The RCT simulation used in this study.

<p>1) Real seizure burden (SB) time courses were initially modelled with a lognormal function. The probability density function (PDF) of the lognormal function parameters across a cohort of 41 neonates was estimated. 2) RCT parameters including the AED protocol, delay between seizure onset and intervention (T_d), outcome measure (OM), and level of AED efficacy were selected. A lognormal SB time course for each RCT arm was simulated by selecting lognormal parameters for each arm of the RCT from a multi-variate random variable with the PDF defined previously. The effect of the trial AED (and any other effects according to the AED protocol) was then applied to the SB time course in the intervention arm. The OM was calculated from the SB time course for each RCT arm. This process was iterated across 50000 simulated neonates and the mean and the standard deviation of the OM was then calculated and used to estimate the effect and sample size.</p

The effect of trial design on effect size with an assumed trial drug efficacy of 80% reduction in seizure burden for 12h.

<p>Outcome measures (OM) are total seizure burden (tSB) in minutes, post-intervention seizure burden (pSB) in minutes measured over a duration specified by the subscript in hours, seizure burden response (rSB) in minutes per hour, 1h pre-intervention vs. a post-intervention duration specified by the subscript in hours. The maximum possible effect for each trial is given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165693#pone.0165693.t004" target="_blank">Table 4</a>. The effect size of tSB and pSB₁₂ are equal as the assumed efficacy of the trial AED effect is 12h. The effect size of pSB₁ and rSB₁ are also equal as the rSB is measured in minutes per hour and not as a proportion. Results are presented as effect size (95% CI).</p

Seizure time courses and neonatal seizure treatment trial designs.

<p>The simulated seizure time courses used to estimate the sample size for various designs of a randomized control trial. A) An example smoothed seizure time course from the cohort of Lynch et al. (2015) plotted over the corresponding real seizure time course [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165693#pone.0165693.ref010" target="_blank">10</a>]. B) All 41 smoothed seizure time courses from the cohorts of Lynch et al. (2012 and 2015) showing the variability of seizures in neonates (the black line in A and B refers to the same neonate) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165693#pone.0165693.ref007" target="_blank">7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165693#pone.0165693.ref010" target="_blank">10</a>]. SB/h is seizure burden in minutes per hour; it is a measure of the short term intensity of seizures. Time is measured with respect to seizure onset. C) The use of different AED protocols and outcome measures in RCT design. Each row defines a common outcome measure and each column defines an AED protocol. Outcome measures are defined by the shaded areas: tSB row–total SB (blue shaded area), pSB row–post-intervention SB (blue shaded area), and rSB row–SB response (blue shaded area subtracted from the red shaded area). Treatment delay is the difference between seizure onset and the initiation of the trial protocol and is 2h in these examples. The level of AED efficacy in these examples is an immediate 90% reduction in seizure burden. The trial drug is denoted as Dx. Existing or positive control anti-epileptic drug (AED) effect was based on phenobarbitone: a 75% reduction in seizure burden for 3h. The asterisk denotes the cessation of the existing AED effect (seizure reoccurrence).</p

The effect of trial design on effect size with an assumed trial drug efficacy of 100% reduction in seizure burden for 72h (the maximum possible effect).

<p>Outcome measures (OM) are total seizure burden (tSB) in minutes, post-intervention seizure burden (pSB) in minutes measured over a duration specified by the subscript in hours, seizure burden response (rSB) in minutes per hour, 1h pre-intervention vs. a post-intervention duration specified by the subscript in hours. The effect size of pSB₁ and rSB₁ are equal as the rSB is measured in minutes per hour and not as a proportion. Results are presented as effect size (95% CI).</p

The effect of trial design on sample size with an assumed trial drug efficacy of 80% reduction in seizure burden for 12h.

<p>Outcome measures (OM) are total seizure burden (tSB) in minutes, post-intervention seizure burden (pSB) in minutes measured over a duration specified by the subscript in hours, seizure burden response (rSB) in minutes per hour, 1h pre-intervention vs. a post-intervention duration specified by the subscript in hours. Results are presented as sample size (95% CI).</p

Averaged DFA plots and comparison with shuffled distributions for fixed length IEI sequences.

<p>(<b>A</b>) Average (across channels) DFA plots of window size, n, against root mean square fluctuation, F(n), for each subject for the first 1000 IEIs. DFA is calculated up to a maximum box size of 1/4 of the length of the signal i.e. 250. Exponents are as indicated. (<b>B,C</b>) Averaged DFA (<b>B</b>) and Whittle (<b>C</b>) exponents (vertical lines) for each subject from sequences of the first 1000 IEIs, along with the probability distributions of the shuffled data.</p

Subject Information.

<p><b>1</b> Subject information. Ages are given as weeks+days. Two different bipolar montages were used. 1 = F4-C4, C4-O2, F3-C3, C3-O1, T4-C4, C4-Cz, Cz-C3, C3-T3, 2 = F4-C4, C4-P4, P4-O2, F3-C3, C3-P3, P3-O1, T4-C4, C4-Cz, Cz-C3, C3-T3. Remaining electrodes indicates those electrodes used for analysis after artefact rejection and removal of short (<1000) IEI sequences – see methods. Electrodes are numerically indexed corresponding to the list here. Number of events per hour is averaged across all (remaining) electrodes. Subjects with haemorrhages (*) had significantly lower number of events per hour – two sample t-test, P = 1.4×10⁻¹⁴. Ultrasound details are indicated by N = normal, A = intraventricular/parenchymal haemorrhage (grade 4) left hemisphere, intraventricular haemorrhage (grade 3) right hemisphere, B = left germinal matrix haemorrhage, grade 4, C = bilateral germinal matrix haemorrhage grade 1 and cystic changes in post ventricular white matter and D = bilateral intraventricular haemorrhage involving parenchyma on right, grade 3 left, grade 4 right. Further details provide known follow up details for each subject. One child (subject 7) died of non-neurological complications of prematurity.</p

Comparison of the DFA and Whittle exponents with the shuffled data.

<p>The DFA (<b>A</b>) and Whittle (<b>B</b>) exponents for each subject (vertical lines, averaged across channels) are clearly distinct from the pooled probability distributions of exponents formed from 5000 shuffled sequences. The exponents from the IEI sequences were found to be significantly different from the shuffled distributions (P<0.001) using the one sample Wilcoxon signed rank test, indicating LRTCs in the IEI sequences of all subjects studied. The data is plotted here using the same format as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031543#pone-0031543-g004" target="_blank">Figure 4 </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031543#pone.0031543-LinkenkaerHansen4" target="_blank">[10]</a>.</p

Hurst exponent estimates in relation to subject age.

<p>Whittle (stars) and DFA exponents (circles) were calculated for each subject for the whole IEI sequence (<b>A</b>) and fixed length sequences of the first 1000 IEIs (<b>B</b>). Each point is the exponent calculated for a single IEI sequence from a single channel. These are plotted against the corrected age (gestational plus time since birth). Arrows indicate subjects with intracranial haemorrhages (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031543#pone-0031543-t001" target="_blank">Table 1</a>). DFA exponents are calculated using a maximum window size of 1/4.</p

Inter-event interval (IEI) sequence and detrended fluctuation analysis (DFA) estimates.

<p>(<b>A</b>) An example of an IEI sequence – produced from the sequential ordering of IEI. This sequence is from the C3-O1 bipolar recording of subject 5. Index indicates the sequential order of the IEI. (<b>B</b>) An example of a randomly shuffled sequence for the data set shown in (<b>A</b>). (<b>C</b>) DFA plot for both sequences with window size, n, against root mean square fluctuation, F(n), open circles - DFA of the actual IEI sequence, filled circles - DFA of the shuffled sequence shown in (<b>B</b>). DFA was calculated with a maximum window size of 1/10 of the length of the sequence. For each the line of best fit is shown (green for the actual IEI sequence, red for the shuffled sequence). The Hurst exponent is estimated by the slope of the line of best fit which in these cases were H = 0.66 and H = 0.49 for the IEI and shuffled sequences respectively.</p