Study design and subjects
This prospective case–control study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board of Incheon St. Mary’s Hospital (Incheon, South Korea). Also, written informed consent was obtained from all participants. Patients were recruited at the Cornea Service of Incheon St. Mary’s Hospital. Patients who wore contact lenses or had any of the following conditions or treatments that could affect eye dryness were excluded: meibomian gland dysfunction above grade 2, allergic conjunctivitis, ocular surgery within the last 6 months, punctal plug insertion, or topical treatments other than artificial tears in the 3 months before this study. Patients who were taking medication for a systemic disease such as autoimmune diseases, diabetes, hypertension, thyroid disease, allergic disease, or a depressive disorder were also excluded.
A total of 120 subjects were included in this study. In total, 15 subjects were male and 115 subjects were female. All of the study subjects had been referred to ophthalmology departments to investigate a diagnosis of primary SS and reported having dry eyes (e.g., dryness, itching, or the sensation of a foreign body) and a dry mouth (e.g., frequent thirst, dry feeling in the mouth and throat, burning or tingling sensation in the mouth and tongue, or cracked lips). We used the American College of Rheumatology/European League Against Rheumatism diagnostic criteria announced in 2016 to diagnose SS.43 In total, 48 patients (group A) were diagnosed with primary SS and the remaining 72 subjects (group B) were classified as having dry eyes and dry mouth symptoms that were unrelated to SS. Patients who had at least two of the following three characteristics were diagnosed as having SS: positive serum anti-SSA/Ro and/or anti-SSB/La (i.e., positive rheumatoid factor and antinuclear antibodies titer ≥ 1:320), labial salivary gland biopsy exhibiting focal lymphocytic sialadenitis with a focus score ≥ focus/4 mm2, or keratoconjunctivitis sicca with OSS.
Assessment of dry eye clinical parameters
OSS that included corneal and conjunctival staining scores, Schirmer’s test, and TBUT were evaluated by the same ophthalmologist (HBH). Dry eye symptoms were graded numerically from 0 to 4 using the OSDI, and the sum of these scores was used in the analyses. The OSDI, developed by the Outcomes Research Group at Allergan (Irvine, CA, USA), is a 12-item questionnaire that assesses vision-related functioning. Each item is scored on a 5-point scale, resulting in a total OSDI score ranging from 0 (no symptoms) to 100 (maximal symptoms).44 To measure TBUT, fluorescein was instilled from a fluorescein strip in a drop of saline (Haag-Streit, Koeniz, Switzerland). The fluorescein was applied to the superior-temporal bulbar conjunctiva with participants instructed to gaze infero-nasally, and the TBUT was measured using a slit-lamp under a cobalt blue light.45 The mean value of three TBUT measurements was used in this study. The OSS was measured in accordance with the SICCA registry ocular examination protocol.46 Corneal punctate epithelial erosions (PEEs) were evaluated and scored after staining with a fluorescent dye. Corneal scores were assigned as follows: PEEs absent = 0 points; one to five PEEs = 1 point; six to 30 PEEs = 2 points; and more than 30 PEEs = 3 points. An additional point was assigned if more than one patch of confluent aggregated staining was found in the pupillary area or if filaments present on the cornea were stained. The maximum possible score for each cornea was 6 points. To assess conjunctival staining scores, fluorescein was washed out with non-preserved saline solution, after which 1% lissamine green dye (Leiter’s Pharmacy, San Jose, CA, USA) was applied to the inferior conjunctival fornix. After several blinks, conjunctival staining scores in the temporal and nasal bulbar conjunctiva were evaluated separately as follows: up to nine dots = 0 points; 10 to 32 dots = 1 point; 33 to 100 dots = 2 points; and more than 100 dots = 3 points. The maximum possible score for the conjunctiva (i.e., temporal and nasal regions) was 6 points. For Schirmer’s test, standard Schirmer strips (Eagle Vision, Memphis, TN, USA) were placed in the lateral one-third of the lower eyelid, without topical anesthesia. After 5 min, the length of the wet portion of the strip was measured.
Sample collection and DNA extractions
The sampling and DNA extraction procedures used in this study were the same as those used in our previous research.14 In the absence of topical anesthesia, sterile dry cotton swabs (MEDIUS Corp., Tokyo, Japan) were used to sample four different locations: left upper bulbar conjunctiva, left lower bulbar conjunctiva, right upper bulbar conjunctiva, and the right lower bulbar conjunctiva. Each cotton swab was placed into a 1.5 mL sterilized Eppendorf microtube and frozen at –70℃ until DNA extraction. Nucleic acids were extracted from the swabs using an i-genomic Soil DNA Extraction Mini Kit (iNtRON, Seoul, South Korea) with a bead-beating apparatus, in accordance with the manufacturer’s instructions with the following method alterations. To enhance cell lysis, approximately 1.5 × lysis buffer was added to the samples for 30 min. DNA concentrations were measured using a Qubit fluorometer (Invitrogen, Gaithersburg, MD, USA).
16S rRNA gene library preparation
The hypervariable region 4 (V4) of the 16S rRNA gene was amplified from the extracted DNA using the 515F (5ʹ–GTGYCAGCMGCCGCGGTAA–3ʹ) and 806R (5ʹ–GGACTACNVGGGTWTCTAAT–3ʹ) primers.47 The polymerase chain reaction (PCR) conditions were 94°C for 3 min, followed by 35 cycles of 94°C for 45 s, 50°C for 60 s, and 72°C for 90 s, followed by a final extension step at 72°C for 10 min. Each sample was amplified in three replicate 25-µL PCR reactions, purified using the UltraClean PCR clean-up kit (Mo Bio Laboratories, Solana Beach, CA, USA), and combined into a single tube. Purified DNA was quantified by incorporating Picogreen (Invitrogen), in accordance with the manufacturer’s instructions.
Sequencing data analyses
Libraries for the V4 region alone were generated, and 16S rRNA-based MiSeq sequencing was conducted using standard Earth Microbiome Project protocols (http://www.earthmicrobiome.org/emp-standardprotocols/) at the Argonne National Laboratory (Lemont, IL, USA). QIIME version 1.9.1 was used for processing all sequencing reads.48 Sequences were de-multiplexed according to their barcodes, merged, and quality-filtered using the default parameters. OTUs were clustered using the latest Greengenes 13_8 reference sequences at 97% similarity (approximately corresponding to species-level OTUs), using the UCLUST algorithm. To remove chimera sequences, UCHIME within USEARCH was used.49 Then, sequences were aligned using PyNAST software.50 Representative OTU sequences were evaluated taxonomically using the ribosomal database project classifier,51 retrained on the Greengenes database (13_8),52 and mitochondrial sequences were filtered out of the OTU table. OTU reads found in negative control samples were removed from the OTU table.
To calculate species diversity and richness, alpha diversity analyses using the Shannon and Chao1 indexes were processed using the QIIME script. To measure the similarity among communities, beta diversity was calculated and MFA plots were constructed.
All experimental procedures including sampling, DNA extraction, preparation, and sequencing were implemented in a consistent manner. DNA extraction and PCR amplification of samples from eyes and unused sterile cotton swabs were all performed using exactly the same procedures. When DNA was extracted from the unused sterile cotton swabs, the quantity of DNA produced was very low (i.e., less than 0.05 ng DNA µL-1), and no DNA was amplified by the bacteria-specific primers (i.e., 27F and 1494R did not generate a PCR band). To minimize variability from any batch effects during different sequencing runs, all samples were sequenced using the same sequencing run. Negative controls were generated in reaction mixtures containing no template. During the sequence data analyses, OTU reads identified in DNA extractions of negative controls were removed from the sequencing data using metadata-based filtering.
Diagnostic testing procedure
The testing procedure for ophthalmic examinations was as follows:
- Subjective interview regarding symptoms of dry eyes (i.e., OSDI) and recording the patient’s medical history (HBH and KDK).
- Slit-lamp evaluation of the cornea, conjunctiva, eyelids, and Meibomian glands (HBH).
- Schirmer’s test (without topical anesthesia; HBH).
- TBUT test using fluorescein dye, repeated 3 times (HBH).
- Fluorescein and lissamine green staining of the cornea and conjunctiva (HBH).
Thereafter, subjects returned after 2 weeks and were classified into two groups according to whether the rheumatologist confirmed a diagnosis of SS.
- Conjunctival swab sampling (HBH, KDK and JMY).
- DNA extraction and sequencing analysis (BNK).
All data are expressed as means ± SD. Statistical analyses were performed using JAMOVI open-source (http://www.jamovi.org) and XLSTAT (Addinsoft, Paris, France) software. Group differences in age and dry eye parameters were evaluated using Student’s t-test after confirming that the data were normally distributed using the Kolmogorov–Smirnov test. Group differences in alpha diversity (i.e., Shannon and Chao1 indexes) were analyzed using a GLM by controlling for age and dry eye parameters as covariates. Before the GLM analysis, we confirmed that the data were normally distributed using the Q–Q plotting method. MFA was based on PCA and was used to create a visual representation of the data, with enrolled subjects and the various microbial strains arranged into two groups. To compare the microbial communities associated with the two groups, an ANOSIM was performed using Bray–Curtis dissimilarities after 9999 permutations and the “vegan” package in ANOSIM. We used Student’s t-test to compare species richness between the groups. A p-value < 0.05 was considered statistically significant.