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Laryngopharyngeal Reflux: Symptoms, Signs,
and Presence of Pepsin in Saliva - A Reliable
Diagnostic Triad
Shilpa Divakaran1Sivaa Rajendran2Roshan Marie Thomas3Jaise Jacob2Mary Kurien2
1Department of ENT, NMC Specialty Hospital, Muscat, Oman
2Department of Biochemistry, Pondicherry Institute of Medical
Sciences, Kalapet, Puducherry, India
3Department of ENT, City Hospital , Kochi, Kerala, India
Int Arch Otorhinolaryngol
Address for correspondence Shilpa Divakaran, MS ENT, MRCS Edin
(ENT), Specialty Doctor, Department of ENT, Sandwell and West
Birmingham NHS trust, Dudley Road, Birmingham, United Kingdom
(e-mail: dr.d.shilpa@gmail.com).
Introduction
Gastroesophageal reflux disease (GERD) is defined as the
retrograde flow of gastric contents into the esophagus or
above. Laryngopharyngeal reflux (LPR) is the condition aris-
ing from the retrograde flow of gastric contents into the
larynx/pharynx, thus causing tissue damage that results in a
wide array of clinical symptoms and signs. Though LPR and
GERD are an extension of similar diseases, they differ con-
siderably in the pathophysiology, clinical presentation, diag-
nosis, and treatment. Gastroesophageal reflux disease is said
to occur due to dysfunction of the lower esophageal sphinc-
ter whereas in LPR, the up per esophageal sphincter is at fault.
The main symptom of GERD is heartburn due to esophagitis.
In LPR, only 25% of the patients have esophagitis, and around
40% complain of heartburn.1
Keywords
►reflux findings score
►reflux symptoms
index
►saliva
►pepsin
►laryngopharyngeal
reflux
Abstract Introduction Twenty-four-hour multichannel intraluminal impedance with double
probe pH monitoring (MII-pH), though considered the most sensitive tool for the
diagnosis of gastroesophageal reflux disease (GERD), is invasive, time consuming, not
widely available, and unable to detect non-acid reflux. In contrast, the presence of
pepsin in the saliva would act as a marker for reflux, considering that pepsin is only
produced in the stomach.
Objective To evaluate the predictive value of salivary pepsin in diagnosing laryng-
opharyngeal reflux (LPR) as suggested by the results of reflux symptom index
(RSI >13), reflux finding score (RFS >7), and positive response to treatment with a
4-week course of proton-pump inhibitors.
Methods This prospective study was done at a tertiary care hospital on 120 adult patients
attending ENT OPD with clinicaldiagnosis of LPR. The presence of pepsin in theirpharyngeal
secretions and saliva using a lateral flow device, the Peptest, was compared with RSI, RFS,
and with the response to medical treatment using the Chi-squared test.
Results Salivary pepsin was found to be positive in 68% of the patients, with 87.5% of
them showing positive response to treatment. Chi-squared analysis showed a signifi-
cant association between positive salivary pepsin and RFS >7, RSI >13, a combination
of RFS >7andRSI>13 as well as with response to treatment (p<0.0001).
Conclusion When considered along with the clinical indicators of RFS and RSI of more
than 7 and 13, respectively, and/or with a response to treatment, a positive salivary
pepsin test indicates statistically significant chance of presence of LPR.
received
October 27, 2019
accepted
March 4, 2020
DOI https://doi.org/
10.1055/s-0040-1709987.
ISSN 1809-9777.
Copyright © by Thieme Revinter
Publicações Ltda, Rio de Janeiro, Brazil
THIEME
Original Research
Published online: 2020-06-30
The major symptoms of LPR are globus sensation, throat
irritation, dysphagia, frequent throat clearing, drynessof throat,
chronic cough, hoarseness of voice, and voice fatigue. These
symptoms are long-term and cause intermittent concern to the
patient. If severe, there can be vocal fold/subglottic edema
andendolaryngeal mucus/granuloma formation.2It has been
shown that mixed as well as non-acid reflux contribute to most
of the symptoms of LPR as compared with purely acid reflux, in
the case of GERD.3Pepsin is an enzyme in gastric juice that has
been implicated in thepathogenesisof LPR as it can damage the
laryngeal mucosa, even at mild acidic or alkaline pH.4–6
The diagnosis of LPR based on symptoms and laryngeal
findings alone has poor sensitivity and specificity as most of
the abnormal findings are also seen in around 86% of healthy
individuals.7The mainstay of the diagnosis of LPR at present is
a combination of symptoms, fiberoptic endoscopic findings,
and 24-hour multichannel intraluminal impedance (MII) com-
bined with double-probe pH monitoring (MII-pH). Multichan-
nel intraluminal impedance-pH monitoring is now considered
the most sensitive tool for the diagnosis and characterization
of GERD and its laryngopharyngeal symptoms. It detects both
acid and non-acid gastric reflux and assesses the proximal
extent and nature of refluxate. However, MII-pH monitoring is
invasive, time consuming and not available in many centers,
with additional patient intolerability.8–11 Moreover, non-acid
reflux cannot be detected by pH monitoring alone. Recent
insight intothe pathophysiology of LPRwith the help of MII-pH
studies has demonstrated non-acid and mixed reflux to be
more common than acid reflux.3Pepsin plays a major role in
the pathogenesis of LPR. Pepsin can damage the laryngeal and
pharyngeal mucosa at both acidic and alkaline pH, as it shows
some activity even at pH 8.12 Johnston et al did a prospective
translational study in established porcine in vitro model to
examine the effect of active/inactive pepsin on laryngeal CAIII
and Sep70 protein levels. They reported detectable levels of
pepsin in laryngeal epithelia after a reflux event.12 Normally,
pepsin in this site would be enzymatically inactive, as the
mean pH of the laryngopharynx is 6.8. Significantly, pepsin
would be reactivated by a subsequent decrease in pH, such as
would occur during an acidic reflux event or possibly after
uptake into intracellular compartments of lower pH. Since
pepsin is produced only in the stomach, its presence in the
saliva would act as a marker for reflux.13 Pepsin, thus, has the
potential to overcome the invasive,time-consuming,and ex-
pensive MII-pH studies, as it can be easily detected in the
pharyngeal secretions and saliva using a lateral flow device
such a Peptest.
There is a paucity of reports from the Indian subcontinent
on this easily available, relatively less expensive, less time-
consuming and non-invasive test utilizing salivary pepsin in
the diagnosis of LPR. Hence, this study was undertaken.
Materials and Methods
This prospective study was performed at a tertiary carehospital
following institutional research and ethical committee approval
(No. RC/16/142). The objective of the present study was to
evaluate the diagnostic value of pepsin in saliva in the diagnosis
of LPR. Written informed consent was obtained from all
patients. All adult patients attending the ear, nose and throat
outpatient department with a history of change in voice/
burning sensation in the substernal or epigastric region/regur-
gitation/dysphagia/throat pain/cough/foreign body sensation
in throat/frequent throat clearing for more than 4 weeks with
clinical diagnosis of LPR were selected for the study. They were
then asked to complete a questionnaire with various possible
symptoms suggestive of LPR for calculating reflux symptom
index (RSI). The RSI is a 9-point questionnaire, each rated on a
Likert scale from 0 to 5,considering a score >13 suggestive of
LPR.8All patients then underwent flexible nasopharyngolar-
yngoscopy for assessing reflux findings score (RFS), which is an
8-component assessment tool for quantifying thevseverity of
laryngeal inflammation. An RFS score >7issuggestiveofLPR.
9
Those with previous laryngeal surgery, neoplasm of the phar-
ynx and larynx, chronic granulomatous lesion of the larynx/
pharynx, and use of proton pump inhibitors in the previous
month were excluded.
Saliva samples were collected 1 hour after meals. Patients
were instructed to cough up saliva from the back of their throat
and spit into 30-ml standard tubes containing 0.5mL of
0.01 mol/L citric acid at pH 2.5 to preserve the action of any
pepsin present. The samples were refrigerated at 4°C. Immu-
noserologic pepsin analysis was performed within a week of
collecting the sample, using a Peptest lateral flow device (LFD)
(RD Biomed Ltd, Hull,UK) by one of the coinvestigatorswho was
blinded to the clinical data. The collection tubes were centri-
fuged at 4,000 rpm for 5 minutes. EightyμL of the supernatant
were drawn up using standardmicropipettes and transferred to
a clear screwtop microtube containing 240 μL of migration
buffer solution. This tube was mixed with a vortex mixer for
10 seconds. Using a dual bulb pipette, 80 μL of this sample was
placed in the well of the lateral flow device (LFD). The control
line appeared within a few minutes and the test line appeared
within 5 to 15 minutes if the result was positive.
All patients were given proton pump inhibitor (PPI –eso-
meprazole 40 mg twice a day) and prokinetic (domperidone
10 mg twice a day) for 4 weeks and the response to treatment
was assessed with RSI reduction by 50%. The patients were
divided into 3 groups for analysis and response to treatment
groupA (with RFS >7); Group B (RFS >7 and RSI >13); Group C
(RFS >7, RSI >13,and positive responseto treatment) (►Fig. 1)
Results
A total of 120 patients were recruited for the study, of which
there were 53 men and 67 women. The age range was from 21
to 68, with the average agebeing 40 years. The average age was
39 in those with positive salivary pepsin and 43 in those with a
negative test. The most common symptoms were throat
irritation (58.3%), globus sensation (46%), dry cough (24%),
and regurgitation (15%). The mean RSI was 14.86 overall, 16.61
in the pepsin-positive, and 12.14 in the pepsin-negative
patients. The mean RFS was 8.53 overall, 9.01 in the pepsin-
positive, and 7.89 in the pepsin-negative patients. The general
characteristic of both groups (pepsin-positive and negative)
are shown in ►Table 1.
International Archives of Otorhinolaryngology
Laryngopharyngeal Reflux: Symptoms, Signs and Presence of Pepsin in Saliva Divakaran et al.
Of the 120 participants who enrolled in the study, 97
(80.8%) had RFS >7, and 72 (60%) had RSI >13. On combining
these two parameters, there were a total of 68 (56.7%) partic-
ipants who had both RFS >7andRSI>13, whereas only 19
(15.8%) had both RFS and RSI below this cut off. Salivary pepsin
was found to be positive in 82 of the 120 subjects (68.1%).
Seventy-two ofthe patientsshowed a positive response to the
treatment, of which 63 had a positive Peptest, whereas 48
participants showed no response to the treatment,29 of whom
tested negative for pepsin (►Tables 2 &3).
The chi-squared analysis showed a significant association
between positive salivary pepsin and RFS >7 as well as with
RSI >13. Similarly, positive associations were observed for
positive pepsin and a combination of RFS >7andRSI>13
alone as well as with response to treatment (►Table 4).
Discussion
Laryngopharyngeal reflux has become a highly prevalent
condition, causing considerable concern both for the patient
and the otolaryngologist.10 It presents a diagnostic challenge
owing to atypical presentation and lack of readily available
sensitive diagnostic tests. Mo re often than not, the treatment
is started empirically with PPIs. In a common clinical setting,
LPR is diagnosed based on symptoms and laryngoscopic
findings. Reflux symptom score (RSI) is a commonly used
Fig. 1 Flowchart showing study procedures.
Table 1 Comparison of general characteristics of pepsin-
positive and pepsin-negative groups
Characteristic Pepsin (þ)Pepsin(-)
Age 39 43
Men 29 (55%) 24 (45%)
Women 44 (66%) 23 (34%)
Mean RSI 16.61 12.14
Mean RFS 9.01 7.89
Mean RSI after
treatment
10.34 9.53
Response to
treatment
54 (45%) 19 (16%)
Most common
item in RSI
Globus
sensation
(54%)
Globus
sensation
(38%)
Excess throat
mucus (23%)
Dry cough
(18%)
Most common
sign in RFS
Arytenoid
congestion
(67%)
Arytenoid
congestion
(47%)
Thick
endolaryngeal
mucus (34%)
Vocal cord
erythema
(26%)
Abbreviations: RFS, reflux findingscore;RSI,reflux symptom index.
International Archives of Otorhinolaryngology
Laryngopharyngeal Reflux: Symptoms, Signs and Presence of Pepsin in Saliva Divakaran et al.
tool, which helps in keeping a record of symptoms for
reassessment after treatment. An RSI score >13 is highly
suggestive of LPR.9In our study, the me an RSI score was 14.86
(overall), which showed improvement with treatment in 60%
of patients (►Table 1).
Several studies have been conducted evaluating pepsin as
a marker for LPR, all of them showing considerable variation
in the sensitivity and specificity of Peptest. A cross-sectional
study conducted by Ocak e al,14 in which a two-channeled
24-hour esophageal p H monitoring catheter was placed i n 20
patients with a suspicion of LPR, and each patient gave one
sample of sputum for the immunoserologic pepsin detection
test. This test was noted to have a sensitivity of 30% and a
specificity of 100%, with a positive predictive value of 100%.
The low sensitivity was attributed to single sample collec-
tion. In addition, this study also noted the proximal probe of
the pepsin positive patients having an apparent acidic pH as
compared with the pepsin-negative group (pH: 3.26 versus
Table 2 Distribution of different groups based on salivary
pepsin
Group Pepsin þPepsin -
RFS >77423
RFS <7815
RSI >13 68 4
RSI <13 14 34
RFS >7&RSI>13 65 3
RFS <7&RSI<13 5 14
Positive Response to treatment 63 9
No Response to treatment 19 29
RFS >7&RSI>13 & positive
response to treatment
56 1
Abbreviations: RFS, reflux findingscore;RSI,reflux symptom index.
Table 3 Latent class distribution
Group RFS >7RSI>13 RESPONSE þPEPSIN þFREQ CUMULATIVE FREQ
1þ 15 15
2þ 116
3 þ 218
4 þ 523
5þþ 225
6þ þ 530
7þ þ 535
8þ þ 035
9þ þ 035
10 þ þ 035
11 þþ þ 136
12 þþ 945
13 þ þ þ 449
14 þ þ þ 352
15 þþ þ þ 56 108
16 12 120
Abbreviations: FREQ, frequency; RFS, reflux findingscore;RSI,reflux symptom index.
Table 4 Chi-squared analysis between positive pepsin and the various clinical parameters
RFS >7
N(%)
RSI >13
N(%)
RFS >7&RSI>13
N(%)
RFS >7&RSI>13 &
positive response to
treatment
N(%)
YES NO YES NO YES NO YES NO
PEPSIN þ74 (76.3) 8(34.7) 68(94.4) 14(29.2) 65 (95.6) 5(26.3) 56 (98.2) 5(29.4)
PEPSIN - 23 (23.7) 15 (65.3) 4(5.6) 34(70.8) 3 (4.4) 14(73.7) 1 (1.8) 12(70.6)
TOTAL 97 23 72 48 68 19 57 17
Chi-squared statistic (pValue) 14.8
(<0.00019)
56.7
(<0.0001)
45.3
(<0.0001)
42.8
(<0.0001)
Abbreviations: RFS, reflux findingscore;RSI,reflux symptom index.
International Archives of Otorhinolaryngology
Laryngopharyngeal Reflux: Symptoms, Signs and Presence of Pepsin in Saliva Divakaran et al.
pH: 6). They thus suggest that a positive pepsin test in a
patient clinically suspected to have LPR can be a cost-effec-
tive, accurate, and alternative diagnostic method.12 Another
study by Alexander et al15 suggests that salivary pepsin has a
sensitivity of 78% and specificity of 53% for predicting an RFS
>7. Wang et al16 conducted a meta-analysis to assess the
diagnostic value of pepsin in saliva for LPR. The pooled
sensitivity and specificity were 64% and 68%, respectively.
They concluded that salivary pepsin has moderate value in
diagnosing LPR and requires further studies to optimize the
method of detection of pepsin. The reason for such varied
results across various studies could be differences in sample
size, number of salivary samples collected, timing of sample
collection, method of pepsin detection and criteria for diag-
nosis of LPR. As there is a wide range of concentration of
salivary pepsin observed in an individual over a period of
24 hours, samples collected soon after reflux event are more
likely to be positive.16 In our study, 82 patients (68.1%) were
positive for salivary pepsin. Similar results were observed in
a study by Ianella et al to assess the correlation between
obstructive sleep apne a (OSA) and LPR, where salivar y pepsin
was used to confirm the diagnosis of clinically suspected LPR
based on positive RSI & RFS. 66.6% of the patients with
clinical LPR tested positive for salivary pepsin.17
There are a few studies comparing treatment response to
salivary pepsin, but none have studied the correlation be-
tween pepsin and combined RSI, RFS, and a positive response
to treatment. A study by Wang et al noted significant
association between strongly positive salivary pepsin and a
good treatment response.18 Another study by Alexander et al
showed significant correlation between RFS scores and posi-
tive Peptest, but no correlation between RSI and a positive
Pepstest.15 Sereg-Bahar et al studied pepsin and bile acids in
the saliva of patients with LPR and reported a significant
correlation between the RSI and RFS scores and the level of
total pepsin and bile acids in the saliva.19 There was a
significant association between a positive Peptest and
RSI >13 and RFS >7, individually, as well as on combining
RSI and RFS together. The association was significant even
when combining RFS, RSI and response to treatment (p<
0.05), in our study (►Table 4), making our study the first to
compare associations between these parameters together.
This prospective study indicates that salivary pepsin test
is indeed a very useful test in the diagnosis of LPR, especially
in a set up in which MII-pH is not available. A positive pepsin
test in combination with RSI, RFS and trial of response to
treatment revealed significant association.
Conclusion
In the clinical presentation of a constellation of symptoms
and signs in LPR, RSI)and RFS)are to be considered. The
present prospective study of immunoserologic pepsin anal-
ysis in the saliva of patients revealed that a positive salivary
pepsin test along with RSI >7, RFS >13, and positive
response to treatment with PPIs indicates statistically signif-
icant chance of presence of LPR. Hence, salivary pepsin test, a
relatively inexpensive, less time-consuming and patient
friendly non-invasive test is being suggested as part of
armamentarium in the diagnosis of LPR.
Limitations
The diagnosis of LPR could not be confir med with MII-pH due
to non-availability of the equipment.
Only a single sample of saliva was tested. The number of
positives may have been more if there were multiple sam-
ples. However, multiple samples could not be obtained due to
limited funds. There was a lack of control group.
Funding
The present study was possible with internal funding
provided by the research committee of the Pondicherry
Institute of Medical Sciences, Puducherry, India.
Conflict of Interests
The authors have no conflict of interests to declare.
References
1Koufman JA. The otolar yngologic manifestations of gastroesoph-
ageal reflux disease (GERD): a clinical investigation of 225
patients using ambulatory 24-hour pH monitoring and an exper-
imental investigation of the role of acid and pepsin in the
development of laryngeal injury. Laryngoscope 1991;101(4 Pt
2, Suppl 53):1–78
2Koufman JA, Aviv JE, Casiano RR, Shaw GY. Laryngopharyngeal
reflux: position statement of the committee on speech, voice, and
swallowing disorders of the American Academy of Otolaryngolo-
gy-Head and Neck Surgery. Otolary ngolHea d NeckS urg 2002;127
(01):32–35
3Lee JS, Jung AR, Park JM, Park MJ, Lee YC, Eun YG. Comparison of
Characteristics According to Reflux Type in Patients With Lar-
yngopharyngeal Reflux. Clin Exp Otorhinolaryngol 2018;11(02):
141–145
4Bulmer DM, Ali MS, Brownlee IA, Dettmar PW, Pearson JP.
Laryngeal mucosa: its susceptibility to damage by acid and
pepsin. Laryngoscope 2010;120(04):777–782
5Jiang A, Liang M, Su Z, et al. Immunohistochemical detection of
pepsin in laryngeal mucosa for diagnosing laryngopharyngeal
reflux. Laryngoscope 2011;121(07):1426–1430
6Hicks DM, Ours TM, Abelson TI, Vaezi MF, Richter JE. The preva-
lence of hypopharynx findings associated with gastroesophageal
reflux in normal volunteers. J Voice 2002;16(04):564–579
7Hila A, Agrawal A, Castell DO. Combined multichannel intra-
luminal impedance and pH esophageal testing compared to pH
alone for diagnosing both acid and weakly acidic gastroesopha-
geal reflux. Clin Gastroenterol Hepatol 2007;5(02):172–177
8Belafsky PC, Postm a GN, Koufman JA. Validity and reliabilit y of the
reflux symptom index (RSI). J Voice 2002;16(02):274–277
9Belafsky PC, Post ma GN, Koufman JA. T he validity and reliabilit y of
the reflux finding score (RFS). Laryngoscope 2001;111(08):
1313–1317
10 Altman KW, Stephens RM, Lyttle CS, Weiss KB. Changing impact of
gastroesophageal reflux in medical and otolaryngology practice.
Laryngoscope 2005;115(07):1145–1153
11 Knight J, Lively MO, Johnston N, Dettmar PW, Koufman JA.
Sensitive pepsin immunoassay for detection of laryngopharyng-
eal reflux. Laryngoscope 2005;115(08):1473–1478
12 Johnston N, Dettmar PW, Bishwokarma B, Lively MO, Koufman JA.
Activity/stability of human pepsin: implications for reflux attrib-
uted laryngeal disease. Laryngoscope 2007;117(06):1036–1039
International Archives of Otorhinolaryngology
Laryngopharyngeal Reflux: Symptoms, Signs and Presence of Pepsin in Saliva Divakaran et al.
13 Bardhan KD, Strugala V, Dettmar PW. Refluxrevisited:advancing the
role of pepsin. Int J Otolaryngol 2012;2012:646901. Doi: 10.1155/
2012/646901
14 Ocak E, Kubat G, Yorulmaz İ. Immunosero logic pepsin detection in
the saliva as a non-invasive rapid diagnostic test for laryngophar-
yngeal reflux. Balkan Med J 2015;32(01):46–50
15 Spyridoulias A, Lillie S, Vyas A, Fowler SJ. Detecting laryngophar-
yngealreflux in patients with upper airways symptoms: Symptoms,
signs or salivary pepsin? Respir Med 2015;109(08):963–969
16 Wang J, Zhao Y, Ren J, Xu Y. Pepsin in saliva as a diagnostic
biomarker in laryngophar yngeal reflux: a meta-analysis. Eur Arch
Otorhinolaryngol 2018;275(03):671–678
17 Iannella G, Vicini C, Polimeni A, et al. Laryngopharyngeal
Reflux Diagnosis in Obstructive Sleep Apnea Patients Using
the Pepsin Salivary Test. Int J Environ Res Public Health 2019;
16(11):2056
18 Wang CP, Wang CC, Lien HC, et al. Saliva Pepsin Detection and
Proton Pump Inhibitor Response in S uspected Laryngopharyngeal
Reflux. Laryngoscope 2019;129(03):709–714
19 Sereg-Bahar M, Jerin A, Jansa R, Stabuc B, Hocevar-Boltezar I,
Sereg-Bahar M. Pepsin and bile acids in saliva in patients with
laryngopharyngeal reflux - a prospective comparat ive study. Clin
Otolaryngol 2015;40(03):234–239
International Archives of Otorhinolaryngology
Laryngopharyngeal Reflux: Symptoms, Signs and Presence of Pepsin in Saliva Divakaran et al.
