Saliva Samples Research Articles

A handheld electrochemical bacterial sensor based on multifunctional composite hydrogels and DNA biomimetic nanowalls for accurate detection of Porphyromonas gingivalis

Porphyromonas gingivalis (Pg) is not merely one of the important periodontal pathogens, but also exhibits a profound and intimate connection with various systemic diseases, such as cancer, cardiovascular disease, and Alzheimer’s disease. Hence, a handheld electrochemical bacterial sensor (Hebs) is developed for point-of-care testing of Pg in saliva based on an enhanced personal glucose meter platform. In the Hebs, a novel multifunctional composite hydrogel (MFCHgel) is prepared with outstanding conductivity, electrical activity, adhesion and modifiability. In particular, its superior adhesion effectively addresses the inherent issue of electrode-modified material being prone to detachment while ensuring excellent conductivity, which is extremely beneficial for the stability of biosensor. In addition, by assembling DNA biomimetic nanowalls (DBNWs), the surface negative charge and steric hindrance of Pg are enhanced significantly, leading to a remarkable improvement in the electrical impedance response of Pg. Based on their cooperation, the Hebs is capable of effectively detecting Pg within the range of 10 to 1 × 107 CFU/mL, with an impressive limit of detection as low as 6 CFU/mL. Significantly, Hebs exhibits remarkable accuracy in analyzing 40 clinical saliva samples and offers distinct advantages in terms of time consumption and portability as compared to the ELISA kit. This work successfully develops a Hebs for point-of-care testing of Pg, thereby presenting a novel perspective for the advancement of portable bacterial detection devices.

Salivary Molecular Spectroscopy with Machine Learning Algorithms for a Diagnostic Triage for Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a genetic disease characterized by poor formation of tooth enamel. AI occurs due to mutations, especially in AMEL, ENAM, KLK4, MMP20, and FAM83H, associated with changes in matrix proteins, matrix proteases, cell-matrix adhesion proteins, and transport proteins of enamel. Due to the wide variety of phenotypes, the diagnosis of AI is complex, requiring a genetic test to characterize it better. Thus, there is a demand for developing low-cost, noninvasive, and accurate platforms for AI diagnostics. This case-control pilot study aimed to test salivary vibrational modes obtained in attenuated total reflection fourier-transformed infrared (ATR-FTIR) together with machine learning algorithms: linear discriminant analysis (LDA), random forest, and support vector machine (SVM) could be used to discriminate AI from control subjects due to changes in salivary components. The best-performing SVM algorithm discriminates AI better than matched-control subjects with a sensitivity of 100%, specificity of 79%, and accuracy of 88%. The five main vibrational modes with higher feature importance in the Shapley Additive Explanations (SHAP) were 1010 cm-1, 1013 cm-1, 1002 cm-1, 1004 cm-1, and 1011 cm-1 in these best-performing SVM algorithms, suggesting these vibrational modes as a pre-validated salivary infrared spectral area as a potential biomarker for AI screening. In summary, ATR-FTIR spectroscopy and machine learning algorithms can be used on saliva samples to discriminate AI and are further explored as a screening tool.

Impact of Stimulation Duration in taVNS-Exploring Multiple Physiological and Cognitive Outcomes.

Transcutaneous auricular vagus nerve stimulation (taVNS) is a non-invasive neuromodulation technique that modulates the noradrenergic activity of the locus coeruleus (LC). Yet, there is still uncertainty about the most effective stimulation and reliable outcome parameters. In a double blind, sham-controlled study including a sample of healthy young individuals (N = 29), we compared a shorter (3.4 s) and a longer (30 s) stimulation duration and investigated the effects of taVNS (real vs. sham) on saliva samples (alpha amylase and cortisol concentration), pupil (pupillary light reflex and pupil size at rest) and EEG data (alpha and theta activity at rest, ERPs for No-Go signals), and cognitive tasks (Go/No-Go and Stop Signal Tasks). Salivary alpha amylase concentration was significantly increased in the real as compared to sham stimulation for the 30 s stimulation condition. In the 3.4 s stimulation condition, we found prolonged reaction times and increased error rates in the Go/No-Go task and increased maximum acceleration in the pupillary light reflex. For the other outcomes, no significant differences were found. Our results show that prolonged stimulation increases salivary alpha-amylase, which was expected from the functional properties of the LC. The finding of longer response times to short taVNS stimulation was not expected and cannot be explained by an increase in LC activity. We also discuss the difficulties in assessing pupil size as an expression of taVNS-mediated LC functional changes.

Expression Analysis of Circulating microRNAs in Saliva and Plasma for the Identification of Clinically Relevant Biomarkers for Oral Squamous Cell Carcinoma and Oral Potentially Malignant Disorders.

This study aims to evaluate the expression of salivary and plasmatic miRNAs as diagnostic biomarkers in patients with oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs). A total of 25 patients were divided into three groups, according to their diagnosis: OSCC patients (n = 14); OPMDs patients (n = 6); and healthy controls (n = 5). At the time at diagnosis/enrolment, patients underwent salivary and plasmatic collection. The expression of miRNA -21, -31, -138, -145, -184, and -424 were evaluated by real-time PCR. An F-test and ANOVA test were performed to evaluate the miRNA levels (significance at p < 0.05). By comparing miRNA expression levels from saliva, a statistically significant difference emerged in the expression of miR-138 and miR-424 between the three groups (p < 0.05). In particular, these two miRNAs showed decreased expression levels in saliva samples from OSCC and OPMD patients compared to those from healthy controls. On the other hand, miRNA expression levels in plasma were low in all the groups, and no statistically significant differences were found. Overall, our results showed that liquid biopsy from saliva may be a useful tool for the identification of diagnostic molecular biomarkers in OSCC and OPMDs.

Analysis of Salivary Neuropeptides in Anxiety and Depression Using the Luminex MAGPIX® System.

Anxiety and depressive disorders are highly prevalent mental health conditions, affecting millions worldwide.Advancements in neurobiology have identified the effects of various neuropeptides in modulating mood and stress responses.Some of the well-researched neuropeptides in plasma are oxytocin (OXT), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-endorphin, neurotensin, and substance P.In this study, we used methods of liquid biopsy to acquire saliva samples to analyze the concentrations of neuropeptides associated with depression. The study was conducted in Bratislava, Slovakia, from January to June 2022. Participants were 20 subjects treated for depression and anxiety without medication; the control group consisted of 20 healthy individuals with no personal history of depression or anxiety. Salivary samples were collected using buccal swabs to measure the concentrations of the examined neuropeptides. Laboratory analysis was based on detecting fluorescent signals performed on the Luminex MAGPIX® System (Luminex Corporation, Austin, Texas).Means and standard deviations were calculated for individual neuropeptide levels.To determine if there are statistically significant differences in neuropeptide levels between individuals with and without depression, independent t-tests and a one-way ANOVA were conducted. Our findings indicate a significant decrease in all studied neuropeptides in subjects compared to healthy controls. Reductions in mean levels were observed for OXT (7.3), alpha-MSH (3.9), beta-endorphin (2.9), neurotensin (15.1), and a 6.9-fold decrease for substance P.Alpha-MSH and beta-endorphin showed higher variability in measured levels within both groups. The results of this study indicate that the levels of the studied salivary neuropeptides, OXT, alpha-MSH, beta-endorphin, neurotensin, and substance P, are statistically significantly reduced in individuals with depression compared to healthy controls.

An automated syringe-based PoC RT-LAMP LFB platform for infectious disease detection from saliva

Decentralized Point-of-Care (PoC) diagnostics hold momentous potential for rapid and accessible viral infection disease detection. Presented is a unique design application of an easy-to-use (plug-and-play) platform for viral detection. The platform leverages a simplified multiplex Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Lateral Flow Biosensor (LFB) assay with a lyophilized master mix, eliminating the need for RNA isolation or special reporting equipment. A user-friendly Saliva Measuring Tube (SMT) ensures accurate saliva volume self-collection, and a Syringe-based PoC (SPoC) platform automates sample treatment, reagent mixing, and temperature control using readily available components and consumables. The platform’s performance was evaluated by multiplexed detection of the SARS-CoV-2 N2 target gene and human ACTB gene from saliva samples. The SPoC platform achieved a detection limit of spiked 500 copies/mL for SARS-CoV-2 and consistent internal control readout. The presented PoC system offers a promising initial step for further development toward a decentralized solution for viral infection testing.

Altered circadian expression of clock genes and clock-regulatory epigenetic modifiers in saliva of children with fetal alcohol spectrum disorders

Prenatal alcohol-exposed (AE) infants and children often demonstrate disrupted sleep patterns, including more frequent awakenings, reduced total sleep time, and more night-to-night sleep variability. Despite the strong connection between sleep patterns and circadian rhythmicity, relatively little is known about circadian rhythm disruptions in individuals with AE. Recently, several reports demonstrated that evaluating the expression patterns of human clock genes in biological fluids could reveal an individual’s circadian phenotype. Human saliva offers an emerging and easily available physiological sample that can be collected non-invasively for core-clock gene transcript analyses. We compared the expression patterns of core-clock genes and their regulatory genes in salivary samples of children aged 6–10 years-old with and without AE during the light cycle between ZT0-ZT11. We isolated the RNA from the samples and measured the expression patterns of core clock genes and clock regulating genes using the human specific primers with quantitative real-time PCR. Analysis of core clock genes expression levels in saliva samples from AE children indicates significantly altered levels in expression of core-clock BMAL1, CLOCK, PER1-3 and CRY1,2, as compared to those in age-matched control children. We did not find any sex difference in levels of clock genes in AE and control groups. Cosinor analysis was used to evaluate the rhythmic pattern of these clock genes, which identified circadian patterns in the levels of core clock genes in the control group but absent in the AE group. The gene expression profile of a salivary circadian biomarker ARRB1 was rhythmic in saliva of control children but was arhythmic in AE children. Altered expression patterns were also observed in clock regulatory genes: NPAS2, NFL3, NR1D1, DEC1, DEC2, and DBP, as well as chromatin modifiers: MLL1, P300, SIRT1, EZH2, HDAC3, and ZR1D1, known to maintain rhythmic expression of core-clock genes. Overall, these findings provide the first evidence that AE disturbs the circadian patten expression of core clock genes and clock-regulatory chromatin modifiers in saliva.

A Pilot Study of Saliva MicroRNA Signatures in Children with Moderate-to-Severe Traumatic Brain Injury.

Background/Objectives: Traumatic brain injury (TBI) is a leading cause of death and disability in children. Currently, no biological test can predict outcomes in pediatric TBI, complicating medical management. This study sought to identify brain-related micro-ribosomal nucleic acids (miRNAs) in saliva associated with moderate-to-severe TBI in children, offering a potential non-invasive, prognostic tool. Methods: A case-control design was used, enrolling participants ≤ 18 years old from three pediatric trauma centers. Participants were divided into moderate-to-severe TBI and non-TBI trauma control groups. Saliva samples were collected within 24 h of injury, with additional samples at 24-48 h and >48 h post-injury from the TBI group. miRNA profiles were visualized with partial least squares discriminant analysis (PLSDA) and hierarchical clustering. Mann-Whitney testing was used to compare miRNAs between groups, and mixed models were used to assess longitudinal expression patterns. DIANA miRPath v3.0 was used to interrogate the physiological functions of miRNAs. Results: Twenty-three participants were enrolled (14 TBI, nine controls). TBI and control groups displayed complete separation of miRNA profiles on PLSDA. Three miRNAs were elevated (adj. p < 0.05) in TBI (miR-1255b-5p, miR-3142, and miR-4320), and two were lower (miR-326 and miR-4646-5p). Three miRNAs (miR-3907, miR-4254, and miR-1273g-5p) showed temporal changes post-injury. Brain-related targets of these miRNAs included the glutamatergic synapse and GRIN2B. Conclusions: This study shows that saliva miRNA profiles in children with moderate-to-severe TBI may differ from those with non-TBI trauma and exhibit temporal changes post-injury. These miRNAs could serve as non-invasive biomarkers for prognosticating pediatric TBI outcomes. Further studies are needed to confirm these findings.

Unveiling the potential of the capillary-driven microfluidic paper-based device integrated with smartphone-based for simultaneously colorimetric salivary ethanol and △9-tetrahydrocannabinol analysis

Monitoring various biomarkers in saliva samples emerges as a dynamic and non-invasive method. However, the high viscosity of saliva presents a distinct challenge when integrating paper-based platforms for on-site analysis. In addressing this challenge, we introduced the capillary-driven microfluidic paper-based analytical devices (μCD-PAD) designed for user-friendly and simultaneous detection of ethanol and tetrahydrocannabinol (THC) in saliva without a sample preparation step. Employing a colorimetric approach, we quantified both analytes. Synthetic salivas of varying viscosity flowed seamlessly to the detection zone without needing a sample preparation step, and no impact on colorimetric detection due to viscosity was observed (RSD <5 %). Within 10 min after the solution reached the detection zone, the device produced a homogeneous color signal, easily analyzed by a smartphone camera. To extend the application for determination to cover a legal limit concentration of ethanol and concentration of salivary THC even 24 h after marijuana consumption, the detection time of 30 min was optimized. Moreover, a saliva sample containing both analytes was used to demonstrate the capability of the developed device to detect ethanol and THC simultaneously. No cross-talk between ethanol and THC occurred and showed recovery in the 98–102 % for ethanol and 95–105 % for THC with acceptable accuracy. This developed device exhibits excellent potential for forensic applications, providing a user-friendly, cost-effective, and real-time screening tool for detecting ethanol and THC in saliva.

Exploring HLA-C methylation patterns and nutritional status in Kichwa mothers and infants from Tena, Ecuador.

Environment and lifestyle can affect the epigenome passed down from generation to generation. A mother's nutrition can impact the methylation levels of her offspring's epigenome, but it's unclear which genes may be affected by malnutrition during gestation or early development. In this study, we examined the levels of methylated GC in the promoter region of HLA-C in mothers and infants from the Kichwa community in Ecuador. To do this, we analyzed saliva samples using bisulfite DNA sequencing. While we did not observe any significant differences in the mean methylation percentages in exon 1 of HLA-C between mothers and their infants after the first two years of lactation and life, respectively, we did find that infants tended to increase their methylation level during the first two years of life, while mothers tended to decrease it after the first two years of breastfeeding. When we compared methylation levels between mothers and infants using an ANOVA/posthoc Tukey test, we found that the average methylation for the entire population was less than 3% at T1 and T2. Although there was a tendency for infants to have higher methylation levels during their first two years of life and for mothers to have lower methylation levels after the first two years of breastfeeding, the mean values were not significantly different. However, we found a significant difference when we contrasted the data using a Kruskal-Wallis test at 0.05 for T1 AND T2 (p-value: 0.0148). Specifically, mothers had an average of X̅ = 2.06% and sons had X̅ = 1.57% at T2 (p-value: 0.7227), while the average for mothers was X̅ = 1.83% and for sons X̅ =1.77%. Finally, we identified three CpG motif nucleotide positions (32-33, 43-44, and 96-97) along the 122 bp analysis of HLA-C exon one, which was found to retain methylation patterns over time and is inherited from mother to offspring. Finally, our small pilot study did not reveal significant correlations between maternal and offspring nutritional status and DNA methylation levels of HLA-C exon one.

Social and clinical drivers of stress responses in African American breast cancer survivors

Racial differences in breast cancer morbidity and mortality have been examined between Black/African American women and White women as part of efforts to characterize multilevel drivers of disease risk and outcomes. Current models of cancer disparities recognize the significance of physiological stress responses, yet data on stress hormones in Black/African American women with breast cancer and their social risk factors are limited. We examined cortisol levels in Black/African American breast cancer patients and tested their association with social and clinical factors to understand the relationship between stress responses and women’s lived experiences. Seventy-two patients who completed primary surgical treatment were included in this cross-sectional study. Data on sociodemographic characteristics and chronic diseases were obtained by self-report. Breast cancer stage and diagnosis date were abstracted from electronic health records. Cortisol levels were determined from saliva samples. Compared to those without hypertension, patients with hypertension were 6.84 (95% CI 1.33, 35.0) times as likely to have high cortisol (p = 0.02). The odds of having high cortisol increased by 1.42 (95% CI 1.03, 1.95, p = 0.03) times for every point increase in negative life events. Hypertension and negative life events are associated with high cortisol levels in Black/African American patients. These findings illustrate the importance of understanding the lived experiences of these patients to enhance cancer health equity.

What could be the role of genetic tests and machine learning of AXIN2 variant dominance in non-syndromic hypodontia? A case-control study in orthodontically treated patients

BackgroundHypodontia is the most prevalent dental anomaly in humans, and is primarily attributed to genetic factors. Although genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNP) associated with hypodontia, genetic risk assessment remains challenging due to population-specific SNP variants. Therefore, we aimed to conducted a genetic analysis and developed a machine-learning-based predictive model to examine the association between previously reported SNPs and hypodontia in the Saudi Arabian population. Our case–control study included 106 participants (aged 8–50 years; 64 females and 42 males), comprising 54 hypodontia cases and 52 controls. We utilized TaqManTM Real-Time Polymerase Chain Reaction and allelic genotyping to analyze three selected SNPs (AXIN2: rs2240308, PAX9: rs61754301, and MSX1: rs12532) in unstimulated whole saliva samples. The chi-square test, multinomial logistic regression, and machine-learning techniques were used to assess genetic risk by using odds ratios (ORs) for multiple target variables.ResultsMultivariate logistic regression indicated a significant association between homozygous AXIN2 rs2240308 and the hypodontia phenotype (ORs [95% confidence interval] 2.893 [1.28–6.53]). Machine-learning algorithms revealed that the AXIN2 homozygous (A/A) genotype is a genetic risk factor for hypodontia of teeth #12, #22, and #35, whereas the AXIN2 homozygous (G/G) genotype increases the risk for hypodontia of teeth #22, #35, and #45. The PAX9 homozygous (C/C) genotype is associated with an increased risk for hypodontia of teeth #22 and #35.ConclusionsOur study confirms a link between AXIN2 and hypodontia in Saudi orthodontic patients and suggests that combining machine-learning models with SNP analysis of saliva samples can effectively identify individuals with non-syndromic hypodontia.

Assessing oral and toothbrush microbial profiles among high-altitude individuals with and without periodontal disease: a case-control study

BackgroundPeriodontitis is the sixth-most common disease worldwide. The oral microbiome composition and its association with Periodontal disease (PD) have been largely explored; however, limited studies have explored the microbial profiles of both oral and toothbrushes in patients with PD. Thus, this study aimed to ascertain the oral and toothbrushes microbial composition in high-altitude populations, hypothesizing that their correlation with periodontal health would differ from those at lower altitudes, potentially indicating links between environmental factors, microbial colonization patterns, and periodontal health in distinct geographic contexts.MethodsIn the present study, we enrolled 35 individuals including 21 healthy and 14 diagnosed with PD from the Lhasa region of Tibet, China. Saliva and toothbrush samples were collected from each participant to assess the association between toothbrush usage and oral microbiome with PD using 16 S rRNA gene-specific V3-V4 regions sequencing. To assess the oral and toothbrush microbiome composition and diversity and its possible link to PD.ResultsSignificantly higher Alpha diversity (Shannon index) was observed between the PD group and PD toothbrushes (p = 0.00021) and between the PD group and Healthy toothbrushes (p = 0.00041). The predominant species were Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Fusobacteria, with genera Pseudomonas, Veillonella, Neisseria, Acinetobacter, and Haemophilus. In addition, PICRUST2 analysis unveiled 44 significant pathways differentiating the disease and healthy groups, along with 29 pathways showing significant differences between their respective toothbrush microbial profiles. The distinct oral and toothbrush microbial composition among high-altitude populations suggests potential adaptations to the challenges of high-altitude environments.ConclusionThis study emphasizes the importance of tailored dental care strategies, accounting for altitude and racial factors, to effectively manage periodontal health in these communities. Further research is warranted to investigate the specific microbial mechanisms and develop targeted interventions for optimizing oral health in populations across varying altitudes.

Saliva-STAT: Sample-to-answer saliva test for COVID-19

Highly accessible and highly accurate diagnostics are necessary to combat rapidly-spreading infectious diseases, such as the recent COVID-19 pandemic. While lateral flow antigen tests have become pervasive, they are insufficiently sensitive to detect early or asymptomatic disease. Nucleic acid amplification tests provide the needed sensitivity, but accessibility of these tests continues to be a challenge due to the need for precise sample processing steps. Here we report a sample-to-answer test for saliva samples (saliva-STAT) that utilizes a battery-powered handheld instrument and a low-cost easily-manufacturable sample cassette to perform a nucleic acid amplification test for viral pathogens. To enable a completely automated assay, we leverage thermally responsive alkane partitions (TRAPs) and paramagnetic beads for virus purification and concentration, as well as reagent addition and mixing. Notably, the saliva STAT easily accommodates directly-dispensed saliva samples (in contrast to microfluidic devices), which is necessary for self-testing. Using the saliva-STAT platform, we demonstrate detection of down to 0.2 copies/μL of SARS-CoV-2 virus in saliva samples. We envision that the saliva-STAT could be used in walk-in clinics, mobile clinics, public testing locations, and in the home. With minor adjustments to the assay, the saliva-STAT platform can easily be adapted for other respiratory viruses, such as Influenza.

Functionalized N95 Face Mask with a Chemical-Free Paper-Based Collector for Exhaled Breath Analysis: SARS-CoV-2 Detection with a Printed Immunosensor as a Case Study.

Exhaled breath electrochemical sensing is a promising biomedical technology owing to its portability, painlessness, cost-effectiveness, and user-friendliness. Here, we present a novel approach for target analysis in exhaled breath by integrating a comfortable paper-based collector into an N95 face mask, providing a universal solution for analyzing several biomarkers. As a model analyte, we detected SARS-CoV-2 spike protein from the exhaled breath by sampling the target analyte into the collector, followed by its detection out of the N95 face mask using a magnetic bead-based electrochemical immunosensor. This approach was designed to avoid any contact between humans and the chemicals. To simulate human exhaled breath, untreated saliva samples were nebulized on the paper collector, revealing a detection limit of 1 ng/mL and a wide linear range of 3.7-10,000 ng/mL. Additionally, the developed immunosensor exhibited high selectivity toward the SARS-CoV-2 spike protein, compared to other airborne microorganisms, and the SARS-CoV-2 nucleocapsid protein. Accuracy assessments were conducted by analyzing the simulated breath samples spiked with varying concentrations of SARS-CoV-2 spike protein, resulting in satisfactory recovery values (ranging from 97 ± 4 to 118 ± 1%). Finally, the paper-based hybrid immunosensor was successfully applied for the detection of SARS-CoV-2 in real human exhaled breath samples. The position of the collector in the N95 mask was evaluated as well as the ability of this paper-based analytical tool to identify the positive patient.

Effect of adiposity status on salivary Streptococcus mutans and Streptococcus sobrinus.

To investigate the effect of adiposity status, salivary physicochemical parameters, and inflammatory biomarkers on the salivary abundance of the two main cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus among Hong Kong adolescents. This study included 180 adolescents aged 12-15 years from Hong Kong local secondary schools. Anthropometric measurements and oral health examinations were performed. Saliva samples were collected to measure salivary physicochemical parameters, protein biomarker levels, and salivary abundance of Streptococcus mutans, Streptococcus sobrinus, and total bacteria. Adolescents with general overweight/obesity or central obesity had significantly higher salivary Streptococcus mutans abundance and total bacterial load, lower stimulated salivary flow rate, and lower secretory phospholipase A2 group IIA levels than the lean controls. Adolescents with general overweight/obesity (OR = 3.33; 95% CI: 1.28-8.65) and those with central obesity (OR = 2.47; 95% CI: 1.02-6.00) had a significantly higher chance of having high salivary abundance of Streptococcus mutans than the lean controls after adjusting for confounders, with a similar detection rate of salivary Streptococcus sobrinus. General overweight or obesity and central obesity were associated with a high salivary abundance of Streptococcus mutans but had no significant effect on salivary Streptococcus sobrinus.

Periodontal treatment causes a longitudinal increase in nitrite-producing bacteria.

The oral microbiome-dependent nitrate (NO3 -)-nitrite (NO2 -)-nitric oxide (NO) pathway may help regulate blood pressure. NO2 --producing bacteria in subgingival plaque are reduced in relative abundance in patients with untreated periodontitis compared with periodontally healthy patients. In periodontitis patients, the NO2 --producing bacteria increase several months after periodontal treatment. The early effects of periodontal treatment on NO2 --producing bacteria and the NO3 --NO2 --NO pathway remain unknown. The aim of this study was to determine how periodontal treatment affects the oral NO2 --producing microbiome and salivary NO3 - and NO2 - levels over time. The subgingival microbiota of 38 periodontitis patients was analysed before (baseline [BL]) and 1, 7 and 90 days after periodontal treatment. Changes in NO2 --producing bacteria and periodontitis-associated bacteria were determined by 16s rRNA Illumina sequencing. Saliva samples were collected at all-time points to determine NO3 - and NO2 - levels using gas-phase chemiluminescence. A significant increase was observed in the relative abundance of NO2 --producing species between BL and all subsequent timepoints (all p<0.001). Periodontitis-associated species decreased at all timepoints, relative to BL (all p<0.02). NO2 --producing species negatively correlated with periodontitis-associated species at all timepoints, with this relationship strongest 90 days post-treatment (ρ=-0.792, p<0.001). Despite these findings, no significant changes were found in salivary NO3 - and NO2 - over time (all p>0.05). Periodontal treatment induced an immediate increase in the relative abundance of health-associated NO2 --producing bacteria. This increase persisted throughout periodontal healing. Future studies should test the effect of periodontal treatment combined with NO3 - intake on periodontal and cardiovascular health.

The effect of novel vitamin D3 compounds on saliva samples from COVID-19 patients: a lab study

Vitamin D has shown antimicrobial effects. This study aimed to explore the antiviral effects of vitamin D3 on saliva samples collected from patients with coronavirus disease-19 (COVID-19) and compare saliva and swab results to aid in policy development. Saliva and swab samples were collected from adult patients with a positive test for COVID-19 at the King Faisal Specialist Hospital and Research Centre, Jeddah. Patients who were immunocompromised and pregnant and aged < 18 years were excluded. Vitamin D3 compound (100, 300, 800, and 1,200 IU) was added to the first saliva sample in the laboratory (n = 20); the rest of the swab specimens were compared with the saliva samples via real-time polymerase chain reaction. Of the 257 patients, 236 (94.8%) had positive saliva sample test results, 7 (2.8%) had errors, and 6 (2.4%) had negative results. Of the 236 positive tests, 235 (99.6%) had a cycle threshold (Ct) indicating strong positive reactions, and only one (Ct = 28.86) was weak. Among the 236 positive results, 235 (99.6%) exhibited robust positive reactions, indicating a substantial positive sample size. Thus, saliva might be a dependable alternative testing tool when obtaining swab samples from patients is inconvenient or challenging.

SARS-CoV-2 infections before, during, and after the Omicron wave: a 2-year Indian community cohort study

We measured the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and re-infections in an adult community-based cohort in southern India. We conducted a 2-year follow-up on 1229 participants enrolled between May and October 2021. Participants provided vaccination histories, weekly saliva samples, and blood samples at 0, 6, 12, and 24 months. Salivary reverse transcription polymerase chain reaction (RT-PCR) and Meso-Scale Discovery panels were used for SARS-CoV-2 detection and anti-spike, anti-nucleocapsid immunoglobulin G quantification. Whole genome sequencing was performed on a subset of positive samples. SARS-CoV-2 infection incidence was measured across Pre-Omicron (May-December 2021), Omicron-I (December 2021-June 2022), and Omicron-II (July 2022-October 2023) periods. In total, 1166 (95%) participants with 83% seropositivity at baseline completed the follow-up, providing 2205 person-years of observation. Utilizing both RT-PCR and serology we identified 1306 infections and yielded an incidence rate of 591.3 per 1000 person-years (95% confidence interval, 559.6-624.3), which peaked during Omicron-I at 1418.1 per 1000 person-years (95% confidence interval, 1307.4-1535.6). During Omicron-I and II, neither prior infection nor vaccination conferred protection against infection. Overall, 74% of infections were asymptomatic. Integrated RT-PCR and serology revealed significant SARS-CoV-2 infection frequency, highlighting the prevalence of asymptomatic cases among previously infected or vaccinated individuals. This underscores the effectiveness of combining surveillance strategies when monitoring pandemic trends and confirms the role of non-invasive sampling in ensuring participant compliance, reflecting national transmission patterns. The study was funded by the Bill and Melinda Gates Foundation.

Nanopore-based detection of periodontitis biomarker miR31 in saliva samples.

MicroRNAs (miRNAs) play important roles in posttranscriptional gene regulation. Aberrations in the miRNA levels have been the cause behind various diseases, including periodontitis. Therefore, sensitive, specific, and accurate detection of disease-associated miRNAs is vital to early diagnosis and can facilitate inhibitor screening and drug design. In this study, we developed a label-free, real-time sensing method for the detection of miR31, which has been frequently linked to periodontitis, using an engineered protein nanopore and in the presence of a complementary ssDNA as a molecular probe. Our method is rapid and highly sensitive with nanomolar concentration of miR31 that could be determined in minutes. Furthermore, our sensor showed high selectivity toward the target miR31 sequence even in the presence of interfering nucleic acids. In addition, artificial saliva and human saliva samples were successfully analyzed. Our developed nanopore sensing platform could be used to detect other miRNAs and offers a potential application for the clinical diagnosis of disease biomarkers.