Study participants
This comparative cross-sectional study was performed at the Outpatient Clinic of the Institute of Rheumatology, University of Belgrade, Serbia. The period of recruitment was from the end of 2017 till the beginning of 2019. Fifty eight patients with primary SS, all fulfilling the American–European Consensus (AEC) classification criteria [6]. These criteria include subjective presence of ocular dryness; subjective presence of oral dryness; objective measures of ocular dryness by Schirmer’s test or corneal staining; focus score > 2 in a salivary gland biopsy; salivary scintigraphy showing reduced salivary flow (1.5 mL in 15 minutes) and/or diffuse sialectasias and positive autoantibodies against SS-A and/or SS-B. Primary SS is diagnosed when 4 out of 6 items are present; either salivary gland pathology or the presence of autoantibodies against SS-A/SS-B is mandatory. If the patients have been diagnosed with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) or scleroderma prior to developing their sicca symptoms the diagnosis of secondary SS is made.
Fifty five healthy controls of similar age and gender were enrolled in this comparative cross-sectional study. The majority of the healthy volunteers were recruited from the healthy staff of the Institute of Rheumatology. Together with other recruited healthy subjects they completed a health questionnaire. All study participants had given their informed consent according to the Declaration of Helsinki, and the study was approved by the Local Ethics Committee at the Institute of Rheumatology. Material preparation, data collection and analysis were performed by [Mirjana Šijan Gobeljić], [Vera Milić] and [Nemanja Damjanov].
The patients with pSS were aged from 25 to 77 years and were randomly and continuously recruited into the study from the Outpatient clinic of the Institute of Rheumatology. The exclusion criteria for the patients were inflammatory rheumatic diseases or systemic connective tissue diseases, active infections, malignant diseases, metabolic diseases or any other condition that may affect patient capability to participate in the study by the investigator’s opinion. The exclusion criteria for the healthy controls were subjective mouth and eye dryness, presence of chronic rheumatic, metabolic or malignant diseases.
Clinical assessment
Patients’ disease characteristics (activity of pSS, extraglandular manifestations), medical history, chronic diseases, use of medications, and lifestyle habits (such as smoking) were recorded. Objective xerophthalmia was assessed by Schirmer’s I test and Rose Bengal score determination. Oral involvement was evaluated by salivary scintigraphy of the major salivary glands. Salivary gland scintigraphy was performed with radioactive technetium-99m (Tc99m) pertechnetate. The difference between the maximum and minimum excretion after being stimulated by vitamin C divided by the maximum counts was defined as the excretion rate. It was classed as normal if excretion level was ≥50% or dysfunctional if excretion level was <50% [24, 25].
Labial salivary gland (LSG) biopsies were performed in patients with pSS. The changes observed in 4mm2 of salivary gland tissue were scored from 0 to 4, according to the semiquantitative scoring method of Chisholm and Mason [26]. Grade 0 was given based on absence of inflammatory infiltrate; grade 1 on the presence of slight infiltrate; grade 2 on the presence of moderate infiltrate of focus score < 1 (focus score is defined as number of aggregates of ≥ 50 lymphocytes per 4 mm2 of tissue). Grades 3 and 4 were given if focus scores were ≥ 1. Grades 3 and 4 were defined as pathological findings.
Laboratory assessment
Routine laboratory and immunoserological tests were carried out on all patients; antinuclear antibodies (ANA; positive if titre>1:80) were measured by indirect
immunofluorescence on the HEp-2 cell line substrate (Organtec Diagnostica, Germany). The serum levels of rheumatoid factor (IgM-RF) were determined by laser nephelometry whereas anti-extractable nuclear antigen antibodies anti-Ro/SS-A and anti-La/SS-B were detected by enzyme-linked immunosorbent assay (ELISA; Organtec Diagnostica) [24, 25].
Assessment of chemosensory and oral disorders
The participants were instructed not to eat, drink, or smoke for 1 h before their appointment at the Institute of Rheumatology. A detailed medical history was recorded and participants were examined by specialists in rheumatology and dentistry.
The olfactory and gustatory assessments were carried out as described below. Before olfactory testing, subjects were asked to score their own general subjective smell perception on a visual analogue scale (VAS) from 0 to 10, where self-reported smell score 0 = no smell perception and 10 = very good smell perception. In cognitive evaluation of olfactory function an identification test with 12 odor pens (Sniffin’Sticks-Screening; BurghartMesstechnik, Wedel, Germany) was used. The pens were positioned under the subject’s nose, approximately 2 cm from either nostril, for a maximum of 4sec. The subjects were instructed to choose from the three possible answers (anosmic/hyposmic – 0 points, or normosmic–1 point) for each of a 12 odors on a multiple-choices scoring card [27]. The answers chosen by every individual were recorded on a protocol sheet, and the data were scored for each of them. A normative classification was used to define anosmic (score: 0–5), hyposmic (score: 6–9), and normosmic (score: 10–12) subjects.
Before gustatory testing, subjects were asked to score their subjective taste perception on a VAS of 0–10, where self-reported taste score 0 = no taste perception and score 10 = very good taste perception. A gustatory assessment was performed after the subjects were given a detailed explanation of the testing procedure. Gustatory function was evaluated using taste strips with four basic taste qualities sweet, sour, salty and bitter [28]. Taste strips (length 8 cm, tip area2 cm2; Burghart Messtechnik, Wedel, Germany) were gently rubbed onto the anterior tip of the extended tongue. The taste qualities were presented in a random manner. A chart with names of the four taste qualities was placed in front of the subjects during testing in order to ask the subjects to identify the taste of the strip. The subjects were allowed to rinse their mouths with water during the gustatory testing. The semi-quantitative evaluation for each of four taste qualities was performed as follows: 0=loss of ability to taste, 1 = reduced ability to taste and 2 = normal ability to taste. This protocol resulted in a total of maximum score 8, for each subject.
The subjects of this study completed a questionnaire for the assessment of dysgeusia, burning sensation in the tongue (BST), and halitosis. In addition they described their experience of these conditions using open-ended questions. They completed oral health-related quality of life (OHRQoL) questionnaire using the 14-item short form of the Oral Health Impact Profile (OHIP-14) [29-31]. Serbian source version of OHIP-14 was produced after the questionnaire had undergone back translation, linguistic and cultural validation. Serbian version of the OHIP 14 was linguistically validated (and psychometrically tested) since no Serbian version was available. Specifically, questionnaire has been translated from English to Serbian by two independent translators. The resulting questionnaire was pretested in 10 patients before its use in this study in order to avoid any misunderstandings regarding meaning of each question. The translated version was considered to be well understood and accurate from the patients, and no further cultural modification seemed necessary.
The total OHIP-14 sum score ranges from 0 to56, giving an overall indication of the patient’s OHRQoL. A high OHIP-14 score indicates a poor OHRQoL.
Disease activity and the presence of extraglandular manifestations were estimated using the EULAR index of disease activity (ESSDAI, range 0-123) [32, 33]. The subjective evaluation of the dryness intensity, joint pain and fatigue were assessed using EULAR SS Patient Reported Index (ESPRI, range 0-10) [31].
Statistical analyses
Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) version 16.0. The sample sizing procedure was as follows: we defined our Population Size and that our Confidence Level should be 95% which corresponds to a Z-score of 1.96. Based on the relevant literature, we used 0.5 for SD value, the number that fairly enough ensures that our sample will be large enough. After defining these values, we used the Sample size formula: Necessary Sample Size = (Z-score)2 * StdDev*(1-StdDev) / (margin of error)2.
Normality of the data was assessed using Kolmogorov-Smirnov normality test. Independent samples t-test was used for comparing normally distributed continuous variables in both patient and control groups. A chi-square test was used to compare dichotomous variables. Odds ratios (ORs) with 95% confidence intervals (CIs) for smell or taste alterations in patients with pSS and healthy controls were determined. P values < 0.05 were considered statistically significant.