Use of saliva and RT-PCR screening for SARS-CoV-2 variants of concern: Surveillance and monitoring.

Genomic surveillance has been applied since the beginning of the COVID-19 pandemic to track the spread of the virus, leading to the characterization of multiple SARS-CoV-2 variants, including variants of concern (VOC). Although sequencing is the standard method, a rapid molecular test for screening and surveillance of VOC is considered for detection. Furthermore, using alternative saliva as specimen collection facilitates the implementation of a less invasive, self-collected sample. In this study, we applied a combinatory strategy of saliva collection and reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 VOC detection. Saliva samples from patients attending a tertiary hospital with suspected COVID-19 were collected and SARS-CoV-2 RNA was detected using SARS-CoV-2 RT-qPCR reagent kit (PerkinElmer). Positive saliva samples were screened for SARS-CoV-2 VOC with previously described RT-PCR for Alpha, Beta, and Gamma variants. Saliva samples were positive in 171 (53%) of 324 tested. A total of 108 (74%) from positive samples were also positive for VOC by RT-PCR screening. Those samples were found between January and August 2021. This approach allowed us to successfully use an alternative and complementary tool to genomic surveillance to monitor the circulation of SARS-CoV-2 VOC in the studied population.

Patient-reported pain after surgical removal of leukoplakia - an observational 1-year follow-up study.

OBJECTIVE: Oral leukoplakia (OL) presents as a white lesion of the oral mucosa and is not typically associated with the sensation of pain. OL should be surgically removed when possible because it is considered a potentially malignant oral disorder (PMOD). This study assessed the pain sensations experienced by patients in association with the occurrence and surgical treatment of OL. METHODS: Inclusion criteria were: a clinical diagnosis of OL; biopsy excision; and observation for at least 12 months in the ORA-LEU-CAN study. At the first visit, all the patients were asked about the occurrence of symptoms within the lesion. Ninety-four subjects were assessed over a period of 1 year. All patients underwent complete removal of OL. The patient cohort was divided into three sub-groups: (i) no pain before excision and at the 1-year follow-up; (ii) pain before excision; and (iii) pain at the 1-year follow-up. RESULTS: Overall, pain was reported by 21.3% of the patients at the study start whereas 13.8% of the patients reported pain 1 year after surgical treatment. Patient-reported pain from the lesion at study inclusion was significantly associated with lesions found on the lateral side of the tongue (p=.002). Pain reported by patients one year after surgery was significantly related to female gender (p=.038) and the presence of epithelial cell dysplasia (p=.022). CONCLUSION: We conclude that surgical removal of OL results in a low risk of long-term post-surgical pain. However, OL located on the lateral side of the tongue and in OL with dysplasia are more likely to be associated with pain.

Infrared spectroscopy as a predictive tool for the severity of COVID-19 using patient's saliva: A strategy to avoid hyperinflammation.

Discriminate the severity level of COVID-19 disease is still a challenge. Here we investigate the capability of micro-infrared absorption spectroscopy (micro-FTIR) to probe COVID-19 severity level and predict hyperinflammation, correlating the assigned vibrational data to relevant biomolecules related to the immune system. Saliva of 184 patients was analysed by ELISA assay (Hepcidin) and micro-FTIR. Vibrational bands related to IgM and IgA can discriminate healthy from Severe individuals (sensitivity ≥ 0.749, specificity ≥ 0.945) and are less effective in discriminating Mild or Moderate individuals from the Severe group (sensitivity ≥ 0.628, specificity ≥ 0.867). Analysis of the second derivative of spectra probed increased levels of IL-6 in the saliva a key additional information for the degree of severity prediction. Because the model discriminates all the groups regarding the Severe group, it predicts an intense state of inflammation based on FTIR analysis. It is a powerful tool for predicting hyperinflammation conditions related to SARS-CoV-2 infection and may be an ally in implementing drugs or therapeutic approaches to manage COVID-19 in the Severe stage in healthcare facilities.

Investigation of Oral Shedding of Torquetenovirus (TTV) in Moderate-to-Severe COVID-19 Hospitalised Patients.

BACKGROUND: Torquetenovirus (TTV) is a small DNA virus constituting the human virome. High levels of TTV-DNA have been shown to be associated with immunosuppression and inflammatory chronic disorders. AIM: To assess the possible association between the salivary viral load of TTV-DNA in patients hospitalised due to COVID-19 and disease severity. METHODS: Saliva samples collected from 176 patients infected with SARS-CoV-2 were used to investigate the presence of SARS-CoV-2 and TTV-DNA by use of real-time RT-PCR. RESULTS: The majority of patients were male with severe COVID-19. Presence of SARS-CoV-2 was observed in the saliva of 64.77% of patients, showing TTV-DNA in 55.68% of them. Patients with impaired clinical conditions (p < 0.001), which evolved to death (p = 0.003), showed a higher prevalence of TTV-DNA. The median viral load in patients with severe condition was 4.99 log10 copies/mL, in which those who were discharged and those evolving to death had values of 3.96 log10 copies/mL and 6.27 log10 copies/mL, respectively. A statistically significant association was found between the distribution of TTV-DNA viral load in saliva samples and severity of COVID-19 (p = 0.004) and disease outcomes (p < 0.001). CONCLUSIONS: These results indicate that TTV-DNA in saliva could be a useful biomarker of COVID-19 severity and prognosis.

Lack of direct association between oral mucosal lesions and SARS-CoV- 2 in a cohort of patients hospitalised with COVID-19.

Background: COVID-19 is a disease affecting various human organs and systems, in which the virus seeks to interact with angiotensin-converting enzyme 2 receptors. These receptors are present in the oral cavity, but the direct relationship between such an interaction and possible oral manifestations of COVID-19 is still unclear. Aim: The present study evaluated oral manifestations in a cohort of COVID-19 patients during the period of hospitalisation. Methods: In total, 154 patients presenting moderate-to-severe forms of COVID-19 had their oral mucosa examined twice a week until the final outcome, either discharge or death. The oral alterations observed in the patients were grouped into Group 1 (pre-existing conditions and opportunistic oral lesions) and Group 2 (oral mucosal changes related to hospitalization). Results: Oral lesions found in the patients of Group 1 are not suggestive of SARS-CoV-2 infection as they are mainly caused by opportunistic infections. On the other hand, oral alterations found in the patients of Group 2 were statistically (P < 0.001) related to intubation and longer period of hospitalisation. Conclusion: It is unlikely that ulcerative lesions in the oral cavity are a direct manifestation of SARS-CoV-2 or a marker of COVID-19 progression.

MALDI-TOF mass spectrometry of saliva samples as a prognostic tool for COVID-19.

BACKGROUND: The SARS-CoV-2 infections are still imposing a great public health challenge despite the recent developments in vaccines and therapy. Searching for diagnostic and prognostic methods that are fast, low-cost and accurate are essential for disease control and patient recovery. The MALDI-TOF mass spectrometry technique is rapid, low cost and accurate when compared to other MS methods, thus its use is already reported in the literature for various applications, including microorganism identification, diagnosis and prognosis of diseases. METHODS: Here we developed a prognostic method for COVID-19 using the proteomic profile of saliva samples submitted to MALDI-TOF and machine learning algorithms to train models for COVID-19 severity assessment. RESULTS: We achieved an accuracy of 88.5%, specificity of 85% and sensitivity of 91.5% for classification between mild/moderate and severe conditions. When we tested the model performance in an independent dataset, we achieved an accuracy, sensitivity and specificity of 67.18, 52.17 and 75.60% respectively. CONCLUSION: Saliva is already reported to have high inter-sample variation; however, our results demonstrates that this approach has the potential to be a prognostic method for COVID-19. Additionally, the technology used is already available in several clinics, facilitating the implementation of the method. Further investigation using a larger dataset is necessary to consolidate the technique.

Salivary Microbial Dysbiosis Is Associated With Peri-Implantitis: A Case-Control Study in a Brazilian Population.

Background and Objectives: The aim of this study was to examine the salivary microbiome in healthy peri-implant sites and those with peri-implantitis. Methods: Saliva samples were collected from 21 participants with healthy peri-implant sites and 21 participants with peri-implantitis. The V4 hypervariable region of the 16S rRNA gene was sequenced using the Ion Torrent PGM System (Ion 318™ Chip v2 400). The NGS analysis and composition of the salivary microbiome were determined by taxonomy assignment. Downstream bioinformatic analyses were performed in QIIME (v 1.9.1). Results: Clinical differences according to peri-implant condition status were found. Alpha diversity metrics revealed that the bacterial communities of participants with healthy peri-implant sites tended to have a richer microbial composition than individuals with peri-implantitis. In terms of beta diversity, bleeding on probing (BoP) may influence the microbial diversity. However, no clear partitioning was noted between the salivary microbiome of volunteers with healthy peri-implant sites or volunteers with peri-implantitis. The highest relative abundance of Stenotrophomonas, Enterococcus and Leuconostoc genus, and Faecalibacterium prausnitzii, Haemophilus parainfluenzae, Prevotella copri, Bacteroides vulgatus, and Bacteroides stercoris bacterial species was found in participants with peri-implantitis when compared with those with healthy peri-implant sites. Conclusion: Differences in salivary microbiome composition were observed between patients with healthy peri-implant sites and those with peri-implantitis. BoP could affect the diversity (beta diversity) of the salivary microbiome.