Sexually Transmitted Diseases:
The Effect of Urine Testing in Evaluations of the Sensitivity of the Gen-Probe APTIMA®Combo 2 Assay on Endocervical Swabs for Chlamydia trachomatis and Neisseria gonorrhoeae: The Infected Patient Standard Reduces Sensitivity of Single Site Evaluation
MONCADA, JEANNE MT*; SCHACHTER, JULIUS PHD*; HOOK, EDWARD W. III MD†; FERRERO, DENNIS MPH‡; GAYDOS, CHARLOTTE DRPH§; QUINN, THOMAS C. MD§; WILLIS, DEAN PhD||; WEISSFELD, ALICE PhD¶; MARTIN, DAVID H. MD**
From the *University of California, San Francisco, California; the †University of Alabama, Birmingham, Alabama; ‡San Joaquin County Public Health Service, Stockton, California; §Johns Hopkins University, Baltimore, Maryland; || Florida Department of Heath, Jacksonville, Florida; ¶Microbiology Specialist Inc., Houston, Texas; and **Louisiana State University, New Orleans, Louisiana
This work was supported by Gen-Probe, Inc. (San Diego, CA).
Correspondence: Jeanne Moncada, MT, Chlamydia Laboratory at SFGH, UCSF Department of Laboratory Medicine, Bldg. 30, Rm. 416, San Francisco, CA 94110. E-mail: firstname.lastname@example.org
Received for publication August 5, 2003, revised October 21, 2003, and accepted December 18, 2003.
Background: The Gen-Probe APTIMA Combo 2 (AC2) assay is a second-generation transcription-mediated amplification assay for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG).
Goal: The goal of this study was to evaluate AC2 performance of endocervical (cx) swabs for the detection of CT and NG using either a specimen or an infected patient standard.
Study Design: In a multicenter clinical study, we compared AC2 with Abbott’s ligase chain reaction (LCR) and Roche’s polymerase chain reaction (PCR; Amplicor or COBAS) for CT, and we compared AC2 with Abbott’s LCR and culture for NG. A total of 1569 females were enrolled in the study; we collected cx and first-catch urine (FCU) specimens.
Results: CT prevalence was 13.3% for cx specimens and 13.7% for FCU specimens. NG prevalence was 8.7% and 7.9% for cx and FCU specimens, respectively. When based only on cx specimens, AC2, LCR, and PCR sensitivities for CT were 99.4%, 95.6%, and 95.6%, respectively. However, cx sensitivity for CT was reduced to 92.1%, 86.6%, and 87.1% for each respective assay when based on both cx and FCU specimen results (infected patient standard). NG sensitivities for AC2, LCR, and culture based solely on cx specimen results were 99.2%, 96.1%, and 85.9%, respectively. Based on infected patient standard, the sensitivities of each respective assay were 98.5%, 93.9%, and 84.0%.
Conclusions: The infected patient standard reduces the sensitivity of the endocervical evaluation because some infected patients are positive only with FCU. The reduction in sensitivity is greater when testing for CT. Specificities improved slightly, because some unique cx positives, initially classified as false-positive were confirmed by a positive FCU result. Sensitivity of AC2 was higher than LCR, PCR, and culture. Specificity was slightly lower, but discrepant analysis (using alternate TMA targets) of apparent AC2 false-positives showed that 75% to 80% were true-positives.
CHLAMYDIA TRACHOMATIS (CT) and Neisseria gonorrhoeae (NG) are the 2 most common bacterial causes of sexually transmitted disease (STD) in the United States. Approximately 3 million new cases of C. trachomatis infections and 1 million new cases of gonorrhea occur annually in the United States. 1 Asymptomatic infections are common in both men and women. If untreated, these infections can lead to epididymitis in men and pelvic inflammatory disease and infertility in women. In addition, newborn infants exposed during passage through an infected birth canal can develop chlamydial pneumonia. Currently, the Centers for Disease Control and Prevention (CDC) recommends that sexually active teen and adult females 24 years of age or younger be routinely screened for both organisms. Accurate laboratory tests for the detection of CT and NG are needed.
Although there are several laboratory methods used to detect these organisms, historically, the gold standards were isolation of the organisms. For CT, this was isolation in tissue culture (TC), a labor-intensive, time-consuming and costly test. 2,3 For NG, selective media were used to isolate oxidase-positive Gram-negative diplococci that were then identified as NG by sugar utilization or other confirmatory tests. 4 Specimen handling is critical for successful isolation of these organisms. Optimal NG culture requires incubation (36°C) of Thayer-Martin plates in CO2 or a candle jar immediately after specimen collection, and maintenance of a cold chain and a sensitive cell line are essential for CT culture. A major disadvantage of both procedures is that neither is suited for mass screening. For CT diagnosis, enzyme immunoassay, direct fluorescent antibody, and DNA hybridization tests provided more rapid and less expensive alternatives to TC. 5–7 However, these assays are less sensitive than optimally performed culture.
With the introduction of nucleic acid amplified tests (NAATs), there was a marked improvement in the detection of CT and NG. Several NAATS such as polymerase chain reaction (PCR), strand displacement amplification (SDA), and transcription-mediated amplification (TMA) tests are commercially available. 8–11 The ligase chain reaction (LCR) assay has recently been withdrawn by Abbott Laboratories (Abbott Park, IL). These NAATs are more sensitive than previously available diagnostic tests for CT or NG. Furthermore, with NAATs, 1 urogenital specimen can be used to screen for both CT and NG. Outstanding performance profiles have been seen with both endocervical (cx) and male urethral swab specimens. 12,13 First-catch urine (FCU), a noninvasive alternative to swab specimens, has also been found to be an appropriate specimen for NAAT testing in asymptomatic males and females, 9,14–18 and its use in screening has become routine.
Although NAATs are highly sensitive and specific, each NAAT has limitations. PCR can be affected by inhibitors that reduce sensitivity in endocervical specimens, 19 and all of the NAATs have shown reduced sensitivities with FCU specimens. 20 Optional use of internal controls (IC) with PCR and SDA has helped to identify inhibitory specimens. LCR had low throughput, which restricted its use in high-volume laboratories (this test was withdrawn because of manufacturing and quality-control problems). The Gen-Probe APTIMA Combo 2 (AC2) assay (Gen-Probe Inc., San Diego, CA) is a second-generation TMA assay for the detection of CT and NG. 15,21 It has incorporated the technology of target capture to isolate the target from inhibitors present in the sample. 22 In this assay, target rRNA molecules are released into the transport medium and then isolated from the specimen by capture onto magnetic particles. The TMA reaction replicates a specific region of the 23S rRNA from C. trachomatis and the 16S rRNA from N. gonorrhoeae through DNA intermediates. Detection of the product sequences occurs by hybridization with single-stranded chemiluminescent DNA probes.
Chlamydiae and gonococci can infected multiple anatomic sites. Therefore, single-site specimen evaluations (tests performed on only 1 type of specimen) will overestimate the performance of a test in identifying infected individuals, whereas a reference standard that includes both swab and FCU (tests performed on 2 different types of specimens) will give a better indication of the performance of a test. The latter is known as the “infected patient” standard. Some clinical evaluations have taken this into account. However, other early clinical evaluations compared NAAT performances with culture from a single site and discrepant analysis. Although this method has been criticized as statistically biased, 23,24 the CDC 455 studies 12,13 confirmed the outstanding NAAT performance using independent reference standards.
In a multicenter evaluation, we compared AC2 with LCR (LCx; Abbott Laboratories) and PCR (Amplicor CT/NG or COBAS; Roche Diagnostics, Branchburg, NJ) for CT, and with LCR and culture for NG. Overall performance with cx swabs was calculated 2 ways, first based on cx specimen standard and then on the subject’s infected patient standard, which was based on the combined results of cx and FCU specimens.
Materials and Methods
Symptomatic and asymptomatic female patients were screened at 7 geographically diverse clinical sites across the United States. Locations were in Stockton and San Francisco, CA, Birmingham, AL, Baltimore, MD, Jacksonville, FL, Houston, TX, and New Orleans, LA. Patients were seen at STD, family planning, and OB/GYN clinics with high and low prevalence of CT and NG infections. From March to August 2000, a total of 1569 females were enrolled in the study.
Either verbal or written informed consent was obtained from each subject. Thereafter, each enrolled subject provided approximately 25 mL of FCU in a urine container. Then, the endocervical canal was cleaned by the removal of discharge with large swabs or sponges. Four cx swabs were collected. The first cx swab (cotton), for NG culture, was streaked onto a Thayer-Martin plate that was put into a candle jar at 36°C. The remaining 3 cx swabs (Dacron) were randomized for AC2, LCR, and PCR. Each of the Dacron swabs was rubbed against the endocervical canal, avoiding contact with any vaginal surface. These NAAT samples were placed into the appropriate transport media and held at 4°C for ≤72 hours until transport to the laboratory for testing. Technologists performing all laboratory testing were masked to the results of other assays.
Neisseria gonorrhoeae Culture
Thayer-Martin plates were read within 48 hours. Oxidase-positive colonies yielding Gram-negative diplococci were subcultured to chocolate agar plates. Isolates were confirmed as NG by either sugar utilization tests (API QuadFerm, Analytab Products), fluorescent antibody (Syva MicroTrak N. gonorrhoeae Culture Confirmation Reagent, Behring Diagnostics Inc.), or HNID (Dade Berhring Microscan).
Ligase Chain Reaction Assay
The LCR (LCx; Abbott Laboratories) reaction has been previously described. 11,25 The targets are the cryptic plasmid of CT and the Opa gene of NG. Specimens were tested in batches within 4 days of collection. For sample preparation, FCUs were microfuged and the pellet resuspended in buffer. The processed FCU and cx swab specimens were placed in a heat block at 100°C for 15 minutes. For DNA amplification, 100 μL of each sample and controls were added to microfuge tubes containing a predispensed LCx reaction mix of 4 oligonucleotide probes, polymerase, and a thermostable enzyme (ligase). A separate amplification was performed for CT and for NG. The tubes were inserted into a Perkin-Elmer thermocycler programmed for 40 cycles. Separate microparticle enzyme immunoassays were then run to detect amplicons for CT and NG.
Polymerase Chain Reaction Assay
There are 2 versions of the PCR test (Amplicor CT/NG and COBAS; Roche Diagnostics). Two sites used the COBAS instrument application and 5 sites used the Amplicor CT/NG test with microwell plates. All test platforms used the inhibition control version of the CT test. 17 Specimens were tested within 7 days of collection. Processed FCUs, swabs, and controls were tested according to the manufacturer’s specifications. Briefly, 50 μL of controls and specimens were placed into an amplification plate or an “A” ring along with the master mix containing primers, nucleotides, and enzymes. The CT primers consisted of a 207-nucleotide sequence within the cryptic plasmid of CT. The internal control was a DNA plasmid with primer-binding regions identical to those of the CT target sequence. For Amplicor, the samples were placed in a GeneAmp PCR System 9600 for 36 cycles. After thermocycling, amplicons were denatured and hybridized to specific oligonucleotide probes. Two separate detections (CT and IC) were done using avidin– horseradish peroxidase as the conjugate and tetramethylbenzidine as the substrate. The plates were read at 450 nm. For COBAS, the automated instrument performed the thermocyling, hybridization, and amplicon detection.
Gen-Probe APTIMA Combo 2 Assay
AC2 swabs and FCU specimens were tested according to Gen-Probe’s specifications. 15,21 A 2-mL aliquot of FCU was transferred into an AC2 urine transport tube within 24 hours of collection. Swab and FCU specimens were kept at either room temperature or 4°C. Specimens were tested within 7 days of collection. Briefly, 400 μL of controls and specimens were placed in a tube containing target capture reagent. After incubation and magnetic separation, the amplification reagent that targets CT and NG was added. Tubes were incubated at 61°C for 10 minutes and then transferred to 42°C for 5 minutes. The enzyme reagent (reverse transcriptase and RNA polymerase) was added; tubes were shaken and reincubated at 42°C for 60 minutes. Gen-Probe’s standard Hybridization Protection Assay was performed. The chemiluminescent detection reaction was read on a Leader HC+ luminometer. The differences in kinetic profiles of the CT and NG probes enabled simultaneous readings as a dual kinetic assay. Curve shape of the light-off reaction, as well as total relative light units, was used to determine the presence of CT and NG.
Determination of Endocervical Specimen Standard
CT true-positives (TP) were defined as cx specimens that were positive by any 2 of the 3 amplification tests (LCR, PCR, or AC2.) NG true-positives were defined by cx specimens that were either culture-positive or positive with both the LCR and AC2 amplification tests.
Determination of Infected Patient Standard
A patient was considered infected with CT when 2 or more positive results were obtained by 2 different amplification methods with either the cx swab or FCU specimen. Thus, to be scored as a positive, a patient had to have, as a minimum, FCU and swab or 2 FCUs or 2 swabs positive (at least 2 of the 6 possible tests), and each of the 2 positive tests had to be with at least 2 of LCR, PCR, or AC2. A patient was considered infected with NG when either the culture result was positive or there was at least 1 LCR-positive (cx or FCU) and 1 AC2-positive (cx or FCU) test result. Patients who were culture-negative, or who had a cx or FCU positive in a single amplification test or 2 positive results by only 1 amplification test were considered to be “uninfected.”
Apparent AC2 CT and NG false-positives (AC2+/LCR- and PCR- or culture-) were tested by another TMA assay. These specimens along with masked positive and negative controls were sent to Gen-Probe for testing. The confirmatory TMA targets an alternate rRNA molecule (16S) for CT and the 23S and 16S for NG.
Of the 1569 female subjects enrolled in the study, we had complete specimen and test results for 1489 NG patients and for 1411 CT patients. Of these subjects, 59.8% (890 of 1489) were symptomatic and 40.2% (599 of 1489) were asymptomatic. CT and NG prevalence was 13.3% (188 of 1411) and 8.7% (130 of 1489) for cx; and 13.7% (193 of 1411) and 7.9% (118 of 1489) for FCU specimens, respectively.
Tables 1 and 2, respectively, show the CT and NG performance profiles obtained with the cx swabs by specimen standard or infected patient standard. Based on specimen standard, there were 183 chlamydia cx swab-positive results. Thirty-two cx specimens tested positive only by AC2, 7 cx specimens tested positive only by LCR, and 9 cx specimens tested positive only by PCR. By definition, these results were categorized as false-positive (FP) for the specimen performance calculation. For the infected patient calculations, 5 of the cx specimens were classified as true-positives based on the FCU specimen result. Fourteen additional infected subjects were also identified, because the FCU specimen for these subjects tested positive in 2 NAATs. Thus, a total of 19 positives were added. This in effect lowered the sensitivity of each test to detect CT in cx specimens by 7% to 9% per test. Overall, specificities changed slightly.
There were 128 NG cx swab-positive results based on specimen standard. Nineteen cx specimens tested positive only by AC2 and 4 cx specimens tested positive only by LCR. By definition, these were categorized as apparent FPs. For the infected patient evaluation, 2 of these cervical specimens were classified as positive based on the FCU specimen result. One additional positive FCU specimen was also identified. Thus, a total of 3 positive specimens were added. When compared with the infected patient standard, NG sensitivity was lowered slightly (1–2% per test) and specificities barely changed. The greatest sensitivity was seen with the AC2 assay.
Discrepant testing on cx AC2 specimens was performed on 28 apparent FP results for CT and 17 apparent FP results for NG. By alternate target TMA, 21 of 28 cx specimens were CT-positive. Based on these findings, sensitivity and specificity for AC2 compared with the CT-infected patient standard was 92.8% (207 of 223) and 99.4% (1181 of 1188), respectively. By alternate target TMA, 14 of 17 cx specimens were positive for NG. Based on these findings, the sensitivity and specificity for AC2 compared with the NG-infected patient standard was 98.6% (143 of 145) and 99.8% (1341 of 1344), respectively. Discrepant analysis was not done on the few LCR or PCR false-positives.
Table 3 shows the breakdown of CT and NG TP results by specimen site. There were 202 CT TP (>2 positive results with LCR, PCR, or AC2 assays in any combination). AC2 identified 200 of the CT TP, whereas LCR identified 198 and PCR identified 194. There were 131 NG TP. AC2 and LCR each missed 1 NG culture-positive specimen. FCU-only TP specimens were seen more often with CT testing as opposed to NG testing (approximately 10% vs. approximately 2%).
The current study demonstrates that the performance profile of diagnostic tests for CT or NG will vary depending on whether the evaluation is based on a single specimen standard or an infected patient standard. The infected patient standard reduces the sensitivity compared with a specimen from any single site (in this case the endocervix) evaluation because some infected patients test positive only at another site (in this case FCU). However, specificities can improve, because some unique cx swab positives, classified as FP for the cx swab evaluation, were confirmed as TP by the FCU specimen result used to define the infected patient. The improvement in specificity was greatest for CT, because more solitary FCU positives were identified (see Table 3). There were an additional 7% more positive subjects identified by testing FCU specimens for CT and only approximately 1% more positive FCU specimens were found for NG. Endocervical swabs detected the majority of NG infections. Thus, we observed only a small difference in NG test performance between the specimen and infected patient standards (1–2%). Table 2 shows that both AC2 and LCR were more sensitive than NG culture, with AC2 showing the greatest sensitivity.
Our results are based on a definition of a TP that requires any 2 amplification tests be positive. In a NAAT evaluation of CT tests, Johnson et al. 13 used several different reference standards to identify a CT-positive patient. In that study, no significant differences were reported when using a “rotating” standard versus a standard of any 2-reference positive test results.
With CT evaluations, the definition of a standard of infection affects sensitivity calculations more than specificity. Significant differences were observed in the comparison of a specimen versus an infected patient standard for AC2, LCR, and PCR (all P values were <0.002). Table 1 shows that CT sensitivities of the cervical swabs with the NAATs dropped by 7% to 9%. These findings are similar to Black et al., 12 in which NAAT sensitivities for cervical swabs dropped approximately 10% when compared with a 2-specimen reference standard, and specificities for LCR and PCR were 99.0% to 99.7%, respectively. Clinical evaluations should include 2 specimen types (swab and urine) to give a more accurate NAAT performance in identifying patients needing treatment. Use of just the specimen standard inflates sensitivities and misses some infected patients. Because NAAT specificities are a concern with CT testing, the use of >2 NAATs is recommended for future CT/NG evaluations. By doing so, more positives will be confirmed and higher (and more accurate) specificities will be calculated.
Of the 3 tests, the AC2 assay had the best CT sensitivity with LCR and PCR having overall lower but similar performances. AC2 specificities were somewhat lower than seen with the LCR and PCR tests. This pattern is typically seen when a new test is more sensitive than the tests used for its evaluation. Additional testing of the apparent false-positive specimens with alternate TMA amplification found many of the AC2 apparent false-positives to be true-positives (21 of 28 for CT and 14 of 17 for NG). Thus, overall CT and NG sensitivities (92.8% and 98.6%) and specificities (99.4% and 99.8%), respectively, increased for the AC2 assay, making the AC2 specificities comparable to the other NAATs. These results also confirm that the Gen-Probe APTIMA Combo 2 assay is a highly sensitive and specific DNA amplification assay for the detection of CT and NG in cx specimens.
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