Name: Examination of Oral Candida Infection in Primary Sjögrens Syndrome Patients (Video) | JoVE Journal
Uploaded: 2024-03-01T14:25:00.000+00:00
Duration: 5 min 26 s
Description: Learn a protocol for detecting oral Candida infection in primary Sjögren's syndrome patients using saliva collection and diagnostic agar methods. This approach enables timely treatment and prevents complications.

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5 minutes

This research was supported by the National Natural Science Foundation of China (82201084), China Postdoctoral Science Foundation (2022M722232), Beijing Postdoctoral Research Foundation (2023-ZZ-020), Miaopu Project of Beijing Tiantan Hospital, Capital Medical University (2023MP10) and General Research Fund, Hong Kong Research Grants Council (27111820 and 17116521).

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
<ol>
	<li>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 &#8805; 1 foci/4 mm2, each scoring 3; an abnormal Ocular Staining Score of &#8805; 5 (or van Bijsterveld score of &#8805; 4), a Schirmer&#39;s test result of &#8804; 5 mm/5 min and an unstimulated salivary flow rate of &#8804; 0.1 mL/min, each scoring 1. Individuals with signs and/or symptoms suggestive of SS who have a total score of &#8805; 4 for the above items meet the criteria for pSS.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Sample collection
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Sample processing
<ol>
	<li>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.</li>
</ol>
4. Candida culture
<ol>
	<li>To make the Sabourand&#39;s Agar (SDA) medium, dissolve 70 g SDA (see Table of Materials) in 1000 mL of double distilled water.</li>
	<li>Sterile with an autoclave at 121 &#176;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.</li>
	<li>Take 200 &#181;L 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.</li>
	<li>Incubate for 48 h at 37 &#176;C, and the Candida colony will mature. Temporarily store the SDA dish with Candida colonies at 4 &#176;C. The Candida colonies cultured on the SDA dish can be stored for 2-4 weeks.</li>
</ol>
5. Candida strain identification
<ol>
	<li>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 &#176;C.</li>
	<li>When the medium is slightly boiling, plate 10 mL in a 10 cm microbiological culture dish and allow to cool.</li>
	<li>Dissolve the Candida colonies in 500 &#181;L of SDA medium (step 4), suspend and dissociate by pipetting.</li>
	<li>Plate 50 &#181;L of the sample with Candida colonies (step 5.3) onto the medium prepared in step 5.2. Incubate for 48 h at 37 &#176;C until the Candida colonies are mature.</li>
	<li>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.</li>
</ol>
6. Candida biofilm formation assay
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Filter the solutions with a 0.22 &#181;m syringe filter (see Table of Materials). The filtered YNB-50G and YNB-100G medium can be stored at 4 &#176;C for 3 months.</li>
	<li>Pick the 3 (or more) colonies from the same strain on the dishes in step 5 and transfer them into 200 &#181;L 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 &#176;C with shaking at 200 rpm.</li>
	<li>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.</li>
	<li>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 &#181;L 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 &#176;C.</li>
	<li>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 25th 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.</li>
	<li>Put 100 &#181;L of the cell suspension in YNB-100G into a 96-well plate and incubate at 37 &#176;C for 2 h while shaking at 200 rpm.</li>
	<li>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.</li>
</ol>
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).
<ol>
	<li>Resuspend the yeast cell suspension in step 6.6 and adjust the cell concentration to 1 x 106 cells/mL with YNB-100G medium.</li>
	<li>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 &#176;C for further experiment.</li>
	<li>Transfer 2 &#181;L of Candida cell suspension diluted in 200 &#181;L of RPMI 1640 complete growth medium into a 96-well plate and culture overnight at 37 &#176;C to let the hyphae and yeast cells form.</li>
</ol>
8. KOH smear test and CFW staining assay
<ol>
	<li>Take out 1 &#181;L 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.</li>
	<li>To make 10% KOH solution, weigh 10 g KOH powder (see Table of Materials) and dissolve it in 100 mL of double-distilled water.</li>
	<li>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.</li>
	<li>Visualize the hyphae and yeast cells under the microscope.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
9. Antifungal susceptibility test
NOTE: This test is performed according to the manufacturer&#39;s instructions for the Yeast-Like Fungal Susceptibility Kit (Microdilution; see Table of Materials).
<ol>
	<li>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.</li>
	<li>Pipette 135 &#181;L 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 &#176;C.</li>
	<li>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.</li>
</ol>

Diagnosis and Strain Identification of pSS-Related Oral Candida

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<li>Serrano, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Risk+factors+related+to+oral+candidiasis+in+patients+with+primary+Sj%C3%B6gren%27s+syndrome%5BTitle%5D)+AND+%22Med+Oral+Patol+Oral+Cir+Bucal%22%5BJournal%5D)">Risk factors related to oral candidiasis in patients with primary Sjögren's syndrome.</a> Med Oral Patol Oral Cir Bucal. 25 (5), e700-e705 (2020).</li>
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<li>Mathews, S. A., Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+manifestations+of+Sj%C3%B6gren%27s+syndrome%5BTitle%5D)+AND+%22J+Dent+Res%22%5BJournal%5D)">Oral manifestations of Sjögren's syndrome.</a> J Dent Res. 87 (4), 308-318 (2008).</li>
<li>Leung, K. C. M., McMillan, A. S., Cheung, B. P. K., Leung, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sj%C3%B6gren%27s+syndrome+sufferers+have+increased+oral+yeast+levels+despite+regular+dental+care%5BTitle%5D)+AND+%22Oral+Dis%22%5BJournal%5D)">Sjögren's syndrome sufferers have increased oral yeast levels despite regular dental care.</a> Oral Dis. 14 (2), 163-173 (2008).</li>
<li>Lundstrom, I. M., Lindstrom, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subjective+and+clinical+oral+symptoms+in+patients+with+primary+Sj%C3%B6gren%27s+syndrome%5BTitle%5D)+AND+%22Clin+Exp+Rheumatol%22%5BJournal%5D)">Subjective and clinical oral symptoms in patients with primary Sjögren's syndrome.</a> Clin Exp Rheumatol. 13 (6), 725-731 (1995).</li>
<li>Alam, M. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Candida+identification%3A+a+journey+from+conventional+to+molecular+methods+in+medical+mycology%5BTitle%5D)+AND+%22World+J+Microbiol+Biotechnol%22%5BJournal%5D)">Candida identification: a journey from conventional to molecular methods in medical mycology.</a> World J Microbiol Biotechnol. 30 (5), 1437-1451 (2014).</li>
<li>Bauters, T. G., Nelis, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+chromogenic+and+fluorogenic+membrane+filtration+methods+for+detection+of+four+Candida+species%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Comparison of chromogenic and fluorogenic membrane filtration methods for detection of four Candida species.</a> J Clin Microbiol. 40 (5), 1838-1839 (2002).</li>
<li>Soto-Rojas, A. E., Villa, A. R., Sifuentes-Osornio, J., Alarcón-Segovia, D., Kraus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+candidiasis+and+Sj%C3%B6gren%27s+syndrome%5BTitle%5D)+AND+%22J+Rheumatol%22%5BJournal%5D)">Oral candidiasis and Sjögren's syndrome.</a> J Rheumatol. 25 (5), 911-915 (1998).</li>
<li>Gilchrist, J. E., Campbell, J. E., Donnelly, C. B., Peeler, J. T., Delaney, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spiral+plate+method+for+bacterial+determination%5BTitle%5D)+AND+%22Appl+Microbiol%22%5BJournal%5D)">Spiral plate method for bacterial determination.</a> Appl Microbiol. 25 (2), 244-252 (1973).</li>
<li>Biomerieux. ATB FUNGUS 3 Sensitivity test for yeast-like fungi. REF 14204. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Biomerieux%22">Biomerieux</a>. , (2014).</li>
<li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+widely+used+ATB+FUNGUS+3+automated+readings+in+China+and+its+misleading+high+MICs+of+Candida+spp.+to+Azoles%3A+Challenges+for+developing+countries%27+clinical+microbiology+labs%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The widely used ATB FUNGUS 3 automated readings in China and its misleading high MICs of Candida spp. to Azoles: Challenges for developing countries' clinical microbiology labs.</a> PLoS One. 9 (12), e114004(2014).</li>
<li>Hu, L., Zhou, P., Zhao, W., Hua, H., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+staining+vs.+routine+KOH+smear+for+rapid+diagnosis+of+oral+candidiasis-A+diagnostic+test%5BTitle%5D)+AND+%22Oral+Dis%22%5BJournal%5D)">Fluorescence staining vs. routine KOH smear for rapid diagnosis of oral candidiasis-A diagnostic test.</a> Oral Dis. 26 (5), 941-947 (2020).</li>
<li>Samaranayake, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Candida+krusei+infections+and+fluconazole+therapy%5BTitle%5D)+AND+%22Hong+Kong+Med+J%22%5BJournal%5D)">Candida krusei infections and fluconazole therapy.</a> Hong Kong Med J. 3 (3), 312-314 (1997).</li>
<li>Lee, Y., Puumala, E., Robbins, N., Cowen, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antifungal+drug+resistance%3A+Molecular+mechanisms+in+Candida+albicans+and+beyond%5BTitle%5D)+AND+%22Chem+Rev%22%5BJournal%5D)">Antifungal drug resistance: Molecular mechanisms in Candida albicans and beyond.</a> Chem Rev. 121 (6), 3390-3411 (2021).</li>
<li>Rihab, B., Lina, E. H., Noémie, S. T., Jean, S., Marjolaine, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+experience+of+dry+mouth+and+screening+for+Sjogren%27s+syndrome+by+the+dentist%3A+patient-reported+experiences%5BTitle%5D)+AND+%22BMC+Oral+Health%22%5BJournal%5D)">The experience of dry mouth and screening for Sjogren's syndrome by the dentist: patient-reported experiences.</a> BMC Oral Health. 23 (1), 1010(2023).</li>
<li>Panpetch, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+Candida+administration+in+a+Clostridium+difficile+mouse+model+worsens+disease+severity+but+is+attenuated+by+Bifidobacterium%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Oral Candida administration in a Clostridium difficile mouse model worsens disease severity but is attenuated by Bifidobacterium.</a> PLoS One. 14 (1), e210798(2019).</li>
<li>Alwaily, E. R., Abood, M. S., Al Uobody, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+of+oral+candidiasis+in+patients+under+12+years%3A+18S+rRNA+as+a+marker+of+molecular+characterization+of+Candida+tropicalis%5BTitle%5D)+AND+%22Arch+Razi+Inst%22%5BJournal%5D)">Diagnosis of oral candidiasis in patients under 12 years: 18S rRNA as a marker of molecular characterization of Candida tropicalis.</a> Arch Razi Inst. 78 (1), 475-483 (2023).</li>
<li>Chen, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Candida+infection+as+an+early+sign+of+subsequent+Sj%C3%B6gren%27s+syndrome%3A+A+population-based+matched+cohort+study%5BTitle%5D)+AND+%22Front+Med+%28Lausanne%29%22%5BJournal%5D)">Candida infection as an early sign of subsequent Sjögren's syndrome: A population-based matched cohort study.</a> Front Med (Lausanne). 8, 796324(2022).</li>
</ol>

The authors have no conflicts of interest to declare.

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 without culturing, can rapidly and preliminarily determine the presence of oral Candida infection in pSS patients. However, these methods have a relatively high false-negative rate16. If it is difficult to confirm the infection, Candida culturing is essential. Drug resistance is a factor that has to be considered during the treatment; some Candida strains are naturally resistant to some antifungal drugs, e.g., Candida krusei is naturally resistant to the treatment with Fluconazole17. Therefore, sometimes, the strain of Candida needs to be identified. In this case, the Candida strain can be identified by a Candida diagnostic agar. Currently, the drug-resistant Candida is getting increasing attention18. The antifungal susceptibility test can guide treatment and thereby avoid drug resistance as much as possible during the treatment. The Candida biofilm formation assay and hyphal formation assay are suited for research related to oral Candida infection.
Oral Candida infection is one of the most frequent complications in pSS patients4, significantly impacting their quality of life due to the array of symptoms it causes. Notably, pSS patients are highly susceptible to this infection, and effective treatment often requires systemic therapy and patient cooperation19. Consequently, oral Candida infections are often recurrent in this population. It is crucial to identify oral candidiasis and perform antifungal susceptibility tests to guide treatment. Among the methods detailed in this manuscript, the collection of oral samples for Candida detection and subsequent culturing are the most critical steps.
Furthermore, Candida that has hyphal formation (such as Candida albicans) can be easily detected by the fast KOH smear test and CFW staining assay using collected samples without culturing. Though most of the oral Candida infections can be detected and the Candida strains can be identified through the methods introduced in this manuscript, some Candida strains cannot be identified from others specifically only by methods such as SDA culture, Candida diagnostic agar culture, Candida biofilm formation assay, and hyphal formation assay. Additionally, some Candida strains cannot be induced to produce hyphae, such as Candida glabrata. The 18s rDNA RT-PCR-based species identification can accurately identify Candida species based on their genetic similarity10,20,21. If needed, the 18s rDNA RT-PCR-based species identification can be utilized, e.g., in research about oral Candida infection in pSS patients.
Intraorally, the manifestation of Candida infection may present as erythematous mucosal lesions, denture stomatitis, and the fissuring of the tongue. In the perioral area, the colonization of Candida causes a dry, cracked, and erythematous appearance7. The reported prevalence of clinical oral candidiasis varies widely in SS patients, ranging from 0% to 80%. The possible reasons for the large variation of oral candidiasis include the lack of clear symptomatology and, more importantly, the different criteria used for the diagnosis of oral candidiasis in literature8,9,12. Moreover, patients with Candida infection are reported to have a higher risk of SS, and Candida infection can act as an early sign of subsequent SS22. Early diagnosis of pSS is crucial for the treatment and the pathological study. Therefore, it is imperative to provide universal, convenient, and feasible methods to identify oral Candida infection in pSS patients.
Though dysbiosis in the gut, oral, and vaginal microbiome is frequent in pSS patients, oral samples demonstrate the greatest extent of microbial variation1. Under normal conditions, the average daily flow of whole saliva is 1 to 1.5 L2. Up to 99% of the saliva is made up of water, and the remaining 1% is composed of proteins and ions. Saliva is reported to play an important role in the determination of the composition and activity of the oral microbiota for multiple reasons3. Saliva not only continuously cleanses the teeth, performing crucial oral hygiene functions in healthy conditions, but it also forms a conditioning film on oral surfaces. This film provides receptors for bacteria attachment, aids in the growth of bacteria by supplying them with nutrients from glycoproteins and maintains a suitable pH for bacterial growth. Additionally, saliva contains antimicrobial and anti-candidal factors that regulate and shape the microbiota. Furthermore, saliva contains antimicrobial and anti-candidal factors, such as adrenomedullin, statherin, histatins, &#945;-defensins, lysozyme, histatins, lactoferrin, and calprotectin, that regulate and shape the microbiota4. pSS is characterized by autoimmune deregulation and glandular hypofunction. Hyposalivation, a reduction in salivary flow, is one of the most prevalent symptoms of pSS. Decreased salivary pH levels and flow are significant risk factors for oral Candida infections5. Furthermore, Candida infections are reported as the second most common orofacial complication associated with SS6. The authors suggest that the increased Candida infections in pSS patients may be linked not only to decreased saliva production but also to the immunological deregulation inherent in SS. 
To conclude, practical and reproducible methods for oral Candida infection and antifungal susceptibility tests introduced in this manuscript hold promise for enhancing clinical identification and treatment of Candida infections. Furthermore, these standardized procedures can minimize inconsistencies in research studies related to oral candidiasis.

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 Sj&#246;gren'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.

<table><tbody><tr><td>0.22 &amp;micro;m Syringe Filter</td><td>MERCK</td><td>SLGV004SL</td><td></td></tr><tr><td>1.5 mL Centrifuge Tube</td><td>axygen</td><td>MCT-150-C-S</td><td></td></tr><tr><td>10 cm Microbiological Culture Dishes</td><td>Jet Bio-Filtration Co. Ltd</td><td>TCD000100</td><td></td></tr><tr><td>15 mL Centrifuge Tube</td><td>Jet Bio-Filtration Co., Ltd</td><td>CFT011150</td><td></td></tr><tr><td>50 mL Centrifuge Tube</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>96-well Microplates</td><td>Corning</td><td>3599</td><td></td></tr><tr><td>API 0.85% NaCl ampule</td><td>BIOMERIEUX</td><td>20070</td><td></td></tr><tr><td>Calcofluor White Stain</td><td>Sigma-Aldrich</td><td>18909-100ML-F</td><td></td></tr><tr><td>CHROMagar Candida</td><td>CHROMagar</td><td>P002860</td><td>&lt;em&gt;Candida &lt;/em&gt;diagnostic agar</td></tr><tr><td>D(+)-Glucose Monohydrate</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10010518</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>26170035</td><td></td></tr><tr><td>McFarland Standards Kit</td><td>BIOMERIEUX</td><td>70900</td><td></td></tr><tr><td>Nunc precision molded loops&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>254399</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco</td><td>C10010500BT</td><td></td></tr><tr><td>Potassium Hydroxide</td><td>Aladdin</td><td>P112284-500g</td><td></td></tr><tr><td>PowerSoil DNA Isolation Kit&amp;nbsp;</td><td>MO BIO</td><td>12888-50</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>11875119</td><td></td></tr><tr><td>Sabourand&#039;s Agar Medium</td><td>Solarbio</td><td>S9710</td><td></td></tr><tr><td>Yeast Nitrogen Base Without Amino acids</td><td>Solarbio</td><td>Y8040</td><td></td></tr><tr><td>Yeast-like Fungal Susceptibility Kit (Microdilution)</td><td>BIOMERIEUX</td><td>14204</td><td></td></tr></tbody></table>

examination of oral candida infection in primary sj&#246;gren&#39;s syndrome patients

Examination of Oral Candida Infection in Primary Sj&#246;gren&#39;s Syndrome Patients

Primary Sj&#246;gren'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.

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), lingual papillae atrophy and bright red dorsal mucosa of the tongue (Figure 1C).
Among these 12 patients involved in the current study, 8 of them had oral Candida infection, which was confirmed by the growth of Candida colonies on the SDA dishes (Figure 2A-C and Supplementary Figure 1). The diagnostic agar showed that oral Candida infections of Candida albicans, Candida krusei, and Candida glabrata exist among these pSS patients (Figure 2D-F, Supplementary Figure 2A,B). In some cases, more than one species of Candida was detected, such as a combination infection with Candida albicans and Candida krusei, as well as a combination infection with Candida albicans and Candida glabrata (Figure 2D, Supplementary Figure 2A,B).
The KOH smear test and the subsequent CFW staining assay, utilizing patient saliva or oral mucosal swab samples, can rapidly determine whether the patient has oral Candida infection. In this study, the presence of hyphae was observed in both oral samples from pSS patients (Figure 3A,B). The finding was further validated by the CFW staining assay, which exhibited fluorescent positive staining of the hyphae (Figure 3C,D). Additionally, the hyphae of a pSS patient infected with oral Candida albicans were inducible through the hyphal formation assay (Figure 4A), and the induced hyphae were observed under the fluorescence microscope in the CFW staining assay (Figure 4D). Furthermore, the KOH smear test and CFW staining assay indicated positive fungal spores and pseudohypha from a pSS patient with Candida krusei infection (Figure 4B,E), and fungal spores from a pSS patient with Candida glabrata infection (Figure 4C,F), after the formation of the Candida biofilm.
Antifungal susceptibility testing is crucial in guiding the clinical management of oral Candida infections in patients with pSS. Here, three representative patients were tested for antifungal susceptibility to five commonly prescribed agents: Fluconazole, Itraconazole, Amphotericin B, Voriconazole, and 5-Fluconazole. The results were interpreted according to the manufacturer's guidelines. pSS patients 1 and 3 exhibited sensitivities to all the antifungal agents tested (Table 1)14,15. pSS patient 2 demonstrated a dose-dependent intermediate response to Fluconazole and Voriconazole, a dose-dependent, intermediate to resistant sensitivity towards Itraconazole, resistance to 5-fluconazole, and sensitivity to Amphotericin B treatment (Table 2)14,15.
<img alt="Oral examination photos showing lips, gums, cavities, and tongue with dental abnormalities." class="xfigimg" src="/files/ftp_upload/66430/66430fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Representative oral manifestations of oral Candida infection. (A) Angular cheilitis (rhagades at the corners of the mouth, desquamation, and hyperemia). (B) An erythematous lesion with edema on the alveolar gingival mucosa, with a yellowish-white stringy pseudo-membrane. (C) Atrophic dorsum of lingual papillae with dispersed and irregular reddened and circumscribed atrophic erythema lesion. Black arrows point to the lesions. <a href="https://www.jove.com/files/ftp_upload/66430/66430fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Candida species growth on SDA and diagnostic agar; diagram; showcases yeast culture identification." class="xfigimg" src="/files/ftp_upload/66430/66430fig02v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Sabouraud dextrose agar (SDA) and Candida strain identification. (A-C) The SDA culture of oral samples (saliva, PBS mouthwash, or oral mucosa swap) from 3 representative pSS patients with Candida infection. The Candida strain identification assay using a Candida diagnostic agar shows pSS patients with oral (D) Candida albicans plus Candida Krusei infections, (E) Candida albicans infection, and (F) Candida glabrata infection. <a href="https://www.jove.com/files/ftp_upload/66430/66430fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="KOH smear test and CFW staining assay micrographs for Candida albicans detection in patients." class="xfigimg" src="/files/ftp_upload/66430/66430fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Instant potassium hydroxide (KOH) smear test and calcofluor white (CFW) staining assay. The assay was performed using oral samples (saliva, PBS mouthwash, or oral mucosa swap) from 2 representative pSS patients. KOH smear test using the oral sample from (A) pSS patient 1 and (B) pSS patient 2. Black, hollow arrows point to the hyphae. CFW staining assay using the oral sample from (C) pSS patient 1 and (D) pSS patient 2. White hollow arrows point to the hyphae and fungal spores. Scale bar = 50 µm. <a href="https://www.jove.com/files/ftp_upload/66430/66430fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="KOH smear test and CFW staining assay; microscopy of Candida species; fungal identification method." class="xfigimg" src="/files/ftp_upload/66430/66430fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Potassium hydroxide (KOH) smear test, calcofluor white (CFW) staining assay of samples after culture. KOH smear test using saliva from pSS patient with (A) Candida albicans infection, (B) pSS patient with Candida krusei infection, and (C) pSS patient with Candida glabrata infection. CFW staining assay of induced hyphae from pSS patient with (D) Candida albicans infection, fungal spores, and pseudo-hypha from (E) pSS patient with Candida krusei infection, and fungal spores from (F) pSS patient with Candida glabrata infection. White hollow arrows point to the hyphae; white solid arrows point to the pseudo-hypha. Scale bar = 50 µm. <a href="https://www.jove.com/files/ftp_upload/66430/66430fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Representative Sabouraud dextrose agar (SDA) culture using pSS patient oral samples (saliva, PBS mouthwash, or oral mucosa swap). (A-C) SDA culture of Candida using oral samples from 3 representative pSS patients. <a href="https://www.jove.com/files/ftp_upload/66430/Supplementary%20Figure%201.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Candida strain identification. The identification of the possible infected Candida from the 12 pSS patients using a Candida diagnostic agar. (A) The identification of Candida strain using oral samples from pSS patients 1-6. pSS patient 1: Candida albicans, 2: Candida albicans, 3: Candida albicans and Candida krusei, 4: Candida albicans, 5: Candida albicans and Candida glabrata, 6: no oral Candida infection. (B) The identification of Candida strain using oral samples from pSS patients 7-12. pSS patient 7: Candida albicans, 8: Candida albicans and Candida glabrata, 9: no oral Candida infection, 10: Candida albicans, 11: no oral Candida infection, 12: no oral Candida infection. <a href="https://www.jove.com/files/ftp_upload/66430/Supplementary%20Figure%202.zip" target="_blank">Please click here to download this File.</a>
Table 1: Information of primary Sjögren's syndrome patients. <a href="https://www.jove.com/files/ftp_upload/66430/Table%201_RE.xls" target="_blank">Please click here to download this Table.</a>
Table 2: Antifungal susceptibility test results of the patients14,15. Abbreviations: S = sensitive, I = intermediate, R = resistant, SDD = dose-dependent sensitive, C = concentration. <a href="https://www.jove.com/files/ftp_upload/66430/Table%202_RE.xls" target="_blank">Please click here to download this Table.</a>

Here, we present a protocol for the examination of oral Candida infection in patients with primary Sj&#246;gren's syndrome, which can be used for timely treatment and, thereafter, avoiding related complications.

Primary Sj&#246;gren's syndrome (pSS) is an autoimmune disease characterized by symptoms such as dry mouth, dry eyes, and other systematic symptoms. Due ...

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