DARWIN, LING H. MS, MBA*; CULLEN, ALLISON P. MS*; CROWE, SUSANNE R. MHA†; MODARRESS, KEVIN J. PhD*; WILLIS, DEAN E. DrPH, MPH, MS†; PAYNE, WILLIAM J. PhD*
RECOGNIZED AS TWO of the most common sexually transmitted diseases, Chlamydia trachomatis and Neisseria gonorrhoeae infections are a worldwide public health problem. Infection by either or both bacterial pathogens may lead to serious, irreversible complications if left untreated. 1 In the United States, the direct and indirect costs for chlamydial illnesses are estimated to exceed $2.4 billion annually, whereas the costs for gonococcal illnesses are estimated at $1 billion. 2 To prevent and control the spread of C trachomatis and N gonorrhoeae infections and thereby reduce the economic burden, early detection followed by proper treatment is critical. Because of the asymptomatic nature of chlamydial and gonococcal infections, routine screening of both men and women at high risk of infection is the most effective way to reduce the reservoir of infections and the risk for complications and sequelae. 1,3 Therefore, a rapid, accurate, reliable, and cost-effective diagnostic test for both C trachomatis and N gonorrhoeae infections is needed.
Conventional culture methods were once considered the most accurate means of detecting these organisms. However, culture is labor-intensive, time-consuming, and technique-dependent, all of which can have an impact on culture sensitivity. 4,5 Recently many new technologies have been developed to detect C trachomatis and N gonorrhoeae infections. Commercially available nucleic acid detection kits such as GenProbe (San Diego, CA) PACE 2 hybridization system, signal amplification–based tests such as Digene's Hybrid Capture 2 (HC2; Digene, Gaithersburg, MD), and target amplification–based tests such as PCR and LCR have been shown to give equivalent or superior performance to culture while overcoming many of the disadvantages of culture methods. 6,7 Both of the tests compared in this study, the PACE 2C and HC2 CT/GC DNA tests can be used to detect the presence of C trachomatis (CT) and/or N gonorrhoeae (GC) with use of a single specimen, which offers a cost-saving alternative for testing low-prevalence populations.
A previous study compared the HC2 CT/GC DNA tests to the PACE 2 tests for detection of C trachomatis and N gonorrhoeae in cervical swab specimens collected in GenProbe PACE 2C Transport Medium. The study showed that the HC2 tests could be performed with use of the GenProbe collection medium and were significantly more sensitive for the detection of C trachomatis than the PACE 2C test. 8 No statistical difference was observed in the detection of N gonorrhoeae.
In this study, we compared the performance of Digene's HC2 CT/GC DNA tests with the GenProbe PACE 2C tests for the detection of C trachomatis and N gonorrhoeae in male urethral swab specimens collected in GenProbe PACE 2C Transport Medium. For adjudication, each test result (HC2 and PACE 2) was evaluated by comparison with a consensus result.
Materials and Methods
Overview of Testing Algorithm and Procedure
A total of 1202 male urethral swab specimens were collected in PACE 2C specimen collection medium and tested with the PACE 2C system at the Florida Department of Health (FDH) by means of standard laboratory procedures. Specimens were obtained from a number of STD clinics, with no limitation toward symptomatic or asymptomatic patients. All specimens were first tested for C trachomatis and N gonorrhoeae with the PACE 2C test, followed by PACE 2 CT and PACE 2 GC testing of all PACE 2C–positive specimens. After completion of the PACE 2 CT and PACE 2 GC tests, the residual specimen was stored at −20 °C and shipped to Digene on dry ice for masked HC2 CT/GC testing. After comparison of the HC2 and PACE results, aliquots from specimens with discrepant CT results were shipped to the Oregon State Public Health Laboratory and tested with the GenProbe AMP CT assay, a transcription-mediated amplification (TMA) technique. A PCR/SHARP method conducted at Digene was used for resolving discrepant results for N gonorrhoeae and for those specimens that did not have sufficient residual volume for performance of the GenProbe TMA test for C trachomatis.
PACE 2 Testing
PACE 2C testing was performed at FDH on all specimens according to the manufacturer's instructions. The PACE 2C procedure was performed by pipetting a 100-μl aliquot from each GenProbe specimen into a clean tube and adding 100 μl of probe reagent, followed by a 60-minute incubation at 60 °C. After mixing with separation reagent, specimens were placed in the magnetic separation unit for 5 minutes. Each residual pellet was then washed with wash solution. Finally, after addition of the detection reagents, specimens were read on a luminometer.
The PACE 2C test results were calculated on the basis of the difference between the response in relative light units (RLU) of the specimen and the mean of the three negative reference replicates. A specimen was considered positive if the difference between the response in RLU of the specimen and the mean of the negative reference was greater than or equal to 300 RLU. All PACE 2C–positive specimens were reanalyzed with the PACE 2 CT and PACE 2 GC tests. Specimens with a volume no less than 220 μl remaining after the completion of PACE 2 tests were stored at −20 °C and shipped to Digene on dry ice for masked HC2 CT/GC DNA testing.
HC2 CT/GC Testing
Digene's HC2 is a second-generation, chemiluminescent, signal amplification–based system. All three HC2 DNA tests (CT/GC, CT-ID, and GC-ID) were performed at Digene on each of the PACE 2 specimens according to the manufacturer's instructions, with slight modification of the standard sample-processing procedure as reported previously. 8 In brief, a 220-μl aliquot from each GenProbe specimen was removed and transferred into a 2.0-ml screw-cap centrifugation tube. After the addition of 110 μl of HC2 CT/GC denaturation reagent, each specimen was denatured at 65 °C for 45 minutes. Three 75-μl aliquots from each denatured PACE 2 specimen were then transferred into 3 microtubes. Specific RNA probe cocktails were added to hybridize with denatured specimens at 65 °C for 60 minutes. Hybridization mixes were then transferred to a 96-well microplate precoated with specific antibodies that reacted with RNA:DNA hybrids during a 60-minute room temperature incubation with shaking. Immobilized complexes were secondarily bound by alkaline phosphatase–conjugated antibodies during a 30-minute incubation at room temperature. After washing of the plate to remove unbound enzyme conjugate, the RNA:DNA hybrids complexed to the enzyme were detected by measuring the amount of light emitted following the enzymatic cleavage of a chemiluminescent substrate.
To interpret the results generated with HC2 testing, the signal recorded as RLU for each specimen was divided by the positive cutoff (CO), calculated from the average RLU value of three positive calibrator replicates. Specimens are normally considered positive if the RLU/CO ratio is equal to or greater than 1.0. For specimens collected in PACE Transport Medium and tested with the HC2 tests, the CO value was adjusted by multiplying with a conversion factor of 0.75 to account for a slight signal suppression by the PACE medium. Therefore, specimens with RLU/0.75 CO values equal to or greater than 1.0 were considered positive for CT and/or GC and were subsequently verified with the HC2 CT-ID and GC-ID DNA tests.
After completion of both HC2 CT/GC and PACE 2 testing, the PACE test results obtained by FDH were forwarded to Digene. Discrepant results observed between PACE 2 and HC2 CT/GC DNA tests were resolved by either the GenProbe AMPLIFIED Chlamydia trachomatis Assay (AMP CT) or Digene PCR/SHARP assay. Two additional specimens were resolved by repeated HC2 testing (one HC2 false-positive and one HC2 false-negative).
The GenProbe AMP CT assay was performed by the Oregon State Public Health Laboratory (OSPHL). The AMP CT assay uses transcription-medicated amplification (TMA) to amplify 23S rRNA and to qualitatively detect C trachomatis. A 22-μl aliquot from each discrepant specimen (in nondenatured GenProbe collection medium) was transferred into a 0.5-ml screw-cap centrifuge tube and shipped to OSPHL on dry ice. In addition, a number of negative and positive GenProbe specimens with concordant PACE 2 and HC2 CT/GC DNA test results were sent to OSPHL for use as TMA assay controls.
The Digene PCR/SHARP assay was used for N gonorrhoeae adjudication and for those C trachomatis–discrepant specimens lacking sufficient specimen volume for TMA testing. A 75-μl aliquot from each of the denatured specimens was purified by phenol extraction, followed by ethanol precipitation. 8 After resuspension of the DNA pellet in 50 μl of TE buffer, 1-μl aliquots from each specimen were processed for PCR amplification. The two primers for C trachomatis PCR were used to amplify a 201-bp DNA fragment of the cryptic plasmid. 9 The primers for N gonorrhoeae PCR were used to amplify a 390-bp DNA sequence of the cryptic plasmid and a chromosomal sequence. 10 The PCR amplification involved 40 cycles. Each cycle consisted of three steps: a denaturation reaction for 1 minute at 95 °C, a primer annealing for 2 minutes at 55°C, and an extension reaction for 1.5 minutes at 72 °C. The SHARP Signal System (Digene) was used to detect C trachomatis and/or N gonorrhoeae amplicons according to the manufacturer's instructions.
Specimens that tested positive by the HC2 CT/GC DNA test and were verified to contain C trachomatis and/or N gonorrhoeae DNA by at least one of the HC2 CT-ID and GC-ID DNA tests were considered HC2 algorithm–positive for the specific organisms identified. Similarly, specimens that tested positive by the PACE 2C assay and by at least one of the two follow-up tests in individual C trachomatis and N gonorrhoeae assays were reported as PACE 2–positive for C trachomatis or/and N gonorrhoeae. Specimens that tested positive by at least two of the three tests (HC2, PACE 2, and TMA or PCR/SHARP) were considered consensus-positive. McNemar paired test was used for statistical evaluation, and a P value ≤0.05 was considered statistically significant.
A total of 1202 male urethral swab specimens collected in GenProbe transport medium were tested in this study by means of the three HC2 CT/GC tests (CT/GC, CT-ID, and GC-ID) and the three PACE System tests (PACE 2C, PACE 2 CT, and PACE 2 GC). As part of the routine practice at the Florida Department of Health, all 1202 specimens were initially tested with the PACE 2C test. Of those, 240 specimens were further tested by PACE 2 CT and GC tests. Among the 240 PACE 2C–positive specimens, 54 were positive for C trachomatis, 140 were positive for N gonorrhoeae, and 35 were positive for both organisms (Table 1). Eleven PACE 2C–positive specimens were not identified as either C trachomatis- or N gonorrhoeae–positive by the individual PACE 2 assays. In comparison, the HC2 CT/GC DNA test detected a total of 263 positive specimens from among the 1202 specimens analyzed, and 254 of them were verified by either CT-ID and/or GC-ID (Table 1). Of the HC2 ID test–positive specimens, 81 were positive for C trachomatis, 154 were positive for N gonorrhoeae, and 19 were positive for both organisms. Nine HC2 CT/GC–positive specimens were not identified as positive by either of the HC2 ID tests.
With the initial testing results, the positive and negative agreement between the HC2 and PACE systems for CT detection were 75.3% and 97.0%, respectively, with an overall agreement of 95.4% (Table 2). The HC2 CT/GC assay detected 67 of the 89 PACE 2 CT–positive specimens and identified as positive an additional 33 PACE 2 CT–negative specimens. Among the 33 PACE 2 CT–negative and HC2 CT–positive specimens, 31 were positive for C trachomatis when retested with an alternate method (26 were TMA-positive and 5 were PCR-positive). Among the 22 PACE 2 CT–positive and HC2 CT–negative specimens, 19 were negative by either TMA or PCR testing (12 were TMA-negative and 7 were PCR-negative) and 3 were positive for C trachomatis. On the basis of the consensus result, 70 of the 89 PACE 2 CT–positive and 98 of the 100 HC2 CT–positive specimens were true-positives. Thus, the relative sensitivity of the HC2 CT test on the basis of the consensus result was 97.0%, and the relative specificity was 99.8%. The relative sensitivity and specificity for PACE 2 CT were 69.3% and 98.3% (Table 3), respectively. The positive predictive values and negative predictive values were 98.0% and 99.7% for HC2 CT and 78.7% and 97.2% for PACE 2 CT, respectively (Table 3). For each specimen, the consensus result was determined by the interpretation common to at least two of the three testing methods (PACE 2, HC2, and TMA or PCR). The difference between the HC2 and PACE 2 CT was extremely significant in terms of relative sensitivity (P ≤ 0.0001) and specificity (P ≤ 0.0005). The prevalence rate for C trachomatis in this study population was 8.4% on the basis of the consensus result.
For detection of N gonorrhoeae, PACE 2 GC and HC2 GC had similar performance, with an initial overall agreement of 99.6%. The positive and negative agreements were 98.3% and 99.8%, respectively. HC2 CT/GC testing was able to detect 172 of 175 PACE 2 GC–positive specimens and detected 2 additional specimens positive for N gonorrhoeae. Compared with the adjudicated results, only 2 false-GC-positive specimens (1 by PACE 2 GC and 1 by HC2 GC) were identified (Table 1). Thus, the relative sensitivities and specificities were 98.9% and 99.9% for HC2 GC and 99.4% and 99.9% for PACE 2 GC, respectively (Table 3), with a 14.6% prevalence rate. The positive and negative predictive values for both methods were greater than 99%.
The results of this study demonstrated that the relative sensitivity of the HC2 tests for CT was significantly greater (P < 0.0001) than that of the PACE 2 system, and the relative sensitivity of the two methods was equivalent for GC when the same male urethral swab specimens were tested. These results are similar to those of a previous study of cervical swab specimens. 8 Among the 33 PACE 2 CT–negative and HC2 CT–positive specimens, 31 were found positive by retesting with an alternate method (26 were TMA-positive and 5 were PCR-positive). The major factor contributing to the difference in sensitivity observed between the two methods might be that the HC2 test utilizes a signal amplification–based DNA detection system. The CT probe cocktail used in the HC2 system is complementary to approximately 46,500 bp, or 5%, of the C trachomatis genome (1 × 106 bp), 11 of which an RNA probe complementary to the 7.5-kb cryptic plasmid is included. This cryptic plasmid is present in the majority of the chlamydial strains at approximately 10 copies per genome. 12–14 In contrast, the GC probe cocktail recognizes 13,800 bp, or 0.7%, of the N gonorrhoeae genome (1.9 × 106bp), 15 including an RNA probe complementary to the 4.2-kb cryptic plasmid that is present in approximately 25 copies per genome in most N gonorrhoeae strains. 16 The combination of large base coverage of the genome and the cryptic plasmid of the RNA probe cocktail and signal amplification technology enables the HC2 CT test to demonstrate clinical sensitivity equivalent to that of PCR-based tests, such as the commercial AMPLICOR Test (Roche, Branchburg, NJ). 17 In contrast, the PACE 2 system relies on detecting multicopy rRNA target sequences by hybridization only, with no amplification of the signal or target.
Although only one study of direct comparison of HC2 and PACE 2 assays has been published, 8 several studies evaluating these two assays in comparison with an adjudicated culture result have been reported. The sensitivities for the detection of C trachomatis varied from 67% to 96%5,18–20 for the PACE 2 CT test, whereas the sensitivity of HC2 CT-ID was 95% or higher. 17,21 The sensitivities for the detection of N gonorrhoeae were between 88% and 98%22–24 for PACE 2 GC and approximately 92% for HC2 GC-ID. 21,25
The relative specificities of the HC2 and PACE 2 tests for CT were significantly (P ≤ 0.0005) different (Table 3). Among 22 PACE 2 CT–positive and HC2 CT–negative specimens, 12 were TMA-negative and 7 were PCR-negative, which led to a false-positivity rate with the PACE 2 CT test of 21.3% (PPV = 78.7%). Furthermore, all 19 GenProbe CT false-positive results were for one of the 35 specimens initially characterized as CT- and GC-positive. There was no apparent reason for these observations. In comparison, two specimens identified as positive for C trachomatis by the HC2 tests were consensus-negative. The false-positivity rate for the HC2 test was therefore 2% (PPV = 98.0%). This high rate of false-positivity with the PACE CT test was not seen in the previous study with use of cervical specimens, perhaps because of the difference between the two patient populations. The prevalence rates for this study (8.4% for CT and 14.6% for GC) are significantly higher than for the previously tested population (4.2% for CT and 1.8% for GC). In addition, in the previous study, procedures at one of the two participating laboratory sites required that positivity of specimens be confirmed by means of the GenProbe Competition Assay (PCA), which may explain the differences in the results of this study and the previous study.
In conclusion, HC2 CT/GC testing of male urethral swab specimens collected in GenProbe PACE 2 medium offers a convenient means for comparing the HC2 test to the PACE 2 assay. The use of specimens collected in GenProbe PACE 2 medium requires only a slight modification of the specimen-processing procedure for the HC2 CT/GC tests. The findings of this study further demonstrate the greater sensitivity of the HC2 test in comparison with the PACE 2 test for detection of C trachomatis.
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