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. 2026 Feb 23;21(2):e0342439. doi: 10.1371/journal.pone.0342439

Children with autism spectrum disorder and attention deficit hyperactivity disorder have evidence of sensory nerve degeneration

Hoda Gad 1, Abdulla AlObaidi 1, Madeeha Kamal 2, Saba F Elhag 2, Marian Aden 3, Adnan Khan 1, Areej Baraka 1, Srividya Bennabhaktula 1, Sara Joseph 1, Aisha Yaseen Alhamadi 4, Mohammed A Tolefat 4, Abdulla A Alhothi 3, Rayaz A Malik 1,5,*
Editor: Shivanand Kattimani6
PMCID: PMC12928439  PMID: 41729841

Abstract

An increasing body of evidence supports the role of altered sensory processing in autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD). This exploratory study undertook corneal confocal microscopy (CCM) to assess for differences in corneal nerve fiber density (CNFD), branch density (CNBD) and fiber length (CNFL) in relation to ASD severity in children with ASD+ADHD (n = 21), ASD (n = 6) and healthy controls (HC) (n = 15). CNBD was significantly lower in children with ASD+ADHD (P = 0.002) and ASD (P < 0.001) and CNFL was significantly lower in children with ASD (P = 0.02) compared to HC. However, CNFD (mean difference = 2.28, 95% CI [−5.84, 10.40], P > 0.99) CNBD (mean difference = 14.1, 95% CI [−8.22, 36.4], p = 0.37) and CNFL (mean difference = 2.46, 95% CI [−2.28, 7.20], p = 0.61) did not differ between ASD+ADHD and ASD. CNFD (P = 0.876), CNBD (P = 0.405) and CNFL (P = 0.606) did not correlate with the severity of ASD. Corneal confocal microscopy reveals sensory nerve degeneration in children with ASD+ADHD and ASD alone compared to controls. Larger studies integrating CCM with sensory and cognitive assessment are required to determine the utility of CCM as a clinical screening strategy for neurodegneration in ASD, ADHD and ASD-ADHD combined.

Introduction

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition defined by altered social communication and interaction with restricted and repetitive behaviour or interests. It can vary in its presentation, may be associated with anxiety and depression and shaped by age, gender, race, ethnicity, and developmental stage [1]. Attention deficit hyperactivity disorder (ADHD) is also a neurodevelopmental disorder characterized by persistent inattention, hyperactivity, and impulsivity that disrupts daily functioning [2]. Both ASD and ADHD have a high chance of co-occurrence with deficits in social communication and interaction, restricted, repetitive behaviour [3], inattention, hyperactivity, and impulsivity, respectively [4]. Most research has focused on brain-centric mechanisms in ASD and ADHD. However, there is increasing recognition of abnormal sensory responses in ASD [5–10] which relate to quantitative autism traits [7] such that they constitute one of the four sub-criteria under the restricted, repetitive behaviors domain in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) [11]. Sensory alterations have been documented using clinical instruments such as the Autism Diagnostic Interview-Revised (ADI-R) scale and sensory abnormalities may underlie agitation in ADHD or interpreted as atypical sensory behavior in ASD [12].

Participants with ASD have normal pain detection thresholds and pain tolerance, but report diminished subjective pain intensity and heightened sensitivity consistent with variability in nociceptive processing and cognitive pain modulation [13]. Hyperresponsiveness of the auditory domain has been shown in 43.8% of children with ASD with hyperresponsiveness, hyporesponsiveness, and sensory-seeking behavior in the visual and proprioceptive domains [8]. Intraepidermal nerve fibre density was reduced in 53.1% and thermal thresholds were elevated in 28% of adults with ASD [14].

Ophthalmologic abnormalities occur in 48.4% of patients with ASD and include hyperopia, astigmatism and myopia with strabismus in 15.4% [15]. Youth with ASD have an increased prevalence of amblyopia, anisometropia, astigmatism and hypermetropia [16]. Corneal confocal microscopy (CCM) is a rapid non-invasive ophthalmic imaging technique that has identified corneal nerve loss in diabetic [17] and other peripheral neuropathies [18], dementia [19], multiple sclerosis [20] epilepsy [20], Parkinson’s disease [21] and ASD [22].

A growing body of evidence shows altered sensory processing in ADHD [4,23–27]. In adults with ADHD, self-reported tactile sensitivity was associated with symptom severity, a lower tolerable threshold for electrical radial nerve stimulus, and greater reduction in cortical SEP amplitudes [4]. In a resting-state functional magnetic resonance imaging (rs-fMRI) study, adults with ADHD had an increased pattern of functional connectivity between sensory seed regions and the rest of the brain and decreased connectivity between sensory seeds and the default-mode network which was related to clinical severity of ADHD [25]. Individuals with higher levels of ADHD traits report hyper- and hypo-sensitivity, determined with the Glasgow Sensory Questionnaire (GSQ) and Adult ADHDH Self Report Scale (ASRS) [28].

Recent studies have shown peripheral [14] and corneal [22] nerve loss and correlations between sensory and social symptoms in individuals with ASD [29]. The aim of this pilot study was to assess for differences in sensory nerve degeneration between children with ADHD and ASD compared to ASD alone.

Methods

Participants and study design

This was a pilot study involving participants with ASD+ADHD (n = 21), ASD-alone (n = 6) and 15 healthy controls aged 7–18 years from Hamad Medical Corporation (HMC) and Shafallah Center for Persons with Disabilities, recruited between June 2021 to October 2024. Parents of eligible participants were informed of the study by pediatricians specializing in ASD and ADHD. Healthy controls (HC) were recruited from children attending the pediatric clinic for follow up of acute conditions, without neurodevelopmental or chronic medical conditions.

Participants had mild to moderate ASD, as CCM was not possible in non-verbal individuals with severe ASD. Participants with any other cause of neuropathy, malignancy, deficiency of B12 or folate, chronic renal failure, liver failure, connective tissue, or systemic disease (rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, systemic scleroderma, Raynaud phenomenon), previous corneal trauma or systemic disease affecting the cornea, and corneal surgery within 6 months of enrollment, were excluded. All participants provided written assent and parental informed consent, and the research adhered to the tenets of Declaration of Helsinki and was approved by the Weill Cornell Medicine-Qatar (WCM-Q) (19–00016) and Medical Research Center at HMC (MRC-01-20-761) Research Ethics Committees.

Autism diagnostic criteria

ASD and ADHD were diagnosed by consultant pediatricians with a specialist interest in behavioral pediatrics, ADHD and ASD, according to the Diagnostic and Statistical Manual of Mental Disorders – version 5 (DSM-5) [30]. Individuals exhibited restricted, repetitive patterns of behavior, interests, or activities, such as stereotyped movements or speech, insistence on sameness, highly restricted interests, or hyper-or hyporeactivity to sensory input. Individuals with ASD met at least 3 of the criteria of social communication deficits and 2 out of 4 of the criteria for repetitive behaviour. The diagnosis of ADHD was based on DSM-5, which captured inattention, hyperactivity and impulsivity [31]. We did not have access to standardized diagnostic tools or questionnaires and data on diagnosis was extracted from the electronic medical records.

Corneal confocal microscopy

Corneal confocal microscopy was undertaken using the Heidelberg Retina Tomograph III Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany). One eye was anaesthetized with 2 drops of Bausch & Lomb Minims® (Oxybuprocaine hydrochloride 0.4% w/v). A drop of hypotears gel (Carbomer 0.2% eye gel) was placed on the tip of the objective lens and a sterile disposable TomoCap was placed over the lens, allowing optical coupling of the objective lens to the cornea. We acquired a minimum of 6 images per eye to allow selection of 3 images of the central sub basal nerve plexus (SBNP) with the best quality based on an established protocol [32]. Corneal nerve fiber density (CNFD) (fibers/mm2) corneal nerve branch density (CNBD) (branches/mm2), and corneal nerve fiber length (CNFL) (mm/mm2) were quantified manually by HG using CCMetrics, who was blinded to the study group.

Statistical analysis

As this was a pilot study, no formal sample size calculation was performed. Statistical analyses were performed using IBM SPSS Statistics software Version 30 and P < 0.05 was considered statistically significant. Normality of the data was assessed using the Shapiro-Wilk test and Q-Q plots. As data were normally distributed, they were expressed as mean ± standard deviation. Comparison between participants with ASD+ADHD, ASD and HC was performed using ANOVA with Bonferroni adjustment. Point estimates using Eta-squared (η²) or Cohen’s d and 95% Confidence Intervals (CI) were reported as appropriate. Adjusted analyses were performed using ANCOVA, controlling for age as a confounding variable. Graph prism version 10 was used to create figures.

Results

Children with ASD had a DSM-5 score of 5.85 ± 1.26 and children with ASD+ADHD had a DSM-5 score of 5.92 ± 1.36. Age differed between ASD, ASD+ADHD and HC groups (15.0 ± 1.79 vs. 12.0 ± 3.21 vs. 14.1 ± 2.07, P = 0.02) (Table 1).

Table 1. Age, DSM-5 and corneal confocal microscopy metrics in patients with ASD, ASD+ADHD and HC.

Variables HC (n = 15) ASD-alone (n = 21) ASD+ADHD (n = 6) η² 95% CI P-value
Age (years) 14.13 ± 2.07 15.00 ± 1.79 12.00 ± 3.21 0.18 0.003–0.36 0.02
DSM-5 – 5.50 ± 0.84 5.92 ± 1.36 0.36 −0.56–1.26 0.45
CNFD (no./mm2) 32.36 ± 7.17 27.08 ± 4.17 29.36 ± 7.45 0.07 0.0–0.22 0.24
CNBD (no./mm2) 73.47 ± 16.01 35.42 ± 16.46 49.50 ± 21.83 0.36 0.11–0.52 <0.001*
CNFL (mm/mm2) 23.37 ± 4.21 17.56 ± 3.66 20.02 ± 4.12 0.21 0.01–0.38 0.01*
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HC: healthy control, ASD: autism spectrum disorder, ASD+ADHD: autism spectrum disorder with attention deficit hyperactivity disorder; CI: confidence intervals; DSM-5: Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition; CNFD: corneal nerve fiber density; CNBD: corneal nerve branch density; CNFL: corneal nerve fiber length. Bonferroni adjusment was used and P-value was significant if <0.016. Point estimate was reported as η²: Eta-squared for age, CNFD, CNBD and CNFL and Cohen’s d for DSM-5.

CCM was not possible in three children with ASD+ADHD. CNFD did not differ between children with ASD+ADHD, ASD and HC (P = 0.24) (Table 1, Figs 1A, 2). CNBD was significantly lower in children with ASD+ADHD (P = 0.002) and ASD (P < 0.001) compared to HC (Figs 1B, 2). CNFL was significantly lower in children with ASD (P = 0.02) (Figs 1C, 2) with a borderline reduction in those with ASD+ADHD (P = 0.06), compared to HC. After adjusting for age as a covariate, the overall group effect on CNFD was not statistically significant (F = 2.18, P = 0.13), while both CNBD (F = 10.24, P=<0.001) and CNFL (F = 5.05, P < 0.01) remained significantly different between groups. Among the CCM parameters, CNBD and CNFL showed the largest group differences and were significantly lower in children with ASD and ASD+ADHD compared to HC, while CNFD did not differ significantly between groups. There was no difference in CNFD (mean difference = 2.28, 95% CI [−5.84, 10.40], P > 0.99) CNBD (mean difference = 14.1, 95% CI [−8.22, 36.4], p = 0.37) and CNFL (mean difference = 2.46, 95% CI [−2.28, 7.20], p = 0.61) between children with ASD+ADHD and ASD. DSM-5 did not correlate with CNFD (P = 0.876), CNBD (P = 0.405) or CNFL (P = 0.606).

Fig 1. CNFD (A), CNBD (B) and CNFL (C) in children with ASD+ADHD, ASD and HC.

Fig 1

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HC: healthy control, ASD: autism spectrum disorder, ADHD: attention deficit hyperactivity disorder, CNFD: corneal nerve fiber density; CNBD: corneal nerve branch density; CNFL: corneal nerve fiber length.

Fig 2. CCM images in children with ASD+ADHD (A), ASD-alone (B) and healthy controls (C).

Fig 2

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Discussion

This study shows evidence of corneal nerve loss in children with ASD+ADHD and ASD, extending our previous findings in children with ASD [22]. It confirms the presence of small nerve fibre damage, as evidenced by a lower intraepidermal nerve fiber density (IENFD) and elevated warm detection thresholds in adults with ASD [14]. These findings add to the evidence supporting the utility of CCM in identifying neurodegeneration in diabetic neuropathy [17], chemotherapy induced peripheral neuropathy, chronic inflammatory demyelinating neuropathy and HIV-neuropathy [18], dementia [19] and multiple sclerosis [33].

ASD and ADHD combined may exacerbate morbidity [34], but did not show evidence of greater neurodegeneration, although the cohort size was small, and we lacked a group with ADHD alone. Furthermore, direct subgroup comparisons between children with ASD+ADHD and ASD revealed no differences in corneal nerve fiber parameters, although the directionality of the findings indicated higher CNBD and CNFL values in the ASD+ADHD group. The lack of difference between children with ASD+ADHD and ASD could be attributed to the difference in age between groups as older age has been associated with a decline in corneal nerve function [35] and corneal nerve fiber density [36]. Additionally, treatment with stimulants such as Methylphenedate (MPH) may improve neuroplasticity and has been shown to modulate sensory thresholds in children with ADHD [37]. Moreover, both ASD and ADHD exhibit substantial heterogeneity in hyper- and hypo-responsivity, which may obscure differences [38].

There was no association between corneal nerve loss and the severity of ASD or ASD+ADHD, possibly reflecting limitations of the DSM-5-based severity scale, which primarily captures social-communication difficulties and restricted/repetitive behaviors, rather than underlying sensory deficits [39,40]. Atypical sensory processing is a non-specific marker of multiple neurodevelopmental conditions, and in the multicEntric Longitudinal study of childrEN with ASD (ELENA) [41] there were no signficant differences between children with ASD, ADHD and ASD+ADHD [23]. In a diffusion tensor imaging (DTI) study comparing adults with ASD and ADHD, voxel-wise fractional anisotropy (FA) and radial diffusivity (RD) were equally abnormal in the corpus callosum and related to sensory symptoms [42]. Participants with ADHD, self-reported greater tactile sensitivity and ADHD symptom severity, a lower tolerable threshold for electrical radial nerve stimulus and greater reduction in cortical SEP amplitudes to tactile stimuli which correlated with ADHD symptom severity [4]. A recent study in adults with ADHD demonstrated an increased risk of developing dementia [43], and we have previously shown corneal nerve loss in participants with mild cognitive impairment and dementia [19]. Neurofilament light chain (NfL), a marker of neuroaxonal damage is increased in a range of peripheral and central neurodegenerative diseases [44] including dementia [45] and is also increased in children with ASD [46], but not in adults with ADHD [47]. Furthermore, methylphenidate may be neuroprotective as treatment of children with ADHD showed enhanced spontaneous neural activity in the nucleus accumbens [48], whilst in animal studies it reduced mechanical and cold hyperalgesia and allodynia [49].

Limitations of this study include the small sample size of the ASD group and unequal group sizes, which may limit the statistical power. Differences in age between the study groups could also confound group comparisons. Specifically, the effect size for CNFD was small, suggesting minimal difference, although CNBD and CNFL showed moderate effect sizes indicating detectable group differences. The lack of a group with ADHD limits our ability to determine whether corneal nerve loss is related to ASD or may also occur in ADHD. The lack of sensory testing and objective measures of ASD severity limits our ability to show an association with the severity of neurodegeneration. The lack of data on confounding factors such as concomitant medications, comorbidities, sleep disorders and ophthalmological disorders is also a limitation, as they may influence CCM outcomes.

These findings highlight the potential value of combining a comprehensive evaluation of motor coordination, visual–perceptual and sensory abilities alongside CCM into screening protocols to identify subtle neurodevelopmental difficulties in children with ASD or ASD and ADHD.

Supporting information

S1 File. Study raw data.

(XLSX)

pone.0342439.s001.xlsx (10.8KB, xlsx)

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was funded by the Autism Research Institute.

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Decision Letter 0

Shivanand Kattimani

9 Oct 2025

Dear Dr. Malik,

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

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Shivanand Kattimani

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: I Don't Know

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: This is an interesting and potentially important pilot study investigating corneal confocal microscopy (CCM) measures of sensory nerve degeneration in children with ASD, with and without ADHD. The topic is novel, as few studies have examined peripheral neurodegeneration in pediatric neurodevelopmental conditions. The manuscript is generally well written and clearly structured, but there are several major and minor issues that should be addressed to strengthen the scientific rigor and clarity.

________________________________________

Major Comments

1. The study included 21 children with ASD+ADHD, 6 with ASD only, and 15 healthy controls. The very small sample size, especially for the ASD-only group, raises concerns about statistical power and generalizability. The authors should acknowledge this limitation more explicitly and discuss the implications for interpretation. A priori or post hoc power analysis would be helpful to clarify what effect sizes could reasonably be detected with the available sample, and to aid in the interpretation of null findings.

2. The design lacks a group of children with ADHD only, which limits the ability to disentangle whether the observed differences are specific to ASD or also present in ADHD. This gap should be acknowledged not only in the limitations but also in the rationale for study design, with an explanation of why the study remains valuable as an exploratory investigation. Where possible, the authors may cite existing literature on sensory nerve changes in ADHD.

3. Beyond age and sex, it is unclear whether other potential confounding factors were considered, such as medication use, comorbid conditions, sleep problems, or ophthalmological disorders. These factors could plausibly affect corneal confocal microscopy (CCM) outcomes and should be addressed either in the methods or the discussion.

4. Statistical analysis details

The manuscript requires more detail on the statistical procedures. Specifically:

(1)Were normality and homogeneity of variances checked?

(2)Given the small sample sizes, what statistical tests were applied (e.g., t-test, Mann–Whitney, ANOVA)?

(3)Was any correction for multiple comparisons (e.g., Bonferroni or FDR) performed?

(4)Were effect sizes or confidence intervals reported?

These details are essential for evaluating the reliability of the results.

5. Additional methodological detail on CCM protocols is warranted. For example, how many images were acquired per participant, and what criteria were used to select images (random vs. best quality)? Were images analyzed by a single rater or multiple raters, and was intra-rater or inter-rater reliability assessed? These factors are important for assessing the validity and reproducibility of the findings.

6. While statistically significant reductions in CNBD and CNFL were reported, the clinical meaning of these changes is not well elaborated. The authors should discuss whether the degree of reduction is likely to have functional consequences, and how CCM could potentially be translated into clinical practice for ASD/ADHD populations.

7. The lack of correlation between ASD severity scores and nerve measures is an important negative finding. This deserves more in-depth discussion. Does this suggest that sensory nerve degeneration is independent of behavioral symptom severity, or could it be related to limitations of the DSM-5-based severity scale?

8. The conclusion states that ADHD does not confer additional neurodegeneration in children with ASD. Given the small subgroup sizes (n=6 ASD-only, n=21 ASD+ADHD), such conclusions may be premature. The authors should temper their interpretation and frame the results as preliminary.

________________________________________

Minor Comments

1. Abstract: The abstract is clear but should briefly state the small sample size and the absence of an ADHD-only group to frame the findings appropriately.

2. Typographical correction in group labels: In the tables, the group name “ASD-AHDH” appears, which seems to be a typographical error for “ASD-ADHD.” Please correct this and ensure consistent labeling of groups across all tables, figures, and text.

3. Methods: The description of statistical tests should be expanded (e.g., correction for multiple comparisons? effect size reporting?).

4. Consistency of group labeling: The group names are not entirely consistent between the tables and figures (e.g., “ASD only” vs. “Autism,” “ASD+ADHD” vs. “Comorbid group”). The authors should standardize terminology across the text, tables, and figures to avoid confusion for readers. Using uniform labels (e.g., “ASD only,” “ASD+ADHD,” “Controls”) throughout the manuscript is strongly recommended.

In the tables, the group name “ASD-AHDH” appears, which seems to be a typographical error for “ASD-ADHD.” Please correct this and ensure consistent labeling of groups across all tables, figures, and text.

5. Discussion: The discussion could better integrate existing literature on peripheral nerve abnormalities in ASD (e.g., Chien et al., 2020, Neurology) and ADHD-related sensory processing studies.

6. References: Ensure all references are updated and formatted consistently.

Reviewer #2: Major Comments

Introduction

- Lines 76–78: The phrasing is awkward, as ASD and ADHD are “bundled together” in a way that blurs their distinct definitions. This opening sentence should be rewritten to introduce each condition clearly before addressing their overlap.

- Lines 80–82: The statement about atypical sensory features being a “core diagnostic criterion in DSM-5” is not entirely correct. While sensory peculiarities are explicitly acknowledged in the DSM-5 criteria, they were already well established in diagnostic tools such as the ADI-R and other scales. I agree that sensory issues are central in ASD, but the nuance is missed here: they are often underestimated (e.g., agitation misattributed to ADHD rather than sensory seeking) or overestimated (any sensory atypicality being interpreted as ASD). This complexity should be better reflected.

- Line 84: Rather than describing individuals as hyposensitive to subjective pain intensity, it would be more accurate to state that they show particularities in pain perception, as both hyper- and hyposensitivity are documented.

- Lines 96–97: The claim of corneal nerve loss in ASD relies on a single study authored by the same group. This should be presented more cautiously, with balanced language, rather than as established fact.

- Overall: The introduction is underdeveloped. It introduces non-consensual concepts (e.g., peripheral sensory nerve involvement) but does not defend them sufficiently. The innovative potential of these hypotheses needs to be articulated more clearly in relation to existing research that remains predominantly brain-centric. This contextualization would help readers understand why this line of inquiry matters.

- Lines 105–106: The manuscript lacks a clear hypothesis. Presenting corneal confocal microscopy (CCM) results in isolation risks being purely descriptive. The authors should articulate how detecting corneal alterations could improve understanding of patient experience, symptoms, or clinical care.

Methods

- The description of participants should clarify whether the ASD group included children with intellectual disability, as this is highly relevant to interpretation.

- Greater transparency is needed about recruitment strategy, representativeness, and diagnostic validation.

Results

- The small sample sizes (ASD without ADHD: n=6) limit statistical validity. Reporting a non-significant p=0.08 as evidence of no difference between groups is misleading. With such underpowered comparisons, absence of significance cannot be taken as equivalence.

General Assessment

The study proposes a potentially innovative shift from brain-centric to peripheral mechanisms in ASD/ADHD, but the introduction is too thin, the hypothesis is not clearly posed, and the interpretation is overstated given the sample size and reliance on prior self-citations. The paper would be strengthened by:

1. Rewriting the introduction for clarity and nuance (particularly around diagnostic criteria and sensory features).

2. Explicitly stating a testable hypothesis with clinical or mechanistic relevance.

3. Tempering claims where evidence is preliminary.

4. Expanding on the rationale for corneal measures—what do they add to patient understanding or care?

Reviewer #3: The authors are correct to highlight the fact that the changes to diagnostic categorisation of autism in the DSM-5 in 2013 resulted in a new separate criterion relating to atypical/unusual responses to sensory stimuli. Furthermore, prior to DSM-5, it was not possible to diagnose autism if ADHD were indicated. With advances in the literature, the common co-occurring nature of these two conditions suggests clinical research should address both presentations in single diagnostic groups and those with multiple diagnoses. This pilot study might contribute to knowledge in this field.

1. The authors need to be clear from the outset that this is a pilot study.

2. The abstract requires greater clarity about the aims of the study rather than simply what was done.

3. Terminology is unclear, i.e., when authors refer to neurodegeneration, do they mean loss of functioning/deterioration or ophthalmologic abnormality? The former would lead me to assume the pilot study aims to review participants over time rather than comparing clinical and non-clinical groups. Cf p2 lines 47 and 56 and p3 line 96.

Methodology

1. The current study description is not adequate to understand the processes or participant selection and hence to replicate the study.

2. Please capitalise the names of the recruiting centres and explain their remit/role.

3. Furthermore, how were the healthy controls selected?

4. How many families were approached before the final sample was selected? Did anyone drop out?

5. The authors have not explained participant characteristics, including whether the children had an existing diagnosis and, if so, how this was ratified, such as brief assessment, questionnaires or tasks. For instance, in lines 80-81, the authors state ‘there is an increasing recognition of abnormal sensory responses in ASD …which relate to quantitative autism traits… such that it now constitutes a core diagnostic [criterion] in DSM-5…’

6. There is no description of validation of the autism diagnoses, how children were identified or recruited, and the opportunity was lost to investigate any quantitative traits, such as those identified via completion of questionnaires and establishing whether the sensory differences criterion was met for all the autistic children and young people.

7. Symptom severity for both autism and ADHD require operational definitions.

8. The pilot study was small, with little detail about the range of participants, including cognitive ability.

9. Whilst age differences were not statistically significant, the non-autistic children were in general older than the autistic children. A table summarising the age ranges for all groups would be helpful.

Diagnostic criteria

1. It is not clear from this section whether the children were assessed and diagnosed as part of this study or whether they were recruited via a clinic where they already had received a diagnosis.

2. It is not noted how they were assessed, and which, if any tools supported the assessment and diagnostic process. Did the researchers have access to the clinical diagnostic assessment details?

Corneal Confocal Microscopy

1. Who conducted this?

2. At the end of the paragraph, it is noted that the investigator was blind to the images but does not say who conducted the procedure.

Discussion

1. P7 line 196 - This is not my field of expertise, but do the results demonstrate ‘loss’ or difference/pathology between groups?

2. P8 line 202 – refer to ELENA cohort earlier in the paper or explain it here.

3. The authors only identify two limitations, including the importance of objective measures of severity – this, in my mind is significant. I would argue that the findings are limited, and it is unclear what the next steps might be or implications of future research or indeed clinical impact.

Typos

1. P3 line 82 criteria should read criterion

2. P5, line 121, capitalise the full name of the manual and ensure that throughout the manuscript it is written correctly

3. P6 line 163 correlate

4. P7 Table 1 typos ADHD rather than AHDH

5. P8 ‘additonal’ should read additional?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Decision Letter 1

Shivanand Kattimani

12 Nov 2025

Dear Dr. Malik,

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

Please submit your revised manuscript by Dec 27 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Shivanand Kattimani

Academic Editor

PLOS ONE

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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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Reviewers' comments:

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #3: I Don't Know

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: The revision effectively clarifies methodology, improves the discussion’s conceptual integration, and adopts a balanced interpretation of results. Minor linguistic polishing and the addition of a brief statement on the clinical relevance of sensory and motor assessments in future screening would enhance the paper’s translational impact. Below are some minor suggestions.

1. Abstract and Introduction: The introduction is concise and sets up the rationale well. However, the research objectives would benefit from a brief mention that this is a preliminary exploratory study aiming to inform future mechanistic investigations and clinical screening strategies.

2. Methods: The methodological transparency has improved, but minor editorial polishing is needed for sentence flow. Clarifying how “routine clinical care” diagnoses were confirmed (e.g., by pediatric neurologist or psychiatrist) would strengthen credibility.

3. Results: the presentation of the results could be strengthened by providing greater quantitative detail and statistical context to assist readers in interpreting the effect magnitude and reliability. Specifically:

(1) Group comparison values (e.g., mean ± SD, p-values, or effect sizes such as Cohen’s d or η²) should be explicitly reported in-text or summarized in a table. This will allow readers to gauge the robustness of the observed differences beyond descriptive trends.

(2) It would be valuable to indicate whether the observed corneal nerve loss differed significantly between the ASD and ASD+ADHD subgroups, given the study’s stated aim of exploring comorbid ADHD effects. Even if the difference did not reach significance, reporting the directionality and confidence intervals would increase transparency.

(3) The authors may consider briefly stating which specific CCM parameters (e.g., corneal nerve fiber density, length, branch density) contributed most strongly to the group differences.

(4) Including a visual representation (figure or boxplot) summarizing group distributions could substantially enhance clarity and reader engagement, particularly for an exploratory study with small sample sizes.

4. Discussion: The discussion could be further strengthened by adding a short clinical implication statement, for example:

“In future screening and follow-up protocols, it may be valuable to include comprehensive evaluations of motor coordination and visual–perceptual abilities, allowing for the early identification of subtle neurodevelopmental difficulties that could precede or co-occur with psychosocial challenges in children with ASD or ASD+ADHD.”

5. Limitation and conclusion: The conclusion could end on a more forward-looking note, highlighting that future multimodal investigations combining corneal confocal microscopy with sensory and cognitive assessments could elucidate mechanisms of neurodevelopmental comorbidity.

Reviewer #3: 1. I continue to believe the authors have not clarified enough the likely benefit of the findings of the current pilot or potential benefits if a larger study were conducted. Despite the revision of the abstract I am still unclear.

2. lines 59 -60 ' Children with ASD+ADHD with and without ADHDASD-alone have evidence of sensory nerve

degeneration'. - typos

3. lines 86-88 'It is highly heterogeneous being varied in its presentation, co-occurring with conditions like intellectual

disability, and is shaped by factors such as age, gender, race/ethnicity, and developmental stage[1]' - this reads as thought ID commonly co-occurs with autism, whereas it is more likely that autistic people do not have additional ID. mental health conditions might be more relevant to add here.

4.Lines 95-98 'Most research has focused on brain centric mechanisms in both ASD and ADHD, however, there iss an increasing recognition of abnormal sensory responses in ASD [5-10] which relate to quantitative autism traits [7] such that it now constitutes one of the four sub-criteria under the restricted, repetitive behaviors domain a core diagnostic criteria in DSM-5 diagnostic criteria [11], but however this does no’t take into account the atypical sensory features of ASD' - this sentence should be rephrased and shortened/separated into two sentences and typos corrected.

5. 'Subjects with ASD' - check for consistency in terminology - subjects, patients or participants

6. ' Eligible participants with ASD were referred' - is this the case? did paediatricians actually refer patients or did they provide information about the research?

7. 'healthy controls were selected from the general pediatrics clinicstudy' -- this is unclear to me. Do all children regularly visit paediatric clinics for review or evaluation or are they referred if there are concerns? this may be different across international systems.

8. 'ASD was diagnosed as part of clinical care' - it still reads as though the children were assessed during the study - if they had already been assessed - i.e., some months or years earlier this should be clarified.

9. 'DSM5' to be written as DSM-5.

10. Overall, I am uncertain of the conclusions being drawn, and ability to replicate this study, given the methodology.

**********

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Reviewer #1: No

Reviewer #3: No

**********

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Decision Letter 2

Shivanand Kattimani

11 Dec 2025

Dear Dr. Malik,

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

Please submit your revised manuscript by Jan 25 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #1: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #3: I Don't Know

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: The revised version of the manuscript is much improved, with clearer presentation and a more focused interpretation. However, regarding the initial hypothesis that ASD+ADHD would show greater corneal nerve degeneration than ASD alone, the Discussion does not yet fully explain why this expectation was not supported. Beyond noting the small sample size and the absence of an ADHD-only comparison group, the authors could strengthen the Discussion by briefly considering several additional factors that may account for the non-significant findings:

1. Age differences between groups and developmental variation in corneal nerve parameters that could influence group comparisons;

2. The potential influence of ADHD neurobiology or stimulant medications, which may modulate peripheral nerve function and confound direct comparisons;

3. Heterogeneity in sensory-processing profiles across ASD and ADHD, which may result in distinct peripheral nerve patterns not captured by group-level analyses;

4. Interpretation of effect sizes and confidence intervals, to clarify whether the lack of significant differences reflects a true absence of group effects or insufficient statistical power.

The author may incorporate these points would provide a more comprehensive explanation for why the initial hypothesis was not confirmed and would help contextualize the findings within broader neurodevelopmental and sensory-processing frameworks.

Reviewer #3: I am satisfied that the authors have addressed the core concerns. I only have two minor typos to highlight:

p3 . It can vary in its presentation may be associated with anxiety and depression and shaped by age, gender,

race/ethnicity, and developmental stage [1]. COMMA MISSING

P4 Parkinson disease - Parkinson's disease

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #3: No

**********

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Decision Letter 3

Shivanand Kattimani

19 Jan 2026

Dear Dr. Malik,

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

Please submit your revised manuscript by Mar 05 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

  • A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Shivanand Kattimani

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

kindly address comments to authors by reviwer 3

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #3: I Don't Know

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: I am satisfied with the revised manuscript. Thank you for the authors’ efforts, and I look forward to seeing the paper published.

Reviewer #3: Thanks you for addressing the comments. I have two minor points to add with respect to your responses to Reviewer 1's comments.

Abstract needs to acknowledge the groups studied, particularly since an ADHD group alone was not included: 'a clinical screening strategy for neurodegneration in ASD and ADHD' should this read and ASD-ADHD combined?

Page 10 outlines the limitations of the exploratory study including group size. Since the groups differed with respect to age, should this also be added?

Many thanks

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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

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

Reviewer #1: No

Reviewer #3: No

**********

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

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Decision Letter 4

Shivanand Kattimani

22 Jan 2026

Children with Autism Spectrum Disorder and Attention Deficit Hyperactivity Disorder have Evidence of Sensory Nerve Degeneration

PONE-D-25-44922R4

Dear Dr. Malik,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Shivanand Kattimani

Academic Editor

PLOS One

Acceptance letter

Shivanand Kattimani

PONE-D-25-44922R4

PLOS One

Dear Dr. Malik,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

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* There are no issues that prevent the paper from being properly typeset

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If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Shivanand Kattimani

Academic Editor

PLOS One

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File. Study raw data.

    (XLSX)

    pone.0342439.s001.xlsx (10.8KB, xlsx)
    Attachment

    Submitted filename: Response to reviewers.pdf

    pone.0342439.s003.pdf (593.3KB, pdf)
    Attachment

    Submitted filename: Response_to_reviewers_auresp_2.pdf

    pone.0342439.s004.pdf (507.7KB, pdf)
    Attachment

    Submitted filename: Response_to_reviewers_auresp_3.pdf

    pone.0342439.s005.pdf (478.3KB, pdf)
    Attachment

    Submitted filename: Response_to_reviewers_auresp_4.pdf

    pone.0342439.s006.pdf (472.1KB, pdf)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLOS One are provided here courtesy of PLOS

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