencies on full-field

electroretinography (Figure 6). Case 3 had follow-up imaging performed over the course 24 months, and

there was no deterioration in visual acuity, retinal sensitivity on microperimetry or progression of the retinal

lesions on examination and AF imaging (Figure 7).

DISCUSSION

Fundus AF is a useful diagnostic tool in the differential diagnosis of various types of pigmentary retinopathies

such as inherited retinal dystrophy, immune or infectious chorioretintis and drug toxicities.5 Signal generated

from the retina in this imaging modality is based on the autofluorescent properties of molecules such as

fluorophores within degraded photoreceptor outer segments in the subretinal space or lipofuscin and melanin

within the RPE and melanin within choroidal melanocytes.8,9 Although fundus AF in congenital rubella has

been described, this was limited to the use of blue light excitation in the macular region and there was no

correlation with photoreceptor cell function and structure. Using multimodal imaging, we demonstrated

speckled hypo-AF with blue, green and near-infrared light excitation in patients with rubella retinopathy. On

wide-field imaging, we documented extension of these AF abnormalities to the equator temporally. There

was a significant difference in the pattern of AF between excitation wavelengths and the lesions on AF did

not correlate with ellipsoid or interdigitation zone attenuation on SD-OCT. Focal and punctate hyper-AF

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correlated with pseudo-vitelliform lesions and hyper-reflective deposits over the RPE. Microperimetry

demonstrated normal retinal sensitivity and AO imaging showed wave-guiding cones over areas of hypo-AF.

The 4 cases were referred with varied diagnoses ranging from idiopathic retinal pigmentation (case

1), presumed retinitis pigmentosa (case 2), cutaneous melanoma chemotherapy toxicity screening (case 3)

and diabetic retinopathy screening (case 4). The first 3 cases had confirmed history of maternal gestational

rubella consistent with vertical transmission but none of them had cataract or a history of cardiac disease

and only two had sensorineural hearing impairment. Retinal toxicity from inhibitors of the serine/threonine-

protein kinase B-RAF (dabrafenib) and mitogen-activated protein kinase (trametinib) have been

described10,11. While the former is known to cause uveitis, the latter has been associated with central serous

retinopathy, retinal vein occlusion and pigment epithelial detachment; none of these features were present in

Case 3. Case 4 has retinal features of a “salt-and-pepper” retinopathy and congenital hearing impairment

without confirmed history of maternal gestational rubella infection. Although congenital syphilis is a possible

differential diagnosis, he had no other features of the Hutchinson triad such as interstitial keratitis, saddle

nose, mulberry molars and peg-shaped incisors. We did not have the opportunity to exclude this diagnosis

with serology. None of these patients had nyctalopia or loss of the photoreceptor layer on SD-OCT to

suggest that they may have a rod cone dystrophy associated with Usher syndrome. The individual referred

with presumed retinitis pigmentosa also had normal full-field electrophysiology, making the diagnosis of a rod

cone dystrophy very unlikely.

Goldberg et al. illustrated unique short wavelength (blue light) AF features in 4 cases of presumed

rubella retinopathy aged 14 to 40 years old4. Perifoveal stippled AF was noted and one case had subfoveal

choroidal neovascularisation. We broadened the description of AF imaging features in rubella retinopathy by

illustrating extension of speckled AF signal loss to the temporal equatorial region of the retina on green light

excitation using the Optos wide field camera. Furthermore, near-infrared AF (successfully acquirerd in 3 of

the 4 cases) showed more numerous and larger hypo-AF lesions in the macular region compared to blue

light AF. Discordance in the distribution of hypo-AF lesions between blue and near-infrared light excitation

has been reported in age-related macular degeneration7,12, inherited retinal dystrophy13–16, central serous

retinopathy17,18 and inflammatory retinopathy19. Most of these studies also demonstrated a relationship

between hypo-AF and the loss of ellipsoid zone integrity and reduced function on microperimetry or visual

field examinations12–14,16,20. Duncker et al. postulated that preservation of blue light autofluorescence in

regions of reduced near-infrared AF signal in Stargardt macular dystrophy is due to photoreceptor debris

over a region where RPE has been lost.14 9,21–23. Conversely, the reduced near-infrared AF outside the foveal

island of vision may be due to loss of melanin and melanolysosomes in RPE adjacent to residual cone cell

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bodies without outer segments or synaptic pedicles15,23,24. If this hypothesis also holds true for rubella

retinopathy, we would expect to see reduced retinal sensitivity on microperimetry, lack of wave-guiding

cones on AO imaging and loss of the ellipsoid and interdigitation zones on SD-OCT in the regions of hypo-

AF on near-infrared excitation.

Surprisingly, our result demonstrated that regions with hypo-AF are compatible with preserved retinal

sensitivity on microperimetry and presence of cones as indicated on AO imaging and en face OCT. However,

there were subtle abnormalities detected in the ellipsoid and interdigitation zones as visualized in the en face

reflectivity map. This feature may indicate abnormal interaction between the photoreceptor cell and the RPE.

It is not possible to visualize rods using the rtx1 AO camera but given the preserved scotopic response on

ERG, it is unlikely that there is significant loss of rod photoreceptors. Localized loss of rods in regions with

hypo-AF cannot be excluded and this will require further investigation using a scotopic microperimetry device

such as the MP1-S (Nidek, Padova, Italy) and a scanning laser system AO device.

What could be the cellular mechanism for the speckled hypo-AF in rubella retinopathy? Rubella virus

replication may impact on normal host cell function through mitochondrial abnormalities, disruption of the

host cell cytoskeleton, altered cell membrane fatty acid composition and changes to apoptosis pathways25,26.

Histopathology of rubella retinopathy has been described as pleomorphic, but the appearance can be highly

variable from case to case. The most frequent change is found in the RPE with atrophy and focal areas of

hypo- and hyper-pigmentation. There was no choroidal inflammation associated with these pigment epithelial

findings.3 In vitro modelling of persistent rubella virus infection of cultured human RPE demonstrated

defective phagocytosis of latex beads26. Electron microscopy and gas liquid chromatography demonstrated

that infected cells had abnormal distribution of microvilli, increased palmitic acid and odd-numbered long-

chain carbon atom fatty acids that are not normally found in human cell membranes. It is not known whether

rubella virus can also infect cones and rods thus altering the lipid composition of their outer segments as

well. The abnormal AF signal arising from melanin (near-infrared excitation) and lipofuscin (blue and green

light excitation) may be due to subtle abnormalities in outer segment metabolism leading to defective

melanin and lipofuscin distribution or defective pigment synthesis in clusters of persistently infected RPE

cells. By co-registering macular images from various modalities, we were able to show that hypo-AF lesions

on near-infrared excitation were hyper-reflective on near-infrared reflectance imaging. It has been reported

that melanin pigments contribute significantly to the pattern of near-infrared reflectance imaging, 27 where

RPE melanin tends to absorb and choroidal melanin tends to backscatter near-infrared light.28 Therefore, the

co-localization between reduced near-infrared AF and increased near-infrared backscatter signal suggest

that loss of RPE melanin is the most likely explanation and this is in keeping with the previously described

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histological finding of altered pigmentation in the RPE following infection with rubella virus. 3 Future

histological studies of rubella oculopathy should incorporate near-infrared and blue light AF imaging to

identify these RPE changes and to confirm co-localisation with rubella-infected cells.

Despite the novelty of our multimodal imaging findings, there are several limitations in this study. The

number of patients described here was small and this was a retrospective review of the clinical data and

images. We also did not perform fluorescein angiography in any of the patients and follow up was short. In

addition, it was not possible to ascertain or verify a history of maternal rubella infection from the fifty-year-old

male because his parents were deceased. Although, we did not perform treponemal serology to rule out

congenital syphilis, we believed this was an unlikely diagnosis given the absence of other systemic features

of this condition.

It has been over 70 years since the Australian ophthalmologist Norman McAlister Gregg published

his observations of infants with the triad of cataracts, congenital heart defects and deafness.29 However, it

was Aileen Mitchell who was the first to describe pigmentary retinopathy associated with CRS.29 Although the

rubella virus was isolated 20 years later, the exact cellular mechanism for rubella retinopathy remains

unknown. Recent developments in high-resolution and wide-field retinal imaging offer an opportunity to study

cellular responses to rubella infection in vivo. This study extends the description of AF imaging in rubella

retinopathy by illustrating altered AF signal in the perimacular and equatorial regions using wide-field imaging

and the greater disturbance in AF signal seen with near-infrared compared to blue light excitation. We

demonstrated relative preservations of the cone ellipsoid zone, wave-guiding cone tips and retinal function in

regions of hypo-AF in the maculae of patients with rubella retinopathy. Combined with subtle disturbance of

the interdigitation zone and formation of pseudo-vitelliform lesions, these fundus AF abnormalities in

congenital rubella may indicate abnormal interaction between photoreceptor and the RPE and melanin

metabolism. Having observed these findings in our series, it must nevertheless be noted that the number of

patients described here is small and the data and images have been retrospectively gathered. Additionally, it

was not possible to ascertain or verify a history of rubella infection in the mother of the 50yo male during

pregnancy as his parents were deceased and treponemal serology had not performed to rule out congenital

syphilis.Higher resolution retinal imaging using scanning laser-based AO systems to examine foveal cones

and rods, histological examination and longer term follow up of these lesions in a larger number of affected

patients may clarify the pathophysiology of these lesions and determine if they are indeed non-progressive.

 

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REFERENCES

1. Lambert N, Strebel P, Orenstein W, Icenogle J, Poland G a. Rubella. Lancet. 2015;6736(14):1–11.

2. Wolff SM. The ocular manifestations of congenital rubella. Trans Am Ophthalmol Soc. 1972;70:577–614.

3. Boniuk M, Zimmerman LE. Ocular pathology in the rubella syndrome. Arch Ophthalmol. 1967;77(4):455–73.

4. Goldberg N, Chou J, Moore A, Tsang S. Autofluorescence Imaging in Rubella Retinopathy. Ocul Immunol Inflamm. 2009;17(6):400–2.

5. Schmitz-Valckenberg S, Holz FG, Bird AC, Spaide RF. Fundus autofluorescence imaging: review and perspectives. Retina. 2008;28(3):385–409.

6. Staurenghi G, Sadda S, Chakravarthy U, Spaide RF. Proposed lexicon for anatomic landmarks in normal posterior segment spectral-domain optical coherence tomography: the IN•OCT consensus. Ophthalmology. 2014;121(8):1572–8.

7. Chen FK, Khoo YJ, Tang I. Near-Infrared Autofluorescence Imaging in Geographic Atrophy Using Spectralis Single and Combined Wavelength Modes. Asia-Pacific J Ophthalmol. 2015 Sep;

8. Delori FC, Dorey CK, Staurenghi G, Arend O, Goger DG, Weiter JJ. In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. Invest Ophthalmol Vis Sci. 1995;36(3):718–29.

9. Keilhauer CN, Delori FC. Near-infrared autofluorescence imaging of the fundus: visualization of ocular melanin. Invest Ophthalmol Vis Sci. 2006;47(8):3556–64.

10. Hauschild A, Grob JJ, Demidov L V., Jouary T, Gutzmer R, Millward M, et al. Dabrafenib in BRAF-mutated metastatic melanoma: A multicentre, open-label, phase 3 randomised controlled trial. Lancet. Elsevier Ltd; 2012;380(9839):358–65.

11. Infante JR, Fecher L a., Falchook GS, Nallapareddy S, Gordon MS, Becerra C, et al. Safety, pharmacokinetic, pharmacodynamic, and efficacy data for the oral MEK inhibitor trametinib: A phase 1 dose-escalation trial. Lancet Oncol. 2012;13(8):773–81.

12. Pilotto E, Vujosevic S, Melis R, Convento E, Sportiello P, Alemany-Rubio E, et al. Short wavelength fundus autofluorescence versus near-infrared fundus autofluorescence, with microperimetric correspondence, in patients with geographic atrophy due to age-related macular degeneration. Br J Ophthalmol. 2011;95(8):1140–4.

13. Duncker T, Tabacaru MR, Lee W, Tsang SH, Sparrow JR, Greenstein VC. Comparison of near-infrared and short-wavelength autofluorescence in retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2013;54(1):585–91.

14. Duncker T, Marsiglia M, Lee W, Zernant J, Tsang SH, Allikmets R, et al. Correlations Among Near-Infrared and Short-Wavelength Autofluorescence and Spectral-Domain Optical Coherence Tomography in Recessive Stargardt Disease. Invest Ophthalmol Vis Sci. 2014;55(12):8134–43.

15. Kellner U, Kellner S, Weber BHF, Fiebig B, Weinitz S, Ruether K. Lipofuscin- and melanin-related fundus autofluorescence visualize different retinal pigment epithelial alterations in patients with retinitis pigmentosa. Eye (Lond). 2009;23(6):1349–59.

16. Yee C, Ivanova E, Sagdullaev BT. Homologous network interactions between AII amacrine cells are essential for aberrant activity in RD. Invest Ophthalmol Vis Sci. 2015;56(7):3230.

17. Ayata A, Tatlipinar S, Kar T, Unal M, Ersanli D, Bilge AH. Near-infrared and short-wavelength autofluorescence imaging in central serous chorioretinopathy. Br J Ophthalmol. 2009;93(1):79–82.

18. Kim SK, Kim SW, Oh J, Huh K. Near-infrared and short-wavelength autofluorescence in resolved central serous chorioretinopathy: Association with outer retinal layer abnormalities. Am J Ophthalmol. 2013;156(1):157–64.e2.

19. Koizumi H, Maruyama K, Kinoshita S. Blue light and near-infrared fundus autofluorescence in acute Vogt-Koyanagi-Harada disease. Br J Ophthalmol. 2010;94(11):1499–505.

20. Oishi M, Oishi A, Ogino K, Makiyama Y, Gotoh N, Kurimoto M, et al. Wide-field fundus autofluorescence abnormalities and visual function in patients with cone and cone-rod dystrophies. Invest Ophthalmol Vis Sci. 2014;55(6):3572–7.

21. Bunt-Milam AH, Kalina RE, Pagon RA. Clinical-ultrastructural study of a retinal dystrophy. Invest Ophthalmol Vis Sci. 1983;24(4):458–69.

10

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2

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78

910

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1920

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242526

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22. Szamier RB, Berson EL, Klein R, Meyers S. Sex-linked retinitis pigmentosa: ultrastructure of photoreceptors and pigment epithelium. Invest Ophthalmol Vis Sci. 1979;18(2):145–60.

23. Szamier RB, Berson EL. Retinal ultrastructure in advanced retinitis pigmentosa. Invest Ophthalmol Vis Sci. 1977;16(10):947–62.

24. Milam AH, Jacobson SG. Photoreceptor Rosettes with Blue Cone Opsin Immunoreactivity in Retinitis Pigmentosa. Ophthalmology. 1990;97(12):1620–31.

25. Lee J-Y, Bowden DS. Rubella Virus Replication and Links to Teratogenicity. Clin Microbiol Rev. 2000;13(4):571–87.

26. Williams LL, Lew HM, Davidorf FH, Pelok SG, Singley CT, Wolinsky JS. Altered membrane fatty acids of cultured human retinal pigment epithelium persistently infected with rubella virus may affect secondary cellular function. Arch Virol. 1994;134(3-4):379–92.

27. Elsner AE, Burns SA, Weiter JJ, Delori FC. Infrared imaging of sub-retinal structures in the human ocular fundus. Vision Res. 1996;36(1):191–205.

28. Cideciyan A V, Swider M, Jacobson SG. Autofluorescence imaging with near-infrared excitation:normalization by reflectance to reduce signal from choroidal fluorophores. Invest Ophthalmol Vis Sci. 2015;56(5):3393–406.

29. Gregg NM, Banatvala JE. Congenital cataract following German measles in the mother. Trans opthalmological Soc Aust. 1942;3:35–46.

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FIGURE LEGENDS

Figure 1Wide-field pseudo-color retinal imaging of Case 1 showed salt and pepper appearance in both fundi (A, B)

with subtle speckled hypo-AF and hyper-AF lesions (C, D). Cases 2, 3 and 4 (rows 2, 3 and 4 respectively)

also showed bilateral salt and pepper appearance on pseudo-color wide-field retinal imaging (E,F, I, J, M, N),

but they had more prominent speckled hypo-AF and hyper-AF lesions on wide-field green light excitation (G,

H, K, L, O, P).

Figure 2Multimodal imaging in Cases 2 (A-D) and 4 (E-G) showing the origin of hyper-AF signals. In case 2, there

was marked speckled hypo-AF and hyper-AF on blue light (A) and near-infrared excitation (B). The larger

hyper-AF lesions (larger white box) were associated with the presence of subretinal pseudo-vitelliform

lesions on SD-OCT (C). The punctate hyper-AF lesions highlighted by the smaller box corresponded to the

presence of hyperreflective subretinal deposit (D, double-headed arrow). Case 4 had a speckled pattern of

abnormal AF on blue light excitation (E) and near-infrared excitation (F). The hyper-AF lesion outlined in the

white box was a small drusen-like lesion on SD-OCT (G). White single-headed arrows (C, G) indicate the

location of the RPE. Note the difference between the pseudo-vitelliform lesion (brighter on blue light AF, RPE

below lesion) and the drusen-like lesion (brighter on near-infrared AF, RPE elevated).

Figure 3Macular images of the right and left eyes of case 1 showed that there was a speckled pattern of abnormal

reflectance in both eyes (A, B). Green AF showed relatively uniform pattern of AF (C, D), but there was

prominent speckled hypo-AF on near-infrared AF imaging (E, F). Some of these hypo-AF lesions

corresponded to hyper-reflective lesions on near-infrared reflectance imaging (white arrows).

Figure 4Near-infrared fundus autofluorescence (AF) images (A, B) showed focal hypo-AF lesions scattered

throughout the macular region of Case 2. Minimum intensity projection en face optical coherence

tomography (OCT) of the ellipsoid zone (see insert) showed patches of focal attenuation (C, D) that are not

as widespread as is shown in the near-infrared AF image. Minimum intensity project en face OCT of the

interdigitation zone (see insert) also showed similar distribution of attenuated signal (E, F) compared to the

ellipsoid zone en face map. Oval regions of ellipsoid and interdigitation zone loss in the left eye (D, F)

corresponded to pseudo-vitelliform lesions.

Figure 5Microperimetry overlay on blue light (A) and near-infrared AF (B) fundus images of the right eye of Case 2.

Inserts show relative preservation of retinal sensitivity in regions of hypo-AF. Adaptive optics cone mosaic

montage overlay on blue (C) and near-infrared AF (D) fundus images of the right eye of Case 2. Inserts

shows wave-guiding cones in regions of hypo-AF. Cone density map of the right eye of Case 2 (E) showing

overall reduced density due to failure by AOdetect software to identify cones with poor signal.

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Figure 6Rod specific electroretinography (ERG) showed b-wave amplitudes of 147 and 132 µV (normal range: 102-

308) and latencies of 91 and 87 ms (81-100) for right and left eyes respectively. Mixed rod-cone ERG a-

wave amplitudes were 127 and 137 µV (123–234) and latencies were 22 and 23 ms (21-23). B-wave

amplitudes were 194 and 212 µV (214-475) and latencies were 54 and 53 ms (48-55) for right and left eyes.

Cone ERG a-wave amplitudes were 19 and 23 µV (18-39) and latencies were 14 and 15 ms (14-17). B-wave

amplitudes were 92 and 102 µV (77-174) and latencies were 29 ms (28-31) for right and left eyes. 30 Hz

flicker ERG amplitudes were 57 and 58 µV (54-150) and latencies were 28 and 29 ms (25-28) for right and

left eyes. Electro-oculography (EOG) showed normal light rise with Arden ratios of 2.1 and 2.3 for right and

left eyes respectively (Normal range: 1.9 – 3.6).

Figure 7Blue light autofluorescence of the right and left eyes of Case 3 in 2013 (A, B) with follow-up performed in

2015 (C, D) shows no significant progression of hypo-AF lesions. Microperimetry of the central 20° shows

preserved retinal sensitivities throughout the macula of each eye (E, F) and this is confirmed on histogram

analysis of the retinal sensitivity measurements (G,H)

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