**5. Precautions for testing new devices** *in vivo*

Finally, the investigators must be aware of the biases frequently encountered in clinical trials that evaluate microbial contamination and colonization of oral devices and prosthesis. Recommendations and guidelines to evaluate the benefits of prophylactic anti-*Candida* procedures are similar to these advocated for any oral care product. Two important biases that must be taken into account are the influence of investigators on patients' hygiene behavior (Grimoud et al., 2005) and the galenic formulation of products lacking the active molecule. Evaluation of dentifrice efficiency for denture hygiene also needs other controls: one testing the product without brushing and one testing the mechanical brushing alone. The abrasive effect of the product must be evaluated, and the abrasiveness of saliva itself is another concern to be considered.

Quantification of the *Candida* biomass that is adherent to the device is difficult in practice. Yeast samples from the oral environment can be collected by rinsing, imprinting, or swabbing. Swabs and imprints are more suitable for gathering yeasts attached to surfaces, and swabbing is easier for clinical studies on a larger scale. Procedures to quantify yeast biomass *in vitro* are not applicable *in vivo* for epidemiological studies and hygiene purposes, particularly since denture-wearers are not always compliant. Dentures can be rinsed in saline and brushed in standard condition to harvest microbial cells. The suspension is thereafter serially diluted for counting (Panzeri et al., 2009).

Another study (Vanden Abbeele et al., 2008) documented the reliability of oral swabbing to investigate yeast carriage on denture. Sampling dentures for *Candida* is more than just a diagnostic tool: it could present an opportunity to verify the patient's compliance with hygiene advice as well as the efficiency of new topical antifungals. Yeast counts after swab

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**8. References** 

MDER

culture reflect the biomass present on the oral surfaces, but this is not the number of yeast cells included in oral biofilms. Colony forming units (CFU) counted on the agar medium represented only a small part of the cells harvested by the cotton, as assessed by three successive spreadings of the same cotton that provided similar data (in the range interval of 0.1 logarithmic units). Furthermore, two successive swabs of the same oral surface yielded similar quantities of yeast cells (in the range interval of 0.5 logarithmic units).

Finally, investigators themselves can influence the hygiene behavior of the subjects under study. The study previously quoted (Vanden Abbeele et al., 2008) also analyzes the effect of the oral care program. In the absence of any hygiene advice, a second denture swabbing taken in 46 patients after an interval of one week demonstrated only minor variations, thus minimizing the hygiene-stimulation effect produced by pursuing the collection of samples. Repeated sampling (at one-week intervals) of 46 different healthy denture-wearers demonstrated yeast counts remaining in the same range (±1 logarithmic unit) for more than 85% of the denture swabs and mucosa samples. Values below the lower limit (-1 logarithmic unit) occurred in less than 15% of denture and mucosa swabs. This was attributed to behavioral changes in hygiene practice following the investigators' first visit. By contrast, a hygiene program including a placebo oral gel (tested to be inactive *in vitro*) led to a decrease of yeast carriage after two weeks.
