A New Objective Diagnostic Tool for Attention-Deficit Hyper-Activity Disorder (ADHD): Development of the Distractor-Embedded Auditory Continuous Performance Test
Abstract
:1. Introduction
- Development and validation of the da-CPT, a novel tool that integrates auditory stimuli with distractors to enhance ecological validity.
- Demonstration of the da-CPT’s effectiveness in providing a more accurate real-world assessment of ADHD symptoms, particularly in environments with auditory distractions.
- Provision of evidence with high corelation WISC-R test supporting the da-CPT’s clinical utility as a reliable and objective diagnostic tool for ADHD in children.
2. Materials and Methods
2.1. Participants
2.2. Distractor-Embedded Auditory Continuous Performance Test (da-CPT)
2.2.1. MOXO Continuous Performance Test (MOXO-CPT)
2.2.2. Integrated Visual and Auditory Continuous Performance Test, Version 2 (IVA-2)
2.2.3. Wechsler Intelligence Scale for Children-Revised (WISC-R)
2.2.4. Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version-DSM-5-Turkish Version (K-SADS-PL-DSM-5-T)
2.2.5. Conners’ Parent Rating Scale-Revised Short Form (CPRS-R:S)
2.3. Procedure
2.4. Statistical Analysis
3. Results
3.1. Participant Demographics
3.2. Diagnostic Accuracy of da-CPT
3.3. Correlation Between da-CPT Sub-Indices and ADHD Severity
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ADHD | Attention-Deficit/Hyperactivity Disorder |
AUC | Area Under the Curve |
CI | Confidence Interval |
CPRS-R:S | Conners’ Parent Rating Scale—Revised Short Form |
CPT | Continuous Performance Test |
da-CPT | Distractor-embedded Auditory Continuous Performance Test |
DSM-5 | Diagnostic and Statistical Manual of Mental Disorders, 5th Edition |
K-SADS-PL-DSM-5-T | Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version, DSM-5-Turkish Version |
MOXO-CPT | MOXO Continuous Performance Test |
PPV | Positive Predictive Value |
ROC | Receiver Operating Characteristic |
SD | Standard Deviation |
SPSS | Statistical Package for the Social Sciences |
WISC-R | Wechsler Intelligence Scale for Children-Revised |
YI | Youden Index |
Appendix A
Appendix A.1. Technological Infrastructure
Appendix A.2. Participant Registration and Demo
Appendix A.3. Main Test Structure
- Correct Attention Responses: The participant is expected to press the space key on the keyboard as soon as they hear the target sound (e.g., the cat meowing). Correct responses are recorded when the participant presses the key while the target sound is active or immediately afterward in the designated blank interval.
- Timing: Timing is assessed by evaluating the precision of the participant’s response, particularly how quickly they react to the target stimulus and maintain consistent reaction times throughout the test. The first key press while the target sound is in progress is used to calculate the timing. Moreover, da-CPT measures response time variability, which includes fluctuations in reaction time between stages. This is a very important measure for the estimation of periods of inattention, under conditions with auditory and visual distractors. In this respect, the test gives a sensitive measure of the lapses in attention so commonly seen in people with ADHD, thus ensuring that periods of inattention and/or delayed responses will be featured in the final outcome of the test.
- Impulsivity: Impulsivity is measured by recording instances where the participant presses the key in response to non-target sounds (e.g., the duck quacking or donkey braying).
- Hyperactivity: Hyperactivity is calculated based on repeated or inappropriate key presses, particularly when the participant reacts to the same target or non-target stimulus multiple times.
Appendix A.4. Stages and Stimuli
- Stage 1: Target and non-target sounds only—Participants are introduced to the target sound (cat meow) and non-target sounds (e.g., other animal sounds) without any distractors.
- Stage 2: Auditory distractors added—Target and non-target sounds are presented along with auditory distractors (e.g., phone ringing, baby crying, crowd noise, motorcycle/car sounds).
- Stage 3: Two auditory distractors—Participants hear two auditory distractors simultaneously with target and non-target sounds. For example, combination of a cat meowing (target), a phone ringing (distractor-1) and a baby crying (distractor-2), with the blank screen.
- Stage 4: Visual distractors introduced—A visual distractor (e.g., animated animal) is added alongside the target and non-target sounds.
- Stage 5: Two visual distractors—Two visual distractors (e.g., cat chasing mouse, eagle flying above a howling wolf) are added, increasing visual complexity.
- Stage 6: Mixed auditory and visual distractors—One auditory and one visual distractor are combined with target and non-target sounds.
- Stage 7: Multiple auditory and visual distractors—Two auditory and two visual distractors are presented together with target and non-target sounds.
- Stage 8: Return to basic level—Only target and non-target sounds are presented again without any distractors, to assess performance consistency.
Appendix A.5. Reporting and Analysis
Appendix A.6. Public Demo Access for Researchers and the Public
References
- American Psychiatric Association. Neurodevelopmental disorders. In Diagnostic and Statistical Manual of Mental Disorders, 5th ed.; Text Revision (DSM-5-TR); American Psychiatric Association: Washington, DC, USA, 2022. [Google Scholar] [CrossRef]
- Willcutt, E.G. The prevalence of DSM-IV attention-deficit/hyperactivity disorder: A meta-analytic review. Neurotherapeutics 2012, 9, 490–499. [Google Scholar] [CrossRef] [PubMed]
- Ercan, E.S.; Polanczyk, G.; Akyol Ardıc, U.; Yuce, D.; Karacetin, G.; Tufan, A.E.; Tekden, M. The prevalence of childhood psychopathology in Turkey: A cross-sectional multicenter nationwide study (EPICPAT-T). Nord. J. Psychiatry 2019, 73, 132–140. [Google Scholar] [CrossRef] [PubMed]
- MacDonald, L.; Sadek, J. Management Strategies for Borderline Personality Disorder and Bipolar Disorder Comorbidities in Adults with ADHD: A Narrative Review. Brain Sci. 2023, 13, 1517. [Google Scholar] [CrossRef] [PubMed]
- Hall, C.L.; Valentine, A.Z.; Groom, M.J.; Walker, G.M.; Sayal, K.; Daley, D.; Hollis, C. The clinical utility of the continuous performance test and objective measures of activity for diagnosing and monitoring ADHD in children: A systematic review. Eur. Child Adolesc. Psychiatry 2016, 25, 677–699. [Google Scholar] [CrossRef]
- Lange, K.W.; Hauser, J.; Lange, K.M.; Makulska-Gertruda, E.; Takano, T.; Takeuchi, Y.; Tucha, O. Utility of cognitive neuropsychological assessment in attention-deficit/hyperactivity disorder. ADHD Atten. Deficit Hyperact. Disord. 2014, 6, 241–248. [Google Scholar] [CrossRef]
- Serra-Pinheiro, M.A.; Mattos, P.; Angélica Regalla, M. Inattention, hyperactivity, and oppositional–defiant symptoms in Brazilian adolescents: Gender prevalence and agreement between teachers and parents in a non–English speaking population. J. Atten. Disord. 2008, 12, 135–140. [Google Scholar] [CrossRef]
- Ginsberg, Y.; Quintero, J.; Anand, E.; Casillas, M.; Upadhyaya, H.P. Underdiagnosis of attention-deficit/hyperactivity disorder in adult patients: A review of the literature. Prim. Care Companion CNS Disord. 2014, 16, PCC.13r01600. [Google Scholar] [CrossRef]
- Quinn, P.O.; Madhoo, M. A review of attention-deficit/hyperactivity disorder in women and girls: Uncovering this hidden diagnosis. Prim. Care Companion CNS Disord. 2014, 16, 27250. [Google Scholar] [CrossRef]
- Stokes, J.D.; Rizzo, A.; Geng, J.J.; Schweitzer, J.B. Measuring Attentional Distraction in Children with ADHD Using Virtual Reality Technology with Eye-Tracking. Front. Virtual Real. 2022, 3, 855895. [Google Scholar] [CrossRef]
- Riccio, C.A.; Reynolds, C.R. Continuous performance tests are sensitive to ADHD in adults but lack specificity. A review and critique for differential diagnosis. Ann. New York Acad. Sci. 2001, 931, 113–139. [Google Scholar] [CrossRef]
- Berger, I.; Slobodin, O.; Cassuto, H. Usefulness and Validity of Continuous Performance Tests in the Diagnosis of Attention-Deficit Hyperactivity Disorder Children. Arch. Clin. Neuropsychol. 2017, 32, 81–93. [Google Scholar] [CrossRef] [PubMed]
- Roebuck, H.; Freigang, C.; Barry, J.G. Continuous Performance Tasks: Not Just About Sustaining Attention. J. Speech Lang. Hear. Res. 2016, 59, 501–510. [Google Scholar] [CrossRef] [PubMed]
- Schmidt, S.L.; Simões, E.D.N.; Novais Carvalho, A.L. Association Between Auditory and Visual Continuous Performance Tests in Students with ADHD. J. Atten. Disord. 2019, 23, 635–640. [Google Scholar] [CrossRef] [PubMed]
- Simões, E.N.; Carvalho, A.L.N.; Schmidt, S.L. The Role of Visual and Auditory Stimuli in Continuous Performance Tests: Differential Effects on Children with ADHD. J. Atten. Disord. 2021, 25, 53–62. [Google Scholar] [CrossRef]
- Corkum, P.V.; Siegel, L.S. Is the Continuous Performance Task a valuable research tool for use with children with Attention-Deficit-Hyperactivity Disorder? J. Child Psychol. Psychiatry 1993, 34, 1217–1239. [Google Scholar] [CrossRef]
- Ballard, J.C. Computerized assessment of sustained attention: A review of factors affecting vigilance performance. J. Clin. Exp. Neuropsychol. 1996, 18, 843–863. [Google Scholar] [CrossRef]
- Riccio, C.A.; Reynolds, C.R.; Lowe, P.; Moore, J.J. The continuous performance test: A window on the neural substrates for attention? Arch. Clin. Neuropsychol. 2002, 17, 235–272. [Google Scholar] [CrossRef]
- Ogundele, M.O.; Ayyash, H.F.; Banerjee, S. Role of computerised continuous performance task tests in ADHD. Prog. Neurol. Psychiatry 2011, 15, 8–13. [Google Scholar] [CrossRef]
- Lehman, E.B.; Olson, V.A.; Aquilino, S.A.; Hall, L.C. Auditory and visual continuous performance tests: Relationships with age, gender, cognitive functioning, and classroom behavior. J. Psychoeduc. Assess. 2006, 24, 36–51. [Google Scholar] [CrossRef]
- Shapiro, S.K.; Herod, L.A. Tasks in the Assessment of Attention-Deficit Hyperactivity. In Disruptive Behavior Disorders in Childhood; Academic Press: Cambridge, MA, USA, 2013; p. 87. [Google Scholar]
- Pelham, W.E., Jr.; Waschbusch, D.A.; Hoza, B.; Gnagy, E.M.; Greiner, A.R.; Sams, S.E.; Vallano, G.; Majumdar, A.; Carter, R.L. Music and video as distractors for boys with ADHD in the classroom: Comparison with controls, individual differences, and medication effects. J. Abnorm. Child Psychol. 2011, 39, 1085–1098. [Google Scholar] [CrossRef]
- Hong, N.; Kim, J.J.; Kwon, J.H.; Eom, H.; Kim, E. Effect of Distractors on Sustained Attention and Hyperactivity in Youth with Attention Deficit Hyperactivity Disorder Using a Mobile Virtual Reality School Program. J. Atten. Disord. 2022, 26, 358–369. [Google Scholar] [CrossRef] [PubMed]
- Baijot, S.; Slama, H.; Söderlund, G.; Dan, B.; Deltenre, P.; Colin, C.; Deconinck, N. Neuropsychological and neurophysiological benefits from white noise in children with and without ADHD. Behav. Brain Funct. 2016, 12, 11. [Google Scholar] [CrossRef] [PubMed]
- Cassuto, H.; Ben-Simon, A.; Berger, I. Using environmental distractors in the diagnosis of ADHD. Front. Hum. Neurosci. 2013, 7, 805. [Google Scholar] [CrossRef] [PubMed]
- Berger, I.; Goldzweig, G. Objective measures of attention-deficit/hyperactivity disorder: A pilot study. Isr. Med. Assoc. J. 2010, 12, 531–535. [Google Scholar] [PubMed]
- Moreno-García, I.; Delgado-Pardo, G.; Roldán-Blasco, C. Attention and response control in ADHD. Evaluation through integrated visual and auditory continuous performance test. Span. J. Psychol. 2015, 18, E1. [Google Scholar] [CrossRef]
- Savaşır, I.; Şahin, N. Wechsler Çocuklar Için Zeka Ölçeği (WISC-R) el Kitabı; Türk Psikologlar Derneği Yayınları: Ankara, Turkey, 1995. [Google Scholar]
- Unal, F.; Oktem, F.; Cetin Cuhadaroglu, F.; Cengel Kultur, S.E.; Akdemir, D.; Foto Ozdemir, D.; Cak, H.T.; Unal, D.; Tiras, K.; Aslan, C.; et al. Reliability and Validity of the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version, DSM-5 November 2016-Turkish Adaptation (K-SADS-PL-DSM-5-T). Turk. J. Psychiatry 2019, 30, 42–50. [Google Scholar] [CrossRef]
- Kaner, S.; Buyukozturk, S.; Iseri, E. Conners parent rating scale-revised short: Turkish standardization study/Conners anababa dereceleme olcegi-yenilenmis kisa: Turkiye stardardizasyon calismasi. Arch. Neuropsychiatry 2013, 50, 100–110. [Google Scholar] [CrossRef]
- Safari, S.; Baratloo, A.; Elfil, M.; Negida, A. Evidence based emergency medicine; part 5 receiver operating curve and area under the curve. Emergency 2016, 4, 111–113. [Google Scholar]
- Wu, D.; Akl, E.A.; Guyatt, G.H.; Scott, I.; Oderda, G.M. Methodological Survey of Designed Uneven Randomization Trials (DU-RANDOM): A Protocol. Trials 2014, 15, 33. [Google Scholar] [CrossRef]
- Cortese, S.; Solmi, M.; Michelini, G.; Bellato, A.; Blanner, C.; Canozzi, A.; Eudave, L.; Farhat, L.C.; Højlund, M.; Köhler-Forsberg, O.; et al. Candidate diagnostic biomarkers for neurodevelopmental disorders in children and adolescents: A systematic review. World Psychiatry 2023, 22, 129–149. [Google Scholar] [CrossRef]
- Bellato, A.; Hall, C.L.; Groom, M.J.; Simonoff, E.; Thapar, A.; Hollis, C.; Cortese, S. Practitioner Review: Clinical utility of the QbTest for the assessment and diagnosis of attention-deficit/hyperactivity disorder—A systematic review and meta-analysis. J. Child Psychol. Psychiatry 2023, 65, 845–861. [Google Scholar] [CrossRef] [PubMed]
- Huang-Pollock, C.L.; Karalunas, S.L.; Tam, H.; Moore, A.N. Evaluating vigilance deficits in ADHD: A meta-analysis of CPT performance. J. Abnorm. Psychol. 2012, 121, 360–371. [Google Scholar] [CrossRef] [PubMed]
- Tallberg, P.; Råstam, M.; Wenhov, L.; Eliasson, G.; Gustafsson, P. Incremental clinical utility of continuous performance tests in childhood ADHD—An evidence-based assessment approach. Scand. J. Psychol. 2019, 60, 26–35. [Google Scholar] [CrossRef] [PubMed]
- Arrondo, G.; Mulraney, M.; Iturmendi-Sabater, I.; Musullulu, H.; Gambra, L.; Niculcea, T.; Banaschewski, T.; Simonoff, E.; Döpfner, M.; Hinshaw, S.P.; et al. Systematic Review and Meta-analysis: Clinical Utility of Continuous Performance Tests for the Identification of Attention-Deficit/Hyperactivity Disorder. J. Am. Acad. Child Adolesc. Psychiatry 2023, 63, 154–171. [Google Scholar] [CrossRef]
- Berger, I.; Cassuto, H. The effect of environmental distractors incorporation into a CPT on sustained attention and ADHD diagnosis among adolescents. J. Neurosci. Methods 2014, 222, 62–68. [Google Scholar] [CrossRef]
- De Rossi, P.; Pretelli, I.; Menghini, D.; D’Aiello, B.; Di Vara, S.; Vicari, S. Gender-Related Clinical Characteristics in Children and Adolescents with ADHD. J. Clin. Med. 2022, 11, 385. [Google Scholar] [CrossRef]
- Forbes, G.B. Clinical utility of the Test of Variables of Attention (TOVA) in the diagnosis of attention-deficit/hyperactivity disorder. J. Clin. Psychol. 1998, 54, 461–476. [Google Scholar] [CrossRef]
- McGee, R.A.; Clark, S.E.; Symons, D.K. Does the Conners’ Continuous Performance Test aid in ADHD diagnosis? J. Abnorm. Child Psychol. 2000, 28, 415–424. [Google Scholar] [CrossRef]
- Kim, J.; Lee, Y.; Han, D.; Min, K.; Kim, D.; Lee, C. The utility of quantitative electroencephalography and Integrated Visual and Auditory Continuous Performance Test as auxiliary tools for the Attention Deficit Hyperactivity Disorder diagnosis. Clin. Neurophysiol. 2015, 126, 532–540. [Google Scholar] [CrossRef]
- Lasee, M.J.; Choi, H. Evidence of Reliability and Validity for a Children’s Auditory Continuous Performance Test. SAGE Open 2013, 3, 2158244013511828. [Google Scholar] [CrossRef]
- Özaslan, A.; İşeri, E. Özel Öğrenme Bozukluklarında Prognoz, Prognostik Faktörler ve Erken Tedavinin Etkisi. In Çocuk ve Gençlik Çağı Ruhsal Hastalıklarında İzlem ve Süreç: Prognostik Faktörler ve Erken Tedavi Yaklaşımlarının Etkisi; Erermiş, H.S., Ed.; Türkiye Klinikleri: Ankara, Turkey, 2019; pp. 32–37. [Google Scholar]
- Romero-Ayuso, D.; del Pino-González, A.; Torres-Jiménez, A.; Juan-González, J.; Celdrán, F.J.; Franchella, M.C.; González, P. Enhancing Ecological Validity: Virtual Reality Assessment of Executive Functioning in Children and Adolescents with ADHD. Children 2024, 11, 986. [Google Scholar] [CrossRef] [PubMed]
- Tyburski, E.; Mak, M.; Sokołowski, A.; Starkowska, A.; Karabanowicz, E.; Kerestey, M.; Jansari, A.S. Executive Dysfunctions in Schizophrenia: A Critical Review of Traditional, Ecological, and Virtual Reality Assessments. J. Clin. Med. 2021, 10, 2782. [Google Scholar] [CrossRef] [PubMed]
- Elbaum, T.; Braw, Y.; Lev, A.; Rassovsky, Y. Attention-Deficit/Hyperactivity Disorder (ADHD): Integrating the MOXO-dCPT with an Eye Tracker Enhances Diagnostic Precision. Sensors 2020, 20, 6386. [Google Scholar] [CrossRef]
Characteristic | ADHD Group (n = 80) | Control Group (n = 80) | p-Value |
---|---|---|---|
Age (months) | 111.08 ± 21.55 | 112.06 ± 21.03 | 0.821 1 |
Gender | 0.282 2 | ||
Boys (%) | 62 (77.5%) | 56 (70.0%) | |
Girls (%) | 18 (22.5%) | 24 (30.0%) |
da-CPT (n = 160) | MOXO (n = 87) | IVA-2 (n = 73) | |
---|---|---|---|
Sensitivity | 91.25% | 86.05% | 86.11% |
(CI: 82.80–96.41%) | (CI: 72.07–94.70%) | (CI: 70.50–95.33%) | |
Specificity | 83.75% | 84.09% | 86.49% |
(CI: 73.82–91.05%) | (CI: 69.93–93.36%) | (CI: 71.23–95.46%) | |
Youden Index | 0.75 | 0.71 | 0.73 |
Test | AUC (95% CI) | Cut-Off Scores | p-Value | Sensitivity (%) | Specificity (%) |
---|---|---|---|---|---|
da-CPT Inattention | 0.881 (0.824–0.938) | 72.6 | 0.001 | 87.5 | 83.5 |
da-CPT Timing | 0.812 (0.743–0.881) | 47.89 | 0.001 | 80.0 | 72.5 |
da-CPT Impulsivity | 0.760 (0.685–0.836) | 9.84 | 0.001 | 72.5 | 71.2 |
da-CPT Hyperactivity | 0.742 (0.662–0.822) | 9.50 | 0.001 | 70.0 | 67.2 |
MOXO-CPT | 0.850 (0.790–0.910) | 50.75 | 0.002 | 82.0 | 78.0 |
IVA-2 | 0.820 (0.760–0.880) | 48.5 | 0.002 | 80.0 | 75.0 |
Variables | ADHD Index Score | Inattention | Timing | Impulsivity | Hyperactivity |
---|---|---|---|---|---|
ADHD Index Score | 1 | ||||
Inattention | −0.494 ** | 1 | |||
Timing | −0.474 ** | 0.805 ** | 1 | ||
Impulsivity | 0.285 ** | −0.350 ** | −0.329 ** | 1 | |
Hyperactivity | 0.271 ** | −0.314 ** | −0.213 ** | 0.535 ** | 1 |
Variable | Groups | N | Mean | SD | t | p * |
---|---|---|---|---|---|---|
da-CPT Inattention | ADHD | 80 | 58.12 | 14.06 | −8.84 | <0.001 |
Control | 80 | 75.73 | 10.94 | |||
da-CPT Timing | ADHD | 80 | 35.01 | 13.23 | −7.90 | <0.001 |
Control | 80 | 50.32 | 11.19 | |||
da-CPT Impulsivity | ADHD | 80 | 15.22 | 8.70 | 6.21 | <0.001 |
Control | 80 | 8.57 | 3.99 | |||
da-CPT Hyperactivity | ADHD | 80 | 21.46 | 17.24 | 6.10 | <0.001 |
Control | 80 | 8.53 | 7.90 |
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Özaslan, A.; Sevri, M.; İşeri, E.; Karacan, B.; Cengiz, M.; Karacan, H.; Sarıpınar, E.G.; Dikmen, A.U.; Güney, E. A New Objective Diagnostic Tool for Attention-Deficit Hyper-Activity Disorder (ADHD): Development of the Distractor-Embedded Auditory Continuous Performance Test. J. Clin. Med. 2024, 13, 6438. https://doi.org/10.3390/jcm13216438
Özaslan A, Sevri M, İşeri E, Karacan B, Cengiz M, Karacan H, Sarıpınar EG, Dikmen AU, Güney E. A New Objective Diagnostic Tool for Attention-Deficit Hyper-Activity Disorder (ADHD): Development of the Distractor-Embedded Auditory Continuous Performance Test. Journal of Clinical Medicine. 2024; 13(21):6438. https://doi.org/10.3390/jcm13216438
Chicago/Turabian StyleÖzaslan, Ahmet, Mehmet Sevri, Elvan İşeri, Barış Karacan, Mehmet Cengiz, Hacer Karacan, Esin Gökçe Sarıpınar, Asiye Uğraş Dikmen, and Esra Güney. 2024. "A New Objective Diagnostic Tool for Attention-Deficit Hyper-Activity Disorder (ADHD): Development of the Distractor-Embedded Auditory Continuous Performance Test" Journal of Clinical Medicine 13, no. 21: 6438. https://doi.org/10.3390/jcm13216438
APA StyleÖzaslan, A., Sevri, M., İşeri, E., Karacan, B., Cengiz, M., Karacan, H., Sarıpınar, E. G., Dikmen, A. U., & Güney, E. (2024). A New Objective Diagnostic Tool for Attention-Deficit Hyper-Activity Disorder (ADHD): Development of the Distractor-Embedded Auditory Continuous Performance Test. Journal of Clinical Medicine, 13(21), 6438. https://doi.org/10.3390/jcm13216438