肺孢子虫病实验与临床研究 Ⅱ．免疫印迹试验检测人血清肺孢子虫抗体

采用ＳＤＳ－ＰＡＧＥ分析鼠源卡氏肺孢子虫包囊可溶性抗原呈现２０条以上多肽区带，主带分子量分别为＞１００、８５－９４、６７、５２和３４ｋＤａ。ＥＩＴＢ检测表明重庆地区健康人血清中存在识别１１０、１０５、８５、６７、５２和４６ｋＤａ的ＩｇＧ型抗体，抗体阳性率为５６．７％（５９／１０４）。其中８５ｋＤａ抗原能与抗人源肺孢子虫单克隆抗体２Ｇ２起反应。健康人血清抗８５ｋＤａ抗原的抗体阳性率为３７．５％（３９／１０４），但ＩｇＭ型抗体均为阴性。另外检测７例确诊的卡氏肺孢子虫病患者，４例血清ＩｇＧ型抗体阳性，其中３例抗８５ｋＤａＩｇＧ型抗体阳性。     

Occurrence of Pneumocystis carinii in HIV-positive patients with suspected pulmonary tuberculosis in Ethiopia

OBJECTIVE: To investigate the prevalence of Pneumocystis carinii in consecutive HIV-positive patients with suspected pulmonary tuberculosis (PTB) attending a university hospital in Ethiopia. METHODS: A PCR for P. carinii and an indirect immunoflorescence (IF) assay were performed on expectorated sputum samples from: 119 HIV-1-positive patients with negative smears and sputum cultures for Mycobacterium tuberculosis; 96 HIV-1-positive patients with culture-verified PTB; and 97 HIV-negative patients with negative mycobacterial cultures and 72 HIV-negative patients with culture-verified PTB, serving as controls. Outcome of PCR and IF were compared with the chest radiographic (CXR) and initial clinical diagnosis. RESULTS: In the HIV+PTB- group, P. carinii was found in 10.9% by IF, 8.4% by single PCR (sPCR) and 30.3% by nested PCR (nPCR). In the HIV+PTB+ group, 3.1% were P. carinii positive by IF and sPCR and 13.5% by nPCR. All IF- and sPCR-positive samples were nPCR positive. In the HIV-PTB+ and HIV-PTB- groups, 4.2% and 3.1% were nPCR positive, respectively. Six out of eight HIV+PTB- patients with CXR suggesting P. carinii pneumonia (PCP) were IF and/or nPCR positive for P. carinii. In the IF-positive and nested PCR-positive HIV+PTB- patients more than one-third were interpreted as PTB by CXR whereas only one patient was diagnosed with clinical PCP. CONCLUSIONS: P. carinii is prevalent in HIV-positive PTB suspects, suggesting that PCP may be an important, but not well recognized, differential diagnosis. Our findings have implications for treatment and primary prophylaxis for PCP in Ethiopia.     

Humoral and cellular responses to Pneumocystis carinii, CMV, and herpes simplex in patients with AIDS and in controls

The titers of IgG and IgA to Pneumocystis carinii in 36 AIDS patients did not differ significantly from those in 31 controls. Only 2/15 patients (13%) with P. carinii pneumonia (PCP) had titers of IgM antibodies greater than or equal to 5, which is significantly less frequent than in 32 controls (62%) and in 21 AIDS patients without PCP (43%). The risk of PCP was 5 times higher in patients without IgM antibodies to P. carinii than in patients who had these antibodies. A significantly higher percentage of those without PCP (57%) showed increasing titers of IgM antibodies to P. carinii in the second of paired samples taken about 6 months apart, compared with whose with PCP (9%; p = 0.05). All patients had high titers of antibodies to CMV and HSV and normal total concentrations of immunoglobulins. None of the patients responded in lymphocyte transformation to P. carinii, CMV, or HSV antigens. There is no obvious explanation to the selective lack of IgM antibodies to P. carinii in patients with PCP. Lack of IgM antibodies may be a marker for an immunodeficiency to P. carinii.     

A longitudinal study of human cytomegalovirus serology and viruria fails to detect active viral infection in 20 systemic lupus erythematosus patients

In this study, we investigated whether active human cytomegalovirus infection could be detected in 20 systemic lupus erythematosus (SLE) patients over a one-year observation period by polymerase chain reaction on serial urine specimens and by monitoring of IgG and IgM HCMV-specific antibody profiles in serial serum samples. Of 788 urine samples analysed for the presence of human cytomegalovirus DNA, only 2 specimens (0.25%) collected from two different patients contained genuine human cytomegalovirus sequences as determined by polymerase chain reaction and subsequent sequencing of the PCR products. These two patients had one positive sample out of 36 samples or 40 samples, respectively. Nineteen of the patients (95%) possessed IgG antibodies against human cytomegalovirus, while 9 (45%) produced IgM antibodies. However, none of the patients showed signs of an active virus infection as judged by the stable anti-HCMV IgG or IgM antibody levels during the observation period, nor was any correlation between disease activity and HCMV serology/viruria observed. Of single serum samples of 26 age- and sex-matched blood donors, 21 (81%) were HCMV IgG positive and 1 (3.8%) was IgM seropositive. In conclusion, our data fail to establish an active human cytomegalovirus infection in SLE patients.     

Detection of Pneumocystis carinii by DNA amplification in patients with connective tissue diseases: re-evaluation of clinical features of P. carinii pneumonia in rheumatic diseases

OBJECTIVES: We evaluated the polymerase chain reaction (PCR) detection of Pneumocystis carinii DNA in induced sputum of patients with connective tissue diseases and assessed the clinical features of patients positive for P. carinii. METHODS: Sputum was induced by nebulization in 29 in-patients with various connective tissue diseases who presented with symptoms suggestive of P. carinii pneumonia (PCP), and was examined by PCR. RESULTS: Detection of P. carinii DNA by PCR was significantly more sensitive than cytology; 54.5% patients were positive by PCR and only 4.5% by cytology. The prevalence of PCP was higher than previously considered and was especially high in patients receiving > 30 mg/day prednisolone with or without other immunosuppressants. P. carinii-positive patients had significant lymphocytopenia and a low serum IgG level compared with P. carinii-negative patients. P. carinii disappeared within 7-10 days after therapy with trimethoprim/sulfamethoxazole. CONCLUSION: We propose that the use of PCR for detection of P. carinii using induced sputum is a useful and non-invasive method that has high sensitivity and specificity for the early diagnosis of PCP.     

聚合酶链反应方法检测诱导排痰标本对卡氏肺孢子虫肺炎的诊断价值

目的 评价聚合酶链反应方法检测诱导排痰标本中卡氏肺孢子虫DNA对卡氏肺孢子虫肺炎（PCP）的诊断意义。方法 分别用姬姆萨和六胺银（GMS）两种染色方法和mt-rRNA-PCR方法检测痰液中的卡氏肺孢子虫。结果 化学染色法检测16例临床拟诊PCP的患者痰标本。结果 8例阳性,20例非PCP患者痰标本均为阴性,化学染色方法的敏感性和特异性分别为50％和100％。PCR方法检测16例临床拟诊PCP患者痰液中卡氏肺孢子虫,14例阳性,20例非PCP患者痰标本均为阴性,mt-rRNA-PCR方法的敏感性和特异性分别为88％和100％。结论 姬姆萨和GMS两种细胞化学染色方法联合检测痰标本卡氏肺孢子虫,特异性高,但敏感性偏低。mt-rRNA-PCR检测痰标本中卡氏肺孢子虫DNA方法敏感性高于化学染色法且特异性高,更适于临床应用。     

用半巢式PCR检测无创性标本诊断卡氏肺孢子虫肺炎的实验研究

目的无创性诊断卡氏肺施子虫肺炎。方法采用一对半引物进行半巢式DHFR－PCR检测实验大鼠无创性标本——支气管液、血清及活检标本——肺及肝脏组织中的卡氏肺孢子虫DNA。结果从59只经病理学检查证实的卡氏肺孢子虫肺炎大鼠中采集到的上述4种标本，卡氏肺孢子虫DNA的半巢式DHFR－PCR检出率分别为72.7％（32／44），37.3％（22／59），94．9％（56／59）和36．4％（8／22），而DHFR－PCR的检出率则仅为25％，13．6％，71．2％和9．1％。12只轻度感染的PCP大鼠的无创性标本中卡氏肺孢子虫DNA的检出率由刚提高至41．7％。正常鼠标本及各种病原体对照的半巢式DHFR－PCR均为阴性。结论用半巢式DHFR－PCR无创性地诊断大鼠卡氏肺孢子虫肺炎具有敏感、特异、省时、省力等优点；本文在血和肝中检出卡氏肺孢子虫DNA，提示在卡氏肺孢子虫肺炎大鼠模型中存在虫血症和肺外卡氏肺孢子虫感染。     

弓形虫既往感染在孕期内对胎儿的影响

目的探讨既往感染者孕期内弓形虫的活动情况及对胎儿的影响。方法对68例抗弓形虫抗体IgG阳性、IgM阴性孕妇的血清、脐血采用酶联免疫吸附试验检测弓形虫抗体IgG、IgM、弓形虫循环抗原（CAg）,PCR法检测虫体DNA;胎盘样本采用直接涂片、匀浆涂片及PCR法观察弓形虫感染情况。结果68例弓形虫抗体IgG阳性孕妇中脐血弓形虫抗体IgG阳性28例,IgG胎盘垂直传播率41.2%;脐血弓形虫DNA阳性6例,宫内感染发生率8.8%;胎盘组织中虫体DNA阳性9例,宫内感染发生率13.2%。结论既往弓形虫感染孕期内仍可导致垂直传播。     

Search for primary infection by Pneumocystis carinii in a cohort of normal, healthy infants.

To determine whether Pneumocystis carinii is associated with clinical illness in the competent host, 107 normal, healthy infants were enrolled in a 2-year prospective cohort study in Chile. P. carinii was identified by specific stains and nested--deoxyribonucleic acid (DNA) amplification of the large subunit mitochondrial ribosomal ribonucleic acid gene of P. carinii f. sp. hominis, and seroconversion was assessed by enzyme-linked immunosorbent assay of serum samples drawn every 2 months. P. carinii DNA was identified in nasopharyngeal aspirates obtained during episodes of mild respiratory infection in 24 (32%) of 74 infants from whom specimens were available for testing. Three (12.5%) of those 24 infants versus 0 of 50 infants who tested negative for P. carinii had apnea episodes. Seroconversion developed in 67 (85%) of 79 infants who remained in the study by 20 months of age and occurred in the absence of any symptoms of disease in 14 (20.8%). The study indicates that P. carinii DNA can be frequently detected in healthy infants, and it raises the hypothesis that they may be an infectious reservoir of P. carinii in the community. Further investigation is needed to identify whether P. carinii causes overt respiratory disease in infants.     

Reduced concentrations of IgG antibodies to Pneumocystis carinii in HIV-infected patients during active Pneumocystis carinii infection and the possibility of passive immunisation

The relationship between Pneumocystis carinii antibody concentrations and acute Pneumocystis infection was investigated by testing sequential samples of serum from HIV antibody-positive patients with respiratory symptoms and HIV-negative immunocompromised patients by means of an indirect immunofluorescence assay for specific IgG antibodies to P. carinii. Loss of circulating antibody at the time of active Pneumocystis infection was observed in five patients with proven infection. Three others showed recovery of antibody coinciding with treatment and clinical recovery from infection. Concentrations of specific IgG antibody against P. carinii were measured in 40 blood donors and in six different batches of an intravenous immunoglobulin (IV Ig) preparation. Titres greater than 128 were found in the IV Ig batches examined. The use of IV Ig, either alone or in conjunction with other therapeutic agents, should therefore be considered in patients suffering from acute infection with P. carinii.     

Serology and serum DNA detection in shingles

AIM: To investigate the sensitivity of various laboratory approaches in the diagnosis of herpes zoster from patient serum. METHODS: Paired sera from 53 consecutive adult patients with acute herpes zoster were tested for the presence of varicella-zoster virus (VZV) antibodies. All acute sera were tested subsequently by real-time polymerase chain reaction (PCR) for the presence of VZV DNA. In addition, convalescent sera of patients who tested initially positive for VZV DNA underwent PCR analysis. RESULTS: VZV IgM antibodies were found by enzyme immunoassay (EIA) in 5 acute (9%) and 20 convalescent (38%) zoster sera. VZV DNA was detected by PCR in 21 (40%) acute zoster sera and was no longer detectable in the convalescent samples. A seroconversion or a fourfold or greater titre increase was found by complement fixation (CF) test in 41 (77%), by IgG indirect fluorescent antibody assay (IgG IFA) in 43 (81%) and by CF and IgG IFA combined in 45 of 53 (85%) paired zoster sera. The combination of all serological methods detected 51 (96%) and PCR combined with serology identified 52 (98%) of 53 patients. CONCLUSIONS: Optimal laboratory sensitivity in the diagnosis of herpes zoster from serum can be achieved by the combination of PCR and serology of paired serum samples. Serological methods alone are of limited value for early diagnosis of zoster when therapy can be initiated, because CF and IgG IFA need convalescent serum and IgM test sensitivity is insufficient. Early diagnosis of VZV reactivation is possible from serum by PCR in the first days of illness and test sensitivity needs further improvement. The findings highlight the need for future studies into the usefulness of PCR and serology in atypical cases of VZV reactivation.     

PCR检测大鼠卡氏肺孢子虫的研究

目的探讨PCR技术检测大鼠卡氏肺孢子虫的应用价值。方法SD大鼠和Wistar大鼠均随机分成实验组和对照组,实验组每周两次皮下注射醋酸可的松,诱导产生卡氏肺孢子虫;8week后,收集肺组织和支气管肺泡灌洗液(BALF),用PCR技术检测卡氏肺孢子虫DNA,并与Giemsa染色法比较。结果实验组两种大鼠肺组织卡氏肺孢子虫DNA阳性率分别为9643%和100%,BALF阳性率亦分别为9643%和100%,它们之间均无显著性差异(P>005);BALF的PCR阳性检出率显著高于Giemsa病原染色法,肺组织的两种方法检出率无显著性差异。结论PCR是一种检出率较高的方法,可作为早期诊断PCP的常规方法,特别适用于BALF检测卡氏肺孢子虫DNA。     

云南西部边境地区健康人群登革热血清学调查

目的了解云南西部边境地区健康人群登革热抗体水平状况,为制定有效登革热防治对策提供依据。方法在具有登革热本地感染病例的不同地区采集健康人群血清,并采用间接酶联免疫吸附试验分别检测登革病毒IgG和IgM抗体。结果采集的740份中国籍血清标本登革病毒IgG和IgM抗体阳性率分别为10.9%（81/740）和5.7%（42/740）,两种抗体同为阳性的阳性率为0.81%（6/740）;缅甸籍学生这两种登革抗体阳性率分别为12.5%（13/104）和2.9%（3/104）。登革IgG/IgM抗体阳性率在不同年龄组、性别、民族、不同国籍学生中的分布无统计学差异（P〉0.05）;务农人群IgG抗体阳性率高于非农人群（P〈0.05）;盈江县IgG抗体阳性率较高。结论云南省边境地区人群广泛存在登革病毒的既往和新近隐性感染,建议加强对边境口岸和边境地区的登革热疫情监测,开展登革热防治知识的宣传和提高当地医疗机构的诊疗水平。     

An analysis of the course of experimental pneumocystosis in rats using some serological methods

Since serological methods used in the diagnosis of pneumocystosis may be helpful to a various extent, depending on the stage of a diagnosed infection, it was decided to evaluate the usefulness of some the methods in the course of infection. Wistar rats were used in the experiments. The animals were administered hydrocortisone injections for 12 weeks to induce immunosuppression and activate naturally occurring asymptomatic infections with Pneumocystis carinii. Blood samples and specimes of lung tissues were collected, then they were examined for the presence of specific IgG and IgM antibodies, circulating antigen of Pneumocystsis carinii and circulating immune complexes using immunoenzymatic assays. The results of the above experiments indicated, that in an early stage of infection, the examinations of serum samples for circulating immune complexes were helpful, particularly for these with IgM antibodies     

用PCR扩增技术检测卡氏肺孢子虫DNA

本文报道了用ＰＣＲ扩增技术检测卡氏肺孢子虫感染大鼠的肺组织，支气管灌洗液和外周血病原体ＤＮＡ，并和肺印片结果进行比较。共检测了实验感染鼠３４只及正常大鼠６只，肺印片阳性者１８例，ＰＣＲ检测肺组织样品出现特异的扩增条带者１９例，检测支气管灌洗液样品出现特异的扩增条带者１６例，外周血样品全部阴性。根据实验结果，认为引法用于检测卡氏肺孢子虫病原体ＤＮＡ有很好的应用前景。     

Serologic responses to Pneumocystis carinii antigens in health and disease

Serum antibodies to human Pneumocystis carinii antigens were measured in greater than 400 specimens from different population groups by the immunoblotting technique. Serologic responses varied during the first 2 years of life, but in children greater than or equal to 2 1/2 years and in adults antibodies to a 40-kDa band were found in greater than 85% of the specimens; antigens to bands of 66, 92, and 116 kDa were also detected frequently. The prevalence of serum antibodies in immunosuppressed patients varied at different institutions and was usually lower than that of healthy controls. Seven (41%) of 17 patients with single episodes of pneumocystosis and 13 (93%) of 14 patients with recurrent episodes followed sequentially developed active serum IgM and/or IgG antibody responses to the 40-kDa antigen. Serologic responses to P. carinii were also detected, though less frequently, by immunofluorescence. These data suggest that the 40-kDa antigen is a major marker of P. carinii infection and that immunoblotting is useful in measuring serum antibody responses to the organism in both normal and immunocompromised hosts.     

Diagnosis of Pneumocystis carinii pneumonia: immunofluorescence staining, simple PCR or nPCR.

OBJECTIVES: to compare immunofluorescence (IF) test, routinely used in the department for the detection of Pnemocystis carinni with simple polymerase chain reaction (PCR) and nested PCR (nPCR) METHODS: Bronchoalveolar lavage (BAL) and induced sputum (IS) specimens from HIV-positive (39), lung transplant ssart transplant (2), and one each from non-Hodgkin's lymphoma, drug addict and a premature baby were screened by IF test, simple PCR and nPCR for the presence of P.carinii. RESULTS: of the 46 specimens tested, two (4.3%) were positive by IF, 11 (23.9%) by simple PCR and 21 (45.6%) by nPCR. Both simple and nPCR amplified those found positive by IF test. Analysis of the clinical data revealed both IF positive, 10 of the simple PCR and 15 of the nPCR group were strongly suspected of P. carinii pneumonia (PCP). Two specimens, one from a patient where chest X-ray was suggestive of PCP and the other where post-mortem histology revealed the presence of PCP, were negative by IF test. CONCLUSION: simple PCR detection may be considered for patients where PCP is suggestive clinically and the specimen is negative by IF test.     

Early diagnosis of scrub typhus in Thailand from clinical specimens by nested polymerase chain reaction

The early detection of scrub typhus in Thailand by nested polymerase chain reaction (PCR) is presented. The diagnosis of scrub typhus, from clinical samples obtained from hospitals in the northern part of Thailand, by nested PCR was compared to immunofluorescence (IF) and Weil-Felix (WF) tests. The primer pairs used for the nested PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen, and RFLP analysis was used for identification. Clotted blood from 80 patients suspected of scrub typhus infection were tested. With the IF test, antibodies for Orientia tsutsugamushi were observed in 38 patients checking IgM and IgG titers. Only 21 patients showed positive seroconversion while 17 patients were negative. For the WF test, only 13 patients gave a positive seroconversion. In the early stage of infection, 19, 13 and 3 patients were detected with a sensitivity of 90.47% (19/21), 61.90% (13/21) and 14.28% (3/21) by the nested PCR, IF and WF test respectively. Two patients who were negative for seroconvesion by IF and WF were positive by nested PCR. Therefore, this suggests that nested PCR is applicable for specific rapid diagnosis at an early stage of scrub typhus in endemic regions.     

免疫感受态小鼠感染卡氏肺孢子虫的研究

目的;研究成年感受态CB17小鼠对卡氏肺孢子虫（Pneumocystis carinii,Pc)的敏感性,探讨其在Pc传播中的作用。方法：使用免疫机能正常的健康成年CB17小鼠,使其中一部分小鼠与Pc( )-SCID小鼠接触1周,另一部分持续接触至被处时为止,分别在第3、5、6周检查肺内Pc包囊或PcDNA。观察病原体和Pc DNA出现和持续时间。结果：接触传染源的动物,不论是接触1周,还是持续接触至被处死,肺内均查到Pc包囊或PcDNA。其中,3周时,PCR法查到PcDNA,第5周时,PCR法和病原学染色法均查到Pc,第6周时,虫体减少到可检出阈值以下。结论：免疫感受态宿主可通过接触方式感染Pc,接触传染源1周即可被感染,感染状态可持续5周以上。期间,这种病原携带状态的小鼠在Pc传播中存在潜在的危险。此状态宿主在流行病学中的意义有待于进一步研究证明。     

PCR-ELISA检测大鼠卡氏肺孢子虫DNA的研究

目的　建立卡氏肺孢子虫 (P.c )DNA的聚合酶链反应-酶联免疫吸附测定(PCR-ELISA)方法 ,并探讨其应用价值。　方法　实验组患卡氏肺孢子虫肺炎的SD大鼠和Wistar大鼠各 2 8只 ,采用PCR法扩增大鼠肺组织DNA和支气管肺泡灌洗液 (BALF)DNA ,用PCR ELISA检测其扩增产物。 2 8只患病大鼠分别制作肺组织印片及BALF涂片 ,姬姆萨 (Giemsa)染色镜检 10 0个视野中有无P .c包囊 (或滋养体 ) ,与PCR ELISA检测扩增产物结果比较。　结果　两种方法检测大鼠肺组织DNA阳性率及BALFDNA阳性率 ,结果相同 ,均分别为 96.4% (27/28)和100% (28/28)。Giemsa染色镜检P.c包囊 (或滋养体 ) ,结果为阳性的大鼠 ,PCR ELISA检测扩增产物结果也均为阳性。阴性对照组 ,两种大鼠的肺组织和BALF各10份标本 ,均有1只大鼠阳性。　结论　PCR ELISA检测大鼠卡氏肺孢子虫DNA ,敏感性较高 ,特异性较好 ,操作简便 ,具有实用价值。