Biochemical analysis of oral fluids for disease detection.

The field of diagnostics using invasive blood testing represents the majority of diagnostic tests used as part of routine health monitoring. The relatively recent introduction of salivary diagnostics has lead to a major paradigm shift in diagnostic analyses. Additionally, in this era of big data, oral fluid testing has shown promising outcomes in a number of fields, particularly the areas of genomics, microbiomics, proteomics, metabolomics, and transcriptomics. Despite the analytical challenges involved in the interpretation of large datasets generated from biochemical studies involving bodily fluids, including saliva, many studies have identified novel oral biomarkers for diagnosing oral and systemic diseases. In this regard, oral biofluids, including saliva, gingival crevicular fluid (GCF), peri-implant crevicular fluid (PICF), dentinal tubular fluid (DTF), are now attracting increasing attention due to their important attributes, such as noninvasive sampling, easy handling, low cost, and more accurate diagnosis of oral diseases. Recently, the utilization of salivary diagnostics to evaluate systemic diseases and monitor general health has increased in popularity among clinicians. Saliva contains a wide range of protein, DNA and RNA biomarkers, which assist in the diagnosis of multiple diseases and conditions, including cancer, cardiovascular diseases (CVD), auto-immune and degenerative diseases, respiratory infections, oral diseases, and microbial (viral, bacterial and fungal) diseases. Moreover, due to its noninvasive nature and ease-of-adoption by children, it is now being used in mass screening programs, oral health-related studies and clinical trials in support of the development of therapeutic agents. The recent advent of highly sensitive technologies, such as next-generation sequencing, mass spectrometry, highly sensitives ELISAs, and homogeneous immunoassays, suggests that even small quantities of salivary biomarkers are able to be assayed accurately, providing opportunities for the development of many future diagnostic applications (including emerging technologies, such as point-of-care and rapid molecular technologies). The present article explores the omics and biochemical compositions of various oral biofluids with important value in diagnostics and monitoring.

Effect of mindfulness intervention versus health education program on salivary Aß-42 levels in community-dwelling older adults with mild cognitive impairment: A randomized controlled trial.

BACKGROUND: Few randomized controlled trials have investigated the effects of mindfulness intervention on older adults diagnosed with mild cognitive impairment (MCI). Specifically, scarce literature exists on the potential benefits of mindfulness intervention on biomarkers representing AD hallmarks. Our previous studies showed the potential of Mindful Awareness Practice (MAP) in improving multiple biomarkers of gut microbiota, systemic inflammation, and synaptic functions. Extending these findings, in this study, we conducted analysis on bio-banked saliva samples, examining whether MAP improved salivary amyloid beta-42 (Aß-42) levels in community-dwelling older adults diagnosed with MCI. We also explored the moderating role of education level, an indicator of cognitive reserve, on intervention effect. METHODS: A total of 55 community-dwelling older adults diagnosed with MCI were randomized into either the treatment arm, MAP, or the active control arm, the health education program (HEP). Interventions were performed for a total of nine months. Field and laboratory investigators who were blinded to the treatment allocations collected saliva samples at baseline, 3-month, and 9-month follow-ups. Salivary Aß-42 levels were quantified using a commercial assay. Linear-mixed models were used to examine the effect of MAP on salivary Aß-42 levels. RESULTS: Compared to the HEP arm, MAP participants had no significantly modified Aß-42 levels throughout the 9-month intervention period, regardless of subgroup analyses stratified by either sex or MCI-subtypes (amnestic and non-amnestic). Exploring the moderating effect of education, participants in the HEP arm with higher education levels had significantly lower salivary Aß-42 at 3-month time-point. DISCUSSION: Taken together with our previous findings and other mindfulness interventional studies failing to find a significant effect on peripheral Aß-42, we conclude the non-significant effects of mindfulness intervention on ameliorating peripheral Aß-42 levels. Conversely, participants in the HEP arm with higher cognitive reserve had significantly improved salivary Aß-42, highlighting the role of cognitive reserve in moderating treatment response in MCI.

A saliva-based rapid test to quantify the infectious subclinical malaria parasite reservoir.

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy. Plasmodium falciparum gametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples.

Role of Salivary Biomarkers in Oral Cancer Detection.

Oral cancers are the sixth most frequent cancer with a high mortality rate. Oral squamous cell carcinoma accounts for more than 90% of all oral cancers. Standard methods used to detect oral cancers remain comprehensive clinical examination, expensive biochemical investigations, and invasive biopsy. The identification of biomarkers from biological fluids (blood, urine, saliva) has the potential of early diagnosis. The use of saliva for early cancer detection in the search for new clinical markers is a promising approach because of its noninvasive sampling and easy collection methods. Human whole-mouth saliva contains proteins, peptides, electrolytes, organic, and inorganic salts secreted by salivary glands and complimentary contributions from gingival crevicular fluids and mucosal transudates. This diagnostic modality in the field of molecular biology has led to the discovery and potential of salivary biomarkers for the detection of oral cancers. Biomarkers are the molecular signatures and indicators of normal biological, pathological process, and pharmacological response to treatment hence may provide useful information for detection, diagnosis, and prognosis of the disease. Saliva's direct contact with oral cancer lesions makes it more specific and potentially sensitive screening tool, whereas more than 100 salivary biomarkers (DNA, RNA, mRNA, protein markers) have already been identified, including cytokines (IL-8, IL-1b, TNF-α), defensin-1, P53, Cyfra 21-1, tissue polypeptide-specific antigen, dual specificity phosphatase, spermidine/spermineN1-acetyltransferase , profilin, cofilin-1, transferrin, and many more. However, further research is still required for the reliability and validation of salivary biomarkers for clinical applications. This chapter provides the latest up-to-date list of known and emerging potential salivary biomarkers for early diagnosis of oral premalignant and cancerous lesions and monitoring of disease activity.

Quantitative Lateral Flow Assays for Salivary Biomarker Assessment: A Review.

Saliva is an emerging biofluid with a significant number of applications in use across research and clinical settings. The present paper explores the reasons why saliva has grown in popularity in recent years, balancing both the potential strengths and weaknesses of this biofluid. Focusing on reasons why saliva is different from other common biological fluids such as blood, urine, or tears, we review how saliva is easily obtained, with minimal risk to the donor, and reduced costs for collection, transportation, and analysis. We then move on to a brief review of the history and progress in rapid salivary testing, again reviewing the strengths and weaknesses of rapid immunoassays (e.g., lateral flow immunoassay) compared to more traditional immunoassays. We consider the potential for saliva as an alternative biofluid in a setting where rapid results are important. We focus the review on salivary tests for small molecule biomarkers using cortisol as an example. Such salivary tests can be applied readily in a variety of settings and for specific measurement purposes, providing researchers and clinicians with opportunities to assess biomarkers in real time with lower transportation, collection, and analysis costs, faster turnaround time, and minimal training requirements. We conclude with a note of cautious optimism that the field will soon gain the ability to collect and analyze salivary specimens at any location and return viable results within minutes.

Salivary Diagnostics Using Purified Nucleic Acids.

Saliva is an easily accessible fluid that has led to increasing interest in the development of salivary diagnostics. This chapter describes some of the newer tools and procedures for collection, stabilization, and storage of oral fluid matrices that aid in the successful use of saliva as a test specimen. This chapter focuses particularly on nucleic acid components for downstream molecular diagnostic (MDx) testing, since this is probably the area where saliva is likely to have the greatest impact in improving healthcare for the general population.

Human Saliva Collection Devices for Proteomics: An Update.

There has been a rapid growth in the interest and adaptation of saliva as a diagnostic specimen over the last decade, and in the last few years in particular, there have been major developments involving the application of saliva as a clinically relevant specimen. Saliva provides a "window" into the oral and systemic health of an individual, and like other bodily fluids, saliva can be analyzed and studied to diagnose diseases. With the advent of new, more sensitive technologies to detect smaller concentrations of analytes in saliva relative to blood levels, there have been a number of critical developments in the field that we will describe. In particular, recent advances in standardized saliva collection devices that were not available three to four years ago, have made it easy for safe, simple, and non-invasive collection of samples to be carried out from patients. With the availability of these new technologies, we believe that in the next decade salivary proteomics will make it possible to predict and diagnose oral as well as systemic diseases, cancer, and infectious diseases, among others. The aim of this article is to review recent developments and advances in the area of saliva specimen collection devices and applications that will advance the field of proteomics.

Salivary cortisol results obtainable within minutes of sample collection correspond with traditional immunoassays.

PURPOSE: Cortisol is frequently assayed as a stress-responsive biomarker which changes over the course of minutes to meet the demands of a person's social context. Salivary cortisol is often used as a noninvasive sampling method that possesses important health implications. A critical barrier to psychobiological research that involves salivary cortisol is a time delay of days to months before cortisol results are obtained via immunoassay, long after the person is no longer proximate to the social context in which they provided the sample. The present study was designed to address this critical barrier through creation of a lateral flow test (LFT) cortisol device capable of measuring salivary cortisol within minutes of sample collection. The LFT is frequently used within commercial point-of-care settings to obtain rapid answers to the presence/absence of a biomarker. The present study extends the LFT into the research domain by presenting performance characteristics of a quantitative LFT that measures salivary cortisol within 20 minutes of sample collection. METHODS: Saliva samples from 29 adults (15 men) were obtained in the morning and afternoon by using Passive Drool and then the Super·SAL Extra Collection Device (hereafter Super·SAL) and later assayed with LFT and a commercially available enzyme immunoassay. FINDINGS: Results indicate the LFT correlated well with these collection methods (R = 0.872 with Super · SAL, R = 0.739 with Passive Drool, P < 0.0001) and at comparable levels to correspondence of Super · SAL with Passive Drool (R = 0.798, P < 0.0001) which were measured with the same assay. IMPLICATIONS: These results open an exciting new possibility to integrate this technologic advance into stress research, including knowing and potentially changing the person's social context in a time-sensitive manner. Methodological improvements such as this have the possibility of refining conceptual models of stress reactivity and regulation.

New techniques for augmenting saliva collection: bacon rules and lozenge drools.

PURPOSE: Saliva is a reliable, noninvasive, and cost-effective alternative to biomarkers measured in other biological fluids. Within certain populations, saliva sampling may be difficult because of insufficient saliva flow, which may compromise disease diagnosis or research integrity. Methods to improve flow rates (eg, administering citric acid, chewing gum, or collecting cotton) may compromise biomarker integrity, especially if the methods involve the presence of a collection aid in the oral cavity. Anecdotal strategies (eg, looking at pictures of food or imagining food) have not been evaluated to date. In this study, we evaluate whether 2 novel collection techniques improve saliva flow or interfere with assay of common biomarkers (ie, cortisol, dehydroepiandrosterone, and testosterone). We evaluate an over-the-counter anhydrous crystalline maltose lozenge intended to increase saliva production for patients with xerostomia long after the lozenge dissolves. We then evaluate whether the smell of freshly cooked bacon stimulates a pavlovian-type reflex. METHODS: Saliva was collected from 27 healthy young adults (aged 20-34 years; 12 men) on a basal day and a lozenge day, providing 5 samples at 15-minute intervals. Twenty participants then returned for the bacon day condition, providing 2 saliva samples with an interval of 15 minutes between samples. Collection times required to generate 2 mL of saliva across collection strategies were recorded, and then saliva samples were assayed for cortisol, dehydroepiandrosterone, and testosterone. FINDINGS: Repeated analysis of variance measures revealed that both the lozenges and bacon significantly decreased collection time compared with the passive drool collection on the basal day. No significant effects were found related to the quantification of cortisol, testosterone, or dehydroepiandrosterone when comparing lozenge or bacon to the basal day. In addition, bivariate correlations revealed that concentrations from time-matched control samples correlated significantly with concentrations from the lozenge and bacon conditions. IMPLICATIONS: These results indicate that both the lozenge and smelling bacon improve saliva collection times and that neither technique interferes with salivary hormone concentrations. This study reveals new methods to augment saliva collection strategies.

RNAPro•SAL: a device for rapid and standardized collection of saliva RNA and proteins.

The stabilization and processing of salivary transcriptome and proteome biomarkers is a critical challenge due to the ubiquitous nature of nucleases and proteases as well as the inherent instability of these biomarkers. Furthermore, extension of salivary transcriptome and proteome analysis to point-of-care and remote sites requires the availability of self-administered ambient temperature collection and storage tools. To address these challenges, a self-contained whole saliva collection and extraction system, RNAPro•SAL, has been developed that provides rapid ambient temperature collection along with concurrent processing and stabilization of extracellular RNA (exRNA) and proteins. The system was compared to the University of California, Los Angeles (UCLA) standard clinical collection process (standard operating procedure, SOP). Both systems measured total RNA and protein, and exRNA IL-8, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ß-actin and ribosomal protein S9 (RPS9) by qPCR. Proteome analysis was measured by EIA analysis of interleukin-8 (IL-8), and ß-actin, as well as total protein. Over 97% of viable cells were removed by both methods. The system compared favorably to the labor-intensive clinical SOP, which requires low-temperature collection and isolation, yielding samples with similar protein and exRNA recovery and stability.

Commercial saliva collections tools.

Saliva has been used as a specimen for diagnostics purposes for many years, but it has only been in the last 10 years that a number of new tools have been developed that promise to greatly increase the use of oral specimens for broad-based diagnosis and potentially screening applications. This article focuses on tools that are commercially viable or can play a role in whole saliva collection and future testing for critical diseases.

Tumor-suppressor Gene Promoter Hypermethylation in Saliva of Head and Neck Cancer Patients.

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80% (with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high κ concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.