Scent dog identification of samples from Covid-19 patients – a pilot study

Sample acquisition

Saliva samples and tracheobronchial secretion samples were collected from hospitalised COVID-19 patients that showed clinical symptoms and were diagnosed as SARS-CoV-2 positive using nasopharyngeal swab samples. Negative control samples were obtained from SARS-CoV-2 RT-PCR negative people with no previous history of COVID-19, nor had the individuals any history of a recent cold or infection. None of the samples were screened for different human coronaviruses like beta coronavirus HCoV-OC43 or alpha coronavirus HCoV-229E. After the sample acquisition, the anonymised samples were transported to the University of Veterinary Medicine Hannover.

Sample preparation

All collected samples were confirmed as positive or negative using the RT-PCR SARS-CoV-2-IP4 assay from Institut Pasteur (recommended by the World Health Organization [10, 11], including an internal control system and protocol as described [12, 13]. Samples from COVID-19 patients (irrespective of the final RT-PCR result) were further subjected to virus quantification (end point dilution assay) and virus isolation analysis using Vero E6 cells under biosafety level 3 conditions. The cell layers were assessed for cytopathic effects and final results were obtained 7 days after cell infection. Since dogs are susceptible to SARS-CoV-2 [14] all samples from COVID-19 patients were inactivated using beta propiolactone (BPL) in order to protect the dogs and their handlers from infection during training. Briefly, samples and reagents were kept at 4 °C, 20 μl/ml NaHCO₃ (7.5%) was added, and samples were incubated for 10 min at 4 °C. After addition of 10 μl/ml of 10% BPL, samples were incubated at 4 °C for 70 to 72 h. Hydrolysis of BPL was conducted at 37 °C for 1 to 2 h. Samples that showed a cytopathic effect before BPL inactivation using virus isolation or end point dilution assay were tested again after BPL inactivation and were confirmed to be inactivated. Only BPL inactivated samples from COVID-19 patients were used for the dog training. Furthermore, detection dogs were provided both negative control samples with and without previous BPL treatment to exclude hydrolysed BPL as a potential distracting reagent.

For the dog training, a volume of 100 μl per sample was pipetted onto a cotton pad, which was placed into a 4 ml glass tube.

Dog training and study design

The presentation of the samples to the dogs was conducted via a device called Detection Dog Training System (DDTS; Kynoscience UG, Germany), which can present samples in a randomised automated manner without trainer interference. For a short video sequence, see Additional file 1. DDTS was utilised for training and testing. The device is composed of seven scent holes. Behind each hole two tubes are leading to two metal containers. In the study, the first container enclosed the target sample and the second one carried the control sample. Only one container is presented in each sniffing hole at any given time as the pairs of containers are situated on movable slides inside the device. The metal containers were covered with grids, which allowed the odour to escape and reach the sniffing hole. Each tube extension was identical and L-shaped, which prevented dogs from physical contact with the samples and excluded any visual cues that may have enabled further detection capabilities. For each trial run, only one hole presented a SARS-CoV-2 positive sample at a time while the other six holes presented negative samples. After the indication of the hole with the positive sample, the dog was automatically rewarded by the device with food or ball. The indication time was changed during successful training from 1 s to 2 s. While the reward was eaten, the device’s software randomly and automatically assigned new positions to the slides for the following session with again only one hole presenting the positive odour sample.

The dog, its handler and a person observing the study were blinded during the double-blinded study. All personnel stood behind the dog during the test runs to avoid distraction. The device recorded automatically the number and time length of each nose dip into the scent holes and the location of the positive and negative samples. This was verified by manual time-stamped video analysis.

Analysis of sensitivity and specificity

The diagnostic sensitivity (Se = true positive (‘TP’) /[TP + false negative (‘FN’)]), diagnostic specificity (Sp = true negative (‘TN’) /[TN + false positive (‘FP’)]), positive predictive values (PPV = TP/[TP + FP]) and negative predictive values (NPV = TN/[TN + FN]) were calculated according to Trevethan [15].

from Hacker News https://ift.tt/2OQyTqT