聚合酶链反应对胆囊癌组织中细菌DNA片段的检测研究

目的　研究胆囊癌组织及相应胆汁中的细菌菌谱,以探讨胆囊癌的发生与细菌感染的关系。方法　采用聚合酶链反应（ＰＣＲ） ,对３７ 例胆囊癌组织标本及９ 例相应胆汁中的细菌ＤＮＡ 片段进行分子生物学扩增。结果　３７ 例胆囊癌组织标本中细菌ＤＮＡ 片段检出率为７８－３７％ （２９／３７）,９ 例相应胆汁中细菌ＤＮＡ检出率为７７－８％ （７／９）。结论　胆囊粘膜癌变可能与厌氧菌尤其是产气荚膜梭菌感染存在联系,厌氧菌与需氧菌协同作用,长期刺激胆囊粘膜可能是引起癌变的主要因素。     

Detection of Brucella spp. DNA in the semen of seronegative bulls by polymerase chain reaction

Semen samples from 88 reproductively mature bulls were screened to detect the presence of Brucella spp. by polymerase chain reaction. Twenty‐seven samples were found to be positive, underscoring the importance of researching brucellosis in males and the need for greater care in the selection of sperm‐donating bulls for semen centres.     

Generic polymerase chain reaction followed by DNA sequencing as a means of diagnosing bacteraemia

There is increasing use of polymerase chain reaction techniques to diagnose infection. We report the use of polymerase chain reaction using a generic section of the bacterial 16S rDNA gene--followed by nucleotide sequencing--to determine the species of the infecting bacteria. In the first case, the clinical and microbiological diagnosis of meningococcal septicaemia was in agreement with the results from polymerase chain reaction technique. In the second case, a Yersinia enterocolitica bacteremia was detected by the polymerase chain reaction technique, but missed with conventional blood culture techniques.     

Diagnosis of parapneumonic pleural effusion by polymerase chain reaction in children

Purpose

Most pleural effusions are associated with bacterial pneumonia, and the identification of the pathogen will assist the therapeutic decision. A specific method that is not affected by previous antibiotic therapy is sought to detect the main causative agents of pneumonia in infants and children (Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus). The aim of the present study was to compare the polymerase chain reaction (PCR) technique with standard culture methods in identifying bacterial infections in infants' and children's pleural effusion.

Methods

Samples obtained from pediatric patients (n = 37) with a diagnosis of pneumonia associated to pleural effusion, submitted to thoracentesis, were analyzed by PCR with specific primers.

Results

The PCR technique identified the presence of bacterial infection in a larger proportion (95.2%) than the standard culture method (33.3%) on complicated pleural effusion samples. The microorganism detection on uncomplicated pleural effusion samples was positive only by the PCR method (31.3%). The frequencies of microorganisms identified on complicated pleural effusion were 57.1% of all patients for methicillin-resistant Staphylococcus; 52.4%, S pneumoniae; 28.6%, S aureus; and 23.8%, H influenzae. The previous use of antibiotics interferes with standard culture method, but it did not interfere with the PCR results.

Conclusions

The molecular diagnosis by PCR method could improve the etiologic diagnosis and might help to guide the treatment of parapneumonic effusion in children.     

精细活检针在结核性胸膜炎诊断中的应用价值

结核性胸膜炎患者以往通常是以X射线、胸部CT的影像学诊断为依据,结合常规胸水检查,如胸水常规、生化、培养、涂片找抗酸杆菌,PCR检测结核分枝杆菌以及结核抗体等实验室检查结果,行诊断性抗结核治疗。如治疗无效则需重新检查以明确诊断,常常导致病情延误。抗结核药物的不良反应较多,诊断性抗结核治疗经常导致药物性肝脏损害、药物性胃炎和药物过敏等。     

Cytomegalovirus infection of renal allografts. Detection by polymerase chain reaction.

Early laboratory diagnosis of acute cytomegalovirus infection in renal transplant recipients is desirable but often difficult. The polymerase chain reaction (PCR) technique for detecting CMV DNA, although it promises a high sensitivity, risks the possibility of detecting latent CMV infection and leading to false-positive results. To address this issue and the feasibility of applying PCR to renal biopsy specimens, we analyzed 37 renal allografts by PCR. Formalin-fixed or Bouin-fixed paraffin-embedded materials were employed, and primers from the LA (late-antigen) region of CMV were used. Amplified products were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot analysis. Of 21 nephrectomy samples, three showed CMV-specific amplified products by PCR, but CMV inclusion bodies were detected histologically in only one of the three. Of 16 renal biopsies, three specimens were positive by PCR, with rare viral inclusions histologically identified in only one. All PCR-positive patients had clinically significant CMV disease as evidenced by positive CMV culture and/or seroconversion. In contrast, all CMV-seropositive patients without active viral disease had PCR-negative allografts. We conclude that PCR positivity in the renal allograft strongly correlates with active CMV disease but not latent infection. For the diagnosis of active CMV disease in patients with a renal allograft, PCR provides a means that is more sensitive and objective than histologic examination, more specific than serology, and faster than viral culture.     

应用聚合酶链反应检测生殖道溶脲脲原体的研究

采用培养法和聚合酶链反应（ＰＣＲ）检测了溶脲脲原体（Ｕｕ）标准菌株纯培养物、模拟临床生殖道Ｕｕ感染样本和５２例不育症病人的精液样本。结果培养法阳性的样本ＰＣＲ亦为阳性，并比培养法高出２～３个稀释度。若以培养法作为标准，ＰＣＲ的敏感性为１００％，特异性为８８％，准确性为９０％，提示ＰＣＲ是一种特异、灵敏和快速检测Ｕｕ感染的诊断方法。     

PCR检测尿结核杆菌的临床应用价值

评价聚合酶链反应（ＰＣＲ）诊断泌尿系结核的临床应用价值，采用ＰＣＲ检测经病理证实为泌尿系结核９例，并报告１７例尿ＰＣＲ阳性的可疑肾结核抗痨治疗前后的临床情况，结果：９例泌尿系结核者，ＰＣＲ检测阳性７例；１７例尿ＰＣＲ阳性的可凝肾结核者经抗痨治疗３个月后，有１４例症状完全消失，尿常规转为正常，认为ＰＣＲ是临床快速诊断泌尿系结核的有效方法。     

多聚酶链反应检测尿中结核杆菌的临床意义

应用多聚酶链反应（ＰＣＲ）对８６例患者（１０例经病理诊断为肾结核，６９例可疑肾结核，７例单纯附睾结核）和３０例健康对照者进行连续２日晨尿结核菌检测。１０例肾结核患者检出均阳性；可疑肾结核者第一次检出９例，第二次为６例；７例附睾结核者两次无一阳性；对照组有１例二次检查均为阳性。认为ＰＣＲ对尿中结核杆菌检出率高、准确、快速，值得在临床上推广应用。     

Eikenella corrodens-induced spondylitis. Detection with 16s-RNA polymerase chain reaction

Isolation of the relevant organism in patients with spondylitis even after an open biopsy is successful only in 75-90%. The rare case of an Eikenella corrodens-induced spondylitis is presented, which could only be identified using 16S ribosomal DNA polymerase chain reaction following unsuccessful microbiological cultivation. Eikenella corrodens is a facultative anaerobic gram-negative organism, which is mostly found in the oropharynx of healthy patients.     

Comparison of polymerase chain reaction amplification of two mycobacterial DNA sequences, IS6110 and the 65kDa antigen gene, in the diagnosis of tuberculosis.

BACKGROUND: Knowledge of the sequences of mycobacterial genes and the availability of DNA amplification techniques have raised the possibility that identification of mycobacterial DNA may offer a rapid and specific diagnostic test for tuberculosis. The correlation between the presence of Mycobacterium tuberculosis DNA and clinical tuberculosis, however, is not known. This study compared the results of polymerase chain reaction amplification of two M tuberculosis DNA sequences, IS6110 and the gene encoding the 65kDa heat shock protein (65kDa Ag), from sputum, bronchoscopy washings, and bronchoalveolar lavage fluid and related these findings to the presence of active and past tuberculosis. METHODS: Highly specific primers were used for amplification of IS6110 and 65kDa Ag DNA. Analysis was performed on one or more samples from 87 patients. RESULTS: IS6110 DNA was identified in samples from all six patients with active tuberculosis, from 15 to 18 patients with past tuberculosis, from five of nine contacts of patients with tuberculosis, and from nine of 54 patients with lung disease unrelated to tuberculosis. The 65kDa Ag DNA was identified in samples from all patients with active and past tuberculosis, from contacts of patients with tuberculosis, and from 14 of 42 patients with non-tuberculous lung diseases. CONCLUSION: These data suggest that the presence of IS6110 DNA correlates more closely with a tuberculosis related diagnosis than that of 65kDa Ag DNA and that both DNAs are found in most subjects with past tuberculosis or contacts of patients with tuberculosis. This may limit the clinical usefulness of these tests.     

Detection of fungi in the sinus mucosa using polymerase chain reaction.

OBJECTIVE: To compare the presence of fungi in the sinus mucosa of patients with and without chronic rhinosinusitis. STUDY DESIGN AND SETTING: Prospective observational study using polymerase chain reaction and conventional culture to detect fungi in the sinus mucosa. Middle meatus mucosal samples were collected from 31 patients with chronic rhinosinusitis and 14 control subjects. RESULTS: Fungi were detected in 6.5% of subjects with chronic rhinosinusitis and in none of the control subjects using polymerase chain reaction. Fungi were detected in 29% of subjects with the combination of inhalant allergies, nasal polyposis, and asthma. Fungi were detected in none of the subjects without the combination of these three comorbidities (P = 0.03). CONCLUSION: Polymerase chain reaction assay appears to be able to detect fungi in chronic rhinosinusitis. SIGNIFICANCE: Fungi may not be implicated in the pathogenesis of most chronic rhinosinusitis. EBM rating: B-3b.     

Direct detection by ligase chain reaction of Mycobacterium tuberculosis complex in pleural fluid

Our purpose was to investigate the utility of the DNA amplification by ligase chain reaction (LCR) for the direct detection of Mycobacterium tuberculosis complex (MTC) in pleural fluid specimens from patients suspected for tuberculous pleural effusion. We have used the LCx M. tuberculosis kit (Abbott) which uses the amplification of the gene that encodes for antigen b. We have examined 81 pleural fluid specimens by isolation (on L?wenstein-Jensen medium and MB/BacT system) and by LCR. Out of 10 positive specimens in culture, 4 were also positive by LCR; out of 71 negative specimens in culture, 8 were positive by LCR. We have re-evaluated the LCR results according to the clinical diagnosis, sustained by the successful therapy, and to the pathological diagnosis on the pleural biopsy. The sensitivity and specificity of LCR in the diagnosis of tuberculous pleural effusion were 31.5% and 100%. This commercial LCR kit is a rapid, specific, but less sensitive test for the routine diagnosis of the tuberculous pleural effusion.     

Detection of Brucella abortus B19 strain DNA in seminal plasma by polymerase chain reaction in Brazil

A total of 27 seminal plasma samples from cattle‐breeding farms or semen centres located in Minas Gerais, Brazil, previously negative by serological and tested positive for Brucella spp. with primer specific for the amplification of the gene virb5 by polymerase chain reaction (PCR ) were analysed for the detection of Brucella abortus DNA by PCR . It was found that nine samples (33.33%) contained B. abortus B19 strain DNA , two (7.40%) contained B. abortus DNA and five (18.51%) contained both DNA . The larger number of samples with B. abortus B19 strain DNA would explained by the environmental contamination by vaccinated females with persistent excretion or some illegal vaccination process. It is first reported of male bovines detected with both DNA .     

Detection of human papillomavirus in the prostate by polymerase chain reaction and in situ hybridization.

Human papillomavirus is associated with a variety of anogenital lesions, including genital warts, precancers and cancers. In male patients human papillomavirus has been identified in proliferative lesions ranging from penile and urethral warts to penile and prostatic cancers. We examined the association of human papillomavirus deoxyribonucleic acid (DNA) in 84 prostate tissue specimens. Specimens were selected from radical prostatectomy, transurethral resection or transrectal biopsy procedures. A total of 60 formalin-fixed, paraffin-embedded tissues (24 prostate cancer specimens, 16 benign prostatic hyperplasia specimens and 20 normal specimens) was examined by polymerase chain reaction and in situ hybridization. Also, 24 gelatin-embedded frozen prostate cancer specimens were examined for human papillomavirus DNA by polymerase chain reaction. Of the specimens 69 were deemed adequate for polymerase chain reaction analysis, whereas all 60 paraffin-embedded tissues were sufficient for in situ hybridization. Human papillomavirus DNA was detected in 2 normal tissues and 6 prostate cancers using polymerase chain reaction. None of the benign prostatic hyperplasia specimens was positive for human papillomavirus. Human papillomavirus typing results indicated that virus type 16 was present in each of the 8 positive specimens. Confirmation of the presence of human papillomavirus was obtained for 1 of the prostate cancers by nonisotopic in situ hybridization with biotinylated human papillomavirus genomic probes. The low prevalence of human papillomavirus in this study population does not strongly support an etiological role for the virus in prostate cancer.     

Molecular biology: the polymerase chain reaction.

Upper aerodigestive tract (UADT) cancers provide an excellent carcinogenesis model for a number of reasons: they are accessible to observation, are usually associated with a known environmental carcinogen (tobacco by-products), are sometimes associated with a tumorigenic DNA virus (HPV), and fall along a spectrum of progressive disease from normal mucosa through leukoplakia and verrucous carcinoma to invasive and metastatic carcinoma. Despite the presence of this unique model, the field of head and neck oncology, as a whole, has been slow in establishing an efficient 2-way conduit between the bedside and the laboratory. Such open communication is important as current evidence suggests that future staging and therapy of head and neck tumors will depend not only on familiar macroscopic and light microscopic criteria, but also on factors that are currently identifiable only in the basic science laboratory. To have a significant impact on the direction and relevance of basic research, clinicians should become knowledgeable and conversant in the vocabulary and general concepts of basic science. The goal of this section is to facilitate communication between the basic researcher and the clinician, thereby promoting clinically relevant basic research. This is to be achieved by fostering understanding of the power, limitations, scope, and horizons of current basic research concepts and techniques. Subsequent articles will review current research topics germane to head and neck cancer, such as oncogenes and tumor suppressor genes, mechanisms of metastasis, tumor immunology and its modulation, and virology.