# The Use of Biological Sensors and Instrumental Analysis to Discriminate COVID-19 Odor Signatures
## 2 Materials and Methods
### Canine Odor Discrimination Methods
#### 2.2.5 Odor Learning Phase  
In the initial phase of the study, dogs were rewarded for sniffing the COVID-19 positive sample. Dogs were presented with jars containing the sleeves of shirts worn by COVID-19 positive patients as a target odor. When the dog sniffed this target odor, the behavior was marked with a conditioned secondary reinforcer (i.e., noise generated by a \"clicker\") and paired with a food or toy reward. The target jars were covered with a mesh lid, allowing full odor release but preventing access to the material. In the same session, dogs were presented with jars containing the sleeves of shirts worn by people who were  
\begin{table}
\begin{tabular}{l c c c c c} \hline \hline \multirow{2}{*}{**Phase**} & \multirow{2}{*}{\begin{tabular}{c} **Holes in** \\ **Negative Lid** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Sensitivity** \\ **Required to** \\ **Advance** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Specificity** \\ **Required to** \\ **Advance** \\ \end{tabular} } & \multirow{2}{*}{\
\begin{tabular}{c} **Minimum \#** \\ **Sessions** \\ **Required to** \\ **Advance** \\ \end{tabular} } & \multirow{2}{*}{
\begin{tabular}{c} **\# COVID-19 (+)** \\ **Samples/Session** \\ **Samples/Session** \\ \end{tabular} } \\ \hline
**Imprinting** & 1 & 9/10 trials overall correct & 1 & 1 & 1 \\ \hline
**Phase 1** & 1 & 80\% (5/6 correct) & 80\% (10/12 correct) & 3 in a row & 2 & 4 \\ \hline
**Phase 2** & 4 & 80\% (5/6 correct) & 80\% (10/12 correct) & 3 in a row & 2 & 4 \\ \hline
**Phase 3** & 16 & 80\% (5/6 correct) & 80\% (10/12 correct) & 3 in a row & 2 & 4 \\ \hline
**Phase 4** & Fully open & 80\% (5/6 correct) & 80\% (10/12 correct) & 3 in a row & 2 & 4 \\ \hline  & \multicolumn{4}{c}{Sensitivity = TP/(TP + FN) Rate of alert to positive samples} \\  & \multicolumn{4}{c}{Specificity = TN/(FP + TN) Rate of no alert to negative/blank samples} \\ \hline \hline \end{tabular}
\end{table}
Table 3: Training progression criteria.  
COVID-19 negative. Dogs were not rewarded, nor was their behavior marked for sniffing the negative sample.  
During this phase, the COVID-19 negative jars were covered by a lid with 1 hole in the top, as compared to the fully open COVID-19 positive jars. This approach allowed the dogs to utilize two differences in odor for differentiation: first, the scent profile difference between COVID-19 negative and COVID-19 positive, and second, a concentration difference where the COVID-19 positive odor, covered with a mesh lid, was stronger than the COVID-19 negative odor, covered with a single-hole punch lid. The concentration difference functioned as \"training wheels\", which were phased out as the dogs' training progressed. This method is known as \"errorless learning\" [37] and has been used in a previous COVID-19 detection study from the PVWDC [20].  
At the beginning of each training session during the odor learning phase, the dogs were presented with the COVID-19 positive and COVID-19 negative jars on the floor. The location was randomized--either left or right--across ten trials. The trainer clicked when the dog sniffed or showed a trained final response on the COVID-19 positive jar and was ignored when the dog sniffed or showed a trained final response on the COVID-19 negative jar.