Study design
This study was based on a hypothesis that assumes the sniffer dogs are able to detect metabolic changes in human body, which is caused by the pathological activities of SARS-Cov-2 virus. For this purpose, we designed a training process to condition six male and female dogs from five different breeds including German shepherd, German black, Labrador, Golden retriever, and Border gypsy. The study was conducted into two different training sectors. One study on participants’ pharyngeal secretions samples tested by RT-PCR as the gold standard method to detect positive and negative cases and another study on participants’ face masks and clothes using , who were already tested positive or negative using RT-PCR. Patents were from cases hospitalized in different ICUs across city of Tehran. Negative cases were healthy individuals who were RT-PCR negative for SARS.CoV-2 virus. Finally, the verification test was carried out in a single-blind process.
This study was approved by Iran’s Ministry of Health, Treatment, and Medical Education (IR.AJAUMS.REC.139.055). All dog trainers were fully equipped with proper Personal Protection Equipment (PPE) when working with dogs and the samples.
Sniffer dogs’ details
Study dogs belonged to the SK9 Dogs Training School. Each dog’s training background, age, and gender had to be taken into account. Since there was no evidence on the success rate of species for detecting the COVID-19 virus, our study dogs were selected from different species and ages. The dogs’ characteristics are shown in Table 1.
Table 1. Dogs’ characteristics
Name
|
Age ( year)
|
Gender
|
Species
|
Lexi
|
1
|
Female
|
Labrador
|
Sami
|
2
|
Male
|
Border gypsy
|
Saray (SY)
|
2
|
Female
|
German shepherd
|
Kuzhi (KZH)
|
1.5
|
Female
|
German black
|
Marco (MRC)
|
1.5
|
Male
|
Labrador
|
Zhico
|
3
|
Male
|
Golden retriever
|
Dog training
Three dogs including one German shepherd, one Labrador, and one Border gypsy were intensively trained by the classical conditioning method for seven weeks (31-33). They were introduced to the pharyngeal secretions of both and COVID-19 patients and healthy individuals. The specimens were daily transported from the hospital laboratories to the training site under safe and standard conditions. These samples were placed in groups of 10 consisting of one to three positive samples for COVID-19 inside a dogs training wheel. Positive samples were obtained from the patients admitted to the ICU and were taken from both male and female patients at different ages before and after taking medicines. It should be noted that some patients were completely healthy before contracting COVID-19, while some had underlying diseases such as diabetes, coronary diseases, heart failure, renal failure, respiratory disease such as asthma and Chronic Obstructive Pulmonary Disease (COPD), and Senecavirus A (SVA). In the second section of the study, the training set included face masks and clothes and another three dog were trained using 1300 clothes and 1300 face masks of Covid-19 patients. These stuffs had been worn for 24 hours before being used as samples . The control samples for this section included the same hospital clothes and masks from the patients who were admitted in the hospital but were proven by RT-PCR to be not COVID-19 case.
Safety and protection protocols
Testing: Before startingproject, all training team members as well as dogs were tested for COVID-19 using RT-PCR method to make sure that they were not themselves infected by the virus. In the course of the study also, the training team members were tested frequently on days 1, 21, 35, and 49. Two weeks after the final verifications, all team members were tested again to make sure they were infected during the past week. Also all protection measures were taken to protect the dogs. Dogs were quarantined for 2 weeks after the testing events. They underwent RT-PCR tests two weeks before and after the testing event and all were negative for SAR-CoV-2 contamination.
Personal protection: All team members were highly required to use th PPE, face masks, and shields similar to those used in ICUs.
Quarantine: All training team members stayed 24hours/7days in the training site during the whole study period of time.
Samples delivery: Pharyngeal secretion samples, face masks, and clothes were safely delivered to the site in sealed boxes.
Sanitizing: Training site, training equipment and the dormitory of the team were sanitized twice a day during the training period.
Dogs condition: The dogs were kept in standard cages under standard conditions and were fed with high-quality dog foods. It should be noted that no forceful training equipment were utilized.
Verification test
In late April 2020, following seven weeks of intensive training where each dog underwent the training process for averagely about 1000 times, and nearly 120 tests were performed during the course, the verifying tests were conducted for both groups of dogs.. For the verification test of the first group of dogs, 80 samples of pharyngeal secretions that 26 and 54 were positive and negative respectively (Negative samples were from healthy individuals) were used in a single-blind fashion. Fr the second group of the dogs 120 samples consisting of clothes and face masks were used that of them 50 and 70 were positive and negative respectively. Negative cases were identified by PCR. However, the negative symptoms of the subjects were checked 2 weeks before and after the test. Notable to mention that the samples were taken from different medical centers and people with various levels of disease severity. The samples were used from the population of Tehran in Iran, and considering that Tehran is a homogeneous sample of the whole country and to be considered socio-economic; positive samples were collected from several different hospitals for maximum diversity.
At the end of the study, none of the dogs had COVID-19 and were completely healthy.
Statistical analysis
Epidemiological and statistical tests including sensitivity or True Positive Rate (TPR), specificity or True Negative Rate (TNR), positive condition, negative condition, False Negative Rate (FNR), False Positive Rate (FPR), Positive and Negative Likelihood Ratios (LR+/LR-) were performed and the data were analyzed using the SPSS software, version 23.