Examination of Oral Candida Infection in Primary Sjögren's Syndrome Patients

Summary

Here, we present a protocol for the examination of oral Candida infection in patients with primary Sjögren's syndrome, which can be used for timely treatment and, thereafter, avoiding related complications.

Abstract

Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by symptoms such as dry mouth, dry eyes, and other systematic symptoms. Due to the hyposalivation experienced by pSS patients, oral dysbacteriosis often occurs. A common complication of pSS is the oral Candida infection. In this article, the authors describe systematic methods that can effectively diagnose oral Candida infection and identify the Candida strains using saliva, oral mucosal swabs, or mouthwash from pSS patients. The Sabouraud's Dextrose Agar (SDA), hyphal formation assay, potassium hydroxide (KOH) smear test, and calcofluor white (CFW) staining assay are used for the diagnosis of oral Candida infection. A Candida diagnostic agar is used for the identification of Candida strains. Finally, antifungal susceptibility testing is used to determine appropriate antifungal drug treatment. This standardized method can enhance the diagnosis, treatment, and future research of pSS-related oral Candida infections. Early diagnosis, using this method, can also prevent any complications arising due to delay in receiving appropriate treatment.

Introduction

Candidiasis is caused by Candida spp., which are opportunistic pathogens. Common strains include Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, and Candida dubliniensis¹. Opportunistic infections caused by Candida spp. include superficial Candida infections and invasive candidiasis. Invasive candidiasis mainly happens in immunocompromised individuals; for example, in patients with acquired immunodeficiency syndrome (AIDS), invasive candidiasis can significantly threaten the quality of life or even the life span². Superficial Candida infection, such as mucocutaneous candidiasis, is more common in patients³.

Oral candidiasis, a superficial Candida infection, is the most common human fungal infection. Candida albicans is a normal commensal of the mouth; the carriage rate of Candida spp. has been reported to range from 20% to 75% in the general population without any symptoms⁴. Overgrowth of Candida can lead to local discomfort in patients, such as the altered taste of sensation, burning sensation in the mouth, dysphagia due to poor nutrition, delayed recovery, and prolonged hospital stay. Long-term oral candidiasis can lead to severe invasive candidiasis, resulting in significant morbidity and mortality⁵. Oral candidiasis can also increase the risk of oral cancer⁵. Impaired salivary gland function is among the risk factors for oral candidiasis⁶. The initiation of infection is the adhesion of Candida to epithelial cell walls. This is followed by the proliferation and filamentation of Candida and the formation and maturation of biofilm⁷. Biofilms are extremely difficult to eradicate and are resistant to conventional antifungal treatment, making clinical treatment a challenge for biofilm-associated oral Candida infection⁷.

Primary Sjögren's syndrome (pSS) is an autoimmune disease that is characterized by dry mouth, dry eyes, and other systematic symptoms⁸. Dry mouth is the most frequent symptom of pSS. Saliva has important physiological antifungal functions. On the one hand, it can dilute and scour the mouth and thereafter remove organisms from the mucosa. On the other hand, saliva contains antimicrobial proteins, such as lactoferrin, sialoperoxidase, lysozyme, histidine-rich polypeptides, and specific anti-candidal antibodies, which can interact with the oral mucosa and prevent the overgrowth of Candida⁶. The reduced saliva flow may predispose pSS patients to oral candidiasis. An observational cross-sectional study conducted in 61 pSS patients found that 13.1% of pSS patients presented oral signs of candidiasis and colony forming unit (CFU)/mL of Candida albicans was found significantly and negatively correlated with levels of unstimulated whole saliva (UWS) and stimulated whole saliva (SWS)⁹.

The methods introduced in this manuscript can identify oral Candida infection based on characterized morphological attributes (e.g., the hyphae and yeast cells) in direct smear or after culturing¹⁰. The strain identification of the Candida using a Candida diagnostic agar is based on the formation of different colored colonies with varied morphology, which result from the cleavage of chromogenic substrates by species-specific enzymes¹¹. In this manuscript, systematic methods for detecting oral Candida infection are introduced, including traditional oral Candida culture and rapid examination by potassium hydroxide (KOH) smear test and calcofluor white (CFW) staining assay¹⁰. Additionally, the antifungal susceptibility test, which can guide clinical oral Candida infection treatment, has been introduced. In actual situations, it is not necessary to perform all of the introduced methods in this manuscript; the oral Candida detection method(s) can be selected according to the purpose. The examination of oral Candida infection and the determination of Candida strains in pSS patients not only benefit the disease treatment but also help assess the characterization of oral candidiasis and the species profiles.

Protocol

All procedures described below were approved by the Ethical Committee of Beijing Tiantan Hospital, Capital Medical University (NO. KY2023-177-01), and all patients involved in this study provided informed consent.

1. Patient inclusion and exclusion criteria

1. Inclusion criteria: Classify patients as pSS patients if they fulfill the 2016 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria¹². Specifically, base the final classification criteria on the weighted sum of five items: anti-SSA/Ro antibody positivity and focal lymphocytic sialadenitis with a focus score of ≥ 1 foci/4 mm², each scoring 3; an abnormal Ocular Staining Score of ≥ 5 (or van Bijsterveld score of ≥ 4), a Schirmer's test result of ≤ 5 mm/5 min and an unstimulated salivary flow rate of ≤ 0.1 mL/min, each scoring 1. Individuals with signs and/or symptoms suggestive of SS who have a total score of ≥ 4 for the above items meet the criteria for pSS.
2. Exclusion criteria: Exclude patients who cannot cooperate with sample collection and patients who are receiving treatment for oral Candida infection.
   NOTE: It is essential to note that the methods described below are also suitable for the comprehensive characterization of oral Candida infections in individuals with secondary SS.
3. To prevent potential contaminations during the collection, transportation, and culturing of Candida samples, take meticulous precautions. This includes meticulous experimental preparation, use of single-use and sterile materials, proper labeling, secure sealing of sampling tubes, and conducting experimental procedures in a professional biosafety cabinet.

2. Sample collection

1. Saliva collection: Instruct the patients to hold the tube with a funnel and let the saliva flow gradually into the tube along the lower lip. At the end of the collection, ask the patient to spit all the remaining saliva in the mouth into the tube. Collect the saliva for 15 min.
2. Mouthwash and swab collection: If the patient has no or extremely low unstimulated saliva flow (less than 0.03 mL/min), swab the oral mucosal area with suspected oral Candida infection at least 10x, then ask the patient to use 5 mL of phosphate-buffered saline (PBS; see Table of Materials) to rinse the mouth for 1 min. Store the mouthwash in a 50 mL sterile tube and put the swab into the mouthwash tube.
   NOTE: For pSS patients with unstimulated saliva flow of more than 0.03 mL/min, step 2.2 can still be used to collect samples for oral Candida infection detection.

3. Sample processing

1. Add 1 mL of PBS to the collected saliva. Do not add PBS into the mouthwash with PBS. Vibrate the mouthwash tube using a vortex for 1 min.

4. Candida culture

1. To make the Sabourand's Agar (SDA) medium, dissolve 70 g SDA (see Table of Materials) in 1000 mL of double distilled water.
2. Sterile with an autoclave at 121 °C for 15 min, plate 10 mL of this SDA medium in a 10 cm microbiological culture dish (see Table of Materials) and cool down.
3. Take 200 µL of sample prepared in step 3 and streak it onto the 10 cm microbiological culture dishes prepared with 10 mL of SDA medium in step 4.2.
4. Incubate for 48 h at 37 °C, and the Candida colony will mature. Temporarily store the SDA dish with Candida colonies at 4 °C. The Candida colonies cultured on the SDA dish can be stored for 2-4 weeks.

5. Candida strain identification

1. To make the medium, take out 47.7 g of Candida diagnostic agar (see Table of Materials) powder, put it into 1000 mL of double distilled water, and heat it to 100 °C.
2. When the medium is slightly boiling, plate 10 mL in a 10 cm microbiological culture dish and allow to cool.
3. Dissolve the Candida colonies in 500 µL of SDA medium (step 4), suspend and dissociate by pipetting.
4. Plate 50 µL of the sample with Candida colonies (step 5.3) onto the medium prepared in step 5.2. Incubate for 48 h at 37 °C until the Candida colonies are mature.
5. According to the color and the patterns of the Candida colonies, determine the strain(s) of the acquired Candida from pSS patients. Use the following Candida strain identification criteria: the emerald green colony is Candida albicans, the blue-gray colony is Candida tropicalis, the pink colonies with fuzzy edges, and microvilli are Candida krusei, the purplish red small colony is Candida glabrata, the milky white colony contains several strains including Candida parapsilosis.

6. Candida biofilm formation assay

1. To make the Yeast Nitrogen Base (YNB)-50 glucose (G) medium, weigh 1.34 g YNB (see Table of Materials) and 1.98 g D (+)-Glucose (see Table of Materials). Dissolve them in 200 mL of double distilled water.
2. To make the YNB-100G medium, take out 1.34 g YNB and 3.96 g D (+)-Glucose and dissolve them in 200 mL of double distilled water.
3. Filter the solutions with a 0.22 µm syringe filter (see Table of Materials). The filtered YNB-50G and YNB-100G medium can be stored at 4 °C for 3 months.
4. Pick the 3 (or more) colonies from the same strain on the dishes in step 5 and transfer them into 200 µL of YNB-50G medium (steps 6.1 and 6.3) in a 96-well plate (see Table of Materials). Do this for the strains observed in the dish. Culture overnight at 37 °C with shaking at 200 rpm.
5. Aspirate the Candida suspension, add 1 mL of PBS, centrifuge the yeast cells at 1500 x g for 5 min at room temperature, and discard the supernatant. Repeat this step 2x in the same tube.
6. Resuspend the cells in 1 mL of YNB-100G medium and prepare different dilutions (1:10, 1:100, and 1:1000) of cells. Take 200 µL of gradient cell suspension and plate it onto the 10 cm SDA medium by the spiral plate method¹³. The plating is done using a machine that deposits a known volume of sample on a rotating agar plate in an ever-decreasing amount in the form of an Archimedes spiral. Culture for 24-48 h at 37 °C.
7. Calculate the concentration of cells in different dilutions according to the number of CFUs in the SDA plate based on a standard method¹³. Briefly count the well-separated colonies on each plate. Count the colonies in one octant from the outer edge towards the center until at least 25 colonies are observed. Also, count the remainder of that arc where the 25^th count occurred. Calculate the concentration of the cells by dividing the colony count by the liquid volume corresponding to the area over which the colony count was obtained¹³. Adjust the concentration of cells to 1 x 10⁷ cells/mL.
8. Put 100 µL of the cell suspension in YNB-100G into a 96-well plate and incubate at 37 °C for 2 h while shaking at 200 rpm.
9. Cautiously take out the supernatant, wash 2x with PBS, and get away the floating yeast cells. The Candida biofilm forms at the bottom of the 96-well plate.

7. Hyphal formation assay

NOTE: Only Candida that can form hyphae (such as Candida albicans) can be induced for hyphal formation. Go to step 8 for further characterization of Candida that cannot form hyphae (such as Candida glabrata).

1. Resuspend the yeast cell suspension in step 6.6 and adjust the cell concentration to 1 x 10⁶ cells/mL with YNB-100G medium.
2. To prepare the RPMI 1640 complete growth medium, add 50 mL of Fetal Bovine Serum (see Table of Materials) into 450 mL of RPMI 1640 medium (see Table of Materials) and store at 4 °C for further experiment.
3. Transfer 2 µL of Candida cell suspension diluted in 200 µL of RPMI 1640 complete growth medium into a 96-well plate and culture overnight at 37 °C to let the hyphae and yeast cells form.

8. KOH smear test and CFW staining assay

1. Take out 1 µL of samples from step 3 or step 6 or step 7 using a precision molded loop (see Table of Materials) and smear an area of 1 cm x 1 cm on the glass slides. Wait for it to dry at room temperature.
2. To make 10% KOH solution, weigh 10 g KOH powder (see Table of Materials) and dissolve it in 100 mL of double-distilled water.
3. Heat the slide slightly with an alcohol lamp until the specimen dissolves. Stain the slides prepared in step 8.1 with 10% KOH (step 8.2), and cover with a cover slip. Slightly press the cover slip to make the sample transparent.
4. Visualize the hyphae and yeast cells under the microscope.
5. Stain slides prepared in step 8.1 with a drop of CFW (see Table of Materials), cover with a coverslip, and wait for 1 min.
6. Visualize the hyphae using a fluorescence microscope at the magnifications of 100x and 200x. CFW is excited at 355 nm and detected at 300-440 nm. Photograph the images under the exposure time. Repeat 3x to avoid false positive results.

9. Antifungal susceptibility test

NOTE: This test is performed according to the manufacturer's instructions for the Yeast-Like Fungal Susceptibility Kit (Microdilution; see Table of Materials).

1. Prepare Candida colonies (step 4) less than 4 days old with the 0.85% NaCl ampule (see Table of Materials) and acquire the suspension having the turbidity equivalent to 2 McFarland. Determine the turbidity according to the McFarland Standards Kit (see Table of Materials). Use the prepared Candida suspension instantly.
2. Pipette 135 µL of ATB F2 medium in the kit containing 3 x 10⁴ yeast cells/mL into each cupule in the test strip and put the lid on. Put the test strip in a sealed container. Culture for 24 h at 35 °C.
3. Observe the growth of the Candida with visual interpretation (refer to the user manual). For the reference value (reference range), refer to the user manual. Check whether the control hole is growing adequately. If the growth of the control hole is insufficient or does not grow, then the results cannot be read and need another 24 h of cultivation.

Results

In this study, 12 patients with pSS were selected as representative patients and screened for oral Candida infection (Table 1). Among these patients, some patients exhibited typical oral lesions of Candida infection, including angular cheilitis (characterized by cracks at the corners of the mouth, desquamation, and hyperemia, Figure 1A), bright red edema on the gingival mucosa, yellowish-white stringy pseudo-membrane (Figure 1B...

Discussion

In the current manuscript, we provide a series of systematic, simple, and feasible methods for detecting oral Candida infections, identifying Candida strains, and testing the antifungal susceptibility of commonly used drugs in patients with pSS.

In practical settings, not all the methods introduced in this manuscript are necessary. The oral Candida detection method(s) can be chosen based on the specific purpose. KOH smear tests and CFW staining assays, using samples...

Materials

Name	Company	Catalog Number	Comments
0.22 µm Syringe Filter	MERCK	SLGV004SL
1.5 mL Centrifuge Tube	axygen	MCT-150-C-S
10 cm Microbiological Culture Dishes	Jet Bio-Filtration Co. Ltd	TCD000100
15 mL Centrifuge Tube	Jet Bio-Filtration Co., Ltd	CFT011150
50 mL Centrifuge Tube	Corning	430290
96-well Microplates	Corning	3599
API 0.85% NaCl ampule	BIOMERIEUX	20070
Calcofluor White Stain	Sigma-Aldrich	18909-100ML-F
CHROMagar Candida	CHROMagar	P002860	Candida diagnostic agar
D(+)-Glucose Monohydrate	Sinopharm Chemical Reagent Co.,Ltd	10010518
Fetal Bovine Serum	Gibco	26170035
McFarland Standards Kit	BIOMERIEUX	70900
Nunc precision molded loops	ThermoFisher Scientific	254399
Phosphate Buffered Saline	Gibco	C10010500BT
Potassium Hydroxide	Aladdin	P112284-500g
PowerSoil DNA Isolation Kit	MO BIO	12888-50
RPMI 1640 Medium	Gibco	11875119
Sabourand's Agar Medium	Solarbio	S9710
Yeast Nitrogen Base Without Amino acids	Solarbio	Y8040
Yeast-like Fungal Susceptibility Kit (Microdilution)	BIOMERIEUX	14204