| Background and ObjectiveLaryngopharyngeal reflux(LPR)is an inflammatory disease of the upper airway and upper digestive tract tissues caused by the reflux of stomach contents or duodenal contents into the throat.LPR is characterized by chronic inflammation of the throat and may affect a wide range of upper airway and upper digestive tract tissues.Common clinical symptoms include a sensation of foreign body in the throat,throat clearing,speech difficulties,excessive mucus in the throat,and post-nasal drip.Due to the lack of specific symptoms and signs for LPR,as well as a lack of sensitive and specific objective diagnostic methods,and a lack of a "gold standard" diagnosis,diagnosing LPR is currently a bottleneck that needs to be overcome.Multiple studies suggest that testing for pepsin in saliva or throat tissue may become a reliable diagnostic marker for LPR,but there is debate regarding the diagnostic threshold.This study aims to explore the diagnostic threshold for pepsin in saliva and tissue samples collected from patients with LPR-related diseases,in order to provide valuable reference values for using pepsin in saliva or throat tissue as a sensitive and specific diagnostic marker for LPR.Research objects and inclusion and exclusion criteriaStudy subjects:1.Patients highly suffering from Laryngopharyngeal Reflux Disease(LPR)related diseases that needed surgery from June 2019 to April 2022;2.Thirty-five volunteers recruited by the Ear,Nose,and Throat Department of Southern Hospital from June 2018 to December 2018.All study subjects were asked about their medical history in detail,underwent electronic laryngoscopy,completed RFS/RSI questionnaires,and had 24-hour Dx-pH monitoring.Saliva samples were collected at four time points:upon waking up,1 hour after breakfast,30 minutes before dinner,and 1 hour after dinner.Inclusion criteria:1.Patients who were diagnosed to have LPR related disease:RSI score>13 or RFS score>7;no history of taking acid-suppressing drugs in the past two weeks;2.Healthy volunteers:aged between 18 and 70 years old;RSI score<3 and RFS score<1;no LPR-related symptoms,acute inflammation in the throat,other underlying diseases or history of PPI drug use.Exclusion criteria:Acute inflammation of the upper airway and upper digestive tract,anatomical variations in the upper airway and upper digestive tract,a history of malignant tumors or chemotherapy/radiotherapy,and mental illness.Volunteers with positive Dx-pH monitoring results were excluded.All study subjects provided a small amount of post-cricoid mucosa tissue.This study was approved by the Ethics Committee of Southern Hospital,and all study subjects were informed and agreed to participate in the study.Research methods1.24-hour oropharyngeal pH monitoringThe Dx-pH monitoring system(Restech3.2,USA)was used to monitor the dynamic changes of oral and pharyngeal pH for 24 hours in the subjects.Patients were advised to accurately record the start and end times of meals and lying down.After completing 24 hours of continuous monitoring,the Date View V3.2 software was used for data analysis,which yielded the dynamic pH waveform,related pH parameters,and Ryan index results of the oral and pharyngeal regions over the 24-hour period.2.Salivary PeptestAll enrolled subjects were instructed to provide saliva samples at four different time points:in the morning after fasting,1 hour after breakfast,30 minutes before dinner,and 1 hour after dinner.The collected saliva was stored in liquid nitrogen and analyzed using the Peptest within 2 months.For analysis,the saliva sample was removed from liquid nitrogen and gradually warmed up,then centrifuged at high speed(1500 rpm)for 5 minutes at 4℃.The clear supernatant from the top layer of the saliva was extracted with a pipette and mixed with 240 μl of migration buffer in a transparent microtube with a screw cap.The mixture was vortexed for 10 seconds,and then 80μL of the mixed sample was dropped into the sample slot of the test strip.After waiting for 15 minutes,the test strip was placed in a reader for measurement..3.Immunohistochemical staining and scoringThe post-pyloric mucosal tissue paraffin blocks were cut into slices,baked,deparaffinized,antigen retrieved under high pressure,blocked,incubated with pepsin antibody,stained with DAB,counterstained with hematoxylin,washed,blued,dehydrated,and sealed.The immunohistochemical staining results were observed under a light microscope and evaluated based on the following scoring criteria:The depth of staining in the cytoplasm of epithelial tissue cells was used to assess the degree of staining intensity,which was classified as negative(-),weak positive(+),positive(++),and strong positive(+++).If no staining was present,the score was 0.Pale yellow staining was considered weakly positive(1 point),brown staining was positive(2 points),and granular dark brown staining was strongly positive(3 points).The staining area was also scored as follows:<25%staining area received 1 point,25%-50%received 2 points,51%-75%received 3 points,and>75%received 4 points.Finally,the expression level of pepsin was divided into four grades based on the product of the staining area score and staining intensity score:Grade 0 indicated negative expression,Grades 1-4 indicated weakly positive expression,Grades 5-8 indicated positive expression,and Grades 9-12 indicated strongly positive expression.4.Statistical analysisSPSS version 22.0 was used for data analysis.If the continuous data followed a normal distribution,they were expressed as means ± standard deviation and independent t-tests were used.If not,non-parametric tests were used.The categorical data were described by percentages and tested using the chi-square test or Fisher’s exact test.Pearson’s correlation analysis was used to analyze the correlation between continuous variables,while Spearman’s rank correlation analysis was used for ordinal variables.A significance level of P<0.05 was considered statistically significant.Results1.Observation of Oropharyngeal pH Monitoring in Different DiseasesA total of 153 patients with Laryngopharyngeal Reflux Disease(LPR)-related diseases and 76 healthy volunteers without LPR-related symptoms were included in this study.The overall positive rate of Dx-pH monitoring was 36.6%,which was significantly higher than that in the healthy control group(10.8%).Among the eight LPR-related diseases included,the positive rates of Dx-pH monitoring were 39.4%(28/71)for vocal polyps,33.3%(6/18)for vocal cord cysts,35%(7/20)for vocal cord leukoplakia,20.8%(5/24)for laryngeal cancer,50%(2/4)for laryngeal contact granuloma,50%(5/10)for epiglottic cysts,and 60%(3/5)for retrocricoid space edema.Statistical analysis results showed that compared with the healthy control group,the positive rates of Dx-pH monitoring were significantly higher in several diseases,including vocal polyps,vocal cord cysts,vocal cord leukoplakia,laryngeal contact granuloma,epiglottic cysts,and retrocricoid space edema.The reflux event frequency,reflux exposure time,and other Dx-pH parameters were also significantly higher in these groups than in the control group.However,there was no significant difference in the positive rates of Dx-pH monitoring for laryngeal cancer compared to the control group.2.Threshold study of salivary pepsinogen for LPR diagnosisThe study included 60 patients with LPR,48 patients without LPR,and 35 healthy controls.A total of 572 saliva samples were collected and tested,with 384 samples testing positive for pepsinogen,resulting in an overall positivity rate of 67.1%.Among the LPR patient group,83.3%had detectable levels of pepsinogen(>16ng/ml)at least once in their saliva,which was significantly higher than the non-LPR group(60.4%)and the healthy control group(49.0%).However,there was no significant difference in the salivary pepsinogen positivity rates between the non-LPR group and the healthy control group.The median and percentile values for the highest concentration of pepsinogen measured in saliva collected from the LPR patient group at four time points were 87.2(21.4-214.2)ng/ml,which was significantly higher than that of the non-LPR patient group(27.2[0.0-68.6]ng/ml)and the healthy control group(0.0[0.0-40.0]ng/ml).There was no significant difference in salivary pepsinogen levels between the non-LPR group and the healthy control group.Among the four time points,only the LPR patient group had significantly higher levels of salivary pepsinogen after waking up,1 hour after breakfast,and 1 hour after dinner compared to the other two groups.There was no significant difference in salivary pepsinogen levels before dinner among the groups.ROC curve analysis showed that the diagnostic performance of Peptest for LPR was optimal when using a postprandial saliva level of ≥49 ng/ml,with a Youden index of 0.46,sensitivity of 60.0%,specificity of 86.7%,positive predictive value of 66.0%,negative predictive value of 83.3%,positive likelihood ratio of 4.48,and negative likelihood ratio of 0.46.3.The Consistency between the Expression of Pepsin in Tissue,Dx-pH Monitoring,and Salivary Pepsin.A total of 99 suspected LPR patients and 16 healthy controls were included in this study.Among the 99 patients,12 had strong positive expression of pepsinogen in their post-cricoid tissues,50 had positive expression,24 had weak positive expression,and 13 had negative expression.In the healthy control group,6 had weak positive expression and 10 had negative expression,with no positive or strong positive expression.Based on the IHC results,all subjects were divided into four groups:strong positive,positive,weak positive,and negative.It was found that the Dx-pH monitoring positivity rate and parameters were significantly higher in the strong positive and positive groups than in the weak positive and negative groups,with no significant difference between the strong positive and positive groups,or between the weak positive and negative groups.The Dx-pH positivity rate and reflux-related parameters were significantly positively correlated with the IHC score(correlation coefficient=0.582,P=0.00).Conclusion1.There is a significant correlation(P=0.00)between the occurrence of vocal cord polyps,contralateral reactive hyperplasia,and LPR,suggesting that LPR may play a role in the development of these conditions.Patients with laryngeal cysts,atypical proliferation of vocal cords,contact granulomas,vallecular cysts,and aryepiglottic space edema also have a higher incidence of reflux,suggesting a possible association with LPR.2.The salivary pepsin concentration in LPR patients was significantly higher(P=0.00)in the morning and after meals compared to non-LPR patients and healthy controls.Saliva sampling after meals had the highest diagnostic value,and the best diagnostic efficacy for LPR was achieved by using a diagnostic threshold of ≥49ng/ml for Peptest(AUC=0.78,Youden index=0.46).3.The expression level of tissue pepsin was significantly positively correlated(P<0.05)with the number of reflux events,reflux exposure time,and salivary pepsin level.When the moderate positive IHC score was used as the diagnostic threshold,there was weak agreement(Kappa<0.2)with Dx-pH monitoring results and Peptest positivity rate. |
PDF Full Text Request