Detection of Chlamydia trachomatis by polymerase chain reaction]

A polymerase chain reaction (PCR) procedure was developed for detection of Chlamydia trachomatis. Two oligonucleotide primers based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used. A single DNA fragment was amplified, when C. trachomatis DNA was template for the PCR. No amplified product was detected in Chlamydia psittaci DNA, Chlamydia pneumoniae DNA or other bacterial DNAs. The amplified DNA fragment was detected, when DNA of greater than or equal to 10(2) C. trachomatis per reaction was used as template for the PCR. Thus, the PCR was shown to be specific for C. trachomatis and more sensitive than the enzyme immunoassays for detection of chlamydial antigen and the chlamydial rRNA:DNA probe hybridization method.     

Detection of cytomegalovirus, Helicobacter pylori and Chlamydia pneumoniae DNA in carotid atherosclerotic plaques by the polymerase chain reaction

OBJECTIVE: Cytomegalovirus (CMV), Helicobacter pylori (H. pylori) and Chlamydia pneumoniae (C. pneumoniae) have been associated with human atherosclerosis. The reported rates of detection within atherosclerotic lesions by PCR vary widely for all of these pathogens.We investigated their presence in carotid atherosclerotic plaques. METHODS AND RESULTS: Eighty-three carotid atherosclerotic specimens were available for examination. The highly sensitive polymerase chain reaction method was employed with primers specific for each agent. The presence of CMV DNA was not detected in any of the examined samples (0%), whereas the presence of H. pylori DNA was observed in two out of eighty-three cases (2.4%). C. pneumoniae DNA was found in eighteen (21.6%) of the plaques studied. CONCLUSIONS: H. pylori DNA was detected in a very small subset of atherosclerotic plaques, whereas CMV DNA was not detected in any of the plaques studied. C. pneumoniae DNA was found in a significant number of our atherosclerotic plaques. Further studies are needed to clarify the possible causal relation between infection by these organisms and atherosclerosis.     

Detection of Chlamydia pneumoniae and Helicobacter pylori in atherosclerotic plaques of carotid artery by polymerase chain reaction.

OBJECTIVES: A possible role of some microorganisms has been proposed in the pathogenesis of atherosclerosis, but it is still an unresolved issue. We investigated the presence of Chlamydia pneumoniae and Helicobacter pylori DNA in carotid artery atherosclerotic plaques by using PCR. METHODS: One hundred and four patients with atherosclerotic diseases were included. The study group consisted of 52 atherosclerotic plaque specimens obtained from the carotid arteries of patients who had carotid endarterectomy and the control group consisted of 52 specimens obtained from the macroscopically healthy regions of ascending aorta in patients who had undergone coronary artery bypass grafting. The presence of C. pneumoniae and H. pylori DNA in endarterectomy specimens were demonstrated by PCR. RESULTS: C. pneumoniae DNA was detected in 16 of 52 (30.8%) atherosclerotic plaques and 1 of 52 (1.9%) macroscopically healthy ascending aorta wall specimens (P < 0.001). H. pylori DNA was detected in 9 of 52 (17.3%) atherosclerotic plaques and none of the controls (P = 0.003). CONCLUSIONS: The higher incidence of C. pneumoniae and H. pylori DNA in atherosclerotic plaques suggests that these microorganisms may play a role in the pathogenesis of atherogenesis.     

Temporal arteritis associated with Chlamydia pneumoniae DNA detected in an artery specimen

Temporal arteritis is a clinical manifestation of giant cell arteritis. The etiology of this disease is still unknown. Sudden onset and wide variations of incidence are reported in different parts of the world. Acute onset is often associated with flu-like symptoms, indicating that infectious factors probably act as precipitating agents. We describe a 72-year-old man referred to our department in January 1999 for unremitting fever and temporal arteritis associated with Chlamydia pneumoniae infection.     

Detection of Mycoplasma pneumoniae and Chlamydia trachomatis in Iranian children with acute lower respiratory infections by polymerase chain reaction

ObjectiveTo determine the frequency of Mycoplasma pneumoniae (M. pneumoniae)and Chlamydia trachomatis (C. trachomatis) in young children with community acquired pneumonia (CAP) and detect C. trachomatis in the subgroup of infants under 1 year of age in Tehran, Iran.MethodsThis cross-sectional study was designed to detect M. pneumoniae from all children (<5 years of age) presenting with CAP, admitted to a tertiary care children's hospital affiliated with the Shaheed Beheshti University of Medical Sciences in Tehran during a period of 14 months, from November 2010 to December 2011. Nasopharyngeal and oropharyngeal swabs were collected from 102 children during the study period. Pathogens were detected using polymerase chain reaction and confirmed with real-time polymerase chain reaction.ResultsOnly one case of M. pneumoniae was isolated from 102 children (1%). C. trachomatis was not detected in any of the 69 infants (<1 year of age).ConclusionsAccording to our findings, M. pneumoniae is an uncommon cause of CAP in children under 5 years old and C. trachomatis could not be listed as causing CAP in infants in our study population. However, more studies with a larger sample size are needed to confirm this observation.     

Detection of Chlamydia pneumoniae by polymerase chain reaction-enzyme immunoassay in intestinal mucosal biopsies from patients with inflammatory bowel disease and controls

BACKGROUND AND AIM: It has been suggested that Chlamydia is an organism that may have the potential to cause inflammatory bowel disease (IBD) in susceptible individuals. Chlamydia pneumoniae has emerged as an important human pathogen in the last decade. The objective of the present study was to investigate the frequency of the presence of C. pneumoniae DNA in intestinal biopsies from patients with IBD and from non-IBD controls. METHODS: The DNA was extracted from 222 colonoscopic biopsies, which were obtained from 11 patients with Crohn's disease (CD), 18 patients with ulcerative colitis (UC) and from 37 non-IBD control patients. The presence of the C. pneumoniae omp1 gene and C. trachomatis 16S rRNA gene was determined using a rapid and sensitive polymerase chain reaction-enzyme immunoassay (PCR-EIA). RESULTS: The C. pneumoniae-specific DNA was detected in 32 (14.4%) of 222 endoscopic biopsies. Among them, C. pneumoniae DNA were found in nine of 42 (21.4%) biopsies from patients with CD, nine of 59 (15.3%) biopsies from patients with UC, and 14 out of 122 (11.4%) biopsies from non-IBD control patients, respectively. Moreover, the percentage of patients with at least one biopsy positive for C. pneumoniae was higher, although not statistically significant, in CD (36.4%) and UC patients (38.9%) compared to non-IBD controls (16.2%). In contrast, C. trachomatis DNA was detected in only two of 222 (0.9%) biopsy samples. CONCLUSION: The C. pneumoniae DNA was detected in the intestine of both patients with IBD and in non-IBD control patients, probably reflecting the high prevalence of this organism in the environment. The moderate yield of positive biopsies in our IBD patients and the fact that the detection rate of C. pneumoniae DNA was similar in endoscopic biopsies from IBD patients and non-IBD controls does not support a direct role for this organism in the pathogenesis of IBD.     

No detection of Chlamydia pneumoniae in normal and atherosclerotic femoral arteries by polymerase chain reaction (PCR)--an autopsy study

Since Saikku's investigations in 1988 a direct relation between atherosclerosis and infection with Chlamydia pneumoniae has been suspected. The aim of the present study was to examine histologically normal and atherosclerotic femoral arteries of autopsy cases and to correlate histological findings with the presence of Chlamydia pneumoniae which was tested with nested polymerase chain reaction. The study included all of the femoral arteries (of which 16 male, 15 female, aged between 50 and 88 years) obtained by autopsy from the cases occurring between July 1, 1999 and August 31, 1999 in the Medical Clinic Darmstadt. Each paraffin block supplied us with six consecutive sections of which the first two sectional areas were histologically stained, while the other four were used in PCR examination for DNA extraction. The polymerase chain reaction did not reveal presence of Chlamydia pneumoniae in any of the cases, neither in atherosclerotic lesions, nor in normal arteries. Because of the negative results, an additional PCR test was performed with primers that amplify another region of the Chlamydia genome, but the result did not change. The negative result of our prospective study does not contradict the assumption that infection with Chlamydia pneumoniae may enhance the progress of atherosclerosis in single cases. However, the results do not favour the concept of atherosclerosis as an infectious disorder caused by Chlamydia pneumoniae.     

Optimised sample DNA preparation for detection of Chlamydia trachomatis in synovial tissue by polymerase chain reaction and ligase chain reaction

OBJECTIVE: Molecular biology techniques such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) are routinely used in research for detection of C trachomatis DNA in synovial samples, and these methods are now in use in some clinical laboratories. This study aimed at determining the method best suited to molecular diagnosis of C trachomatis by examining four standard DNA preparation methods using chlamydia spiked synovial tissue and chlamydia infected monocytes. METHODS: Synovial tissue from a chlamydia negative patient with rheumatoid arthritis was spiked with defined numbers of C trachomatis elementary bodies (EB). Purified human peripheral monocytes from normal donors were infected with the organism at a multiplicity of infection 1:1 in vitro and harvested after four days. DNA was prepared from all samples by four methods: (1) QIAmp tissue kit; (2) homogenisation in 65 degrees C phenol; (3) incubation at 97 degrees C; (4) proteinase K digestion at 97 degrees C. DNA from methods 1 and 2 was subjected to PCR using two different primer sets, each targeting the C trachomatis omp1 gene. LCR was done on DNA prepared by each method. RESULTS: In synovial tissue samples spiked with EB, and in monocytes persistently infected with the organism, preparation of template using the QIAmp tissue kit (method 1) and the hot phenol extraction technique (method 2) allowed sensitive detection of C trachomatis DNA. These methods also produced template from both sample types for LCR. DNA prepared by heat denaturation (method 3) allowed only low sensitivity chlamydia detection in LCR and did not work at all for PCR. Proteinase K digestion plus heat denaturation (method 4) gave template that did not allow amplification in either PCR or LCR assays. CONCLUSIONS: The sensitivity of detection for C trachomatis DNA in synovial tissue by PCR and LCR depends strongly on the method used for preparation of the amplification template. LCR targeting the multicopy chlamydial plasmid and two nested PCR assay systems targeting the single copy omp1 gene showed roughly equivalent sensitivity. Importantly, template preparation method and the specific PCR primer system used for screening must be optimised in relation to one another for highest sensitivity.     

An improved polymerase chain reaction assay to detect Trypanosoma cruzi in blood.

Amplification by the polymerase chain reaction of Trypanosoma cruzi satellite DNA was used to enhance sensitivity in the detection of the parasite in blood, with the ultimate goal of improving diagnosis of the chronic phase of Chagas' disease. Two contiguous oligonucleotides were synthesized corresponding to the most conserved region of the 195-basepair repeated sequence and used as primers for the amplification reaction. Nineteen femtograms of parasite DNA that was amplified in the presence of 15 micrograms of human or mouse DNA produced a visible band upon electrophoresis in agarose gels and staining with ethidium bromide. In reconstitution experiments, one parasite in 10 ml of blood could be unambiguously determined when the DNA was isolated from nuclei after the blood was treated with NP40 and centrifuged. Polymerase chain reaction assays were carried out to detect T. cruzi in chronically infected mice. Most mice were parasite-positive when organs or tissues were tested, but all were negative when total blood was tested.     

Polymerase chain reaction detection of Chlamydia pneumoniae in circulating white blood cells

Several recently published studies suggest that Chlamydia pneumoniae may represent a risk factor for atherosclerosis or its complications. In order to establish whether C. pneumoniae is causally linked to atherosclerotic diseases, clinical intervention trials may be needed. However, to identify eligible subjects with a persistent C. pneumoniae infection and to monitor the effect of antibiotic therapy, there is a need for a reliable diagnostic marker. Blood-based polymerase chain reaction assays may be of value for identifying patients persistently infected with C. pneumoniae and for assessing the microbiologic efficacy of antichlamydial therapy in clinical intervention trials.     

应用聚合酶链反应快速检测临床标本中的肺炎支原体

应用聚合酶链反应（ＰＣＲ）技术检测临床标本中的肺炎支原体（ＭＰ）。在１４０例非细菌性肺炎患者的标本中，ＰＣＲ试验阳性３０例。其中间接血凝试验阴性面ＰＣＲ试验阳性者２１例。ＰＣＲ阳性标本经ＭＰ５－４寡核苷酸探针检测验证，均为阳性结果。     

Detection of Chlamydia trachomatis from urethral swab in male urethritis by polymerase chain reaction]

A polymerase chain reaction (PCR) with Chlamydia trachomatis-specific primers was applied for detection of C. trachomatis from urethral swab in male urethritis. The results were compared with those of culture method for detection of C. trachomatis. Of 18 clinical specimens tested in this study, inclusion bodies of C. trachomatis were detected in 11 specimens by the culture method. For PCR, sample DNA was prepared from transport medium in which urethral smear was suspended and two oligonucleotides based on sequences within the major outer membrane protein gene from C. trachomatis serovar L2 were used as extension primers. In 12 of the 18 specimens, 242bp DNA fragment was amplified by PCR and demonstrated to be the DNA fragment of C. trachomatis by Southern blot hybridization. No DNA of 242bp was amplified by PCR from five specimens in which any inclusion bodies of C. trachomatis were observed or from a specimen in which one inclusion body per cover slip was detected by culture method. C. trachomatis DNA of 242bp was amplified from all specimens in which 14 and more inclusion bodies per cover slip were detected by culture method. In two specimens concluded s negative by culture method, amplified C. trachomatis DNA were detected by PCR. Thus, the PCR would be a more simple and sensitive method for detection of C. trachomatis, compared with the culture method.     

Failure to detect hepatitis C virus genome in human secretions with the polymerase chain reaction.

Although hepatitis C infection has been clearly demonstrated to be transmitted through blood products or blood contamination, most cases of sporadic hepatitis C infection are unassociated with parenteral risk factors, and it is unclear how infection might be acquired by nonparenteral means. One potential mode of nonparenteral transmission is through body secretions. We used a highly sensitive and specific polymerase chain reaction assay to determine whether hepatitis C viral genomic RNA could be detected in secretions obtained from nineteen individuals with chronic hepatitis C virus infection. Although hepatitis C genomic RNA was found in all 19 sera, hepatitis C virus RNA was not detected in any samples of saliva, semen, urine, stool or vaginal secretions from these patients. Viral titers in serum ranged from 10(2) to 10(7) polymerase chain reaction units/ml. The sensitivity of our polymerase chain reaction assay indicates that, if hepatitis C virus were in secretions, it would be present in amounts less than 1 to 4 polymerase chain reaction units/ml. This contrasts with hepatitis B virus infection, in which serum titers frequently are in excess of 10(9) copies of hepatitis B genomes/ml. Body secretions have been found to contain up to 10(6) copies of hepatitis B genomes/ml. Our findings support seroepidemiological studies indicating that nonparenteral transmission of hepatitis C through secretions is uncommon and probably much less efficient than hepatitis B virus infection.     

Rapid diagnosis of Mycoplasma pneumoniae by polymerase chain reaction in community-acquired lower respiratory tract infections

Two hundred children hospitalized for community-acquired lower respiratory tract infections (LRTIs) were investigated for Mycoplasma pneumoniae employing serological tests and a P1 adhesin gene-based polymerase chain reaction assay (PCR) on nasopharyngeal aspirates. Serological evidence of M. pneumoniae infection was observed in 68 (34%) patients and PCR was positive in 20 (10%) children. Together PCR and/or enzyme immuno assay detected M. pneumoniae in 71(35.5%) children. Our data underline the role of M. pneumoniae in Indian children with community-acquired LRTIs even in children aged < 24 months.     

Diagnosis of Mycoplasma pneumoniae pneumonia in pediatric patients by polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) testing was performed on respiratory tract specimens obtained by throat swab in 21 children admitted to the hospital with suspected Mycoplasrna pneurnoniae pneumonia. Of 13 patients with a clinical condition compatible with mycoplasma infection and an immunological response to M. pneurnoniae, 11 were positive by PCR. Eight patients were negative by serology and/or had a clinical condition not compatible with mycoplasma infection, and all were negative by PCR. The antibody response to M. pneurnoniae was delayed for a week or more in 3 (23%) of the 13 patients with documented mycoplasma infection. These results suggest that PCR performed on a respiratory tract specimen obtained by a throat swab may be useful in the initial evaluation of children with suspected M. pneurnoniae pneumonia, especially in patients in whom the serological response is delayed. Pediatr Pulmonol. 1995; 20:297–300 . © 1995 Wiley-Liss, Inc.     

Detection of Chlamydia trachomatis in first-voided urine sediments from male urethritis by polymerase chain reaction]

Chlamydia trachomatis was detected from first-voided urine sediments of 97 male patients with urethritis by polymerase chain reaction (PCR). Since urine and urinary sediments only treated with proteinase K inhibited DNA amplification by PCR, DNA was further purified by phenol extraction and concentrated. Two oligonucleotides based on sequences within the major outer membrane gene from C. trachomatis serovar L2 were used as primers. A DNA fragment of 242 bp specific for C. trachomatis was amplified by PCR and detected by agarose gel electrophoresis. The DNA fragment was amplified by PCR in all specimens of urine sediments from 50 patients with Chlamydiazyme-positive urethral swab. In 38 specimens of urine sediments from 47 patients with Chlamydiazyme-negative urethral swab, PCR was negative. The overall coincidence rate between the PCR for detecting C. trachomatis in first-voided urine sediments and Clamydiazyme in urethral swab was 90.7% (88/97). Detection of C. trachomatis from first-voided urine sediments by PCR was considered to be noninvasive and useful for the diagnosis of male urethritis due to C. trachomatis.     

Temperature stability of vaginal specimens for Chlamydia trachomatis detection by Amplicor polymerase chain reaction assay

Vaginal introital specimens were collected from 17 women - 10 positive and 7 negative for Chlamydia trachomatis, and kept in Amplicor collection medium at ambient temperature. Aliquots were removed at intervals for up to 34 days and frozen at -80 degrees C. Samples were thawed and assayed for C. trachomatis by polymerase chain reaction (PCR). The results of all specimens remained unchanged over this time interval.