Comparison of performances of two commercially available tests, a PCR assay and a ligase chain reaction test, in detection of urogenital Chlamydia trachomatis infection

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.     

The surgical treatment of disorders in the formation and fusion of the lumbar vertebrae in children

OBJECTIVE: To investigate the value of polymerase chain reaction (PCR) for follow-up patients infected by Chlamydia trachomatis. METHODS: Follow-up specimens were collected from 30 patients. Chlamydia trachomatis positive were detected by PCR and direct fluorescence assay test (DFA) in the 30 patients before therapy. 15 patients were treated with minocycline (100 mg twice daily) for 10 days, and 15 patients were treated with 1.0 g of azithromycine as a single oral dose. RESULTS: After 1-2 weeks of antimicrobial therapy, all patients had negative DFA for Chlamydia trachomatis, but 9 had positive Chlamydia trachomatis DNA as detected by PCR. CONCLUSIONS: The 9 specimens were not confirmed to livae viable organisms of Chlamydia trachomatis. The debris of nonviable Chlamydia trachomatis DNA was excluded from urinogenital tract at about one month.     

Head-to-head evaluation of five chlamydia tests relative to a quality-assured culture standard

Nucleic acid amplification tests offer superior sensitivity for the detection of Chlamydia trachomatis infection, but many laboratories still use nonamplification methods because of the lower cost and ease of use. In spite of their availability for more than a decade, few studies have directly compared the nonamplification tests. Such comparisons are still needed in addition to studies that directly compare individual nonamplification and amplification tests. The purpose of this study was to evaluate and compare the performance characteristics relative to culture of five different tests for the detection of C. trachomatis with and without confirmation of positive results. The tests were applied to endocervical specimens from 4,980 women attending family planning clinics in the northwestern United States. The five nonculture tests included Chlamydiazyme (Abbott), MicroTrak direct fluorescent antibody (DFA) (Syva), MicroTrak enzyme immunoassay (EIA) (Syva), Pace 2 (Gen-Probe), and Pathfinder EIA (Sanofi/Kallestad). All positive results obtained with a nonculture test (except MicroTrak DFA) were confirmed by testing the original specimens with a blocking antibody test (Chlamydiazyme), a cytospin DFA (MicroTrak EIA and Pathfinder EIA), and a probe competition assay (Pace 2). The prevalence of culture-proven chlamydia was 3.9%. The sensitivities of the nonculture tests were in a range from 62 to 75%, and significant differences between tests in terms of sensitivity were observed. The positive predictive value for each test was 0.85 or higher. The specificities of the nonculture tests without performance of confirmations were greater than 99%. Performing confirmatory tests eliminated nearly all of the false positives.     

Detection of Chlamydia trachomatis in urine samples by nucleic acid tests: comparison with culture and enzyme immunoassay of genital swab specimens

Two commercially available nucleic acid-based tests, ligase chain reaction (LCR; Abbott Laboratories) and PCR (Roche Diagnostics), for the detection of Chlamydia trachomatis in male and female urine samples were compared with culture and enzyme immunoassay (EIA) (Microtrak; Syva) for C. trachomatis detection in genital samples. The samples were collected from 1,005 patients who attended a sexually transmitted disease clinic. In this study population, the prevalence of the infection was 4%. Specimens which were reactive in any of the tests were retested with a different PCR test using primers directed against the major outer membrane protein gene. With a "gold standard" of a positive culture, or any other positive test result if it was confirmed by an independent test, the Roche PCR (95% sensitive, 99.9% specific) was more sensitive than the LCR (75% sensitive, 100% specific) (chi2, P < 0.0001) while both tests were more sensitive than culture (58% sensitive, 100% specific) or EIA (45% sensitive, 100% specific) (chi2, P < 0.001). The Roche PCR and Abbott LCR tests of urine identified 65% and 30% more positive patients, respectively, than did testing by culture of urethral or cervical specimens. Nucleic acid testing of urine specimens for C. trachomatis is a more sensitive and convenient method for the detection of genital infection.     

Antigen retrieval: p53 staining in benign, pre-malignant and malignant tissues of the larynx

Verification of specimens positive for Chlamydia trachomatis by enzyme immunoassay (EIA) has been recommended when testing low prevalence populations. This study compared direct fluorescent antibody (DFA) and blocking antibody (BLA) verification assays in specimens presumptively positive for C. trachomatis by the Syva Microtrak II EIA. Of 1785 specimens originally tested by EIA, 96 were presumptively positive for C. trachomatis. Verification assays were concordant in 86 specimens (69 positive, 17 negative); nine of the remaining samples gave positive results in a second EIA and one was unresolved. Both verification assays gave some false-negative results. When initial EIA absorbance values were correlated with verified results, all EIA false positive results had absorbances in the low range (less than a three-fold increase over assay cut-off values). Verification of EIA results by both DFA and BLA was effective in detecting false positive results, but confining verification to low-value positive specimens could be considered for cost effective C. trachomatis testing.     

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.     

Detection of Chlamydia trachomatis infection by PCR in first voided urines in men

The goal of this study was to evaluate whether the new commercially available PCR-based assay Amplicor C. trachomatis (Roche Molecular Systems) could improve the diagnosis of chlamydial urogenital infections in men, compared with cell culture of C. trachomatis considered as the reference method. A total of 466 men attending the STD clinic were tested by the Amplicor test in urine and by cell culture in urethra. The prevalence of C. trachomatis was 13.7% (64/466) by cell culture and 14.4% (67/466) by the Amplicor test. After resolution of the discrepant results, the sensitivity of culture was 91.4% in male urethral specimens. The resolved sensitivity of the PCR assay was 92.7% in male urine and the specificity was 99.5%. We concluded that this rapid PCR-based assay showed an improvement in quality for diagnosing C. trachomatis infections in men.     

Improvement of cervical Chlamydia detection in asymptomatic family planning attendees by using Amplicor Chlamydia trachomatis assay

We evaluated the Amplicor C. trachomatis PCR assay (Roche Molecular Systems, N.J.) for the diagnosis of cervical infection in asymptomatic women attending a family planning clinic, aged between 18 and 25 years. Culture onto McCoy cells with fluorescent monoclonal staining was the reference system. Cervical specimens from 485 women were tested. The prevalence of C. trachomatis was 10.5% by culture and 11% by Amplicor. No specimen was positive by culture and negative by PCR. Three PCR-positive, culture-negative specimens were positive by MOMP-PCR and a second plasmid-based PCR. The resolved sensitivity of PCR and culture were 100% and 94.5%, respectively. Specificities for both were 100%, positive and negative predictive values for culture were 100% and 99.3%. Total test efficiency was 99.4%. The Amplicor C. trachomatis assay gave very clear results, quite above or below the cut-off value, and showed high sensitivity and specificity, improved ease of handling and represented a good alternative to culture for large scale diagnosis of asymptomatic C. trachomatis infection.     

Evaluation of sensitivity of 10 diagnostic assays for Chlamydia trachomatis by use of a simple laboratory procedure

AIMS: To determine the sensitivity of commercially available diagnostic assays for Chlamydia trachomatis using a simple method. METHODS: Nine commercial assays and an "in-house" polymerase chain reaction (PCR) were evaluated using serial dilutions of a laboratory grown H serovar--four of them using a laboratory grown E serovar. Seven of the assays were further tested using dilutions of several cervical samples known to contain chlamydiae. RESULTS: The most sensitive assays were the MicroTrak direct fluorescent antibody (DFA) test (Syva) and the PCR which detected C trachomatis at a 10(-8) dilution of the H serovar, while the two least sensitive, Clearview (Unipath) and TestPack (Abbott), were positive only at 10(-4) and 10(-3) dilutions, respectively. A range of enzyme immunoassays (EIAs) and a nucleic acid hybridisation test were of intermediate sensitivity. The results with serovar E were consistent with these. When clinical samples were examined, the DFA test detected C trachomatis in dilutions at least 10-fold greater than any other assay. CONCLUSIONS: The range of sensitivity of diagnostic assays determined by the laboratory dilution procedure is very wide. Sensitivity assessed in this way, however, reflects the ability of the assays to detect C trachomatis in large scale clinical trials. The dilution procedure, which is simple to undertake, could therefore be applied by any laboratory before a new diagnostic method is considered for routine use.     

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.     

Confirmatory testing of Chlamydia trachomatis Syva enzyme immunoassay gray zone specimens by Syva direct fluorescent antibody test

A series of gray zone specimens with sample to cutoff ratio of 0.7 to 0.99 encountered in routine use of the Syva Microtrak (Syva, Inc.) Chlamydia trachomatis enzyme immunoassay (EIA) test for urogenital specimens were subjected to repeat testing in duplicate and high-speed centrifugation with direct fluorescent antibody testing of the centrifugate. Immunofluorescent C. trachomatis elementary bodies were observed in high-speed centrifugates in more than 40% of two series of gray zone specimens examined indicating that a C. trachomatis gray zone result would require confirmatory testing.     

Detection of Chlamydia trachomatis in first-void urine from men and women as an alternative to swabs

BACKGROUND: Ideally, an effective preventive strategy for the control of Chlamydia trachomatis infection should take into account the following attributes: rapid and simple specimen collection, low cost and noninvasive test processing. Therefore, we compared the performance profile of urine-based detection of C. trachomatis antigen in first-void urine with that of testing urethral and endocervical samples in men and women. MATERIAL AND METHODS: Urethral and endocervical samples and first-void urine from 285 men and 192 women attending the Sexually Transmitted Diseases Outpatient Clinic at the Medical University in Plovdiv, Bulgaria were tested using direct immunofluorescence assay (DFA) (MicroTrak, Syva, Palo Alto, CA, USA). RESULTS: Seventy (25%) of all men tested were positive for C. trachomatis antigen in either urethral or urine samples. 65 men (93%) had both a positive urethral and urine sample, three men (4%) had only a positive urethral sample and two (3%) had only a positive urine sample. Thirty-five women (18%) had C. trachomatis infection. Twenty-six women (74%) had both a positive endocervical and urethral sample, 6 (17%) had only a positive endocervical sample and 3 (8%) had only a positive urethral sample. All women with positive urethral samples tested positive on their urine samples. Two of the women with a negative urethral sample and a positive endocervical sample had a positive urine sample. CONCLUSIONS: These results show that using direct immunofluorescence assay on first-void urine samples is a reliable noninvasive method which can replace urethral swabs in the diagnosis of C. trachomatis infection in symptomatic men. Urine-based strategies are also an acceptable alternative for the diagnosis of C. trachomatis infection in symptomatic women when it is not possible to obtain an urogenital sample.     

Preconditioning with cromakalim improves long-term myocardial preservation for heart transplantation

Polymerase chain reaction (PCR) and ligase chain reaction (LCR) were compared for the diagnosis of Chlamydia trachomatis infections by testing urine specimens from 408 high school female students. After therapy, sequential urine specimens were tested to determine persistence of chlamydial DNA in urine. Baseline PCR of cervical specimens was positive in 53 (13.0%) students, and PCR and LCR of urine specimens were positive in 63 (15.4%) and 60 (14.7%), respectively. After discrepant analysis, 64 (15.7%) patients could be confirmed as truly infected. Follow-up urine specimens from 33 infected patients demonstrated that at 1-3 days after therapy, PCR and LCR were positive for 40% and 73.3%, respectively. Only at 15 days after therapy did all specimens test negative. Urine tests for Chlamydia organisms should not be used as a test of cure within 3 weeks after treatment. Use of urine assays for screening sexually active adolescents has the potential to significantly improve control of chlamydial infections.     

Evaluation of 2-SP transport medium for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by two automated amplification systems and culture for chlamydia

AIMS: To assess the performance of 2-sucrose-phosphate based transport medium (2-SP) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by an automated commercial polymerase chain reaction (PCR) and ligase chain reaction (LCR) compared to centrifugation culture on McCoy cells for C trachomatis. Second, to compare both amplification systems for initial diagnostic testing of a low prevalence population for sexually transmitted diseases. METHODS: Four hundred and eighty one consecutive urogenital and conjunctival specimens were examined. All tests were performed on the same specimen collected with a dacron swab and transported in 2-SP medium. Samples that were positive by culture or by both PCR and LCR were considered to be true positives. RESULTS: The prevalences of C trachomatis and of N gonorrhoeae were 2.7% and 0.4%, respectively. PCR had a resolved sensitivity and specificity of 100% and 99.8%, respectively, for C trachomatis, and 100% and 98.9%, respectively, for N gonorrhoeae. LCR was 100% sensitive and specific for both pathogens. The resolved sensitivity of the shell vial assay was 85%. No culture positive sample would have been missed by PCR or LCR. The inhibition rate for PCR was 4.8%. CONCLUSIONS: 2-SP medium proved to be suitable for both PCR and LCR. It is not limited to any one test manufacturer and allows the performance of amplification techniques and viral and chlamydia culture from the same specimen. The LCR was more reliable than PCR on initial testing. However, hands on time is longer, and no amplification control is available for LCR.     

Effects of SL-probiotic preparation on the body weight and phagocytosis of white mice

Specimens from 15 young patients presenting with acute epididymitis were tested for the presence of Chlamydia trachomatis by an enzyme immunoassay (EIA), polymerase chain reaction (PCR), and for other bacteria by standard laboratory techniques. C. trachomatis urethral infection was detected in 3 patients by an EIA test of the urethral swabs (20%) and in 13 patients by the PCR (87%). This difference in detection rate was statistically significant (p < 0.005). Thirteen specimens were positive by the PCR, but only three of them were positive by the EIA method. These findings indicate that the PCR assay is a highly sensitive assay for the detection of C. trachomatis in male urine specimens and provides a noninvasive technique for routine screening of chlamydia infection in the patient with acute epididymitis.     

An examination of the occurrence of surgical wound infection following equine orthopaedic surgery (1981-1990)

First-void urine specimens, collected from 309 military recruits, 246 male adolescent gymnasium students and 194 patients consulting venereal disease clinics, were studied for the presence of Chlamydia trachomatis with the use of antigen detection tests--two enzyme immunoassays (EIA) and a direct immunofluorescence test (DIF; Syva MicroTrak). Urethral swabs were collected when discrepancies between the EIA and DIF tests were detected. The patient was regarded as positive when the culture result was positive or when two antigen detection tests corraborated one another. The Syva MicroTrak EIA and DIF tests were more sensitive than the Orion EIA, i.e. 98.5%, 99.2% and 74%, respectively. This was true when testing both low- and high-risk groups, with a prevalence of chlamydial infection ranging from 0.4% to 58.6%. All three tests were highly specific. The positive predictive values for the Syva MicroTrak EIA, the DIF and the Orion EIA were 99.2%, 100% and 100%, respectively and the negative predictive values 99.8%, 99.8% and 94.8%, respectively.     

Multiplex AMPLICOR PCR screening for Chlamydia trachomatis and Neisseria gonorrhoeae in women attenting non-sexually transmitted disease clinics. The European Chlamydia Epidemiology Group

A new PCR kit (AMPLICOR CT/NG; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was used as a screening tool for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in first-void urine (FVU) specimens from 3,340 asymptomatic women attending European health care units for contraceptive advice or pregnancy termination. All samples were kept frozen (-20 degrees C) prior to testing. Chlamydia-positive samples were retested once by the plasmid-based PCR kit and also by a major outer membrane protein (MOMP) primer-based PCR. Discrepancies were resolved by using the direct immunofluorescence test (DIF) with the centrifuged sediment of the FVU specimens. Samples positive for N. gonorrhoeae were retested by chromosomal primer-based PCR and verified by a 16S RNA PCR. Of the samples tested, 1.8% were considered inhibitory by using the internal amplification control. Of 81 samples positive for C. trachomatis, 74 samples were positive by both plasmid- and MOMP-based PCRs, 6 samples were positive by plasmid-based PCR and DIF, and one sample was positive by both MOMP-based PCR and DIF. Nine samples (0.3%) were positive for N. gonorrhoeae by the chromosomal primer-based PCR; however, none of the results could be confirmed. The test offers the unique ability to identify inhibition of amplification with the optional internal control.     

Use of a single swab in multi-microbe or flex trans transport medium for detection of Chlamydia trachomatis by Roche Amplicor PCR and culture in specimens from two different patient populations

The Roche Amplicor PCR increased the detection of Chlamydia trachomatis compared with culture in promptly processed clinical specimens from a local clinic (100 and 86.5%, respectively) and in samples with delayed processing transported from distant facilities (100 and 72.7%, respectively). A single swab collected in culture transport medium was used. Two media, Multi-Microbe and Flex Trans, were tested and found to be equally acceptable.     

Lowering the cut off value of an automated chlamydia enzyme immunoassay and confirmation by PCR and direct immunofluorescent antibody test

AIMS: To increase the sensitivity of an automated chlamydia enzyme immunoassay by significantly lowering its cut off value, and to maintain specificity by confirmation with polymerase chain reaction (PCR) and direct immunofluorescent antibody test (DFA). METHODS: Over five months, the cut off value of the enzyme immunoassay used to screen urogenital samples for chlamydia antigen was reduced from 80 to 10. Samples with a test value of 10 or above were further tested with a commercial PCR assay. All samples during the first three months and discrepant samples during the last two months of the study were also tested with the DFA. RESULTS: 3250 urogenital swabs (1246 urethral, 1335 endocervical, 669 pooled urethral/endocervical) from 1246 males and 2004 females were processed. Using the manufacturer's recommended cut off of 80, the enzyme immunoassay identified chlamydia antigen in 134 samples (4.1%). Using the lower cut off value of 10 and either PCR or DFA as the confirmatory test, Chlamydia trachomatis was identified in 178 samples (5.5%). Thus, 45 additional positive samples were identified and the confirmed detection rate was increased by 33.8% (45/133). Excluding equivocal PCR results, the concordance between DFA and PCR was 91.8%. This strategy increased the detection rate by 2.1% in men and 0.9% in women (significant only in men). In female patients, pooled urethral/endocervical swabs as a specimen gave a significantly higher yield than endocervical swabs regardless of whether the lower cut off strategy was used. CONCLUSIONS: This strategy of significantly lowering the cut off test value with confirmation on the same specimen by either PCR or DFA is feasible and cost effective. The use of pooled urethral/ endocervical specimens in females should be considered routinely as detection rate was significantly improved.