CLINICAL SCIENCE
Utility of Assessing Nerve Morphology in Central Cornea Versus Whorl Area for Diagnosing Diabetic Peripheral Neuropathy Nicola Pritchard, PhD,* Cirous Dehghani, MSc,* Katie Edwards, PhD,* Edward Burgin, BVisSc, MOptom,* Nick Cheang, BVisSc, MOptom,* Hannah Kim, BVisSc, MOptom,* Merna Mikhaiel, BVisSc, MOptom,* Gemma Stanton, BVisSc, MOptom,* Anthony W. Russell, MBBS, PhD,†‡ Rayaz A. Malik, MBChB, PhD,§¶ and Nathan Efron, PhD, DSc*
Purpose: To compare small nerve fiber damage in the central cornea and whorl area in participants with diabetic peripheral neuropathy (DPN) and to examine the accuracy of evaluating these 2 anatomical sites for the diagnosis of DPN.
Methods: A cohort of 187 participants (107 with type 1 diabetes and 80 controls) was enrolled. The neuropathy disability score (NDS) was used for the identification of DPN. The corneal nerve fiber length at the central cornea (CNFLcenter) and whorl (CNFLwhorl) was quantified using corneal confocal microscopy and a fully automated morphometric technique and compared according to the DPN status. Receiver operating characteristic analyses were used to compare the accuracy of the 2 corneal locations for the diagnosis of DPN. Results: CNFLcenter and CNFLwhorl were able to differentiate all 3 groups (diabetic participants with and without DPN and controls) (P , 0.001). There was a weak but significant linear relationship for CNFLcenter and CNFLwhorl versus NDS (P , 0.001); however, the corneal location · NDS interaction was not statistically significant (P = 0.17). The area under the receiver operating characteristic curve was similar for CNFLcenter and CNFLwhorl (0.76 and 0.77, respectively, P = 0.98). The sensitivity and specificity of the cutoff points were 0.9 and 0.5 for CNFLcenter and 0.8 and 0.6 for CNFLwhorl. Conclusions: Small nerve fiber pathology is comparable at the central and whorl anatomical sites of the cornea. Quantification of CNFL from the corneal center is as accurate as CNFL quantification of the whorl area for the diagnosis of DPN. Received for publication January 19, 2015; revision received February 25, 2015; accepted February 27, 2015. Published online ahead of print April 24, 2015. From the *Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Australia; †School of Medicine, University of Queensland, St Lucia, Australia; ‡Department of Diabetes and Endocrinology, Princess Alexandra Hospital, Woolloongabba, Australia; §Center for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester, United Kingdom; and ¶Weill Cornell Medical College in Qatar, Doha, Qatar. Supported by grants from the National Health and Medical Research Council of Australia (497230) and the JDRFI (8-2008-362). The authors have no funding or conflicts of interest to disclose. Reprints: Nathan Efron, PhD, DSc, Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland 4059, Australia (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Key Words: corneal confocal microscopy, corneal subbasal nerve plexus, diabetic peripheral neuropathy (Cornea 2015;34:756–761)
T
he corneal subbasal nerve plexus (SNP) is located between the Bowman layer and the corneal basal epithelium and originates from stromal nerves penetrating the Bowman layer, predominantly in the peripheral area.1,2 At this interface, subbasal nerves anastomose extensively and form a dense homogenous nerve network that radiates toward a whorl-like assembly (whorl area), usually 1 to 2 mm inferior to the corneal apex, as described in ex vivo histological studies1,2 and in vivo corneal confocal microscopy (CCM) investigations.3,4 The transparency of the cornea and unique attributes of the SNP, such as its parallel arrangement in relation to the ocular surface, provide an opportunity for the noninvasive assessment of this nerve plexus using CCM. Quantification of the SNP using CCM as a noninvasive and reiterative modality has been shown to be a promising marker for the detection and stratification of diabetic peripheral neuropathy (DPN) in several studies over the past decade.5–11 Because of the convenience and ease of imaging from the central cornea, this region has been the main focus in almost all of these studies. However, recent investigations in diabetic patients4 and animals12 have provided evidence of more pronounced and earlier corneal neural loss at the inferior whorl. Given that diabetic neuropathy affects the most distal axons first and that the nerves at the whorl are distal to the central cornea, one may expect more marked pathology at this site.13 The corneal nerve fiber length (CNFL) is the most reliable14,15 and frequently reported morphological parameter of the SNP obtained using CCM. The aim of this study was to quantify and compare this parameter at the corneal apex (central cornea) and whorl-like area (whorl) in diabetic individuals with and without neuropathy and healthy participants. Furthermore, the capability of CNFL in the center and whorl for diagnosing DPN was determined.
MATERIALS AND METHODS This study was conducted in accordance with the Declaration of Helsinki and was approved by ethics Cornea Volume 34, Number 7, July 2015
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Cornea Volume 34, Number 7, July 2015
committees of Queensland University of Technology, Princess Alexandra Hospital, and Mater Hospital in Brisbane, Australia. All participants provided written informed consent. Based on the inclusion and exclusion criteria, 187 participants were enrolled. All participants were assessed for the presence of any corneal compromise. Participants were excluded if they had a history of ocular trauma or surgery, ocular disease or systemic disease with potential corneal effects, and systemic disease (other than diabetes). Other causes of neuropathy were excluded. The cohort included 107 participants with type 1 diabetes who were recruited from the Diabetes and Endocrinology Research Center at Princess Alexandra and Mater Hospitals and the general population in Brisbane. Eighty healthy participants, without peripheral neuropathy and/or diabetes, were also recruited as controls. The neuropathy disability score (NDS),16 which is a scale of 0 to 10, was used to define the neuropathy status for all participants. This measure included assessment of vibration, pin-prick, and temperature perception as well as the presence or absence of ankle reflexes in both lower limbs. An NDS $ 3 was defined as the presence of neuropathy. The methods used during this study to quantify corneal nerve morphology using CCM and a fully automated segmentation technique have been reported in detail elsewhere17 and are described only briefly here. CCM was conducted using a Heidelberg Retina Tomograph (HRT 3) with Rostock Corneal Module (Heidelberg Engineering GmbH, Heidelberg, Germany) on the cornea of the dominant-hand side of the participants. Participants were instructed to fixate on the near target attached to the CCM instrument during the examination. The location of the whorl area was facilitated by gently moving this target and CCM in X and Y directions until the whorl pattern was in view. For each participant, multiple images of the SNP were captured from the central cornea and whorl area. From digitally saved images, 8 central and 3 whorl images of the SNP were chosen and analyzed using a fully automated algorithm18 to quantify CNFL. For each participant, CNFL assessed at the central cornea (CNFLcenter) and whorl (CNFLwhorl) was the average value obtained from the chosen images (8 and 3, respectively) and expressed in the unit of mm/mm2. SPSS (version 21) was used to analyze the data. A univariate analysis of variance (ANOVA) was performed to examine the possible interaction between the neuropathy status of the participants (control, diabetes without neuropathy, and diabetes with neuropathy) and their assignment group (controls and diabetes) and quantified CNFL values from central corneal and whorl regions. To establish the association with the severity of neuropathy and CNFL from the central cornea and whorl area, the CNFL values were plotted against the respective NDS values. A mixed-model analysis was conducted to assess the interaction between CNFL from the 2 corneal locations and NDS. In this model, CNFL was considered as the response variable, and the effects of corneal location (center vs. whorl), NDS, and corneal location · NDS interaction were examined. Receiver operating characteristic (ROC) curve analyses were used to compare the capability of CNFLcenter and CNFLwhorl for diagnosing DPN. The area under the curve (AUC), which represents a measure of accuracy,19 was Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Corneal Nerves in Diabetic Neuropathy
estimated. Using the Youden index,20 the cutoff values were determined for the presence of neuropathy (NDS $ 3). Finally, the sensitivity, specificity, positive likelihood ratio (+LR), and negative likelihood ratio (2LR) of CNFLcenter and CNFLwhorl were calculated. In healthy corneas, Patel and McGhee3 reported a whorl/ center CNFL ratio of 1.2 using laser-scanning CCM. In the absence of previous quantitative data of the whorl area versus center in DPN, we anticipated 25% lower whorl/center CNFL ratio in individuals with DPN. Assuming this difference, a = 0.05 and 0.9% power, the required sample size was determined to be 76 diabetic participants including 25 with DPN. Enrollment continued until the DPN group contained 25 participants, resulting in a total of 107 diabetic participants. A cohort of 80 healthy controls was also enrolled for comparison.
RESULTS
In the entire cohort, the mean 6 SD for CNFLcenter and CNFLwhorl was 17.5 6 3.8 and 19.5 6 4.6 mm/mm2, respectively (P , 0.001). CNFLcenter and CNFLwhorl were significantly lower in participants with diabetes compared with the control group (CNFLcenter, 16.1 6 3.7 vs. 19.3 6 3.0 mm/mm2 and CNFLwhorl, 17.5 6 4.2 vs. 22.1 6 3.9 mm/ mm2, respectively, P , 0.01). Although the whorl/center CNFL ratio was slightly lower in the diabetic group (1.09) compared with controls (1.14), the univariate ANOVA showed no significant interaction between the corneal location (center vs. whorl) and assignment group of the participants (P = 0.1). The mean age was 37.0 6 17.8 years for controls and 48.3 6 15.1 years for the diabetic group. Of the 107 participants with type 1 diabetes, 25 (23%) were diagnosed with DPN. Ten participants had mild (3 # NDS # 5), 12 had moderate (6 # NDS # 8), and 3 had severe (NDS $ 9) neuropathy. Figure 1 depicts representative images from central corneal and whorl regions in controls and diabetic participants with and without neuropathy. There was a significant difference between groups (control, diabetes without neuropathy, and diabetes with neuropathy) for both CNFLcenter and CNFLwhorl (P , 0.001) (Table 1). Diabetic participants with and without neuropathy showed significantly lower CNFLcenter compared with controls (14.2 6 3.5 and 16.7 6 3.5 mm/mm2 vs. 19.3 6 3.0 mm/mm2, respectively, P , 0.01). Similarly, CNFLwhorl was significantly lower in diabetic participants with (15.4 6 4.4 mm/mm2) and without (18.2 6 3.9 mm/mm2) neuropathy versus control group (22.1 6 3.9 mm/mm2) (P , 0.01). However, the univariate ANOVA showed that the neuropathy status · corneal location term was not significant for estimated CNFL (P = 0.1). Figure 2 illustrates the relationship between CNFL from the central cornea and whorl area against NDS. There was a weak but significant linear relationship for CNFLcenter and CNFLwhorl versus NDS (R2: 0.08 and 0.09, respectively, P , 0.001). The calculated slopes of the regression lines were 20.5 for CNFLcenter and 20.6 for CNFLwhorl. Mixed model analysis revealed significant effects of corneal location (P , 0.001) and NDS (P , 0.001). However, the corneal www.corneajrnl.com |
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FIGURE 1. Representative images of the SNP from the 3 study groups. Examples of central corneal nerves are shown in a control participant (A), a diabetic participant without neuropathy (B), and a diabetic participant with severe neuropathy (C). Example images of the whorl area for respective participants are shown in (D–F). All images are 400 · 400 mm.
location · NDS interaction was not statistically significant (P = 0.17). Figure 3 shows the ROC curves for CNFL center and CNFL whorl among the participants for the identification of DPN cases according to NDS. The AUC for the presence of DPN (NDS $ 3) was 0.76 for CNFLcenter and 0.77 for CNFLwhorl . The 2 estimated AUCs were statistically similar (P = 0.98). The results of the calculated Youden cutoff values, sensitivity, specificity, +LR, and
2LR associated with the cutoff points are presented in Table 2.
DISCUSSION Detection of nerve pathology at the earliest stage of DPN is crucial as patients may benefit substantially from prompt management to halt or slow the changes and/or to avoid serious consequences such as foot ulceration or
TABLE 1. Comparison of CNFL From Center (CNFLcenter) and Whorl (CNFLwhorl) of the Cornea in the Study Participants According to the Presence (DPN+ve) and Absence (DPN2ve) of Neuropathy Defined by NDS $ 3 Group Parameter
Controls (n = 80)
DPN2ve (n = 82)
DPN+ve (n = 25)
CNFLcenter, mm/mm2
19.3 6 3.0
16.7 6 3.5
14.2 6 3.5
CNFLwhorl, mm/mm2
22.1 6 3.9
18.2 6 3.9
15.4 6 4.4
P, Group Difference ,0.001*, controls vs. DPN2ve, DPN+ve†; DPN2ve vs. DPN+ve† ,0.001*, controls vs. DPN2ve, DPN+ve†; DPN2ve vs. DPN+ve†
Outcomes are presented as mean 6 SD. *1-way ANOVA test. †Scheffe post hoc test (P , 0.05).
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Corneal Nerves in Diabetic Neuropathy
FIGURE 2. Scatter plot illustrates the relationship of CNFL values quantified from the central cornea (CNFLcenter) and whorl area (CNFLwhorl) with their NDS values. The dashed line indicates the linear regression for CNFLcenter (b = 20.5, P , 0.01), and the solid line indicates the linear regression for CNFLwhorl (b = 20.6, P , 0.01). No significant difference was found between the 2 slopes (P = 0.17).
amputation.21,22 Morphometric assessment of the SNP using CCM at the central cornea has a demonstrated capacity for the detection and severity stratification of DPN.6,7,9,23 There is also some evidence of identification of earlier small nerve fiber damage associated with diabetes using CCM at the central cornea.10,24 Assessment of the SNP microstructures using in vivo CCM has been shown to be reliable, repeatable, and reproducible for the detection of small nerve injury in diabetes, particularly in the context of DPN.7,14,15,25 Akin to the length-dependent process of DPN whereby the most distal and thinner small fibers are the earliest nerves to be damaged,26,27 the hypothetically optimal approach would be to examine the most distal nerves on the SNP (whorl-like
FIGURE 3. ROC curves for CNFL quantified from the central cornea (solid line) and whorl area (dotted line) against NDS $ 3. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
assembly) to detect the earliest and perhaps more prominent nerve damage. In this study, we report data from a large cohort of patients with type 1 diabetes and control participants who underwent assessment of corneal nerve morphology at the central cornea and whorl area. Quantified CNFL from the whorl area (CNFLwhorl) was found to be approximately 2 mm/mm2 higher than that at the corneal center (CNFLcenter) for the entire cohort. The higher nerve tissue density at the whorl area compared with the center of the cornea has previously been reported in healthy corneas using in vivo CCM3 and highlights the differences in nerve tissue at these 2 anatomical sites. Depletion of small nerve fibers was evident at the corneal center and whorl area in participants with diabetes. However, in this study, corneal location did not affect the observed CNFL deficit in diabetic participants. Our findings showed that both CNFLcenter and CNFLwhorl were capable of differentiating diabetic participants without and with neuropathy from controls, and the differentiation was not affected by corneal location. Although there was a negative relationship between NDS versus CNFLcenter and CNFLwhorl, the lack of significant interaction between corneal location and NDS indicated that the rate of change of CNFLcenter and CNFLwhorl in relation to NDS (ie, the slopes in Fig. 2) was not different. This outcome demonstrated that the damage to small nerves of the SNP assessed using in vivo CCM was equivalent at the central and whorl areas of the cornea, regardless of the severity of neuropathy. To further explore the diagnostic accuracy of CNFL pertaining to the 2 corneal locations in assessing participants with (NDS $ 3) and without (NDS # 2) neuropathy, ROC analysis was used. The AUC, an overall performance measure of the test, was similar for CNFLcenter and CNFLwhorl. Using the Youden index, the estimated optimal cutoff points that identified the presence of DPN were 17.9 mm/mm2 for CNFLcenter and 18.6 mm/mm2 for CNFLwhorl. Compared with CNFLwhorl, CNFLcenter showed higher sensitivity (0.9 vs. 0.8) and lower specificity (0.5 vs. 0.6); however, the difference between likelihood ratios—an expression of the probability of test results given the correct diagnosis in the presence or absence of disease19—was trivial. Overall, it can be concluded www.corneajrnl.com |
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TABLE 2. Detection Capacity of CNFL Pertaining to the Central (CNFLcenter) and Whorl Area (CNFLwhorl) of the Cornea for the Presence of Neuropathy as Defined by NDS $ 3 Parameter
AUC
P
Youden Cutoff Point
Sensitivity (95% CI)
Specificity (95% CI)
+LR
2LR
CNFLcenter CNFLwhorl
0.76* 0.77
,0.001 ,0.001
#17.9 #18.6
0.9 (0.7–1.0) 0.8 (0.6–1.0)
0.5 (0.5–0.6) 0.6 (0.5–0.7)
2.0 2.2
0.2 0.3
*No significant difference compared with CNFLwhorl (x2, P = 0.98). +LR, positive likelihood ratio; 2LR, negative likelihood ratio.
from a comparison of ROC plots and AUC results that there was little difference in diagnostic accuracy based on the assessment of nerve morphology at the 2 locations. Although we postulated that the whorl area, which is 1 to 2 mm more distal than the central cornea, would demonstrate more nerve depletion in participants with DPN, the findings of this study do not support this notion. A possible explanation for this might be that a 1 to 2 mm difference in distal nerve extension is insufficient to observe more pronounced distal axonal neuropathy in the cornea. Furthermore, the possibility remains that the NDS may suffer from a lack of adequate proficiency in diagnosing and staging the severity of neuropathy. Our findings of ROC analysis for the diagnostic performance of CNFLcenter are different from previous studies. Tavakoli et al6 used the definition (NDS $ 3) as in this study to define DPN and reported a cutoff point of 3.4 mm/mm2 with a sensitivity of 0.6 and specificity of 0.8 in a cohort of 101 diabetic participants (66% with DPN) and 17 healthy controls. However, they used a first-generation slitscanning CCM, which has limited resolution. Ahmed et al7 used laser-scanning CCM, as in this study, and reported a threshold value of 14.0 mm/mm2 with a sensitivity of 0.8 and specificity of 0.8 in a cohort of 89 participants with type 1 diabetes in which 37% had neuropathy. However, their definition of neuropathy was based on clinical and neurophysiological criteria. Indeed, in a recent study deploying different cutoffs for neurophysiology and thermal thresholds to define DPN, the sensitivity ranged from 0.6 to 0.8 and the specificity ranged from 0.6 to 0.7 for automated CNFL.28 These discrepancies can be attributed to differences in the definitions of neuropathy used, the proportion of participants with DPN and their neuropathy severity, and methodological differences in image acquisition protocols, image analysis techniques, and the number of images evaluated. The strengths of this study are the inclusion of a relatively large study population, using an established protocol for the number of images to best represent the central cornea,29 and the application of a fully automated technique for CNFL quantification. We acknowledge limitations of this study. Our diabetic and control participants were not age matched; however, our results are unlikely to be confounded by this discrepancy in view of the negligible impact of age on CNFL.30 Including only participants with type 1 diabetes with predominantly mild and moderate neuropathy may limit the generalizability of these findings. This was a cross-sectional study comparing the morphology of the SNP in 2 corneal locations, and only a prospective comparative longitudinal study can reveal sequential changes.
1. He JC, Bazan NG, Bazan HEP. Mapping the entire human corneal nerve architecture. Exp Eye Res. 2010;91:513–523. 2. Marfurt CF, Cox J, Deek S, et al. Anatomy of the human corneal innervation. Exp Eye Res. 2010;90:478–492. 3. Patel DV, McGhee CNJ. Mapping of the normal human corneal subbasal nerve plexus by in vivo laser scanning confocal microscopy. Invest Ophthalmol Vis Sci. 2005;46:4485–4488. 4. Edwards K, Pritchard N, Gosschalk K, et al. Wide-field assessment of the human corneal subbasal nerve plexus in diabetic neuropathy using a novel mapping technique. Cornea. 2012;31:1078–1082. 5. Malik RA, Kallinikos P, Abbott CA, et al. Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients. Diabetologia. 2003;46:683–688. 6. Tavakoli M, Quattrini C, Abbott C, et al. Corneal confocal microscopy: a novel noninvasive test to diagnose and stratify the severity of human diabetic neuropathy. Diabetes Care. 2010;33:1792–1797. 7. Ahmed A, Bril V, Orszag A, et al. Detection of diabetic sensorimotor polyneuropathy by corneal confocal microscopy in type 1 diabetes: a concurrent validity study. Diabetes Care. 2012;35:821–828. 8. Edwards K, Pritchard N, Vagenas D, et al. Utility of corneal confocal microscopy for assessing mild diabetic neuropathy: baseline findings of the LANDMark study. Clin Exp Optom. 2012;95:348–354. 9. Pritchard N, Edwards K, Dehghani C, et al. Longitudinal assessment of neuropathy in type 1 diabetes using novel ophthalmic markers (LANDMark): study design and baseline characteristics. Diabetes Res Clin Pract. 2014;104: 248–256. 10. Ziegler D, Papanas N, Zhivov A, et al. Early detection of nerve fiber loss by corneal confocal microscopy and skin biopsy in recently diagnosed type 2 diabetes. Diabetes. 2014;63:2454–2463. 11. Halpern EM, Lovblom LE, Orlov S, et al. The impact of common variation in the definition of diabetic sensorimotor polyneuropathy on the validity of corneal in vivo confocal microscopy in patients with type 1 diabetes: a brief report. J Diabetes Complications. 2013;27:240–242. 12. Davidson EP, Coppey LJ, Kardon RH, et al. Differences and similarities in development of corneal nerve damage and peripheral neuropathy and in diet-induced obesity and type 2 diabetic rats. Invest Ophthalmol Vis Sci. 2014;55:1222–1230. 13. Tesfaye S. Recent advances in the management of diabetic distal symmetrical polyneuropathy. J Diabetes Investig. 2011;2:33–42. 14. Hertz P, Bril V, Orszag A, et al. Reproducibility of in vivo corneal confocal microscopy as a novel screening test for early diabetic sensorimotor polyneuropathy. Diabet Med. 2011;28:1253–1260. 15. Wu T, Ahmed A, Bril V, et al. Variables associated with corneal confocal microscopy parameters in healthy volunteers: implications for diabetic neuropathy screening. Diabet Med. 2012;29:e297–e303. 16. Young MJ, Boulton AJM, Macleod AF, et al. A multicenter study of the prevalence of diabetic peripheral neuropathy in the United Kingdom hospital clinic population. Diabetologia. 1993;36:150–154. 17. Dehghani C, Pritchard N, Edwards K, et al. Fully automated, semiautomated, and manual morphometric analysis of corneal subbasal
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In conclusion, although the whorl area represents a more distal extension of nerves than the central cornea, our findings indicate the comparable deficits of small nerve fibers at both corneal anatomical sites. Furthermore, automated quantification of the CNFL from the center is comparable to the whorl area in the diagnosis of DPN. REFERENCES
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nerve plexus in individuals with and without diabetes. Cornea. 2014;33: 696–702. Dabbah MA, Graham J, Petropoulos IN, et al. Automatic analysis of diabetic peripheral neuropathy using multi-scale quantitative morphology of nerve fibres in corneal confocal microscopy imaging. Med Image Anal. 2011;15:738–747. Zweig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clin Chem. 1993;39: 561–577. Youden WJ. Index for rating diagnostic tests. Cancer. 1950;3:32–35. Dyck PJ, Norell JE, Tritschler H, et al. Challenges in design of multicenter trials. Diabetes Care. 2007;30:2619–2625. Gaede P, Lund-Andersen H, Parving HH, et al. Effect of a multifactorial intervention on mortality in type 2 diabetes. N Engl J Med. 2008;358: 580–591. Sivaskandarajah GA, Halpern EM, Lovblom LE, et al. Structurefunction relationship between corneal nerves and conventional small-fiber tests in type 1 diabetes. Diabetes Care. 2013;36: 2748–2755.
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24. Quattrini C, Tavakoli M, Jeziorska M, et al. Surrogate markers of small fiber damage in human diabetic neuropathy. Diabetes. 2007;56:2148–2154. 25. Efron N, Edwards K, Roper N, et al. Repeatability of measuring corneal subbasal nerve fiber length in individuals with type 2 diabetes. Eye Contact Lens. 2010;36:245–248. 26. Malik RA, Veves A, Tesfaye S, et al. Small fibre neuropathy: role in the diagnosis of diabetic sensorimotor polyneuropathy. Diabetes Metab Res Rev. 2011;27:678–684. 27. Sumner CJ, Sheth S, Griffin JW, et al. The spectrum of neuropathy in diabetes and impaired glucose tolerance. Neurology. 2003;60:108–111. 28. Petropoulos IN, Alam U, Fadavi H, et al. Rapid automated diagnosis of diabetic peripheral neuropathy with in vivo corneal confocal microscopy. Invest Ophthalmol Vis Sci. 2014;55:2071–2078. 29. Vagenas D, Pritchard N, Edwards K, et al. Optimal image sample size for corneal nerve morphometry. Optom Vis Sci. 2012;89:812–817. 30. Dehghani C, Pritchard N, Edwards K, et al. Natural history of corneal nerve morphology in mild neuropathy associated with type 1 diabetes: development of a potential measure of diabetic peripheral neuropathy. Invest Ophthalmol Vis Sci. 2014;55:7982–7990.
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Utility of Assessing Nerve Morphology in Central Cornea Versus Whorl Area for Diagnosing Diabetic Peripheral Neuropathy Nicola Pritchard, PhD,* Cirous Dehghani, MSc,* Katie Edwards, PhD,* Edward Burgin, BVisSc, MOptom,* Nick Cheang, BVisSc, MOptom,* Hannah Kim, BVisSc, MOptom,* Merna Mikhaiel, BVisSc, MOptom,* Gemma Stanton, BVisSc, MOptom,* Anthony W. Russell, MBBS, PhD,†‡ Rayaz A. Malik, MBChB, PhD,§¶ and Nathan Efron, PhD, DSc*
Purpose: To compare small nerve fiber damage in the central cornea and whorl area in participants with diabetic peripheral neuropathy (DPN) and to examine the accuracy of evaluating these 2 anatomical sites for the diagnosis of DPN.
Methods: A cohort of 187 participants (107 with type 1 diabetes and 80 controls) was enrolled. The neuropathy disability score (NDS) was used for the identification of DPN. The corneal nerve fiber length at the central cornea (CNFLcenter) and whorl (CNFLwhorl) was quantified using corneal confocal microscopy and a fully automated morphometric technique and compared according to the DPN status. Receiver operating characteristic analyses were used to compare the accuracy of the 2 corneal locations for the diagnosis of DPN. Results: CNFLcenter and CNFLwhorl were able to differentiate all 3 groups (diabetic participants with and without DPN and controls) (P , 0.001). There was a weak but significant linear relationship for CNFLcenter and CNFLwhorl versus NDS (P , 0.001); however, the corneal location · NDS interaction was not statistically significant (P = 0.17). The area under the receiver operating characteristic curve was similar for CNFLcenter and CNFLwhorl (0.76 and 0.77, respectively, P = 0.98). The sensitivity and specificity of the cutoff points were 0.9 and 0.5 for CNFLcenter and 0.8 and 0.6 for CNFLwhorl. Conclusions: Small nerve fiber pathology is comparable at the central and whorl anatomical sites of the cornea. Quantification of CNFL from the corneal center is as accurate as CNFL quantification of the whorl area for the diagnosis of DPN. Received for publication January 19, 2015; revision received February 25, 2015; accepted February 27, 2015. Published online ahead of print April 24, 2015. From the *Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Australia; †School of Medicine, University of Queensland, St Lucia, Australia; ‡Department of Diabetes and Endocrinology, Princess Alexandra Hospital, Woolloongabba, Australia; §Center for Endocrinology and Diabetes, Institute of Human Development, University of Manchester, Manchester, United Kingdom; and ¶Weill Cornell Medical College in Qatar, Doha, Qatar. Supported by grants from the National Health and Medical Research Council of Australia (497230) and the JDRFI (8-2008-362). The authors have no funding or conflicts of interest to disclose. Reprints: Nathan Efron, PhD, DSc, Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, Queensland 4059, Australia (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Key Words: corneal confocal microscopy, corneal subbasal nerve plexus, diabetic peripheral neuropathy (Cornea 2015;34:756–761)
T
he corneal subbasal nerve plexus (SNP) is located between the Bowman layer and the corneal basal epithelium and originates from stromal nerves penetrating the Bowman layer, predominantly in the peripheral area.1,2 At this interface, subbasal nerves anastomose extensively and form a dense homogenous nerve network that radiates toward a whorl-like assembly (whorl area), usually 1 to 2 mm inferior to the corneal apex, as described in ex vivo histological studies1,2 and in vivo corneal confocal microscopy (CCM) investigations.3,4 The transparency of the cornea and unique attributes of the SNP, such as its parallel arrangement in relation to the ocular surface, provide an opportunity for the noninvasive assessment of this nerve plexus using CCM. Quantification of the SNP using CCM as a noninvasive and reiterative modality has been shown to be a promising marker for the detection and stratification of diabetic peripheral neuropathy (DPN) in several studies over the past decade.5–11 Because of the convenience and ease of imaging from the central cornea, this region has been the main focus in almost all of these studies. However, recent investigations in diabetic patients4 and animals12 have provided evidence of more pronounced and earlier corneal neural loss at the inferior whorl. Given that diabetic neuropathy affects the most distal axons first and that the nerves at the whorl are distal to the central cornea, one may expect more marked pathology at this site.13 The corneal nerve fiber length (CNFL) is the most reliable14,15 and frequently reported morphological parameter of the SNP obtained using CCM. The aim of this study was to quantify and compare this parameter at the corneal apex (central cornea) and whorl-like area (whorl) in diabetic individuals with and without neuropathy and healthy participants. Furthermore, the capability of CNFL in the center and whorl for diagnosing DPN was determined.
MATERIALS AND METHODS This study was conducted in accordance with the Declaration of Helsinki and was approved by ethics Cornea Volume 34, Number 7, July 2015
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Cornea Volume 34, Number 7, July 2015
committees of Queensland University of Technology, Princess Alexandra Hospital, and Mater Hospital in Brisbane, Australia. All participants provided written informed consent. Based on the inclusion and exclusion criteria, 187 participants were enrolled. All participants were assessed for the presence of any corneal compromise. Participants were excluded if they had a history of ocular trauma or surgery, ocular disease or systemic disease with potential corneal effects, and systemic disease (other than diabetes). Other causes of neuropathy were excluded. The cohort included 107 participants with type 1 diabetes who were recruited from the Diabetes and Endocrinology Research Center at Princess Alexandra and Mater Hospitals and the general population in Brisbane. Eighty healthy participants, without peripheral neuropathy and/or diabetes, were also recruited as controls. The neuropathy disability score (NDS),16 which is a scale of 0 to 10, was used to define the neuropathy status for all participants. This measure included assessment of vibration, pin-prick, and temperature perception as well as the presence or absence of ankle reflexes in both lower limbs. An NDS $ 3 was defined as the presence of neuropathy. The methods used during this study to quantify corneal nerve morphology using CCM and a fully automated segmentation technique have been reported in detail elsewhere17 and are described only briefly here. CCM was conducted using a Heidelberg Retina Tomograph (HRT 3) with Rostock Corneal Module (Heidelberg Engineering GmbH, Heidelberg, Germany) on the cornea of the dominant-hand side of the participants. Participants were instructed to fixate on the near target attached to the CCM instrument during the examination. The location of the whorl area was facilitated by gently moving this target and CCM in X and Y directions until the whorl pattern was in view. For each participant, multiple images of the SNP were captured from the central cornea and whorl area. From digitally saved images, 8 central and 3 whorl images of the SNP were chosen and analyzed using a fully automated algorithm18 to quantify CNFL. For each participant, CNFL assessed at the central cornea (CNFLcenter) and whorl (CNFLwhorl) was the average value obtained from the chosen images (8 and 3, respectively) and expressed in the unit of mm/mm2. SPSS (version 21) was used to analyze the data. A univariate analysis of variance (ANOVA) was performed to examine the possible interaction between the neuropathy status of the participants (control, diabetes without neuropathy, and diabetes with neuropathy) and their assignment group (controls and diabetes) and quantified CNFL values from central corneal and whorl regions. To establish the association with the severity of neuropathy and CNFL from the central cornea and whorl area, the CNFL values were plotted against the respective NDS values. A mixed-model analysis was conducted to assess the interaction between CNFL from the 2 corneal locations and NDS. In this model, CNFL was considered as the response variable, and the effects of corneal location (center vs. whorl), NDS, and corneal location · NDS interaction were examined. Receiver operating characteristic (ROC) curve analyses were used to compare the capability of CNFLcenter and CNFLwhorl for diagnosing DPN. The area under the curve (AUC), which represents a measure of accuracy,19 was Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Corneal Nerves in Diabetic Neuropathy
estimated. Using the Youden index,20 the cutoff values were determined for the presence of neuropathy (NDS $ 3). Finally, the sensitivity, specificity, positive likelihood ratio (+LR), and negative likelihood ratio (2LR) of CNFLcenter and CNFLwhorl were calculated. In healthy corneas, Patel and McGhee3 reported a whorl/ center CNFL ratio of 1.2 using laser-scanning CCM. In the absence of previous quantitative data of the whorl area versus center in DPN, we anticipated 25% lower whorl/center CNFL ratio in individuals with DPN. Assuming this difference, a = 0.05 and 0.9% power, the required sample size was determined to be 76 diabetic participants including 25 with DPN. Enrollment continued until the DPN group contained 25 participants, resulting in a total of 107 diabetic participants. A cohort of 80 healthy controls was also enrolled for comparison.
RESULTS
In the entire cohort, the mean 6 SD for CNFLcenter and CNFLwhorl was 17.5 6 3.8 and 19.5 6 4.6 mm/mm2, respectively (P , 0.001). CNFLcenter and CNFLwhorl were significantly lower in participants with diabetes compared with the control group (CNFLcenter, 16.1 6 3.7 vs. 19.3 6 3.0 mm/mm2 and CNFLwhorl, 17.5 6 4.2 vs. 22.1 6 3.9 mm/ mm2, respectively, P , 0.01). Although the whorl/center CNFL ratio was slightly lower in the diabetic group (1.09) compared with controls (1.14), the univariate ANOVA showed no significant interaction between the corneal location (center vs. whorl) and assignment group of the participants (P = 0.1). The mean age was 37.0 6 17.8 years for controls and 48.3 6 15.1 years for the diabetic group. Of the 107 participants with type 1 diabetes, 25 (23%) were diagnosed with DPN. Ten participants had mild (3 # NDS # 5), 12 had moderate (6 # NDS # 8), and 3 had severe (NDS $ 9) neuropathy. Figure 1 depicts representative images from central corneal and whorl regions in controls and diabetic participants with and without neuropathy. There was a significant difference between groups (control, diabetes without neuropathy, and diabetes with neuropathy) for both CNFLcenter and CNFLwhorl (P , 0.001) (Table 1). Diabetic participants with and without neuropathy showed significantly lower CNFLcenter compared with controls (14.2 6 3.5 and 16.7 6 3.5 mm/mm2 vs. 19.3 6 3.0 mm/mm2, respectively, P , 0.01). Similarly, CNFLwhorl was significantly lower in diabetic participants with (15.4 6 4.4 mm/mm2) and without (18.2 6 3.9 mm/mm2) neuropathy versus control group (22.1 6 3.9 mm/mm2) (P , 0.01). However, the univariate ANOVA showed that the neuropathy status · corneal location term was not significant for estimated CNFL (P = 0.1). Figure 2 illustrates the relationship between CNFL from the central cornea and whorl area against NDS. There was a weak but significant linear relationship for CNFLcenter and CNFLwhorl versus NDS (R2: 0.08 and 0.09, respectively, P , 0.001). The calculated slopes of the regression lines were 20.5 for CNFLcenter and 20.6 for CNFLwhorl. Mixed model analysis revealed significant effects of corneal location (P , 0.001) and NDS (P , 0.001). However, the corneal www.corneajrnl.com |
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Cornea Volume 34, Number 7, July 2015
Pritchard et al
FIGURE 1. Representative images of the SNP from the 3 study groups. Examples of central corneal nerves are shown in a control participant (A), a diabetic participant without neuropathy (B), and a diabetic participant with severe neuropathy (C). Example images of the whorl area for respective participants are shown in (D–F). All images are 400 · 400 mm.
location · NDS interaction was not statistically significant (P = 0.17). Figure 3 shows the ROC curves for CNFL center and CNFL whorl among the participants for the identification of DPN cases according to NDS. The AUC for the presence of DPN (NDS $ 3) was 0.76 for CNFLcenter and 0.77 for CNFLwhorl . The 2 estimated AUCs were statistically similar (P = 0.98). The results of the calculated Youden cutoff values, sensitivity, specificity, +LR, and
2LR associated with the cutoff points are presented in Table 2.
DISCUSSION Detection of nerve pathology at the earliest stage of DPN is crucial as patients may benefit substantially from prompt management to halt or slow the changes and/or to avoid serious consequences such as foot ulceration or
TABLE 1. Comparison of CNFL From Center (CNFLcenter) and Whorl (CNFLwhorl) of the Cornea in the Study Participants According to the Presence (DPN+ve) and Absence (DPN2ve) of Neuropathy Defined by NDS $ 3 Group Parameter
Controls (n = 80)
DPN2ve (n = 82)
DPN+ve (n = 25)
CNFLcenter, mm/mm2
19.3 6 3.0
16.7 6 3.5
14.2 6 3.5
CNFLwhorl, mm/mm2
22.1 6 3.9
18.2 6 3.9
15.4 6 4.4
P, Group Difference ,0.001*, controls vs. DPN2ve, DPN+ve†; DPN2ve vs. DPN+ve† ,0.001*, controls vs. DPN2ve, DPN+ve†; DPN2ve vs. DPN+ve†
Outcomes are presented as mean 6 SD. *1-way ANOVA test. †Scheffe post hoc test (P , 0.05).
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Cornea Volume 34, Number 7, July 2015
Corneal Nerves in Diabetic Neuropathy
FIGURE 2. Scatter plot illustrates the relationship of CNFL values quantified from the central cornea (CNFLcenter) and whorl area (CNFLwhorl) with their NDS values. The dashed line indicates the linear regression for CNFLcenter (b = 20.5, P , 0.01), and the solid line indicates the linear regression for CNFLwhorl (b = 20.6, P , 0.01). No significant difference was found between the 2 slopes (P = 0.17).
amputation.21,22 Morphometric assessment of the SNP using CCM at the central cornea has a demonstrated capacity for the detection and severity stratification of DPN.6,7,9,23 There is also some evidence of identification of earlier small nerve fiber damage associated with diabetes using CCM at the central cornea.10,24 Assessment of the SNP microstructures using in vivo CCM has been shown to be reliable, repeatable, and reproducible for the detection of small nerve injury in diabetes, particularly in the context of DPN.7,14,15,25 Akin to the length-dependent process of DPN whereby the most distal and thinner small fibers are the earliest nerves to be damaged,26,27 the hypothetically optimal approach would be to examine the most distal nerves on the SNP (whorl-like
FIGURE 3. ROC curves for CNFL quantified from the central cornea (solid line) and whorl area (dotted line) against NDS $ 3. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
assembly) to detect the earliest and perhaps more prominent nerve damage. In this study, we report data from a large cohort of patients with type 1 diabetes and control participants who underwent assessment of corneal nerve morphology at the central cornea and whorl area. Quantified CNFL from the whorl area (CNFLwhorl) was found to be approximately 2 mm/mm2 higher than that at the corneal center (CNFLcenter) for the entire cohort. The higher nerve tissue density at the whorl area compared with the center of the cornea has previously been reported in healthy corneas using in vivo CCM3 and highlights the differences in nerve tissue at these 2 anatomical sites. Depletion of small nerve fibers was evident at the corneal center and whorl area in participants with diabetes. However, in this study, corneal location did not affect the observed CNFL deficit in diabetic participants. Our findings showed that both CNFLcenter and CNFLwhorl were capable of differentiating diabetic participants without and with neuropathy from controls, and the differentiation was not affected by corneal location. Although there was a negative relationship between NDS versus CNFLcenter and CNFLwhorl, the lack of significant interaction between corneal location and NDS indicated that the rate of change of CNFLcenter and CNFLwhorl in relation to NDS (ie, the slopes in Fig. 2) was not different. This outcome demonstrated that the damage to small nerves of the SNP assessed using in vivo CCM was equivalent at the central and whorl areas of the cornea, regardless of the severity of neuropathy. To further explore the diagnostic accuracy of CNFL pertaining to the 2 corneal locations in assessing participants with (NDS $ 3) and without (NDS # 2) neuropathy, ROC analysis was used. The AUC, an overall performance measure of the test, was similar for CNFLcenter and CNFLwhorl. Using the Youden index, the estimated optimal cutoff points that identified the presence of DPN were 17.9 mm/mm2 for CNFLcenter and 18.6 mm/mm2 for CNFLwhorl. Compared with CNFLwhorl, CNFLcenter showed higher sensitivity (0.9 vs. 0.8) and lower specificity (0.5 vs. 0.6); however, the difference between likelihood ratios—an expression of the probability of test results given the correct diagnosis in the presence or absence of disease19—was trivial. Overall, it can be concluded www.corneajrnl.com |
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Cornea Volume 34, Number 7, July 2015
Pritchard et al
TABLE 2. Detection Capacity of CNFL Pertaining to the Central (CNFLcenter) and Whorl Area (CNFLwhorl) of the Cornea for the Presence of Neuropathy as Defined by NDS $ 3 Parameter
AUC
P
Youden Cutoff Point
Sensitivity (95% CI)
Specificity (95% CI)
+LR
2LR
CNFLcenter CNFLwhorl
0.76* 0.77
,0.001 ,0.001
#17.9 #18.6
0.9 (0.7–1.0) 0.8 (0.6–1.0)
0.5 (0.5–0.6) 0.6 (0.5–0.7)
2.0 2.2
0.2 0.3
*No significant difference compared with CNFLwhorl (x2, P = 0.98). +LR, positive likelihood ratio; 2LR, negative likelihood ratio.
from a comparison of ROC plots and AUC results that there was little difference in diagnostic accuracy based on the assessment of nerve morphology at the 2 locations. Although we postulated that the whorl area, which is 1 to 2 mm more distal than the central cornea, would demonstrate more nerve depletion in participants with DPN, the findings of this study do not support this notion. A possible explanation for this might be that a 1 to 2 mm difference in distal nerve extension is insufficient to observe more pronounced distal axonal neuropathy in the cornea. Furthermore, the possibility remains that the NDS may suffer from a lack of adequate proficiency in diagnosing and staging the severity of neuropathy. Our findings of ROC analysis for the diagnostic performance of CNFLcenter are different from previous studies. Tavakoli et al6 used the definition (NDS $ 3) as in this study to define DPN and reported a cutoff point of 3.4 mm/mm2 with a sensitivity of 0.6 and specificity of 0.8 in a cohort of 101 diabetic participants (66% with DPN) and 17 healthy controls. However, they used a first-generation slitscanning CCM, which has limited resolution. Ahmed et al7 used laser-scanning CCM, as in this study, and reported a threshold value of 14.0 mm/mm2 with a sensitivity of 0.8 and specificity of 0.8 in a cohort of 89 participants with type 1 diabetes in which 37% had neuropathy. However, their definition of neuropathy was based on clinical and neurophysiological criteria. Indeed, in a recent study deploying different cutoffs for neurophysiology and thermal thresholds to define DPN, the sensitivity ranged from 0.6 to 0.8 and the specificity ranged from 0.6 to 0.7 for automated CNFL.28 These discrepancies can be attributed to differences in the definitions of neuropathy used, the proportion of participants with DPN and their neuropathy severity, and methodological differences in image acquisition protocols, image analysis techniques, and the number of images evaluated. The strengths of this study are the inclusion of a relatively large study population, using an established protocol for the number of images to best represent the central cornea,29 and the application of a fully automated technique for CNFL quantification. We acknowledge limitations of this study. Our diabetic and control participants were not age matched; however, our results are unlikely to be confounded by this discrepancy in view of the negligible impact of age on CNFL.30 Including only participants with type 1 diabetes with predominantly mild and moderate neuropathy may limit the generalizability of these findings. This was a cross-sectional study comparing the morphology of the SNP in 2 corneal locations, and only a prospective comparative longitudinal study can reveal sequential changes.
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In conclusion, although the whorl area represents a more distal extension of nerves than the central cornea, our findings indicate the comparable deficits of small nerve fibers at both corneal anatomical sites. Furthermore, automated quantification of the CNFL from the center is comparable to the whorl area in the diagnosis of DPN. REFERENCES
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Cornea Volume 34, Number 7, July 2015
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