The goal of this project is to validate respiratory and neurologic disease testing using a targeted nextgeneration sequencing (tNGS) panel we previously developed. We expect this assay, which includes 34 different pathogens known to be associated with reproductive/neurologic disease in dogs, will perform as well as individual real-time PCR assays designed to detect each of these pathogens individually. This type of comprehensive testing takes the guesswork out of choosing which individual tests or small panel of tests to run to diagnose disease.
We have now completed testing 100% of the pathogens/DNA representing the pathogens that are detectable with this assay, and all but 1 could be detected. The pathogen missed was Campylobacter jejuni. This one was included for its possible role in reproductive disease, but it is more likely to cause diarrhea in dogs. Therefore, we will not redesign the assay at this point.
We performed some additional testing of sensitivity (limit of detection) using synthetic DNA to represent 12 additional pathogens in the assay, for which we could not obtain clinical samples. We determined for 11 of these that we could detect approximately 100-1000 copies of organism in the sample. One of the fungal pathogens had a lower sensitivity, requiring approximately 10,000 copies in the sample to be detected. We can increase the sensitivity of the detection for the fungal pathogen by adding more primers to the panel for this organism.
We performed some additional diagnostic sensitivity and specificity testing using 20 canine abortion cases we received at the Purdue Animal Disease Diagnostic Lab for necropsy. Fetal/neonatal tissues are tested by a real-time PCR panel and an aerobic culture is performed as part of the necropsy, looking for infectious organisms associated with abortion in dogs. Based on comparison between these tests and our tNGS assay, the diagnostic sensitivity and specificity for the tNGS assay were 100%. There were 6 positive and 14 negative samples. To date, we have tested 42/50 samples we planned to test for this aim. We will try to find more positive samples for testing to complete this aim.
We evaluated the data from the blinded panel provided by UC Davis. Unfortunately, they did not have enough positive neurologic samples for us to test. They sent us 7 canine neurologic samples and 11 canine respiratory samples. The point of this testing is to evaluate reproducibility of the tNGS assay between labs. Our collaborator at U Penn has these samples and plans to test them by our tNGS method in September. It will be interesting to see if our collaborator can reproduce our results using our method.
We are on target to complete all the work for the remaining aims by the end of the contract period.