Single center clinical analysis of metagenomic next-generation sequencing for clinical diagnosis of Pneumocystis jirovecii Pneumonia after allogeneic hematopoietic stem cell transplantation
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摘要: 目的 探讨异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation，allo-HSCT)后经宏基因组二代测序(metagenomic next-generation sequencing，mNGS)协助诊断的耶氏肺孢子菌肺炎(Pneumocystis jirovecii pneumonia，PJP)患者的临床主要特征。方法 2020年10月—2023年3月allo-HSCT后的19例发热患者，完善常规微生物学检测及影像学检查无法明确诊断，经验性抗感染治疗无效时，通过mNGS检测临床诊断为PJP。分析患者的主要临床表现，mNGS结果、细胞免疫功能、炎症因子、血气分析、G-试验等，肺部高分辨CT、支气管镜结果以及治疗和转归等临床特征。结果 Allo-HSCT后PJP发生的中位时间为6个月，发热和呼吸困难是PJP的主要临床症状。19例患者均出现发热，以持续中度发热为主，安静状态下呼吸困难患者10例，3例患者为活动后出现呼吸困难；CD4+T淋巴细胞计数的中位值为148(34~394)个/μL，其中12例CD4+T淋巴细胞计数 < 200个/μL，10例患者血气分析提示Ⅰ型呼吸衰竭，氧分压的中位值为59.5(36.8~74.0) mmHg (1 mmHg=0.133 kPa)，双变量相关分析显示CD4+T淋巴细胞计数与患者氧分压呈正相关(r=0.468，P=0.043)。9例(47.36%)PJP患者G试验阳性；肺部CT主要以多发磨玻璃样密度增高影为主；17例PJP患者行支气管镜并肺泡灌洗液(BALF)检测mNGS，耶氏肺孢子菌核酸序列数的中位数为487(178~131 444)条，中位相对丰度为56.9%(26.0%~99.5%)，双变量相关分析显示患者氧分压与PJP序列数呈显著负相关(r=-0.498，P=0.042)。2例患者行外周血耶氏肺孢子菌核酸序列数分别为2条和4条；19例患者中12例发生PJP时存在慢性移植物抗宿主病，13例患者发生PJP前接受剂量不等的糖皮质激素治疗。诊断PJP后，患者主要接受复方磺胺甲噁唑(SMZ)/甲氧苄啶(TMP)联合卡泊芬净或米卡芬净治疗，1例患者因呼吸功能衰竭死亡，其余患者均治愈。结论 Allo-HSCT后发生PJP的患者主要以发热和呼吸困难为主要临床表现，CT影像学特征主要为双肺磨玻璃影，通过BALF和全血的mNGS有助于早期临床诊断。
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关键词:
- 异基因造血干细胞移植 /
- 耶氏肺孢子菌肺炎 /
- 宏基因组二代测序
Abstract: Objective To explore the clinical characteristics of Pneumocystis jirovecii Pneumonia(PJP) patients diagnosed with metagenomic next-generation sequencing(mNGS) assisted by allogeneic hematopoietic stem cell transplantation(allo-HSCT).Methods From October 2020 to March 2023, 19 patients with fever were diagnosed with PJP through mNGS testing after allo-HSCT at our transplantation center, as routine microbiological and imaging examinations were unable to determine the diagnosis, and empirical anti-infective treatment was ineffective. The main clinical manifestations of the patients were analyzed, including mNGSresults, cellular immune function, inflammatory factors, blood gas analysis, G-experiments, high-resolution CT and bronchoscopyresults of the lungs, as well as clinical features such as treatment and prognosis.Results The median time of occurrence of PJP after allo-HSCT was 6 months, and fever and dyspnea were the main clinical symptoms of PJP. All 19 patients experienced fever, with persistent moderate fever being the main cause. 10 patients had difficulty breathing in a quiet state, and 3 patients had difficulty breathing after activity. The average CD4+T lymphocyte count was 148(34-394)/μL. Among them, 12 cases had CD4+T lymphocyte counts < 200/μL. Blood gas analysis of 10 patients showed type I respiratory failure, with an average oxygen partial pressure of 59.5(36.8-74.0) mmHg. Bivariate correlation analysis showed a positive correlation between CD4+T lymphocyte count and patient oxygen partial pressure(r=0.468, P=0.043). 9 PJP patients(47.37%) showed positive G-testresults; The CT imaging of the lungs mainly shows multiple ground glass like high-density shadows; 17 PJP patients underwent bronchoscopy and alveolar lavage fluid(BALF) to detect mNGS. The median nucleic acid sequence number of Pneumocystis jirovecii Pneumonia was 487(178-131 444), and the median relative abundance was 56.9%(26.0%-99.5%). Bivariate correlation analysis showed a significantly negative correlation between patient oxygen partial pressure and PJP sequence number(r=-0.498, P=0.042). The number of nucleic acid sequences of Pneumocystis jirovecii pneumonia in the peripheral blood of two patients was 2 and 4, respectively. Among the 19 patients, 12 cases had cGVHD when PJP occurred, and 13 patients received varying doses of glucocorticoid therapy before PJP occurred. After the diagnosis of PJP, the patient mainly received treatment with compound sulfamethoxazole(SMZ)/trimethoprim(TMP) combined with caspofungin or micafungin. One patient died of respiratory failure, and the remaining patients were cured.Conclusion The main clinical manifestations of patients with PJP after allo-HSCT are fever and dyspnea. The CT imaging features are mainly ground glass shadows in both lungs, and early clinical diagnosis is facilitated by BALF and whole blood mNGS. -
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表 1 患者基本情况及临床数据
病例号 性别 年龄/岁 疾病 PJP发生时间 发病时免疫抑制剂使用 GVHD PJP发生前糖皮质激素使用时间 临床表现 PJP治疗方案 转归 1 男 68 MDS-EB2 +5月 他克莫司 无 未使用 发热、咳嗽、咳痰、气短 SMZ/TMP，米卡芬净 治愈 2 男 25 ALL +6月 环孢素A cGVHD 7 d 发热、咳嗽、咳痰、气短 SMZ/TMP，卡泊芬净 治愈 3 男 27 AML +4.5月 环孢素A GVHD 8 d 发热，咳嗽、咳痰、活动后气短 SMZ/TMP，卡泊芬净 治愈 4 男 29 AML +5.5月 麦考酚钠肠溶片 无 未使用 发热 SMZ/TMP，米卡芬净 治愈 5 女 62 AML +5月 环孢素A cGVHD 14 d 发热、气短 SMZ/TMP，米卡芬净 治愈 6 男 44 AML +12月 环孢素A cGVHD 12 d 发热、咳嗽、咳痰、气短 SMZ/TMP，米卡芬净 治愈 7 女 44 ALL +4.5月 环孢素A 无 未使用 发热、咳嗽、咳痰、气短 SMZ/TMP，卡泊芬净 治愈 8 女 38 AML +6月 他克莫司 cGVHD 15 d 发热、咳嗽、咳痰、活动后气短 SMZ/TMP，卡泊芬净 治愈 9 男 54 AML +22月 他克莫司 cGVHD 9 d 发热、咳嗽、咳痰 SMZ/TMP，米卡芬净 治愈 10 男 33 CMML +14月 他克莫司 cGVHD 9 d 发热 SMZ/TMP，卡泊芬净 治愈 11 女 60 MDS-EB2 +7月 他克莫司 无 14 d 发热、气短 SMZ/TMP，卡泊芬净 死亡 12 男 15 B-ALL +5.5月 环孢素A cGVHD 9 d 发热 SMZ/TMP，卡泊芬净 治愈 13 男 59 B-ALL +4.5月 环孢素A 无 7 d 发热、咳嗽、咳痰、气短 SMZ/TMP，卡泊芬净 治愈 14 女 34 B-ALL +4月 环孢素A 无 未使用 发热、咳嗽咳痰、活动后气短 SMZ/TMP，卡泊芬净 治愈 15 男 38 CMML +19月 无 无 未使用 发热 SMZ/TMP，卡泊芬净 治愈 16 男 27 AML +17月 无 cGVHD 7 d 发热、咳嗽咳痰、气短 SMZ/TMP，卡泊芬净 治愈 17 男 49 B-ALL +9月 环孢素A cGVHD 10 d 发热、咳嗽咳痰、气短 SMZ/TMP，卡泊芬净 治愈 18 男 32 T-ALL +12月 环孢素A cGVHD 13 d 发热、咳嗽、咳痰、气短 SMZ/TMP，卡泊芬净 治愈 19 男 52 MDS-EB2 +12月 他克莫司 cGVHD 未使用 发热 SMZ/TMP，卡泊芬净 治愈 
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表 2 患者实验室检查结果
病例号 白细胞计数/ (×109/L) CD4+T淋巴细胞计数 C反应蛋白/(mg/L) 白细胞介素-6 /(pg/mL) 降钙素原/(ng/mL) G-试验 氧分压/mmHg 二氧化碳分压/mmHg mNGS病原体核酸序列数(相对丰度)/条(%) 1 6.86 198 91.0 60.1 0.09 52.8 59.5 46.9 433(50.5) 2 5.39 135 34.0 10.2 0.05 289.6 53.5 47.7 435(50.0) 3 4.96 233 38.3 10.2 0.03 61.2 68.0 36.8 289(36.4) 4 6.86 394 48.3 23.4 0.05 46.7 77.7 33.7 1 539(75.5) 5 5.33 331 8.7 1.9 0.02 无 36.8 41.2 808(90.8) 6 4.09 68 37.1 7.7 0.10 181.1 52.3 48.1 608(48.0) 7 0.66 39 80.0 77.1 0.04 108.1 58.2 32.0 6 926(99.5) 8 2.65 69 65.4 10.1 0.10 367.3 63.0 31.0 356(64.0) 9 6.50 275 79.3 41.7 0.06 37.5 80.0 33.0 568(94.4) 10 8.80 211 90.0 107.0 0.12 83.5 73.0 37.0 487(88.8) 11 2.41 35 90.0 14.9 0.03 71.5 32.1 28.1 131 444(99.6) 12 2.05 102 49.7 126.2 0.03 109.2 76.0 31.0 2(0.6) 13 5.75 327 82.7 81.6 0.12 42.6 57.0 42.4 899(38.0) 14 8.67 190 47.1 23.2 0.08 37.5 64.0 40.0 288(53.6) 15 4.80 381 74.4 19.5 0.06 37.5 83.4 34.8 276(26.2) 16 7.67 34 80.0 1 220.0 0.61 37.5 48.0 33.0 2 692(51.0) 17 6.48 50 90.0 397.0 0.24 37.5 47.0 30.0 297(700.0) 18 3.10 92 72.8 78.2 0.16 40.1 57.0 21.0 78(56.9) 19 6.69 148 31.2 43.1 0.09 37.5 84.0 31.0 4(1.0)
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表 3 患者肺部CT及支气管镜检查结果
病例号 CT检查结果 支气管镜结果 1 双肺呈弥漫性磨玻璃样密度增高影 支气管炎症改变(左下肺明显) 2 双肺多发磨玻璃密度增高影 左侧支气管炎症改变 3 双肺可见多发大小不一的结节、斑片状及片絮状渗出 右肺上叶及左肺上叶气管黏膜炎症 4 双肺多发渗出性病灶，双肺可见多发斑片磨玻璃密度影 支气管炎症改变 5 双肺可见散在片状、小斑片状及结节样稍高密度影 右中肺叶炎症改变 6 双肺多发斑片状磨玻璃密度增高影 支气管炎症改变 7 双肺多发磨玻璃密度增高影 气管镜检查未见异常 8 双肺多发斑片状磨玻璃密度增高影，双侧胸腔积液 支气管炎症改变 9 双肺多发斑片渗出影 气管、支气管未见明显异常 10 双肺多发结节状及斑片状模糊灶 支气管炎症改变 11 双肺多发斑片状密度增高影，部分实变，双侧胸腔积液 支气管炎症改变 12 双肺多发斑片状、团片状高密度渗出影 无 13 双肺多发斑片及结节状渗出 气管、支气管未见明显异常 14 双肺多发斑片状及结节状密度增高影 各级支气管轻度炎症改变 15 左肺尖可见磨玻璃密度结节 支气管镜下未见明显异常 16 双肺渗出影，部分组织实变，可见多发斑片磨玻璃影 支气管镜下未见明显异常 17 双肺多发斑片状、结节样磨玻璃样密度增高影及少许索条影 支气管镜下未见明显异常 18 双肺多发磨玻璃密度斑片状渗出影 支气管炎症改变 19 双肺多发斑片状、结节样高密度影 无
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