Five-year follow-up of fundus autofluorescence and retinal sensitivity in the fellow eye in exudative age-related macular degeneration in Japan.

PURPOSE: To assess the 5-year change in abnormal fundus autofluorescence (FAF) patterns and retinal sensitivity in the fellow eye of Japanese patients with unilateral exudative age-related macular degeneration (AMD).
METHODS: Patients with unilateral exudative AMD who developed abnormal FAF in the fellow eyes were enrolled. FAF imaging and microperimetry were performed at baseline and follow-ups. FAF findings were classified into 8 patterns based on the International Fundus Autofluorescence Classification Group to assess retinal sensitivity. Forty-five points covering the central 12 degrees on microperimetry were superimposed onto the FAF images. Each point was classified depending on the distance from the abnormal FAF. "Close" was defined as the portion within 1 degree from the border of any abnormal FAF, and "Distant" was defined as the portion over 1 degree from the border of abnormal FAF. To investigate the association between the retinal sensitivity and distance from the abnormal FAF, hierarchical linear mixed-effect models were used with the distance, time and time squared from baseline (months), and angle (degrees) as fixed effects. Differences among patients, eyes, and test point locations were considered successively nested random effects.
RESULTS: We studied 66 fellow eyes with abnormal FAF. Twenty-seven eyes were followed-up during the 5 years. In the 13 of 27 eyes (48%), the abnormal FAF patterns had changed during the 5 years. We found retinal sensitivity was associated significantly with the distance from the abnormal FAF ("Distant": p<0.001, time2 from baseline: p<0.001, angle: p<0.001). The mean retinal sensitivity of the "Close" tended to deteriorate after the third year and eventually showed the similar sensitivity as the portion within the abnormal FAF.
CONCLUSION: FAF patterns can change about half during the 5 years and the retinal sensitivity near abnormal FAF tends to deteriorate after the third year.

Introduction

Age-related macular degeneration (AMD) is a progressive retinal degenerative disease in the elderly [1]. There are several classifications of AMD in the literature [2]. In this study, we use the classification according to the Age-Related Eye Disease Study (AREDS) category [3]. According to the AREDS, no AMD (AREDS category 1) represents the control group; it is characterized by no or few small drusen (<63 μm in diameter). Early AMD (AREDS category 2) is characterized by a combination of multiple small drusen, few intermediate drusen (63–124 μm in diameter), or mild RPE abnormalities. Intermediate AMD (AREDS category 3) is characterized by any of the following features: numerous intermediate drusen, at least one large drusen (>125 μm in diameter), or geographic atrophy (GA). Advanced AMD (AREDS category 4) is characterized by one or more of the following in one eye: GA of the RPE involving the foveal center and neovascular maculopathy. Previous studies reported that the fellow eye in unilateral exudative AMD represents a risk factor for the development of choroidal neovascularization (CNV) or GA involving the center of the macula [4-6]. In intermediate-to-late AMD, patients are recommended to quit smoking [7,8] and maintain a balanced and healthy diet [9]. Other risk factors such as hypertension, atherosclerosis, overweight [10], and genetic factors [11] are also reported. Therefore, the detection of early-to-intermediate AMD is important for the prophylactic guidance to patients. Microperimetry is a useful tool to detect a slight functional change and fundus autofluorescence (FAF) is a useful tool to detect a slight fundus change non-invasively.

Many studies related to microperimetry have been performed [12-14]. Clinically, microperimetry is used for diseases with visual impairment, such as AMD [15,16], central serous chorioretinopathy [17,18], angioid streaks [19], and retinal dystrophy including choroideremia [20,21].

FAF imaging with a short wavelength detects the lipofuscin distribution in the retinal pigment epithelium (RPE) cell monolayer noninvasively [22,23]. Therefore, FAF imaging supports a quantifiable assessment of GA through detection of a hypofluorescent atrophic area [24, 25]. In addition, various abnormal FAF patterns have been reported in the eyes with early-to-intermediate AMD. Our previous study demonstrated that the prevalence of abnormal FAF patterns and clinical features of late AMD were different among races [9].

The association between microperimetry and FAF imaging has been reported with a short-duration follow-up [9,26]. We previously reported the one-year results [9]. However, long-duration follow-ups and the clinical course of retinal sensitivity and FAF pattern changes have not been reported.

Our aim is to demonstrate the clinical course of retinal sensitivity and FAF pattern changes without treatment in the fellow eye of patients with unilateral exudative AMD in a Japanese population with a long-duration follow-up.

Materials and methods

Study design

The Japanese Fundus Autofluorescence and Microperimetry in Early Age-Related Maculopathy (JFAM) study group conducted this study from December 2006 to March 2014 at 5 university hospitals in Japan. Data of all consecutive patients with unilateral exudative AMD who developed abnormal FAF in the fellow eyes were prospectively studied.

Ethical statement

This study adhered to the tenets of the Declaration of Helsinki. The Institutional Ethics Committees of the Nagoya City University Graduate School of Medical Sciences, Nihon University School of Medicine, Osaka University Graduate School of Medicine, Fukushima Medical University School of Medicine, and Kyushu University Graduate School of Medical Sciences reviewed and approved the study protocol (University Hospital Medical Information Network approval number: R000043372/UMIN000038050). Patients provided written informed consent before participating in the study.

Study patients and protocol

The eligible patients were Japanese, aged ≥50 years, and had unilateral advanced AMD with exudative choroidal neovascularization. The inclusion criterion was hyper- or hypofluorescence on FAF imaging in the fellow eye without exudative choroidal neovascularization. FAF imaging was performed with the Heidelberg Retina Angiogram Digital Angiography System (HRA) or HRA2 (Heidelberg Engineering, Heidelberg, Germany). The major exclusion criteria were the presence of exudative findings in the fellow eyes, including CNV, hemorrhage, serous RPE detachment, serous retinal detachment, and hard exudates, diabetic retinopathy, uveitis, high myopia (less than -8.0 diopters), retinal vein occlusion, hazy media interfering with fundus examinations, or a history of laser photocoagulation. In this study, the fellow eyes had drusen and/or pigmentary abnormalities (n = 65) and extrafoveal geographic atrophy (n = 1) at initial visit [9].

The participants were monitored during the 5 years. Each participant underwent measurements of the best-corrected visual acuity (BCVA), binocular funduscopy, and color fundus photography at baseline and at the 3, 6, 9, 12, 24, 36, 48, and 60 months. FAF imaging and microperimetry were performed at baseline and at the 6, 12, 24, 36, 48 and 60 months.

Macular sensitivity was measured with a microperimetry device (MP-1, Nidek Technologies, Padova, Italy) using the Nidek Advanced Vision Information System software (NAVIS; Nidek Technologies). We adopted the same parameters as previously reported, a customized radial grid of 45 stimuli covering the central 12 degrees, stimuli size Goldman III with a presentation time of 200 ms, and 0–20 dB stimulus light intensity of 4-asb white light as the background. A 4–2 stimuli strategy was used [9].

Image analysis

FAF patterns were determined based on the classification system provided by the International Fundus Autofluorescence Classification Group (IFAG) [27,28]. Four authors (ST, MS, YO, and TY) graded the images into the following 8 patterns: minimal change, focal increase, focal plaque-like, patchy, linear, lace-like, reticular, and speckled. The JFAM study group created a flow chart to facilitate the pattern classification, which was based on the IFAG definition [9]. We used the flow chart to evaluate the FAF patterns.

The decision of the area on FAF was made by one retina expert (TY) for all cases. Hyperfluorescent and/or hypofluorescent portions were demarcated manually. Subsequently, these images were overlaid on fundus photographs to assess colocalization of the hyperfluorescent and/or hypofluorescent spots with the findings on color funduscopy. The baseline retinal sensitivity maps on microperimetry were overlaid on the baseline FAF images using the dedicated Nidek Advanced Vision Information System software. Retinal points tested with microperimetry were classified into 3 groups at baseline based on the distance from the abnormal FAF: “Within” (within abnormal FAF), “Close” (within 1 degree from the border of abnormal FAF), and “Distant” (over 1 degree from abnormal FAF). Those test patches were followed-up until the 5th year with baseline classifications. The mean retinal sensitivity in each group was calculated at each time point. The average change from baseline to each time point was also assessed.

Statistical analysis

The paired t-test was used to compare the retinal sensitivity at each year compared to baseline by each pattern of abnormal FAF during the 5 years. P < 0.05 was considered significant.

To investigate the association between the retinal sensitivity and distance from the abnormal FAF, hierarchical linear mixed-effect models were used with the distance, time and time squared from baseline (months), and angle (degrees) as fixed effects. Differences among patients, eyes, and test point locations were considered successively nested random effects, i.e., test point locations nested within eyes within patients.

We used two models, the first of which included a random intercept for each patient, each eye nested within each patient, and each test point location nested within each eye within each patient (model 1). The second model added random slopes to time and time squared from baseline (model 2). Akaike’s information criterion (AIC) and Bayesian information criterion (BIC) were used to compare the statistical models used. Both criteria are parsimony-adjusted indices useful for examining the fit of competing models. These criteria are based on the log likelihood value of a given model and impose a penalty on over-parameterized models. With these statistics, the preferred model is associated with lower relative values [28].

Analysis was performed with the nlme package in R [Jose Pinheiro DB, DebRoy Saikat, Sarkar Deepayan, the R Core team (2018). nlme: Linear and Nonlinear Mixed Effects Models].

Results

Patient characteristics

Sixty-six fellow eyes with abnormal FAF were enrolled in this study. The baseline color fundus photographs of 62 eyes and FAF images of 66 eyes were of sufficient quality [9]. No eyes with a normal pattern were enrolled in this study. Exudative AMD occurred in 14 (21.2%) of 66 eyes during the 5 years in fellow eyes, with the occurrence in 6 eyes within 1 year and in 8 eyes between the 2- and 5-year follow-ups. Advanced AMD such as polypoidal choroidal vasculopathy (PCV), typical AMD, and retinal angiomatous proliferation were detected in 4, 9, and 1 eye, respectively (the six eyes are written in Table 4 of our previous report [9], and the other 8 eyes are written in Table 1 of this manuscript).

Table 1

Characteristics of eight eyes with progression to exudative AMD.

Case	Age	Month with withdrawal	Mean retinal sensitivity		Abnormal FAF pattern	Hard drusen	Soft drusen	Confluent drusen	Hyper pigmentation	Hypo pigmentation
			Baseline	Final Examination
1	88	17	14.0	14.0	Patchy	+	+	-	+	-
2	72	35	15.6	8.5	Patchy	+	+	+	-	-
3	81	30	12.3	17.5	Patchy	-	+	+	-	-
4	69	42	16.1	11.8	Patchy	-	+	+	-	-
5	80	14	13.9	16.6	Minimal change	+	+	-	-	+
6	80	22	10.1	12.4	Reticular	-	+	+	+	-
7	80	39	9.7	7.4	Focal increase	-	+	+	+	-
8	77	26	12.8	14.4	Patchy	-	+	+	-	-

Of the remaining 52 eyes, the number of patients’ loss to follow-up was 2, 5, 6, 3, and 3 at the 1-, 2-, 3-, 4-, and 5-year follow-ups. Thirty-three eyes completed the 5-year follow-up. However, six eyes were followed-up with only microperimetry or FAF. Twenty-seven eyes completed 5 years of follow-up with both microperimetry and FAF imaging (right eyes were 12 (44.4%) (Table 2).

Table 2

Baseline profiles of study patients (n = 27).

Age (mean ± SD, years)	71.2 ± 7.2 (54–85)
Gender
Male	19 (70.4%)
Female	8 (29.6%)
Best-Corrected Visual Acuity, logMAR (Snellen)	-0.02 ± 0.11 (20/20)
Mean retinal sensitivity	14.1 ± 4.3
Type of drusen—number of eye (%) (Some findings are overlapped)
Hard drusen	7 (25.9%)
Soft drusen [with confluent]	25 (92.6%) [12 (44.4%)]
Pigmentary abnormalities	16 (59.3%)
Diagnosis of fellow eye—no. (%)
Typical neovascular AMD	19 (70.4%)
PCV	8 (29.6%)

SD: standard deviation; LogMAR: logarithm of the minimum angle of resolution.

Fundus findings and patterns of abnormal FAF

FAF patterns of sixty-six eyes when enrolling had 8 patterns. However, 27 eyes which were followed-up during the 5 years had only 7 patterns. Twenty-seven eyes visualized on microperimetry at baseline were classified into the following 7 patterns: minimal change 1, focal increase 10, linear 2, focal plaque-like 4, patchy 6, lace-like 3, and speckled 1 (Fig 1).

Fig 1

The proportions of FAF patterns.

Top: all FAF patterns of 66 eyes at baseline. Middle: FAF patterns at baseline of 27 eyes which were followed-up during the 5 years. Bottom: the final FAF patterns of 27 eyes at the 5th year. The numbers within graphs show the actual number of the eye.

Transition to other FAF patterns

In 13 of 27 eyes (48%), the abnormal FAF patterns changed at the 5th year (Fig 1 and Table 3).

Table 3

Pattern alterations observed during follow-up.

Initial FAF pattern	Last FAF pattern	Number of eyes, n (%)
Focal increase	minimal change	1 (7.7%)
	linear	1 (7.7%)
	patchy	2 (15.4%)
	lace-like	2 (15.4%)
Linear	lace-like	1 (7.7%)
Focal plaque-like	focal increase	2 (15.4%)
	patchy	1 (7.7%)
Patchy	lace-like	1 (7.7%)
Lacelike	patchy	2 (15.4%)
Total		13 (100%)

Visual acuity and retinal sensitivity

The mean baseline logarithm of the minimum angle of resolution (logMAR) BCVA of 66 eyes was -0.011 ± 0.128. The mean logMAR BCVA of the 27 eyes that underwent both microperimetry and FAF decreased from -0.023 ± 0.108 at baseline to 0.058 ± 0.188 at the 5th year, which showed significance (p = 0.026, paired t-test).

The retinal sensitivity within 12 degrees was measurable during the 5 years in 27 eyes (1,215 points on microperimetry in total, others were not eligible for analysis). The baseline mean retinal sensitivity was 14.1 ± 4.3 dB, which decreased to 13.5 ± 5.8 dB at the 5th year. The mean baseline retinal sensitivity of the 14 eyes that developed AMD was 12.9 ± 4.4 dB (630 points in total). The mean retinal sensitivity in eyes with each abnormal FAF pattern differed among the abnormal FAF patterns. The speckled FAF pattern had the lowest retinal sensitivity at the 5th year. Although linear, focal plaque-like, patchy and speckled FAF pattern deteriorated at the 5th year compared to baseline, lace-like pattern improved at the 5th year (Fig 2).

Fig 2

Transition of mean retinal sensitivity by each pattern of abnormal FAF during the 5 years.

The number of each pattern differs at each time-point compared to baseline (cf. Fig 1). The sensitivity of the linear, focal plaque-like, patchy, and speckled FAF pattern decreased significantly compared to the baseline at the 5th year. The speckled FAF pattern at the 5th year had the lowest mean retinal sensitivity. The sensitivity of lace-like increased significantly compared to baseline at the 5th year. Data are expressed as mean ± standard error (SE). * P < 0.05 compared to baseline, paired t-test. BL: baseline.

Retinal sensitivity and the distance

In total, 1,215 points on microperimetry in 27 eyes were followed-up during the 5 years and were classified into 3 groups (Within, Close, and Distant) based on the distance from the abnormal FAF. The mean retinal sensitivities were 15.0 ± 0.8, 12.3 ± 0.9 and 10.6 ± 0.9 dB in the Distant, Close and Within group, respectively, at baseline. “Close” showed deterioration in sensitivity after the 3rd year (p < 0.05) compared to baseline and resulted in 10.7 ± 1.3 dB at the 5-year follow-up. The mean retinal sensitivity of the “Close” tended to deteriorate after the third year and showed eventually the similar sensitivity as the portion within the abnormal FAF (Fig 3).

Fig 3

The association between the mean retinal sensitivity and the distance from the abnormal FAF (1,215 points per year).

Total 1,215 points were classified as “Within” (within abnormal FAF), “Close” (within 1 degree from the border of abnormal FAF), and “Distant” (over 1 degree from the border of abnormal FAF). Data are expressed as mean ± SE.

First, we fitted a random intercept model to account for a three-level nested data structure. i.e., test point locations nested within eyes within patients (model 1). We also fitted a random intercept and slope model to assess possible variation on the effect of time and time squared from baseline across patients, eyes nested within patients and test point locations nested within eyes within patients (model 2). In a comparison of the two models, model 2, provided a better fit than model 1, with an AIC and BIC of 30897.95 and 30964.84 in model 1, and 30155.77 and 30262.79 in model 2, respectively. Thus, we examined the association between the retinal sensitivity and the distance from the abnormal FAF based on model 2 and found the retinal sensitivity was associated significantly with the distance from the abnormal FAF (Table 4).

Table 4

Results of the random intercept and slope model.

	Estimate	SE	P-value
Distance: Within	Reference
Close	0.48	0.40	0.227
Distant	2.34	0.37	<0.001
Time	0.04	0.01	<0.001
Time2	<-0.01	<0.01	<0.001
Angle	0.41	0.04	<0.001

Fig 4 is a representative case of FAF imaging and color fundus imaging.

Fig 4

A representative case of FAF imaging and color fundus imaging.

A 66-year-old man. The focal increase FAF pattern was seen at baseline, and the mean retinal sensitivity was 12.2 dB. In 3 years, the portion showing hyperfluorescence at baseline changed to hypofluorescence in part, and the hyperfluorescence expanded and changed to the patchy pattern. In the fundus photograph, drusen increased during the 5 years. The mean retinal sensitivity was 13.4 dB at the 3rd year and 9.5 dB at the 5th year.

Discussion

In the current study, we studied 66 eyes with unilateral exudative AMD, which showed an abnormal FAF in the contralateral eye. In total, 14 out of 66 eyes (21.2%) progressed to exudative AMD during the 5-year follow-up. Patients with advanced AMD or vision loss due to non-advanced AMD in 1 eye (Category 4) reportedly have a 43% expected probability of progression to advanced AMD in the fellow eye after 5 years [6]. CNV reportedly develops in the other eye in 12.3% of the Japanese population by 5 years [5]. Our result showed a higher incidence than that reported 20 years before in Japan. This may be influenced by westernization of the diet.

In this study, seven (50%) of 14 eyes which progressed to exudative AMD during the 5 years showed the patchy FAF pattern. The remaining patchy FAF pattern (6 eyes (22.2%) at baseline) showed no significant change in retinal sensitivity until the 3rd year but showed a significant decrease in retinal sensitivity at the 3rd, 4th and 5th year compared to baseline (10 eyes (37.0%) at the 5th year) (Fig 2). Although the retinal sensitivity of patchy, linear, focal plaque-like and speckled pattern deteriorated at the 5th year compared to baseline, the lace-like pattern improved at the 5th year in this study. The mechanism of recovery of retinal sensitivity is unknown. It is possible that the lace-like pattern may be associated with RPE hyperplasia or other transient stressed condition. Because the lace-like pattern was not co-localized with drusen. In our previous report, we demonstrated that antioxidant supplementation might affect the recovery of retinal sensitivity [9]. Therefore, supplementation and smoking cessation may be related to the recovery of this specific pattern of abnormal FAF. Possible other reasons may involve the learning effect and the inaccuracy of auto-tracking of the microperimeter.

FAF pattern changes in this study was partially similar to other report [29]. Further study is needed to ascertain whether these pattern changes are reversible or not.

In general, retinal sensitivity in areas showing an abnormal FAF pattern was low from the start. In this study, test patches within 1 degree from the border of an abnormal FAF pattern deteriorated during the 5-year course. Retinal sensitivity decreased significantly in the Close group after the 3-year follow-up compared to baseline, probably as a result of the expansion of abnormal FAF with time. The test patches which were “Close” group may change to “Within” group during the 5 years. This indicates that a more careful follow-up is necessary for the “Close” (within 1 degree from the border of abnormal FAF) group. On the other hand, areas over 1 degree from the border of an abnormal FAF pattern showed good retinal sensitivity during the 5-year course.

This study has some limitations, including the absence of systematic evaluation of other known risk factors for AMD such as hypertension, atherosclerosis, overweight [10], and genetic factors [11]. Some older subjects may get weaker and less cognitive during the 5-year follow-up period even though they are perfectly healthy at the time of enrollment. An age-matched control group is necessary in the future study.

Conclusions

The fellow eyes of patients with unilateral exudative AMD were studied to assess the 5-year change. Our results imply that FAF patterns can change about half during the 5-year and the retinal sensitivity within 1 degree from the border of abnormal FAF tends to deteriorate after the third year. Microperimetry and FAF are presumed to be useful for long-term changes in retinal sensitivity and prediction of prognosis.

30 Oct 2019

PONE-D-19-28095

Five-Year Follow-Up of Fundus Autofluorescence and Retinal Sensitivity in the Fellow Eye in Age-Related Macular Degeneration in Japan

PLOS ONE

Dear Dr. Yasukawa

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by December 31, 2109. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. You do not need agree with all the points raised by the reviewers.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Yuhua Zhang

Academic Editor

PLOS ONE

Journal requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We understand this manuscript may be closely relate to a previous publication, written by you/you co-authors:

Fundus autofluorescence and retinal sensitivity in fellow eyes of age-related macular degeneration in Japan.

https://doi.org/10.1371/journal.pone.0213161

If the participant populations of the two studies overlap please include this information in the Methods section.

3. Thank you for stating the following in the Financial Disclosure section:

"Santen Pharmaceutical Co., Ltd. provided support in the form of honoraria for speaking and/or organizing at meetings (AS, MS, RM, TS, Yuji Oshima, TI, Yuichiro Ogura, FG, TY), but did not have any additional role in the study design, data analysis, decision to publish, or preparation of the manuscript. This work was also supported by JSPS KAKENHI Grant Number JP19K18893.".

We note that you received funding from a commercial source: [Santen Pharmaceutical Co., Ltd]

* Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.

* Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

* Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

Reviewer #3: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. P9 line 137: What do you mean “hyper- or hypofluorescence on FAF imaging in the fellow eye”? The follow eye has hypofluorescence but not (dry) AMD? Or it may has (dry) AMD also? If both eye has been diagnosed to have AMD, which eye would be considered as fellow eye? Although it has been stated in the previous study in the series, it is still worth a brief description in this paper.

2. Figure 1: standard deviation bar is missing. Also mark how many eyes and percentage of the 27 included for each pattern.

3. Figure 2: mark how many spots and also percentage of total analyzed spots for distant, close and within.

4. Sadly the only FAF pattern (speckled) shows worst progression and no pattern change has only 1 eye under study. Though the study cannot be re-done, yet the conclusion may not be convincing based on this very small sample size.

5. P19 Line293 In terms of the pattern change happened in this study during the 5 year, there is a brief description in the discussion but including a graph could be more helpful. How’s your pattern change trends compared to other studies?

6. P20 Line303 How would you explain the “Close” pattern the sensitivity slightly “recovers” between 3 and 5 years? Worth more discussion.

7. P20 Line316 The reversible sensitivity also appears in lace-like pattern, what do you (and others) say about it?

Reviewer #2: This prospective, observational study assessed the 5-year changes of FAF and retinal sensitivity evaluated by microperimetry in the fellow eyes of Japanese patients with unilateral

nAMD. I have the following questions:

1. Line 205-210, in this paragraph the authors described the data of the 14 eyes which progressed to nAMD (6 within Year 1 and 8 between Year 2 to Year 5), but in Table 2 the authors listed only the data of the 8 eyes progressed between Year 2 to Year 5, which made some data appear to be unmatched and confusing. Please clarify this.

2. Have the authors tried to compare the demographics, the FAF patterns, mean retinal sensitivity, as well as the % of “within”, “close”, and “distant” spots between the 8 eyes progressed to nAMD between 2-5 years and the other 6 eyes progressed within 1 year?

3. The exclusion criteria based on refractive error was < -8D, which was looser than most studies appoint -6D as the diagnostic criteria of high myopia. Previous studies showed macular sensitivity decreases with spherical equivalent. Given that East Asian countries have more high myopic patients than anywhere else over the world, I have a concern that if too many subjects in this study had relatively high myopia (between -6D to -8D), the retinal sensitivity results could be biased. Please comment on this point and if possible, exclude those with relatively high myopia.

4. Line 218-222, is there any statistical significance between the baseline mean sensitivity of the 27 eyes without progression and the 14 eyes with progression, and between the baseline and Year 5 mean sensitivity of the 27 eyes? What’s the sensitivity of the 14 eyes at the endpoint? Or the last measurement before those eyes became wet?

5. Line 238-239, “…deteriorated after 3 years compared to the other patterns (p < 0.001). *P < 0.05 compared to baseline, paired t-test.” Can the authors clarify what’s the statistical method used and how the comparisons were made here? Is it ever possible to use paired t-test to do this comparison (speckled VS other patterns)? Similarly, in Line 246-247, the method for conducting the statistical analysis should also be clearly stated in the Methods Section.

6. With the disease progression during the 5 years, I think it is likely that the prior “distant” point may later turn “close” or “within”, and the prior “close” point may later become “within”. If such alteration happened, how were the points classified? In Figure 2, the authors mentioned that paired t test was used. Does it mean that even a point has altered over time, it still belongs to the initial group?

7. Is it possible to divide each group (within, close, and distant) into 2 subgroups: progressive and non-progressive, and compare their sensitivity?

8. Line 252-255, unfortunately, the statistical methods were inappropriate no matter whether it was paired or unpaired t test, as the authors described in the Methods Section. When analyzing the 1215 points obtained from 27 eyes during multiple follow-ups, the data was not independent (multiple points from one eye) and repeated measurements were taken. Therefore, a mixed-effects model or a generalized estimating equation should be used. Also, the deterioration rate of the retinal sensitivity can be modeled by this means.

9. The test statistics (e.g., F, t) should be shown before the P value.

10. Line 266-269, please clearly describe how many cases have changed from one pattern to another, with more detailed demographic features and the converted time of those eyes. A table is preferred.

11. The authors stated that the lace-like FAF pattern may be associated with retinal sensitivity reservation over time. However, there was only one such case involved in the study, and the slight increase of retinal sensitivity at Year 3 can also be attributed to the learning effect of the patient. More careful investigations should be conducted before this conclusion is made.

12. Figure 1: Each line on this grey scale figure is very difficult to distinguish. Perhaps the authors may want to use a color figure for better illustration. Also, please add tick marks of each axis. If possible, data obtained at Year 2 & Year 4 could also be added.

13. As a psychophysics test, the results of microperimetry may be significantly affected by the examinees’ cognitive status, which is highly associated with older age and systemic health. It is not surprising that some older subjects get weaker and less cognitive during the 5-year follow-up period even though they are perfectly healthy at the time of enrollment. That’s why an age-matched control group is necessary. The lack of a control group should be discussed as a limitation of this study.

14. Minor comments:

• Careful language polish may be needed. For example, in multiple places (e.g., Line 190, Line 233, Line 234 …) “for” 5 years should be replaced by “during the”; Line 230 “up to the 5-year follow-up” should better be replaced by “during the 5 years”; Line 236-237, “after 5 years” could be “The speckled FAF pattern had the lowest retinal sensitivity compared to the baseline at Year 5/at the 5th year”. Line 245, “at 5 years” should be “at the 5th year”. Line 246, “after 3 years” should be “after the 3rd year”. The expression of “The speckled FAF pattern at 5 years was the least sensitive” should be “The speckled FAF pattern at the 5th years had the lowest mean retinal sensitivity”. Line 252, “more” should be “better”. There are more similar errors, the authors may need to carefully check it thoroughly.

• Caption of Figure 2: there is no data are expressed as mean ± standard error, in the figure.

• Line 198-199: need reformatting.

• Line 205-205: Polypoidal choroidal vasculopathy, wet AMD, and retinal angiomatous proliferation were detected in 4, 9, and 1 eyes, respectively (Table 1). But Table 1 didn’t contain such information.

• Line 209: lost should be loss.

• Line 209-210, “The number of patients lost to follow-up was 2, 5, 6, 3, and 3 at the 1-, 2-, 3-, 4-, and 5-year follow-ups”, please move this sentence to Line 191, after “Of the remaining 191 eyes”, in order to minimize any confusion.

• Line 310, “after” 5 years may be inaccurate.

Reviewer #3: General comments

The authors report on a comparison between microperimetry and FAF patterns in a cohort of patients followed longitudinally for five years. Several are lost to follow-up or convert to CNV in the fellow eye, leaving 27 total eyes that were followed fully for five years. The authors show that test patches ‘close’ to abnormal areas seen on FAF decrease over three years. The authors show that one FAF pattern ‘speckled’ significantly decreased between both 3 and 5 years from baseline. Unfortunately, they only had one participant that exhibited this pattern, making it difficult to make generalizable conclusions based on a single observation. Another major drawback is that the authors only looked at FAF and did not assess how other pathology like large drusen at the test patch locations could have altered sensitivity in those areas, especially as drusen are dynamic and could change over the five years. The manuscript could be improved by better weighting of this finding with respect to the rest of the results as it seems like there is too much emphasis placed on this point. It is also difficult to understand how the FAF image classification changed over time because the authors state that for 14/27 followed for five years that they changed over time but it doesn’t mention how they changed from year to year – this is particularly confusing with respect to figure 1 as this figure implies that the pattern stayed the same over time. This point needs to be clarified. There are also several specific items that could be altered to improve the overall presentation of the work, described below.

Specific comments

Abstract, lines 60-62: The wording of the descriptions of the classification of test points is awkward, please consider rephrasing.

Abstract, lines 69-71: This is confusing as written. Please rephrase from “…lost statistical significance…” to make this clearer.

Abstract, lines 72-73: The emphasis here on the speckled pattern relies on an n=1, perhaps the authors can add some additional points to this conclusion statement to less heavily weight this finding based on a single eye.

Introduction, line 77: The phrasing “…in the elderly in developed countries.” Suggests that AMD only exists in developed countries, please rephrase.

Introduction, line 78: Some classification systems designate AMD as being AMD only if it is in persons >55 years of age, the authors should state the AMD classification system they are using here (e.g. Beckman, AREDS, etc.).

Introduction, line 84: Again, state the scale you are using to define intermediate AMD, etc.

Introduction, line 84-87: This entire sentence needs to be re-written as it is difficult to follow. Certain classification systems outline clearly what constitutes early, intermediate and advanced AMD, the authors should pick one to use and then clearly state the differences between the different stages here.

Introduction, line 91: As written, this is awkwardly phrased, particularly “…overweight, and genetic factors are considerable.”

Introduction, line 95: It is unclear what the authors are intending to state here as microperimetry give a functional change, not a ‘fundus change’. Please rephrase or reword this sentence.

Introduction, lines 100-101: This sentence does not seem to add anything and can be omitted.

Methods, lines 169-170: Who did the image grading? Was it always the same person? What criterion did they use to demarcate the areas manually? Were comparisons made between graders. It would be important for you to include enough detail here for someone else to replicate this experiment.

Results, general: The order the results are presented in does not make sense. The discussion of the transition between

patterns (lines 265-269) needs to be described in better detail and should come before the presentation of the (lines 232-239) of three- and five-year findings.

Results, line 192: Replace “…autofluorescent examination…” with “…FAF imaging…”.

Results, line 198: This sentence appears on its own and should be a part of the preceding paragraph. It should be restated here that only 27 eyes were followed for 5 years – that is how you get 44% from just 12 eyes.

Results, line 199-201: This sentence also could be merged into the preceding paragraph – it doesn’t need to be its own paragraph.

Results, lines 233-239: Are these results for participants patterns only at the last timepoint? How did they change from the first timepoint, was there a difference between those whose patterns changed and those that stayed the same?

Results, 266-269, 279-281: This is confusing and needs to be completely rewritten. The authors may need to make a table or something describing the different transitions between different patterns. It cannot be understood fully from this prose how the patterns of 66 eyes changed over 5 years.

Discussion, line 292: The word “also” can be omitted from this sentence.

Discussion, lines 299-300: What is meant by the “low-fluorescent portion”? I think we need to see these images.

Discussion, lines 300-302: Do the authors mean progress to GA? They all have dry AMD so this does not make sense.

Discussion, line 304: It seems like the authors have the data to answer this question. Are the test patches that were ‘close’ now ‘within’ abnormal areas at year 3 and is this why they have the same sensitivity as ‘within’ patches from 3-5 years?

Discussion, line 311: Do the authors mean that it is a risk factor for AMD progression?

Discussion, line 313: What is meant by ‘…consistent with pigmentation…”?

Discussion, line 316: We need more information on what the patterns transitioned to so that this point can be better understood. Did the lace pattern transition to a less altered pattern?

Discussion, line 317: Do the authors mean “areas” here rather than “lesions”?

Discussion, line 318 & 319: Do the authors mean “test patches” here rather than “lesions”?

Discussion, line 325: The authors should state ‘…”Close” test points…” rather that ‘…”Close” patterns…’

Conclusions, general: This section needs to be completely rewritten, there is too much emphasis on the speckled pattern finding and there are some points that don’t make sense (see specific comment below).

Conclusions, line 330: These patients all have dry AMD – see comment on this above.

Figure 1: Are these results for subjects whose patterns were always consistently the same at each timepoint? It is unclear what the number of subjects is in each pattern at each timepoint – maybe that could be included? Why do we have only 3 and 5 year timepoints shown here but we see sensitivity at a more granular level compared in figure 2?

Figure 2: The figure caption needs to be improved so it is clearer what is being tested for significance here at each timepoint (or if it is being tested to the first timepoint?). The (*) is compared to baseline timepoint but the (#) is compared to close at the same timepoint or at baseline? I think that a bar chart with lines showing comparisons may be needed to show exactly what was tested here. I think it would be useful for the authors to state whether the ‘close’ points at baseline would be graded as ‘within’ at the year 3 timepoint – it seems to be what is implied here. Are these test patches just now within abnormal areas or are they still ‘close’?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: review of PONE-D-19-28095.pdf

Click here for additional data file.

24 Jan 2020

We appreciate the comments from all reviewers. We carefully responded to all of comments raised by three reviewers. Now we believe that all issues are addressed in the revised version of the manuscript. The part of sentences changed is written in red. We reanalyzed our results according to the reviewers’ questions. Therefore, we added new author (Takeshi Nishiyama) who analyzed the results. I would be pleased to respond to any questions or comments you may have. We re-wrote the title and introduction for better understanding this manuscript. NOTE: The order of results, references and figures have been changed.

Submitted filename: Responses to reviewers 20200123.docx

Click here for additional data file.

12 Feb 2020

Five-Year Follow-Up of Fundus Autofluorescence and Retinal Sensitivity in the Fellow Eye in Exudative Age-Related Macular Degeneration in Japan

PONE-D-19-28095R1

Dear Dr. Yasukawa,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Yuhua Zhang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

24 Feb 2020

PONE-D-19-28095R1

Five-Year Follow-Up of Fundus Autofluorescence and Retinal Sensitivity in the Fellow Eye in Exudative Age-Related Macular Degeneration in Japan

Dear Dr. Yasukawa:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Yuhua Zhang

Academic Editor

PLOS ONE