本研究得到国家自然科学基金（82201084）、中国博士后科学基金（2022M722232）、北京市博士后科研基金（2023-ZZ-020）、首都医科大学附属北京天坛医院苗浚项目（2023MP10）和香港研究资助局优配研究金（27111820和17116521）资助。

下述所有程序均经首都医科大学附属北京天坛医院伦理委员会批准（编号：KY2023-177-01），参与本研究的所有患者均提供知情同意书。
1. 患者纳入和排除标准
<ol><li>纳入标准：如果患者满足 2016 年美国风湿病学会 （ACR）/欧洲抗风湿病联盟 （EULAR） 标准12，则将患者分类为 pSS 患者。具体来说，最终分类标准基于五个项目的加权总和：抗 SSA/Ro 抗体阳性和局灶性淋巴细胞性唾液腺炎，焦点评分为 ≥ 1 个病灶/4 mm2，每个评分为 3;异常眼部染色评分为 ≥ 5（或 van Bijsterveld 评分为 ≥ 4），Schirmer 试验结果为 ≤ 5 mm/5 min，未刺激唾液流速为 ≤ 0.1 mL/min，每项评分为 1。具有提示 SS 的体征和/或症状且上述项目总分为 ≥ 4 分的个人符合 pSS 的标准。</li>
	<li>排除标准： 排除无法配合样本采集的患者和正在接受口腔 念珠菌 感染治疗的患者。 注意：必须注意的是，下面描述的方法也适用于继发性SS个体口腔 念珠菌 感染的综合表征。</li>
	<li>为防止在念 珠菌 样本的收集、运输和培养过程中可能发生的污染，请采取细致的预防措施。这包括细致的实验准备、使用一次性和无菌材料、适当的标签、采样管的安全密封以及在专业的生物安全柜中进行实验程序。</li>
</ol>2. 样本采集
<ol><li>唾液收集：指导患者用漏斗握住管子，让唾液沿着下唇逐渐流入管子。在收集结束时，要求患者将口腔中所有剩余的唾液吐入管中。收集唾液 15 分钟。</li>
	<li>漱口水和拭子采集：如果患者没有未受刺激的唾液流量或未受刺激的唾液流量极低（小于 0.03 mL/min），则对疑似口腔 念珠菌 感染的口腔粘膜区域进行至少 10 次拭子测定，然后要求患者使用 5 mL 磷酸盐缓冲盐水 （PBS;参见 材料表） 漱口 1 分钟。将漱口水存放在 50 mL 无菌管中，然后将棉签放入漱口水管中。 注意：对于未刺激的唾液流量超过 0.03 mL/min 的 pSS 患者，步骤 2.2 仍可用于收集样本进行口腔 念珠菌 感染检测。</li>
</ol>3.样品处理
<ol><li>向收集的唾液中加入 1 mL PBS。请勿将 PBS 添加到含有 PBS 的漱口水中。使用涡旋振动漱口水管 1 分钟。</li>
</ol>4. 念珠菌 培养
<ol><li>要制备 Sabourand 琼脂 （SDA） 培养基，将 70 g SDA（参见 材料表）溶解在 1000 mL 双蒸水中。</li>
	<li>在121°C的高压灭菌器中无菌15分钟，将10mL该SDA培养基置于10cm微生物培养皿中（参见 材料表）并冷却。</li>
	<li>取步骤 3 中制备的 200 μL 样品，将其划线到步骤 4.2 中用 10 mL SDA 培养基制备的 10 cm 微生物培养皿上。</li>
	<li>在37°C孵育48小时， 念珠菌 菌落将成熟。将带有 念珠菌 菌落的SDA培养皿暂时储存在4°C。 在 SDA 培养皿上培养的 念珠菌 菌落可以储存 2-4 周。</li>
</ol>5. 念珠菌 菌株鉴定
<ol><li>为了制备培养基，取出47.7g 念珠菌 诊断琼脂（见 材料表）粉末，将其放入1000mL双蒸水中，并将其加热至100°C。</li>
	<li>当培养基稍微沸腾时，将 10 mL 放入 10 cm 微生物培养皿中并冷却。</li>
	<li>将 念珠菌 菌落溶解在 500 μL SDA 培养基中（第 4 步），悬浮并通过移液解离。</li>
	<li>将 50 μL 含有 念珠菌 菌落的样品（步骤 5.3）接种到步骤 5.2 中制备的培养基上。在37°C孵育48小时，直到 念珠菌 菌落成熟。</li>
	<li>根据 念珠菌 菌落的颜色和模式，确定从 pSS 患者那里获得的 念珠菌 的菌株。使用以下念 珠菌 菌株鉴定标准：翠绿色菌落为 白色念珠菌，蓝灰色菌落为 热带念珠菌，边缘模糊的粉红色菌落和微绒毛 为克鲁氏念珠菌，紫红色小菌落为 光滑念珠菌，乳白色菌落包含包括 副念珠菌在内的几种菌株。</li>
</ol>6. 念珠菌 生物膜形成测定
<ol><li>要制备酵母氮碱（YNB）-50葡萄糖（G）培养基，称取1.34g YNN（参见 材料表）和1.98g D（+）-葡萄糖（参见 材料表）。将它们溶解在 200 mL 双蒸水中。</li>
	<li>要制备 YNB-100G 培养基，取出 1.34 g YNB 和 3.96 g D （+）-葡萄糖，并将它们溶解在 200 mL 双蒸水中。</li>
	<li>用0.22μm注射器过滤器过滤溶液（参见 材料表）。过滤后的YNB-50G和YNB-100G培养基可在4°C下储存3个月。</li>
	<li>在步骤 5 中从培养皿上的相同菌株中挑选 3 个（或更多）菌落，并将它们转移到 96 孔板中的 200 μL YNB-50G 培养基（步骤 6.1 和 6.3）中（参见 材料表）。对培养皿中观察到的菌株执行此操作。在37°C下培养过夜，以200rpm振荡。</li>
	<li>吸出 念珠菌 悬浮液，加入 1 mL PBS，在室温下以 1500 x g 离心酵母细胞 5 分钟，并弃去上清液。在同一管中重复此步骤 2 次。</li>
	<li>将细胞重悬于 1 mL YNB-100G 培养基中，并制备不同稀释度（1：10、1：100 和 1：1000）的细胞。取 200 μL 梯度细胞悬浮液，并通过螺旋板法将其接种到 10 cm SDA 培养基上13。电镀是使用一台机器完成的，该机器将已知体积的样品以阿基米德螺旋的形式以不断减少的量沉积在旋转的琼脂平板上。在37°C下培养24-48小时。</li>
	<li>根据标准方法13，根据SDA板中CFU的数量计算不同稀释液中细胞的浓度。简要计算每个板上分离良好的菌落。从外缘到中心以一个八分之一的菌落计数，直到观察到至少 25 个菌落。此外，计算发生第 25次 计数的该弧的剩余部分。通过将菌落计数除以对应于获得菌落计数的区域的液体体积来计算细胞的浓度13。将细胞浓度调节至 1 x 107 个细胞/mL。</li>
	<li>将 100 μL YNB-100G 中的细胞悬浮液放入 96 孔板中，并在 37 °C 下孵育 2 小时，同时以 200 rpm 振荡。</li>
	<li>小心地取出上清液，用PBS洗涤2次，并去除漂浮的酵母细胞。 念珠菌 生物膜在 96 孔板的底部形成。</li>
</ol>7. 菌丝形成测定
注意：只有可以形成菌丝的 念珠菌 （例如 白色念珠菌）才能诱导菌丝形成。转到步骤 8 以进一步表征不能形成菌丝的 念珠菌 （例如 光滑念珠菌）。
<ol><li>在步骤6.6中重悬酵母细胞悬浮液，并用YNB-100G培养基将细胞浓度调节至1×106 个细胞/ mL。</li>
	<li>为了制备RPMI 1640完全生长培养基，将50mL胎牛血清（参见 材料表）加入450mL RPMI 1640培养基（参见 材料表）中，并在4°C下储存以进行进一步实验。</li>
	<li>将2μL在200μLRPMI 1640完全生长培养基中稀释的 念珠菌 细胞悬浮液转移到96孔板中，并在37°C下培养过夜，让菌丝和酵母细胞形成。</li>
</ol>8. KOH涂片试验和CFW染色测定
<ol><li>使用精密模制环（参见 材料表）从步骤 3 或步骤 6 或步骤 7 中取出 1 μL 样品，并在载玻片上涂抹 1 cm x 1 cm 的区域。等待它在室温下干燥。</li>
	<li>要制备 10% KOH 溶液，称取 10 g KOH 粉末（参见 材料表）并将其溶解在 100 mL 双蒸水中。</li>
	<li>用酒精灯轻轻加热载玻片，直到标本溶解。用10%KOH（步骤8.2）染色步骤8.1中制备的载玻片，并用盖玻片覆盖。轻轻按压盖玻片，使样品透明。</li>
	<li>在显微镜下观察菌丝和酵母细胞。</li>
	<li>在步骤8.1中用一滴CFW（参见 材料表）染色载玻片，用盖玻片覆盖，并等待1分钟。</li>
	<li>使用荧光显微镜在100x和200x的放大倍率下观察菌丝。 CFW在355nm处激发，在300-440nm处检测。在曝光时间下拍摄图像。重复 3 次以避免假阳性结果。</li>
</ol>9.抗真菌药敏试验
注意：该测试是根据制造商的酵母样真菌敏感性试剂盒（微量稀释;参见 材料表）的说明进行的。
<ol><li>用0.85%NaCl安瓿（参见材料表）制备不到4天的念珠菌菌落（步骤4），并获得浊度相当于2McFarland的悬浮液。根据麦克法兰标准品套件测定浊度（参见材料表）。立即使用准备好的念珠菌悬浮液。</li>
	<li>将含有 3 x 104 个酵母细胞/mL 的试剂盒中的 135 μL ATB F2 培养基移液到试纸条中的每个杯中，并盖上盖子。将试纸放入密封容器中。在35°C下培养24小时。</li>
	<li>通过视觉解释观察 念珠菌 的生长（请参阅用户手册）。有关参考值（参考范围），请参阅用户手册。检查控制孔是否充分增长。如果控制孔的生长不足或没有生长，则无法读取结果，需要再培养24小时。</li>
</ol>

pSS 相关口腔念珠菌的诊断和菌株鉴定

<ol>
	<li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+dysbiosis+in+primary+Sj&#246;gren's+syndrome+and+the+ameliorative+effect+of+hydroxychloroquine.">Microbiota dysbiosis in primary Sj&#246;gren's syndrome and the ameliorative effect of hydroxychloroquine.</a> Cell Rep. 40 (11), 111352 (2022).</li><li>Humphrey, S. P., Williamson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+saliva:+normal+composition,+flow,+and+function.">A review of saliva: normal composition, flow, and function.</a> J Prosthet Dent. 85 (2), 162-169 (2001).</li><li>Marsh, P. D., Do, T., Beighton, D., Devine, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+saliva+on+the+oral+microbiota.">Influence of saliva on the oral microbiota.</a> Periodontol 2000. 70 (1), 80-92 (2016).</li><li>Vila, T., Rizk, A. M., Sultan, A. S., Jabra-Rizk, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+power+of+saliva:+Antimicrobial+and+beyond.">The power of saliva: Antimicrobial and beyond.</a> PLoS Pathog. 15 (11), e1008058 (2019).</li><li>Serrano, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+related+to+oral+candidiasis+in+patients+with+primary+Sj&#246;gren's+syndrome.">Risk factors related to oral candidiasis in patients with primary Sj&#246;gren's syndrome.</a> Med Oral Patol Oral Cir Bucal. 25 (5), e700-e705 (2020).</li><li>Nape&#241;as, J. J., Rouleau, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+complications+of+Sj&#246;gren's+syndrome.">Oral complications of Sj&#246;gren's syndrome.</a> Oral Maxillofac Surg Clin North Am. 26 (1), 55-62 (2014).</li><li>Mathews, S. A., Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+manifestations+of+Sj&#246;gren's+syndrome.">Oral manifestations of Sj&#246;gren'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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sj&#246;gren's+syndrome+sufferers+have+increased+oral+yeast+levels+despite+regular+dental+care.">Sj&#246;gren'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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subjective+and+clinical+oral+symptoms+in+patients+with+primary+Sj&#246;gren's+syndrome.">Subjective and clinical oral symptoms in patients with primary Sj&#246;gren'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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+identification:+a+journey+from+conventional+to+molecular+methods+in+medical+mycology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+chromogenic+and+fluorogenic+membrane+filtration+methods+for+detection+of+four+Candida+species.">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., Alarc&#243;n-Segovia, D., Kraus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+candidiasis+and+Sj&#246;gren's+syndrome.">Oral candidiasis and Sj&#246;gren'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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiral+plate+method+for+bacterial+determination.">Spiral plate method for bacterial determination.</a> Appl Microbiol. 25 (2), 244-252 (1973).</li><li>Biomerieux. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATB+FUNGUS+3+Sensitivity+test+for+yeast-like+fungi.+REF+14204.">ATB FUNGUS 3 Sensitivity test for yeast-like fungi. REF 14204.</a> Biomerieux. , (2014).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+staining+vs.+routine+KOH+smear+for+rapid+diagnosis+of+oral+candidiasis-A+diagnostic+test.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+krusei+infections+and+fluconazole+therapy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+drug+resistance:+Molecular+mechanisms+in+Candida+albicans+and+beyond.">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., No&#233;mie, S. T., Jean, S., Marjolaine, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+experience+of+dry+mouth+and+screening+for+Sjogren's+syndrome+by+the+dentist:+patient-reported+experiences.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Candida+administration+in+a+Clostridium+difficile+mouse+model+worsens+disease+severity+but+is+attenuated+by+Bifidobacterium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+oral+candidiasis+in+patients+under+12+years:+18S+rRNA+as+a+marker+of+molecular+characterization+of+Candida+tropicalis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+infection+as+an+early+sign+of+subsequent+Sj&#246;gren's+syndrome:+A+population-based+matched+cohort+study.">Candida infection as an early sign of subsequent Sj&#246;gren's syndrome: A population-based matched cohort study.</a> Front Med (Lausanne). 8, 796324 (2022).</li></ol>

提交人无需声明任何利益冲突。

在目前的手稿中，我们提供了一系列系统、简单、可行的方法，用于检测口腔 念珠菌 感染、鉴定 念珠菌 菌株以及检测pSS患者常用药物的抗真菌敏感性。 
在实际环境中，并非本手稿中介绍的所有方法都是必需的。口腔 念珠菌 检测方法可以根据具体目的进行选择。KOH 涂片试验和 CFW 染色试验使用未经培养的样本，可以快速、初步地确定 pSS 患者是否存在?...

念珠菌病是由 念珠菌属引起的，念珠菌 属是机会性病原体。常见菌株包括 白色念珠菌、克鲁塞念珠菌、光滑念珠菌、副念珠 菌和 都柏林念珠菌1。 念珠菌 属引起的机会性感染包括浅表 念珠菌 感染和侵袭性念珠菌病。侵袭性念珠菌病主要发生在免疫功能低下的个体中;例如，在患有获得性免疫缺陷综合症 （AIDS） 的患者中，侵袭性念珠菌病会严重威胁生活质量甚至寿命2。浅表 念珠菌 感染，如皮肤黏膜念珠菌病，在患者中更常见3. 
口腔念珠菌病是一种浅表性 念珠菌 感染，是最常见的人类真菌感染。 白色念珠菌 是口腔的正常共生;据报道，在没有任何症状的一般人群中 ，念珠菌 属的携带率为 20%-75%4。 念珠菌 的过度生长会导致患者的局部不适，例如感觉味觉改变、口腔灼热感、营养不良导致的吞咽困难、恢复延迟和住院时间延长。长期口服念珠菌病可导致严重的侵袭性念珠菌病，导致严重的发病率和死亡率5.口腔念珠菌病也会增加患口腔癌的风险5.唾液腺功能受损是口腔念珠菌病的危险因素之一6.感染的开始是 念珠菌 粘附在上皮细胞壁上。其次是 念珠菌 的增殖和丝化以及生物膜7 的形成和成熟。生物膜极难根除，并且对常规抗真菌治疗具有抵抗力，这使得临床治疗成为生物膜相关口腔 念珠菌 感染的挑战7.
原发性干燥综合征 （pSS） 是一种自身免疫性疾病，其特征是口干、眼睛干涩和其他全身症状8.口干是pSS最常见的症状。唾液具有重要的生理抗真菌功能。一方面，它可以稀释和冲刷口腔，然后从粘膜中清除微生物。另一方面，唾液中含有抗菌蛋白，如乳铁蛋白、唾液过氧化物酶、溶菌酶、富含组氨酸的多肽和特异性抗念珠菌抗体，它们可以与口腔粘膜相互作用，防止 念珠菌6的过度生长。唾液流量减少可能使 pSS 患者易患口腔念珠菌病。一项对 61 例 pSS 患者进行的观察性横断面研究发现，13.1% 的 pSS 患者出现念珠菌病的口腔体征，发现 白色念珠 菌菌落形成单位 （CFU）/mL 与未刺激的全唾液 （UWS） 和刺激的全唾液 （SWS） 水平显着且呈负相关9。
本手稿中介绍的方法可以根据直接涂片或培养后的表征形态属性（例如菌丝和酵母细胞）识别口腔念珠菌感染10。使用念珠菌诊断琼脂对念珠菌进行菌株鉴定是基于具有不同形态的不同颜色菌落的形成，这些菌落是由物种特异性酶裂解显色底物引起的11。在本文中，介绍了检测口腔念珠菌感染的系统方法，包括传统的口腔念珠菌培养和氢氧化钾（KOH）涂片试验和Calcofluor White（CFW）染色试验的快速检查10。此外，还引入了抗真菌药敏试验，可指导临床口腔念珠菌感染治疗。在实际情况下，没有必要执行本手稿中介绍的所有方法;口腔念珠菌检测方法可根据目的选择。口腔念珠菌感染的检查和 pSS 患者念珠菌菌株的测定不仅有利于疾病治疗，而且有助于评估口腔念珠菌病的特征和物种特征。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;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>Candida 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&#160;</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&#160;</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&#160;</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&#39;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

原发性干燥综合征 （pSS） 是一种自身免疫性疾病，其特征是口干、眼睛干涩和其他全身症状等症状。由于pSS患者所经历的唾液分泌减退，口腔菌群失调经常发生。pSS 的一个常见并发症是口腔 念珠菌 感染。在本文中，作者描述了可以有效诊断口腔 念珠菌 感染并使用唾液、口腔粘膜拭子或 pSS 患者漱口水识别 念珠菌 菌株的系统方法。Sabouraud 葡萄糖琼脂 （SDA）、菌丝形成试验、氢氧化钾 （KOH） 涂片试验和钙荧光白 （CFW） 染色试验用于诊断口腔 念珠菌 感染。 念珠菌 诊断琼脂用于鉴定 念珠菌 菌株。最后，使用抗真菌药敏试验来确定适当的抗真菌药物治疗。这种标准化方法可以促进pSS相关口腔 念珠菌 感染的诊断、治疗和未来研究。使用这种方法进行早期诊断还可以防止由于延迟接受适当治疗而出现的任何并发症。

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.

作者聚焦：口腔 念珠菌 诊断推进 pSS 患者的临床治疗方案

原发性干燥综合征患者口腔 念珠菌 感染的检查

In this research, we present a protocol for the examination of oral candida infection in patients with primary Sjogren syndrome, which can be used for timely treatment to avoid infection-related complications. 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 primary Sjogren's syndrome. Maybe our focus on the role of oral candida infection in the onset and the progression of primary Sjogren's syndrome pathologists in our future studies.

本研究选取12例pSS患者作为代表性患者，筛查口腔 念珠菌 感染（表1）。在这些患者中，一些患者表现出 念珠菌 感染的典型口腔病变，包括口角炎（以口角裂纹、脱屑和充血为特征， 图1A）、牙龈粘膜上鲜红色水肿、黄白色细状假膜（图1B）、舌萎缩和舌背粘膜鲜红色（图1C）。
在?...

Watch this Scientific Journal Video about Author Spotlight: Oral Candida Diagnosis to Advance Clinical Treatment Regimen for pSS Patients at JoVE.com

在这里，我们提出了一种检查原发性干燥综合征患者口腔 念珠菌 感染的方案，该方案可用于及时治疗，此后避免相关并发症。

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 ...

在这项研究中，我们提出了一种用于原发性干燥综合征患者口腔念珠菌感染检查的方案，该方案可用于及时治疗以避免与感染相关的并发症。我们为原发性干燥综合征患者口腔念珠菌感染的检测、念珠菌菌株的鉴定、常用药物的抗真菌敏感性等提供了一系列系统、简便、可行的方法。也许我们在未来的研究中关注口腔念珠菌感染在原发性干燥综合征病理学家的发病和进展中的作用。

examination-oral-candida-infection-primary-sjgren-s-syndrome

Research

JoVE Journal

Medicine

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

562 次观看.  Capital Medical University. 在这项研究中，我们提出了一种用于原发性干燥综合征患者口腔念珠菌感染检查的方案，该方案可用于及时治疗以避免与感染相关的并发症。我们为原发性干燥综合征患者口腔念珠菌感染的检测、念珠菌菌株的鉴定、常用药物的抗真菌敏感性等提供了一系列系统、简便、可行的方法。也许我们在未来的研究中关注口腔念珠菌感染在原发性干燥综合征病理学家的发病和进展中?...

视频: 作者聚焦：口腔 念珠菌 诊断推进 pSS 患者的临床治疗方案

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.

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.

科研

医学

Sample Collection for Diagnosis of Oral Candida in pSS Patients

To begin, select the participants based on the framed inclusion criteria. For saliva collection, instruct the patient to hold the tube with a funnel and let the saliva flow gradually into the tube along the lower lip for 15 minutes. At the end, ask the participant to spit all the remaining saliva into the tube.In case of low, unstimulated saliva flow, swab the oral mucosa in infection-suspected areas at least 10 times, and let the patient rinse their mouth with five milliliters of PBS for one minute. Then collect the mouthwash in a 50-milliliter sterile tube and put the swab into it. Add one milliliter of PBS to the collected saliva and vortex the tube for one minute.

作者聚焦：口腔 念珠菌 诊断推进 pSS 患者的临床治疗方案

作者聚焦：口腔念珠菌诊断推进 pSS 患者的临床治疗方案