For all tested agents, the sensitivity (defined as a “low percentage of false negative results”) of sampling and testing with the Sentinel EAD capture system (holder location, design and electrostatic capture medium) and analysis by Real-Time PCR has been repeatedly demonstrated as at least equal or, most of the time, much more sensitive than usual sampling methods used with direct and indirect methods, particularly when relying on the use of sentinel animals.
It is important to point out that the sensitivity (capture efficiency) appears to be similar after about 4 weeks (usually the first screening during quarantine) and 12 weeks (quarterly colony screening). The electrostatic capture medium is not getting saturated by dust way more than 3-4 months.
At this stage, it is worth reminding that a solution has been found to the usual drawback of PCR samples taken directly from animals: even if shedding is transitory, e.g. of coronaviruses in immunocompetent animals, the capture medium will collect the particles of interest and allow a reliable easy detection. Of course, the overall sensitivity performance requires, at diagnostic lab level, fully validated methods of NA extraction and PCR amplification.
The second critical question to be addressed when assessing and validating a new screening approach is about the specificity (defined as a “low percentage of false positive results”) of the combined sampling / testing methodology. As for the sensitivity assessment, the “real condition” testing conducted in North America and in Europe (about 50 research institutes) allowed gathering very useful feedback from users, demonstrating an outstanding specificity, i.e. the absence of false positive results.
The first fear is to deal with dead / residual nucleic acids (NA) present in feed or bedding, even if irradiated or autoclaved. A second concern is the risk of interference due to residual NA present in dust accumulated in the exhaust plenum.
Over more than two years of data collection, no false positive due to “contaminated” bedding or feed was reported. The cause was attributed to the real-time / quantitative PCR, which allows discriminating (by setting a “CT” value between positive or borderline results) a very low level of NA vs. the level generated with a shedding by animals, even if low and transient.
In addition, it appeared very quickly that no difference could be observed between racks housing mice from the same group and with an identical health status, if the racks were thoroughly cleaned before placing the animals or if they were not cleaned for a long time (up to one year). It allowed demonstrating that detected biological particles from agents were actually originating directly from the cages, not from accumulated dust in the plenum. This appeared even more obvious with the results of comparative screening results between plenum swabbing and Sentinel EAD capture: false positives (historical contaminations) were the rule with plenum swabs, while it appeared that Sentinel EAD captured exclusively biological particles with past or ongoing shedding by mice, originating directly from cages.
Further to these observations, which allowed concluding about the lack of interference of residual NA and the specificity of the “Sentinel” sampling method, we also gathered very positive feedback related to rack sanitation. As “dead” / residual NA are not fully destroyed by chlorine dioxide, hydrogen peroxide or even by autoclaving, most users stress on the need to guarantee a very efficient cleaning phase, allowing removing and eliminating these highly resistant chemical structures, rather than trying to fully denaturing NA. This approach is neither new nor unusual, as it is already recommended to eliminate highly resistant contaminants such as parasite eggs or parvoviruses, prior to rack final disinfection. The user feedback was about the easiness and efficiency of cleaning of the rack exhaust system, thanks to the horizontal manifolds, easy to hose through lateral openings and to the door giving full access to the vertical plenum.
Sentinel is a trademark of Allentown, Inc., EAD is a registered trademark of Charles River Laboratories, Inc.