Ethics

The study received approval by the institutional review board of OHS de Lorraine for collection techniques used to obtain odour samples from patients with epilepsy. All institutional (Medical Mutts) and published guidelines (Assistance Dogs International) for the use of positive reinforcement in the care and use of the dogs were followed. The Animal Ethics Committee of Rennes was consulted for this study and confirmed there was no need to request an authorization for this research, since it did not affect dog welfare.

The present research was non-invasive and did not involve pharmacological interventions. Hence, in accordance with the Ethics Committee’s guidelines, parents gave only an informed written consent to allow the child’s participation in the experiment prior to their inclusion in the study.

Subjects

Five trained dogs participated in this study (Supplementary Table S2). All subjects lived in Medical Mutts facilities (USA) and had never been exposed to the experimental five patient odours. Medical Mutts selected dogs from shelters to be trained by professional trainers as service dogs. Selection criteria included temperament traits compatible for service dog training40 and physical aptitudes. Dogs are trained for obedience for certification for public access in addition to scent discrimination. Dogs spent one hour per day, five days per week in a training session15.

Odour samples

Patients were recruited in the Healthcare Centre OHS Flavigny, Flavigny sur Moselle, France. Ten participants were initially invited to participate after medical recommendations. One declined and the other 4 did not experience a seizure through the sample collection time of one week in December 2017. Thus, odours were collected from 5 females who were residents of the Healthcare Centre OHS. All had a confirmed diagnosis of epilepsy (See Supplementary Table S3) and none experienced psychogenic non-epileptic seizures. Psychogenic non-epileptic seizures (exclusive or in addition to epileptic seizures) were actually a factor of exclusion when recruiting patients on the basis of their medical diagnosis. Since they all lived in a medicalized structure at the time of the sampling, the diagnosis was further confirmed.

They received the same food without dietary restrictions. Since the seizures could be experienced at any time, the collection of epilepsy seizure samples could occur randomly at any time (night or day), whereas all other samples were collected during day. Patients were not given any specific recommendation (e.g. regarding hormonal contraception, perfume, smoking etc.), so as to maintain an “ecological” individual variability.

Odour sampling procedure

Three types of samples were collected: (a) a “seizure sample”, collected during or right after (<5 min) an ictal event; (b) a “physical exercise sample”, collected right after (<5 min) moderate exercise (defined as 1 min during which the patient ran approximately 30 m, and did leg and arm movements as step-touch and punches). Physical exercise samples served as control for movements or arousal that can be made or experienced during a seizure41,42. And (c) a “day sample” was collected at a pseudo-random moment during calm activity (e.g. drawing, paper crafting) in day time. The non-seizure samples were collected a minimum of 6 hours before or after a seizure in order to avoid a pre- or post-ictal collection.

For each patient, two physical exercise samples were collected on two different days and four day samples on four different days.

Odour collection was performed in accordance with the procedure used at Medical Mutts for training assistance dogs15: Patients were instructed to use a sterile cotton pad (10*10 cm, 4 fold) to wipe their hands, forehead and back of the neck, allowing a multiplicity of odour origins. The cotton pad was then placed into a zip-locked bag (Ziplock brand, SC Johnson, Racine, WI, USA) and the patient was instructed to exhale into the bag before sealing it. Two cotton pads were collected from each patient, for each of the 7 types of odour. Each bag was labelled with the patient’s initials, day and time of collection and the corresponding odour type (normal, physical exercise or seizure). The samples were stored in the freezer (Temperature −12 °C) then shipped from France to USA at ambient temperature and stored again in the freezer until use (average of 47 days, SD = 18). Freezing has been shown to preserve samples of human body odour without altering perceptions of these odours and freeze–thaw cycles to not affect sample quality43.

Training

The dogs that participated in the study were trained using positive reinforcement methods [12, 13]. They underwent the general training for assistance dog (30 commands, staying under control in public areas, etc.).

More specifically, the training for SAD required three main steps. First a positive association is created between the scent of a seizure and treats in order to induce a conditioned emotional response. Then the dog learns to go to the scent and to stand still above it. This indication behaviour (dog standing above the scent) is developed by adding distance and duration and using the command “Check”. The second step consisted in a discrimination task, for additional scents (distractors, inter-ictal scent) were progressively added and the dog learned to only indicate the seizure scent.

In the third phase of training, the seizure scent was placed on a person. The ‘stay-stand’ indication was replaced by teaching the dog to alert by a poke of the nose on the person’s body.

The odours used for training were not from the patients. Therefore, the dogs included in the present study were completely unfamiliar with these particuliar odours.

Experimental Setup

Tests took place from January 2018 to February 2018, in a closed room (9.1 × 6.1 m, 4 m high, 21 °C in average) (Supplementary Fig. S1) in the training facility. No experimenter was present in the testing room during a trial (see also15). Experimenter was in a room nearby, separated from the first one by a wooden door. One camera was linked to a monitoring system, and another recorded the trials. At the beginning of a trial, the room was open, the trainer gave the command (“Check”), then closed the door and stayed out of view or reach in the second room. The dog was free to explore the experimental set-up until the correct identification of the seizure sample. It was then rewarded with a treat from an automatic dispenser remotely activated by the trainer (Pet Tutor®). Then the trainer opened the door and called the dog out of the room. The dog stayed in the second room in between two trials, while the experimenter changed the samples and put the cans in the right locations for the next trial.

The experimental set-up consisted of seven steel cans (18 cm high × 17 cm in diameter), each containing one small canister (5.2 cm in diameter, 2 cm high) in which one odour sample was placed. These canisters prevented the dog from being in direct contact with samples. The cans were in a semicircle arrangement, equidistant (2.08 m) from the Pet Tutor®. Each can contained an odour sample that could be either a seizure, physical exercise or calm activity sample. Compared to the literature on similar experiments, this is a high average for the number of samples presented in line-up methods and the distance between cans (about 50 centimeters) was comparable8,15,26.

At each test, all seven cans contained an odour from the same person: one seizure sample, two physical exercise samples and four calm sample.

Test procedure

Trials took place as follows: each dog had the first 5 trials using the same person’s odour, a 2-hour break, and then one trial with each of the 4 remaining patients (See Fig. 2). Thus, each dog had 9 trials in one day. Each test trial lasted a maximum of 5 minutes, with a 2 min break between each trial in order to remove and replace the cans. Odour locations were pseudo-randomly determined prior to the sessions and changed between trials, with the constraint that the same location could not be used more than three times in a row for the same type of odour in order to avoid learning bias and inducing A-not-B errors (Smith et al., 1999).

Behavioural coding

Each trial was videotaped (30 fps, Sony Handycam HDR-CX380) and coded using frame-by-frame playback for accuracy of determination of investigatory behaviour. The latency of first response was measured as well as the time spent by the dog in the olfactory investigation of cans (i.e. nose at less than ten centimeters from the cans44,45,46). Given the indication behavior (remain standing with the head above the cans), an indication was counted if it lasted more than 3 seconds. The same person (A.C.), blind to the cans’ contents, coded all trials. Another coder, unfamiliar with the goals of the study, verified the reliability of this coding on all trials. Inter-observer agreement was high (κ = 0.91).

Statistical analyses

Sensitivity and specificity were calculated after pooling the data across all trials for all dogs in accordance with15.

In addition, as the data were not normally distributed, non-parametric statistical tests were used47. Performances for individual dogs were statistically assessed with binomial tests. A Friedman test compared the time spent exploring the different sample categories, with all dogs and trials pooled. To assess potential differences between the odours of each patient, all dogs were pooled on the cumulated exploration time for the five different patients (first presentation of the repeated person and the last four persons). The concordance among dogs’ performances according to persons was estimated using a Kendall test by permutation.