A Morphological Study of Retinal Changes in Unilateral Amblyopia Using Optical Coherence Tomography Image Segmentation
2014

A Szigeti
E Tátrai
A Szamosi
P Vargha
Z Z Nagy
A Morphological Study of Retinal Changes in Unilateral Amblyopia Using Optical Coherence Tomography Image Segmentation

PLoS ONE
92883632014Received October 5, 2013; Accepted January 12, 2014;Editor: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The University of Miami and Dr. Cabrera DeBuc hold a pending patent used in the study and have the potential for financial benefit from its future commercialization. The remaining authors declared that no competing interests exist. The data of the patent are the following: Name: System and method for early detection of diabetic retinopathy using optical coherence tomography Pub. No.: WO/2010/080576 International Application No.: PCT/US2009/ 068653. This does not alter the authors9 adherence to all the PLoS ONE policies on sharing data and materials. *
Objective: The purpose of this study was to evaluate the possible structural changes of the macula in patients with unilateral amblyopia using optical coherence tomography (OCT) image segmentation.Patients and Methods: 38 consecutive patients (16 male; mean age 32.4617.6 years; range 6-67 years) with unilateral amblyopia were involved in this study. OCT examinations were performed with a time-domain OCT device, and a custombuilt OCT image analysis software (OCTRIMA) was used for OCT image segmentation. The axial length (AL) was measured by a LenStar LS 900 device. Macular layer thickness, AL and manifest spherical equivalent refraction (MRSE) of the amblyopic eye were compared to that of the fellow eye. We studied if the type of amblyopia (strabismus without anisometropia, anisometropia without strabismus, strabismus with anisometropia) had any influence on macular layer thickness values.Results: There was significant difference between the amblyopic and fellow eyes in MRSE and AL in all subgroups. Comparing the amblyopic and fellow eyes, we found a statistically significant difference only in the thickness of the outer nuclear layer in the central region using linear mixed model analysis keeping AL and age under control (p = 0.032). There was no significant difference in interocular difference in the thickness of any macular layers between the subgroups with one-way between-groups ANCOVA while statistically controlling for interocular difference in AL and age.Conclusions: According to our results there are subtle changes in amblyopic eyes affecting the outer nuclear layer of the fovea suggesting the possible involvement of the photoreceptors. However, further studies are warranted to support this hypothesis.

Introduction

Amblyopia remains an important cause of low visual acuity, affecting 2% to 6% of the general population [1][2][3][4][5]. Unilateral amblyopia is defined as reduced best-corrected visual acuity (BCVA) secondary to an abnormal visual experience during the critical period of visual development. Classic causes include strabismus, anisometropia, form deprivation or a combination of these factors [6].

The neural sites that are influenced by visual deprivation are still under investigation. Nevertheless, it has been reported by several studies in humans [7,8] and also in animal species [9][10][11][12] that visual deprivation has an effect on the cell growth in the lateral geniculate body that receives input from the amblyopic eye and on the shift in the dominance pattern in the visual cortex [13], Banko et al revealed that latencies of the event-related potential components increased and were more variable in the amblyopic eye compared to the fellow eye [14], although the initial neural site of the visual deficit in this condition is still under investigation.

Evidences for direct retinal changes in amblyopic eyes are still inconclusive and controversial [15][16][17], although electroretinograms elicited by patterned stimuli in humans with various types of amblyopia were found to be significantly reduced [18,19]. Studies using optical coherence tomography (OCT) imaging of the retina have produced discordant results, some investigators have found an increased peripapillary retinal nerve fiber layer (cpRNFL) [20][21][22] or/and macular thickness [20,[23][24][25][26] in amblyopic eyes, whereas others have found no significant differences between amblyopic and healthy eyes [27][28][29][30][31][32].

Yen et al. hypothesized that the normal postnatal reduction (apoptosis) of retinal ganglion cells is arrested in amblyopia and predicted that this would cause increased cpRNFL thickness [21]. If this does indeed occur, it is likely that the arrest of normal postnatal changes would result not only in increased RNFL thickness but also would affect the normal maturation of the macula, including movement of Henle's fibers away from the foveola and a decrease in foveal cone diameter. This would result in increased foveal thickness [21]. Furthermore, because of the reduced apoptosis of retinal ganglion cells, the thickness of the ganglion cell layer in the macula would also be increased.

The purpose of this study was to evaluate the structural changes of the macula in patients with unilateral amblyopia using OCT image segmentation.

Materials and Methods

Subjects

This prospective study was performed at the Department of Ophthalmology, Semmelweis University, Budapest, Hungary, on 38 consecutive Caucasian patients (16 male; mean age 32.4617.6 years; range 6-67 years) with unilateral amblyopia who presented at the Orthoptics Outpatient Clinic of the department for examination. Only amblyopic eyes with BCVA of 20/30 or worse were included in the study. The BCVA of the fellow eye was 20/ 20 in each patient.

Patients with a history of intraocular surgery, retinal or neurological disease (e. g. multiple sclerosis), optic nerve disease including glaucoma, nystagmus, laser treatment or any ocular media opacities including cataract were excluded from the study. All participants were treated in accordance with the tenets of the Declaration of Helsinki. Institutional Review Board approval was obtained for all study protocols (Semmelweis University Regional and Institutional Committee of Sciences and Research Ethics). Written informed consent was obtained from all participants in this study. In case of subjects under 18 years of age, written informed consent was obtained from a parent or a legal guardian.

In order to study the effect of the type of amblyopia on interocular difference (IOD) in the different macular layers, patients were divided into 3 study groups: strabismic patients without anisometropia (Group S), anisometropic patients without strabismus (Group A) and combined amblyopic patients with strabismus and anisometropia (Group AS). Anisometropia was defined as an IOD of at least 1.5 diopters of the manifest spherical equivalent refraction (MRSE). For the group descriptions see Table 1.

All patients underwent a comprehensive eye examination, including BCVA (measured with Snellen chart adjusted at 5 m, converted to logMAR values for analysis), cover and coveruncover test, duction and version testing, measurement of the intraocular pressure, slit-lamp and indirect fundus examination after pupil dilation and determining manifest refraction and cycloplegic refraction after pupil dilation with 3 times cyclopentolate 1% drops.

Spherical equivalent was defined as the spherical power plus half of the minus cylinder power (sphere+ K cylinder).

The axial length (AL) of the eyes was measured using a LenStar LS 900 device (LS 900H Haag-Streit AG, Koeniz, Switzerland).

Optical Coherence Tomography

OCT examinations were performed on each eye using a timedomain (TD) Stratus OCT device (Carl Zeiss Meditec, Dublin, CA), by the same operator through dilated pupils at least 5 mm in diameter. ''Fast RNFL map protocol'' consisting of three circular scans with diameters of 3.4 mm centered on the optic disc was performed along with the ''Macular Thickness Map'' protocol consisting of six radial scan lines centered on the fovea, each having a 6 mm transverse length. In order to obtain the best image quality, focusing and optimization settings were controlled and scans were accepted only if the signal strength (SS) was .6 (preferably 9-10). Scans with foveal decentration [i.e., with center point thickness standard deviation (SD) .10%] were repeated. Patients who had poor fixation cooperation due to poor vision or low age were excluded from the study.

The mean overall cpRNFL thickness values measured by the built-in software of Stratus OCT were recorded for each eye. In order to assess the thickness of intraretinal layers, the raw data of all 6 OCT images were exported from the device and processed using a custom-built image analysis software (OCTRIMA) [33,34]. The OCTRIMA software's methodology includes the removal of speckle noise followed by the automatic detection of the boundaries of the cellular layers of the retina. Manual correction of the boundary detection is enabled if segmentation errors are present. The repeatability and reproducibility of the thickness measurements was assessed earlier and was found to be very good [35,36]. All the scans were segmented by the same blinded operator to reduce the possible bias caused by different operators and unblinded analysis. (Figure 1). The thickness values for the RNFL, ganglion cell layer and inner plexiform layer complex (GCL+IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), retinal pigment epithelium (RPE) and the total retina measured by OCTRIMA were recorded for each eye in the nine regions of the macula defined by the Early Treatment Diabetic Retinopathy Study (ETDRS) Group. [37] The distribution of the regions is the following: 1 central, 2 inner superior, 3 inner nasal, 4 inner inferior, 5 inner temporal, 6 outer superior, 7 outer nasal, 8 outer inferior, 9 outer temporal. The thickness of the ganglion cell complex (GCC) composed by the RNFL and GCL+IPL consisting of distal and proximal parts of the retinal ganglion cells was also determined to determine the integrity of the ganglion cells.

In order to assess the intraretinal layers in various eccentricities from the fovea, the mean thickness of the layers for the central (R1), pericentral (R2-R5) and peripheral (R6-R9) ETDRS regions of the macula were calculated. Since the number of sampling points is different at the central, pericentral and peripheral regions because of the radial spoke pattern used in the scanning protocol of Stratus OCT, a weighted mean thickness was calculated for the total macula instead of averaging retinal thickness results in the 9 macular regions [38]. The weighted mean thickness was generated using the following equation:
WMT~R 1 36 z R2zR3zR4zR5 18 z (R6zR7zR8zR9)x3 16

Statistical Analysis

The magnitude of anisometropia and IOD in BCVA, AL and macular thickness were obtained by subtracting the value obtained in the fellow eye from the value obtained in the amblyopic eye.

The distributions of MRSE, BCVA, AL and macular thickness were confirmed as normally distributed by the Shapiro-Wilks W and Kolmogorov-Smirnov tests and therefore macular thickness, AL and MRSE of the amblyopic eye was compared to that of the fellow eye using the paired t-test.

In order to prevent multicollinearity we examined the Pearson correlation between the independent variables (AL, BCVA, MRSE) before the multiple regression analysis. The IOD in BCVA did not correlate either with IOD in AL or with IOD in MRSE (Pearson correlation IOD in BCVA vs. AL r = 0.217 p = 0.218 n = 34; Pearson correlation IOD in BCVA vs. MRSE r = 0.015 p = 0.929 n = 38), while IOD in MRSE (i.e. anisometropia) correlated with the IOD in AL (Pearson correlation r = 2 0.602, p,0.0001, n = 34). Thus, we excluded anisometropia as an independent variable from the multiple regression model.

Multiple regression was performed between interocular difference in macular layers as dependent variable and IOD in AL and BCVA, which showed statistically significant correlation between IOD in the thickness of most retinal layers and IOD in AL. (p, 0.05), while IOD in BCVA did not correlate with IOD in most retinal layer thickness data. (p.0.05) (see Table 2).

Linear correlation was calculated between intraretinal layer thickness data and age in the control eyes which revealed significant correlation for the ONL, RPE layers and the total macula in the central region (see Table 3), therefore we controlled our further analyses for age as well.

One-way between-groups ANCOVA was used to test if the type of amblyopia (Group S, A, AS) had influence on the IOD in macular layer thickness between amblyopic and fellow eyes. Because of the above lack of correlation between IOD in BCVA and IOD in retinal layer thickness, the test was controlled for IOD in AL and age. In order to compare macular thickness data linear mixed model was used, keeping AL and age under control.

Statistical analyses were performed using SPSS software program (Statistical Package for Social Sciences, SPSS version 16.0; SPSS Inc., Chicago, IL, USA). A p value of ,0.05 was considered statistically significant.

Results

A total of 38 patients with unilateral amblyopia were enrolled in this study. Altogether 17 patients (44.7%) had strabismic amblyopia, 11 patients (28.9%) had anisometropic amblyopia, and 10 patients (26.3%) had combined amblyopia (strabismus and anisometropia). Of the patients with anisometropia, spherical hyperopia was noted in 9 cases and myopia was present in 2 cases. Among patients with strabismus, esotropia was noted in 12 cases with a mean of 22614 (range: 6-40) prism diopters (PD) deviation, and exotropia was noted in 5 cases with a mean of 35612 (range: 14-40) PD deviations. In the mixed cases (Group AS), there were 5 cases in which spherical anisometropic hyperopia and exotropia (mean deviation: 28610 PD, range: 14-40 PD) were concomitantly present, and there were 5 cases with anisometropic hyperopia combined with exotropia (mean deviation: 30610 PD, range: 20-40 PD). (Table 1) There were 12 right and 26 left amblyopic eyes.

The mean BCVA was +0.6960.39 logMAR in all amblyopic eyes involved in the study and 0.00 in all fellow eyes. In the S, A and AS subgroups the mean BCVAs were +0.6760.44 logMAR; + 0.7560.34 logMAR; and +0.6460.38 logMAR, respectively. The mean MRSE in the amblyopic eyes was +2.9762.39 diopters (D) and in the normal fellow eyes was +1.6561.87 D. The mean AL was 22.5161.08 mm in the amblyopic eyes and 22.9661.03 mm in the fellow eyes. Table 4 shows the MRSE and AL values in the different subgroups. There was statistically significant difference between the amblyopic and fellow eye in MRSE and AL in all subgroups. Two patients with strabismic amblyopia and two patients with anisometropic amblyopia did not undergo axial length measurements due to technical reasons.

The mean macular layer thickness and cpRNFL thickness measurements are shown in Table 5. Comparing the amblyopic and fellow eyes without including any other parameters in the test there was a slight statistically significant difference in the GCC layer in the pericentral region and in the OPL layer calculated for the total macula and measured in the peripheral region (paired t test; p = 0.018; p = 0.014). However, after implementing a linear   We studied whether the type of amblyopia had any influence on the IOD in macular layers between amblyopic and fellow eye with one-way between-groups ANCOVA while statistically controlling for IOD in AL and age. There was no significant difference in IOD in macular layers and cpRNFL between Group S, A and AS.

Discussion

In this study we assessed the retinal structural changes of the macula in patients with unilateral amblyopia using OCT imaging and image segmentation methodology.

Amblyopia occurs during the period when the neuronal network between the retina and the cerebral cortex is developing and maturing. The neural sites that are influenced by visual deprivation are still under investigation. However, some animal studies demonstrated abnormal findings in retinal microstructures, including degeneration of retinal ganglion cells [39,40], decreased nucleolar volume and cytoplasmic cross-sectional area of RGCs [16], an increased number of amacrine synapses in the IPL [41,42], a reduction in the number of bipolar synapses in the IPL  [41], thinning of the IPL [16,43], and a decrease in the density of Müller fibres [43]. Evidence for direct retinal changes in amblyopic eyes remain inconclusive and controversial. Yen et al. hypothesized that amblyopia may affect the postnatal maturation of the retina, including the postnatal reduction of retinal ganglion cells, which would lead to a measurable increase in the thickness of the retinal nerve fiber layer in amblyopic eyes [21]. If this indeed occur, it is likely that the arrest of normal postnatal changes would result not only in increased RNFL thickness but also would affect the normal maturation of the macula, including movement of Henle's fibers away from the foveola and a decrease in foveal cone diameter, and would result in increased foveal thickness [21]. According to this assumption and the above mentioned animal studies we could reasonably hypothesize that some anatomic rearrangement could be present in the retina.

A small number of previous studies that aimed the assessment of retinal structural changes in amblyopia has been reported. Enoch et al. were the first of many authors to suggest a specific cause for an organic anomaly affecting the retina in amblyopia [44]. More recently, using a third-generation nerve fiber analyzer (GDx, Laser Diagnostic Technologies, San Diego, CA), Colen et al. measured RNFL thickness in strabismic amblyopia and reported no significant difference between amblyopic and sound eyes [45]. In 2005, Altintas et al. carried out OCT examination on 14 unilateral strabismic amblyopic patients and no difference was seen in macular and cpRNFL thickness or macular volume [27]. Kee et al. enrolled 26 unilateral amblyopic children (6 strabismic, 15 anisometropic, 5 combined amblyopes), and found no difference in cpRNFL in any of examined 4 quadrants (superior, inferior, nasal, temporal) and foveal thickness between neither the amblyopic eye and fellow eye, nor between values of these amblyopic patients and 42 normal control children using timedomain OCT [29]. However, they found statistically significant difference in mean thickness values of the fovea and the retinal nerve fiber layer of the amblyopic eyes of the children with anisometropic amblyopia (n = 15) and strabismic amblyopia (n = 6) (146.5 vs. 173.1 mm p = 0.046, cpRNFL 112.9 vs. 92.8 mm p = 0.034) [29]. They did not measure the AL, and in the anisometropic group 10 of 15 children were myop.

Repka et al. completed studies in 2006 and 2009 evaluating 17 patients aged 5-28 years and subsequently 37 amblyopic children and found no difference in cpRNFL thickness between amblyopic and sound eyes using TD-OCT [30,31]. Similarly, in 2011, Walker et al. investigated 30 adults (mean age: 56 years) with amblyopia (using Cirrus HD-OCT) and found no statistically significant difference in RNFL thickness of any peripapillary quadrants and macular thickness in any anatomical location [32].

In 2004, Yen et al. used 2nd generation optical coherence tomography to measure cpRNFL in 38 patients (mean age 26.4, range 6-75 years) with unilateral amblyopia (strabismic and refractive amblyopia) and found no significant difference between strabismic amblyopic and normal eyes [21]. However, the cpRNFL was significantly thicker in eyes with refractive amblyopia compared with the fellow eye and the differences were significant in the multivariate regression analysis as well with adjustment for AL, SE, age and sex [21]. Yoon et al had similar findings in a study of 31 hyperopic anisometropic children regarding cpRNFL thickness (115.2 vs. 109.6 mm, p = 0.019) but found no difference in mean macular retinal thickness (252.5 vs. 249.7 mm) [22]. They did not measure the AL.

In the Sydney Childhood Eye Study, Huynh et al. tested 48 unilateral amblyopes (17 strabismic, 19 hyperopic anisometropia) and reported that amblyopic eyes had slightly greater foveal minimum thickness than the normal fellow eye (by 5.0 mm) and the right eyes of non-amblyopic children (by 10 mm) [25]. This difference was more pronounced in 6-year old children (6.9 mm) than 12-year old children (4.2 mm) [25]. The IOD in foveal minimum thickness was greater in children who did not receive any treatment for unilateral amblyopia [25]. Foveal minimum thickness remained significantly greater in amblyopic than nonamblyopic eyes, after adjusting for amblyopia severity and IOD in AL (p = 0.01). In their study the inner macular ring was significantly thinner in amblyopic children, and there were no significant differences in outer macular ring thicknesses, central and total macular volume or in cpRNFL [25].

In 2011, Alotaibi et al. evaluated 93 unilateral amblyopic eyes (36 strabismic, 33 anisometropic, 24 combined) and found significantly thicker RNFL (259.3 vs. 255.6 mm, p,0.0001) in the overall amblyopic group, and no significant difference in macular and foveal thickness [20]. There was slightly higher macular and foveal thickness only in the anisometropic amblyopic group (macular thickness: 256.76 vs. 246.61 mm p = 0.050; foveal thickness: 187.12 vs. 177.61 mm p = 0.039) [20]. However, they did not measure the AL either.

In the studies by Dickmann et al. a significant difference between the amblyopic and the fellow eye was found in mean macular thickness only in the strabismic amblyopic group, and there was no difference for the refractive amblyopic group, similarly to the cpRNFL in any of the amblyopic groups [23,24]. Alotaibi and Dickmann suggested based on their findings that amblyopia of different etiologies is associated with the loss of different neural cells [20,23,24]. Later, in 2012, Dickmann evaluated 15 strabismic (esotropic) and 15 anisometropic amblyopic patients, and found no intereye differences in cpRNFL, macular thickness and foveal volume in neither group using spectral-domain (SD) OCT [28].

In 2011, Pang et al. investigated 31 myopic children with unilateral amblyopia [26]. The refractive error in spherical equivalent in the amblyopic eyes was 210.7963.40 diopters and in the normal fellow eyes was 21.6762.90 D. The mean magnitude of anisometropia was 9.1263.53 D, ranging from 3.63 to 17.50 D. They found a statistically significant difference in macular thickness between amblyopic and fellow eyes, with amblyopic eyes having greater foveal thickness but reduced inner and outer macular thickness [26]. No statistically significant differences were indentified in the macular thicknesses between subgroups (purely myopic anisometropia n = 24, combined mechanism amblyopia n = 7). IOD in AL (measured AL with A-scan ultrasound biometry) showed a moderate correlation with the nasal, superior and temporal outer macular thickness [26].

To the best of our knowledge, there are three recent studies that employed some form of OCT image segmentation in amblyopia so far. Al-Haddad et al used one single horizontal SD-OCT scan for the manual segmentation of six layers of the central 1000 mm diameter area and found an increase in the INL and a decrease in the ONL in the temporal area in amblyopic eyes compared to the fellow eyes, while the mean foveal thickness was increased in amblyopic eyes (228.56620.2 vs 221.7615.3 mm) [46]. Tugcu et al. used the built-in analysis option of the RTVue OCT platform to measure the thickness of the GCC and found an increase in strabismic amblyopia (99.29 vs. 103.08 mm, p = 0.019, amblyopic vs. nonamblopic eye) while there was no such difference for the anisometropic or combined subgroups [19]. Park et al. enrolled 20 unilateral amblyopic children (16 strabismus, 2 anisoastigmatism, 2 unilateral ptosis) with a mean age of 9.064.03 (4-19 years) and examined horizontal and vertical spectral-domain OCT scans through the fovea [47]. Thickness Table 6. Summary of previous studies employing optical coherence tomography of the retina in patients with amblyopia.      values were measured at the foveal centre and in 500 and 1500 mm distance from the foveal centre in all 4 quadrants (superior, inferior, nasal, temporal). The thickness of each retinal layer (GCL+IPL, INL, OPL, ONL, IS, OS, RPE) was measured manually using the calipers provided with the SD-OCT instrument [47]. They found significantly decreased thickness in the thickness of the GCL+IPL at all four nasal and temporal macular locations and at the outer superior and inferior locations [47]. The ONL was thinner at the inner and outer temporal locations and thicker at the inner and outer superior and inner nasal locations. The NFL and OPL were thicker in the amblyopic eyes than in the fellow eyes in some areas and thinner in other areas [47]. It should be noted that none of these studies corrected their results for either age or axial length.

In the present study we used OCT image segmentation methodology involving the entire macular area, extracting seven retinal layers. We found that subtle changes may be present in the retina in unilateral amblyopia. As there was evidence showing the confounding effect of AL and age we used state-of-the-art statistical methodology in order to keep these variables under control. As most of the similar previous studies in the field were using basic comparisons, we also performed such analyses. Interestingly, the basic pairwise comparisons were indicating significant changes in the GCC layer in the pericentral region and in the OPL layer calculated for the total macula and measured in the peripheral region. However, after applying rigorous statistical methodology in order to account for the effects of AL and age, these differences disappeared, while a significant difference was revealed for the central (foveal) ONL. This implies that the photoreceptors and not the ganglion cells could be affected by amblyopia, this being the opposite of what was earlier speculated.

Previous studies reported that there was a correlation between retinal thickness and AL, age, or even race in healthy eyes [48][49][50][51][52] and that AL may have an effect on the thickness of the intraretinal layers in the macula [53]. Song et al. found that axial length correlated negatively with average outer macular thickness, overall average macular thickness and macular volume [51].

In unilateral amblyopia, we found significant difference in AL between amblyopic and fellow eyes (not only in the anisometropic subgroup), and multiple regression showed a statistically significant correlation between IOD in the thickness of most retinal layers and IOD in AL. For this reason, the effect of AL must be taken into consideration in statistical analyses to obtain reliable results, just as we did in our study. In contrast, most of the previous studies about amblyopic retinal OCT measurements were not taking into account this potential effect of AL, which could influence their results. A summary of these studies, also mentioned above, can be found in Table 6.

In support of our results, there is early evidence showing that photoreceptors may be affected in amblyopia. First, Enoch suggested that photoreceptor orientation is abnormal in amblyopic eyes using the Stiles-Crawford function [44], while others did not found indication of retinal dysfunction at the level of the cone photoreceptors in amblyopic eyes. Later, three groups described electro-oculographic abnormalities in amblyopic patients, with their results providing evidence for a retinal abnormality in amblyopia [54][55][56]. These results were also suggestive of the retinal pigment epithelium being involved in the process. Indeed, the RPE plays an important role in maintaining visual pigment density and perhaps also in the maintenance of photoreceptor orientation.

Leone et al. reviewed the literature on measuring macular thickness in amblyopes and proposed that increased macular thickness found by several studies may be due to inadvertent measurement of a parafoveal eccentric point in amblyopia [57]. To address this issue, central fixation was confirmed in each subject in our study by ensuring the location of the foveal depression at the center of the macular scan. Furthermore, interocular mean differences of macular thickness in nonamblyopic patients have been investigated and were minimal, even if degrees of asymmetry existed in considered individual patients.

There are several limitations to our study, namely, the small number of patients, wide age range (6-67 years), and the lack of a control group of non-amblyopic healthy patients. First, the number of patients involved in the study is comparable to those reported in most papers in the field; however, a larger study is warranted to confirm our results. Second, we were able to use the healthy eye in each patient as a control. The use of TD-OCT could limit the thickness measurements of the outermost retinal layers (e.g. the RPE) performed by OCTRIMA as compared to the improved resolution of (SD-OCT) devices. We have previously shown the high reproducibility and repeatability of OCTRIMA measurements and their comparability with SD-OCT results, therefore, we believe that our results would be similarly valid if performed by a newer generation of OCT devices [34][35][36]. Additionally, SD-OCT could detect the fine structural changes by the improved resolution and thus better segmentation of the photoreceptor outer segments. Finally, axial length could also influence the measured thickness of the retinal structures due to the reflectance directionality which can degrade the accuracy of technologies assessing RNFL thickness [58].

In addition, we would like to point out that retinal thickness measured by SD-OCT differs from that measured by TD-OCT because the delineation of the outer boundary of the retina differs in the two instruments; which corresponds to the inclusion of the outer segment-RPE-Bruch's membrane-choriocapillaris complex in the measurements. It is well known that segmentation errors are less frequent with SD-OCT. This could be attributed to the greater resolution and acquisition speed of SD-OCT. Therefore, the custom-built software used in our study facilitates the manual correction of segmentation errors by the operators; which eliminates the uncertainty of erroneous thickness calculations due to artifacts. We would like to note that a recently published study that evaluated macular thickness, agreement and intraclass repeatability in eyes with pathologies obtained by three optical coherence tomography devices (Stratus TD-OCT and two SD-OCTs, Spectralis and Cirrus OCT), revealed that although each OCT device has a unique method of defining algorithms and cannot be used interchangeably, each device has the ability to measure the retinal thickness accurately and repeatedly [59].

To the best of our knowledge, this is the first study to measure the macular thickness in unilateral amblyopia using OCT imaging and image segmentation of the entire macula area. According to our results patient age and axial length should be taken into consideration in segmentation studies in amblyopia. Using our methodology we observed subtle changes in amblyopic eyes affecting the outer nuclear layer of the fovea suggesting the possible involvement of the photoreceptors. However, further studies are warranted to support this hypothesis.

Figure 1 .
1Macular image segmentation using OCTRIMA software. Top: the image of a healthy macula scanned by Stratus OCT. Bottom: the same OCT scan processed using OCTRIMA software. Abbreviations: GCL+IPL, ganglion cell layer and inner plexiform layer complex; INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RNFL, retinal nerve fiber layer; RPE, retinal pigment epithelium. doi:10.1371/journal.pone.0088363.g001

(
Abbreviations: ND: no data, FT: foveal thickness, FV: foveolar volume, MT: macular thickness, FMT: foveal minimum thikness; type of amblyopia: anisometropic amblyopia: A, strabismic amblyopia: S, combined amblyopia patients with strabismus and anisometropia: AS, TD-OCT(2): second generation time-domain OCT, TD-OCT(3): third generation time-domain OCT, SD-OCT: spectral domain OCT). doi:10.1371/journal.pone.0088363.t006

Table 1 .
1Descriptive statistics and distribution of study participants. (SD: standard deviation).Amblyopic groups
Number
Gender (m/f)
Age (years) mean ± SD (range)
Characteristic
Number

Group S
17 (44.7%)
6/11
31.2614.9
(8-56)

Esotropia
12

Exotropia
5

Group A
11 (28.9%)
5/6
39.9624.2
(6-67)

spherical hyperopia
9

spherical myopia
2

Group AS
10 (26.3%)
5/5
37.9618.0
(12-67)

spherical hyperopia+ esotropia
5

spherical hyperopia+ exotropia
5

spherical myopia+esotropia
0

spherical myopia +exotropia
0

Total
38 (100%)
16/22
32.4617.6
(6-67)

(Group S: strabismic patients without anisometropia; Group A: anisometropic patients without strabismus; Group AS: combined amblyopic patients with strabismus and
anisometropia).
doi:10.1371/journal.pone.0088363.t001

Table 2 .
2Multiple regression analysis between interocular difference (IOD) in macular layers as dependent variable and IOD in axial length (AL) and best-corrected visual acuity (BCVA).Adjusted R square, which indicates the strength of the model, standardised regression coefficients (beta) and p values. GCL+IPL: ganglion cell layer and inner plexiform layer complex; GCC: ganglion cell complex; INL: inner nuclear layer, OPL: outer plexiform layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium. doi:10.1371/journal.pone.0088363.t002IOD in retinal
layers

Model
adjusted
R square Independent variable

IOD in AL
IOD in BCVA

beta
p
beta
p

Mean thickness

RNFL
0.287
0.309
0.049
0.422
0.009

GCL+IPL
0.094
20.376
0.034
20.038
0.822

GCC
0.040
20.134
0.448
0.314
0.082

INL
0.127
20.423
0.016
0.187
0.270

OPL
0.112
20.298
0.086
0.349
0.046

ONL
0.377
20.549
0.000
20.238
0.101

RPE
20.018
20.203
0.267
20.023
0.900

total macula
0.158
20.461
0.008
0.181
0.279

Central region

ONL
0.146
20.364
0.035
0.345
0.044

RPE
0.094
20.260
0.135
20.234
0.177

total macula
0.078
0.197
0.258
0.268
0.127

Pericentral region

RNFL
0.012
0.182
0.312
0.162
0.369

GCL+IPL
0.090
0.364
0.041
0.059
0.732

GCC
0.160
0.404
0.019
0.147
0.375

INL
0.058
0.339
0.059
0.004
0.980

OPL
0.020
20.064
0.721
0.289
0.112

ONL
0.343
20.489
0.002
20.288
0.055

RPE
20.020
20.168
0.358
20.087
0.631

total macula
20.057
20.060
0.746
0.074
0.690

Peripheral region

RNFL
0.322
0.311
0.042
0.453
0.004

GCL+IPL
0.204
20.487
0.005
20.056
0.728

GCC
0.084
20.309
0.080
0.286
0.103

INL
0.291
20.586
0.000
0.209
0.173

OPL
0.162
20.398
0.021
0.335
0.049

ONL
0.356
20.559
0.000
20.191
0.193

RPE
20.024
20.199
0.278
0.023
0.901

total macula
0.247
20.550
0.001
0.181
0.250

cpRNFL
0.029
20.199
0.284
0.269
0.152

Table 3 .
3Linear correlation for intraretinal layer thickness data and age in the non-amblyopic eyes.Control eyes
Pearson correlation
p

mean thickness

ONL
20.349
0.032

RPE
0.615
0.000

central region

RPE
0.355
0.029

total macula
0.416
0.009

pericentral region

RPE
0.594
0.000

peripheral region

ONL
20.385
0.017

RPE
0.578
0.000

Only layers with a statistically significant correlation are shown.
doi:10.1371/journal.pone.0088363.t003

Table 4 .
4Manifestrefraction in spherical equivalent (MRSE)
and axial length in the study subjects compared by paired t-
test.

MRSE (D)
N

Amblyopic eye
mean ± SD
(range)

Fellow eye
mean ± SD
(range)
P values

All amblyopic
patients

38
2.9762.39
(22.75-8.88)

1.6561.83
(21.38-6.88)

0.000

Group S
17
2.3161.96
(21.00-7.50)

1.8862.03
(21.38-6.88)

0.007

Group A
11
2.7463.06
(22.75-8.88)

0.9961.11
(20.5-3.13)

0.001

Group AS
10
4.3661.78
(1.5-7.5)

1.9862.06
(20.5-5.5)

0.000

Axial length
(mm)

N
Amblyopic eye
mean ± SD
(range)

Fellow eye
mean ± SD
(range)

P values

All amblyopic
patients

34
22.5161.08
(20.78-25.68)

22.9661.03
(20.95-25.08)

0.001

Group S
15
22.4960.88
(21.02-23.94)

22.7260.96
(21.38 to 24.38)

0.028

Group A
9
22.8661.69
(20.78-25.68)

23.1161.35
(20.95-25.08)

0.002

Group AS
10
22.2160.58
(21.34 23.08)

23.1760.82
(22.1 24.36)

0.003

Note that two patients with strabismic amblyopia and two patients with
anisometropic amblyopia did not undergo axial length measurements due to
technical reasons.
doi:10.1371/journal.pone.0088363.t004

Table 5 .
5Macular layer thickness values and cpRNFL (mm) data in the study eyes with p values for the comparisons between amblyopic and fellow eyes by two different methods.Data are presented as mean 6 SD. Comparisons were performed by paired t-test and a linear mixed model keeping axial length and age under control. doi:10.1371/journal.pone.0088363.t005Amblyopic eye (n = 38)
Macular layer thickness (mm)
mean ± SD

Fellow eye (n = 38)
Macular layer thickness (mm)
mean ± SD

P values
Paired t test
(n = 38)

P values
Mixed model (controlled
age and AL) n = 34

Mean thickness

RNFL
36.5363.14
36.9562.78
0.265
0.583

GCL+IPL
71.365.89
71.3665.12
0.912
0.860

GCC
107.8366.9
108.3166.75
0.361
0.772

INL
34.5162.32
34.4861.76
0.928
0.978

OPL
33.4361.65
32.8761.43
0.032
0.844

ONL
81.9866.93
81.9266.38
0.898
0.083

RPE
12.361.34
12.4861.32
0.332
0.205

Total retina
296.39613.74
296.05612.28
0.766
0.379

Central Region

ONL
120.0268.69
119.78610.38
0.836
0.032

RPE
14.9861.61
14.9461.45
0.861
0.410

Total retina
242.22619.68
241.34622.35
0.631
0.985

Pericentral Region

RNFL
23.2963.15
23.8662.52
0.191
0.266

GCL+IPL
93.9566.49
95.0266.26
0.079
0.360

GCC
117.2368.02
118.8867.62
0.018
0.873

INL
38.6763.22
39.0162.41
0.277
0.936

OPL
38.8462.66
38.3762.33
0.320
0.471

ONL
90.9268.42
91.0368.73
0.892
0.328

RPE
12.5461.71
12.4961.72
0.842
0.284

Total retina
323.6615.26
324.64613.77
0.272
0.341

Peripheral Region

RNFL
41.8163.55
42.263.35
0.365
0.805

GCL+IPL
67.2366.6
66.9965.51
0.863
0.932

GCC
109.0467.46
109.1967.28
0.819
0.846

INL
34.5562.49
34.4261.97
0.645
0.957

OPL
33.0761.61
32.4661.47
0.014
0.481

ONL
77.9266.73
77.9366.1
0.989
0.242

RPE
12.1261.31
12.4061.31
0.167
0.247

Total retina
290.29614.45
289.61612.63
0.601
0.413

CpRNFL
106.10610.37
104.4368.74
0.182
0.098

PLOS ONE | www.plosone.org
February 2014 | Volume 9 | Issue 2 | e88363

17th annual Frank Costenbader Lecture. Amblyopia revisited. J T Flynn, J Pediatr Ophthalmol Strabismus. 28Flynn JT (1991) 17th annual Frank Costenbader Lecture. Amblyopia revisited. J Pediatr Ophthalmol Strabismus 28: 183-201.

Evidence-based guidelines for amblyogenic risk factors. T D France, Am Orthopt J. 56France TD (2006) Evidence-based guidelines for amblyogenic risk factors. Am Orthopt J 56: 7-14.

Epidemiology of strabismus. P A Graham, Br J Ophthalmol. 58Graham PA (1974) Epidemiology of strabismus. Br J Ophthalmol 58: 224-231.

Neural mechanisms underlying amblyopia. L Kiorpes, S P Mckee, Curr Opin Neurobiol. 9Kiorpes L, McKee SP (1999) Neural mechanisms underlying amblyopia. Curr Opin Neurobiol 9: 480-486.

Current concepts of amblyopia: a neuro-ophthalmology perspective. G Roper-Hall, Am Orthopt J. 57Roper-Hall G (2007) Current concepts of amblyopia: a neuro-ophthalmology perspective. Am Orthopt J 57: 2-11.

Mechanisms of amblyopia. G K Noorden, Adv Ophthalmol. 34Noorden GK (1977) Mechanisms of amblyopia. Adv Ophthalmol 34: 93-115.

The lateral geniculate nucleus in human strabismic amblyopia. G K Von Noorden, M L Crawford, Invest Ophthalmol Vis Sci. 33von Noorden GK, Crawford ML (1992) The lateral geniculate nucleus in human strabismic amblyopia. Invest Ophthalmol Vis Sci 33: 2729-2732.

The lateral geniculate nucleus in human anisometropic amblyopia. G K Von Noorden, M L Crawford, R A Levacy, Invest Ophthalmol Vis Sci. 24von Noorden GK, Crawford ML, Levacy RA (1983) The lateral geniculate nucleus in human anisometropic amblyopia. Invest Ophthalmol Vis Sci 24: 788- 790.

Cellular changes in the lateral geniculate nucleus of infant monkeys after suture of the eyelids. M P Headon, T P Powell, J Anat. 116Headon MP, Powell TP (1973) Cellular changes in the lateral geniculate nucleus of infant monkeys after suture of the eyelids. J Anat 116: 135-145.

Behavioral and morphological evidence for binocular competition in the postnatal development of the dog's visual system. S M Sherman, J R Wilson, J Comp Neurol. 161Sherman SM, Wilson JR (1975) Behavioral and morphological evidence for binocular competition in the postnatal development of the dog's visual system. J Comp Neurol 161: 183-195.

Histological studies of the visual system in monkeys with experimental amblyopia. G K Von Noorden, Invest Ophthalmol. 12von Noorden GK (1973) Histological studies of the visual system in monkeys with experimental amblyopia. Invest Ophthalmol 12: 727-738.

Effects of visual deprivation on morphlology and physiology of cells in the cats lateral geniculate body. T N Wiesel, D H Hubel, J Neurophysiol. 26Wiesel TN, Hubel DH (1963) Effects of visual deprivation on morphlology and physiology of cells in the cats lateral geniculate body. J Neurophysiol 26: 978- 993.

Effect of citicoline on visual acuity in amblyopia: preliminary results. E C Campos, C Schiavi, P Benedetti, R Bolzani, V Porciatti, Graefes Arch Clin Exp Ophthalmol. 233Campos EC, Schiavi C, Benedetti P, Bolzani R, Porciatti V (1995) Effect of citicoline on visual acuity in amblyopia: preliminary results. Graefes Arch Clin Exp Ophthalmol 233: 307-312.

Amblyopic deficits in the timing and strength of visual cortical responses to faces. E M Banko, J Kortvelyes, J Nemeth, B Weiss, Z Vidnyanszky, Cortex. 49Banko EM, Kortvelyes J, Nemeth J, Weiss B, Vidnyanszky Z (2013) Amblyopic deficits in the timing and strength of visual cortical responses to faces. Cortex 49: 1013-1024.

Failure to demonstrate changes in the visual system of monkeys kept in darkness or in colored lights. K L Chow, J Comp Neurol. 102Chow KL (1955) Failure to demonstrate changes in the visual system of monkeys kept in darkness or in colored lights. J Comp Neurol 102: 597-606.

Altered structure and composition of retinal cells in darkreared mammals. E Rasch, H Swift, A H Riesen, K L Chow, Exp Cell Res. 25Rasch E, Swift H, Riesen AH, Chow KL (1961) Altered structure and composition of retinal cells in darkreared mammals. Exp Cell Res 25: 348-363.

Effect of light deprivation on the postnatal development of the optic nerve. Wendell-Smith Cp, Nature. 204707Wendell-Smith CP (1964) Effect of light deprivation on the postnatal development of the optic nerve. Nature 204: 707.

Pattern ERG in amblyopia. G B Arden, S L Wooding, Invest Ophthalmol Vis Sci. 26Arden GB, Wooding SL (1985) Pattern ERG in amblyopia. Invest Ophthalmol Vis Sci 26: 88-96.

The morpho-functional evaluation of retina in amblyopia. B Tugcu, B Araz-Ersan, M Kilic, E T Erdogan, U Yigit, Curr Eye Res. 38Tugcu B, Araz-Ersan B, Kilic M, Erdogan ET, Yigit U, et al. (2013) The morpho-functional evaluation of retina in amblyopia. Curr Eye Res 38: 802- 809.

Unilateral amblyopia: Optical coherence tomography findings. A G Alotaibi, Al Enazi, B , Saudi Journal of Ophthalmology. 25Alotaibi AG, Al Enazi B (2011) Unilateral amblyopia: Optical coherence tomography findings. Saudi Journal of Ophthalmology 25: 405-409.

Retinal nerve fiber layer thickness in unilateral amblyopia. M Y Yen, C Y Cheng, A G Wang, Invest Ophthalmol Vis Sci. 45Yen MY, Cheng CY, Wang AG (2004) Retinal nerve fiber layer thickness in unilateral amblyopia. Invest Ophthalmol Vis Sci 45: 2224-2230.

Thicknesses of macular retinal layer and peripapillary retinal nerve fiber layer in patients with hyperopic anisometropic amblyopia. S W Yoon, W H Park, S H Baek, S M Kong, Korean J Ophthalmol. 19Yoon SW, Park WH, Baek SH, Kong SM (2005) Thicknesses of macular retinal layer and peripapillary retinal nerve fiber layer in patients with hyperopic anisometropic amblyopia. Korean J Ophthalmol 19: 62-67.

A morpho-functional study of amblyopic eyes with the use of optical coherence tomography and microperimetry. A Dickmann, S Petroni, V Perrotta, A Salerni, R Parrilla, J AAPOS. 15Dickmann A, Petroni S, Perrotta V, Salerni A, Parrilla R, et al. (2011) A morpho-functional study of amblyopic eyes with the use of optical coherence tomography and microperimetry. J AAPOS 15: 338-341.

Unilateral amblyopia: An optical coherence tomography study. A Dickmann, S Petroni, A Salerni, R Dell'omo, E Balestrazzi, J AAPOS. 13Dickmann A, Petroni S, Salerni A, Dell'Omo R, Balestrazzi E (2009) Unilateral amblyopia: An optical coherence tomography study. J AAPOS 13: 148-150.

Macular and nerve fiber layer thickness in amblyopia: the Sydney Childhood Eye Study. S C Huynh, C Samarawickrama, X Y Wang, E Rochtchina, T Y Wong, Ophthalmology. 116Huynh SC, Samarawickrama C, Wang XY, Rochtchina E, Wong TY, et al. (2009) Macular and nerve fiber layer thickness in amblyopia: the Sydney Childhood Eye Study. Ophthalmology 116: 1604-1609.

A prospective study of macular thickness in amblyopic children with unilateral high myopia. Y Pang, G W Goodfellow, C Allison, S Block, K A Frantz, Invest Ophthalmol Vis Sci. 52Pang Y, Goodfellow GW, Allison C, Block S, Frantz KA (2011) A prospective study of macular thickness in amblyopic children with unilateral high myopia. Invest Ophthalmol Vis Sci 52: 2444-2449.

Thickness of the retinal nerve fiber layer, macular thickness, and macular volume in patients with strabismic amblyopia. O Altintas, N Yuksel, B Ozkan, Y Caglar, J Pediatr Ophthalmol Strabismus. 42Altintas O, Yuksel N, Ozkan B, Caglar Y (2005) Thickness of the retinal nerve fiber layer, macular thickness, and macular volume in patients with strabismic amblyopia. J Pediatr Ophthalmol Strabismus 42: 216-221.

Measurement of retinal nerve fiber layer thickness, macular thickness, and foveal volume in amblyopic eyes using spectral-domain optical coherence tomography. A Dickmann, S Petroni, V Perrotta, R Parrilla, S Aliberti, J AAPOS. 16Dickmann A, Petroni S, Perrotta V, Parrilla R, Aliberti S, et al. (2012) Measurement of retinal nerve fiber layer thickness, macular thickness, and foveal volume in amblyopic eyes using spectral-domain optical coherence tomography. J AAPOS 16: 86-88.

Thicknesses of the fovea and retinal nerve fiber layer in amblyopic and normal eyes in children. S Y Kee, S Y Lee, Y C Lee, Korean J Ophthalmol. 20Kee SY, Lee SY, Lee YC (2006) Thicknesses of the fovea and retinal nerve fiber layer in amblyopic and normal eyes in children. Korean J Ophthalmol 20: 177- 181.

Retinal nerve fiber layer thickness in amblyopic eyes. M X Repka, N Goldenberg-Cohen, A R Edwards, Am J Ophthalmol. 142Repka MX, Goldenberg-Cohen N, Edwards AR (2006) Retinal nerve fiber layer thickness in amblyopic eyes. Am J Ophthalmol 142: 247-251.

Retinal nerve fiber layer thickness in amblyopic eyes. M X Repka, R T Kraker, S M Tamkins, D W Suh, N A Sala, Am J Ophthalmol. 148Repka MX, Kraker RT, Tamkins SM, Suh DW, Sala NA, et al. (2009) Retinal nerve fiber layer thickness in amblyopic eyes. Am J Ophthalmol 148: 143-147.

Macular and peripapillary retinal nerve fibre layer thickness in adults with amblyopia. R A Walker, S Rubab, A R Voll, V Erraguntla, P H Murphy, Can J Ophthalmol. 46Walker RA, Rubab S, Voll AR, Erraguntla V, Murphy PH (2011) Macular and peripapillary retinal nerve fibre layer thickness in adults with amblyopia. Can J Ophthalmol 46: 425-427.

Automated detection of retinal layer structures on optical coherence tomography images. Cabrera Fernandez, D Salinas, H M Puliafito, C A , Opt Express. 13Cabrera Fernandez D, Salinas HM, Puliafito CA (2005) Automated detection of retinal layer structures on optical coherence tomography images. Opt Express 13: 10200-10216.

Comparison of retinal thickness by Fourier-domain optical coherence tomography and OCT retinal image analysis software segmentation analysis derived from Stratus optical coherence tomography images. E Tatrai, S Ranganathan, M Ferencz, D C Debuc, G M Somfai, J Biomed Opt. 1656004Tatrai E, Ranganathan S, Ferencz M, Debuc DC, Somfai GM (2011) Comparison of retinal thickness by Fourier-domain optical coherence tomog- raphy and OCT retinal image analysis software segmentation analysis derived from Stratus optical coherence tomography images. J Biomed Opt 16: 056004.

Improving image segmentation performance and quantitative analysis via a computer-aided grading methodology for optical coherence tomography retinal image analysis. D C Debuc, H M Salinas, S Ranganathan, E Tatrai, W Gao, J Biomed Opt. 1546015Debuc DC, Salinas HM, Ranganathan S, Tatrai E, Gao W, et al. (2010) Improving image segmentation performance and quantitative analysis via a computer-aided grading methodology for optical coherence tomography retinal image analysis. J Biomed Opt 15: 046015.

Reliability and reproducibility of macular segmentation using a custom-built optical coherence tomography retinal image analysis software. D C Debuc, G M Somfai, S Ranganathan, E Tatrai, M Ferencz, J Biomed Opt. 1464023DeBuc DC, Somfai GM, Ranganathan S, Tatrai E, Ferencz M, et al. (2009) Reliability and reproducibility of macular segmentation using a custom-built optical coherence tomography retinal image analysis software. J Biomed Opt 14: 064023.

Early Treatment Diabetic Retinopathy Study design and baseline patient characteristics. ETDRS report number. 98Early Treatment Diabetic Retinopathy Study design and baseline patient characteristics. ETDRS report number 7. (1991) Ophthalmology 98: 741-756.

Reproducibility of retinal mapping using optical coherence tomography. P Massin, E Vicaut, B Haouchine, A Erginay, M Paques, Arch Ophthalmol. 119Massin P, Vicaut E, Haouchine B, Erginay A, Paques M, et al. (2001) Reproducibility of retinal mapping using optical coherence tomography. Arch Ophthalmol 119: 1135-1142.

Degeneration of retinal ganglion cells in infant chimpanzees reared in darkness. K L Chow, A H Riesen, F W Newell, J Comp Neurol. 107Chow KL, Riesen AH, Newell FW (1957) Degeneration of retinal ganglion cells in infant chimpanzees reared in darkness. J Comp Neurol 107: 27-42.

Effect of lid suture on retinal ganglion cells in Macaca mulatta. G K Von Noorden, M L Crawford, P R Middleditch, Brain Res. 122Von Noorden GK, Crawford ML, Middleditch PR (1977) Effect of lid suture on retinal ganglion cells in Macaca mulatta. Brain Res 122: 437-444.

Effect of visual deprivation and light on synapses of the inner plexiform layer. E Fifkova, Exp Neurol. 35Fifkova E (1972) Effect of visual deprivation and light on synapses of the inner plexiform layer. Exp Neurol 35: 458-469.

Increase in number of synapses in the inner plexiform layer of light deprived rat retinae: quantitative electron microscopy. L Sosula, P H Glow, J Comp Neurol. 141Sosula L, Glow PH (1971) Increase in number of synapses in the inner plexiform layer of light deprived rat retinae: quantitative electron microscopy. J Comp Neurol 141: 427-451.

Sensory deprivation and the cat's optic nervous system. L Weiskrantz, Nature. 181Weiskrantz L (1958) Sensory deprivation and the cat's optic nervous system. Nature 181: 1047-1050.

Receptor amblyopia. J M Enoch, Am J Ophthalmol. 483Enoch JM (1959) Receptor amblyopia. Am J Ophthalmol 48(3), Part 2: 262- 274.

Retinal nerve fiber layer thickness in human strabismic amblyopia. T P Colen, J T De Faber, H G Lemij, Binocul Vis Strabismus Q. 15Colen TP, de Faber JT, Lemij HG (2000) Retinal nerve fiber layer thickness in human strabismic amblyopia. Binocul Vis Strabismus Q 15: 141-146.

Macular ultrastructural features in amblyopia using high-definition optical coherence tomography. C E Al-Haddad, El Mollayess, G M Mahfoud, Z R Jaafar, D F Bashshur, Z F , Br J Ophthalmol. 97Al-Haddad CE, El Mollayess GM, Mahfoud ZR, Jaafar DF, Bashshur ZF (2013) Macular ultrastructural features in amblyopia using high-definition optical coherence tomography. Br J Ophthalmol 97: 318-322.

Analysis of spectral-domain optical coherence tomography measurements in amblyopia: a pilot study. K A Park, D Y Park, S Y Oh, Br J Ophthalmol. 95Park KA, Park DY, Oh SY (2011) Analysis of spectral-domain optical coherence tomography measurements in amblyopia: a pilot study. Br J Ophthalmol 95: 1700-1706.

Retinal thickness decreases with age: an OCT study. B Alamouti, J Funk, Br J Ophthalmol. 87Alamouti B, Funk J (2003) Retinal thickness decreases with age: an OCT study. Br J Ophthalmol 87: 899-901.

Retinal thickness analysis by race, gender, and age using Stratus OCT. A H Kashani, I E Zimmer-Galler, S M Shah, Dustin L Do, D V , Am J Ophthalmol. 149Kashani AH, Zimmer-Galler IE, Shah SM, Dustin L, Do DV, et al. (2010) Retinal thickness analysis by race, gender, and age using Stratus OCT. Am J Ophthalmol 149: 496-502 e491.

Macular thickness assessment in healthy eyes based on ethnicity using Stratus OCT optical coherence tomography. P J Kelty, J F Payne, R H Trivedi, J Kelty, E M Bowie, Invest Ophthalmol Vis Sci. 49Kelty PJ, Payne JF, Trivedi RH, Kelty J, Bowie EM, et al. (2008) Macular thickness assessment in healthy eyes based on ethnicity using Stratus OCT optical coherence tomography. Invest Ophthalmol Vis Sci 49: 2668-2672.

Macular thickness variations with sex, age, and axial length in healthy subjects: a spectral domainoptical coherence tomography study. W K Song, S C Lee, E S Lee, C Y Kim, S S Kim, Invest Ophthalmol Vis Sci. 51Song WK, Lee SC, Lee ES, Kim CY, Kim SS (2010) Macular thickness variations with sex, age, and axial length in healthy subjects: a spectral domain- optical coherence tomography study. Invest Ophthalmol Vis Sci 51: 3913-3918.

Relationship of gender, body mass index, and axial length with central retinal thickness using optical coherence tomography. A C Wong, C W Chan, S P Hui, Eye (Lond). 19Wong AC, Chan CW, Hui SP (2005) Relationship of gender, body mass index, and axial length with central retinal thickness using optical coherence tomography. Eye (Lond) 19: 292-297.

The Effect of Axial Length on the Thickness of the Intraretinal Layers in the Macula. E Tatrai, A Szigeti, J Nemeth, D Debuc, G M Somfai, ARVO Meeting Abstracts. 534102Tatrai E, Szigeti A, Nemeth J, DeBuc D, Somfai GM (2012) The Effect of Axial Length on the Thickness of the Intraretinal Layers in the Macula. ARVO Meeting Abstracts 53: 4102.

Central and peripheral retinal photoreceptor orientation in amblyopic eyes as assessed by the psychophysical Stiles-Crawford function. H E Bedell, Invest Ophthalmol Vis Sci. 19Bedell HE (1980) Central and peripheral retinal photoreceptor orientation in amblyopic eyes as assessed by the psychophysical Stiles-Crawford function. Invest Ophthalmol Vis Sci 19: 49-59.

Photoreceptor function in unilateral amblyopia. P J Delint, C Weissenbruch, T T Berendschot, D V Norren, Vision Res. 38Delint PJ, Weissenbruch C, Berendschot TT, Norren DV (1998) Photoreceptor function in unilateral amblyopia. Vision Res 38: 613-617.

Electro-oculographic abnormalities in amblyopia. C Williams, D Papakostopoulos, Br J Ophthalmol. 79Williams C, Papakostopoulos D (1995) Electro-oculographic abnormalities in amblyopia. Br J Ophthalmol 79: 218-224.

Macular and retinal nerve fibre layer thickness in strabismus and anisometropic amblyopia. J Leone, K Koklanis, Z Georgievski, R Wilkinson, Binocul Vis Strabismus Q. 23Leone J, Koklanis K, Georgievski Z, Wilkinson R (2008) Macular and retinal nerve fibre layer thickness in strabismus and anisometropic amblyopia. Binocul Vis Strabismus Q 23: 227-234.

An optical model of the human retinal nerve fiber layer: implications of directional reflectance for variability of clinical measurements. R W Knighton, C Qian, J Glaucoma. 9Knighton RW, Qian C (2000) An optical model of the human retinal nerve fiber layer: implications of directional reflectance for variability of clinical measure- ments. J Glaucoma 9: 56-62.

Comparison of time domain and spectral domain optical coherence tomography in measurement of macular thickness in macular edema secondary to diabetic retinopathy and retinal vein occlusion. E Hatef, A Khwaja, Z Rentiya, Ibrahim M Shulman, M , J Ophthalmol. 354783Hatef E, Khwaja A, Rentiya Z, Ibrahim M, Shulman M. et al. (2012) Comparison of time domain and spectral domain optical coherence tomography in measurement of macular thickness in macular edema secondary to diabetic retinopathy and retinal vein occlusion. J Ophthalmol 2012: 354783.

Retinal nerve fibre layer and macular thickness in amblyopia as measured by spectral-domain optical coherence tomography. C E Al-Haddad, G M Mollayess, C G Cherfan, D F Jaafar, Z F Bashshur, Br J Ophthalmol. 95Al-Haddad CE, Mollayess GM, Cherfan CG, Jaafar DF, Bashshur ZF (2011) Retinal nerve fibre layer and macular thickness in amblyopia as measured by spectral-domain optical coherence tomography. Br J Ophthalmol 95: 1696- 1699.


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A M Pierro
Regional Reference Centre for Microbiological Emergencies (CRREM)
Azienda Ospedaliero-Universitaria di Bologna
Microbiology Unit

Policlinico S. Orsola-Malpighi
BolognaItaly

G Rugna
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna
BresciaItaly

V Sambri
Regional Reference Centre for Microbiological Emergencies (CRREM)
Azienda Ospedaliero-Universitaria di Bologna
Microbiology Unit

Policlinico S. Orsola-Malpighi
BolognaItaly

G Squintani
Veterinary and Food Hygiene Service
Emilia-Romagna Region
BolognaItaly

P Macini
Public Health Service
Emilia-Romagna Region
BolognaItaly

West Nile virus circulation in Emilia-Romagna, Italy: the integrated surveillance system 2009
22 April 2010C148C16331A5B1A6F875E121460712E9
Following a large West Nile virus (WNV) epidemic in north-eastern Italy in 2008, human and animal surveillance activities were implemented in Emilia Romagna.Human surveillance was performed by serology or genome detection on blood and cerebrospinal fluid for all suspected cases suffering from acute meningoencephalitis in the regional territory.Animal surveillance consisted of passive and active surveillance of horses and active surveillance of wild birds and mosquitoes.Between 15 June and 31 October 2009, nine of 78 possible cases of West Nile neuroinvasive disease were confirmed (three fatal).From May to October, 26 cases of neurological West Nile disease were confirmed among 46 horses.The overall incidence of seroconversion among horses in 2009 was 13%.In 2009, 44 of 1,218 wild birds yielded positive PCR results for WNV infection.The planned veterinary and entomological surveillance actions detected WNV activity from the end of July 2009, about 2-3 weeks before the onset of the first human neurological case.Passive surveillance of horses seems to be an early and suitable tool for the detection of WNV activity, but it will be less sensitive in the future, because an intensive programme of horse vaccination started in June 2009.

Regional integrated surveillance system

In Italy a national veterinary plan for the surveillance of West Nile virus (WNV) circulation was set up in 2001 under the coordination of the National Reference Centre for Exotic Diseases of animals (CEntro Studi Malattie Esotiche; CESME).During the late summer of 2008 a large epidemic of WNV infection occurred in north eastern Italy over an area exceeding 7,000 km 2 , in three regions, including Emilia-Romagna.Twenty-three horse cases and three human cases of the neuroinvasive form of West Nile disease (WND) were confirmed by laboratory tests [1,2].After the first evidence of WNV circulation in horses was found, additional surveillance on horses, birds and mosquitoes was activated.

In Emilia-Romagna the WNV surveillance plan 2009 adopted locally the surveillance measures indicated by the national plan.In particular, among the surveillance activities, the choice was to monitor wild non-migratory birds, such as corvids (the crow family), considered the most sensitive indicators among birds, which can be captured easily.As regards equine surveillance, the regional plan emphasised the education of veterinary practitioners, focusing on the inclusion of WNV in differential diagnosis and the achievement of rapid reporting.A major feature of this plan was to establish an extremely sensitive system of passive surveillance.In addition to passive surveillance, active monitoring of horses was implemented in the area involved in the 2008 outbreak, including Ferrara and the neighbouring provinces [3].

Evidence of WNV circulation in 2008 was found in animals [1,4], humans [5], and mosquitoes.This highlighted the need to implement an integrated surveillance system which would describe the phenomenon comprehensively.Such a system facilitates the collection of data to evaluate spatial distribution and time trends of viral circulation and shares information.

For this reason the 2009 Regional Surveillance Plan implemented activities beyond those of the National Plan, revised the surveillance system of human cases, activated intensive entomological monitoring, and enlarged the surveillance area to involve all the provinces along the Po River.

Human surveillance

The aim of the human surveillance system was the early detection of infection in humans and the estimation of its diffusion through the systematic analysis of newly emerging clinical cases, in order to manage specific interventions.

The surveillance was performed throughout the regional territory, from 15 June to 31 October 2009, corresponding to the period of vector activity in Emilia-Romagna and adjusted locally according to weather conditions and vector activity reports.In 2009, the 2008 case definition [6] was extended to include cases of all ages and not only those over 15 years of age.

The human surveillance activity was performed by serology or genome detection on blood and cerebrospinal fluid for all suspected cases suffering from acute meningoencephalitis in the regional territory.Active surveillance of people who live or work in areas of documented viral circulation was also performed.In addition blood and cerebrospinal fluid samples from subjects living or staying for at least one night in the regional area were sent to the Regional Reference Centre for Microbiological Emergencies (Centro di Riferimento Regionale per le Emergenze Microbiologiche; CRREM).In selected cases, positive specimens were confirmed by the National Health Institute (Istituto Superiore di Sanità; ISS) and by the National Institute for Infectious Diseases (Istituto Nazionale Malattie Infettive; INMI).

From October 2008 to April 2009 a serosurvey performed on 9,177 healthy blood donors living in the province of Ferrara detected a total of 62 IgG positive subjects, corresponding to a seroprevalence of 0.68%.

Animal surveillance

The regional veterinary WNV surveillance system was activated from May to October, performing passive and active surveillance on horses and on non-migratory wild birds.

Horse passive surveillance

In Italy all suspected signs of WND in horses must be notified to the official veterinary services.Suspected cases were confirmed if resulted positive by reverse transcription -polymerase chain reaction (RT-PCR) performed on central nervous system [7] or to a WNV virus

Horse active surveillance

In the provinces of Ferrara, Bologna, Modena, Ravenna, and Reggio Emilia, every 1,600 km 2 , 28 seronegative unvaccinated equine sentinels, sufficient to detect an incidence above 10% (CI 95%), were selected in the spring of 2009.They were serologically tested twice after the selection, at the beginning of August and the beginning of September.Samples collected were screened by a home-made competitive ELISA [10].

Positive samples were confirmed by VN and IgM ELISA at the CESME in Teramo.A seroconversion was confirmed if VN titre was at least 1:10 and there was evidence of IgM antibodies.

Wild bird surveillance

Monitoring was carried out in all the provinces along the Po River, in the plain area of Emilia-Romagna.Every 1,600 km 2 , a monthly sample of about 40 wild birds caught or shot within specific wildlife population control programmes was collected.Samples of organs (brain, heart, and kidney) of each bird were pooled and examined by RT-PCR [7].

Entomological surveillance

The surveillance system was based on the weekly to monthly (frequency depends on local resources) collection of mosquitoes in fixed stations and in the sites where birds, humans, or horses signalled WNV activity.Mosquito collections for WNV screening were conducted in six provinces: Ferrara, Ravenna, Bologna, Modena, Reggio Emilia, and Parma, using 92 CO 2 baited traps positioned in fixed stations.Moreover, mosquito collections were performed promptly using CO 2 and gravid traps in sites where positive horses and human cases had been detected.

The surveillance system was activated in the period 15 April to 10 October.Collected mosquitoes were pooled (maximum 200) by species, date, and site of collection and examined by RT-PCR [7].In addition, overwintered mosquito females were collected during the period 3 March to 8 April by manual aspirator in rural buildings in the area where WNV was active in 2008.

Virological analysis

Human samples

The detection of WNV genome in human plasma, cerebrospinal fluid, and serum samples obtained from patient suffering from clinical symptoms of meningoecephalitis was performed by an RT-PCR assay based on specific TaqMan probes [7].

Animal samples

In horses RNA was extracted starting from 200 µl of serum or whole blood with EDTA as anticoagulant.In birds RNA was extracted from 200 µl of phosphate buffer saline homogenate (about 20% tissue g / buffer ml) of pooled brain, heart and kidney of each analysed bird.In mosquitoes RNA was extracted from 200 µl of a maximum of 200 pooled mosquitoes manual grinded by using copper stained beads, in 500-800 µl of PBS.

cDNA was submitted to RT-PCR according to the method of Tang and colleagues [7].Positive samples were confirmed by sequencing of partial nucleocapsin and premembrane protein M amplified according to [11].Finally from each RT-PCR positive sample WNV was isolated on Vero and RK13 cell lines.

Results

Results of the integrated surveillance system are mapped in figure 1, with the sequence of events summarised in figure 2 (July-October), and discussed below.

Human cases

Horse cases

Passive surveillance

From May to October, 26 cases of neurological WND were confirmed among 46 horses in which it was suspected.Four of the eight provinces involved in the regional surveillance system had WND cases in horses.

The first symptoms in horses were detected in the second half of July in the provinces of Ferrara and Reggio Emilia, but the most cases were notified between mid-August and mid-September (figure 2).

Active surveillance

Seroconversions in sentinel horses were detected in three provinces (Ferrara, Modena, and Reggio Emilia).

Early seroconversions were registered among the controls examined at the beginning of August.Serological controls around the stables with WND cases also confirmed recent WNV infections in the province of Parma.

The overall incidence of seroconversion among horses in 2009 was 13% (95% CI: 10% to 16%), but in Ferrara it was 28% (95% CI: 19% to 39%).

Wild birds

Six of the eight provinces that took part in the regional surveillance system reported positive birds.In 2009, 44 wild birds out of 1,218 (tested by PCR) yielded positive results for WNV infection.With the exception of a magpie caught in May, positive wild birds were detected from the end of July (figure 2).Most of the infected wild birds were corvids (Pica pica, Corvus corone cornix, Garrulus glandarius), collected within population control programmes in August and September, but WNV was detected also in other species, mainly found dead in wildlife recovery centres (table 1).positive pools/no.of mosquitoes tested) x 1,000) [12] were calculated, with higher MIR values recorded in August in the provinces of Reggio Emilia (3.08) and Modena (1.44).

Referring to the collection of overwintering mosquitoes, three mosquito species were collected: Culex pipiens (516 females, 52%), Anopheles maculipennis s.l.(475 females, 48%), Culiseta annulata (4 females, <1%): all specimens were tested and yielded negative results.

Conclusions

The planned veterinary and entomological surveillance actions detected WNV activity from the end of July 2009, about 2-3 weeks before the onset of the first human neurological case.Figure 2 shows that mosquitoes and birds were the first indicators of WNV circulation.The same figure makes it clear that human cases occurred later in the season, as reported elsewhere [6].Passive surveillance of horses also seems to be an early and suitable tool for the detection of WNV activity, but it will be less sensitive in the future, because an intensive programme of horse vaccination started in June 2009.

More human cases of WNND occurred in 2009 than in 2008, and three were fatal.It is important to note that in 2008 the epidemic became evident in the late summer (beginning of September).In 2009 the first human cases were detected earlier than 2008.It is likely that increased attention of clinicians to this emerging disease improved the surveillance system sensitivity in 2009.

The circulation of WNV in a large area of the Po plain in two consecutive years shows that this territory is becoming suitable to support WNV establishment and possible endemicity.This indicates a need to organise standard surveillance measures to detect WNV activity early and assess risk to public health.

The results of the entomological surveillance confirm that the CO 2 trap is a reliable and valuable tool for early detection of WNV.Culex pipiens, the most abundant mosquito species in the region, is the only vector species incriminated, since no other species collected in the field were found to be infected.

The quick and intensive spread of WNV in the past two years suggests that the whole Po plain may be affected in the future.In forthcoming years, surveillance of wild birds and insects will be used to forecast the extension and spread of WNV.The information gathered will be used to direct or optimise actions intended to prevent virus transmission, such as vector monitoring and control, information campaigns to improve personal protection, and deploy screening tests on blood, tissue, and organs for transplant.

Figure 2
2

Table 1
1
Species distribution of wild birds tested for West Nile virus (n=1,218), Emilia-Romagna, Italy, May-October 2009 living in Ferrara Province and one in Modena.In addition, not reported in figures, the local health units of Parma and Modena notified two other confirmed cases, both 72 year-old women resident in Mantua province (Lombardy region), treated in hospital in Emilia-Romagna.Another case of infection was that of a 78 year-old female liver donor.Before her death, she had spent two weeks visiting relatives in the WNV affected area (Reggio Emilia).
of 31 October 2009, nine out of 78 possible casesof West Nile neuroinvasive disease (WNND) notifiedin Emilia-Romagna have been confirmed (8/9 males;median age 72 years, range: 62-78). Three patients

Table 1 .
1
Species distribution of wild birds tested for
West Nile V (n=1,218), Emilia-Romagna, Italy, May-October 2009MosquitoesAbout 190,000 mosquitoes were collected, pooled andtested using PCR (1,789 pools of ≤200 individuals/pool). Culex pipiens were the most abundant species(81.4%) followed by Aedes caspius (15.4%) and Aedesvexans (2.4%). Other collected species are shown intable 2.
Twenty-seven pools, all consisting of Culex pipiens, yielded positive results for WNV.Early positive pools were collected in the province of Reggio Emilia at the end of July.Minimum infection rates (MIR: (no. of

Table 2
2
Species of mosquitoes tested for West Nile virus (n=190,516), Emilia-Romagna, Italy, May-October 2009

AcknowledgementsThe authors would like to thank Ugo Santucci of the Ministry of Employment, Health and Social Policy and Paolo Calistri of CESME for supporting us in managing veterinary surveillance activities.Giovanni Savini of CESME confirmed by VN and PCR all ELISA positive horse sera.The authors also acknowledge the medical and veterinarian practitioners of the local health units involved in epidemic management and all the collaborators in the surveillance system.
West Nile virus transmission in 2008 in North-Eastern Italy. P Calistri, A Giovannini, G Savini, F Monaco, L Bonfanti, C Ceolin, 10.1111/j.1863-2378.2009.01303.x2010Zoonosis and Public Health57

Indication of Culex pipiens as the main vector of West Nile virus spread in Italy. R Bellini, P Bonilauri, P Angelini, A Albieri, R Veronesi, E Martini, West Nile virus 10 years later. Savannah, GA, USA2008. 2009. Feb 19-2031

Detection of West Nile virus infection in horses. P Macini, G Squintani, A C Finarelli, P Angelini, E Martini, M Tamba, Euro Surveill. 1339September 2008. 2008

West Nile regional surveillance plan. Emilia-Romagna , 2009Italian version

First human case of West Nile virus neuroinvasive infection in Italy. G Rossini, F Cavrini, A Pierro, P Macini, A C Finarelli, C Po, Euro Surveill. 1341September 2008. 2008-case report

West Nile virus transmission with human cases in Italy. C Rizzo, F Vescio, S Declich, A C Finarelli, P Macini, A Mattivi, Euro Surveill. 1440August -September 2009. 2009

Highly sensitive TaqMan RT-PCR assay for detection and quantification of both lineages of West Nile virus RNA. Y Tang, C A Hapip, B Liu, C T Fang, J Clin Virol. 362006

Manual of diagnostic tests and vaccines for terrestrial animals. Cap. 2.1.20 West Nile fever. 2008OIE

Re-emergence of West Nile virus in Italy. F Monaco, R Lelli, L Teodori, C Pinoni, Di Gennaro, A Polci, A , 10.1111/j.1863-2378.2009.01245.xZoonoses and Public Health. 2009

Virus West Nile: caratterizzazione di anticorpi monoclinali e potenziale applicazione nella diagnosi di laboratorio. D Lelli, A Moreno, E Brocchi, E Sozzi, E Canelli, G L Autorino, Proceedings of 3rd national workshop of veterinary virology. 3rd national workshop of veterinary virologyValenzano (Bari), ItalyJun 11-12. 200963

Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase -PCR assay. R S Lanciotti, A J Kerst, R S Nasci, M S Godsey, C J Mitchell, H M Savage, J Clin Microbiol. 38112000

West Nile virus infection rates in pooled mosquito samples and individual. Vector-Borne and Zoonotic Diseases. S A Condotta, F Hunter, M J Bidochka, 20044