干燥综合症合并肺孢子菌肺炎一例并文献复习

目的 探讨干燥综合症合并肺孢子菌肺炎（PCP）的早期诊断方法。方法 对1例干燥综合症合并肺孢子菌肺炎患者的临床资料进行回顾性分析。结果 患者因干燥综合症接受免疫抑制剂治疗两月后出现不明原因发热、干咳,活动憋气。依据患者临床表现、胸部X线和CT征像、痰肺孢子菌六胺银染色镜检和多聚酶链反应（PCR）监测结果而确诊PCP。予克林霉素（患者磺胺过敏）、卡泊芬静治疗6 d后痰液肺孢子菌DNA载量由104/ml减为102/ml,12 d后进一步降至101/ml,经卡泊芬静治疗21 d后痊愈出院。结论 痰液标本肺孢子菌六胺银染色镜检具有高度特异性,但敏感性较低。高敏感性的PCR方法不仅可以用于PCP筛查,还可用于疗效监测。     

肺孢子菌肺炎的临床诊断分析

目的探讨肺孢子菌肺炎的临床诊断方法。方法2010年2月~2013年3月,我院呼吸科有不同程度肺孢子菌肺炎临床症状患者20例,均先行胸部 CT检查,通过影像学显示,拟诊断肺孢子菌肺炎患者19例;行染色镜检法拟诊断肺孢子菌肺炎患者13例,6例行肺组织活体穿刺,综合临床症状和体征进行诊断。结果综合拟诊断20例患者均为肺孢子菌肺炎患者,诊断率为100%,其中胸部CT检查检出率为85%（17/20）;染色镜检查法检出率率为65%（13/20）;肺组织活体穿刺检出率100%（6/6）。结论综合应用多种诊断方法能有效地提高肺孢子菌肺炎的诊断率,提高疾病诊治水平。     

黄芪注射液穴位注射足三里治疗肺孢子菌肺炎的临床研究

目的：通过探讨应用黄芪注射液穴位注射足三里治疗肺孢子菌肺炎的临床疗效，旨在为提高治疗效率和患者生活质量提供理论依据。方法选择2012年1月~2014年5月在我院接受治疗的获得性免疫缺陷综合征合并肺孢子菌肺炎患者30例，随机平均分成研究组和对照组，对照组给予患者口服复方磺胺甲噁唑，研究组在对照组患者治疗的基础上用黄芪注射液穴位注射足三里，对比两组患者治疗前后CD4+T淋巴细胞、肺泡灌洗测肺孢子菌包囊数的变化情况、肺部病灶影像学的吸收情况及治疗有效率。结果治疗后研究组患者CD4+T淋巴细胞为219.34个/μl、肺孢子菌包囊数为1.81个、肺部病灶吸收率为86.67%、治疗有效率为80.0%，与对照组比较差异显著，且<0.05差异有统计学意义。结论黄芪注射液穴位注射足三里能显著提高CD4+T淋巴细胞数和病灶吸收率，减少肺孢子菌包囊数，临床疗效显著。     

艾滋病与非艾滋病病例合并卡氏肺孢子菌肺炎的临床特征对比研究

目的:比较艾滋病相关性卡氏肺孢子菌肺炎与非艾滋病相关性卡氏肺孢子菌肺炎的临床特征及辅助检查,以提高临床医师诊疗水平。方法:收集2018年1月至2021年7月在某三甲医院确诊的卡氏肺孢子菌肺炎17例,按照不同基础疾病将病例分为艾滋病组（7例）与非艾滋病组（10例）,比较两组病例的人口学特征、临床表现、实验室检查、病原学以...     

瑞香素联合母牛分支菌治疗大鼠卡氏肺孢子菌肺炎的实验研究

目的探讨瑞香素和母牛分支菌联合对大鼠卡氏肺孢子菌肺炎(PcP)的治疗作用。方法地塞米松腹股沟皮下注射SD大鼠8周,建立PcP大鼠模型,将其随机分为模型对照组,瑞香素组,母牛分支菌组,瑞香素与母牛分支菌联合用药组,并设立正常对照组。观察肺病理切片、肺印片中每视野肺孢子菌包囊均数、脾细胞增殖能力及血清INF-γ水平变化。结果治疗组肺孢子菌包囊数较模型组明显减少,肺组织损伤较模型组减轻或修复,脾细胞增殖能力及血清INF-γ水平治疗组较模型组有不同程度的提高,其中联合用药组较单用组效果显著。结论瑞香素和母牛分支菌联合用药治疗卡氏肺孢子菌肺炎大鼠较单用疗效显著。     

医学微孢子茵研究概况

微孢子菌病是一种新发现的机会性感染，常见于AIDS感染者、器官移植患者、儿童、旅行者、角膜接触镜佩戴者及老年人，可表现出广泛的临床综合征。本文对医学微孢子菌生物学和种属特征等方面的研究概况进行综述。     

免疫组化和GMS染色对AIDS合并肺孢子菌肺炎的临床诊断价值

目的 探讨免疫组化及六胺银(gomori's methenamine silver nitrate stain,GMS)染色法对AIDS合并肺孢子菌肺炎(pneumocystis pneumonia,PCP)的临床诊断价值.方法 选择50例临床确诊为合并PCP的AIDS患者作为实验组(PCP组)、25例非PCP的呼吸道感染患者作为对照组,分别采用单克隆抗体免疫组化法和GMS染色法对两组患者的支纤镜肺活检标本进行肺孢子菌(pneumocystis jiroveri,Pj)检测,并比较两种方法的敏感性和特异性.结果 免疫组化法检测Pj的敏感性为66%(33/50),特异性为100%(25/25);GMS染色法检测Pj的敏感性为42%(21/50),特异性为92%(23/25).结论 免疫组化法检测Pj的敏感性和特异性均高于GMS染色法,这将有助于提高Pj的检出率及PCP的确诊率.     

实时荧光RT-PCR检测活性单核细胞增生李斯特菌方法的建立

目的 采用逆转录结合实时荧光定量PCR技术,建立一种快速、准确、特异甄别单核细胞增生李斯特菌(Listeria monocytogenes,简称单增李氏菌)死活状态的定量方法.方法 根据单增李氏菌hlyA基因序列设计引物和探针;对实时荧光PCR反应体系进行优化后,提取菌体mRNA,通过随机引物进行逆转录反应;产生的cDNA通过实时荧光定量PCR进行鉴定.进一步评价逆转录结合实时荧光定量PCR方法的特异性、灵敏度、重复性后,对20份模拟双盲样本进行检测.结果 本实验所建立的逆转录结合实时荧光定量PCR方法可准确、特异地检测单增李氏菌,其他菌株和失活的单增李氏菌均无阳性结果出现;该方法检测纯菌和模拟样本的灵敏度分别可达到10 CFU/ml和1000CFU/ml;定量检测的批间和批内的变异系数均小于5%;对20份模拟样本进行检测,其中10份含有活性单增李氏菌样本的检测结果均为阳性,其他含有失活单增李氏菌的5份样本和其他致病菌的5份样本检测结果为阴性.结论 本文建立的检测活性单增李氏菌实时荧光定量PCR方法快速、准确,结果可靠,实用性强,可进行定量分析,为食品安全监测和现场流行病学调查提供较好的分析手段和完整的数据.     

肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

不同免疫状态小鼠烟曲霉感染后肺组织dectin-1 mRNA表达

目的 研究不同免疫状态小鼠感染烟曲霉后肺组织dectin-1 mRNA表达变化.探讨免疫抑制剂对dectin-1表达的影响与疾病发展的相互关系.方法 小鼠分4组:正常对照组、免疫抑制组、免疫抑制种菌组及免疫正常种菌组.采用实时荧光定量PCR法检测各组小鼠不同时间点肺组织dectin-1 mRNA表达.同时进行肺组织真菌载量测定观察疾病发展情况.结果 小鼠感染烟曲霉后第3天,免疫抑制种菌组菌载量明显高于正常种菌组.在感染后第1天及第3天,免疫抑制组小鼠肺组织dectin-1表达较正常对照组明显降低;第3天,免疫正常种菌组表达较正常对照组及免疫抑制种菌组明显升高(P<0.05).结论 对于免疫正常宿主,dectin-1可能在防御烟曲霉感染中起重要作用.环磷酰胺可降低肺组织dectin-1 mRNA表达,可能与其促发侵袭性肺曲霉病有关.     

实验感染大、小鼠和临床疑似病例肺孢子虫mt LSU rRNA基因检测

为探讨PCR技术对实验感染大、小鼠肺孢子虫肺炎(PCP)和临床疑似病例诊断的可行性。用皮下注射地塞米松法诱导SD大鼠和ICR小鼠PCP并收集临床疑似病例24h深部痰液。受试动物解剖后,制备肺印片,经瑞姬氏复合染色镜检确定肺孢子虫(Pc)感染的阳性率;同时,以针对虫体mtLSUrRNA基因设计的引物,PCR扩增鼠肺组织和支气管灌洗液(BALF),以及痰液样本中的DNA;比较实验动物样本镜检和PCR两种方法的检测结果;测定PCR的敏感性和特异性。结果表明,SD大鼠和ICR小鼠肺印片的阳性率分别为68.8%(1116)和85.7%(1821),肺组织PCR检测的阳性率分别为75%(1216)和80.9%(1721),肺泡灌洗液PCR的阳性率分别为81.2%(1316)和23.8%(521)。肺印片镜检和PCR技术检测结果无显著性差别;可见,PCR对虫体mtLSUrRNA基因的检测具有显著的特异性和敏感性,至少可以检测出0.6pg的肺孢子虫DNA;6例临床疑似病例样本中2例显示肺孢子虫PCR阳性反应。     

系统性红斑狼疮并发肺孢子虫肺炎的临床病例报告

目的探讨系统性红斑狼疮并发肺孢子虫肺炎（PCP）的诊断和防治措施。方法研究1例系统性红斑狼疮并发肺孢子虫肺炎患者的临床表现、实验室检查结果、影像学资料以及诊断和治疗的方法。结果患者出现发热、干咳、无痰、胸闷、伴阵发性呼吸困难等症状,血氧饱和度下降,胸部CT显示两肺部毛玻璃状改变,肺泡灌洗液找到肺孢子虫。给予复方新诺明、卡泊芬净、调整免疫抑制剂及其它支持对症治疗,痊愈出院。结论免疫功能抑制患者有发热、干咳、呼吸困难等临床症状,应考虑PCP并及时做病原学检查。     

Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia

Objective   To study the clinical significance of a nested polymerase chain reaction (PCR) method compared to immunofluorescence (IF) for detection of Pneumocystis carinii .
Methods   The medical records of 89 patients with 91 episodes of pneumonia were scrutinised retrospectively. The pneumonia episodes were divided into categories according to the likelihood that the patient had had clinical Pneumocystis carinii pneumonia (PCP). All respiratory tract samples from the 89 patients (34 broncho-alveolar lavage (BAL) and 57 sputa) were tested for Pneumocystis carinii by IF and nested PCR.
Results   Fifteen episodes, as diagnosed by IF, were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 60%, specificity 97%). Among the P. carinii DNA-positive episodes, detected with nested PCR, 24 were classified as true PCP (combination of the groups with definite and probable PCP; sensitivity 96%, specificity 59%), since all IF-positive samples were nested PCR positive. Only one pneumonia episode classified as a probable PCP, was negative with both methods, as applied to a BAL sample.
Conclusions   IF applied to BAL or sputum seems to be the most specific method for diagnosis of clinical PCP. Additional clinical cases can be found by nested PCR, although this then gives a high risk of detecting subclinical colonisation of P. carinii .     

Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia

Pneumonia due to Pneumocystis jirovecii (PCP) is a frequent infection among HIV-positive or other immunocompromised patients. In the past several years, PCR on pulmonary samples has become an essential element for the laboratory diagnosis of PCP. Nevertheless, very few comparative studies of available PCR assays have been published. In this work, we evaluated the concordance between four real-time PCR assays, including three commercial kits, AmpliSens, MycAssay, and Bio-Evolution PCR, and an in-house PCR (J. Fillaux et al. 2008, J Microbiol Methods 75:258–261, doi:http://dx.doi.org/10.1016/j.mimet.2008.06.009), on 148 pulmonary samples. The results showed concordance rates ranging from 81.6% to 96.6% (kappa, 0.64 to 0.93). Concordance was excellent between three assays: the in-house assay, AmpliSens, and the MycAssay PCR (kappa, >0.8). The performances of these PCR assays were also evaluated according to the classification of the probability of PCP (proven, probable, possible, or no final diagnosis of PCP) based on clinical and radiological signs as well as on the direct examination of bronchoalveolar lavage samples. In the proven PCP category, Pneumocystis jirovecii DNA was detected with all four assays. In the probable PCP category, the in-house PCR, AmpliSens, and the MycAssay PCR were positive for all samples, while the Bio-Evolution PCR failed to detect Pneumocystis jirovecii DNA in two samples. In the possible PCP category, the percentage of positive samples according to PCR varied from 54.5% to 86.4%. Detection of colonized patients is discussed. Finally, among the four evaluated PCR assays, one was not suitable for colonization detection but showed good performance in the proven and probable PCP groups. For the three other assays, performances were excellent and allowed detection of a very low fungal burden.     

Pneumocystis carinii in FNA of the thyroid.

We report the diagnosis of Pneumocystis carinii (PC) in a fine-needle aspirate (FNA) from the thyroid of a human immunodeficiency virus infected (HIV+) male receiving aerosolized pentamidine as prophylaxis for Pneumocystis carinii pneumonia (PCP). The clinical diagnosis prior to FNA was multinodular goiter. The patient did not have pulmonary symptoms nor previous diagnosis of PCP at the time of the aspirate diagnosis. Recently, extrapulmonary Pneumocystis carinii (EPC) has been reported with increasing frequency in HIV+ patients receiving prophylactic aerosolized pentamidine. Awareness of extrapulmonary presentations of Pneumocystis carinii infection is a prerequisite for accurate cytologic diagnosis.     

mtLSU-巢式PCR检测实验大鼠卡氏肺孢子虫的实验研究

目的对mtLSU-巢式PCR方法检测大鼠卡氏肺孢子虫的应用价值以及基因序列进行评价。方法采用地塞米松免疫抑制法诱导大鼠感染肺孢子虫;实验组10只,对照组1只;诱导至第7周时收集实验组及对照组大鼠肺组织和支气管肺泡灌洗液（BALF）标本,采用mtLSU-巢式PCR方法对人源与鼠源肺孢子虫共有的基因进行扩增和序列测定,同时采用镜检法对实验组大鼠肺组织和肺泡灌洗液标本进行检测,评估两种方法的敏感性。结果采用mtLSU-巢式PCR方法对实验感染大鼠肺组织和BAL进行检测,卡氏肺孢子虫DNA阳性率分别为100%（10/10）、90%（9/10）。而GMS染色镜检法检测的阳性率分别为80%（8/10）、60%（6/10）。所测Wistar大鼠卡氏肺孢子虫mtLSU基因序列长度为155bp,与GenBank的大鼠源肺孢子虫（U20170）及人源肺孢子虫（DQ473446）同源性均为100%（154/154、155/155）。结论 mtLSU-巢式PCR方法应用于大鼠卡氏肺孢子虫检测敏感性高,特异性强;获得与人源耶氏肺孢子虫相同的Wistar大鼠卡氏肺孢子虫mtLSU的基因序列。     

A highly sensitive novel PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveloar lavage specimens from immunocompromised patients

Pneumocystis jirovecii pneumonia (PCP) is a leading cause of morbidity and mortality in immunocompromised patients. Despite the sensitivity of the commonly used PCR for diagnosing P. jirovecii with primers pAZ102-H/pAZ102-E and pAZ102-X/pAZ102-Y derived from mtLSU rRNA (conventional PCR), some PCP patients who had demonstrable organisms by staining methods failed to give positive PCR results. Herein, we devised a more sensitive PCR assay derived from the same gene target to circumvent these false-negative tests. Single brochoalveolar lavage (BAL) samples were collected from human immunodeficiency virus (HIV)-infected (n = 66) and non-HIV (n = 36) immunocompromised patients presenting with fever, dyspnoea, cough and pulmonary infiltrates. Pneumocystis jirovecii was diagnosed with Giemsa-stained smear, immunofluorescence assay, conventional single-round and nested PCR, and new single-round and nested PCR in 46 (45.1%), 53 (52.0%), 69 (67.6%), 74 (72.6%), 87 (85.3%) and 91 (89.2%) patients, respectively. The new PCR could detect P. jirovecii DNA in BAL fluids two to three orders of magnitude more dilute than conventional PCR. Sequence analysis revealed one to three nucleotide substitutions within the primers for conventional PCR among clinical isolates. Although both conventional and new PCR assays were highly specific for diagnosing P. jirovecii, the new PCR yielded more positive results than conventional PCR among BAL samples that were negative by both Giemsa stain and immunofluorescence assay. Hence, the new PCR offered a more sensitive detection of P. jirovecii infection and colonization than conventional PCR.     

Multicenter, prospective clinical evaluation of respiratory samples from subjects at risk for Pneumocystis jirovecii infection by use of a commercial real-time PCR assay

Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations. Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, ≤ 3.5 copies/μl of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.     

Usefulness of PCR for diagnosis of Pneumocystis carinii pneumonia in different patient groups.

Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.