真菌血清学检测新方法的临床性能

发布时间:2021-03-18       作者:张园       来源:临床实验室        浏览:2658       收藏: 0

作者:张园   杜君洋   李绘宇   贺婷婷   温雅慧   王贺

随着侵入性检查,广谱抗生素,免疫抑制剂,糖皮质激素和肿瘤化疗药物的广泛应用,真菌感染问题逐年增多,引起医学界广泛重视。目前,真菌感染问题影响到10亿人,每年有150万人死于真菌感染[1]。真菌在自然界种类多,分布广,它们通过逃避宿主防御机制得以生存。然而,当机体处于免疫受损的条件下真菌会侵入人体,导致严重感染[2]。流行病学显示念珠菌属、曲霉菌属和隐球菌属是侵袭性真菌病(Invasive fungal disease,IFD)最常见致病菌。

为了采取及时准确的抗真菌治疗,早期的微生物学诊断至关重要。传统的诊断方法,如培养法和组织病理学,存在耗时长、依从性差和敏感性低等局限性。为了解决这些问题,新兴的血清学和分子生物学检测方法成为研究焦点[3-5]。目前,用于临床的血清学检测包括(1-3)-β-D葡聚糖(β-D-Glucan,G试验)泛真菌检测(隐球菌与接合菌除外),念珠菌甘露聚糖检测(Mannan,Mn试验),曲霉菌半乳甘露聚糖检测(Galactomannan,GM试验),隐球菌荚膜多糖检测 (Capsular polysaccharide,GXM试验),曲霉菌IgG抗体检测、念珠菌IgG抗体检测和曲霉菌IgE抗体检测。在纳入指南和临床应用之前上述检测方法都经历漫长的临床验证。评估包括敏感性,特异性,精密度,准确度,定量检测的上限和下限以及线性范围等多项指标。与此同时,PCR和其他分子诊断方法,例如基质辅助激光解吸电离(MALDI)和荧光原位杂交(FISH)和T2-Candida磁共振(T2MR)已经证明在临床检测的前景,但在临床应用普及之前需要进行标准化评价。本综述的目的是概述以上检测方法的性能并讨论它们在不同患者人群中的应用。

一、真菌感染的传统检测方法

念珠菌属(Candida species)是最常见条件致病菌,其所致疾病在IFD中占首位[6]。在念珠菌属中引起人类感染主要为白念珠菌、光滑念珠菌、热带念珠菌、近平滑念珠菌和克柔念珠菌等10余种。目前临床对念珠菌感染的传统检测方法有血培养,镜检等。其中,血培养是念珠菌感染诊断的“金标准”,但血培养花费时间较长,通常需要3-7天,敏感性为21%-71%,存在相当程度的漏诊率[7]。显微镜检查需要检验医师具有好的临床经验,而且敏感性不高。侵袭性真菌感染中,约50%为念珠菌感染,患者死亡率达40%,甚至更高[7-9]。临床研究表明,早期诊断侵袭性念珠菌感染,尽早给予药物干预可降低患者的死亡率[8]。与此同时,在没有采取及时准确的抗真菌治疗,死亡率和住院费用都会显著增加。Morrell等研究证实诊断导致的治疗延误是侵袭性念珠菌感染死亡的高危因素[10-12]

曲霉菌属(Aspergillus species)感染是免疫功能低下患者危及生命的重要原因。高危人群包括长期中性粒细胞减少症,同种异体造血干细胞移植(HSCT),实体器官移植(SOT),遗传性或获得性免疫缺陷和使用皮质类固醇等患者。曲霉菌属中引起人类感染主要为烟曲霉,其次还有黄曲霉,黑曲霉和土曲霉等。目前临床对曲霉菌感染的传统检测方法有培养法、活检标本、组织病理学、细胞病理学、直接镜检等。侵袭性曲霉病的确诊通过组织活检标本中的真菌菌丝得以证实。GMS和PAS是常用的曲霉菌染色法,大多数染色法价格低廉,可以在各种样本完成,如痰,肺泡灌洗液(BALF),病变抽吸物,脑脊液(CSF)和其他组织[13, 14]。组织培养法对诊断曲霉菌病的敏感性低,取决于检测人群。在最近的两项研究,移植受者中分子检测阳性的侵袭性曲霉病患者,其组织培养结果阳性只有25%-50%[15, 16]。此外,培养的阳性预测值在免疫功能低下的患者更高。例如,在一项评估痰液或BALF培养物对不同患者人群的阳性预测值的研究中,HSCT、血液系统恶性肿瘤和粒细胞减少症患者阳性预测值为72%,实体器官移植和接受类固醇治疗的患者为58%,HIV感染患者仅占14%[17]。培养法的另一缺点是延迟种属鉴定,特别是孢子形成缓慢的种属,这可能会延迟选择合适的抗菌药物[18]。最后,回顾性分析1453例HSCT患者的血培养,其中19例患者为曲霉血培养阳性,即使在高危人群侵袭性曲霉血培养阳性率也很低[19]

隐球菌属(Cryptococcus species)主要影响免疫功能低下人群,它是AIDS患者主要的机会致病菌,随着人类免疫缺陷病毒(HIV)的流行,隐球菌病呈增加趋势,据报道约6%-10%的AIDS患者会合并隐球菌感染[20, 21]。在隐球菌属中引起人类感染主要为新生隐球菌和格特隐球菌。隐球菌主要特征是荚膜多糖,其中含有葡糖醛酸甘露聚糖抗原。目前,隐球菌的传统的检测方法包括墨汁染色及培养,其中,脑脊液的墨汁染色可见新型隐球菌酵母细胞周围呈一圈透亮的厚荚膜[22]。脑脊液的隐球菌培养选用沙氏葡萄糖琼脂斜面(SDA),新型隐球菌在SDA上25℃及37℃培养2-4d可见菌落生长,类似于细菌的菌落,湿润、透明,颜色由乳白、奶油逐渐变成橘黄色。

二、真菌感染的血清学检测方法

1. G试验的临床应用:(1-3)-β-D葡聚糖检测简称G试验,占真菌细胞壁成分的50%以上,在酵母菌中的含量最高,其他微生物,动物及人的细胞均不含该成分,是真菌细胞壁上的特有成分,成为真菌检测标志物。当真菌进入血液或深部组织后,经吞噬细胞的吞噬、消化等处理后,(1-3)-β-D葡聚糖从胞壁中释放出来,能特异性激活反应主剂中的G因子,凝固酶原,引起凝固蛋白原转变成凝固蛋白从而引起吸光度变化进行定量测定。然而,由于葡萄糖在环境中广泛存在,患者长期应用纤维素膜进行血液透析,输注免疫球蛋白、白蛋白、凝血因子或血液制品,服用某些抗肿瘤药物和磺胺类药物,标本暴露于纱布等含葡聚糖的材料以及实验过程的污染等均可导致假阳性。2008年,欧洲癌症治疗研究组织和美国国家过敏症与传染病研究所霉菌病研究组(EROTC/MSG)修订了深部真菌感染的标准,首次将G试验耐入微生物学标准[23]

在2012年ESCMID指南中,推荐G试验作为诊断侵袭性念珠菌病的早期指标[24]。多项荟萃研究显示,G试验诊断侵袭性念珠菌病的敏感性在75%-80%,特异性为80%,G试验值可作为抗真菌治疗效果的指标[25-27]。发表的一项荟萃中1,770名血液系统恶性肿瘤患者的研究结果显示,连续2次G实验检测特异性好(98.9%),敏感性较低(49.6%)在用于诊断侵袭性念珠菌感染[28]

在2016年IDSA指南中,推荐G试验作为诊断侵袭性曲霉病的早期指标[29]。血清G试验检测用于诊断血液系统恶性肿瘤和同种异体HSCT等高危人群的侵袭性曲霉感染。目前,对于患有侵袭性曲霉菌病(IA)的恶性血液病患者,G试验的敏感性为55%-95%,特异性为77%-96%[30-33]。侵袭性曲霉感染是免疫功能低下重症监护病房(ICU)患者中第二常见的侵袭性真菌病,ICU患者侵袭性曲霉感染的血清G试验浓度明显高于非侵袭性真菌感染患者,敏感性和特异性分别为85.7%和69.7%[25]

近年来,随着BDG在临床的广泛应用,除对念珠菌和曲霉感染检测中发挥重要的作用外[27]。一项荟萃分析表明,血清样本BDG检测对肺孢子菌肺炎的敏感性和特异性分别为94.8%和86.3%[34]。此外,一项涉及147例疑似患有肺孢子虫感染的前瞻性研究显示BDG检测可以从定植中区分确诊和临床诊断[35]。然而BDG检测不能作为肺孢子虫肺炎患者治疗预后的预测因子,因为该检测值的降低速度明显滞后于临床改善[36]

2. Mn试验的临床应用:念珠菌甘露聚糖(Mannan,Mn)作为念珠菌感染的标志物已被广泛研究。甘露聚糖常以糖蛋白的形式存在于多种微生物中,是念珠菌表面除葡萄糖外的另一重要抗原。甘露聚糖是机体固有免疫和适应性免疫的重要抗原,它能引起很强的抗体反应,IFD约一半为念珠菌感染,其中白念珠菌最为常见,占念珠菌感染比例超过65%。血培养阳性率低,耗时长,G试验又不能实现念珠菌的特异性鉴别诊断,所以Mn试验对侵袭性念珠菌感染的检测有十分重要的意义,多项实验研究肯定甘露聚糖抗原及其抗体在深部真菌感染的诊断价值[37, 38]。2010年,第三届欧洲白血病感染会议建议甘露聚糖抗原和抗甘露聚糖抗体在侵袭性念珠菌病诊断中的应用[39]。近年来念珠菌抗原、抗体检测是的研究焦点。国外研究报道甘露聚糖检测的敏感性为58%、特异性为93%,抗甘露聚糖抗体的敏感性59%,特异性为83%。当抗原和抗体联合检测可提高敏感性为83%,特异性为86%[40]。与培养法相比,甘露聚糖抗原检测阳性结果平均提前6天,抗甘露聚糖抗体平均提前7天[39]。此外,免疫功能低下患者的抗体检测的敏感性低,这是由于该群体通常不能产生针对念珠菌抗原的抗体,因此具有被侵袭性念珠菌病感染的高风险[41]

3. GM试验的临床应用:半乳甘露聚糖(Galactomannan,GM)是曲霉细胞壁的主要成分,被认为是曲霉菌感染后最早释放入血的标志性抗原。目前,GM试验是IA的早期诊断方法,已被国内外专家共识及指南推荐。尽管半乳甘露聚糖也可以在组织胞浆菌和镰刀菌属的细胞壁上发现,但GM检测对于IA的诊断是特异且敏感的[42, 43]。血清、BALF、CSF或胸膜液均可以进行GM试验,其特异性和敏感性从40%-100%不等,并且在很大程度上取决检测人群[44, 45]

2016年美国IDSA指南明确指出推荐使用血清和BALF的GM试验作为恶性血液病和HSCT患者IA的标志物,连续血清GM监测可用于上述疾病进展、治疗效果以及预后评价。不建议对接受抗菌治疗或预防性治疗的病人常规血液筛查GM,建议该类病人的支气管镜样本检查GM,文献报道,骨髓移植患者在给予抗真菌治疗后血清GM敏感性降低至20%以下[46]。不建议对接受SOT或慢性肉芽肿性疾病(CGD)的患者筛查GM[29]。最近发表的一项荟萃中表明血清GM检测在血液系统恶性肿瘤患者或接受过HSCT患者中敏感性高(70-80%)。然而,在SOT或免疫功能正常的患者敏感性低(22%)[47]。此外,检测的性能取决于宿主的免疫反应以及疾病的发病机制,使用类固醇治疗的患者其敏感性低于中性粒细胞减少患者[46]

目前,普遍认为BALF样本的GM试验的敏感性比血清样本高。研究表明,BALF进行GM检测的敏感性高于血清GM检测,其特异性和敏感性从80%-100%不等[48-51]。BALF的假阳性结果可能代表真菌在气道的简单定植,常见于肺移植受者,但这并不会影响GM试验检测的特异性,仍然高于95%[52]。然而,研究结果发现在特定人群HSCT患者的死亡率与血清GM检测相关,而与BALF GM检测没有关系[49-53]。值得注意的是,GM检测通常用于监测对治疗的反应,Fisher等人研究表明血清GM检测具有预后价值,结果显示,GM水平升高与同种异体HCT受者的呼吸道死亡率升高有关[53]。最后,需要注意的是BALF的获取应该尽量标准化,这样才会提高GM试验检测的准确度。

4. 新型隐球菌荚膜多糖(Galactomannan,Gxm)检测的临床应用:目前常用的隐球菌抗原检测方法包括乳胶凝集试验(LA)、酶联免疫分析法(EIA)和侧流免疫层析法(LFA),常规检测的标本包括血清和脑脊液(CSF)。与培养、病理学检查相比,隐球菌抗原检测具有快速简便的优势,同时具有较高的敏感性和特异性。Meta分析发现,LFA诊断隐球菌感染的总体敏感性和特异性分别为97.6%和98.1%[54]。对脑脊液(CSF)样本进行抗原检测具有高敏感性和特异性(97%和93%-100%),当CSF不能用时,可以用血清进行检测,敏感性为87%[55]。由于其优越的性能,在最新的EORTC/MSG指南中将隐球菌抗原检测作为隐球菌性脑膜炎推荐的诊断方法。LA和EIA都是隐球菌抗原检测方法,具有很高的一致性,乳胶凝集往往会产生更多的假阳性结果,特别是在滴度较低时[54]。一种新开发的隐球菌抗原检测方法利用LFA,其性能可与CSF和血清样本的EIA和乳胶凝集试验媲美成本低,简便,准确度高,可在血清和尿液样本量有限的情况下进行,是一种即时诊断方法[56, 57]。对于隐球菌抗原阳性且逆转录病毒治疗不足以控制的HIV感染患者CD4+计数<100个细胞/ul时,及时检测尤为重要[58]。因为,对于这类人群快速隐球菌筛选试验可以及时拯救生命。2019 EORTC/MSG侵袭性真菌病指南明确将脑脊液、血液样本GXM抗原阳性作为隐球菌感染的确诊指标[59]

5. 念珠菌IgG抗体检测的临床应用:念珠Mn检测和抗体IgG检测在欧洲获得了比美国更大的认可。通常,抗原检测受到来自血流的快速清除限制。人们对免疫抑制宿主中抗体检测的可靠性表示担忧,但是对于中性粒细胞减少症和细胞介导的免疫缺陷(包括HSCT和SOT)患者,检测结果表现良好。目前,最好的研究检测是联合抗原抗体检测,已在欧洲获批准使用。曲霉抗原抗体联合检测在侵袭性念珠菌检测的优势已经在前面进行提及。中国人民解放军总医院回顾性对照研究证实,Mn、Mn-IgG以及两者联合检测时敏感性和特异性分别是64.8%和89.2%、74.6%和87.0%、81.7%和81.6%,联合检测的敏感性显著高于单项检测[60]。与培养法相比,Mn检测阳性结果平均提前6天,Mn-IgG抗体平均提前7天[61],对提高治愈率与病死率有重要的临床价值。中国成人念珠菌病诊断与治疗专家共识推荐Mn抗原/Mn-IgG抗体检测用于临床侵袭性念珠菌病的诊断,该产品已获得国家食品药品监督管理总局批准[62]

6. 曲霉IgG抗体检测的临床应用:目前,深部曲霉感染主要包括侵袭性(急性)曲霉病(IA)、慢性肺曲霉病(CPA)和过敏性肺曲霉病(ABPA)。其中GM抗原检测在严重免疫缺陷及中性粒细胞缺乏的IA患者具有高敏感性和特异性,而曲霉IgG抗体检测在非严重免疫抑制的CPA患者的敏感性最高。2016年美国IDSA指南,明确指出曲霉IgG抗体检测为CPA患者首选的微生物学证据。并且,曲霉IgG抗体检测为CPA患者最灵敏的微生物学检测方法[29]。2017年美国ESCMID-ECMM-ERS指南,明确指出曲霉IgG抗体检测(对有空洞或结节的CPA患者)为A级推荐,BALF的GM检测为B级推荐,而不推荐血清GM进行检测[63]。最新流行病学研究证实,每年有20万的IA患者,300万的CPA患者和480万的ABPA患者。一项基于大数据的研究证实,血清GM检测对IPA、CPA和ABPA的敏感性分别为62%、23%和0%,曲霉IgG抗体检测对IA、CPA和ABPA的敏感性分别为65%、100%和65%,曲霉IgE抗体检测对IA、CPA和ABPA的敏感性分别为0%、66%和100%。由此可见,曲霉IgG抗体检测不仅对CPA患者有极高的敏感性同时针对部分的IA和ABPA患者也有一定的检出率,覆盖更广泛的曲霉感染人群[64]。近年来,国内外多家医疗机构采用曲霉IgG抗体国产试剂盒发表多篇研究证实曲霉IgG抗体检测在CPA诊断中的价值,其敏感性在70.0%-84.1%,特异性在82.8%-94.4%。除此之外,研究发现曲霉IgG抗体检测可用区分曲霉的定植和感染、治疗监测和病情转归,并在非粒缺IPA患者也有一定的检测价值[65-68]

表1. 国内外多家医疗机构研究结果

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7. 曲霉IgE抗体检测的临床应用:过敏性支气管肺曲霉病(ABPA)是烟曲霉致敏引起的一种变应性肺部疾病,表现为慢性支气管哮喘和反复出现的肺部阴影,可伴有支气管扩张。在2017年ABPA诊治专家共识的诊断标准中血清曲霉IgE水平升高或曲霉皮试速发反应阳性是诊断ABPA的必需条件之一,是ABPA特征性的诊断指标。有研究显示,曲霉特异性IgE水平是诊断ABPA灵敏度最高的指标,血清烟曲霉特异性IgE抗体诊断ABPA灵敏度达100%[69]

三、真菌感染的其他检测方法

目前,常用的分子生物学方法很多,其中,PCR检测是真菌诊断中最古老和最广泛的分子生物学方法。尽管PCR方法的潜力大,但还是存在几方面的问题。首先,真菌生物具有厚实的细胞壁难以裂解,因此需要复杂和繁琐的DNA分离方法[70]。其次,真菌无处不在需要避免和污染物引起的假阳性[71]。第三,难以区分定植和感染[72]。第四,缺乏国际标准,很难评估来自不同检测数据的一致性。最后,不同引物的选择是改变PCR检测的诊断性能的重要因素。研究结果证实PCR检测在侵袭性念珠菌感染人群中敏感性和特异性范围分别为56.2%-100%和54%-100%。在侵袭性曲霉感染人群中敏感性和特异性范围分别为43%-100%和64%-100%。研究发现BALF样本用于曲霉PCR的检测,结果令人满意。耶氏肺孢子菌不能培养,目前的检测基于呼吸道标本的染色,敏感性低。许多研究已经评估了PCR对呼吸道标本耶氏肺孢子菌检测敏感性高达100%。目前,毛霉菌没有明确的抗原抗体检测,PCR检测也得到进一步研究。T2 Candida磁共振(T2MR)是诊断念珠菌血症的新方法,可在4个小时报告结果,已经被美国FDA批准。西班牙的两项研究表明T2 Candida可作为患者预后判断的工具,其在鉴别复杂念珠菌血症、预测疑似念珠菌血症患者经验性抗真菌治疗解决的能力优于血培养或G实验。美国的一项研究表明,T2 Candida的使用缩短了经验型治疗的时间,使受试患者平均每人节省280美元的抗真菌治疗费用。罗马的一项研究表明,T2 Candida识别感染征象患者的敏感性、特异性均高于血培养,并且其菌种鉴定及报告阴性结果时间也显著减少于血培养,因此前景广阔,但还需要更多的临床数据来验证其性能。

目前,侵袭性真菌传统的检测方法存在很多不足。因此开发快速,准确的检测方法势在必行。新兴的血清学和分子生物学方法检测显示出取代传统诊断检测的潜力。因此,应该努力实现技术的标准化确保其可靠性,以便有效及时的提高真菌病原体的检测能力。新方法的不断涌现,提醒我们真菌诊断还有很大的改进和完善空间。


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