Research
JAMA Ophthalmology | Brief Report
Use of Corneal Confocal Microscopy to Evaluate Small Nerve Fibers in Patients With Human Immunodeficiency Virus Harriet I. Kemp, BM BCh, FRCA; Ioannis N. Petropoulos, PhD; Andrew S. C. Rice, MD, FRCP; Jan Vollert, MSc; Christoph Maier, MD; Dietrich Strum, MD; Marc Schargus, MD, FEBO; Tunde Peto, MD, PhD; Scott Hau, MSc, BSc; Reena Chopra, BSc; Rayaz A. Malik, MB ChB, PhD Supplemental content IMPORTANCE Objective quantification of small fiber neuropathy in patients with human
immunodeficiency virus (HIV)–associated sensory neuropathy (HIV-SN) is difficult but needed for diagnosis and monitoring. In vivo corneal confocal microscopy (IVCCM) can quantify small fiber damage. OBJECTIVE To establish whether IVCCM can identify an abnormality in corneal nerve fibers and Langerhans cells in patients with and without HIV-SN. DESIGN, SETTING, AND PARTICIPANTS This prospective, cross-sectional cohort study was conducted between July 24, 2015, and September 17, 2015. Twenty patients who were HIV positive were recruited from adult outpatient clinics at Chelsea and Westminster Hospital NHS Foundation Trust in England. These patients underwent IVCCM at Moorfields Eye Hospital NHS Foundation Trust in London, England, and the IVCCM images were analyzed at Weill Cornell Medicine–Qatar in Ar-Rayyan, Qatar. Patients were given a structured clinical examination and completed validated symptom questionnaires and the Clinical HIV-Associated Neuropathy Tool. Results from patients with HIV were compared with the results of the age- and sex-matched healthy control participants (n = 20). All participants were classified into 3 groups: controls, patients with HIV but without SN, and patients with HIV-SN. MAIN OUTCOMES AND MEASURES Comparison of corneal nerve fiber density, corneal nerve branch density, corneal nerve fiber length, corneal nerve fiber tortuosity, and corneal Langerhans cell density between healthy controls and patients with HIV with and without SN. RESULTS All 40 participants were male, and most (ⱖ70%) self-identified as white. Of the 20 patients with HIV, 14 (70%) had HIV-SN. This group was older (mean [SD] age, 57.7 [7.75] years) than the group without HIV-SN (mean [SD] age, 42.3 [7.26] years) and the controls (mean [SD] age, 53.8 [10.5] years). Corneal nerve fiber density was reduced in patients with HIV compared with the controls (26.7/mm2 vs 38.6/mm2; median difference, −10.37; 95.09% CI, −14.27 to −6.25; P < .001) and in patients with HIV-SN compared with those without (25.8/mm2 vs 30.7/mm2; median difference, −4.53; 95.92% CI, −8.85 to −0.26; P = .03). Corneal nerve branch density and corneal nerve fiber length were reduced in patients with HIV, but no differences were identified between those with neuropathy and without neuropathy (corneal nerve branch density: 95.83/mm2 for the controls vs 72.37/mm2 for patients with HIV; median difference, −24.53; 95.32% CI, −50.62 to −3.13; P = .01; and corneal nerve fiber length: 28.4 mm/mm2 for the controls vs 21.9 mm/mm2 for patients with HIV; median difference, −5.24; 95.09% CI, −8.83 to −1.38; P = .001). Tortuosity coefficient was increased in patients with HIV compared with controls (16.44 vs 13.95; median difference, 2.34; 95.09% CI, 0.31 to 4.65; P = .03) and in those with HIV-SN compared with those without (17.84 vs 14.18; median difference, 4.32; 95.92% CI, 0.68-9.23; P = .01). No differences were identified in corneal Langerhans cell density (19.84 cells/mm2 for the controls vs 41.43 cells/mm2 for patients with HIV; median difference, 9.38; 95% CI, −12.51 to 26.34; P = .53). CONCLUSIONS AND RELEVANCE In vivo corneal confocal microscopy could be used in the
assessment of HIV-SN, but larger studies are required to confirm this finding. JAMA Ophthalmol. doi:10.1001/jamaophthalmol.2017.1703 Published online June 8, 2017.
Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Author: Rayaz A. Malik, MB ChB, PhD, Institute of Cardiovascular Sciences, Cardiac Centre, School of Medicine, University of Manchester, 46 Grafton St, Manchester M13 9NT, England ([email protected]).
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Research Brief Report
Corneal Confocal Microscopy Use to Evaluate Small Nerve Fibers in Patients With HIV
H
uman immunodeficiency virus–associated sensory neuropathy (HIV-SN) is one of the most frequent neurologic consequences of HIV infection and its treatment.1 Patients with HIV-SN present with pain and paraesthesia, and their quality of life and ability to work are substantially affected.2 Small fiber neuropathy can be accurately assessed by quantifying intraepidermal nerve fiber density but is invasive and does not easily facilitate longitudinal studies.3 In vivo corneal confocal microscopy (IVCCM) is a rapid, noninvasive ophthalmic imaging technique that can quantify small nerve fiber damage in diabetic neuropathy,4 hereditary Charcot-Marie-Tooth type 1A,5 chemotherapy-induced peripheral neuropathy,6 chronic inflammatory demyelinating polyneuropathy,7 and amyloid neuropathy.8 In a nonhuman primate model of HIV-SN, animals with the most severe disease showed a decrease in corneal nerve fiber density,9 and IVCCM has demonstrated a reduction in corneal nerve fiber length in HIV-positive patients.10 Epidermal Langerhans cell (LC) density is reduced in patients with HIV or AIDS,11 but in vivo assessment of corneal LC density has not been reported in HIV. This study undertook IVCCM to quantify corneal nerve fiber structure and LC density in patients with HIV.
Methods Study participants had a diagnosis of HIV, and the exclusion criteria included pregnancy, peripheral vascular disease, severe coincident psychiatric diagnosis, other causes of peripheral neuropathy, nystagmus, or corneal surgery. Patients were recruited from adult outpatient clinics at Chelsea and Westminster Hospital NHS Foundation Trust in the United Kingdom. Images from age- and sex-matched healthy control participants (volunteers) were collected using the same image acquisition protocol as that used for the patient groups.4 The study was conducted from July 24, 2015, to September 17, 2015. Image analysis was performed from April 7, 2016, to April 18, 2016. Ethical approval for this study was granted by the National Ethics Research Service. This study was part of the EuroPain project (http://imieuropain.org) and was conducted in accordance with the Declaration of Helsinki.12 All patients gave written informed consent to take part in the study.
Classification All participants were classified into 3 groups: controls, patients with HIV but without HIV-SN, and patients with HIV-SN. (The presence of 1 sign and at least 1 symptom from the validated Clinical HIV-Associated Neuropathy Assessment Tool indicated neuropathy.13) Patients with HIV underwent a structured clinical examination and completed the Douleur Neuropathique 4 interview (DN4-i; the score range was 0-7, and a DN4-i score higher than 3 was classified as painful neuropathy) as well as the Brief Pain Inventory (the severity range was 0-10 [0.0-7.0 for our patient cohort] with a pain interference scale of 0-10 [1.29-8.57 for our patient cohort]).
Image Acquisition and Quantification Participants underwent IVCCM at Moorfields Eye Hospital NHS Foundation Trust in London, England, and IVCCM images were E2
Key Points Question Can in vivo corneal confocal microscopy be used to assess changes in corneal nerve fiber structure and Langerhans cell density in patients with human immunodeficiency virus (HIV)–associated sensory neuropathy? Findings In this cohort study of 20 patients with HIV, a decrease in corneal nerve fiber density and an increase in corneal nerve tortuosity were observed in patients with HIV-associated sensory neuropathy when compared with patients without neuropathy, with no change in Langerhans cell density. Meaning In vivo corneal confocal microscopy is a noninvasive ophthalmic imaging technique that could aid in the diagnosis and longitudinal monitoring of patients with HIV-associated sensory neuropathy.
analyzed at Weill Cornell Medicine–Qatar in Ar-Rayyan, Qatar.4 Images were acquired using a corneal confocal microscope (Heidelberg Retinal Tomography and Rostock Corneal Module; Heidelberg Engineering). Six images from the right and left eyes were selected for analysis and manually quantified by a single experienced examiner (I.N.P.) using CCMetrics software (M. A. Dabbah, University of Manchester). Corneal nerve fiber density (number per square millimeter), corneal nerve branch density (number per square millimeter), corneal nerve fiber length (in millimeters per square millimeter), corneal nerve fiber tortuosity, and corneal LC density (in cells per square millimeter) were quantified.4 Patients did not undergo any assessment of corneal sensation.
Statistical Analysis A power calculation using SigmaPlot, version 10.0 (Systat) of data from patients with diabetic polyneuropathy revealed a sample size of at least 6 participants per group to identify a difference in corneal nerve fiber length (mean [SD] difference, 1.16 [0.50] mm/mm2; α = 0.05; β = 0.8) and a sample size of 4 participants to identify a difference in corneal LC density (mean [SD] difference, 8.01 [2.7] cells/mm2; α = 0.05; β = 0.8). Statistical analysis was performed using SPSS, version 22.0 (IBM). Data are presented as mean (SD) or median (interquartile range [IQR]). Corneal nerve measures and corneal LC density in patients with HIV were compared with controls using the Wilcoxon rank sum test. The control and HIV groups (HIV without SN, HIV-SN) were compared using the Kruskal-Wallis test for continuous nonparametric data (with post hoc testing for pairwise comparisons). Median differences are reported using a Hodges-Lehmann estimate; because nonparametric data rely on ranks, exact 95% CIs were not always possible (exact CIs stated). To reduce the chance of a type I error, a false detection rate correction was made to the P value using the Benjamini-Hochberg method. Spearman rank correlation was used between corneal nerve fiber measures and symptom severity scores.
Results Twenty male patients with HIV were matched by age and sex to 20 healthy controls (Table). All 40 participants were male,
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Corneal Confocal Microscopy Use to Evaluate Small Nerve Fibers in Patients With HIV
Brief Report Research
Table. Demographics for Healthy Control Participants, Patients With HIV–No SN, and Patients With HIV-SN HIV–No SN (n = 6)
Characteristic Age, mean (SD), y
HIV-SN (n = 14)
42.3 (7.26)
Race/ethnicity, white, No. (%)
5 (83)
Healthy Controls (n = 20)
57.7 (7.75) 13 (93)
53.8 (10.5) 14 (70)
Height, mean (SD), cm
174.2 (4.96)
174.0 (7.95)
167.9 (11.19)
Weight, mean (SD), kg
70.5 (7.53)
74.9 (13.1)
81.6 (14.63)
HIV
12.4 (10.81)
25.3 (4.68)
NA
Medication
10.8 (9.26)
20.7 (3.75)
NA
Duration, mean (SD), y
CD4 count, mean (SD), cells/mm3 Most recent
797.0 (285.51)
685.4 (370.55)
Not measured
Nadir
192.8 (141.74)
154.5 (152.18)
Not measured
Peak viral load, mean (SD), copies/mL
93 137.2 (84 721.61) 59 281.5 (91 369.90) Not measured
CHANT, mean (SD) Symptom score
0.2 (0.41)
Sign score
0.0 (0.0)
1.2 (0.43)
NA
0.3 (0.82)
4.4 (1.40)
NA
DN4-i score, mean (SD)
1.8 (0.43)
NA
BPI, mean (SD) Severity score
0.04 (0.10)
3.5 (3.13)
NA
Interference score
1.3 (0.0)
4.26 (2.33)
NA
Exposure to neurotoxic antiretroviral agents, No. (%)
2 (33)
and most (≥70%) self-identified as white. Patients with HIV-SN were older (mean [SD] age, 57.7 [7.75] years) than those without HIV-SN (mean [SD] age, 42.3 [7.26] years) and the healthy controls (mean [SD] age, 53.8 [10.5] years). Six patients (30%) had no neuropathy (HIV–no SN) and 14 (70%) had neuropathy (HIV-SN). Corneal nerve fiber density was lower in patients with HIV than in the controls (26.7/mm2 vs 38.6/mm2; median difference, −10.37; 95.09% CI, −14.27 to −6.25; P < .001) as well as in patients with HIV-SN than those with HIV–no SN (25.8/mm2 vs 30.7/mm2; median difference, −4.53; 95.92% CI, −8.85 to −0.26; P = .03). Corneal nerve branch density and corneal nerve fiber length were lower in patients with HIV, and no difference was identified between those with neuropathy and those without neuropathy (corneal nerve branch density: 95.83/mm2 for the controls vs 72.37/mm2 for patients with HIV; median difference, −24.53; 95.32% CI, −50.62 to −3.13; P = .01; and corneal nerve fiber length: 28.4 mm/mm2 for the controls vs 21.9 mm/mm2 for patients with HIV; median difference, −5.24; 95.09% CI, −8.83 to −1.38; P = .001). Tortuosity coefficient was increased in patients with HIV compared with controls (16.44 vs 13.95; median difference, 2.34; 95.09% CI, 0.314.65; P = .03) and in patients with HIV-SN compared with those without SN (17.84 vs 14.18; median difference, 4.32; 95.92% CI, 0.68-9.23; P = .01). (Tortuosity coefficient is independent of nerve fiber orientation; a straight nerve equals a tortuosity coefficient of zero and increases with increasing tortuosity.14) No differences were identified in corneal LC density (19.84 cells/mm2 for the controls vs 41.43 cells/mm2 for patients with HIV; median [IQR] difference, 9.38; 95% CI, −12.51 to 26.34; P = .53) or between patients with HIV without SN and those with HIV-SN (54.17 cells/mm2 vs 28.13 cells/mm2; median difference, −25.15; 95.92% CI, −43.75 to 0.42) (Figure 1 and Figure 2 and eTables 1 and 2 in the Supplement). jamaophthalmology.com
12 (86)
NA
Abbreviations: BPI, Brief Pain Inventory; CHANT, Clinical HIV-Associated Neuropathy Tool; DN4-i, Douleur-Neuropathique 4 interview; HIV, human immunodeficiency virus; HIV–No SN, patients with HIV but no neuropathy; HIV-SN, patients with HIV-associated sensory neuropathy; NA, not applicable because the group includes healthy controls only.
Adjustment for Covariates Using corneal measures as dependent factors and group (control, HIV without SN, and HIV-SN) as an independent variable, we found no significant correlation between corneal measures and covariates of age and duration of HIV infection.
Correlations There was a significant correlation between tortuosity coefficient with Brief Pain Inventory severity (r = 0.628; P = .003) and interference scores (r = 0.650; P = .002). There was no correlation between corneal nerve fiber measures and duration of HIV, age, duration of treatment with antiretroviral medication, most recent or nadir CD4 count, or peak viral load.
Discussion Neuropathy is a frequent complication of HIV infection and its treatment, affecting 32% to 67% of patients in 1 study.1 We show that IVCCM can quantify small nerve fiber damage in patients with HIV, particularly patients with HIV-SN. Corneal nerve fiber density was reduced in patients with HIV without neuropathy, indicating subclinical small nerve fiber damage. Patients with HIV-SN demonstrated reduced corneal nerve fiber density and increased tortuosity with more branching. This reduction in corneal nerve fiber measures replicates the findings in simian immunodeficiency virus–infected macaques8 and in patients with HIV,9 diabetic neuropathy,4 CharcotMarie-Tooth type 1A,5 chemotherapy-induced peripheral neuropathy, 6 chronic inflammator y demyelinating polyneuropathy,7 and amyloid neuropathy.8 Langerhans cells modulate the transmission of HIV 11 and may be increased because of anterior segment pathologic changes causing corneal nerve damage.15 Our study (Reprinted) JAMA Ophthalmology Published online June 8, 2017
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Research Brief Report
Corneal Confocal Microscopy Use to Evaluate Small Nerve Fibers in Patients With HIV
Figure 1. Comparison of Corneal Nerve Fiber Measures and Langerhans Cell (LC) Density Between Healthy Controls and Patients With Human Immunodeficiency Virus (HIV), With and Without Neuropathy A Corneal nerve fiber density
B
a,b
d c
30
40
20
0 Controls
HIV−No SN
HIV−SN
Controls
HIV−SN
40
Tortuosity Coefficient
150
CNBD, No./mm2
HIV−No SN
D Tortuosity coefficient
Corneal nerve branch density 200
100
50
0
30 e f
20
10
0 Controls
E
20
10
0
C
Corneal nerve fiber length 40
CNFL, mm/mm2
CNFD, Fibers/mm2
60
HIV−No SN
HIV−SN
Controls
HIV−No SN
HIV−SN
Corneal LC density
Corneal LC Density, Cells/mm2
150
Pairwise comparison after Benjamini-Hochberg correction (significance, P < .017) showed differences between patients with sensory neuropathy (HIV-SN) and patients without sensory neuropathy (HIV–no SN) for the tortuosity coefficient (P = .01). The corneal nerve fiber density (CNFD) did not reach this level of significance (P = .03). CNBD indicates corneal nerve branch density; CNFL, corneal nerve fiber length. a
P < .001, controls vs patients with HIV-SN.
b
P = .03, controls vs patients with HIV–no SN.
c
P = .03, patients with HIV–no SN vs HIV-SN.
d
P = .017, controls vs patients with HIV-SN.
e
P = .003, controls vs patients with HIV-SN.
f
P = .01, patients with HIV–no SN vs patients with HIV-SN.
100
50
0 Controls
HIV−No SN
HIV−SN
Figure 2. Examples of In Vivo Corneal Confocal Microscopy (IVCCM) Images in Control Participant and Patient Groups A Healthy control
B
Patient with HIV−no SN
C
Patient with HIV-SN
A, An IVCCM image of a healthy control. B, An IVCCM image of a patient with HIV but no sensory neuropathy (HIV–no SN). C, An IVCCM image of a patient with sensory neuropathy (HIV-SN) showing a progressive reduction in corneal nerve fiber density and an increase in corneal nerve tortuosity. E4
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Corneal Confocal Microscopy Use to Evaluate Small Nerve Fibers in Patients With HIV
Brief Report Research
demonstrated no change in corneal LC density in patients with HIV, and a recent study of patients with stable CD4 counts showed a low inc idence of anterior segment abnormality.16
functional loss given that intraepidermal nerve fiber density correlates with quantitative sensory testing in HIV.2
Limitations
Conclusions
In healthy controls, advancing age correlates with decreasing corneal nerve fiber density and corneal nerve fiber length and an increasing tortuosity coefficient.17 Although patients with HIV-SN were slightly older than those without HIV-SN, no significant effect of age was demonstrated. Future studies, using esthesiometry, should relate corneal nerve fiber loss with corneal sensation
Corneal confocal microscopy could act as a rapid, noninvasive ophthalmic imaging technique to identify nerve damage in patients with HIV. Larger longitudinal studies are required to establish corneal confocal microscopy as a surrogate end point for diagnosing and monitoring small fiber neuropathy in patients with HIV.
ARTICLE INFORMATION Accepted for Publication: April 1, 2017. Published Online: June 8, 2017. doi:10.1001/jamaophthalmol.2017.1703 Author Affiliations: Imperial College London, London, England (Kemp, Rice); Faculty of Medicine, Weill CornellMedicine-Qatar,Ar-Rayyan,Qatar(Petropoulos, Malik); BG Universitätsklinikum Bergmannsheil GmbH, Ruhr University, Bochum, Germany (Vollert, Maier, Strum); Department of Ophthalmology, University Eye Hospital, Dusseldorf, Germany (Schargus); National Institute for Health Research Biomedical Research Centre, Moorfields Eye Hospital NHS Foundation Trust, London, England (Peto, Hau, Chopra); University College London Institute of Ophthalmology, London, England (Peto, Hau, Chopra); Queen’s University Belfast, Belfast, Northern Ireland (Peto); Institute of Cardiovascular Sciences, Cardiac Centre, School of Medicine, University of Manchester, Manchester, England (Malik). Author Contributions: Drs Kemp and Petropoulos had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Rice, Schargus, Peto, Hau, Malik, Maier, Strum. Acquisition,analysis,orinterpretationofdata:Allauthors. Drafting of the manuscript: Kemp, Petropoulos, Malik. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: Kemp, Petropoulos, Vollert. Obtained funding: Rice, Maier, Peto, Schargus. Administrative, technical, or material support: Kemp, Schargus, Strum, Chopra, Vollert, Hau. Study supervision: Rice, Maier, Schargus, Malik, Peto. Image analysis: Petropoulos. Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Rice reported undertaking paid consultancy and advisory board work for Imperial College Consultants, whose clients in the past 36 months included Spinifex Pharmaceuticals, Abide, Astellas, Neusentis, Merck, Medivir, Mitsubishi, Aquilas, Asahi Kasei, Galapagos, Toray, Relmada, Novartis, and Orion; owning share options in Spinifex from which he accrued personal benefit when it was acquired by Novartis in 2015 and from which he may receive milestone payments in the future; and being coinventor on this patent: Okuse K, Pristera A, inventors; Imperial Limitations Limited, assignee. Methods of treating pain by inhibition of VGF activity. European patent application EP13702262.0/WO2013110945. Approved July 25, 2014. Mr Vollert reported receiving personal fees from Mundipharma. Dr Maier reported receiving
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grants and personal fees from Mundipharma, Astellas, Grünenthal, Epionics, and AstraZeneca. No other disclosures were reported. Funding/Support: The EuroPain project is a publicprivate partnership and has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement No. 115007, resources for which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/20072013) and European Federation of Pharmaceutical Industries and Associations companies in kind contribution. Dr Kemp was also supported by the NeuroPain (European Union’s Seventh Framework Programme grant) No. HEALTH F2-2013-602891. Dr Peto was funded by the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and the University College London Institute of Ophthalmology. Insurance for this study was sponsored by the Imperial College London Joint Research Office. Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. REFERENCES 1. Ellis RJ, Rosario D, Clifford DB, et al; CHARTER Study Group. Continued high prevalence and adverse clinical impact of human immunodeficiency virus–associated sensory neuropathy in the era of combination antiretroviral therapy: the CHARTER Study. Arch Neurol. 2010;67(5):552-558. 2. Phillips TJ, Brown M, Ramirez JD, et al. Sensory, psychological, and metabolic dysfunction in HIV-associated peripheral neuropathy: a cross-sectional deep profiling study. Pain. 2014; 155(9):1846-1860. 3. Polydefkis M, Yiannoutsos CT, Cohen BA, et al. Reduced intraepidermal nerve fiber density in HIV-associated sensory neuropathy. Neurology. 2002;58(1):115-119. 4. Chen X, Graham J, Dabbah MA, et al. Small nerve fiber quantification in the diagnosis of diabetic sensorimotor polyneuropathy: comparing corneal confocal microscopy with intraepidermal nerve fiber density. Diabetes Care. 2015;38(6):1138-1144. 5. Tavakoli M, Marshall A, Banka S, et al. Corneal confocal microscopy detects small fiber neuropathy in Charcot-Marie-Tooth disease type 1A patients. Muscle Nerve. 2012;46(5):698-704. 6. Ferdousi M, Azmi S, Petropoulos IN, et al. Corneal confocal microscopy detects small fibre neuropathy in patients with upper gastrointestinal cancer and nerve regeneration in chemotherapy
induced peripheral neuropathy. PLoS One. 2015;10 (10):e0139394. 7. Stettner M, Hinrichs L, Guthoff R, et al. Corneal confocal microscopy in chronic inflammatory demyelinating polyneuropathy. Ann Clin Transl Neurol. 2015;3(2):88-100. 8. Rousseau A, Cauquil C, Dupas B, et al. Potential role of in vivo confocal microscopy for imaging corneal nerves in transthyretin familial amyloid polyneuropathy. JAMA Ophthalmol. 2016;134(9): 983-989. 9. Dorsey JL, Mangus LM, Oakley JD, et al. Loss of corneal sensory nerve fibers in SIV-infected macaques: an alternate approach to investigate HIV-induced PNS damage. Am J Pathol. 2014;184 (6):1652-1659. 10. Sabato L, Bertazzi L, Pellegrini M, Luccarelli S, Meroni L, Staurenghi G. In vivo confocal microscopy of corneal nerve fibers in HIV+ patients [ARVO abstract 2804]. Invest Opthalmol Vis Sci. 2008;49 (13):2804. 11. Silva RB, Rocha LP, de Souza LR, et al. Morphological and immunological changes in the skin of autopsied women with AIDS. Virchows Arch. 2012;461(4):449-455. 12. World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013;310(20):2191-2194. doi:10.1001/jama.2013.281053 13. Woldeamanuel YW, Kamerman PR, Veliotes DG, et al. Development, validation, and field-testing of an instrument for clinical assessment of HIV-associated neuropathy and neuropathic pain in resource-restricted and large population study settings. PLoS One. 2016;11(10):e0164994. 14. Kallinikos P, Berhanu M, O’Donnell C, Boulton AJ, Efron N, Malik RA. Corneal nerve tortuosity in diabetic patients with neuropathy. Invest Ophthalmol Vis Sci. 2004;45(2):418-422. 15. Bucher F, Schneider C, Blau T, et al. Small-fiber neuropathy is associated with corneal nerve and dendritic cell alterations: an in vivo confocal microscopy study. Cornea. 2015;34(9):1114-1119. 16. Rutar T, Youm J, Porco T, et al. Ophthalmic manifestations of perinatally acquired HIV in a US cohort of long-term survivors. Br J Ophthalmol. 2015;99:650-653. 17. Tavakoli M, Ferdousi M, Petropoulos IN, et al. Normative values for corneal nerve morphology assessed using corneal confocal microscopy: a multinational normative data set. Diabetes Care. 2015;38(5):838-843.
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JAMA Ophthalmology | Brief Report
Use of Corneal Confocal Microscopy to Evaluate Small Nerve Fibers in Patients With Human Immunodeficiency Virus Harriet I. Kemp, BM BCh, FRCA; Ioannis N. Petropoulos, PhD; Andrew S. C. Rice, MD, FRCP; Jan Vollert, MSc; Christoph Maier, MD; Dietrich Strum, MD; Marc Schargus, MD, FEBO; Tunde Peto, MD, PhD; Scott Hau, MSc, BSc; Reena Chopra, BSc; Rayaz A. Malik, MB ChB, PhD Supplemental content IMPORTANCE Objective quantification of small fiber neuropathy in patients with human
immunodeficiency virus (HIV)–associated sensory neuropathy (HIV-SN) is difficult but needed for diagnosis and monitoring. In vivo corneal confocal microscopy (IVCCM) can quantify small fiber damage. OBJECTIVE To establish whether IVCCM can identify an abnormality in corneal nerve fibers and Langerhans cells in patients with and without HIV-SN. DESIGN, SETTING, AND PARTICIPANTS This prospective, cross-sectional cohort study was conducted between July 24, 2015, and September 17, 2015. Twenty patients who were HIV positive were recruited from adult outpatient clinics at Chelsea and Westminster Hospital NHS Foundation Trust in England. These patients underwent IVCCM at Moorfields Eye Hospital NHS Foundation Trust in London, England, and the IVCCM images were analyzed at Weill Cornell Medicine–Qatar in Ar-Rayyan, Qatar. Patients were given a structured clinical examination and completed validated symptom questionnaires and the Clinical HIV-Associated Neuropathy Tool. Results from patients with HIV were compared with the results of the age- and sex-matched healthy control participants (n = 20). All participants were classified into 3 groups: controls, patients with HIV but without SN, and patients with HIV-SN. MAIN OUTCOMES AND MEASURES Comparison of corneal nerve fiber density, corneal nerve branch density, corneal nerve fiber length, corneal nerve fiber tortuosity, and corneal Langerhans cell density between healthy controls and patients with HIV with and without SN. RESULTS All 40 participants were male, and most (ⱖ70%) self-identified as white. Of the 20 patients with HIV, 14 (70%) had HIV-SN. This group was older (mean [SD] age, 57.7 [7.75] years) than the group without HIV-SN (mean [SD] age, 42.3 [7.26] years) and the controls (mean [SD] age, 53.8 [10.5] years). Corneal nerve fiber density was reduced in patients with HIV compared with the controls (26.7/mm2 vs 38.6/mm2; median difference, −10.37; 95.09% CI, −14.27 to −6.25; P < .001) and in patients with HIV-SN compared with those without (25.8/mm2 vs 30.7/mm2; median difference, −4.53; 95.92% CI, −8.85 to −0.26; P = .03). Corneal nerve branch density and corneal nerve fiber length were reduced in patients with HIV, but no differences were identified between those with neuropathy and without neuropathy (corneal nerve branch density: 95.83/mm2 for the controls vs 72.37/mm2 for patients with HIV; median difference, −24.53; 95.32% CI, −50.62 to −3.13; P = .01; and corneal nerve fiber length: 28.4 mm/mm2 for the controls vs 21.9 mm/mm2 for patients with HIV; median difference, −5.24; 95.09% CI, −8.83 to −1.38; P = .001). Tortuosity coefficient was increased in patients with HIV compared with controls (16.44 vs 13.95; median difference, 2.34; 95.09% CI, 0.31 to 4.65; P = .03) and in those with HIV-SN compared with those without (17.84 vs 14.18; median difference, 4.32; 95.92% CI, 0.68-9.23; P = .01). No differences were identified in corneal Langerhans cell density (19.84 cells/mm2 for the controls vs 41.43 cells/mm2 for patients with HIV; median difference, 9.38; 95% CI, −12.51 to 26.34; P = .53). CONCLUSIONS AND RELEVANCE In vivo corneal confocal microscopy could be used in the
assessment of HIV-SN, but larger studies are required to confirm this finding. JAMA Ophthalmol. doi:10.1001/jamaophthalmol.2017.1703 Published online June 8, 2017.
Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Author: Rayaz A. Malik, MB ChB, PhD, Institute of Cardiovascular Sciences, Cardiac Centre, School of Medicine, University of Manchester, 46 Grafton St, Manchester M13 9NT, England ([email protected]).
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Research Brief Report
Corneal Confocal Microscopy Use to Evaluate Small Nerve Fibers in Patients With HIV
H
uman immunodeficiency virus–associated sensory neuropathy (HIV-SN) is one of the most frequent neurologic consequences of HIV infection and its treatment.1 Patients with HIV-SN present with pain and paraesthesia, and their quality of life and ability to work are substantially affected.2 Small fiber neuropathy can be accurately assessed by quantifying intraepidermal nerve fiber density but is invasive and does not easily facilitate longitudinal studies.3 In vivo corneal confocal microscopy (IVCCM) is a rapid, noninvasive ophthalmic imaging technique that can quantify small nerve fiber damage in diabetic neuropathy,4 hereditary Charcot-Marie-Tooth type 1A,5 chemotherapy-induced peripheral neuropathy,6 chronic inflammatory demyelinating polyneuropathy,7 and amyloid neuropathy.8 In a nonhuman primate model of HIV-SN, animals with the most severe disease showed a decrease in corneal nerve fiber density,9 and IVCCM has demonstrated a reduction in corneal nerve fiber length in HIV-positive patients.10 Epidermal Langerhans cell (LC) density is reduced in patients with HIV or AIDS,11 but in vivo assessment of corneal LC density has not been reported in HIV. This study undertook IVCCM to quantify corneal nerve fiber structure and LC density in patients with HIV.
Methods Study participants had a diagnosis of HIV, and the exclusion criteria included pregnancy, peripheral vascular disease, severe coincident psychiatric diagnosis, other causes of peripheral neuropathy, nystagmus, or corneal surgery. Patients were recruited from adult outpatient clinics at Chelsea and Westminster Hospital NHS Foundation Trust in the United Kingdom. Images from age- and sex-matched healthy control participants (volunteers) were collected using the same image acquisition protocol as that used for the patient groups.4 The study was conducted from July 24, 2015, to September 17, 2015. Image analysis was performed from April 7, 2016, to April 18, 2016. Ethical approval for this study was granted by the National Ethics Research Service. This study was part of the EuroPain project (http://imieuropain.org) and was conducted in accordance with the Declaration of Helsinki.12 All patients gave written informed consent to take part in the study.
Classification All participants were classified into 3 groups: controls, patients with HIV but without HIV-SN, and patients with HIV-SN. (The presence of 1 sign and at least 1 symptom from the validated Clinical HIV-Associated Neuropathy Assessment Tool indicated neuropathy.13) Patients with HIV underwent a structured clinical examination and completed the Douleur Neuropathique 4 interview (DN4-i; the score range was 0-7, and a DN4-i score higher than 3 was classified as painful neuropathy) as well as the Brief Pain Inventory (the severity range was 0-10 [0.0-7.0 for our patient cohort] with a pain interference scale of 0-10 [1.29-8.57 for our patient cohort]).
Image Acquisition and Quantification Participants underwent IVCCM at Moorfields Eye Hospital NHS Foundation Trust in London, England, and IVCCM images were E2
Key Points Question Can in vivo corneal confocal microscopy be used to assess changes in corneal nerve fiber structure and Langerhans cell density in patients with human immunodeficiency virus (HIV)–associated sensory neuropathy? Findings In this cohort study of 20 patients with HIV, a decrease in corneal nerve fiber density and an increase in corneal nerve tortuosity were observed in patients with HIV-associated sensory neuropathy when compared with patients without neuropathy, with no change in Langerhans cell density. Meaning In vivo corneal confocal microscopy is a noninvasive ophthalmic imaging technique that could aid in the diagnosis and longitudinal monitoring of patients with HIV-associated sensory neuropathy.
analyzed at Weill Cornell Medicine–Qatar in Ar-Rayyan, Qatar.4 Images were acquired using a corneal confocal microscope (Heidelberg Retinal Tomography and Rostock Corneal Module; Heidelberg Engineering). Six images from the right and left eyes were selected for analysis and manually quantified by a single experienced examiner (I.N.P.) using CCMetrics software (M. A. Dabbah, University of Manchester). Corneal nerve fiber density (number per square millimeter), corneal nerve branch density (number per square millimeter), corneal nerve fiber length (in millimeters per square millimeter), corneal nerve fiber tortuosity, and corneal LC density (in cells per square millimeter) were quantified.4 Patients did not undergo any assessment of corneal sensation.
Statistical Analysis A power calculation using SigmaPlot, version 10.0 (Systat) of data from patients with diabetic polyneuropathy revealed a sample size of at least 6 participants per group to identify a difference in corneal nerve fiber length (mean [SD] difference, 1.16 [0.50] mm/mm2; α = 0.05; β = 0.8) and a sample size of 4 participants to identify a difference in corneal LC density (mean [SD] difference, 8.01 [2.7] cells/mm2; α = 0.05; β = 0.8). Statistical analysis was performed using SPSS, version 22.0 (IBM). Data are presented as mean (SD) or median (interquartile range [IQR]). Corneal nerve measures and corneal LC density in patients with HIV were compared with controls using the Wilcoxon rank sum test. The control and HIV groups (HIV without SN, HIV-SN) were compared using the Kruskal-Wallis test for continuous nonparametric data (with post hoc testing for pairwise comparisons). Median differences are reported using a Hodges-Lehmann estimate; because nonparametric data rely on ranks, exact 95% CIs were not always possible (exact CIs stated). To reduce the chance of a type I error, a false detection rate correction was made to the P value using the Benjamini-Hochberg method. Spearman rank correlation was used between corneal nerve fiber measures and symptom severity scores.
Results Twenty male patients with HIV were matched by age and sex to 20 healthy controls (Table). All 40 participants were male,
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Corneal Confocal Microscopy Use to Evaluate Small Nerve Fibers in Patients With HIV
Brief Report Research
Table. Demographics for Healthy Control Participants, Patients With HIV–No SN, and Patients With HIV-SN HIV–No SN (n = 6)
Characteristic Age, mean (SD), y
HIV-SN (n = 14)
42.3 (7.26)
Race/ethnicity, white, No. (%)
5 (83)
Healthy Controls (n = 20)
57.7 (7.75) 13 (93)
53.8 (10.5) 14 (70)
Height, mean (SD), cm
174.2 (4.96)
174.0 (7.95)
167.9 (11.19)
Weight, mean (SD), kg
70.5 (7.53)
74.9 (13.1)
81.6 (14.63)
HIV
12.4 (10.81)
25.3 (4.68)
NA
Medication
10.8 (9.26)
20.7 (3.75)
NA
Duration, mean (SD), y
CD4 count, mean (SD), cells/mm3 Most recent
797.0 (285.51)
685.4 (370.55)
Not measured
Nadir
192.8 (141.74)
154.5 (152.18)
Not measured
Peak viral load, mean (SD), copies/mL
93 137.2 (84 721.61) 59 281.5 (91 369.90) Not measured
CHANT, mean (SD) Symptom score
0.2 (0.41)
Sign score
0.0 (0.0)
1.2 (0.43)
NA
0.3 (0.82)
4.4 (1.40)
NA
DN4-i score, mean (SD)
1.8 (0.43)
NA
BPI, mean (SD) Severity score
0.04 (0.10)
3.5 (3.13)
NA
Interference score
1.3 (0.0)
4.26 (2.33)
NA
Exposure to neurotoxic antiretroviral agents, No. (%)
2 (33)
and most (≥70%) self-identified as white. Patients with HIV-SN were older (mean [SD] age, 57.7 [7.75] years) than those without HIV-SN (mean [SD] age, 42.3 [7.26] years) and the healthy controls (mean [SD] age, 53.8 [10.5] years). Six patients (30%) had no neuropathy (HIV–no SN) and 14 (70%) had neuropathy (HIV-SN). Corneal nerve fiber density was lower in patients with HIV than in the controls (26.7/mm2 vs 38.6/mm2; median difference, −10.37; 95.09% CI, −14.27 to −6.25; P < .001) as well as in patients with HIV-SN than those with HIV–no SN (25.8/mm2 vs 30.7/mm2; median difference, −4.53; 95.92% CI, −8.85 to −0.26; P = .03). Corneal nerve branch density and corneal nerve fiber length were lower in patients with HIV, and no difference was identified between those with neuropathy and those without neuropathy (corneal nerve branch density: 95.83/mm2 for the controls vs 72.37/mm2 for patients with HIV; median difference, −24.53; 95.32% CI, −50.62 to −3.13; P = .01; and corneal nerve fiber length: 28.4 mm/mm2 for the controls vs 21.9 mm/mm2 for patients with HIV; median difference, −5.24; 95.09% CI, −8.83 to −1.38; P = .001). Tortuosity coefficient was increased in patients with HIV compared with controls (16.44 vs 13.95; median difference, 2.34; 95.09% CI, 0.314.65; P = .03) and in patients with HIV-SN compared with those without SN (17.84 vs 14.18; median difference, 4.32; 95.92% CI, 0.68-9.23; P = .01). (Tortuosity coefficient is independent of nerve fiber orientation; a straight nerve equals a tortuosity coefficient of zero and increases with increasing tortuosity.14) No differences were identified in corneal LC density (19.84 cells/mm2 for the controls vs 41.43 cells/mm2 for patients with HIV; median [IQR] difference, 9.38; 95% CI, −12.51 to 26.34; P = .53) or between patients with HIV without SN and those with HIV-SN (54.17 cells/mm2 vs 28.13 cells/mm2; median difference, −25.15; 95.92% CI, −43.75 to 0.42) (Figure 1 and Figure 2 and eTables 1 and 2 in the Supplement). jamaophthalmology.com
12 (86)
NA
Abbreviations: BPI, Brief Pain Inventory; CHANT, Clinical HIV-Associated Neuropathy Tool; DN4-i, Douleur-Neuropathique 4 interview; HIV, human immunodeficiency virus; HIV–No SN, patients with HIV but no neuropathy; HIV-SN, patients with HIV-associated sensory neuropathy; NA, not applicable because the group includes healthy controls only.
Adjustment for Covariates Using corneal measures as dependent factors and group (control, HIV without SN, and HIV-SN) as an independent variable, we found no significant correlation between corneal measures and covariates of age and duration of HIV infection.
Correlations There was a significant correlation between tortuosity coefficient with Brief Pain Inventory severity (r = 0.628; P = .003) and interference scores (r = 0.650; P = .002). There was no correlation between corneal nerve fiber measures and duration of HIV, age, duration of treatment with antiretroviral medication, most recent or nadir CD4 count, or peak viral load.
Discussion Neuropathy is a frequent complication of HIV infection and its treatment, affecting 32% to 67% of patients in 1 study.1 We show that IVCCM can quantify small nerve fiber damage in patients with HIV, particularly patients with HIV-SN. Corneal nerve fiber density was reduced in patients with HIV without neuropathy, indicating subclinical small nerve fiber damage. Patients with HIV-SN demonstrated reduced corneal nerve fiber density and increased tortuosity with more branching. This reduction in corneal nerve fiber measures replicates the findings in simian immunodeficiency virus–infected macaques8 and in patients with HIV,9 diabetic neuropathy,4 CharcotMarie-Tooth type 1A,5 chemotherapy-induced peripheral neuropathy, 6 chronic inflammator y demyelinating polyneuropathy,7 and amyloid neuropathy.8 Langerhans cells modulate the transmission of HIV 11 and may be increased because of anterior segment pathologic changes causing corneal nerve damage.15 Our study (Reprinted) JAMA Ophthalmology Published online June 8, 2017
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Research Brief Report
Corneal Confocal Microscopy Use to Evaluate Small Nerve Fibers in Patients With HIV
Figure 1. Comparison of Corneal Nerve Fiber Measures and Langerhans Cell (LC) Density Between Healthy Controls and Patients With Human Immunodeficiency Virus (HIV), With and Without Neuropathy A Corneal nerve fiber density
B
a,b
d c
30
40
20
0 Controls
HIV−No SN
HIV−SN
Controls
HIV−SN
40
Tortuosity Coefficient
150
CNBD, No./mm2
HIV−No SN
D Tortuosity coefficient
Corneal nerve branch density 200
100
50
0
30 e f
20
10
0 Controls
E
20
10
0
C
Corneal nerve fiber length 40
CNFL, mm/mm2
CNFD, Fibers/mm2
60
HIV−No SN
HIV−SN
Controls
HIV−No SN
HIV−SN
Corneal LC density
Corneal LC Density, Cells/mm2
150
Pairwise comparison after Benjamini-Hochberg correction (significance, P < .017) showed differences between patients with sensory neuropathy (HIV-SN) and patients without sensory neuropathy (HIV–no SN) for the tortuosity coefficient (P = .01). The corneal nerve fiber density (CNFD) did not reach this level of significance (P = .03). CNBD indicates corneal nerve branch density; CNFL, corneal nerve fiber length. a
P < .001, controls vs patients with HIV-SN.
b
P = .03, controls vs patients with HIV–no SN.
c
P = .03, patients with HIV–no SN vs HIV-SN.
d
P = .017, controls vs patients with HIV-SN.
e
P = .003, controls vs patients with HIV-SN.
f
P = .01, patients with HIV–no SN vs patients with HIV-SN.
100
50
0 Controls
HIV−No SN
HIV−SN
Figure 2. Examples of In Vivo Corneal Confocal Microscopy (IVCCM) Images in Control Participant and Patient Groups A Healthy control
B
Patient with HIV−no SN
C
Patient with HIV-SN
A, An IVCCM image of a healthy control. B, An IVCCM image of a patient with HIV but no sensory neuropathy (HIV–no SN). C, An IVCCM image of a patient with sensory neuropathy (HIV-SN) showing a progressive reduction in corneal nerve fiber density and an increase in corneal nerve tortuosity. E4
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Corneal Confocal Microscopy Use to Evaluate Small Nerve Fibers in Patients With HIV
Brief Report Research
demonstrated no change in corneal LC density in patients with HIV, and a recent study of patients with stable CD4 counts showed a low inc idence of anterior segment abnormality.16
functional loss given that intraepidermal nerve fiber density correlates with quantitative sensory testing in HIV.2
Limitations
Conclusions
In healthy controls, advancing age correlates with decreasing corneal nerve fiber density and corneal nerve fiber length and an increasing tortuosity coefficient.17 Although patients with HIV-SN were slightly older than those without HIV-SN, no significant effect of age was demonstrated. Future studies, using esthesiometry, should relate corneal nerve fiber loss with corneal sensation
Corneal confocal microscopy could act as a rapid, noninvasive ophthalmic imaging technique to identify nerve damage in patients with HIV. Larger longitudinal studies are required to establish corneal confocal microscopy as a surrogate end point for diagnosing and monitoring small fiber neuropathy in patients with HIV.
ARTICLE INFORMATION Accepted for Publication: April 1, 2017. Published Online: June 8, 2017. doi:10.1001/jamaophthalmol.2017.1703 Author Affiliations: Imperial College London, London, England (Kemp, Rice); Faculty of Medicine, Weill CornellMedicine-Qatar,Ar-Rayyan,Qatar(Petropoulos, Malik); BG Universitätsklinikum Bergmannsheil GmbH, Ruhr University, Bochum, Germany (Vollert, Maier, Strum); Department of Ophthalmology, University Eye Hospital, Dusseldorf, Germany (Schargus); National Institute for Health Research Biomedical Research Centre, Moorfields Eye Hospital NHS Foundation Trust, London, England (Peto, Hau, Chopra); University College London Institute of Ophthalmology, London, England (Peto, Hau, Chopra); Queen’s University Belfast, Belfast, Northern Ireland (Peto); Institute of Cardiovascular Sciences, Cardiac Centre, School of Medicine, University of Manchester, Manchester, England (Malik). Author Contributions: Drs Kemp and Petropoulos had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Rice, Schargus, Peto, Hau, Malik, Maier, Strum. Acquisition,analysis,orinterpretationofdata:Allauthors. Drafting of the manuscript: Kemp, Petropoulos, Malik. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: Kemp, Petropoulos, Vollert. Obtained funding: Rice, Maier, Peto, Schargus. Administrative, technical, or material support: Kemp, Schargus, Strum, Chopra, Vollert, Hau. Study supervision: Rice, Maier, Schargus, Malik, Peto. Image analysis: Petropoulos. Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Rice reported undertaking paid consultancy and advisory board work for Imperial College Consultants, whose clients in the past 36 months included Spinifex Pharmaceuticals, Abide, Astellas, Neusentis, Merck, Medivir, Mitsubishi, Aquilas, Asahi Kasei, Galapagos, Toray, Relmada, Novartis, and Orion; owning share options in Spinifex from which he accrued personal benefit when it was acquired by Novartis in 2015 and from which he may receive milestone payments in the future; and being coinventor on this patent: Okuse K, Pristera A, inventors; Imperial Limitations Limited, assignee. Methods of treating pain by inhibition of VGF activity. European patent application EP13702262.0/WO2013110945. Approved July 25, 2014. Mr Vollert reported receiving personal fees from Mundipharma. Dr Maier reported receiving
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grants and personal fees from Mundipharma, Astellas, Grünenthal, Epionics, and AstraZeneca. No other disclosures were reported. Funding/Support: The EuroPain project is a publicprivate partnership and has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement No. 115007, resources for which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/20072013) and European Federation of Pharmaceutical Industries and Associations companies in kind contribution. Dr Kemp was also supported by the NeuroPain (European Union’s Seventh Framework Programme grant) No. HEALTH F2-2013-602891. Dr Peto was funded by the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and the University College London Institute of Ophthalmology. Insurance for this study was sponsored by the Imperial College London Joint Research Office. Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. REFERENCES 1. Ellis RJ, Rosario D, Clifford DB, et al; CHARTER Study Group. Continued high prevalence and adverse clinical impact of human immunodeficiency virus–associated sensory neuropathy in the era of combination antiretroviral therapy: the CHARTER Study. Arch Neurol. 2010;67(5):552-558. 2. Phillips TJ, Brown M, Ramirez JD, et al. Sensory, psychological, and metabolic dysfunction in HIV-associated peripheral neuropathy: a cross-sectional deep profiling study. Pain. 2014; 155(9):1846-1860. 3. Polydefkis M, Yiannoutsos CT, Cohen BA, et al. Reduced intraepidermal nerve fiber density in HIV-associated sensory neuropathy. Neurology. 2002;58(1):115-119. 4. Chen X, Graham J, Dabbah MA, et al. Small nerve fiber quantification in the diagnosis of diabetic sensorimotor polyneuropathy: comparing corneal confocal microscopy with intraepidermal nerve fiber density. Diabetes Care. 2015;38(6):1138-1144. 5. Tavakoli M, Marshall A, Banka S, et al. Corneal confocal microscopy detects small fiber neuropathy in Charcot-Marie-Tooth disease type 1A patients. Muscle Nerve. 2012;46(5):698-704. 6. Ferdousi M, Azmi S, Petropoulos IN, et al. Corneal confocal microscopy detects small fibre neuropathy in patients with upper gastrointestinal cancer and nerve regeneration in chemotherapy
induced peripheral neuropathy. PLoS One. 2015;10 (10):e0139394. 7. Stettner M, Hinrichs L, Guthoff R, et al. Corneal confocal microscopy in chronic inflammatory demyelinating polyneuropathy. Ann Clin Transl Neurol. 2015;3(2):88-100. 8. Rousseau A, Cauquil C, Dupas B, et al. Potential role of in vivo confocal microscopy for imaging corneal nerves in transthyretin familial amyloid polyneuropathy. JAMA Ophthalmol. 2016;134(9): 983-989. 9. Dorsey JL, Mangus LM, Oakley JD, et al. Loss of corneal sensory nerve fibers in SIV-infected macaques: an alternate approach to investigate HIV-induced PNS damage. Am J Pathol. 2014;184 (6):1652-1659. 10. Sabato L, Bertazzi L, Pellegrini M, Luccarelli S, Meroni L, Staurenghi G. In vivo confocal microscopy of corneal nerve fibers in HIV+ patients [ARVO abstract 2804]. Invest Opthalmol Vis Sci. 2008;49 (13):2804. 11. Silva RB, Rocha LP, de Souza LR, et al. Morphological and immunological changes in the skin of autopsied women with AIDS. Virchows Arch. 2012;461(4):449-455. 12. World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013;310(20):2191-2194. doi:10.1001/jama.2013.281053 13. Woldeamanuel YW, Kamerman PR, Veliotes DG, et al. Development, validation, and field-testing of an instrument for clinical assessment of HIV-associated neuropathy and neuropathic pain in resource-restricted and large population study settings. PLoS One. 2016;11(10):e0164994. 14. Kallinikos P, Berhanu M, O’Donnell C, Boulton AJ, Efron N, Malik RA. Corneal nerve tortuosity in diabetic patients with neuropathy. Invest Ophthalmol Vis Sci. 2004;45(2):418-422. 15. Bucher F, Schneider C, Blau T, et al. Small-fiber neuropathy is associated with corneal nerve and dendritic cell alterations: an in vivo confocal microscopy study. Cornea. 2015;34(9):1114-1119. 16. Rutar T, Youm J, Porco T, et al. Ophthalmic manifestations of perinatally acquired HIV in a US cohort of long-term survivors. Br J Ophthalmol. 2015;99:650-653. 17. Tavakoli M, Ferdousi M, Petropoulos IN, et al. Normative values for corneal nerve morphology assessed using corneal confocal microscopy: a multinational normative data set. Diabetes Care. 2015;38(5):838-843.
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