Appropriate antibiotic prescription contributes to reducing bacterial resistance; therefore, it is critical to provide training regarding this challenge. The objective of this study was to develop a virtual learning environment for antibiotic prescription and to determine its impact on dentists’ awareness, attitudes, and intention to practice. First, the learning content on multimedia resources was developed and distributed into three challenges that participants had to overcome. Then, a quasi-experimental study was performed in which the virtual learning environment was implemented on dentists from seven Colombian cities. The median of correct answers and the levels of awareness, attitudes, and intention to practice were compared before, immediately after, and 6-months post-intervention. Wilcoxon signed-rank and McNemar’s tests were used to determine the differences. A total of 206 participants who finished the virtual learning environment activities exhibited a favorable and statistically significant impact on the median of correct answers of awareness (p < 0.001), attitudes (p < 0.001), and intention to practice (p = 0.042). A significant increase occurred in the number of participants with a high level of awareness (p < 0.001) and a non-significant increase in participants with high levels of attitudes (p = 0.230) and intention to practice (p = 0.286). At 6 months, the positive effect on the median of correct answers on awareness and intention to practice persisted (p < 0.001); however, this was not evident for attitudes (p = 0.105). Moreover, there was a significant decrease in the number of participants who showed low levels of awareness (p = 0.019) and a slight increase in those with high levels of the same component (p = 0.161). The use of a virtual learning environment designed for dentists contributed to a rapid improvement in awareness and intention to practice antibiotic prescription; however, their attitudes and information retention need reinforcement.

Dental pain, which is the main reason for patients consulting dentists, is classified as a public health concern. The study of cellular and molecular mechanisms contributing to pain is a fundamental element for developing new analgesics. By using a selective antagonist in an in vitro model, this study aimed to establish the role of TRPV-1 in human odontoblast-like cells (OLCs) as a therapeutic target for dental pain mediated by noxious thermal and osmotic stimuli. OLCs were differentiated from dental pulp mesenchymal cells and TRPV1 expression was evaluated. Activation of TRPV-1 was determined by evaluating changes in calcium concentration after stimulation with mannitol and xylitol hyperosmotic solutions or DMEM heated at 45 °C, using the fluorescent calcium probe Fluo-4 AM. In addition, changes in fluorescence (F/F0) due to calcium flux were evaluated using fluorometry and flow cytometry. Simultaneously, the cells were co-stimulated with the selective antagonist capsazepine (CZP). OLCs expressed DSPP and DMP-1, confirming their cellular phenotype. TRPV1 was expressed, and its activation by different stimuli produced an increase in cytosolic Ca²⁺ which was reduced by the antagonist. Both methods used to evaluate TRPV1 activation through the measurement of calcium probe fluorescence showed similar patterns. These results suggest that TRPV-1 modulation using an antagonist can be implemented as a pharmacological strategy for managing dental pain mediated by hyperosmotic and thermal stimuli.

The human metapneumovirus (hMPV) is an important viral agent associated with severe infections of the upper and lower airways, especially in young children and immunosuppressed subjects. Nevertheless, in vitro studies of hMPV are very difficult due to the little knowledge we have on its laboratory manipulation. Objective: The aim of this study was to isolate and propagate hMPV from patients, and to establish a method to quantify the virus by plaque assay. Method: As part of a Latin American respiratory virus surveillance study, 12 nasal secretion samples – hMPV-positive by direct fluorescence – were inoculated on LLC-MK2 cells to isolate the virus. The supernatants were re-inoculated and the cytopathic effect and syncytium formation were evaluated daily; the infection was confirmed by immunofluorescence and RT-PCR. A protocol to titrate the harvested virus was established inoculating serial dilutions on LLC-MK2 cells, and agarose was then added as an overlay. After different time periods, the monolayers were fixed and stained with Naphthol blue/black or crystal violet and finally the viral titer was obtained. Results: Eight out of 12 hMPV-positive respiratory samples were positive for the isolation and confirmed by RT-PCR and immunofluorescence, but the cytopathic effect and syncytium formation were observed only in 5 cultures. One out of 8 viral isolates was used for propagation and plaque assay standardization. We found that incubation for 7 days in the semisolid overlay yielded plaques with appropriate size and shape to be counted, although crystal violet staining showed slightly larger plaques than those seen with Naphthol blue/black staining. Conclusions: The isolation and propagation from patient-derived hMPV and the standardization of a practical, reliable, and inexpensive method of detection and quantification of hMPV were carried out, without the additional use of antibodies that had not been reported previously. These results offer some important insights for future studies of cellular and molecular biology of hMPV.

PCR and its variants (RT‐PCR and qRT‐PCR) are valuable and innovative molecular techniques for studying nucleic acids. qPCR has proven to be highly sensitive, efficient, and reproducible, generating reliable results that are easy to analyze. During the COVID‐19 pandemic, qPCR became the gold standard technique for detecting the SARS‐CoV‐2 virus that allowed to confirm the infection event, and those asymptomatic ones, and thus save millions of lives. In‐house multiplex qPCR tests were developed worldwide to detect different viral targets and ensure results, follow the infections, and favor the containment of a pandemic. Here, we present the detailed fundamentals of the qPCR technique based on fluorogenic probes and processes to develop and optimize a successful multiplex RT‐qPCR test for detecting SARS‐CoV‐2 that could be used to diagnose COVID‐19 accurately.