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.     

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.     

Is urine leukocyte esterase test a useful screening method to predict Chlamydia trachomatis infection in women?

We evaluated the use of the leukocyte esterase test (LET) on first-catch urine specimens from women as a screening test to predict infection with Chlamydia trachomatis. For diagnosis, we used Abbott's ligase chain reaction (LCR) on urine specimens and isolation by tissue culture (TC) on cervical brushes. Of 4,053 women attending sexually transmitted disease and family planning clinics, 4.3% (n = 174) were positive by TC and 5.9% (n = 239) were positive by LCR. When LET was compared to TC, the sensitivity, specificity, positive predictive value, and negative predictive value were 54.0, 67.0, 6.8, and 97.0%, respectively. The corresponding performance of LET versus LCR was 53.1, 67.3, 10.1, and 95.8%. Almost half of the laboratory-confirmed chlamydial infections were negative by LET. The low specificity probably reflects multiple causes of pyuria in women and results in a low positive predictive value. LET is neither sensitive nor specific as a predictor of chlamydial infection and cannot be recommended for use as a screening test for C. trachomatis with first-catch urine samples from females from low- or moderate-prevalence populations.     

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.     

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.     

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.     

Ligase chain reaction for detection of Neisseria gonorrhoeae in urogenital swabs

The ligase chain reaction (LCR) is an in vitro nucleic acid amplification technique that exponentially amplifies targeted DNA sequences. In a multicenter study, we evaluated the use of a 4-h LCR-based assay for the diagnosis of Neisseria gonorrhoeae infection of the cervix and male urethra. The LCR results were compared with those of culture for N. gonorrhoeae by using selective media. This assay amplifies target sequences within the N. gonorrhoeae opacity gene. Discordant LCR-positive and culture-negative specimens were further evaluated by testing by another LCR assay which used N. gonorrhoeae-specific pilin probe sets. A total of 1,539 female endocervical specimens and 808 male urethral swab specimens were evaluated in the study. An expanded "gold standard" was defined to include all culture-positive as well as culture-negative, confirmed LCR-positive specimens. After resolution of discrepant samples, the sensitivities of the N. gonorrhoeae LCR assays for the female and male specimens were 97.3 and 98.5%, respectively, with specificities of 99.6 and 99.8%, respectively. Resolved culture sensitivities were 83.9 and 96.5% for the female and male specimens, respectively. The LCR assay for gonorrhea is a rapid, highly sensitive nonculture method for detecting gonococcal infection of the cervix and male urethra.     

Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by ligase chain reaction

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a ligase chain reaction (LCR)-based assay and acid-fast stain and culture techniques. Results from the LCR assay (Abbott LCx Mycobacterium tuberculosis [MTB] Assay) were compared to results from standard culture techniques held for 6 weeks. Four hundred ninety-three specimens from 205 patients suspected of pulmonary tuberculosis were included in the prospective study. Thirty-four (6.9%) of the specimens were culture positive for M. tuberculosis, and 13 (38%) of these were also fluorochrome stain positive. LCR sensitivities and specificities compared to culture were 74 and 98%, respectively. LCR sensitivity was 100% for fluorochrome stain-positive specimens and 57% for fluorochrome stain-negative specimens. Nine LCR-negative, culture-positive specimens were the result of low concentrations of M. tuberculosis. No inhibitors were detected in any of these specimens. Of the eight LCR-positive, culture-negative specimens, five were from patients with active tuberculosis. With these considered culture misses, final LCR sensitivity, specificity, positive predictive value, and negative predictive value were 77, 99, 91, and 98%, respectively. The same performance values for the fluorochrome acid-fast bacillus smear were 33, 98, 62, and 94%, respectively. After normal laboratory sputum processing, the Abbott LCx MTB Assay can be completed in 6 h. Thus, it is possible to have results available within 8 h of specimen submission.     

Comparison of the ligase chain reaction with solid and liquid culture media for routine detection of Mycobacterium tuberculosis in nonrespiratory specimens

The aim of this study was to compare the results of a commercial assay based on the ligase chain reaction [(LCR) LCx Probe System MTB; Abbott, USA] with those of culture in liquid medium (Septi-Chek AFB; Becton-Dickinson, USA) and culture on the egg-based L?wenstein-Jensen solid medium for the direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens. The results were analyzed according to the standard definition of a true-positive result. Two hundred thirty-five nonrespiratory samples routinely submitted to rule out tuberculosis were analyzed. All samples were smear-negative. Mycobacterial growth in either culture medium was detected in 18 (7.6%) specimens: Mycobacterium tuberculosis was recovered from seven (38.9%) specimens cultured on L?wenstein-Jensen medium and from 18 (100%) specimens cultured in Septi-Chek AFB. The LCR protocol was positive in 22 specimens. None of the LCR-negative controls showed positive results. All samples positive by culture on L?wenstein-Jensen medium were positive by culture in liquid medium and by the LCR assay. However, Mycobacterium tuberculosis was detected by culture in liquid medium in two specimens that were negative by the LCR assay, whereas six specimens negative by culture in liquid medium were positive by the LCR protocol; three of these were identified as true-positive results of the LCR assay. The sensitivity, specificity, and positive and negative predictive values were 33.3%, 100%, 100%, and 93.8%, respectively, for L?wenstein-Jensen medium; 85.7%, 100%, 100%, and 98.6% for the liquid medium; and 90.4%, 98.5%, 86.3%, and 99% for the LCR assay. These findings indicate that the LCR assay may be a valid method of high diagnostic yield for direct detection of Mycobacterium tuberculosis complex in nonrespiratory specimens.     

Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens

A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.     

Quantification of Chlamydia trachomatis in cervical and urine specimens from women attending a genitourinary medicine clinic: implications for screening strategies

Population screening and intervention programmes can reduce the prevalence and incidence of infection with Chlamydia trachomatis, especially if sensitive molecular diagnostic tests are used. However, diagnostic tests that perform well on genitourinary medicine (GUM) clinic populations may be less useful for screening, particularly if the majority of infected subjects are asymptomatic and their samples contain fewer organisms. We have compared the extent of low organism load in cervical and urine samples from symptomatic and asymptomatic chlamydia-positive women, by using a direct fluorescent antibody staining method and counting the chlamydial elementary bodies (EBs). We have investigated the ability of an enzyme immunoassay (EIA; MicroTrak) and a DNA amplification (ligase chain reaction; LCR) assay to detect low numbers of organisms in cervical samples and the ability of the LCR assay to detect low numbers of organisms in urine. A low organism load (< 10 EBs) was seen by direct fluorescent antibody (DFA) staining in about 30% of cervical samples and in about 75% of urines from chlamydia-positive women; the proportions in symptomatic women were not significantly different from those in asymptomatic women. The EIA identified only 16% of cervical samples that contained < 10 EBs by DFA staining; the LCR identified 100% of cervical samples and 93% of urine samples that contained < 10 EBs by DFA staining. The findings suggest that the ability of chlamydial diagnostic tests to identify positive women should be similar among patients attending a GUM clinic and those taking part in a population screening programme, and that sensitive molecular assays such as the LCR should identify subjects with a low organism load in both groups.     

Interpretation of pooling experiments using the Markov chain Monte Carlo method

This paper describes an effective method for extracting as much information as possible from pooling experiments for library screening. Pools are collections of clones, and screening a pool with a probe determines whether any of these clones are positive for the probe. The results of the pool screenings are interpreted, or decoded, to infer which clones are candidates to be positive. These candidate positives are subjected to confirmatory testing. Decoding the pool screening results is complicated by the presence of errors, which typically lead to ambiguities in the inference of positive clones. However, in many applications there are reasonable models for the prior distributions for positives and for errors, and Bayes inference is the preferred method for ranking candidate positives. Because of the combinatoric complexity of the Bayes formulation, we implemented a decoding algorithm using a Markov chain Monte Carlo method. The algorithm was used in screening a library with 1298 clones using 47 pools. We corroborated the posterior probabilities for positives with results from confirmatory screening. We also simulated the screening of a 10-fold coverage library of 33,000 clones using 253 pools. The use of our algorithm, effective under conditions where combinatorial decoding techniques are imprudent, allows the use of fewer pools and also introduces needed robustness.     

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.     

Thymus size in infants from birth until 24 months of age evaluated by ultrasound. A longitudinal prediction model for the thymic index

To determine whether an absence of leukocytes in semen and urine predicts an absence of Chlamydia trachomatis in asymptomatic infertile men, chlamydial DNA was detected in subjects' semen and urine by ligase chain reaction (LCR). Ninety-eight infertile men were studied, including 39 cases of oligozoospermia, 19 of azoospermia, 16 of asthenozoospermia, and 24 of normozoospermia. None of the subjects had pyospermia or pyuria. C. trachomatis was detected by LCR. Antichlamydial and antisperm antibody were also measured. C. trachomatis was detected by LCR in the semen of only 1 of 98 patients (1.02%), but not in the urine samples. In C. trachomatis-positive patients LCR, IgG, and IgA levels were higher than normal. No antisperm antibody was detected. Even if leukocytes are not observed in the semen and urine of asymptomatic infertile men, the presence of C. trachomatis in semen specimens is rarely observed. Therefore, it should be noted that the presence of C. trachomatis in such cases is addressed in the context of artificial insemination and other assisted reproductive technologies.     

ECS-induced dopamine release: effects of electrode placement, anticonvulsant treatment, and stimulus intensity

BACKGROUND AND OBJECTIVES: The detection of asymptomatic urethritis using a leukocyte esterase (LE) strip may have a role in primary care screening to select men who need diagnostic testing for Chlamydia trachomatis and Neisseria gonorrhoeae. STUDY DESIGN: Eight-hundred and eighty-two men, 16 to 35 years of age were studied when they attended their family physician or university health clinic for nongenitourinary complaints. First void urine (FVU) was tested by an LE strip (Chemstrip 2 LN, Boehringer Mannheim Corp., Indianapolis, IN), Chlamydiazyme (Abbott Laboratories, N. Chicago, IL) enzyme immunoassay with confirmatory blocking and polymerase chain reaction (PCR) with chlamydial plasmid primers. RESULTS: Forty-five men (5.1%) were positive (> trace) by LE strip. Of the LE-positive urines, 9 (20.0%) were positive by EIA or PCR, and none of the LE-negatives were positive by EIA or PCR. Twenty-three LE positives (5 EIA/PCR-positive; 1 PCR-positive; 17 EIA/PCR-negative) were able to be followed with a second urine and 2 urethral swabs. All of the 6 chlamydia-positives who had follow-up tests were positive by both immunoassay and PCR on urine. Based on the FVU results, the prevalence of asymptomatic chlamydial infection was 1.0% (9/88) (95% CL, 0.5 to 1.9) for which the LE urine strip was 100% (9/9) sensitive and 95.9% (837/873) specific. Analyses based on screening 1,000 men, 16 to 25 years of age, showed that the cost per case detected was $192.00 using the LE strip (> 1+) to select urine specimens for EIA testing, compared to $1,326.00 using the EIA to test all urine specimens. CONCLUSION: In this low prevalence, primary care setting, the LE urine strip was an accurate screening test, which if used to preselect urine specimens for subsequent chlamydial testing, would be less costly per case detected than assaying each specimen for chlamydia.     

Diagnostic value of PCR for detection of Borrelia burgdorferi in skin biopsy and urine samples from patients with skin borreliosis

Skin biopsies of 36 patients with erythema migrans and acrodermatitis chronica atrophicans (ACA) before therapy and those of 8 patients after therapy were examined for Borrelia burgdorferi DNA by PCR. Skin biopsies of 27 patients with dermatological diseases other than Lyme borreliosis and those of 10 healthy persons were examined as controls. Two different primer sets targeting 23S rRNA (PCR I) and 66-kDa protein (PCR II) genes were used. PCR was performed with freshly frozen tissue (FFT) and paraffin-embedded tissue (PET). For FFT specimens of erythema migrans, 73% were positive by PCR I, 79% were positive by PCR II, and 88% were positive by combining PCR I and II. For PET specimens, PCR was less sensitive (PCR I, 44%; PCR II, 52%). For FFT specimens of ACA, PCR I was positive for two of five patients and PCR II was positive for four of five patients. B. burgdorferi was cultured from 79% of the erythema migrans specimens but not from any of the ACA lesions. Elevated B. burgdorferi antibodies were detected in sera of 74% of erythema migrans patients and 100% of ACA patients. All urine samples were negative by PCR II, whereas PCR I was positive for 27%. However, hybridization of these amplicons was negative. Sequencing of three amplicons identified nonborrelial DNA. In conclusion, urine PCR is not suitable for the diagnosis of skin borreliosis. A combination of two different primer sets achieves high sensitivity with skin biopsies. In early erythema migrans infection, culture and PCR are more sensitive than serology.     

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.