病原真菌real-time PCR检测对非中性粒细胞缺乏的老年患者肺部真菌病的诊断价值

中国真菌学杂志　 2022, Vol. 17 　Issue (6): 454-460.

病原真菌real-time PCR检测对非中性粒细胞缺乏的老年患者肺部真菌病的诊断价值

蒙星烨¹, 刘晓¹, 万喆¹, 陈伟¹, 阙呈立², 林连君³, 余进¹, 宋营改¹, 李若瑜¹

1. 北京大学第一医院皮肤性病科, 北京大学真菌和真菌病研究中心, 国家皮肤与免疫疾病临床医学研究中心, 皮肤病分子诊断北京市重点实验室, 北京 100034;
2. 北京大学第一医院呼吸内科, 北京 100034;
3. 北京大学第一医院老年病内科, 北京 100034

收稿日期:2022-11-22 发布日期:2023-01-04
通讯作者: 宋营改,E-mail:syg3515@163.com;李若瑜,E-mail:mycolab@126.com E-mail:syg3515@163.com;mycolab@126.com
作者简介:蒙星烨,女(布依族),博士在读,住院医师.E-mail:mxybjmu@163.com
基金资助:
国家重点研发计划（2020YFC2005401）

The real-time PCR of fungal pathogens in bronchoalveolar lavage fluid for the diagnosis of pulmonary fungal diseases in non-neutropenic elderly patients

MENG Xingye¹, LIU Xiao¹, WAN Zhe¹, CHEN Wei¹, QUE Chengli², LIN Lianjun³, YU Jin¹, SONG Yinggai¹, LI Ruoyu¹

1. Departmentof Dermatology and Venerology, Peking University First Hospital;Research Center for Medical Mycology, Peking University;
National Clinical Research Center for Skin and Immune Diseases;Beijing Key Laboratory of Molecular Diagnosis of Dermatoses, Beijing 100034, China;
2. Department of Respiratory Medicine, Peking University First Hospital, Beijing 100034, China;
3. Geriatrics Department, Peking University First Hospital, Beijing 100034, China

Received:2022-11-22 Published:2023-01-04

摘要/Abstract

摘要： 目的评估本实验室设计的肺部常见病原真菌real-time PCR检测支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）标本对非中性粒细胞缺乏的老年人群肺部真菌病的诊断价值。方法回顾性纳入2020年至2021年间于北京大学第一医院就诊的怀疑肺部感染的294名非中性粒细胞缺乏老年患者，采用real-time PCR检测其BALF标本中的病原真菌；根据宿主因素、临床表现和真菌学检查进行诊断分类，27名患者符合确诊或临床诊断的肺部侵袭真菌病，其中7例（25.6%）为多种真菌合并感染，6名患者诊断慢性肺曲霉病。结果曲霉PCR Ct值受试者工作特征曲线下面积为0.936（95%置信区间0.865~1.000），最佳cut-off值为34.8，对肺曲霉病的诊断灵敏度和特异度分别为95.7%和92.1%。耶氏肺孢子菌PCR以Ct值37.1为cut-off值，诊断耶氏肺孢子菌肺炎灵敏度和特异度分别为94.7%和99.7%。隐球菌和毛霉PCR以Ct值35.0为cut-off值，在肺隐球菌病和肺毛霉病中阳性率分别为50%（2/4）和100%（1/1）。病原真菌real-time PCR确定cut-off值后，以PCR检出的真菌与肺部真菌病的致病真菌一致作为阳性，灵敏度、特异度、阳性预测值和阴性预测值分别为86.5%（32/37）、91.0%（253/278）、56.1%（32/57）和98.1%（253/258），总符合率为90.5%（285/315）。结论本研究团队开发的病原真菌real-time PCR体系覆盖常见肺部致病真菌，初步探索了其在BALF标本中的cut-off值，证实该方法在非中性粒细胞缺乏老年患者的肺部真菌病中具有良好的诊断价值。

关键词: 肺部真菌病, real-time PCR, 支气管肺泡灌洗液, 分子诊断

Abstract: Objective To evaluate the diagnostic accuracy of an in-house real-time PCR assay of fungal pathogens performed in bronchoalveolar lavage fluid (BALF) for diagnosing pulmonary fungal diseases in non-neutropenic elderly patients. Methods This one-year (2020-2021) retrospective study included 294 elderly patients suspected pulmonary infection who underwent bronchoscopy (BALF n=325) at Peking University First Hospital. All BALF samples tested with the real-time PCR of fungal pathogens, which covered the Aspergillus, Mucorales, Cryptococcus and Pneumocystis jirovecii and five species of Aspergillus (A. fumigatus, A. flavus, A.terreus, A. niger and A.nidulans). Twenty-seven patients had proven/probable invasive pulmonary fungal diseases, among which 7(25.9%) patients had more than one fungal pathogen. Six patients had chronic pulmonary aspergillosis. Results The area under the receiver operating characteristic curve of Aspergillus PCR cycle threshold (Ct) value was 0.936 (95% confidence interval 0.865-1.000), and the optimal cut-off value was 34.8 which yielded 95.7% sensitivity and 92.1% specificity for aspergillosis. Pneumocystis jirovecii PCR yielded 94.7% sensitivity and 99.7% specificity at the cut-off Ct value of 37.1. With cut-off Ct value 35.0, the positive rates of Cryptococcus and Mucorales PCR were 50%(2/4) and 100%(1/1) in pulmonary cryptococcosis and mucormycosis respectively. Collectively, when positive results defined by the pathogens detected by real-time PCR were consistent with the final diagnosis, the sensitivity specificity, positive and negative predictive value of PCR was 86.5%(32/37), 91.0%(253/278), 56.1%(32/57) and 98.1%(253/258), and the agreement rate was 91.4%. Conclusions The real-time PCR of fungal pathogens covers the most common pathogens of pulmonary fungal infection and is highly specific and sensitive for the diagnosis of pulmonary fungal diseases in non-neutropenic elderly patients with the cut-off value preliminarily explored in our study.

Key words: pulmonary fungal diseases, real-time PCR, bronchoalveolar lavage fluid, molecular diagnosis

中图分类号:

R 519 A

蒙星烨, 刘晓, 万喆, 陈伟, 阙呈立, 林连君, 余进, 宋营改, 李若瑜. 病原真菌real-time PCR检测对非中性粒细胞缺乏的老年患者肺部真菌病的诊断价值[J]. 中国真菌学杂志, 2022, 17(6): 454-460.

MENG Xingye, LIU Xiao, WAN Zhe, CHEN Wei, QUE Chengli, LIN Lianjun, YU Jin, SONG Yinggai, LI Ruoyu. The real-time PCR of fungal pathogens in bronchoalveolar lavage fluid for the diagnosis of pulmonary fungal diseases in non-neutropenic elderly patients[J]. Chinese Journal of Mycology, 2022, 17(6): 454-460.

参考文献

[1] ZHOU L H, JIANG Y K, LI R Y, et al. Risk-based estimate of human fungal disease burden, China[J]. Emerg Infect Dis, 2020, 26(9):2137-2147.
[2] LATGé J P, CHAMILOS G. Aspergillus fumigatus and aspergillosis in 2019[J]. Clin Microbiol Rev, 2019, 33(1):e00140-18.
[3] CHEN K Y, KO S C, HSUEH P R, et al. Pulmonary fungal infection:emphasis on microbiological spectra, patient outcome, and prognostic factors[J]. Chest, 2001, 120(1):177-184.
[4] HOENIGL M, SEIDEL D, SPRUTE R, et al. COVID-19-associated fungal infections[J]. Nat Microbiol, 2022, 7(8):1127-1140.
[5] RAYENS E, RAYENS M K, NORRIS K A. Demographic and socioeconomic factors associated with fungal infection risk, United States, 2019[J]. Emerg Infect Dis, 2022, 28(10):1955-1969.
[6] ADER F, NSEIR S, LE BERRE R, et al. Invasive pulmonary aspergillosis in chronic obstructive pulmonary disease:an emerging fungal pathogen[J]. Clin Microbiol Infect, 2005, 11(6):427-429.
[7] SCHAUWVLIEGHE A, RIJNDERS B J A, PHILIPS N, et al. Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza:a retrospective cohort study[J]. Lancet Respir Med, 2018, 6(10):782-792.
[8] WEI L W, ZHU P Q, CHEN X Q, et al. Mucormycosis in mainland China:a systematic review of case reports[J]. Mycopathologia, 2022, 187(1):1-14.
[9] GHEMBAZA A, VAUTIER M, CACOUB P, et al. Risk Factors and prevention of Pneumocystis jirovecii pneumonia in patients with autoimmune and inflammatory diseases[J]. Chest, 2020, 158(6):2323-2332.
[10] INOUE K, MURAMATSU K, NISHIMURA T, et al. Association between early diagnosis of and inpatient mortality from invasive pulmonary aspergillosis among patients without immunocompromised host factors:a nationwide observational study[J]. Int J Infect Dis, 2022, 122:279-284.
[11] DONNELLY J P, CHEN S C, KAUFFMAN C A, et al. Revision and update of the consensus definitions of invasive fungal disease from the European organization for research and treatment of cancer and the mycoses study group education and research consortium[J]. Clin Infect Dis, 2020, 71(6):1367-1376.
[12] DOBIAS R, JAWORSKA P, TOMASKOVA H, et al. Diagnostic value of serum galactomannan, (1,3)-β-d-glucan, and Aspergillusfumigatus-specific IgA and IgG assays for invasive pulmonary aspergillosis in non-neutropenic patients[J]. Mycoses, 2018, 61(8):576-586.
[13] 中国医药教育协会真菌病专业委员会, 国家皮肤与免疫疾病临床医学研究中心, 国家血液疾病临床医学研究中心. 侵袭性真菌病实验室诊断方法临床应用专家共识[J]. 中华内科杂志, 2022, 61(2):134-141.
[14] KIDD S E, CHEN S C A, MEYER W, et al. A new age in molecular diagnostics for invasive fungal disease:are we ready[J]? Front Microbiol, 2020, 10:2093.DOI:10.3389/fmicb.2019.02903.
[15] EIGL S, HOENIGL M, SPIESS B, et al. Galactomannan testing and Aspergillus PCR in same-day bronchoalveolar lavage and blood samples for diagnosis of invasive aspergillosis[J]. Med Mycol, 2017, 55(5):528-534.
[16] BOCH T, SPIESS B, HEINZ W, et al. Aspergillus specific nested PCR from the site of infection is superior to testing concurrent blood samples in immunocompromised patients with suspected invasive aspergillosis[J]. Mycoses, 2019, 62(11):1035-1042.
[17] BASSETTI M, AZOULAY E, KULLBERG B J, et al. EORTC/MSGERC definitions of invasive fungal diseases:summary of activities of the intensive care unit working group[J]. Clin Infect Dis, 2021, 72(Suppl 2):S121- S127.
[18] DENNING D W, CADRANEL J, BEIGELMAN-AUBRY C, et al. Chronic pulmonary aspergillosis:rationale and clinical guidelines for diagnosis and management[J]. The Eur Respir J, 2016, 47(1):45-68.
[19] LIU X, SONG Y G, LI R Y. The use of combined PCR, fluorescence in situ hybridisation and immunohistochemical staining to diagnose mucormycosis from formalin-fixed paraffin-embedded tissues[J]. Mycoses, 2021, 64(12):1460-1470.
[20] AVNI T, LEVY I, SPRECHER H, et al. Diagnostic accuracy of PCR alone compared to galactomannan in bronchoalveolar lavage fluid for diagnosis of invasive pulmonary aspergillosis:a systematic review[J]. J Clin Microbiol, 2012, 50(11):3652-3658.
[21] RUBIO-PORTILLO E, ORTS D, LLORCA E, et al. The domestic environment and the lung mycobiome[J]. Microorganisms, 2020, 8(11):1717.
[22] IMBERT S, MEYER I, PALOUS M, et al. Aspergillus PCR in bronchoalveolar lavage fluid for the diagnosis and prognosis of aspergillosis in patients with hematological and non-hematological conditions[J]. Front Microbiol, 2018, 9:1877.