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。     

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.     

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.     

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。     

Diagnostic approach to Pneumocystis carinii pneumonia in the setting of prophylactic aerosolized pentamidine

Recurrent Pneumocystis carinii pneumonia is common in patients with the acquired immunodeficiency syndrome who receive prophylaxis with aerosolized pentamidine. In this setting, the number of organisms is reduced and the clinical presentation may be altered. These observations have led to doubts regarding the use of induced sputum to diagnose PCP in patients receiving prophylactic AP. To determine if the examination of induced sputum is useful for patients receiving prophylactic AP, we examined our results over a 12-month period. We also examined several clinical criteria to ascertain if they could predict the likelihood of a positive induced sputum. As assessed by P(A-a)O2, need for admission and mortality, patients receiving AP presented with less severe disease than those not receiving AP. Twelve of 19 (63 percent) patients who developed PCP while receiving prophylactic AP were diagnosed by induced sputum. Induced sputum was positive for 35 of 55 (64 percent) patients who developed PCP and had not been receiving AP. However, there were no clinical characteristics which predicted a positive induced sputum. We conclude that induced sputum is an effective method for diagnosing PCP in patients receiving prophylactic AP.     

Role of multiple induced sputum examination in the diagnosis of Pneumocystis carinii pneumonia

We evaluated the usefulness of repeated nebulized saline induced sputum examinations among 60 HIV infected patients clinically suspected to have Pneumocystis carinii pneumonia (PCP). We found that the first sample was positive for 15 episodes (21.4%); the second sample was positive in 33 episodes (47.1%); the third sample was positive in 22 episodes (31.4%). Repeated nebulized saline induced sputum examination imporved the yield of Pneumocystis carinii and enhanced the sensitivity of a positive result. This technique is simple, cost-effective, non-invasive, and reliable. We recommend the examination of multiple induced samples of nebulized saline induced sputum in all HIV infected patients with suspected PCP. This recommendation may decrease the need for invasive procedures.     

The high burden of Pneumocystis carinii pneumonia in African HIV-1-infected children hospitalized for severe pneumonia.

OBJECTIVE: To evaluate the burden of Pneumocystis carinii pneumonia (PCP) and the usefulness of induced sputum and nasopharyngeal aspirates (NPA) in diagnosing PCP in African children in whom the use of bronchoalveolar lavage is unavailable. DESIGN: Children aged 2-24 months who were either known or suspected of being HIV-1 infected and who were hospitalized for severe pneumonia were investigated for P. carinii using induced sputum and NPA. P. carinii identification was performed using a direct monoclonal antibody immunofluorescent stain. A group of children who subsequently died also had lung biopsies performed. RESULTS: P. carinii cysts were identified in 51 out of 105 (48.6%) children either from induced sputum (37/105, 35.2%) or NPA (26/101, 25.7%) samples, or from both. Neither clinical nor laboratory tests were useful in distinguishing between HIV-1-infected children with and without PCP. Twenty-eight per cent (14/51) of HIV-1-infected children who developed PCP had a history of being on cotrimoxazole prophylaxis at the time of their illness. Mortality rates of HIV-1-infected children with and without PCP were equally high (27.5 and 27.8%, respectively). Histological evidence of PCP and cytomegalovirus pneumonia was observed on post-mortem lung biopsy in eight out of 18 (44.4%) children each. Using post-mortem lung histology as a reference, the sensitivity and specificity for induced sputum and NPA in diagnosing PCP were 75 and 80%, respectively. CONCLUSION: Strategies to reduce the high burden of PCP, which can successfully be diagnosed using NPA and induced sputum, in HIV-1-infected children hospitalized with severe pneumonia are urgently warranted in Africa.     

Rapid detection of dihydropteroate polymorphism in AIDS-related Pneumocystis carinii pneumonia by restriction fragment length polymorphism

Sulpha agents, which act by inhibiting the enzyme dihydropteroate synthase (DHPS), are used widely for the treatment and prophylaxis of Pneumocystis carinii pneumonia (PCP). Recently, we have shown that mutations in the dihydropteroate synthase (DHPS) gene of Pneumocystis carinii f.sp hominis are associated with failure of sulpha prophylaxis and increased mortality in HIV-1 positive patients with PCP, suggesting that DHPS mutations may cause sulpha resistance. To facilitate detection of DHPS mutations we developed a restriction fragment length polymorphism (RFLP) assay, detecting mutations at codon 55 and 57 of the P. carinii DHPS gene. The RFLP-assay was compared with direct DNA sequencing on 27 PCP isolates from HIV-1 positive patients with a mixture of wildtype and mutant DHPS types. In all samples the RFLP-assay correctly identified wildtype or DHPS mutation at codon 55 or 57. Combined with DNA extraction by a Chelex-based method, this method can be performed within 1 d and allows a fast, cost-efficient and reliable method of detection of DHPS mutations in P. carinii.     

The use of oral washes to diagnose Pneumocystis carinii pneumonia: a blinded prospective study using a polymerase chain reaction-based detection system.

Pneumocystis carinii pneumonia (PCP) can be diagnosed by direct microscopic examination of induced sputum or by bronchoalveolar lavage (BAL). However, many institutions have little diagnostic success with induced sputum, and BAL is invasive and expensive. This prospective, blinded study assessed oral washes as a more convenient specimen than either sputum or BAL fluid and used a dissociation-enhanced lanthanide fluoroimmunoassay time-resolved fluorescent hybridization polymerase chain reaction (PCR) detection system that is feasible for clinical laboratories. The study assessed 175 oral washes, each paired with either an induced sputum that was positive for Pneumocystis or a BAL sample. The PCR test based on the Pneumocystis major surface glycoprotein primers had a sensitivity of 91% and a specificity of 94%, compared with a test based on mitochondrial large subunit rRNA primers, which had a sensitivity of 75% and a specificity of 96%. These results suggest that oral washes can provide a useful sample for diagnosis of PCP when a sensitive PCR detection system is used.     

Emerging importance of Pneumocystis carinii among indian immunosuppressed patients

Pneumocystis carinii is known to cause both significant morbidity and mortality in immunosuppressed individuals. In a six and a half year period starting with 1992, a total of 204 samples including bronchial aspirates, induced sputum and suction catheter tips were examined for P. carinii by direct microscopic examination of Giemsa's and toluidine 'O' stained smears. At a later stage of the investigation, immunofluorescent staining using monoclonal reagent (Meriflour, USA) was also used for examination of the specimens. In all 24 (11.8%) of 204 samples were positive for P. carinii. In addition, Pneumocystis carinii pneumonia (PCP) was diagnosed in five (33%) of 15 patients of acquired immunodeficiency syndrome (AIDS) with opportunistic infections. All these 15 patients had CD4 T cell counts of less than 500 T lymphocyte equivalent (TLE) /ml as measured by a Trax ELISA assay. Laboratories in India have to be better equipped for an early and correct diagnosis of PCP that is bound to rise with the increase in the number and variety of immunosuppressed patients.     

Rapid and sensitive diagnosis of cytomegalovirus and Pneumocystis carinii pneumonia in patients with haematological neoplasia by using capillary polymerase chain reaction

We attempted the simultaneous detection of cytomegalovirus DNA (CMV-DNA) and Pneumocystis carinii ( carinii -DNA) in sputum samples obtained from 20 patients with haematological neoplasm with pneumonia, using rapid cycle DNA amplification (capillary PCR). We used a thermal cycler for capillary PCR which featured recirculation of hot air for rapid temperature control of 10 μl reaction samples in thin glass capillary tubes.
We extracted DNA from patients' sputa using a simple method. A comparison of the results obtained using the phenol extraction–ethanol precipitation method and those obtained using our simple method was made, and demonstrated complete agreement between the two. For detection of CMV-DNA and P. carinii -DNA with capillary PCR it was not necessary to vary temperature setting based on the primers used. Therefore, capillary PCR was used for the simultaneous detection of CMV and P. carinii.
After amplification, the total time required for which was 20 min, amplified products were electrophoresed on agarose gels and visualized with ethidium bromide. Product sensitivity was higher with capillary PCR than with conventional PCR.
We conclude that capillary PCR amplification is a valuable tool for rapid and simple diagnosis of CMV and P. carinii pneumonias.     

Analysis of induced sputum in the diagnosis of Pneumocystis carinii pneumonia

Sixty-two patients with possible AIDS-associated Pneumocystis carinii pneumonia (PCP) were studied to determine the diagnostic usefulness of sputum analysis and whether or not the results of sputum analysis are related to severity of disease. Induced sputum was stained with Gomori Methenamine silver and modified Wright Giemsa stains. Indicators of disease severity were: extent of chest roentgenographic infiltrate, serum lactic dehydrogenase activity, alveolar-arterial oxygen tension difference, and total blood lymphocyte count. All patients with sputum negative for Pneumocystis underwent bronchoscopy with bronchoalveolar lavage. Sputum analysis was 71% sensitive and 100% specific for the diagnosis of PCP. The negative predictive value of sputum analysis was 48%. There was no relationship between sputum results and the severity of PCP. This study led to the conclusions that sputum analysis is a sensitive, specific, rapid, and low-cost technique for the diagnosis of PCP, and that the sensitivity of sputum analysis for the detection of PCP is not affected by the severity of PCP.     

妊娠期大鼠对卡氏肺孢子菌的易感性研究

目的研究妊娠期大鼠对卡氏肺孢子菌（Pneumocystiscarinii,PC）的易感性,为妇女孕期保健及优生优育提供参考。方法采用皮下注射地塞米松的方法建立大鼠肺孢子菌肺炎（pneumocystispneumonia,PCP）模型,作为环境中Pc的感染来源。于实验的第6周处死2只PCP大鼠,取肺组织印片,用改良四胺银（GMS）染色,镜检Pc的感染情况。妊娠组、非妊娠组大鼠各取10只,于实验的第6周末将鼠笼置于PCP模型大鼠的鼠笼两侧,实验第7、8、9周分别取3只、3只和4只妊娠组及非妊娠组大鼠,乙醚麻醉下处死并取肺组织,提取DNA,PCR扩增Pc的mtI。SUrRNA。结果PCP模型组大鼠肺印片GMS染色后油镜下观察,可见大量Pc包囊,证明PCP模型建立成功。PCR扩增妊娠组8只大鼠Pc阳性,感染率为80．00％（8／10）;非妊娠组6只大鼠Pc阳性,感染率60．O¨0％（6／10）。经Fisher确切概率法检验,两组大鼠的Pc感染率差异无统计学意义（P〉0．05）。结论健康妊娠期大鼠对外源性Pc普遍易感,但感染率与非妊娠女鼠无明显善别．     

(1-->3)-beta-D glucan is a diagnostic and negative prognostic marker for Pneumocystis carinii pneumonia in patients with connective tissue disease

OBJECTIVE: To assess risk factors for P. carinii pneumonia (PCP) and compare the outcome of survivors and nonsurvivors in patients with various connective tissue diseases (CTD). METHODS: A retrospective study according to medical records recent 5 years was done to recruit patients with a definite diagnosis of PCP with CTD. RESULTS: Fifteen PCP patients were recruited, 10 women and 5 men (mean age: 54.4 +/- 4.69 years). Positive cases of (1-->3)-beta-D-glucan (beta-glucan) were 13 cases out of 15 (86.7%). There was a significant difference between survivors and nonsurvivors in hypoalbuminemia (p = 0.02), and high levels of beta-glucan (p = 0.04). CONCLUSION: These results suggested beta-glucan could be a diagnostic and negative prognostic marker of PCP.     

Occurrence of Pneumocystis carinii DNA and anti-Pneumocystis antibodies in the sera of infants during a period of of physiologic decreased immune response

The polymerase chain reaction (PCR) and indirect immunofluorescent test (IF) were used for examination of serum samples obtained from infants with respiratory tract infections. Sixty (11.9%) out of the 503 examined infant samples were positive for anti-P. carinii IgM and 354 (70.4%) contained anti-Pneumocystis IgG. P. carinii DNA was found in 6 (6.7%) sera from 90 of infected infants. Five out these 6 samples were for anti-Pneumocystis antibodies positive; 4 contained both IgG and IgM classes and one had only IgG. The sixth sample had neither IgG nor IgM, despite of P. carinii DNA presence. The results of the studies indicated that for diagnosis Pneumocystis carinii pneumonia (PCP) in infants on serum specimens detection of antibodies by IF test is of greater value than Pneumocystis DNA amplification by PCR method.     

环介导恒温扩增法检测卡氏肺孢子菌方法的建立

通过建立肺孢子菌肺炎动物模型获取感染卡氏肺孢子菌(Pneumocystis carinii)的大鼠肺组织,用环介导恒温扩增法(LAMP)扩增卡氏肺孢子菌核内核糖体小亚基18s rDNA,扩增产物用限制性内切酶ApalⅠ酶切,观察酶切片段大小。卡氏肺孢子菌核内核糖体小亚基18s rDNA克隆至载体pGEX6p2,筛选阳性克隆。本研究初步建立了检测卡氏肺孢子菌的环介导恒温扩增法。     

卡氏肺孢子虫特异性DNA探针的制备及其实验应用

目的制备特异、敏感的卡氏肺孢子虫(Pc)DNA探针,用于检测卡氏肺孢子虫肺炎(PCP)患者临床标本中的PcDNA。方法PCR扩增获得Pc的线粒体核糖体RNA大亚基基因,以其为模板,采用随机引物法制备半抗原地高辛标记的探针。检测探针的敏感性和特异性后,用斑点杂交法检测实验大鼠肺组织和PCP患者临床标本中的PcDNA。结果敏感性检测显示该探针可检出2pg水平的PcDNA,特异性检测显示该探针仅与PcDNA杂交,而不与伯氏疟原虫、恶性疟原虫、弓形虫、人白细胞及正常大鼠肺组织DNA反应。斑点杂交检测PCP大鼠肺组织中的PcDNA,检出率为73.7%,低于PCR结合斑点杂交的检出率94.7%。用该探针从1例肾移植术并发PCP患者的支气管肺泡灌洗液中检测到PcDNA。结论制备的PcDNA探针具有敏感性高、特异性好、无放射性污染等优点,对PcDNA有良好的检测效果。     

Rapid diagnosis of cytomegalovirus pneumonia and Pneumocystis carinii pneumonia by using polymerase chain reaction DNA amplification]

We attempted to detect cytomegalovirus DNA (CMV-DNA) and Pneumocystis carinii DNA (carinii-DNA) in urine, blood and sputum samples of 16 leukemia patients with pneumonia, using the polymerase chain reaction (PCR). Synthetic oligonucleotide primer pair were used to amplify DNA from the major immediately genes of CMV and genes for the large subunit of mitochondrial ribosomal RNA of P. carinii. Amplified products were detected by gel electrophoresis. In two cases, CMV-DNA was detected at about the time the pneumonia occurred, and in one of the two cases, CMV-DNA was detected in the sputum sample. This patient was treated immediately with ganciclovir. After ganciclovir treatment, clinical and biochemical signs of CMV pneumonia disappeared. In three cases, carinii-DNA was detected in their sputum samples. In their blood and urine samples, carinii-DNA were not detected. This three cases were treated with sulfamethoxazole-trimethoprim and successfully treated episodes of P. carinii pneumonia. We conclude that PCR amplification may be a valuable tool for rapid diagnosing CMV pneumonia and P. carinii pneumonia.     

Biomolecular techniques to detect Pneumocystis carinii f. sp. hominis pneumonia in patients with acquired immunodeficiency syndrome.

OBJECTIVES: To verify the clinical value of two different polymerase chain reactions (PCRs) for noninvasive diagnosis and follow-up during Pneumocystis carinii f. sp. hominis pneumonia (PCP) and to analyze the P. carinii f. sp. hominis genotypes involved. METHODS: Internal transcribed spacers (ITSs) nested PCR was applied to 630 samples (bronchoalveolar lavage, sera, peripheral blood mononuclear cells, and oropharyngeal samples) from 122 patients with acquired immunodeficiency syndrome and pneumonia and 40 control samples from 20 subjects seronegative for human immunodeficiency virus. One hundred and eighty samples also were examined by mt-rRNA PCR. Bronchoalveolar lavage samples and 33 sera were analyzed by type-specific oligonucleotide hybridization. RESULTS: On bronchoalveolar lavage samples, the two PCRs consistently confirmed the morphologic diagnosis of PCP. The sensitivity of ITSs nested PCR versus mt-rRNA PCR was 57.3% versus 14.3% on sera, 32.3% versus 22. 8% on peripheral blood mononuclear cells, and 69.1% versus 48.6% on oropharyngeal samples (garglings). Both PCRs had 100% specificity. Type-specific oligonucleotide hybridization revealed in 72.2% of bronchoalveolar lavage samples a single P. carinii f. sp. hominis genotype, whereas in 27.8% co-infection with more than one strain was detected. CONCLUSION: On noninvasive samples, ITSs nested PCR was more sensitive than mt-rRNA PCR, and it confirmed the diagnosis in all patients with PCP. For each patient with PCP at least one noninvasive sample was positive for P. carinii f. sp. hominis DNA.     

Sputum examination for the diagnosis of Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome

The diagnostic utility of sputum examination in patients with Pneumocystis carinii pneumonia secondary to the acquired immunodeficiency syndrome (AIDS) has so far not been determined. Sputum was induced in 43 patients with AIDS or suspected AIDS just prior to fiberoptic bronchoscopy, scheduled because of an unexplained pulmonary infiltrate on a chest radiograph. Pneumocystis carinii pneumonia was diagnosed by sputum examination and/or by a bronchoscopic procedure in 20 patients. Of these, sputum samples were positive for Pneumocystis organisms in 11 (55%) of 20 patients tested, bronchial washings were positive in 11 (79%) of 14 patients tested, brush biopsies were positive in 9 (53%) of 17 patients tested, and transbronchial lung biopsies were positive in 18 (90%) of 20 patients tested. The presence of P. carinii cysts in sputum did not correlate with the presence of alveolar macrophages in sputum nor with the volume of sputum. Sputum examination for P. carinii organisms, employed as a first diagnostic step in patients with AIDS with pulmonary infiltrates, may frequently obviate the need for bronchoscopy.