Diagnosis of Chlamydia trachomatis infections in asymptomatic men and women by PCR assay.

A PCR assay was evaluated for its ability to detect genital chlamydial infection in asymptomatic men and women. Urethral swab specimens were collected from 472 men for culture and PCR assay, and first-void urine (FVU) specimens were collected from 379 of these men for enzyme immunoassay (EIA) and PCR assay. Cervical swab specimens were collected from 242 women for culture, EIA, and PCR assay. Patients were considered infected if they were culture positive or positive by PCR with both plasmid- and major outer membrane protein-based primers. By using this extended "gold standard," the prevalence of infection in this population was 7.6% for men and 7.9% for women. For men, the sensitivities of urethral swab specimen culture and PCR and FVU specimen EIA and PCR were 61, 72, 55, and 91%, respectively. All assays had specificities of > or = 99.8%. The positive and negative predictive values for PCR testing of FVU specimens were 100 and 99.4%, respectively, compared with values of 96.3 and 97.8%, respectively, for PCR of urethral swab specimens. The sensitivities of cervical swab specimen culture and PCR testing were 42 and 90%, respectively, with corresponding specificities of 100 and 99.3%. All cervical swabs were negative by EIA. Molecular techniques such as PCR assays are valuable tools for the detection of symptomatic genital chlamydial infection. In particular, PCR assays of FVU specimens from men offer a highly sensitive, noninvasive screening tool that will likely improve patient compliance for diagnostic testing.     

Diagnosis of genital Chlamydia trachomatis infections in asymptomatic males by testing urine by PCR.

An enzyme-linked immunosorbent assay (EIA) (MikroTrak; Syva) was compared with PCR (Amplicor; Roche) for detection of Chlamydia trachomatis in first-void urine (FVU) from 184 men attending a skin and venereal disease clinic. The prevalence of C. trachomatis in the population studied was 18.5%. Discrepant results between Syva EIA and Roche PCR were retested by using major outer membrane protein primer-based PCR. After retesting, the sensitivity, the specificity, and the positive and negative predictive values for the Syva EIA were 85.3, 100, 100, and 77.5%, respectively, and those for the Roche PCR 100, 100, 100, and 100%, respectively. It was concluded that PCR provides a highly sensitive and specific noninvasive screening method for genital chlamydial infection in asymptomatic men.     

Detection of Chlamydia trachomatis infections in women by Amplicor PCR: comparison of diagnostic performance with urine and cervical specimens.

We used the Roche Amplicor PCR assay to compare urine and cervical swabs as sample material in the detection of Chlamydia trachomatis causing genital infections. The diagnostic performance of Amplicor PCR was compared with that of cell culture and the Gen-Probe PACE 2 assay with cervical specimens. If discrepant from other results, the specimens negative by PCR were diluted and reanalyzed to reveal PCR inhibitors. Of 666 patients, 39 (5.9%) were confirmed to have chlamydial infection. The respective sensitivity and specificity of Amplicor PCR were as follows: urine specimens, 82.0 and 99.7%; cervical specimens, 82.0 and 99.8%. Those for cell culture with cervical specimens were 84.6 and 100%. For the Gen-Probe PACE 2 assay, the sensitivity and specificity with cervical specimens were 79.5 and 100%, respectively. Without the effect of PCR inhibitors, the sensitivity of PCR with urine would have been 97.4%. Provided that the problems currently caused by inhibitors will be solved, the Amplicor PCR assay with urine specimens offers a tempting alternative for the diagnosis of C. trachomatis infection in women.     

实时荧光定量PCR检测沙眼衣原体的研究

目的采用TaqMan探针建立检测沙眼衣原体的实时荧光定量PCR（real-time PCR）方法。方法根据沙眼衣原体外膜蛋白A的基因（ompA）序列设计引物和探针,以克隆的ompA部分基因片段作DNA模板,建立实时荧光定量检测方法。结果建立的荧光定量PCR检测方法的最低检出限为5 copies/反应,检测线性范围100～107线性关系良好（r2=0.997）,比巢式PCR敏感100倍;且与鹦鹉热衣原体、淋球菌、解脲脲原体、大肠杆菌等病原菌DNA以及人基因组DNA均无交叉反应,表明该方法具有良好的特异性。以巢式PCR作参比,建立的荧光定量PCR法检测沙眼衣原体的阳性符合率为100.00%,阴性符合率为95.09%,总符合率为96.81%。结论建立的检测沙眼衣原体实时荧光定量PCR具有特异性强和敏感性高的特点,可快速检测样本中微量沙眼衣原体DNA,适用于对沙眼衣原体进行大规模筛选。     

Detection of PCR inhibitors in cervical specimens by using the AMPLICOR Chlamydia trachomatis assay.

To determine that susceptibility of AMPLICOR Chlamydia trachomatis PCR to inhibitory factors possibly present in cervical specimens, we obtained cervical specimens from 200 gynecology patients attending our outpatient clinic. The prevalence of C. trachomatis infection was 4.1%, as determined by cell culture. All AMPLICOR specimens were tested in one procedure as described by the manufacturer, and after the specimen was spiked with C. trachomatis, several other pretreatment protocols were used. Complete inhibition of the PCR was observed in 38 (19%) cervical specimens. Heat treatment at 95 degrees C, freeze-thawing, or 10-fold dilution of the samples reduced the initial inhibition to 9, 16, or 9%, respectively. A combination of heat treatment and 10-fold dilution reduced the inhibition to 4% of the samples. A second specimen type (swabs inoculated in 0.2 M sucrose phosphate buffer [2SP]) was also evaluated. A 10-fold dilution of the spiked 2SP specimen resulted in an inhibition rate of 6%, which was comparable to that obtained by centrifugation of the 2SP specimen prior to processing. Furthermore, it was shown that the inhibition was not correlated with blood contamination. Processing the specimens on the day of collection or the day after resulted in a higher inhibition rate than did delayed processing (27.6 versus 15.5%, respectively). An inverse correlation was found between the concentration of C. trachomatis added to the sample and the rate of inhibition observed. The inhibition was partly correlated with the pH of the cervical mucosa. Decreased inhibition was found at pH values of > or = 7.5. The effects of blood, pH, and delay in processing were all evaluated by using the AMPLICOR specimen. We conclude that the susceptibility of AMPLICOR C. trachomatis PCR to inhibiting factors in cervical specimens can be significantly reduced if the pretreatment procedure includes heat treatment or the use of 2SP transport medium. Also, a 10-fold dilution of the clinical specimen followed by heat treatment will largely prevent the inhibition of this PCR.     

Prevalence of Helicobacter pylori in Sri Lanka as determined by PCR

Fifty-seven Sinhalese patients were investigated for the presence of Helicobacter pylori by PCR. A prevalence of 70.1%, with 47.5% positive for cagA, was demonstrated. The most common vacA allele was s1am1. There was no significant association between either the s1 allele or the cagA allele and severe gastroduodenal disease. There was an association between the s1 allele and the cagA locus.     

Pooling of urine specimens for detection of asymptomatic Chlamydia trachomatis infections by PCR in a low-prevalence population: cost-saving strategy for epidemiological studies and screening programs

Pooling, in groups of five, of urine specimens from asymptomatically infected men in a population with 4% prevalence, as determined by case finding, is 100% sensitive and specific and results in a 60.5% reduction in the number of tests needed. Pooling of urine specimens in groups of 10 for the estimation of population-based prevalence is 96.1% sensitive and 100% specific and saves 90% of the test costs.     

Partner notification among asymptomatic Chlamydia trachomatis cases, by means of mailed specimens.

The objective of the study was to evaluate the prevalence of Chlamydia trachomatis infection and the participation, among partners of asymptomatic cases in general practice. Index cases were requested to invite partners for testing by mailed urine samples. One or more partners of 62% of the index cases participated, and the prevalence of infection among partners was 48%. A steady relationship was a determinant of both participation and prevalence. In conclusion, the mailing strategy is an effective strategy for partner notification. A high prevalence wasfound among partners.     

HPV Prevalence in cytomorphologically normal cervical scrapes of pregnant women as determined by PCR: The age-related pattern

Diverging data exist on human papillomavirus (HPV) prevalence in cytomorphologically normal scrapes during pregnancy. The prevalence of HPV was therefore investigated by polymerase chain reaction method (PCR) in cytomorphologically normal scrapes of 709 pregnant women and 3, 948 non-pregnant women visiting the same hospital during the same time period. The prevalence of all types of HPV among pregnant women was 9.6% (68/709) and the high risk HPV types 16 and 18 were found in 3.1% (22/709). In the non-pregnant women the prevalence of all types of HPV was 10.9% (432/3, 948) with 2.9% (116/3, 948) HPV types 16 and 18. The highest prevalence of HPV was present in women at younger ages in both groups. With increasing age the prevalence declines from about 19% (15–25 yrs) to 5% (40–49 yrs). The age-adjusted odds ratio of prevalence of all types of HPV in pregnant versus non-pregnant women was 0.73 (95% CI 0.56–;0.96, P = 0.025) and statistically significant. When HPV types 16 and 18 were considered, significant differences were not found. HPV of all types and types 16/18 prevalence was higher in the second half of pregnancy than in the first part but did not reach statistical significance. High HPV copy numbers in the scrapes were found during the first half of the pregnancy and not during the second half using a semi-quantitative HPV 16/18 PCR detection method. Since the difference in HPV prevalence between non-pregnant and pregnant women is very small, it is concluded that HPV prevalence in cytomorphologically normal smears is hardly influenced by pregnancy. © 1995 Wiley-Liss, Inc. © 1995 Wiley-Liss, Inc.     

Improved PCR sensitivity for direct genotyping of Chlamydia trachomatis serovars by using a nested PCR.

Successful amplification of omp1 DNA by PCR is crucial in the genotyping of Chlamydia trachomatis when directly performed with clinical samples (J. Lan, J. M. M. Walboomers, R. Roosendaal, G. J. van Doornum, D. M. McLaren, C. J. L. M. Meijer, and A. J. C. van den Brule, J. Clin. Microbiol. 31:1060-1065, 1993). Several primers flanking the four variable domains of the omp1 gene were selected and tested for sensitivity in several nested PCRs with serial dilutions of serovar G. The optimal sensitivity obtained was 0.1 to 0.01 inclusion-forming units, similar to that obtained in the C. trachomatis plasmid PCR. With this approach, any C. trachomatis PCR-positive sample can be typed.     

Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis

The aim of this study was to develop and evaluate multiplex and nested PCR-reverse line blot (RLB) hybridization assays for detection and serovar identification of Chlamydia trachomatis. Two sets of primers targeting the VD2 region of the omp1 gene and one set targeting the cryptic plasmid were designed for use in multiplex (both targets) and nested PCR (omp1 only). For the RLB assay, labeled omp1 amplicons were hybridized to a membrane containing probes specific for 15 C. trachomatis serovars. The assays were used to test 429 clinical specimens, which had been previously tested for C. trachomatis using the COBAS AMPLICOR system. Specimens were tested without knowledge of the COBAS AMPLICOR result. Of 205 specimens that were positive by COBAS AMPLICOR, 201 (98%) were positive by multiplex PCR-RLB and 188 (92%) were also positive by omp1 nested PCR-RLB. In addition, three of 224 COBAS AMPLICOR-negative specimens were positive by omp1 nested PCR-RLB. One hundred sixty-six of 191 (87%) specimens in which C. trachomatis serovars were identified contained only one serovar and 25 (13%) contained two or three serovars. Serovars D, E, and F were found in 31 (16%), 83 (43%), and 51 (27%) specimens, respectively. Serovar E (41%) was the most commonly identified single serovar. Serovars J and K were found alone uncommonly (<2% each), but 18 of 25 (72%) specimens with multiple C. trachomatis serovars contained one or both (10 specimens) of these serovars. The nested (ompI) PCR-RLB is a specific and sensitive method for simultaneous detection and serovar identification of C. trachomatis, which can reliably identify mixed C. trachomatis serovars. It is suitable for use in epidemiological studies.     

Influence of endocervical specimen adequacy on PCR and direct fluorescent-antibody staining for detection of Chlamydia trachomatis infections.

The cellular quality of the endocervical swab specimen used for the detection of Chlamydia trachomatis may dramatically impact the sensitivity of the diagnostic assay used. An evaluation of the adequacy of 319 endocervical swab specimens from women attending two inner-city sexually transmitted disease and family planning clinics, as well as five high school-based family planning clinics, was performed, and the resulting data were compared with the diagnostic results obtained by both Amplicor PCR and Microtrak direct fluorescent-antibody (DFA) staining. The swab from each patient was rolled across the open circular area of a DFA slide and then used to inoculate a transport tube for PCR (Roche), after which the swab was discarded. The slides were stained and examined by epifluorescence microscopy for the presence of C. trachomatis elementary bodies and for the presence and number of cell types to determine specimen adequacy. Cellular adequacy for a cervical swab specimen was defined as the presence of one or more columnar epithelial or metaplastic epithelial cells or the presence of more than 100 erythrocytes per high-power microscopic field. Of the 319 specimens read by DFA, 204 (63.9%) were determined to be adequate. There were 34 (10.7%) positive specimens by DFA and/or PCR. Twenty-nine (9.1%) specimens were positive by PCR, 20 (6.3%) specimens were DFA positive, and 15 (4.7%) were concordantly positive by both tests. The prevalence of chlamydia among adequate specimens was 14.2% (29/204), compared to 4.3% (5/115) for inadequate specimens (P < 0.0001). Variations in specimen quality and the sensitivity of the diagnostic assay used have a significant impact on determining the prevalence of C. trachomatis in a population.     

Ocular sensitization of mice by live (but not irradiated) Chlamydia trachomatis serovar A.

Ocular exposure of mice to live elementary bodies of Chlamydia trachomatis serovar A results in immunological sensitization of the mice. This reactivity is manifested by the development of early (5 h) and delayed-type (24 h) dermal reactivity and serovar-specific antibody formation against either live or irradiated (100 kilorads) elementary bodies. Parallel ocular exposure of mice to irradiated elementary bodies does not result in this sensitization. The early and late dermal immune responses induced by ocular exposure to live organisms can be transferred to unexposed mice by serum and lymphoid cell transfers, respectively. It appears that successful murine ocular sensitization by human C. trachomatis serovar A elementary bodies is an ability manifested by live organisms and not by inactivated but antigenic organisms.     

How sensitive is the Papanicolaou smear in the diagnosis of infections with Chlamydia trachomatis?

Fifty-three patients who presented to the sexually transmitted disease clinic at Boston City Hospital had simultaneous cervicovaginal Papanicolaou smears and cultures for Chlamydia trachomatis taken prior to the initiation of antibiotic therapy. Eleven of the chlamydial cultures had positive results. None of the Papanicolaou smears satisfied the morphologic criteria for the diagnosis of chlamydial infection. This suggests that the Papanicolaou smear is an insensitive technic for the diagnosis of chlamydial infection of the cervix.     

Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis.

The synthesis of early proteins from Chlamydia trachomatis serovar L2 was analyzed by two-dimensional gel electrophoresis. By pulse-label experiments, the synthesis of seven proteins was observed at 2 to 8 h postinfection before the major outer membrane protein was detected at 8 to 10 h after infection. The early proteins were synthesized throughout the 30-h period investigated, but the synthesis of three proteins of 75, 62, and 45 kilodaltons decreased from 26 to 30 h postinfection. Pulse-chase analysis showed that the signals from the same three proteins declined 26 to 30 h after infection. Three of the early proteins were identified as the S1 ribosomal protein, the GroEL-like protein, and DnaK-like protein, respectively.     

Ability of commercial ligase chain reaction and PCR assays to diagnose Chlamydia trachomatis infections in men by testing first-void urine.

A total of 287 men (37.6% with symptoms of urethritis) attending a hospital-based sexually transmitted disease clinic had urethral swabs tested by culture and by direct fluorescent-antibody assay. First-void urine (FVU) was tested for Chlamydia trachomatis by commercially available ligase chain reaction (LCR) and PCR assays. By using an expanded reference standard, 35 men (12.2%) were found to be positive. By performing LCR and PCR, the infection prevalence was found to be approximately twice (11.5 and 12.2%, respectively) that determined swab testing. The sensitivity values were 94.3% for LCR and 100% for PCR. One of the two positive specimens missed by LCR contained inhibitors. PCR produced five false-positive results and LCR produced one.     

Current methods of laboratory diagnosis of Chlamydia trachomatis infections.

Infections caused by Chlamydia trachomatis are probably the most common sexually transmitted diseases in the United States. Commonly unrecognized and often inadequately treated, chlamydial infections can ascend the reproductive tract and cause pelvic inflammatory disease, which often results in the devastating consequences of infertility, ectopic pregnancy, or chronic pelvic pain. C. trachomatis infections are also known to increase the risk for human immunodeficiency virus infection. The obligate intracellular life cycle of C. trachomatis has traditionally required laboratory diagnostic tests that are technically demanding, labor-intensive, expensive, and difficult to access. In spite of these historical challenges, however, laboratory diagnosis of C. trachomatis has been a rapidly advancing area in which there is presently a wide array of commercial diagnostic technologies, costs, manufacturers. This review describes and compares the diagnostic methods for C. trachomatis infection that are currently approved for use in the United States, including the newest DNA amplification technologies which are yet to be licensed for commercial use. Issues to consider in selecting a test for purposes of screening versus diagnosis based on prevalence, performance, legal, social, and cost issues are also discussed.