结合外周血宏基因组二代测序诊断的7例重症非人类免疫缺陷病毒感染者肺孢子菌肺炎临床分析

目的探讨外周血病原宏基因组二代测序(mNGS)在重症肺孢子菌肺炎(PCP)诊断中的应用价值, 并提高临床医师对此类疾病的认识。方法本研究为横断面研究。采用非随机抽样法, 分析2020年1月至2022年1月在南京鼓楼医院呼吸与危重症医学科重症监护病房通过外周血mNGS确诊的7例重症非HIV感染者PCP患者的临床资料, 分析临床特征、影像学表现、治疗以及预后。结果 7例患者, 男2例, 女5例, 年龄64(57, 65)岁, 年龄范围为36～66岁, 均有长期服用激素药物史, 其中3例患者同时服用免疫抑制剂, 主要症状包括发热、气喘、咳嗽, 实验室检查显示外周血淋巴细胞绝对计数均降低, 外周血CD4+T淋巴细胞绝对计数均小于0.2×109/L, 外周血CD8+T淋巴细胞绝对计数均小于0.3×109/L, 血清G试验、乳酸脱氢酶均增高, 血气分析均提示Ⅰ型呼吸衰竭, 影像学主要表现为两肺弥漫性磨玻璃影, 并可见囊状影、实变影。7例患者外周血mNGS均检出肺孢子菌, 治疗上7例患者均给予口服复方磺胺甲恶唑并联合克林霉素及卡泊芬净治疗PCP, 同时给予注射用甲泼尼龙琥珀酸钠抗炎平喘, 并联合其他...     

外周血宏基因组二代测序在肺炎中的应用价值

目的 评估外周血宏基因组二代测序(mNGS)技术在肺炎患者中微生物检测方面的应用价值。方法 使用回顾性分析的方法,收集常州市第二人民医院收治的28例使用外周血mNGS技术进行病原学检测的肺炎患者,并与常规微生物学检测(CMT)方法进行比较。结果 在这28例患者中,重症肺炎有19例,非重症肺炎有9例。mNGS对微生物的检出率(78.6%,22/28)与CMT(64.3%,18/28)相比差异无统计学意义(P=0.338)。mNGS对病毒的检出率高于CMT(P=0.004),但是在检出细菌(P=0.625)和真菌(P=0.118)方面无明显优势。进一步在重症肺炎患者的亚组分析中得到了同样的结果。经过综合评估,7例患者通过mNGS确诊病原体,分别为耶氏肺孢子菌(PJ)2例,PJ合并巨细胞病毒(CMV)2例,鹦鹉热衣原体2例和腺病毒1例。结论 尽管血mNGS对病原微生物的检出率并不优于CMT,但是当病毒、PJ或鹦鹉热衣原体感染时,可以考虑使用血mNGS进行病原学诊断。     

宏基因组二代测序辅助诊断鹦鹉热衣原体肺炎一例

<正>鹦鹉热衣原体作为一种革兰氏阴性、胞内寄生的病原体,通常在鹦鹉及鸽子中被发现。由于鹦鹉热的低发病率及无特异性的临床表现,其发生易被忽视。而且目前所存在的传统检测方法检出鹦鹉热衣原体病原体较为困难,然而宏基因组二代测序(metagenomic next-generation sequencing, mNGS)作为一种新型宏基因组二代测序方法,具有快速、准确、全面检测的优势。     

应用宏基因组二代测序诊断的13例肺诺卡菌病患者的临床特征分析

目的 回顾性分析肺诺卡菌病患者的临床资料,以提高对该病的认识,并探讨病原宏基因组二代测序(metagenomic Next-Generation Sequencing, mNGS)在诺卡菌病诊断的应用价值。方法 收集2019年1月～2021年8月厦门大学附属第一医院mNGS确诊为肺诺卡菌病的13例患者的临床资料,分析其临床特征并追踪其治疗与转归。结果 (1)13例患者中,男性5例,女性8例,年龄37～78岁,平均年龄(58±12)岁,体重指数(BMI)为(19.63±3.43)kg/m²,3例有长期吸烟史。(2)13例患者中只有1例无基础疾病,余12例均存在基础疾病,主要合并慢性呼吸系统疾病以及自身免疫性疾病,其中6例患者在进行糖皮质激素或者免疫抑制剂的治疗。(3)患者的主要临床症状包括发热、咳嗽咳痰、呼吸困难等。(4)在所有经mNGS检出的诺卡菌患者中,诺卡菌分型主要为亚洲诺卡菌。(5)9例患者表现为双肺受累,主要表现为斑片或结节状高密度影。(6)所有患者都接受了磺胺甲噁唑治疗,大部分患者在此基础上联合其他药物治疗。结论 肺诺卡菌病多见于慢性呼吸系统疾病以及自身...     

宏基因组二代测序技术在艾滋病合并肺诺卡菌病诊断中的应用（附1例报告）

宏基因组二代测序技术（mNGS）可对感染标本直接进行高通量测序,为复杂性感染提供快速精准诊断。本文分析1例艾滋病（AIDS）合并肺诺卡菌病患者临床表现及诊疗经过,探讨mNGS在诊断中的应用情况,为AIDS合并诺卡菌感染的诊治提供经验思路。患者入院时按常规临床症状、实验室检查等资料初始误诊为细菌性肺炎、肺结核,后结合mNGS诊断为肺诺卡菌感染,经经验性应用复方磺胺甲(口恶)唑联合阿米卡星抗诺卡菌治疗好转出院。该病例提示在免疫缺陷合并感染的患者中,应用mNGS进行病原体检测可指导感染性疾病诊断。     

宏基因组二代测序技术对髋/膝关节结核的诊断价值

目的:评价宏基因组二代测序(metagenomic next generation sequencing, mNGS)技术对髋/膝关节结核的诊断价值。方法:采用回顾性研究方法,收集2019年6月至2021年6月河北省胸科医院收治的34例髋/膝关节感染患者资料,按照病原菌种类分为关节结核组(16例)和非结核感染性疾病组(18例),分别对所有患者感染病灶组织样本进行常规细菌学培养和分枝杆菌培养(MGIT 960培养)、结核分枝杆菌(MTB)-DNA扩增检测及mNGS检测。以临床诊断为参照标准,比较分枝杆菌培养、MTB-DNA扩增和mNGS检测对髋/膝关节结核的诊断效能。结果:mNGS对非结核感染性疾病组中病原体的检出率为100.0%(18/18);对髋/膝关节结核的阳性检出率为93.8%(15/16),明显高于MTB-DNA扩增检测[50.0%(8/16)]和MGIT 960培养[25.0%(4/16)],差异均有统计学意义(χ²=7.575,P=0.015;χ²=15.676,P=0.000)。以临床诊断为参照标准,mNGS诊断髋/膝关节结核的...     

宏基因组二代测序在结核性胸膜炎中的诊断价值

<正>目前结核病疫情仍未得到有效控制,其中印度、印度尼西亚、中国是新发结核最多的3个国家^[1],约1/3的结核杆菌感染表现为结核性胸膜炎^[2]。我国结核性胸膜炎引起的胸腔积液约占胸腔积液所有类型的45%～55%^[3]。结核性胸膜炎确诊主要依赖胸膜或胸腔积液病原学检查结果和病理表现^[4-5],胸腔穿刺及胸腔镜下病理活检阳性率分别为54%～63%和66%^[6],但临床实践中胸膜活检操作难度大、对医师及设备要求高,限制了胸膜活检的应用。而由于胸腔积液的少菌性,病原学检查结果常为阴性,阳性率仅为15%～46%^[7],且耗时长。目前临床诊断主要综合临床表现和胸腔积液细胞学及生化检查结果,本文就结核性胸膜炎患者的临床资料进行前瞻性分析,探索宏基因组二代测序(m NGS)在结核性胸膜炎中的诊断价值。     

基于宏基因组的二代测序技术在重症肺炎患者病原体快速检测中的应用价值探讨

目的探讨基于宏基因组的二代测序技术(mNGS)在重症肺炎患者病原学检测中的应用价值。方法纳入2018-07~2019-05收治的重症肺炎患者26例,比较mNGS与传统检测方法对病原菌的诊断效能,同时评价抗生素暴露对检出率的影响。结果mNGS检出病原菌的阳性率为92.3%,高于传统培养方法的42.3%,差异有统计学意义(P<0.05)。在使用抗生素的前提下,mNGS的阳性率(91.3%)显著高于传统方法(43.5%),差异有统计学意义(P<0.05)。结论mNGS对病原菌鉴定具有较高的灵敏度,而且使用抗生素对mNGS检测结果影响小,对感染患者的诊治及疗效有着重要的临床应用价值。     

宏基因组二代测序对肾脏重症监护病房免疫功能低下患者感染的诊断价值

目的：回顾性分析宏基因组二代测序(mNGS)对肾脏重症监护病房(ICU)免疫功能低下患者感染的诊断价值,评估临床应用前景。方法：选取2018年8月至2022年4月国家肾脏疾病临床医学研究中心ICU收治疑诊感染的慢性肾脏病(CKD)免疫功能低下患者,分为常规微生物学检测(CMT)确诊感染组、临床诊断感染组和排除感染组,对比mNGS与CMT的诊断效能。结果：本研究共纳入303例患者,累计使用2(1, 3)种免疫抑制剂,中位治疗时间7(2, 50)月,中位CD4⁺T细胞计数149.50(81.25, 262.25)个/μL,在院死亡率20.46%。CMT确诊感染组中38.79%的mNGS与CMT结果完全一致,27.88%部分一致,33.33%不一致。临床诊断感染组中57.69%通过mNGS发现感染病原体。mNGS报阳时间更早,检测病原体种类更多(P<0.05),混合感染检出率高(P<0.001),可鉴定出常规培养难以发现的病原体。mNGS对感染病原体检测灵敏度和准确度强于CMT(P=0.014),特异度、阳性及阴性预测值无明显差异。结论：mNGS能增加免疫功...     

肺泡灌洗液宏基因组二代测序辅助诊断肺隐球菌病一例

<正>肺隐球菌病(pulmonary cryptococcosis,PC)的影像学表现多种多样,以单发或多发的周围型结节团块及局限的肺炎样病灶最为多见^[1-2],主要取决于宿主的免疫状态,易被误诊为肺结核、肺癌等疾病。目前针对肺隐球菌病的诊断没有相关的指南推荐意见。临床多采用检测外周血隐球菌荚膜多糖抗原的传统方法,具有快速简便的优势,在孤立性肺隐球菌病的诊断中存在敏感性不足;常规支气管镜下毛刷及肺泡灌洗液培养,因多数肺隐球菌病病灶靠近外周,存在培养阳性率低;组织病理学检查,是肺隐球菌病确诊的“金标准”,需经支气管镜肺活检、或在B超/CT引导下经皮肺穿刺活检以及经电视胸腔镜肺活检等方法获取肺组织标本,存在医院技术条件的限制。本文对我院收治的1例经肺泡灌洗液m NGS辅助诊断肺隐球菌病患者的全过程进行回顾性分析,结合文献对m NGS技术在诊断肺隐球菌病中的价值进行讨论,报告如下。     

Pneumocystis jiroveci pneumonia with cytomegalovirus infection diagnosed by metagenomic next-generation sequencing in a patient with nephrotic syndrome: A case report

Introduction:Opportunistic infection with multiple pathogens currently has become less uncommon since the application of immunosuppressant or corticosteroid in non- Human immunodeficiency virus patients. However, the clinical diagnosis of the co-infection remains difficult since the uncertainty and deficiency of the microbiologic testing methods.Patient concerns:A 66-year-old male patient was admitted to our hospital with chest stuffiness, shortness of breath and elevated body temperature.Diagnosis:He was diagnosed with the co-infection of Pneumocystis jiroveci and cytomegalovirus by metagenomic next-generation sequencing of bronchoalveolar lavage fluid after bronchoscopy.Interventions:The patient was empirically treated with broad-spectrum antibiotics, trimethoprim/ sulfamethoxazole and ganciclovir in the beginning of the admission.Outcomes:The condition of this patient was not improved even with the intervention at the early stage of the disease. His family requested discharge after 24 inpatient days.Lessons:This case highlights the application of metagenomic next-generation sequencing in the clinical diagnosis of pulmonary co-infection. Suitable prophylaxis, necessary clinical awareness and accurate diagnosis are indispensable for immunocompromised patients with pulmonary infection.     

Clinical characteristics of 14 cases of severe Chlamydia psittaci pneumonia diagnosed by metagenomic next-generation sequencing: A case series

Introduction:The objective of this study was to explore the clinical, laboratory, and imaging features of severe Chlamydia psittaci pneumonia in order to improve early diagnosis and treatment success rates.Methods:We conducted a retrospective record review of 14 cases of severe Chlamydia psittaci pneumonia diagnosed by metagenomic next-generation sequencing technology in our hospital. We extracted and analyzed data on the clinical symptoms and signs, contact history, laboratory investigations, chest computed tomography, treatment, and clinical outcomes.Results:Of the 14 patients, 12 (86%) were male and two (14%) were female, with a mean age of 57 years (SD: 7 years). Eleven patients (79%) had a history of poultry contact. The main clinical manifestations were fever (n = 14, 100%), flu-like symptoms (n = 10, 71%), cough, sputum (n = 9, 64%), and dyspnea (n = 5, 36%). Blood tests revealed marked elevation of neutrophil percentage, C-reactive protein, procalcitonin, brain natriuretic peptide, and creatine kinase levels; slight elevation of aspartate aminotransferase, creatinine, urea, fibrinogen, and D-dimer levels; and decreased albumin, sodium, and calcium levels. Chest computed tomography showed bilateral lesions (n = 7, 50%), middle-lower lobe lesions (n = 10, 71%), lesions in multiple lobes (n = 9, 64%), consolidation shadows (n = 11, 79%), and pleural effusions (n = 11, 79%). The median time from disease onset to hospital admission was 4.5 days (interquartile range: 1–17 days); the mean length of hospital stay was 20.9 ± 8.5 days, and the mean time from admission to diagnosis was 5.1 ± 2.6 days. After diagnosis, patients were either treated with doxycycline alone or doxycycline combined with quinolones. All 14 patients developed respiratory failure and received invasive mechanical ventilation; two (14%) received veno-venous extracorporeal membrane oxygenation, four (29%) received continuous renal replacement therapy, and three (21%) died.Discussion and conclusion:A poultry contact history and typical flu-like symptoms are early indicators of Chlamydia psittaci pneumonia. Substantial elevations in procalcitonin, creatine kinase, and brain natriuretic peptide indicate severe disease. Metagenomic next-generation sequencing is useful for diagnosis. Early empirical antibiotic therapy with quinolones can reduce the mortality in critically ill patients.     

Clinical and chest computed tomography features associated with severe Chlamydia psittaci pneumonia diagnosed by metagenomic next-generation sequencing: A multicenter,retrospective, observational study

Chlamydia psittaci pneumonia is a rare disease with varying clinical presentations. Here, we aimed to investigate the clinical and chest computed tomography (CT) features of severe psittacosis pneumonia. Clinical data of 35 patients diagnosed with psittacosis pneumonia were retrospectively analyzed using metagenomic next-generation sequencing. The patients were classified into severe (n = 20) and non-severe (n = 15) groups. The median age of patients was 54 years, and 27 patients (77.1%) had a definite history of bird contact. Severe patients had more underlying comorbidities and were more prone to dyspnea and consciousness disorders than non-severe patients. The neutrophil count and D-dimer, lactate dehydrogenase, interleukin (IL)-2, IL-6, and IL-10 levels were higher, whereas the lymphocyte, CD3⁺T cell, and CD4⁺T cell counts, CD4⁺/CD8⁺T cell ratio, and albumin level were substantially lower in severe patients than in non-severe patients. Chest CT findings of severe patients revealed large areas of pulmonary consolidation, and ground-glass opacities were observed in some patients, with a higher risk of involving multiple lobes of the lungs and pleural effusion. One patient died of multiple organ failure, whereas the condition of the other 34 patients improved, and they were discharged from the hospital. Patients with severe psittacosis pneumonia often have underlying comorbidities and are prone to developing dyspnea, consciousness disorder, and lesions in both lungs. Serum D-dimer, IL-2, IL-6, and IL-10 levels and lymphocyte, CD3⁺T cell, and CD4⁺T cell counts are associated with disease severity.     

1例宏基因二代测序提示钩端螺旋体感染患者的实验室诊断

目的 对一不明原因发热伴呼吸困难患者进行实验室诊断,探讨钩端螺旋体实验室确诊的检测方法。方法 利用宏基因二代测序技术(mNGS)对一不明原因发热伴呼吸困难患者的肺泡灌洗液进行病原体分析,利用实时荧光定量PCR(Real-time PCR)、显微镜凝集试验(MAT)和传统培养方法对患者进行实验室确诊。结果 mNGS对不明原因发热伴呼吸困难患者的肺泡灌洗液进行病原体分析提示患者可能被问号钩端螺旋体感染,Real-time PCR和传统培养方法对患者的肺泡灌洗液、血液和尿液检测均为阴性,血清抗体检测(MAT法)致病性钩端螺旋体澳洲型滴度为1:800。结论 该患者确诊为致病性钩端螺旋体澳洲型感染,mNGS与实验室传统方法的有机结合有助于对不明原因发热伴呼吸困难患者实验室诊断,特别是对钩端螺旋体的实验室诊断具有重要意义。     

A multicenter-retrospective cohort study of chromosome instability in lung cancer: clinical characteristics and prognosis of patients harboring chromosomal instability detected by metagenomic next-generation sequencing

BackgroundThe usefulness of metagenomic next-generation sequencing (mNGS) in identifying the prognosis of lung cancer with chromosomal instability (CIN) remains unclear. We aimed to analyze clinical characteristics and prognosis of patients in patients harboring CIN.MethodsThis retrospective cohort study included 668 patients diagnosed with suspected pulmonary infection or lung cancer whose samples underwent mNGS detection from January 2021 to January 2022. Difference between clinical characteristics were calculated by the Student’s t-test and the chi-square test. The subjects were followed-up from registered to September 2022. Survival curves were analyzed by the Kaplan-Meier method.ResultsOf 619 bronchoalveolar lavage fluid (BALF) samples collected by bronchoscopy, 30 CIN-positive samples were confirmed as malignant on histopathology, with a sensitivity of 61.22%, a specificity of 99.65%, and an 83.17% accuracy [cut-off values were established by the receiver operating characteristic (ROC) area under the curve (AUC) =0.804]. In 42 patients with lung cancer, mNGS detected 24 patients as CIN-positive and 18 as CIN-negative. There were no differences between two groups including ages, pathologic types, stage and metastases. In 25 cases, we detected 523 chromosomal copy number variants (CNVs) with forms including duplication (dup), deletion (del), mosaic (mos), and whole chromosome amplification or loss. A total of 243 duplication variants and 192 deletion variants occurred in all chromosomes. Duplications occurred in most chromosomes except for Chr9 and Chr13, in which CNV tended to delete. The median overall survival (OS) in patients with Chr5p15 duplication was 32.4 months [95% confidence interval (CI), 10.35–54.45 months]. The median OS differed significantly between the 5p15dup+ group and the combined group (32.4 vs. 8.63 months, P=0.049). In 29 patients with unresected lung cancer, the median OS of 18 cases in the CIN-positive group was 32.4 months (95% CI, 14.2–50.6 months) and the median OS of 11 cases in the CIN-negative group was 35.63 months (95% CI, 21.64–49.62 months; Wilcoxon, P=0.227).ConclusionsVarious forms of CIN detected by mNGS may predict prognosis of patients with lung cancer differentially. CIN with duplication or deletion deserves further study to guide clinical treatment.     

Detection of Chlamydia psittaci in both blood and bronchoalveolar lavage fluid using metagenomic next-generation sequencing: A case report

Rationale:Chlamydia psittaci (C psittaci) is a gram-negative obligate intracellular parasite, with birds as main hosts. The main route of infection in humans is inhalation of aerosols from contaminated animal excreta through the respiratory tract. The main manifestation of C psittaci infection is pneumonia. Patients suffering from severe infection are prone to sepsis and multiple organ failure. We report a case of simultaneous detection of C psittaci in blood and bronchoalveolar lavage fluid using metagenomic next-generation sequencing (mNGS) technology.Patient concerns:The 71-year-old male patient was a farmer with a long history of raising poultry and initial symptoms of fever and muscle pain accompanied by limb weakness and paroxysmal cough.Diagnoses:The patient was diagnosed with sepsis, severe pneumonia, and multiple organ failure.Interventions:Anti-infective therapy with doxycycline and meropenem was applied.Outcomes:The patient''s body temperature and infection indicators improved and the chest X-ray findings showed the amelioration of lesions after 18 days of treatment. The patient was discharged without treatment on hospital day 19 due to financial constraints and subsequently died after 7 days.Lessons:mNGS is an excellent diagnostic tool when specific pathogens are undetected by traditional assays.     

肺孢子菌肺炎31例临床分析

目的探讨研究肺孢子菌肺炎患者的临床特点、影像学表现及治疗方法。方法分析在我院诊断的肺孢子菌肺炎的31例患者的临床资料。结果 31例患者中HIV/AIDS患者28例,非AIDS患者3例,其中男性22例,女9例,年龄23~75岁。主要临床表现为咳嗽,气促者28例(90.3%),发热25例(80.6%),咳痰17例(54.8%),乏力,纳差15例(48.3%),胸痛8例(25.8%),腹泻4例(12.9%),反复皮疹3例(9.6%)。3例患者行无创通气,1例因严重肺部感染,低氧血症,行有创机械通气。31例患者胸部CT均表现为典型的双肺弥漫性磨玻璃影,所有患者均选用复方磺胺甲基异噁唑治疗,对吸空气时血氧分压PaO2低于70 mmHg患者给予激素治疗。结论当AIDS患者或免疫抑制患者出现发热,咳嗽,呼吸困难,低氧血症,其胸部CT提示典型的双肺弥漫性磨玻璃影,需考虑PCP的可能,但其病原学检查困难,治疗以复方磺胺甲基异噁唑及激素治疗为主。     

Fast and precise pathogen detection and identification of overlapping infection in patients with CUTI based on metagenomic next-generation sequencing: A case report

Rationale:The gold standard for pathogen detection and identification of complicated urinary tract infection (CUTI) remains quantitative urine culture, however, the diagnostic value of urine culture remains limited due to the time-consuming procedure and low detection rate. Here we report a case of successfully using Metagenomic next-generation sequencing (mNGS) provided fast and precise detection and identification of overlapping infection in patients with CUTI with no similar reports or studies published before to the best of our knowledge.Patient concerns:A 70-year-old male was admitted to hospital due to elevated serum creatinine for 2 weeks.Diagnoses:Acute exacerbation of chronic renal failure and CUTI were the most critical diagnosis on admission.Interventions:Blood purification, bladder irrigation and aggressive anti-infective therapy were administered. But the empirical anti-infection therapy and the adjustment of treatment according to the evidence of urine culture drug sensitivity had no obvious effect. We further carried out urinary PMseq-DNA detection and the results showed overlapping infection with Enterococcus faecium, Enterococcus hirae, Pseudomonas aeruginosa, Pseudomonas denitrificans and Candida albicans. According to the genetic test results, linezolid, meropenem and fluconazole triple anti-infection treatment was given.Outcomes:After adjusting the treatment, the infection was basically controlled in 10 days, and even the renal function was significantly improved, dialysis independence was achieved after 3 months.Lessons:Our case illustrated the potential application of mNGS in detecting pathogenic microorganisms in patients with CUTI especially when overlapping infections are present.