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Background/Objectives Threshold tracking transcranial magnetic stimulation (TMS) is a valuable diagnostic tool for amyotrophic lateral sclerosis (ALS). Recently, various tracking paradigms have been proposed. This study evaluated the diagnostic utility of serial ascending and parallel threshold tracking TMS in ALS.Methods Threshold tracking TMS was performed on 90 prospectively recruited individuals suspected of ALS. Short interval intracortical inhibition (SICI) was recorded using both serial ascending and parallel paradigms between interstimulus intervals (ISI) of 1–7 ms. The primary outcome measure was the difference in diagnostic utility of each paradigm in distinguishing ALS from ALS-mimicking disorders, assessed using receiver operating characteristic (ROC) analysis.Results A reduction in SICI reliably differentiated ALS from mimics across both paradigms, regardless of the threshold tracking paradigm used. However, the serial ascending paradigm demonstrated superior diagnostic accuracy, with a significantly higher mean area under the curve (AUC) for SICI (1–7 ms) (0.81, 95% CI 0.72–0.91) compared to the parallel paradigm (0.72, 95% CI 0.61–0.83, p=0.0065). The advantage of the serial ascending paradigm was evident for ISI 1–5 ms and remained consistent regardless of disease onset site, functional impairment, or lower motor neuron dysfunction. Both paradigms have comparable diagnostic utility in ALS participants who have a relative paucity of upper motor neuron signs.Conclusion Threshold tracking TMS reliably differentiates ALS from mimic disorders, with the serial ascending paradigm offering better diagnostic utility. The serial ascending threshold tracking TMS paradigm should be considered a preferred diagnostic tool for ALS in clinical practice.