Sexually Transmitted Diseases:
Vaginal Swabs Are the Specimens of Choice When Screening for Chlamydia trachomatis and Neisseria gonorrhoeae: Results From a Multicenter Evaluation of the APTIMA Assays for Both Infections
Schachter, Julius PhD*; Chernesky, Max A. PhD†; Willis, Dean E.‡; Fine, Paul M. MD§; Martin, David H. MD∥; Fuller, Deanna¶; Jordan, Jeanne A.**; Janda, William††; Hook, Edward W. III MD‡‡
From the *University of California, San Francisco, California; †St. Joseph’s Healthcare, Hamilton, Ontario, Canada; ‡Florida State Department of Health, Jacksonville, Florida; §Planned Parenthood of Houston and Southeast Texas, Houston, Texas; ∥Louisiana State University Medical Center, New Orleans, Louisiana; ¶Wishard Memorial Hospital, Indianapolis, Indiana; **McGee Women’s Research Institute, Pittsburgh, Pennsylvania; the ††University of Illinois, Chicago, Illinois; and the ‡‡University of Alabama, Birmingham, Alabama
The authors thank Jeannine R. Lane and Songbai Wang (Gen-Probe, Inc.) for their assistance in the data analysis.
Preliminary reports based on parts of this study were presented at the International Society for Sexually Transmitted Diseases Research Congress (July 27–30, 2003, Ottawa, Ontario, Canada), International Society for Infectious Diseases (March 4–7, 2004, Cancun, Mexico), 2004 National STD Prevention Conference (March 8–11, 2004, Philadelphia, Pennsylvania), the 104th General Meeting of the American Society for Microbiology (May 23–27, 2004, New Orleans, Louisiana), the 5th Meeting of the European Society for Chlamydial Research (September 1–4, 2004, Budapest, Hungary), and at the 42nd Annual Meeting of the Infectious Diseases Society of America (September 30–October 3, 2004, Boston, Massachusetts).
The authors acknowledge the funding of Gen-Probe Inc. for this study. The study sponsor had no role in the conduct of the study, the interpretation of the data, or in approval of the manuscript. They provided kits and reagents at no cost to each of the study sites and assisted in the collection and analysis of the data.
Dr. Schachter wrote the manuscript, had full access to all of the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis. Each author is responsible for the results obtained at a participating clinic site and participated in the critical revision of this manuscript.
Correspondence: Julius Schachter, PhD, Chlamydia Research Laboratory, Department of Laboratory Medicine, University of California, San Francisco, 1001 Potrero Avenue, SFGH 3416, San Francisco, CA 94110.
Received for publication January 25, 2005, and accepted April 22, 2005.
Background: Vaginal swabs were recently U.S. Food and Drug Administration-cleared for detecting Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) using Gen-Probe Incorporated’s APTIMA COMBO2 Assay (AC2). We assessed the APTIMA CT Assay (ACT) for CT, APTIMA GC Assay (AGC) for GC, and AC2 for both organisms using patient- and clinician-collected vaginal swabs.
Method: Women attending family planning, obstetrics and gynecology, or sexually transmitted disease (STD) clinics had first-catch urines (FCUs), patient-collected vaginal swabs, clinician-collected vaginal swabs, and endocervical swabs tested by ACT, AGC, and AC2. A second endocervical swab and FCU were tested using BD ProbeTec (Becton Dickinson) for CT and GC. We calculated sensitivity and specificity using vaginal swabs to detect CT and GC.
Results: Of 1464 subjects enrolled, 180 had CT and 78 GC. ACT sensitivities and specificities for patient-collected vaginal swabs were 98.3% and 96.5%, respectively; for clinician-collected vaginal swabs, 97.2% and 95.2%, respectively. AGC sensitivities and specificities for patient-collected vaginal swabs were 96.1% and 99.3%, respectively; for clinician-collected vaginal swabs, 96.2% and 99.3%, respectively. AC2 results were similar. If an FCU tested positive for CT or GC, >94% of matching vaginal swabs were positive. Positive endocervical swabs showed slightly less concordance (>90% and >88%, respectively). More infected patients were identified using vaginal swabs than FCUs. With AC2, 171 CT-infected patients were identified using FCUs and 196 using patient-collected vaginal swabs. This difference was more pronounced for CT than for GC.
Conclusions: Vaginal swab specimens allowed sensitive and specific detection of CT and GC in the APTIMA assays. Vaginal swabs identified as many infected patients as endocervical swabs and more than FCUs, and may well be the specimen of choice for screening.
NUCLEIC ACID AMPLIFICATION TESTS (NAATs) are the most sensitive tests available for the diagnosis of genital tract infections with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC).1 A major advantage of NAATs is the ability to use them with noninvasively collected specimens. When NAATs were introduced, it was shown that they could be used to test first-catch urine (FCU) specimens from men, and that the performance profiles with FCUs were very similar to those seen with urethral swabs.2,3 Shortly thereafter, FCUs from females, when tested by NAATs, were shown to have performance profiles similar to those reported for endocervical swab specimens.4,5 The ability to use FCUs from men and women meant that specimens could be collected in places other than traditional clinical settings. Noninvasive collection of specimens has made it far easier to test asymptomatic individuals and has also made it possible to perform studies aimed at determining population-based prevalence and even incidence of these infections. After FCUs were found to be useful specimens, a number of researchers evaluated vaginal swab specimens, and these too were found to be highly effective for detecting both chlamydial and gonococcal infections using NAATs.6–9 The potential advantage of the vaginal swab specimen is that it requires less processing than FCUs with most NAATs (these typically require centrifugation to sediment the potential target for the NAATs, except for the APTIMA assays, which offer greater stability and do not require centrifugation). A large multicenter trial confirmed the superior performance of NAATs with vaginal swabs, as compared with FCUs, and found that all 3 NAATs that were commercially available at the time (LCR [LCx Probe System, Abbott Laboratories, Abbott Park, IL]; PCR [Amplicor; Roche Molecular Systems, Branchburg, NJ]; TMA [Gen-Probe Amplified CT Assay; Gen-Probe Inc., San Diego, CA]) performed well with vaginal swabs.10
Patients can easily collect vaginal swab specimens, and a number of studies have shown that patient- and clinician-collected specimens are essentially equivalent. In other words, patients are equally adept at collecting vaginal swab specimens as clinicians. Thus, a female patient need not be seen by a clinician to collect the specimens for this sexually transmitted disease (STD) testing. Even if a clinician sees the patient, vaginal swab specimens do not require a pelvic examination. Thus, the use of vaginal swabs would greatly improve the cost benefit in screening for these genital tract infections.11
Recently, Gen-Probe introduced a new family of assays (APTIMA) that use their transcription-mediated amplification (TMA) technology. These assays include the APTIMA CT Assay (ACT), which detects CT, the APTIMA GC Assay (AGC) for detecting GC, and the APTIMA COMBO 2 Assay (AC2), which detects both organisms. These second-generation NAATs add target capture specimen processing (to remove potential inhibitors from samples) to the TMA and the dual kinetic assay (DKA) detection technology. Target capture partially purifies CT and GC rRNA from the specimens, TMA amplifies the rRNA, and DKA detects and discriminates CT and GC amplified product. The ACT and AGC use oligonucleotides that target rRNA sequences different from those of the AC2. The procedure for all 3 tests is the same. Initial evaluations of these tests have shown them to be highly sensitive and specific for diagnosing chlamydial and gonococcal infections.12,13 The purpose of this study was to evaluate vaginal swabs as a specimen to detect CT and GC with Gen-Probe’s APTIMA assays.
One thousand four hundred sixty-four symptomatic and asymptomatic female subjects attending STD, obstetric and gynecology, teen, and family planning clinics were enrolled in this study. Specimens were collected from 9 centers in North America. Subjects were classified as symptomatic if they reported symptoms such as discharge, dysuria, and pelvic pain or as asymptomatic if they did not report symptoms.
Five specimens were collected from each female: an FCU, a patient-collected vaginal swab, a clinician-collected vaginal swab, and 2 endocervical swabs. FCU was collected first, followed by the patient-collected vaginal swab, the clinician-collected vaginal swab, and then 2 randomized endocervical swabs. Each specimen type was tested using the ACT, AGC, and AC2 assays. In addition, the FCU and the second endocervical swab specimen were tested using the BDProbeTec ET System CT/GC Assays (BD) (BD Diagnostic Systems, Sparks, MD). All tests were performed following the manufacturer’s instructions. For the purposes of this evaluation, the subject was considered infected if any 2 of the U.S. Food and Drug Administration (FDA)-cleared tests (BD and AC2) were positive on FCUs or endocervical swabs.
The clinical characteristics and demographics of the subject population are shown in Table 1. Of the 1464 subjects enrolled, there were 13 subjects with unknown CT patient-infected status and 14 subjects with unknown GC patient-infected status as a result of missing results. The results from these patients were not included in the performance calculations. There were 180 CT- (12.4%) and 78 (5.4%) GC-infected subjects using the infected patient definition described here (Table 2).
Table 3shows the sensitivity and specificity of clinician-collected vaginal swabs and patient-collected vaginal swabs in each of the assays for CT and GC infection. There was no difference in results obtained with specimens from symptomatic or asymptomatic subjects. The results obtained with clinician-collected vaginal swabs and patient-collected vaginal swabs were very similar. It should be noted that the sensitivity for each was >95%.
There was good agreement between the results obtained with the vaginal swab and the endocervical swab specimens. If the endocervical swab was chlamydia-positive, >90% of the matching vaginal swab specimens were positive (Table 4). The results with GC were similar, although there was slightly less concordance (>88%) with positive endocervical swab specimens. Agreement was even better with positive FCUs in which there was >95% agreement in most of the comparisons. Results obtained with specimens from symptomatic and asymptomatic individuals were essentially the same (data not shown).
Table 5shows a comparison of the results obtained with the AC2 assay on vaginal swab specimens and the results obtained with the BD assay on FCUs and endocervical swab specimens. There were many positive results on either patient-collected vaginal swabs or clinician-collected vaginal swabs by AC2 (for both CT and GC) that were negative by the BD assay on the matching FCUs or endocervical swab specimens. For example, there were 149 positive concordant results for CT by the AC2 on patient-collected vaginal swabs and BD on FCUs. There were 6 paired specimens in which the BD test on FCUs was positive and the AC2 patient-collected vaginal swab result was negative. However, there were 49 positive AC2 patient-collected vaginal swab specimens with matching FCUs negative by BD. The increment in positive results was greater for CT than was seen with the GC assays.
There were a number of apparent false-positive results with the vaginal swab specimens. These false-positives were observed in samples from patients who were not scored as infected by the definition of infected status that required 2 or more positive results with endocervical swab or FCU specimens. The majority of apparent false-positive ACT patient-collected vaginal swabs (57%; 24 of 42) and clinician-collected vaginal swabs (46%; 28 of 61) or AGC patient-collected vaginal swabs (67%; 6 of 9) and clinician-collected vaginal swabs (78%; 7 of 9) results were also positive in the AC2, which has different targets than the ACT or AGC. The same general pattern of confirmatory results was seen with apparent false-positive results in the AC2 for CT/GC. In this instance, the ACT confirmed 86% (24 of 28) of apparent false-positive patient-collected vaginal swabs and 76% (28 of 37) of clinician-collected vaginal swabs, whereas the AGC confirmed 6 of 6 apparent false-positive patient-collected vaginal swabs and 7 of 7 clinician-collected vaginal swabs.
A similar strategy of confirming apparent false-positives in the AC2 assay with endocervical swabs and FCUs increased the number of true-positive results above the numbers found in Table 2. There were 171 CT-infected women identified by FCU and 194 by endocervical swab, compared with the 196 detected with patient-collected vaginal swab (chi-squared with Yates correction 21, P <0.00001).
The sensitivities and specificities for vaginal swab specimens in the ACT and AGC assays were quite high. There was no difference seen in the performance of these tests with the patient-collected vaginal swab or clinician-collected vaginal swab specimens. Patients were equally adept as clinicians at collecting the specimens. When a subset of patients in this study was queried as to ease of collection and specimen preference, a large majority found it easy to collect and preferred vaginal swabs to FCUs or having a pelvic examination to collect an endocervical swab.14 Results obtained with patient-collected vaginal swabs or clinician-collected vaginal swabs were in high agreement with the positive results obtained with FCUs (>95%) or endocervical swabs (>90%) in the APTIMA assays. There were more positive results obtained with the vaginal swabs than were obtained with either FCUs or endocervical swabs (significantly so for FCUs). Although most studies in the literature have found essential equivalence in the results obtained between vaginal swabs and these other specimens, some have found a higher positivity rate with vaginal swab specimens than with either endocervical swabs or FCUs.15
It is likely that the vaginal swab specimen combines material from 2 potential sites of infection: urine or discharge coming from the urethra and discharge from the endocervix. These anatomic sites (cervix and urethra) can be infected alone or in combination. Specimens collected from the endocervix obviously do not reflect urethral infections, and the converse may also be true. With NAATs, although both FCUs and vaginal swabs are contaminated, there is more target present in vaginal swab specimens than in FCUs.16 This is likely the result of dilution in the FCU and the greater contribution of cervical discharge to the vaginal swab, and probably explains the different rates of positivity. The analysis of the apparent false-positive results obtained with vaginal swab specimens with the APTIMA assays shows that the vaginal swab specimens are identifying infected individuals who are not so classified using the algorithm that requires 2 positive results with endocervical swab and FCU specimens.
Thus, our study is another in a series of reports suggesting that vaginal swabs are appropriate specimens for diagnosing genital chlamydial and gonococcal infections using NAATs. The evidence supporting the use of vaginal swab specimens is impressive, and members of the research community have encouraged manufacturers of diagnostic tests to seek FDA clearance for the use of vaginal swab specimens in their tests.10 Until recently, vaginal swabs had not been cleared for use in any diagnostic test. It is noteworthy that the FDA has cleared use of vaginal swabs for CT and GC detection using the APTIMA COMBO 2 Assay.
The excellent sensitivity and specificity obtained with vaginal swab specimens, the ease of and preference for self-collection, the ease of processing, and the essential equivalence with positive FCU and endocervical swab results all suggest that the vaginal swab specimen is the specimen of choice when screening for these genital infections in women. Symptomatic women will likely require a pelvic examination and cervical specimens will be collected. We found essential equivalence between vaginal swabs and endocervical swabs for detecting these infections. However, a vaginal swab is preferable to FCUs in most settings and could replace endocervical swabs for selected uses, because many, or more, infected women are identified using vaginal swabs as with any other specimen.
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13. Hook EW, Martin DH, Schachter J, et al. Patient- and clinician-collected vaginal swabs: Performance of the Aptima Combo 2 assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. In: Proceedings of the International Society of Sexually Transmitted Disease Research; Ottawa, CA; July 27–30, 2003; Abstract 515.
14. Chernesky MA, Hook EW, Martin DH, et al. Women find it easy and prefer to collect their own vaginal swabs which are effective for the diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae. Sex Transm Dis. In press.
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