Detection of intestinal bacterial translocation in subclinical ischemia-reperfusion using the polymerase chain reaction technique

BACKGROUND/PURPOSE: The purpose of this study was to evaluate the detection of bacterial translocation after subclinical ischemia reperfusion injuries in rats with the polymerase chain reaction (PCR) technique. METHODS: Six-week-old weaning rats were divided into 3 groups. (1) Experiment rats (n = 20) were gavaged with 10(10) Escherichia coli followed by superior mesentery artery occluded for 10 minutes, then reperfused for 30 minutes. (2) Control rats (n = 20) received bacterial gavage. (3) Group 3 were sham rats (n = 20). After the procedure, 3 mL of blood was obtained from the portal vein. The terminal ileum and mesenteric lymph node (MLN) near the terminal ileum were removed. E. coli DNA was detected in blood and MLN samples by PCR, and histological changes were examined. RESULTS: E. coli DNA detection in ischemia-reperfusion (I/R) group animals was 6 of 20 (30%) in the MLN and 2 of 20 (10%) in the blood. PCR was negative in all the rats in the control group and in the sham group (P < .05). There were no significant differences in the histological examination of rat intestines. CONCLUSION: These data suggest that subclinical intestinal I/R injury results in bacterial translocation. Also, PCR is a highly sensitive and rapid method to detect the presence of microbial DNA.     

Detection of human papillomavirus in formalin-fixed, invasive squamous carcinomas using the polymerase chain reaction

We analyzed 88 formalin-fixed, paraffin-embedded invasive squamous carcinomas for human papillomavirus-related DNA sequences (HPV types 16 and 18) following in vitro gene amplification using the polymerase chain reaction. HPV DNA sequences were found in 35 of 50 (70%) carcinomas of the anogenital region, including four of four (100%) anal, six of eight (75%) vulvar, nine of 14 (64%) vaginal, two of five (40%) penile, and 14 of 19 (74%) cervical tumors. Nine of 25 (36%) oropharyngeal squamous carcinomas contained HPV DNA sequences, including four of 10 (40%) laryngeal, three of eight (38%) buccal, and two of seven (29%) glossal tumors. HPV DNA sequences were not found in 13 esophageal carcinomas. Of the 44 cases that contained viral DNA, HPV-16 was detected in 41 cases (93%) and HPV-18 in five cases (11%), while both types were found in two cases (one anal and one vulvar). HPV DNA sequences were found in 43 of 83 (52%) nonverrucous and in one of five (20%) verrucous carcinomas, but this difference was not significant. These findings demonstrate that HPV DNA sequences are more frequently associated with anogenital than oropharyngeal squamous carcinomas and can be readily detected in routinely processed tissues using the polymerase chain reaction.     

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检测非细菌性前列腺炎沙眼衣原体的初步研究

对３０例非细菌性前列腺炎（ＮＢＰ）患者的前列腺按摩液（ＥＰＳ）沙眼衣原体（ＣＴ）进行了聚合酶链反应（ＰＣＲ）检测，并与经二乙氨基葡聚糖处理的ＨｅＬａ细菌培养法进行对比研究，结果发现，６例ＰＣＲ和细胞培养双阳性，２１例ＰＣＲ和细胞培养双阴性，３例两种检测结果不一致，包括２例ＰＣＲ阳性但培养阴性，１例培养阳性但ＰＣＲ阴性，将ＰＣＲ与细胞培养进行比较，ＰＣＲ的敏感性为８５．７％，特异性９１．３％，阳性预     

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.     

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。     

Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primer.

Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression.     

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.     

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.     

用聚合酶链反应诊断男性淋菌性尿道炎

采用聚合酶反应（ＰＣＲ），对１５０例男性淋菌性尿道炎病人进行检测。与直接涂片检查的结果相比较，ＰＣＲ具有快速、特异和敏感的特点。     

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.     

Analysis of aerobic bacterial strains found in chronic rhinosinusitis using the polymerase chain reaction.

INTRODUCTION: Rhinosinusitis is a common disease affecting an estimated 14% of the population. Although there is general agreement in the literature regarding acute rhinosinusitis, chronic rhinosinusitis is not as well studied, and no consensus has been reached regarding the bacterial etiology. The goal of this study was to test chronic rhinosinusitis mucosal specimens using the polymerase chain reaction (PCR) for aerobic bacterial content and to compare the results with standard culture data. RESULTS: Routine culture samples grew 50% aerobic bacteria, whereas PCR detected 62% aerobic bacteria contamination. CONCLUSION: PCR detected more bacteria in mucosal samples than in standard culture, but standard culture of this mucosa reflects the general aerobic bacteria found in chronic rhinosinusitis, with no predominant species of aerobic bacteria. SIGNIFICANCE: The analysis of chronic rhinosinusitis mucosa with the PCR method should give a more accurate picture of the bacteria found in chronic rhinosinusitis so that proper therapy can be instituted.     

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 disseminated tumor cells in the lymph nodes of colorectal cancer patients using a real-time polymerase chain reaction assay

Introduction Postoperative treatment for colorectal cancer depends on tumor stage as defined by the International Union Against Cancer (UICC). Adjuvant chemotherapy is not recommended in patients without lymph node involvement (UICC stages I and II). As many as 20–30% of these patients, however, will develop recurrence. Aims and objectives We conducted this study to determine the presence of disseminated tumor cells in the lymph nodes by quantitative real-time polymerase chain reaction (QRT-PCR) for cytokeratin 20 (CK20) in an attempt to provide supplementary information compared to histopathological findings. Materials and methods Using a standard QRT-PCR assay, we examined primary tumors and 391 lymph nodes from 31 patients with completely resected colorectal cancer. Results Of the 31 primary tumors, 29 were positive for CK20 by QRT-PCR. Discussion An examination of the lymph nodes from the 29 patients with CK20-positive primary tumors revealed that 35 (92.1% sensitivity) of the 38 histopathologically positive lymph nodes and 54 (16.7%) of the 324 histopathologically negative lymph nodes were positive by molecular analysis. CK20 expression was detected in 10 (100%) of 10 patients with a histopathologically positive lymph node status (pN1). In 9 (47.4%) of 19 patients with negative histopathological results (pN0), we detected a CK20 mRNA signal in at least one lymph node. Whereas eight patients with histopathologically negative lymph nodes could be upstaged on the basis of the molecular findings, no patient would be downstaged. Conclusion Our results suggest that QRT-PCR for CK20 is a useful tool for the quantitative detection of micrometastases in the regional lymph nodes. We introduce a standardized procedure that integrates a molecular diagnostic technique in the clinical staging.