Determination of pepsin in human saliva using pepsin-susceptible peptide reporter and colorimetric dipstick assay: a prospective, cross-sectional study.

A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically cleaved by pepsin, was modified with fluorescein isothiocyanate (FITC) and biotin at the N- and C-terminals to be used as a reporter for colorimetric detection of dipsticks. A universal lateral flow dipstick consisting of a streptavidin test line for biotin binding and a sample pad immobilized with a gold-labeled polyclonal (rabbit) anti-FITC antibody was used to verify PSP-based pepsin detection. When the PSP reporter reacts with pepsin in a tube, it cleaves into two fragments, and the cleaved fragments do not display any color on the test line. Therefore, the higher the concentration of pepsin is, the greater is the decrease in test line intensity (IT-line) and the higher is the control line intensity (IC-line). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (IT-line/IC-line) of PSP-based dipstick assay showed a linear relationship with log concentration of pepsin ranging between 4 and 500 ng/mL (R2 = 0.98, n = 6), with a limit of detection of 1.4 ng/mL. It also exhibited high specificity and good reproducibility. Finally, pepsin levels were quantified in saliva samples from healthy controls (n = 34) and patients with laryngopharyngeal reflux (LPR, n = 61). Salivary pepsin levels were higher in patients with LPR than in healthy controls. The salivary pepsin levels correlated with those measured using a conventional enzyme-linked immunosorbent assay kit. Therefore, this PSP-based dipstick assay is a convenient tool for assessing salivary pepsin levels.

Diagnostic Value of Salivary miRNA in Head and Neck Squamous Cell Cancer: Systematic Review and Meta-Analysis.

Several studies have highlighted the diagnostic potential of salivary microRNA (miRNA) in head and neck squamous cell cancer (HNSCC). The purpose of this meta-analysis was to summarize published studies and evaluate the diagnostic accuracy of salivary miRNA in HNSCC detection. In this meta-analysis, we systematically searched PubMed, EMBASE, and Cochrane Library databases for studies on miRNA and HNSCC diagnosis. Pooled sensitivity, specificity, and diagnostic odds ratio (DOR) with a summary receiver-operating characteristic curve were calculated using a bivariate random-effect meta-analysis model. Furthermore, subgroup analyses were conducted to explore the main sources of heterogeneity. Seventeen studies from ten articles, including 23 miRNA and a total of 759 subjects, were included in this meta-analysis. The pooled sensitivity and specificity of salivary miRNA in the diagnosis of HNSCC were 0.697 (95% CI: 0.644-0.744) and 0.868 (95% CI: 0.811-0.910), respectively. The overall area under the curve was 0.803 with a DOR of 12.915 (95% CI: 9.512-17.534). Salivary miRNAs are a promising non-invasive diagnostic biomarker with moderate accuracy for HNSCC. These results must be verified by large-scale prospective studies.

Optimization of Saliva Collection and Immunochromatographic Detection of Salivary Pepsin for Point-of-Care Testing of Laryngopharyngeal Reflux.

Salivary pepsin is a promising marker for the non-invasive diagnosis of laryngopharyngeal reflux (LPR). For reliable results regarding pepsin in saliva, it is critical to standardize the collection, storage, and pre-processing methods. In this study, we optimized the saliva collection protocols, including storage conditions, i.e., solution, temperature, and time, and the pre-processing filter for pepsin. Moreover, we prepared a simple immunochromatographic strip for the rapid detection of pepsin and evaluated its sensing performance. As a result, we selected a polypropylene (PP) filter as the pre-processing filter for salivary pepsin in low resource settings, such as those where point of care testing (POCT) is conducted. This filter showed a similar efficiency to the centrifuge (standard method). Finally, we detected the pepsin using gold nanoparticles conjugated with monoclonal pepsin antibody. Under optimized conditions, the lower limit of detection for pepsin test strips was determined as 0.01 µg/mL. Furthermore, we successfully detected the salivary pepsin in real saliva samples of LPR patients, which were pre-processed by the PP filter. Therefore, we expect that our saliva collection protocol and pepsin immunochromatographic strip can be utilized as useful tools for a non-invasive diagnosis/screening of LPR in POCT.

Development of an effective sample transfer device for biomarker detection in nasal secretions.

Nasal secretions (NS) reflect inflammatory activity of the nasal mucosa and thus can be utilized for disease diagnosis and determining treatment effects in Allergic rhinitis (AR). However, non-standardized collection of samples can affect the measured concentration of inflammatory biomarker in NS. In this study, we aimed to develop and evaluate new devices capable of standardizing the collection, storage, and preprocessing methods of NS samples. First, we chose the best swab as polyester (PE) and selected a stimulation method, twirling for 10 s at 1 Hz, to efficiently release AR biomarkers from a PE swab. Storage of sample solutions at -20 °C was optimal for the stability of biomarkers for the detection of AR. The new swab sample transfer device showed excellent concentration recovery efficiency (90-100%) for tryptase (Trp) and eosinophil cationic protein (ECP) without crosstalk between the two biomarkers. Finally, we compared the concentration of Trp in human NS samples of AR patients (n = 6) pre-processed by the new device with that by centrifuge as a standard method. As a result, the concentrations of Trp in NS were very similar in both groups. Therefore, this device can be utilized as an effective sample transfer and pre-processing device for point-of-care testing of AR.

Comparison of Computed Tomography, Magnetic Resonance Imaging, and Positron Emission Tomography and Computed Tomography for the Evaluation Bone Invasion in Upper and Lower Gingival Cancers.

PURPOSE: Preoperative detection of bone invasion is important in cases of gingival cancer. The aim of this study was to compare the diagnostic value of 3 imaging methods for the detection of bone invasion in upper and lower gingival cancer: computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) and CT. MATERIALS AND METHODS: This retrospective cohort study enrolled patients who underwent a maxillectomy or a mandibulectomy for gingival cancer. Each preoperative image (CT, MRI, or PET/CT) was reviewed for the presence of bone invasion, and the possibility for bone invasion was graded. These results were verified with pathology reports. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of mandibular involvement in alveolar bone were calculated, and a receiver operating characteristics (ROC) curve analysis was performed. RESULTS: Forty patients (27 men and 13 women) were enrolled. Pathologic examination disclosed bone invasion in 25 of the 40 patients. Of these patients, 13 had maxillary and 12 had mandibular alveolus involvement. The diagnostic accuracy of CT (90.0%) was highest among the 3 modalities for the detection of bone invasion. In the ROC curve analysis, values for the area under the curve for upper gingival cancer were lower than those for lower gingival cancer. CONCLUSIONS: The 3 imaging methods were less sensitive for the detection of bone invasion in upper gingival cancer than in lower gingival cancer. Cases of upper gingival cancer should be evaluated more carefully for bone invasion before surgery.

Risk Factors for Complications of Intraoral Removal of Submandibular Sialoliths.

PURPOSE: Intraoral removal of submandibular sialoliths is a surgical technique for the treatment of sialolithiasis and is reported to have excellent outcomes. The aim of this study was to determine the risk factors leading to complications of this procedure. PATIENTS AND METHODS: The medical records of 200 patients who had undergone intraoral removal of sialoliths from January 2006 through June 2015 were retrospectively reviewed. A telephone survey was used to check postoperative symptoms. Dry mouth, wound infection, lingual nerve dysfunction, and recurrence were considered complications. Computed tomograms of the neck were reviewed for location, shape, number, and size of the stone. RESULTS: Forty-four patients reported a complication. The incidence of complications was significantly higher in patients with stones in the proximal region of the salivary duct (proximal group) than in those with middle or distally located stones (middle/distal group; P < .05). The average stone size was larger in the proximal group; the operation time and length of admission also were longer in the proximal group, with a statistically significant difference (P < .05). Complaints of lingual nerve dysfunction were significantly higher in the proximal group than in the middle/distal group (P < .05). CONCLUSION: Patients with proximally located stones had more complications, especially lingual nerve dysfunction, than those with middle or distally located stones. The former group also required a longer operation time and hospital stay.

Label-Free Detection of Salivary Pepsin Using Gold Nanoparticle/Polypyrrole Nanocoral Modified Screen-Printed Electrode.

Detection of salivary pepsin has been given attention as a new diagnostic tool for laryngopharyngeal reflux (LPR) disease, because saliva collection is non-invasive and relatively comfortable. In this study, we prepared polypyrrole nanocorals (PPNCs) on a screen-printed carbon electrode (SPCE) by a soft template synthesis method, using β-naphthalenesulfonic acid (NSA) (for short, PPNCs/SPCE). Gold nanoparticles (GNPs) were then decorated on PPNCs/SPCE by electrodeposition (for short, GNP/PPNCs/SPCE). To construct the immunosensor, pepsin antibody was immobilized on GNP/PPNCs/SPCE. Next, citric acid was applied to prevent non-specific binding and change the electrode surface charge before pepsin incubation. Electrochemical stepwise characterization was performed using cyclic voltammetry, and immunosensor response toward different pepsin concentrations was measured by differential pulsed voltammetry. As a result, our electrochemical immunosensor showed a sensitive detection performance toward pepsin with a linear range from 6.25 to 100 ng/mL and high specificity toward pepsin, as well as a low limit of detection of 2.2 ng/mL. Finally, we quantified the pepsin levels in saliva samples of LPR patients (n = 2), showing that the results were concordant with those of a conventional ELISA method. Therefore, we expect that this electrochemical immunosensor could be helpful for preliminarily diagnosing LPR through the detection of pepsin in saliva.

Prevalence of snoring and high risk of obstructive sleep apnea syndrome in young male soldiers in Korea.

There are little information on prevalence of obstructive sleep apnea syndrome (OSAS) and clinical features in the young military population. The purpose of this study was to estimate the prevalence of snoring and high risk of OSAS in young male soldiers in Korea and to identify the risk factors of OSAS. A total of 665 participants (aged 20-23 yr) who visited the Armed Forces Ildong Hospital for regular physical examination were enrolled. All participants completed the Berlin Questionnaire and underwent a physical examination. The participants with high risk for OSAS completed portable sleep monitoring. The prevalence of snoring and high risk of OSAS in young male soldiers in Korea was 13.5% and 8.1%, respectively. The prevalence of high arched palate, tongue indentation, long uvula, large tonsil and retrognathia was significantly higher in the high risk OSAS group. High arched palate, long uvula or low lying soft palate, tonsil size III or IV, Epworth Sleepiness Scale score > 10 and obesity (BMI > 27 kg/m(2)) were found to independently predict OSAS. For early identification and treatment of young soldiers with OSAS in a military environment, a precise screening by questionnaire and physical examination is needed.

Circulating Tumor DNA in Saliva in an Orthotopic Head and Neck Cancer Mouse Model.

AIM: To evaluate the feasibility of conducting studies of saliva circulating tumor DNA (ctDNA) as a biomarker of metastasis or recurrence in our orthotopic head and neck cancer (HNC) mouse model. MATERIALS AND METHODS: A mouse model of recurrence or metastasis after tongue cancer resection was developed. Blood and saliva were collected at baseline and at the establishment of recurrence or metastasis. Real-time polymerase chain reaction was performed to quantify human long interspersed element (hLINE) in samples to assess the amount of ctDNA. RESULTS: In our model, salivary hLINE increased as the cancer developed and decreased after surgery. Plasma hLINE was significantly elevated in mice with metastasis. The presence of tongue cancer recurrence in mice was more correlated with hLINE concentration in saliva than in plasma. CONCLUSION: In our orthotopic model, salivary ctDNA better reflected tumor development and recurrence than did plasma ctDNA.

Association Between Pepsin in the Saliva and the Subjective Symptoms in Patients With Laryngopharyngeal Reflux.

OBJECTIVES: Our study was designed to further evaluate the relationships between the saliva pepsin level and the symptoms and quality of life of patients with laryngopharyngeal reflux (LPR). STUDY DESIGN: A prospective cohort study without controls. SETTING: Tertiary teaching hospital. SUBJECTS AND METHODS: We analyzed 50 patients diagnosed with LPR by 24-hour multichannel intraluminal impedance pH monitoring. All subjects were instructed to collect saliva samples upon waking in the morning. The saliva pepsin levels were analyzed using enzyme-linked immunosorbent assay. The Reflux Symptom Index, Reflux Finding Score, Laryngopharyngeal Reflux-Health-Related Quality of Life, and Short Form 36 survey were administered. RESULTS: The pepsin was detected in the saliva of 41 patients with LPR (17.15 ± 20.42 ng/mL). Nine patients did not have pepsin in the saliva. There were no significant associations between the pepsin level in the saliva and Reflux Symptom Index, Laryngopharyngeal Reflux-Health-Related Quality of Life, or Short Form 36 of patients with LPR. CONCLUSION: The saliva pepsin level is not significantly correlated with LPR symptoms or quality of life in LPR patients. It may be true that there is no association between pepsin levels and LPR symptoms, but this lack of association does not prove the lack of pathophysiological effect.

Prevalence and Predictors of Sjögren's Syndrome in Patients with Burning Mouth Symptoms.

AIMS: To investigate the prevalence and predictive factors of Sjögren's syndrome (SS) in a cohort of patients with burning mouth symptoms. METHODS: A total of 125 patients with burning mouth symptoms were enrolled in a prospective study and assessed for the presence of SS. The severity of oral symptoms was evaluated by using questionnaires. Salivary flow rates and salivary scintigraphy were used to evaluate salivary function. Patient laboratory work-ups were reviewed, and SS was diagnosed by a rheumatologist based on the American-European Consensus Group criteria. The differences between the SS patient group and the non-SS patient group were analyzed with chi-square test or t test. RESULTS: A total of 12 of the 125 enrolled patients (9.5%) had a positive autoimmune antibody test, and 6 (4.8% of the entire cohort) had SS (4 [3.2%] primary and 2 [1.6%] secondary). Patients with SS exhibited significantly decreased hemoglobin levels, an increased erythrocyte sedimentation rate, and an increased prevalence of autoantibody positive results compared to non-SS patients. Salivary scintigraphy showed that the uptake ratio of the submandibular gland in SS patients was decreased significantly. CONCLUSION: The prevalence of SS in patients with burning mouth symptoms was 4.8%. Therefore, clinicians who treat patients with burning mouth symptoms should evaluate laboratory findings and salivary functions to identify patients with SS.

Optimal timing of saliva collection to detect pepsin in patients with laryngopharyngeal reflux.

OBJECTIVES/HYPOTHESIS: To determine salivary pepsin levels as a function of collection time. STUDY DESIGN: A prospective, case-control study. METHODS: We selected 57 patients with clinical symptoms and signs of laryngopharyngeal reflux who underwent 24-hour multichannel intraluminal impedance-pH (24h MII-pH) monitoring tests, and 12 control subjects without clinical symptoms and signs of laryngopharyngeal reflux. All subjects were instructed to collect saliva samples upon waking, 1 hour after each meal (3 times per day), and upon any occurrence of laryngopharyngeal reflux (LPR) symptoms. The pepsin levels in saliva were measured by using enzyme-linked immunosorbent assay. The Reflux Symptom Index survey was also administered. RESULTS: A total of 50 patients in whom LPR was diagnosed based on results of 24h MII-pH monitoring test were enrolled in the study. The average pepsin level upon waking was 17.2 ng/mL, which was significantly higher than that measured in samples collected at any other time (P < .005). Pepsin levels were higher in patients with LPR than those in controls. The Reflux Symptom Index scores were statistically different between these groups of subjects. CONCLUSIONS: The levels of total pepsin in saliva collected upon waking were significantly higher in the group of patients presenting with LPR symptoms and who demonstrated at least one episode of proximal esophageal reflux during 24h MII-pH monitoring. Furthermore, the average pepsin level upon waking was higher than that measured at any other time. Measuring pepsin levels in the saliva upon waking may be a useful method in the diagnosis of LPR. LEVEL OF EVIDENCE: 3b Laryngoscope, 126:2770-2773, 2016.

Analysis of mandibular structure using 3D facial computed tomography.

OBJECTIVE: This study measured and analyzed the position and dimension of genial tubercle (GT) and mental foramen (MF) STUDY DESIGN: Retrospective chart review study. SETTING: Tertiary care teaching hospital. SUBJECTS AND METHODS: Two hundred ten subjects were included who received 3-dimensional (3D) facial computed tomography (CT), and the GT and MF were evaluated. Subjects were divided into 4 groups by gender and skeletal type. Seven variables were measured: (1) height of GT (GTH), (2) width of GT (GTW), (3) distance from apices of lower incisors to superior border of GT (LI-SGT), (4) distance from inferior border of GT to inferior border of mandible (IGT-IBM), (5) thickness of anterior mandible (MT), (6) distance from symphysis of mandible to MF (S-MF), and (7) distance from superior border of GT to inferior border of mandible (SGT-IBM). RESULTS: All the parameters showed marked differences in individuals. Class I males showed longer GTH, MT, and SGT-IBM than class I females (P < .05). IGT-IBM and S-MF were longer in class II males than in class I females (P < .05). LI-SGT and IGT-IBM also showed personal variation. CONCLUSION: Anatomical features of mandibular structures showed individual variations. GTH, IGT-IBM, MT, S-MF, and SGT-IBM also showed differences between the groups.

Intralesional injection versus mouth rinse of triamcinolone acetonide in oral lichen planus: a randomized controlled study.

OBJECTIVE: To compare the efficacy, relapse, and adverse effects between intralesional injection and mouth rinse of triamcinolone acetonide (TA) in patients with oral lichen planus (OLP). STUDY DESIGN: A randomized controlled study. SETTING: College medical center. SUBJECTS AND METHODS: Forty consecutive patients, who had been diagnosed with OLP, were recruited. Participants were randomly divided into 2 groups using intralesional injection or mouth rinse of TA. The severity of pain and burning sensation on a 10-cm visual analog scale (VAS) and the Oral Health Impact Profile-14 (OHIP-14) were assessed at weeks 0, 1, 2, 3, 4, and 6. The signs of OLP were quantified using a special scoring system for OLP. The rate of relapse and the adverse effects were compared between both groups. RESULTS: The VAS scores for pain and burning mouth sensation and objective scoring for OLP were significantly improved at 1, 2, 3, 4, and 6 weeks in both groups. The changes in the VAS for burning mouth sensation, OHIP-14, and objective scoring for OLP were similar between both groups. The change in the VAS for pain from baseline to week 1 in the intralesional injection group was significantly higher than in the mouth rinse group. The rate of adverse effects was significantly higher in the mouth rinse group than in the intralesional injection group (44.4% vs 5.0%). CONCLUSION: The efficacies of both treatments were similar. The rate of adverse effects was significantly lower for intralesional injection of TA than mouth rinse of TA.

Objective usefulness of thin silastic septal splints after septal surgery.

BACKGROUND: Silastic splints have been used as effective tools for septal support, avoiding adhesion, and mucosal healing after septal surgery. Furthermore, although many surgeons insert septal splints, no well-designed trials exist to support their use. A randomized double-blinded controlled trial was performed. METHODS: We recruited 40 subjects who had undergone septoplasty only without sinus surgery or turbinoplasty. A silastic septal splint was inserted in one side of the nasal cavity at the end of each septoplasty, with the other side serving as a control. The splint side and control side were randomly selected. Nasal discomfort score (10-point scale) and mucosal status (grades 1-4) were surveyed in a blinded setting on postoperative days 7 and 14. RESULTS: Forty of 83 subjects fulfilled the enrollment criteria. On the 7th postoperative day there was no significant difference in nasal discomfort between the splint and control sides (6.2 ± 1.28 and 5.7 ± 1.27, respectively; p = 0.116), but the mucosal status was better on the splint side than on the control side (1.5 ± 0.51 and 2.5 ± 0.85; p < 0.001). At 14 days postoperatively, the symptom score (2.7 ± 1.06 versus 3.8 ± 1.25; p < 0.001) and mucosal status (1.5 ± 0.55 versus 1.9 ± 0.68; p = 0.013) were significantly better on the splint side compared with the control side. CONCLUSION: Insertion of a silastic septal splint after septal surgery should be accepted as a routine procedure.

Advantages of intraoral removal over submandibular gland resection for proximal submandibular stones: a prospective randomized study.

OBJECTIVES/HYPOTHESIS: To compare surgical outcomes after intraoral removal of proximal submandibular stones versus traditional submandibular gland (SMG) resection. STUDY DESIGN: : A prospective randomized study. METHODS: Forty-four consecutive patients were diagnosed with proximal submandibular stones in the hilum of the submandibular gland by ultrasonography or computed tomography. All of the patients were randomized to undergo removal of the stones either by an intraoral approach (IORS group, 22 patients) or through SMG resection (SMGR group, 22 patients). We then compared the surgical outcomes between these two groups. RESULTS: Stones in the IORS group were significantly smaller than those in the SMGR group. There was no significant difference in the distance of the stones from the hilum between groups. The mean operation time in the IORS group was significantly shorter than that of the SMGR group. The mean hospital stay of the IORS group was also significantly shorter than that of the SMGR group, and IORS patients felt significantly less pain than did SMGR patients. No patient experienced any complication after surgery with the exception of a single patient who experienced transient and mild neck swelling. CONCLUSIONS: Intraoral removal of proximal submandibular stones has several advantages over SMG resection. Based on our results, we suggest that our intraoral removal method be selected as the primary procedure for the removal of proximal submandibular stones rather than SMG resection.