Evaluation of the Abbott LCx Ligase Chain Reaction Assay for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine and Genital Swab Specimens from a Sexually Transmitted Disease Clinic Population

The Abbott LCx ligase chain reaction (LCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae was evaluated by using swab and urine specimens from 562 patients. C. trachomatis results by LCR were compared to those by the Gen-Probe PACE 2 assay, whereas N. gonorrhoeae results by LCR were compared to those by culture. The Gen-Probe and LCR assays were performed according to the manufacturers’ instructions. Gram-negative diplococci growing on modified Thayer-Martin medium were confirmed as N. gonorrhoeae by the GonoGen II assay. Supplemental data analysis was performed by major outer membrane protein PCR for C. trachomatis and probes for pilin gene detection for N. gonorrhoeae. A true-positive result for each pathogen was defined as a positive result for all three or two of three assays. Overall agreement among the six assays was 94.8%. C. trachomatis prevalence was 16.2%; N. gonorrhoeae prevalence was 5.5%. The overall sensitivity and specificity, respectively, for each test (after supplemental data analysis) were as follows: for C. trachomatis, Gen-Probe, 65.9 and 100%; LCR on urine, 90.1 and 100%; LCR on swab specimens, 96.7 and 100%; and for N. gonorrhoeae, culture, 80.6 and 100%; LCR on urine, 93.5 and 99.8%; and LCR on swab specimens, 96.8 and 100%. For women, the N. gonorrhoeae culture was very insensitive compared to its performance in men (58.3 versus 94.7%, respectively). For C. trachomatis, the Gen-Probe assay’s sensitivity was lower for men than for women (62.3 versus 71.1%, respectively). The sensitivity for C. trachomatis detection by LCR on urethral and cervical swab specimens was 96.2 and 97.4% for men and women, respectively. For men, swab results were slightly better than urine results for both pathogens (sensitivity for C. trachomatis in swab and urine specimens, 96.2 and 92.5%, respectively; sensitivity for N. gonorrhoeae in swab and urine specimens, 100 and 94.7%, respectively), while for women, cervical swabs were superior in sensitivity to urine samples for detecting C. trachomatis (swab, 97.4%; urine, 81.6%) and equivalent for N. gonorrhoeae (swab, 92.3%; urine, 91.6%). The LCx LCR appears to be both sensitive and specific for the detection of C. trachomatis and N. gonorrhoeae when performed on urine or genital swab samples. Swab samples had better sensitivity than urine samples for the detection of both pathogens.     

Performance of the Abbott RealTime CT/NG for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae

A multicenter clinical study was conducted to evaluate the performance characteristics of the Abbott RealTime CT/NG assay, a multiplex real-time PCR assay, for simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The specimens were collected from a total of 3,832 male and female subjects at 16 geographically diverse sites. Specimens included male and female urine samples, male urethral swabs, female endocervical swabs, and self-collected and clinician-collected vaginal swabs. Specimens were tested with the automated Abbott RealTime CT/NG assay, Aptima Combo 2 assay (Gen-Probe), ProbeTec ET CT/GC assay (Becton Dickinson), and culture for N. gonorrhoeae. The Aptima Combo 2 assay, the ProbeTec assay, and the N. gonorrhoeae culture were used as the reference assays. For each subject, a patient infected status (PIS) was determined based on the combined results from the reference assays. The overall prevalence in female subjects was 8.9% for C. trachomatis and 3.8% for N. gonorrhoeae. The overall male prevalence was 18.2% for C. trachomatis and 16.7% for N. gonorrhoeae. The overall sensitivity and specificity of the Abbott RealTime CT/NG assay were 92.4% and 99.2% for C. trachomatis and 96.9% and 99.7% for N. gonorrhoeae, respectively. In comparison, the sensitivity and specificity, respectively, for the Aptima Combo 2 assay were 94.5% and 99.0% for C. trachomatis and 96.1% and 99.5% for N. gonorrhoeae, and those for the ProbeTec ET assay were 90.3% and 99.5% for C. trachomatis and 92.0% and 97.3% for N. gonorrhoeae in this study. The Abbott RealTime CT/NG assay offers C. trachomatis and N. gonorrhoeae dual detection with high sensitivity and specificity. The automated assay provides a useful alternative nucleic acid amplification assay for clinical laboratories and clinicians.Chlamydia trachomatis and Neisseria gonorrhoeae are the two most prevalent bacterial sexually transmitted infections (STIs) reported to the Centers for Disease Control and Prevention (CDC) (2). Together these two infections accounted for over 1.5 million cases of STIs in 2008. The CDC estimates that sexually transmitted diseases (STDs) cost the health care system $1.5 billion annually. Since these infections, especially chlamydia, are most often asymptomatic, the CDC recommends yearly screening for chlamydia in all sexually active women ages 16 to 25 years. Since coinfections are common, many diagnostic test platforms assay for both organisms. Although there are several commercial assays available for performing nucleic acid amplification tests (NAATs), which are now recommended by the CDC as the test of choice, new assays and new platforms are needed by both private and public health programs.The Abbott RealTime CT/NG assay is a new real-time PCR assay for the direct, qualitative detection of the plasmid DNA of Chlamydia trachomatis and the genomic DNA of Neisseria gonorrhoeae in female endocervical or vaginal swab specimens, in male urethral swab specimens, and in male and female urine specimens from symptomatic and asymptomatic individuals. There were several sets of objectives of this study: (i) to determine the performance characteristics of the Abbott RealTime CT/NG assay compared to those of other amplified assays using specimens obtained from symptomatic and asymptomatic male and female subjects from clinical sites with high or low prevalence of Chlamydia trachomatis and/or Neisseria gonorrhoeae; (ii) to demonstrate the precision of the Abbott RealTime CT/NG assay using a precision panel consisting of combinations of high and low concentrations of Chlamydia trachomatis and Neisseria gonorrhoeae specimens; (iii) to evaluate the clinical sensitivity, clinical specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Abbott RealTime CT/NG assay in symptomatic and asymptomatic individuals from high- and low-prevalence sites; (iv) to compare the performance of the new Abbott assay with that of the Gen-Probe Aptima Combo 2 assay for female endocervical and vaginal swabs, male urethral swabs, and male and female urine specimens; (v) to compare the performance of the Abbott assay with those of the Becton Dickinson ProbeTec ET Chlamydia trachomatis and Neisseria gonorrhoeae amplified DNA assays (ProbeTec ET) for female endocervical swabs and male and female urine specimens; and (vi) to evaluate the procedure for self-collection of a vaginal swab specimen using the Abbott multi-Collect specimen collection kit.     

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.     

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.     

Pooling of Urine Samples for Screening for Neisseria gonorrhoeae by Ligase Chain Reaction: Accuracy and Application

The accuracy of detection of genital Neisseria gonorrhoeae infection in pooled urine samples by ligase chain reaction (LCR) was examined in three populations. Firstly, urine specimens from 300 female military recruits (FMR) were tested by LCR individually and in pools of four and six. Secondly, 300 urine specimens from middle-school students (MSS) were tested individually by LCR, and then the processed specimens were stored frozen for subsequent testing in pools of 4 and 10. Thirdly, 600 frozen urine specimens from high-school students (HSS) were tested by using the LCR pooling algorithm, i.e., testing processed specimens in pools of four in one test unit dose, and retesting individual specimens from positive pools. Finally, the pooling algorithm results were compared to culture results for a subset of 344 students from the original 600 HSS from whom cervical or urethral samples were taken at the discretion of the school nurse practitioners. Compared to individual testing of specimens by LCR in the FMR population, the pooling-by-four algorithm was 100% sensitive (5 of 5) and 100% pool specific (70 of 70), and the pool-by-six algorithm was 100% sensitive (5 of 5) and 100% pool specific (45 of 45). In the MSS population, the pool-by-4 algorithm was 95.8% sensitive (23 of 24) and 100% (52 of 52) pool specific, and the pool-by-10 algorithm was 95.8% sensitive (23 of 24) and 100% (17 of 17) pool specific. In the subset of 344 HSS from whom endocervical or urethral specimens were collected for culture, 31 were positive by LCR in urine and 26 were positive by culture. After results discrepant between culture and LCR were adjudicated by a confirmatory LCR test, the pooling algorithm was 93.8% (30 of 32) sensitive and 99.7% (311 of 312) specific. Culture from these 344 HSS was 81.3% (26 of 32) sensitive. The pooling algorithm reduced the cost of the N. gonorrhoeae LCR assay by 60% compared to individual testing of the HSS specimens and was both sensitive and specific.     

Evaluation of the Abbott LCx Mycobacterium tuberculosis Assay for Direct Detection of Mycobacterium tuberculosis Complex in Human Samples

Seven hundred thirty-seven clinical samples from 460 patients were processed for direct detection of Mycobacterium tuberculosis complex by a semiautomated ligase chain reaction commercial assay, the LCx Mycobacterium tuberculosis Assay (LCx assay) from Abbott Laboratories. Results were compared to those of direct microscopy and standard microbiological culture. Of 26 patients (5.7%) with a culture positive for M. tuberculosis, 22 (84.6%) were found positive by the LCx assay. The sensitivity of the LCx assay was 98% for smear-positive samples and 27% for smear-negative samples. With an overall culture positivity rate for M. tuberculosis of 8.3% (61 of 737 samples) and after resolution of discrepant results according to clinical data, the sensitivity, specificity, and positive and negative predictive values of the LCx assay were 78, 100, 95, and 98%, respectively, compared to 85, 100, 100, and 98%, respectively, for culture and 67, 99, 87, and 97%, respectively, for acid-fast staining. In conclusion, the LCx assay proved satisfactory and appears to be an easy-to-use 1-day test which must be used with standard culture methods but can considerably reduce diagnosis time versus culture. However, its clinical interest appears to be limited in our population with low mycobacterial prevalence because of its cost considering the small gain in sensitivity versus direct microscopy.     

Comparative Evaluation of Chlamydiazyme, PACE 2, and AMP-CT Assays for Detection of Chlamydia trachomatis in Endocervical Specimens

We conducted a comparative evaluation of the Chlamydiazyme (Abbott Laboratories), PACE 2 (Gen-Probe), and AMP-CT (Gen-Probe) assays for the detection of Chlamydia trachomatis in endocervical samples. Specimens from 787 females were included in the study. The sensitivities of the PACE 2 and Chlamydiazyme assays in comparison to the results of the AMP-CT assay were 79.3 and 63.4%, respectively. The specificities of the Chlamydiazyme and PACE 2 assays were 100%. All of the positive specimens detected in this study were positive by the AMP-CT assay. On the basis of the final results of the comparison, the prevalence of C. trachomatis in the population was 10.4%. Retesting of specimens whose results were in the intermediate zone by the PACE 2 assay by a probe competition assay identified some additional true-positive specimens. Amplification assay testing of such specimens did not significantly increase the yield. The majority of specimens which tested positive by the AMP-CT assay only were not in the intermediate zone by the PACE 2 assay. We were unable to identify demographic or clinical factors which could predict those individuals who tested positive by amplified tests but not by nonamplified tests. The Gen-Probe PACE 2 assay proved to be superior to the Chlamydiazyme assay for the screening and diagnosis of C. trachomatis infections in female endocervical specimens.     

Use of Ligase Chain Reaction with Urine versus Cervical Culture for Detection of Chlamydia trachomatis in an Asymptomatic Military Population of Pregnant and Nonpregnant Females Attending Papanicolaou Smear Clinics

Ligase chain reaction (LCR) (Abbott Laboratories, Abbott Park, Ill.) with first-catch urine specimens was used to detect Chlamydia trachomatis infections in 465 asymptomatic military women attending clinics for routine Papanicolaou smear tests. Results were compared to results of cervical culture to determine the sensitivity of the urine LCR and the possible presence of inhibitors of amplification in pregnant and nonpregnant women. Discrepant results for LCR and culture were resolved by direct fluorescent antibody staining of culture sediments, two different PCR assays, and LCR for the outer membrane protein 1 gene. The prevalence of Chlamydia in specimens by urine LCR was 7.3% compared to 5% by culture. For 434 women with matching specimens, there were 11 more specimens positive by LCR than were positive by culture, of which all but one were determined to be true positives. There were four culture-positive, LCR-negative specimens, all from nonpregnant women. The sensitivity, specificity, and positive and negative predictive values of urine LCR after discrepant results were resolved were 88.6, 99.7, 96.9, and 99.0%, respectively. The sensitivity of culture was 71.4%. From the 148 pregnant women (prevalence by LCR, 6.8%), there were no patients who were cervical culture positive and urine LCR negative to indicate the presence in pregnant women of inhibitors of LCR. Additionally, a subset of 55 of the LCR-negative frozen urine specimens from pregnant women that had been previously processed in LCR buffer were inoculated with 5 cell culture inclusion forming units of C. trachomatis each and retested by LCR; all tested positive, indicating the absence of inhibitors of LCR in urine from these pregnant women. The use of LCR testing of urine specimens from asymptomatic women, whether pregnant or not, offers a sensitive and easy method to detect C. trachomatis infection in women.     

Rapid Diagnosis of Extrapulmonary Tuberculosis by Ligase Chain Reaction Amplification

A rapid amplification-based test for the diagnosis of extrapulmonary tuberculosis, the LCx Mycobacterium tuberculosis Assay from Abbott Laboratories, was evaluated. Results from the LCx M. tuberculosis Assay were compared with those from culture and the final clinical diagnosis for each patient. A total of 526 nonrespiratory specimens from 492 patients were tested. The specimens included urine; feces; lymph node exudates; pleural, cerebrospinal, articular, and ascitic fluids; tissue biopsies; gastric aspirates; purulent exudates; blood; and bone marrow aspirates. After combination of the culture results and the patient’s clinical data, a total of 135 specimens were collected from 122 patients with a diagnosis of extrapulmonary tuberculosis. The sensitivity, specificity, and positive and negative predictive values for the LCx M. tuberculosis Assay were 77.7, 98.7, 95.2, and 93.1%, respectively; these values rose in resolved cases of TB to 78.5, 100, 100, and 93.1%, respectively. For 37 (27.4%) specimens from patients smear positive for the disease and 98 (72.6%) specimens from patients smear negative for the disease, the sensitivities of the LCx M. tuberculosis Assay were 100 and 71.1%, respectively. Statistically significant differences (P < 0.01) in sensitivities were found between culture and the LCx M. tuberculosis Assay. These differences were even greater among smear-negative specimens. The results demonstrate that the LCx M. tuberculosis Assay will provide rapid and valuable information for the diagnosis of extrapulmonary tuberculosis.     

Comparative Evaluation of the New Gen-Probe Mycobacterium tuberculosis Amplified Direct Test and the Semiautomated Abbott LCx Mycobacterium tuberculosis Assay for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory and Extrapulmonary Specimens

Two commercial assays that detect Mycobacterium tuberculosis complex (MTB) in clinical specimens by rRNA target amplification (AMTDII) and ligase chain reaction (LCx) were evaluated. The tests were applied to 457 respiratory (n = 273) and extrapulmonary (n = 184) specimens collected from 357 patients. The results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered to be the “gold standard.” Seventy specimens were from patients with pulmonary tuberculosis and 28 specimens were from patients with extrapulmonary tuberculosis. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens were 92.8, 99.4, 98.5, and 97%, respectively, for AMTDII and 75.7, 98.8, 96.4, and 90.5%, respectively, for LCx. With extrapulmonary specimens, the overall sensitivities, specificities, and positive and negative predictive values were 78.6, 99.3, 95.6, and 96.2%, respectively, for AMTDII and 53.6, 99.3, 93.7, and 92.1%, respectively, for LCx. The level of agreement between AMTDII and LCx assay results was 78.2%. We conclude that although both nucleic acid amplification methods are rapid and specific for the detection of MTB in clinical specimens, AMTDII is significantly more sensitive than LCx with both respiratory (P = 0.005) and extrapulmonary (P = 0.048) specimens.     

Rapid Diagnosis of Pulmonary Tuberculosis with the LCx Mycobacterium tuberculosis Assay and Comparison with Conventional Diagnostic Techniques

The LCx MTB amplification assay is a nucleic acid amplification test intended for the direct detection of Mycobacterium tuberculosis complex in respiratory specimens. We evaluated its performance on 2,001 consecutive respiratory specimens; 78 were culture positive for M. tuberculosis. Sensitivity, specificity, and positive and negative predictive values of this assay for all specimens compared to culture results were 88.5, 97.7, 60.5, and 99.5%, respectively. When referred to resolved clinical diagnosis of active tuberculosis, these values improved to 90.2, 98.4, 72.8, and 99.5%, respectively.     

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 Löwenstein-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 Löwenstein-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 Löwenstein-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 Löwenstein-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.     

Genus Level Identification of Mycobacteria from Clinical Specimens by Using an Easy-To-Handle Mycobacterium-Specific PCR Assay

An easy-to-handle Mycobacterium-specific PCR assay for detection of the presence of a wide range of mycobacterial species in clinical samples was evaluated. The performance of the genus probe was compared with the performance of probes specific for Mycobacterium tuberculosis and Mycobacterium avium and with that of standard culture. In addition, the utility of an internal control in monitoring amplification inhibitors was studied. Of 545 respiratory and 325 nonrespiratory specimens (a total of 870 specimens), 58 (6.7%) showed the presence of amplification inhibitors, as determined by a negative result for the internal control. Of these 58 specimens, 31 (53%) were stool specimens; other material, even citrate blood after lysis of erythrocytes, did not pose a problem with regard to inhibition of PCR amplification. Eighty-one of the remaining 812 specimens had a positive Mycobacterium culture result. Of these culture-positive specimens, 58 (71.6%) showed a positive result with the Mycobacterium genus-specific probe. Seventy-two samples had a positive result with the Mycobacterium-specific probe but a negative culture result. Of these 72 samples, 26 samples were regarded as true positive, either because the M. tuberculosis- or M. avium-specific probe was also positive at the same time or because other specimens from the same patient taken at the same time were culture positive. The sensitivity of the Mycobacterium-specific probe was 78.5% and the specificity was 93.5%. This study showed that pretesting of clinical specimens for mycobacteria to the genus level with a Mycobacterium-specific probe offers the routine clinical laboratory the possibility of detecting tuberculous and nontuberculous mycobacteria with one test. Furthermore, specimens testing positive with the genus-specific probe can be immediately identified with species-specific probes.     

Development and evaluation of a molecular assay for detection of nontuberculous mycobacteria by use of the cobas amplicor platform

We have developed and evaluated a semiautomated assay for detection of nontuberculous mycobacteria (NTM) from clinical samples based on the Cobas Amplicor Mycobacterium tuberculosis test (Roche Diagnostics, Switzerland). A capture probe, specific for mycobacteria at the genus level, was linked to magnetic beads and used for the detection of amplification products obtained by the Cobas Amplicor M. tuberculosis assay. We demonstrate that the analytical sensitivity of the genus assay is similar to that of Cobas Amplicor M. tuberculosis detection. Four hundred sixteen clinical specimens were evaluated for the presence of NTM DNA. Sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. Specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. The genus assay is easy to perform, produces reliable results, and was found to be a valuable diagnostic tool for rapid diagnosis of infections with NTM. The genus assay has the potential to detect NTM not routinely recovered by culture and to discover new mycobacterial species.     

Performance Characteristics of the BDProbeTec System for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens

Strand displacement amplification (SDA) technology has been established in a fully automated system known as BDProbeTec. Target sequences of the insertion sequence IS6110 and the 16S rRNA gene are simultaneously amplified, which thus allows the detection of Mycobacterium tuberculosis complex and, as an additional option, of most Mycobacterium species. Detection occurs via a chemiluminescent microwell assay that employs the simultaneous hybridization and capture of SDA products with a biotinylated capture probe and an alkaline phosphatase detector probe. We have evaluated the performance of the BDProbeTec system in detecting M. tuberculosis complex by testing 799 respiratory specimens and comparing the results to those obtained by conventional diagnostic techniques, i.e., microscopy and culture (solid and radiometric media). M. tuberculosis was cultivated from 41 specimens, of which 28 (68.4%) were smear positive and 13 (31.6%) were smear negative. The overall sensitivity of the SDA assay was 97.6% (for smear-positive specimens, 100%; for smear-negative specimens, 92.3%), and specificity was 95.0%. After resolution of the discrepancies by studying the patients’ clinical data, sensitivity and specificity were 97.9 and 96.5%, respectively, and positive and negative predictive values were 63.9 and 99.9%, respectively. These preliminary data demonstrate that the BDProbeTec system has promising performance characteristics with respiratory specimens and that it allows the detection of M. tuberculosis complex within hours.     

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.     

Multicenter Evaluation of the Fully Automated COBAS AMPLICOR PCR Test for Detection of Chlamydia trachomatis in Urogenital Specimens

The fully automated COBAS AMPLICOR CT/NG test for the detection of Chlamydia trachomatis was evaluated in a multicenter trial. Test performance was evaluated for 2,014 endocervical swab and 1,278 urine specimens obtained from women and for 373 urethral swab and 254 urine specimens obtained from men. Culture served as the reference test. Culture-negative, COBAS AMPLICOR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the C. trachomatis major outer membrane protein gene were resolved as true positives. The overall prevalence of chlamydia was 4.3% in cervical swabs and 11.0% in urethral swabs from men. When the results for each specimen type were considered separately, the resolved sensitivities were 96.5% (83 of 86) for endocervical swab specimens, 95.1% (39 of 41) for urine specimens from women, 100.0% (41 of 41) for urethral swab specimens from men, and 94.4% (17 of 18) for urine specimens from men; the resolved specificities were 99.4% (1,912 of 1,924) for endocervical swab specimens, 99.8% (1,204 of 1,207) for urine specimens from women, 98.5% (325 of 330) for urethral swab specimens from men, and 100.0% (236 of 236) for urine specimens from men. For the subset of patients from whom both swab and urine specimens were collected, the combined results for both specimen types were used to identify all infected patients. Using these combined reslts as criteria, the resolved sensitivities for the COBAS AMPLICOR test were 82.6% (38 of 46) for endocervical swab specimens, 84.4% (38 of 45) for urine specimens from women, 84.2% (16 of 19) for urethral swab specimens from men, and 89.5% (17 of 19) for urine specimens from men. In comparison, the sensitivity of culture was only 56.5% (26 of 46) for endocervical specimens and 63.2% (12 of 19) for urethral specimens from men. The internal control provided in the COBAS AMPLICOR test revealed that 2.9% of specimens were inhibitory when they were initially tested. Nevertheless, valid results were obtained for 99.1% of specimens because 68.7% of the inhibitory specimens were not inhibitory when a second aliquot of the original sample was tested. Two additional COBAS AMPLICOR-positive specimens were detected by retesting inhibitory specimens. The COBAS AMPLICOR CT/NG test for the detection of C. trachomatis exhibited equally high sensitivities and specificities with both urogenital swab and urine specimens and, thus, is well-suited for use in screening.     

Evaluation of AMPLICOR Neisseria gonorrhoeae PCR Using cppB Nested PCR and 16S rRNA PCR

Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence (~9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.     

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.