Pre-treatment Saliva Research Articles

The prognostic prediction of periodontal non-surgery therapy in periodontitis patients based on surface-enhanced Raman measurements of pre-treatment saliva

Periodontitis is one of the most prevalent dental diseases, and the patients with periodontitis often suffer from refractory periodontitis or recurrence of disease due to improper or inadequate treatment. In clinical practice, the early and accurate assessment of post-treatment prognosis in periodontitis patients is always very important in order to implement timely interventions. In this study, a pre-treatment saliva SERS based prognostic protocol was explored to predict the prognosis of periodontal non-surgery therapy in periodontitis patients. According to the biomolecular analysis, significant differences in the levels of ascorbic acid, uric acid and glutathione are observed between good prognosis group and poor prognosis group, which are expected to serve as potential prognostic markers. Furthermore, high accuracy, sensitivity and specificity can also be achieved by using the proposed prognostic model. The excellent performance of the proposed method has demonstrated its potential for fast, accurate, and non-invasive prognostic prediction of periodontal non-surgery therapy in periodontitis patients, even at the time before implementing treatment, thus is expected to benefit timely and rational guidance on clinical interventions.

Salivary Electrolytes in Patients Undergoing Fixed Orthodontic Treatment

The base metal alloys are more susceptible to corrosion than nobl ealloys, and fixed orthodontic appliances are generally made of base alloys. Fixed orthodontic appliances in the oral cavity are exposed to destructive physical and chemical agents, resulting in metallic corrosion. The present study aimed to evaluate the concentration of cobalt, zinc, and magnesium in patients' saliva with fixed orthodontic appliances at three different times. Saliva samples from 18 patients (9 males and 9 females) between 15-25 years were taken at three different time points. Group I: Pretreatment saliva sample, group II: one month after appliance placement, Group III: four months after appliance placement. The fixed appliance consists of 20 stainless steel brackets, 4 buccal tubes, and a super elastic nickel-titanium arch wire. Inductively Coupled Plasma Optical Emission Spectrometry analyzed the level of ions in salivary samples. Ions are recorded in parts per billion. Statistical analysis was performed by non-parametric tests (Friedman) and one-way repeated measures ANOVA. Levels of cobalt, magnesium, and zinc ions in saliva were highest in group II, lowest in group I for both zinc and cobalt, and lowest in group III for magnesium. On a pair wise comparison between different groups, it was statistically significant for all groups (<0. 005) except for cobalt and magnesium level between group I and group III and zinc level between groups II and III. Cobalt, magnesium, and zinc levels in saliva were increased after placing a fixed orthodontic appliance.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Oral microbiota of periodontal health and disease and their changes after nonsurgical periodontal therapy

This study examined the microbial diversity and community assembly of oral microbiota in periodontal health and disease and after nonsurgical periodontal treatment. The V4 region of 16S rRNA gene from DNA of 238 saliva and subgingival samples of 21 healthy and 48 diseased subjects was amplified and sequenced. Among 1979 OTUs identified, 28 were overabundant in diseased plaque. Six of these taxa were also overabundant in diseased saliva. Twelve OTUs were overabundant in healthy plaque. There was a trend for disease-associated taxa to decrease and health-associated taxa to increase after treatment with notable variations among individual sites. Network analysis revealed modularity of the microbial communities and identified several health- and disease-specific modules. Ecological drift was a major factor that governed community turnovers in both plaque and saliva. Dispersal limitation and homogeneous selection affected the community assembly in plaque, with the additional contribution of homogenizing dispersal for plaque within individuals. Homogeneous selection and dispersal limitation played important roles, respectively, in healthy saliva and diseased pre-treatment saliva between individuals. Our results revealed distinctions in both taxa and assembly processes of oral microbiota between periodontal health and disease. Furthermore, the community assembly analysis has identified potentially effective approaches for managing periodontitis.

Unclear association between levels of Plasmodium falciparum lactate dehydrogenase (PfLDH) in saliva of malaria patients and blood parasitaemia: diagnostic implications?

BackgroundThe use of saliva in diagnosis of infectious diseases is an attractive alternative to procedures that involve blood drawing. It promises to reduce risks associated with accidental needle pricks and improve patient compliance particularly in malaria survey and drug efficacy studies. Quantification of parasitaemia is useful in establishing severity of disease and in assessing individual patient response to treatment. In current practice, microscopy is the recommended technique, despite its limitations. This study measured the levels of Plasmodium falciparum lactate dehydrogenase (PfLDH) in saliva of malaria patients and investigated the relationship with blood parasitaemia.MethodsMatched pre-treatment blood and saliva samples were collected from patients at Msambweni District Hospital, Kenya. Parasitaemia was determined and only those confirmed to be Plasmodium falciparum mono-infected were recruited. PfLDH was quantified in saliva using a commercial ELISA kit. A total of 175 samples were collected. Relationship between blood parasitaemia and concentration of PfLDH in saliva was determined using Pearson correlation statistics. F test was used to determine whether there is a significant difference between levels of PfLDH in saliva of patients with moderate to high parasitaemia and those with low parasitaemia.ResultsOne-hundred and seventy-five patient samples were positive for malaria by microscopy. Of these, 62 (35%) tested positive for PfLDH in saliva, 113 (65%) were false negatives. For those that tested positive, (53) 85% were from patients with moderate to high parasitaemia while 9 (15%) were from patients with low parasitaemia. A correlation co-efficient of 0.18 indicated a weak positive relationship between the concentration of PfLDH in saliva and blood parasitaemia. There was a marginal difference between levels of PfLDH in saliva of patients with moderate to high parasitaemia and those with low parasitaemia [F (1, 59) = 1.83, p = 0.1807].ConclusionThe results indicate that there is a weak correlation between levels of PfLDH in saliva and blood parasitaemia. This is weak association could be as a result of low sensitivity of the assay used as well as presence of inhibitors and proteases in saliva. Further studies should be focused towards reducing the number of false negatives and developing a customised assay that is specific for detection of PfLDH in saliva.

Saliva testing for human papilloma virus in oropharyngeal squamous cell carcinoma: A diagnostic accuracy study.

New cases of oropharyngeal squamous cell carcinoma (OPSCC) are routinely tested for HPV. HPV in saliva can be detected with PCR, but its clinical applicability in the context of OPSCC remains unknown. Forty-six consecutive patients diagnosed with OPSCC had pre-treatment saliva specimens collected. PCR for HPV on saliva was compared to p16 IHC and HPV DNA in situ hybridisation (ISH) on surgical biopsies. The sensitivity and specificity of saliva testing when compared to the reference test of p16 IHC and HPV DNA ISH were 72.2% and 90%, and positive and negative predictive values were 96.3% and 47.4%. There were no adverse events. Time from last meal, smoking, alcohol drinking and physical exercise did not impact on results. Saliva testing is a promising test to detect HPV in patients with OPSCC. A positive result could avoid the need for surgical biopsies, thereby reducing costs, patient morbidity and expedite treatment.

Immune-related saliva micro-RNA’s to predict clinical response to a combination of cetuximab and low-dose chemotherapy in head and neck cancer (HNSCC) patients (pts) with poor performance status (PS).

e17548 Background: Recurrent/Metastatic HNSCC is an immunosuppressive disease, especially in the group of patients with poor performance status. Recent studies have shown that low dose chemotherapy, which is much better tolerated than full dose cytotoxic chemotherapy, can increase the anti-tumor immune response and cause tumor regression. In this study we assessed the immune-effects of weekly cetuximab in combination with low dose weekly chemotherapy in HNSCC pts with poor PS by measuring saliva and plasma immune modulatory micro RNAs (miRs). Methods: Patients with recurrent/metastatic HNC with an ECOG PS of 2 or 3 were treated with low-dose weekly carboplatin (AUC 1) and paclitaxel (25 mg/m2) along with weekly cetuximab (400mg/m2 followed by 250 mg/m2). Seven immune modulatory miRs (146a, 155, 181, 20a, 223, 335, 125b) were measured in the saliva and plasma by quantitative PCR prior to treatment and at week 5. Results: Between 4/2016 and 1/2017, 30 patients were enrolled in this study. 24 patients were evaluable for response. Mean age was 69 years (range 51-89), Male/Female: 14/8. ECOG PS 2/3: 13/9. Site of disease: oral cavity 7, pharynx 9, larynx 4, para-nasal 2, unknown 2. Treatment was well tolerated. The most common dose limiting toxicity was skin rash in 10/24 pts (41.5%). Response: partial response 12 (50%); stable disease 4 (16.5%); progressive disease 8 (33.5%). Reduction in miR levels by week 5 were associated with clinical response. Reduction in saliva levels of miRs 146a and 181 correlated with clinical benefit (PR+SD). Pre-treatment saliva levels of miR 146a also showed to be a predictor of tumor response to therapy. Plasma levels of the immune modulatory miRs tested were not associated with response to therapy. Conclusions: The combination of cetuximab and low-dose chemotherapy is active and well tolerated in HNSCC patients with poor PS. Reduction in saliva levels of immune modulatory miRs 146a and 181 were associated with clinical benefit to therapy.

Possible role for interleukins as biomarkers for mortality and recurrence in oral cancer.

Salivary and serum levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) have previously been studied in oral cancer with conflicting results. We designed a controlled study to assess the correlation between pretreatment salivary and serum levels of IL-6 and IL-8, and all-cause survival and cancer recurrence in oral cancer patients. Fifty-two oral cancer patients and 52 healthy control cases were selected. In univariate analysis, salivary IL-6 and IL-8 seemed to be more expressed in cases (p<0.001 and p = 0.010, respectively). Multivariate analysis showed that higher pretreatment saliva IL-6 levels were significantly associated with better survival (hazard ratio [HR] = 8.62; 95% confidence interval [95% CI], 1.21-62.50; p = 0.031). To date, this is the largest prospective controlled study that has analyzed the pretreatment salivary and serum levels of IL-6 and IL-8 in oral cancer patients, suggesting salivary IL-6 as a possible prognostic biomarker. But further validation in a larger sample is still necessary.

Saliva and plasma quantitative polymerase chain reaction-based detection and surveillance of human papillomavirus-related head and neck cancer.

Human papillomavirus type 16 (HPV-16) is a major causative factor in oropharyngeal squamous cell carcinoma (OPSCC). The detection of primary OPSCC is often delayed owing to the challenging anatomy of the oropharynx. To investigate the feasibility of HPV-16 DNA detection in pretreatment and posttreatment plasma and saliva and its potential role as a marker of prognosis. This is a retrospective analysis of a prospectively collected cohort. Among a cohort of patients with oropharyngeal and unknown primary squamous cell carcinoma with known HPV-16 tumor status from the Johns Hopkins Medical Institutions and Greater Baltimore Medical Center (from 1999 through 2010), 93 patients were identified with a complete set of pretreatment and posttreatment plasma or saliva samples, of which 81 patients had HPV-16-positive tumors and 12 patients had HPV-16-negative tumors. Real-time quantitative polymerase chain reaction was used to detect HPV-16 E6 and E7 DNA in saliva and plasma samples. Main outcomes included sensitivity, specificity, negative predictive value of combined saliva and plasma pretreatment HPV-16 DNA status for detecting tumor HPV-16 status, as well as the association of posttreatment HPV DNA status with clinical outcomes, including recurrence-free survival and overall survival. The median follow-up time was 49 months (range, 0.9-181.0 months). The sensitivity, specificity, negative predictive value, and positive predictive value of combined saliva and plasma pretreatment HPV-16 DNA status for detecting tumor HPV-16 status were 76%, 100%, 42%, and 100%, respectively. The sensitivities of pretreatment saliva or plasma alone were 52.8% and 67.3%, respectively. In a multivariable analysis, positive posttreatment saliva HPV status was associated with higher risk of recurrence (hazard ratio [HR], 10.7; 95% CI, 2.36-48.50) (P = .002). Overall survival was reduced among those with posttreatment HPV-positive status in saliva (HR, 25.9; 95% CI, 3.23-208.00) (P = .002) and those with HPV-positive status in either saliva or plasma but not among patients with HPV-positive status in plasma alone. The combined saliva and plasma posttreatment HPV-16 DNA status was 90.7% specific and 69.5% sensitive in predicting recurrence within 3 years. Using a combination of pretreatment plasma and saliva can increase the sensitivity of pretreatment HPV-16 status as a tool for screening patients with HPV-16-positive OPSCC. In addition, analysis of HPV-16 DNA in saliva and plasma after primary treatment may allow for early detection of recurrence in patients with HPV-16-positive OPSCC.

OP056: Salivary proteome for diagnosis and prognosis of oral cancer

Purpose The purpose of this study is to identify biomarkers from the salivary proteome for early detection/prognostication of Oral Squamous Cell Carcinoma (OSCC). Materials and methods Patients diagnosed with OSCC and premalignant lesions were recruited for the study and healthy volunteers were used as controls. Pre-treatment saliva samples were collected and processed according to standard protocols. Proteomic profiling of normal and diseased saliva was carried out by multiple methods; mass spectrometric analysis following iTRAQ labeling methods was used for differential profiling. The data from fractions were analyzed using the commercially available MASCOT and SEQUEST software. Results Processing of the salivary supernatant was standardized for the purpose of depleting abundant proteins; starch, trisacryl resin and MARS column based depletion of amylase and albumin respectively was carried out. MS analysis on normal saliva and subsequent comparison with existing markers in the database identified 213 novel proteins. The proteomic profile of the tumor group (buccal mucosal cancer) with metastatic lymph nodes was compared to matched normal controls (N = 5 each). A total of 150 differential proteins were obtained after analysis by the MASCOT software. These proteins included Profilin, Cofilin, S100A9 and MMP9 identified previously in the saliva of head and neck cancer patients along with others associated with epithelial mesenchymal transition and metastasis such as Vimentin. Ingenuity Pathway Analysis (IPA) identified multiple networks which included proteins involved in cellular movement, cell–cell signaling, cell growth and proliferation. Further studies to validate these markers in tissues/saliva are currently ongoing. Proteomic profiling of the other study groups are also underway to identify biomarkers that specify the clinical status of the patients. Discussion Proteomic profiling of the salivary biomarkers contributed towards identifying novel markers in the normal subjects and HNSCC patients with lymphatic metastasis; validation in the saliva/tissues of the patients will enable assessment of their clinical application.

Abstract 723: Detection of TIMP3 promoter hypermethylation in salivary rinse as an independent predictor of local recurrence-free survival in head and neck cancer

Abstract Objective: To validate a panel of methylation-based salivary rinse biomarkers (P16, CCNA1, DCC, TIMP3, MGMT, DAPK, and MINT31) previously shown to be independently associated with poor overall survival and local recurrence in a larger, separate cohort of patients with head and neck squamous cell carcinoma (HNSCC). Methods: One hundred ninety-seven patients were included. All pre-treatment saliva DNA samples were evaluated for the methylation status of the gene promoters by quantitative methylation-specific PCR. The main outcome measures were overall survival, local recurrence-free survival and disease-free survival. Results: In univariate analyses, the detection of hypermethylation of CCNA1, MGMT, and MINT31 was significantly associated with poor overall survival; the detection of hypermethylation of TIMP3 was significantly associated with local recurrence-free survival; and the detection of hypermethylation of MINT31 was significantly associated with poor disease-free survival. In multivariate analyses, detection of hypermethylation at any single marker was not predictive of overall survival in patients with HNSCC; detection of hypermethylation of TIMP3 in salivary rinse had an independent, significant association with local recurrence-free survival (Hazard Ratio, 2.51, 95% CI, 1.10 to 5.68); and none of the studied markers was significantly associated with disease-free survival. Conclusions: The detection of promoter hypermethylation of the seven genes in salivary rinse as an independent prognostic indicator of overall survival in patients with HNSCC was not validated. Detection of promoter hypermethylation of TIMP3 in pretreatment salivary rinse is independently associated with local recurrence-free survival in patients with HNSCC and may be a valuable salivary rinse biomarker for HNSCC recurrence. Such a test could potentially refine our ability to identify HNSCC patients at a high risk for recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 723. doi:1538-7445.AM2012-723

Detection of TIMP3 Promoter Hypermethylation in Salivary Rinse as an Independent Predictor of Local Recurrence-Free Survival in Head and Neck Cancer

To validate a panel of methylation-based salivary rinse biomarkers (P16, CCNA1, DCC, TIMP3, MGMT, DAPK, and MINT31) previously shown to be independently associated with poor overall survival and local recurrence in a larger, separate cohort of patients with head and neck squamous cell carcinoma (HNSCC). One hundred ninety-seven patients were included. All pretreatment saliva DNA samples were evaluated for the methylation status of the gene promoters by quantitative methylation-specific PCR. The main outcome measures were overall survival, local recurrence-free survival, and disease-free survival. In univariate analyses, the detection of hypermethylation of CCNA1, MGMT, and MINT31 was significantly associated with poor overall survival; the detection of hypermethylation of TIMP3 was significantly associated with local recurrence-free survival; and the detection of hypermethylation of MINT31 was significantly associated with poor disease-free survival. In multivariate analyses, detection of hypermethylation at any single marker was not predictive of overall survival in patients with HNSCC; detection of hypermethylation of TIMP3 in salivary rinse had an independent, significant association with local recurrence-free survival (HR = 2.51; 95% CI: 1.10-5.68); and none of the studied markers was significantly associated with disease-free survival. The detection of promoter hypermethylation of the seven genes in salivary rinse as an independent prognostic indicator of overall survival in patients with HNSCC was not validated. Detection of promoter hypermethylation of TIMP3 in pretreatment salivary rinse is independently associated with local recurrence-free survival in patients with HNSCC and may be a valuable salivary rinse biomarker for HNSCC recurrence.

Salivary Epstein-Barr virus DNA level in patients with nasopharyngeal carcinoma following radiotherapy

Nasopharyngeal carcinoma (NPC) is a solid tumor closely associated with Epstein-Barr virus (EBV) infection. The purpose of this investigation was to detect and quantify the EBV DNA level in salivary samples of NPC patients following treatment using real-time PCR. A total of 175 consecutive newly diagnosed NPC patients' whole saliva samples were collected before treatment, and the EBV DNA level was measured by real-time PCR, with the primers and probe targeting the BamHI-W region of the EBV genome. The post-treatment salivary EBV DNA level was also assessed in 46 patients. The change of EBV DNA level before and after treatment and relationship of EBV DNA level to demographic data and tumor staging were tested by Wilcoxon signed-rank test and Mann-Whitney U test, respectively with the level of significance set at 0.05. The EBV detection rate of pre-treatment saliva samples was 80%. The EBV DNA level of post-treatment saliva samples was significantly higher than the pre-treatment ones (P<0.01). There is a trend that patients with advanced-stage showed a higher EBV DNA level than patients with early-stage. The detection of EBV DNA in saliva using real-time PCR might be a feasible and non-invasive method for early diagnosis of NPC.

Detection of promoter hypermethylation in salivary rinses as a biomarker for head and neck squamous cell carcinoma surveillance.

Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP). Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1. We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8-80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2-6.5; P = 0.016). We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients.

Identification of a truncated cystatin SA-I as a saliva biomarker for oral squamous cell carcinoma using the SELDI ProteinChip platform

New and more consistent biomarkers of oral squamous cell carcinoma (OSCC) are needed to improve early detection of the disease and to monitor patient management. The aim of this study was to detect new OSCC tumor markers in saliva. Unstimulated saliva, collected from patients with primary stage I OSCC as matched pre-and post-treatment samples, was used in the analysis. A surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in the saliva samples. This analysis revealed 26 proteins with significantly different expression levels in the pre-and post-treatment samples (P<0.05). A 14 kDa protein detected in pre-treatment saliva from the OSCC patients was identified as a truncated cystatin SA-I, with deletion of three amino acids from the N-terminus. The authors propose that ProteinChip analysis may provide a reliable screening test for early diagnosis of OSCC and that truncated cystatin SA-I might be a useful tumor biomarker for OSCC.

Modeling Plan-Related Clinical Complications Using Machine Learning Tools in a Multiplan IMRT Framework

To predict organ-at-risk (OAR) complications as a function of dose-volume (DV) constraint settings without explicit plan computation in a multiplan intensity-modulated radiotherapy (IMRT) framework. Several plans were generated by varying the DV constraints (input features) on the OARs (multiplan framework), and the DV levels achieved by the OARs in the plans (plan properties) were modeled as a function of the imposed DV constraint settings. OAR complications were then predicted for each of the plans by using the imposed DV constraints alone (features) or in combination with modeled DV levels (plan properties) as input to machine learning (ML) algorithms. These ML approaches were used to model two OAR complications after head-and-neck and prostate IMRT: xerostomia, and Grade 2 rectal bleeding. Two-fold cross-validation was used for model verification and mean errors are reported. Errors for modeling the achieved DV values as a function of constraint settings were 0-6%. In the head-and-neck case, the mean absolute prediction error of the saliva flow rate normalized to the pretreatment saliva flow rate was 0.42% with a 95% confidence interval of (0.41-0.43%). In the prostate case, an average prediction accuracy of 97.04% with a 95% confidence interval of (96.67-97.41%) was achieved for Grade 2 rectal bleeding complications. ML can be used for predicting OAR complications during treatment planning allowing for alternative DV constraint settings to be assessed within the planning framework.

Microfluidic immunoassays as rapid saliva-based clinical diagnostics

At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip). Before anticipatory use of diagnostics for assessment of complex diseases becomes widespread, development of sophisticated bioassays capable of quantitatively measuring disease biomarkers is necessary. Successful translation of new bioassays into clinical settings demands the ability to monitor both the onset and progression of disease. Here we report on a clinical POC diagnostic that enables rapid quantitation of an oral disease biomarker in human saliva by using a monolithic disposable cartridge designed to operate in a compact analytical instrument. Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. Using 20 microl of saliva, we demonstrate rapid (<10 min) measurement of the collagen-cleaving enzyme matrix metalloproteinase-8 (MMP-8) in saliva from healthy and periodontally diseased subjects. In addition to physiologically measurable indicators of periodontal disease, conventional measurements of salivary MMP-8 were used to validate the microfluidic assays described in this proof-of-principle study. The microchip-based POC diagnostic demonstrated is applicable to rapid, reliable measurement of proteinaceous disease biomarkers in biological fluids.

Integrated Microfluidic Platform for Oral Diagnostics

While many point-of-care (POC) diagnostic methods have been developed for blood-borne analytes, development of saliva-based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip-based immunoassays (<3-10 min) with low sample volume requirements (10 microL) and appreciable sensitivity (nM-pM). Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip-based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.

Increased Mitochondrial DNA Content in Saliva Associated with Head and Neck Cancer

Alterations of the mitochondrial DNA (mtDNA) have been described in human tumors and in other tissues in association with smoking exposure. We did quantitative PCR of cytochrome c oxidase I (Cox I) and cytochrome c oxidase II (Cox II) genes on oral rinse samples obtained from 94 patients with primary head and neck squamous cell carcinoma (HNSC) and a control group of 656 subjects. Mitochondrial DNA/nuclear DNA in saliva from HNSC patients and controls in relationship to smoking exposure, ethanol intake, and tumor stage were examined. Mean levels of Cox I and Cox II in saliva samples were significantly higher in HNSC patients: Cox I, 0.076 [95% confidence interval (95% CI), 0.06-0.09] and Cox II, 0.055 (95% CI, 0.04-0.07) in comparison with controls Cox I, 0.054 (95% CI, 0.05-0.06), P < 0.0001 and Cox II, 0.046 (95% CI, 0.04-0.05), P = 0.003 (t test). MtDNA levels were elevated in primary tumors when compared with matched, pretreatment saliva and significant correlation was noted (Cox I, r = 0.30, P = 0.005 and Cox II r = 0.33, P = 0.002, respectively, Pearson's correlation). On univariate analysis, smoking, age, HNSC diagnosis, and advanced stage of HNSC were associated with higher level of mtDNA content in saliva. Multivariate analysis showed a significant and independent association of HNSC diagnosis, age, and smoking with increasing mtDNA/nuclear DNA for Cox I and Cox II. mtDNA content alteration is associated with HNSC independently of age and smoking exposure, can be detected in saliva, and may be due to elevation in mtDNA content in primary HNSC.

Changes in Protein Composition of Saliva from Radiation-induced Xerostomia Patients and Its Effect on Growth of Oral Streptococci

Clarified and dialyzed mixed saliva-water rinses, obtained from patients receiving radiation treatment of the major salivary glands, exhibited quantitative reductions in total protein content and qualitative changes in protein composition. Radiation or post-radiation saliva preparations supported enhanced growth of Streptococcus mutans, but reduced growth of Streptococcus sanguis relative to growth on pretreatment saliva.